Abstract: Early embryonic development is critically dependent on both maternal preparation and embryonic signalling of pregnancy. Matrix metallopeptidases (MMP) contribute to spatial and temporal matrix remodeling in the bovine endometrium. In this study we observed distinct changes in expression of MMP2, MMP14, and the metallopeptidase inhibitor TIMP2 between different phases of the estrous cycle indicating an endocrine regulation. An increase of TIMP2 protein abundance was ascertained in the uterine lumen during the time of embryo elongation. The expression pattern and cellular localization correlate well with the assumed effects of MMPs on release and activation of cytokines and growth factors directing cell migration, differentiation, and vascularization during this pivotal period of development. Specifically, active MMP2 in the endometrium may determine the allocation of growth factors supporting conceptus development. The presence of a day 18 conceptus in vivo and day 8 blastoysts in vitro induced endometrial TIMP2 mRNA expression. The results imply that TIMP2 is involved in very early local maternal recognition of pregnancy. Matrix metallopeptidases are likely to participate in remodeling processes preparing a receptive endometrium for a timely and precise regulation of embryo development.
Abstract: The importance of using techniques that allow the study of pure populations of cells has been increasingly recognized. The authors used laser-assisted microdissection (LAM) in combination with quantitative real-time PCR (qPCR) to assess the relative expression of mRNAs encoding estrogen receptor α (ERα) and progesterone receptor (PR) in the different compartments of the bovine oviduct (epithelium, stroma, smooth muscle coat) during the follicular and mid-luteal phases of the estrus cycle. The localization of receptor mRNA was further studied using non-radioactive in situ hybridization (NISH). A special focus was on whether formalin fixation and paraffin embedding influence the quality and quantity of mRNA obtained from microdissected material. Distinct cyclic changes of the mRNA in the bovine oviduct were observed with elevated levels of PR mRNA transcripts in the epithelium and smooth muscle coat during the follicular phase. The expression of PR mRNA did not vary significantly in the stroma of the bovine oviduct during follicular and mid-luteal phases. In conclusion, the authors found that LAM with qPCR can precisely locate and accurately quantify mRNA expression in specific cell populations from formalin-fixed and paraffin-embedded oviductal tissue.
Abstract: Lentiviral vectors are a powerful tool for the genetic modification of livestock species. We previously generated transgenic founder cattle with lentiviral integrants carrying enhanced green fluorescent protein (EGFP) under the control of the phosphoglycerate kinase (PGK) promoter. In this study, we investigated the transmission of LV-PGK-EGFP integrants through the female and male germ line in cattle. A transgenic founder heifer (#562, Kiki) was subjected to superovulation treatment and inseminated with semen from a non-transgenic bull. Embryos were recovered and transferred to synchronized recipient heifers, resulting in the birth of a healthy male transgenic calf expressing EGFP as detected by in vivo imaging. Semen from a transgenic founder bull (#561, Jojo) was used for in vitro fertilization (IVF) of in vitro matured (IVM) oocytes from non-transgenic cows. The rates of cleavage and development to blastocyst in vitro corresponded to 52.0 +/- 4.1 and 24.5 +/- 4.4%, respectively. Expression of EGFP was observed at blastocyst stage (day 7 after IVF) and was seen in 93.0% (281/302) of the embryos. 24 EGFP-expressing embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos, flushed from the uterus on day 15, two fetuses recovered on day 45, and a healthy male transgenic calf revealed consistent high-level expression of EGFP in all tissues investigated. Our study shows for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. The pattern of inheritance was consistent with Mendelian rules. Importantly, high fidelity expression of EGFP in embryos, fetuses, and offspring of founder #561 provides interesting tools for developmental studies in cattle, including interactions of gametes, embryos and fetuses with their maternal environment.
Abstract: A histochemical study using conventional carbohydrate histochemistry (periodic-acid staining including diastase controls, alcian blue staining at pH 1 and 2.5) as well as using a battery of 14 fluorescein isothiocyanate (FITC)-labelled lectins to identify glycoconjugates present in 10 different areas of the skin of a catfish (Arius tenuispinis) was carried out. The lectins used were: mannose-binding lectins (Con A, LCA and PSA), galactose-binding lectins (PNA, RCA), N-acetylgalactosamine-binding lectins (DBA, SBA, SJA and GSL I), N-acetylglucosamine-binding lectins (WGA and WGAs), fucose-binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E, PHA L). Conventional glycoconjugate staining (PAS staining, alcian blue at pH 1 and 2.5) showed that the mucous goblet cells contain a considerable amount of glycoconjugates in all locations of the skin, whereas the other unicellular gland type, the club cells, lacked these glycoconjugates. The glycoproteins found in goblet cells are neutral and therefore stain magenta when subjected to PAS staining. Alcian blue staining indicating acid glycoproteins was distinctly positive at pH 1, but gave only a comparable staining at pH 2.5. The mucus of the goblet cells therefore also contains acid glycoproteins rich in sulphate groups. Using FITC-labelled lectins, the carbohydrate composition of the glycoproteins of goblet cells could be more fully characterized. A distinct staining of the mucus of goblet cells was found with the mannose-binding lectins LCA and PSA; the galactosamine-binding lectins DBA, SBA and GLS I; the glucosamine-binding lectin WGA; and PHA E which stains glycoproteins with complex carbohydrate configurations. No reaction occurred with the fucose-binding lectin UEA and the sialic acid-specific lectin SNA. In addition, the galactose-binding lectins PNA and RCA showed only a weak or completely negative staining of the mucus in the goblet cells. The specificity of the lectin staining could be proved by inhibiting binding of the lectins by competitive inhibition with the corresponding sugars. From these data, we can conclude that the mucus produced by the epidermal goblet cells of A. tenuispinis is rich in mannose, N-acetylgalactosamine and N-acetylglucosamine residues.
Abstract: Progesterone (P4) is synthesized in the luteal cells of many species. The objective of the present study was to determine the expression pattern of P4 receptor (PR) mRNA and the distribution of PR protein in the bovine corpus luteum (CL) during the estrous cycle. The gene expression of PR in the bovine CL throughout the estrous cycle was determined by real-time PCR analysis, and the PR protein expression was evaluated by immunohistochemistry. Messenger RNA of PR was clearly expressed in the CL throughout the estrous cycle. The level of PR mRNA in the CL was highest at the early stage of the estrous cycle and was higher at the mid and late stages than at the regressed stage (P<0.01). In regard to the distribution of PR, the protein was expressed in both small and large luteal cells and in vascular endothelial cells throughout the estrous cycle. These results suggest that P4 has a role in regulating luteal and endothelial cell function in the bovine CL, especially at the early luteal stage as an autocrine/paracrine regulator.
Abstract: Laser-assisted microdissection (LAM) is an important method to provide new significant insights into many embryological processes. To understand these processes, it is important to obtain specific populations of cells from complex tissue in an efficient and precise manner and to combine with many different molecular biological methods. During the last few years, the sophistication of the techniques of LAM has increased significantly and made the procedure easy to use. New micro-extraction protocols for DNA, RNA and proteins now allow broad downstream applications in the fields of genomics, transcriptomics and proteomics. In this review, we give a short overview of the application of LAM in combination with quantitative qPCR for the analysis of gene expression in mammalian germ cells.
Abstract: The emerging insights into glycan functionality direct increasing attention to monitor core modifications of N-glycans and branch-end structures. To address this issue in histochemistry, a panel of lectins with respective specificities was devised. The selection of probes with overlapping specificities facilitated to relate staining profiles to likely target structures. The experiments on fixed sections of adult murine testis and epididymis were carried out at non-saturating lectin concentrations to visualize high-affinity sites with optimal signal-to-background ratio. They revealed selectivity in lectin reactivity for distinct cell types and segment-dependent staining in the epididymis. Leydig cells, for instance, were reactive with the Sambucus nigra agglutinin and human siglec-2 (CD22), two lectins also separating principal from basal and apical cells in the caput segments I-III of the epididymis. Apical cells were reactive with the Maackia amurensis agglutinin-I, and basal cells with the erythroagglutinin of Phaseolus vulgaris. The reported differences support the concept of lectin staining as cell marker. They thus intimate to study glycogene (genes for glycosyltransferases and lectins) expression and cellular reactivity with tissue lectins. These investigations will be instrumental to assign a role as biochemical signals to the detected staining properties.
Abstract: Thyroid cancer is the second most common malignancy following breast cancer in Arab females. Thioredoxin (TRX) is a small multi-functional redox protein with both intracellular and extracellular functions. The protein exists in either a reduced form (thioredoxin-SH2) or an oxidized form (thioredoxin-S2). TRX acts as an enhancement for growth factors and stimulates the growth of cancer cells. In this study of thyroid neoplasms, involving 121 female and 62 male patients, expression of TRX and TRX-R was studied using purified mouse anti-human TRX monoclonal antibody and anti-human TRX-R antiserum from rabbits, respectively. In order to delineate tumour cell growth, proliferating cell nuclear antigen (PCNA) polyclonal antibody was used. Compared to normal thyroid tissue, expression of TRX and TRX-R was increased in the cytoplasm and nuclei of thyroid cancer cells. Furthermore, TRX expression correlated with that of TRX-R. Of the 183 thyroid neoplasms investigated, overexpression of TRX-R was found in different types of neoplasms. The majority of carcinomas showed a correlation between strongly positive TRX and TRX-R expression and neoplastic cellular proliferation, as measured by PCNA. This indicates that increased TRX and TRX-R expression may be associated with tumourigenesis by acting as an autocrine growth stimulus. This study suggests that TRX immunoreactivity in thyroid tumours is a function of malignancy and cancer progression. In addition, secreted TRX can also act as an extracellular growth factor for both normal and tumour cells and enhance the sensitivity of the cells. Furthermore, this study emphasizes the potential benefits of anti-TRX/TRX-R agents in cancer therapeutics in the treatment of thyroid cancer.
Abstract: To determine which cell types produce tumor necrosis factor-α (TNF) and its receptors (TNFRI and TNFRII) in bovine endometrium, we investigated the expression and cellular localization of their mRNAs and proteins. TNF transcripts and proteins were co-localized in endometrial epithelial cells, glandular epithelial cells and endothelial cells of microvessels but not in the stromal cells. TNF protein was detected in the lysate and the cultured media of epithelial cells, but was only weakly detected in the stromal cells. Both TNFRI (TNFRSF1A) and TNFRII (TNFRSF1B) transcripts were expressed in the epithelial cells, glandular epithelial cells and the stromal cells, whereas their proteins were weakly expressed in the stroma. TNF mRNA and protein expressions in the cultured epithelial cells were increased by TNF and interleukin-1α, and the TNFRII mRNA expressions were stimulated by oxytocin. Together, TNF secreted by the endometrial cells may locally play a role in regulating uterine function throughout the estrous cycle.
Abstract: A histochemical study using fluorescein isothiocyanate (FITC)-labelled lectins to identify glycoconjugates present in the efferent ductules and the three segments of the ductus epididymis (initial, middle and terminal segment) of dogs was carried out. The lectins used were: mannose-binding lectins (Con A, LCA and PSA), galactose-binding lectins (PNA, RCA), N-acetylgalactosamine-binding lectins (DBA, SBA, SJA and GSL I), N-acetylglucosamine-binding lectins (WGA and WGAs), fucose-binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E and PHA L). The lectin-binding pattern in the canine epididymis presents similarities and differences to those observed in other mammalian species. The ductuli efferentes distinctly stained with most of the lectins used, whereas in the ductus epididymis a segment specific staining pattern was observed. Whereas principal cells of the ductus epididymis stained clearly with several FITC-labelled lectins (WGA, UEA and PHA-L), basal cells showed only a significant binding of Con A.
Abstract: Selenium is linked to male fertility. Glutathione peroxidase 4 (GPx4), first described as an antioxidant enzyme, is the predominant selenoenzyme in testis and has been suspected of being vital for spermatogenesis. Cytosolic, mitochondrial, and nuclear isoforms are all encoded by the same gene. While disruption of entire GPx4 causes early embryonic lethality in mice, inactivation of nuclear GPx4 does not impair embryonic development or fertility. Here, we show that deletion of mitochondrial GPx4 (mGPx4) allows both normal embryogenesis and postnatal development, but causes male infertility. Infertility was associated with impaired sperm quality and severe structural abnormalities in the midpiece of spermatozoa. Knockout sperm display higher protein thiol content and recapitulate features typical of severe selenodeficiency. Interestingly, male infertility induced by mGPx4 depletion could be bypassed by intracytoplasmic sperm injection. We also show for the first time that mGPx4 is the prevailing GPx4 product in male germ cells and that mGPx4 disruption has no effect on proliferation or apoptosis of germinal or somatic tissue. Our study finally establishes that mitochondrial GPx4 confers the vital role of selenium in mammalian male fertility and identifies cytosolic GPx4 as the only GPx4 isoform being essential for embryonic development and apoptosis regulation.
Abstract: Frozen-thawed sexed semen from six bulls (Holstein) was used for studying their efficiency in an in vitro fertilization (IVF)-programme and to compare their ultrastructure with in vitro produced bovine blastocysts produced with non-sorted sperm. Progressive motility of sorted spermatozoa, their IVF rate, development of produced blastocysts and the ultrastructure of the blastocysts were analysed. The cleavage rates of sexed sperm of bulls (groups S1, S2 and S4) were significantly lower than that of unsorted control sperm (P < 0.01). Blastocyst development at day 7 of the sexed semen groups varied between 3.5% and 28.8% versus 33.6% for non-sexed semen. The individual blastocyst yield with sexed semen of group S5 (28.8%) was similar to the mean blastocyst production of the non-sexed control spermatozoa (C, 33.6%; P > 0.05). The remaining five sexed sperm groups resulted in significantly lower developmental rates of blastocysts on day 7 (S1, 4.9%; S2, 0%; S3, 0%, S4, 3.5%; S6, 25.8%, P < 0.01). Group S2 showed microbiological contamination in 50% (four of eight) and S3 in 100% of the experiments (eight of eight). Progressive motility of sexed sperm was significantly lower than that of unsorted sperm (S1, 48 +/- 12.0%; S2, 41 +/- 11.9%; S3, 39.0 +/- 9.9%; S4, 42 +/- 4.6%; P < 0.01; S5, 72 +/- 7.1% and S6, 64 +/- 9.3; P < 0.05 versus C 82 +/- 4.6%). The percentage of progressive motile spermatozoa showed a good correlation with the developmental capacity of blastocysts (r(2): >0.70), the regression parameter was significant (P < 0.01). Furthermore, with a straw containing 10 x 10(6) sexed spermatozoa significantly lower number oocytes was fertilized than with the same concentration of non-sexed sperm (P < 0.01). Our results demonstrate that the suitability of sperm sorting for in vitro fertilization (IVF) is lower than no sexed sperm. Our ultrastructural studies showed that blastocysts produced with flow-cytometrically sex-sorted spermatozoa possessed deviations in the number and structure of organelles like mitochondria, rough endoplasmic reticulum (ER) and nuclear envelope. These morphological alterations may be responsible for compromised development that observed in embryos produced with sex-sorted spermatozoa. Thus, we conclude that sperm sex sorting can markedly affect the efficiency of an IVF-programme.
Abstract: The epidermal growth factor receptor (EGFR) and its ligands are emerging as key molecules in regulating female reproduction. Here, we used a transgenic mouse model to evaluate whether and at which level of the reproduction cascade higher-than-normal levels of the EGFR ligand betacellulin (BTC) in the reproductive organs affect fertility. Western blots and immunohistochemistry revealed increased BTC levels in uterus and ovaries from transgenic females, particularly evident in granulosa cells of antral follicles. Onset of puberty, estrous cyclicity, and the anatomy and histology of reproductive organs at puberty were not altered as compared to control females. Fertility tests revealed a reduction (~50%) in litter size as the major reproductive deficit of transgenic females. Embryo implantation was delayed in transgenic females, but this was not the reason for the reduced litter size. Transgenic females produced a normal number of oocytes after natural ovulation. The in vivo fertilization rate was significantly reduced in untreated transgenic females but returned to normal levels after superovulation. Impaired oocyte fertilization in the absence of superovulation treatment was associated with MAPK3/MAPK1 hyperactivation in BTC transgenic ovaries, whereas similar levels of MAPK3/MAPK1 activation were detected in transgenic and control ovaries after superovulation treatment. Thus, tight regulation of MAPK3/MAPK1 activity appears to be essential for appropriate granulosa cell function during oocyte maturation. Our study identified hitherto unknown effects of BTC overabundance in reproduction and suggests BTC as a novel candidate protein for the modulation of fertility.
Abstract: The chance of an intrathyroidal occurrence of a parathyroid gland is about 1-3%. Among the causes of hyperparathyroidism, parathyroid cases occur in less than 1% of patients. Here we present the case of a 63 year old Saudi female suffering from an intrathyroidal parathyroid carcinoma. The suspicion coming from the clinical investigations that the removed tumor tissue may be a parathyroid carcinoma could be confirmed by histology. Additionally non-radioactive in situ hybridization to localize mRNA transcripts for Cyclin D1 and immunohistochemical localization of Cyclin D1 was performed. Although parathyroid adenoma and carcinoma have disparate natural history, it can be difficult to differentiate between the two entities. Clinical presentation, operative findings may raise suspicion, but may not be conclusive especially if there is no evidence of invasion or metastasis, especially if the gland was intrathyroidal.
Abstract: During the oestrous cycle, the bovine endometrium exhibits characteristic morphological and functional changes, which are mainly induced by progesterone (P(4)), oestrogens and oxytocin. We studied the response of the endometrium to this changing hormonal environment at the transcriptome level using a custom-made cDNA microarray. Endometrium samples were recovered from Simmental heifers on days 0 (oestrus), 3.5 (metoestrus), 12 (dioestrus) and 18. The latter group was divided into animals with high (late dioestrus) and low P(4) levels (preoestrus). Significance analysis of microarrays revealed 269 genes exhibiting significant changes in their transcript levels during the oestrous cycle in distinct temporal patterns. Two major types of expression profiles were observed, which showed the highest mRNA levels during the oestrus phase or the highest levels during the luteal phase respectively. A minor group of genes exhibited the highest mRNA levels on day 3.5. Gene ontology (GO) analyses revealed GO categories related to extracellular matrix remodelling, transport, and cell growth and morphogenesis enriched at oestrus, whereas immune response and particular metabolic pathways were overrepresented at dioestrus. Generation of gene interaction networks uncovered the genes possibly involved in endometrial remodelling (e.g. collagen genes, TNC, SPARC, MMP2, MEP1B, TIMP1, TIMP2, HTRA1), regulation of angiogenesis (e.g. ANGPTL2, TEK, NPY, AGT, EPAS1, KLF5 ), regulation of invasive growth (e.g. PCSK5, tight junction proteins, GRP, LGALS1, ANXA2, NOV, PLAT, MET, TDGF1, CST6, ITGB4), cell adhesion (e.g. MUC16, LGALS3BP) and embryo feeding (e.g. SLC1A1, SLC11A2, SLC16A1, SEPP1, ENPP1). Localisation of mRNA expression in the endometrium was analysed for CLDN4, CLDN10, TJP1, PCSK5, MAGED1, and LGALS1.
Abstract: The expression pattern of several intermediate-filament proteins (vimentin, cytokeratin 8, 18 and 19) and the basal lamina component laminin was investigated in the Wolffian and the Müllerian ducts of bovine embryos and fetuses. The material studied comprised sexually undifferentiated stages [crown-rump length (CRL) 0.9 cm/1.0 cm/1.2 cm/1.9 cm/2.5 cm] and female stages (CRL 3.0 cm/4.2 cm/5.1 cm). Laminin could be demonstrated in the basal lamina of the developing Wolffian and Müllerian duct as well as in the stroma surrounding the Müllerian duct. The intermediate-filament protein vimentin was expressed in the mesothelium of the funnel field and in the epithelium of the Müllerian duct in all studied specimens, whereas the epithelial cells of the Wolffian duct only showed vimentin expression from a CRL of 2.2 cm onwards. In the cranial part of the Müllerian ducts only a few cells stained with pan-cytokeratin antibodies, whereas mesothelium and epithelium of the Wolffian duct showed as distinct immunostaining in all investigated stages. Both genital ducts showed no immunostaining with the antibody against cytokeratin 19 at any time of development. We conclude from our immunohistochemical results that the epithelial cells of the Wollfian duct do not contribute cells to the developing Müllerian duct.
Abstract: In this study, we investigated the ultrastructural changes during the prenatal differentiation of oviductal epithelium in 16 bovine embryos and fetuses from CRL of 18.0 cm to a CRL of 94.0 cm. Ciliated and secretory cells of bovine uterine tube, a derivative of the Müllerian duct, differentiate to distinct development stages in the prenatal period. The typical cellular pattern, which is generally characteristic for the adult bovine oviduct, is also obtained during fetal life. In the early stages (CRL 18.0/20.4 cm), the bovine oviductal epithelium appears mostly undifferentiated. The epithelial cells show only a few mitochondria, some cisternae of rough endoplasmic reticulum (rER) and a small Golgi-complex. Most of the cytoplasm is filled with a large amount of glycogen, which decreases during later development. Interspersed between the undifferentiated epithelial cells, a few cells undergoing ciliogenesis can be observed. Ciliogenesis increased significantly during the later prenatal developmental stages. At a CRL of 55.0 cm, ciliated cells appear fully differentiated with mature cells covering their luminal surface. Formation of cilia usually use the acentriolar pathway. Fibrous granules occurred initially in association with the Golgi-apparatus and r(ER) in the supranuclear cytoplasm. Fibrous granules later fuse with deuterosomes and give rise to procentrioles, which are translocated to the luminal plasma membrane. There they become arranged in a line just beneath the apical cell membrane and further differentiate to basal bodies from which the formation of cilia and striated rootlets take place. Clear signs of differentiation of secretory cells were first seen in our material in fetuses with a CRL of 51.0 cm and 64.0 cm. These cells contain a well developed rER and Golgi-apparatus with dilated cisterns. In the supranuclear cytoplasm, the number of secretory granules continuously increases during later development and the cells adapt to the morphology of mature secretory cells at the CRL 94.0 cm.
Abstract: Galectin-3 is a multifunctional protein with modular design. A distinct expression profile was determined in various murine organs when set into relation to homodimeric galectins-1 and -7. Fittingly, the signature of putative transcription-factor-binding sites in the promoter region of the galectin-3 gene affords a toolbox for a complex combinatorial regulation, distinct from the respective sequence stretches in galectins-1 and -7. A striking example for cell-type specificity was the ovary, where these two lectins were confined to the surface epithelium. Immunohistochemically, galectin-3 was found in macrophages of the cortical interstitium between developing follicles and medullary interstitium, matching the distribution of the F4/80 antigen. With respect to atresia and luteolysis strong signals in granulosa cells of atretic preantral but not antral follicles and increasing positivity in corpora lutea upon regression coincided with DNA fragmentation. Labeled galectin-3 revealed lactose-inhibitable binding to granulosa cells. Also, slender processes of vital granulosa cells which extended into the zona pellucida were positive. This study demonstrates cell-type specificity and cycle-associated regulation for galectin-3 with increased presence in atretic preantral follicles and in late stages of luteolysis.
Abstract: In the equine, the zona pellucida (ZP) is the major barrier to successful in vitro fertilization. Therefore the aim of our studies was to analyze species-specific features of the equine ZP in regard to structure and glycoprotein ZPB and ZPC expression sites during oocyte development and embryogenesis. The equine ZP revealed high immunological cross-reactivity to porcine ZPB and ZPC. In the ovary, the distribution of ZPB and ZPC was co-localized and correlated with the developmental stage of the follicle. ZPB and ZPC expression started in the oocyte of the late primordial and primary follicle. In the secondary follicle, both the oocyte and the cumulus cells contributed to ZPB and ZPC synthesis. After in vivo maturation the oocyte stopped ZPB and ZPC production whereas the cumulus cells continued synthesis. Contrary, in vitro matured (IVM) cumulus-oocyte-complexes (COCs) revealed a reverse expression pattern. This was correlated to alterations in the distribution, number, and size of pores in the ZP. In the zona, N-acetylglucosamine residues were co-localized with ZPC. The acellular glycoprotein capsule surrounding early equine embryos was negative for ZPB and ZPC. Our results imply that in the horse ZPB and ZPC glycoprotein expression is differentially regulated during folliculogenesis, oocyte maturation, and embryogenesis. Contrary to the bovine and porcine, zona protein synthesis during in vivo maturation is completely overtaken by the cumulus cells implying that in the horse these cells are crucial for zona integrity. During IVM, the cumulus cells lose their ability to synthesize glycoproteins leading to alterations in the zona structure.
Abstract: The development of somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA, we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses of whole-mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos, even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, whereas later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent but was activated by a second round of SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses, providing an attractive model for systematic investigation of epigenetic reprogramming in large mammals.
Abstract: In degrading the extracellular matrix, matrix metalloproteinases (MMP) and the plasminogen activator (PA) system may play a critical role in extensive remodeling that occurs in the bovine mammary gland during development, lactation, and involution. Therefore, the aim of our study was to investigate the mRNA expression of MMP-1, MMP-2, MMP-14, MMP-19, tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, urokinase-type PA, tissue-type PA, urokinase-type PA receptor, and PA inhibitor-1 by quantitative PCR and to localize with immunohistochemistry MMP-1, MMP-2, MMP-14, and TIMP-2 proteins in the bovine mammary gland during pubertal mammogenesis, lactogenesis, galactopoiesis, and involution. Expression of mRNA for each of the studied factors was relatively lower during galactopoiesis and early involution but was markedly increased during mammogenesis and late involution, 2 stages in which tissue remodeling is especially pronounced. The localization of proteins for MMP-1, MMP-14, and TIMP-2 showed a similar trend with strong staining intensity in cytoplasm of mammary duct and alveolar epithelial cells during pubertal mammogenesis and late involution. Interestingly, MMP-2 protein was localized only in the cytoplasm of endothelial cells during late involution. Our study demonstrated clearly that expression of extracellular matrix-degrading proteinases coincides with a concomitant expression of their inhibitors. High expression levels of MMP, TIMP, and PA family members seem to be a typical feature of the nonlactating mammary gland.
Abstract: Leptin has been shown to exert positive effects during the maturation of bovine oocytes, influencing blastocyst development, apoptosis, and the transcript levels of developmentally important genes. The present study was conducted to characterize further the mechanisms of leptin action on oocytes and the role of cumulus cells (CCs) in this context. In the first series of experiments, cumulus-oocyte complexes (COCs) were matured in serum-free medium that contained 0, 1 or 10 ng/ml leptin or in medium that was supplemented with 10% (v/v) estrus cow serum (ECS). Leptin concentrations of 1 and 10 ng/ml stimulated the meiotic progression of oocytes. Moreover, TUNEL staining demonstrated that these leptin doses reduced the proportion of apoptotic CCs. In the second series of experiments, COCs or denuded oocytes (DOs) were matured in the presence of 0 or 10 ng/ml leptin. The percentages of COCs and DOs with extruded polar bodies were increased by leptin. In contrast, positive effects of leptin on fertilization rates and blastocyst development were only observed after treatment of COCs but not of DOs. Leptin treatment of COCs consistently enhanced blastocyst development even after parthenogenetic activation of oocytes or after the removal of CCs before fertilization. The proportion of polyspermic oocytes was not affected by leptin treatment or oocyte denudation. In the third series of experiments, COCs were matured in the presence of 0, 1 or 10 ng/ml leptin. The transcript levels of specific genes were determined by reverse transcriptase-quantitative PCR (RT-qPCR) analysis of cumulus cells and single oocytes. Leptin treatment increased the levels of FAS, FASLG, and STAT3 transcripts in oocytes, but did not affect the LEPR, BAX, and BIRC4 mRNA concentrations. In cumulus cells, leptin treatment increased the mRNA levels for LEPR, STAT3, BAX, BIRC4, and FAS, but did not alter FASLG mRNA abundance. In conclusion, leptin differentially regulates gene expression in oocytes and cumulus cells. Moreover, leptin enhances both oocyte maturation and developmental capacity via cumulus cell-independent and -dependent mechanisms.
Abstract: Following the detection of individual members of the family of galectins it is an obvious challenge to define the extent of functional overlap/divergence among these proteins. As a step to address this issue a comparative profiling has been started in the mouse as a model organism, combining sequence analysis, expression patterns and structural features in the cases of the homodimeric galectins-1, -2 and -7. Close relationship was apparent at the level of global gene organization. Scrutiny of the proximal promoter regions for putative transcription-factor-binding sites by two search algorithms uncovered qualitative and quantitative differences with potential to influence the combinatorial functionality of regulatory sequences. RT-PCR mapping with samples from an array of 17 organs revealed significant differences, separating rather ubiquitous gene expression of galectin-1 from the more restricted individual patterns of galectins-2 and -7. Using specific antisera obtained by affinity depletion including stringent controls to ascertain lack of cross-reactivity these results were corroborated at the level of galectin localization in fixed tissue sections. Nuclear presence was seen in the case of galectin-1. In addition to nonidentical expression profiles the mapping of the carbohydrate recognition domains of galectins-1 and -7 by homology modelling and docking of naturally occurring complex tetra- and pentasaccharides disclosed a series of sequence deviations which may underlie disparate affinities for cell surface glycans/glycomimetic peptides. In view of applicability the presented data can serve as useful reference to delineate changes with respect to disease and in genetically engineered models. To enable more general conclusions on the galectin network it is warranted to further pursue this combined approach within this lectin family.
Abstract: Currently, our knowledge of early mammalian embryogenesis, stem cell differentiation and development is largely based on studies performed in mouse models. However, in important aspects, e.g. the timing of epigenetic reprogramming and embryonic genome activation, livestock species probably reflect far more closely the situation in men and other non-rodent mammals. A major challenge is the fact that in mammals, the development of individual zygotes is highly variable and vulnerable, and the outcome is uncertain. Valid indicators of the highly heterogeneous development and health status, and the actual developmental potential of individual oocytes, zygotes or embryos would be crucially important to tap the full power of holistic transcriptome and proteome analyses. Fluorescent reporter proteins opened new vistas for embryology and stem cell research: they can be used as reporters for the activity of gene promoters or tagged to functional proteins to study their intracellular localization in living cells, tissues and organisms. Fluorescent reporter genes may be used to microscopically observe key processes of early development. Thus, novel information related to developmental potential can be obtained from living embryos before processing them, e.g. for "-omic" studies. This review summarizes the main current reporter gene techniques and gene transfer approaches, which might be suitable for the investigation of early embryogenesis in livestock mammals. The potential of promoter reporter genes is exemplified by a bovine model system for quantitative monitoring of transcriptional reactivation of the so-called pluripotency gene POU5F1 in cloned bovine embryos.
Abstract: In this study the development of the bovine Fallopian tube was investigated using light microscopic methods. Formation and differentiation of the Müllerian duct were studied in mesonephroi of 16 embryos and fetuses with a crown-rump lengths (CRL) of 0.9-8.4 cm. The funnel field, the rostral beginning of the Müllerian duct was first observed at a CRL of 0.9 cm. It appears as a thickening of the mesothelium on the craniolateral side of the mesonephros. During later development the Müllerian duct emerges by caudal outgrowth from the funnel field. Formation of a common basal lamina surrounding the caudal tips of Müllerian and Wolffian ducts could be observed at all stages up to CRL of 2.7 cm. The mesothelium and the epithelium of the Wolffian duct adjacent to the Müllerian duct showed a modification of epithelium height in all examined stages. Probably the Wolffian duct influences the growth of Müllerian duct by epithelio-mesenchymal interactions. Fetuses from a CRL of 12.0 to 94.0 cm were used for investigation of the prenatal differentiation of the oviductal mucosa. Folding of the oviductal mucosa started at a CRL of 29.0 cm and continued until birth. Individual primary, secondary and tertiary folds are formed in special proliferation zones and epithelium-folding buds. The cellular differentiation of the oviductal epithelium involves the formation of ciliated and secretory cells during different times of prenatal development. Ciliogenesis was first detected at a CRL of 33.0 cm. Active secretory cells could be observed in the oviductal epithelium from a CRL of 64.0 cm onwards.
Abstract: Nitric oxide synthases (NOS) account for the endogenous production of nitric oxide (NO), a small and permeable bioreactive molecule. NO is known to act as a paracrine mediator during various processes associated with female reproduction. In the present study, the mRNA expression of the endothelial (eNOS) and inducible (iNOS) NO synthases were examined in bovine oviduct epithelial cells (BOEC) during the oestrous cycle. In addition, eNOS and iNOS mRNA and protein were localised by in situ hybridisation and immunocytochemistry respectively. Furthermore, the effects of exogenously applied oestradiol-17beta and progesterone on NOS mRNA regulation were studied in a suspension culture of BOEC. The eNOS mRNA abundance was low around ovulation (day 0) and increased significantly until pro-oestrus (day 18) in the ampulla. Immunoreactive protein of eNOS was detected predominantly in endothelial cells as well as in secretory oviduct epithelial cells at pro-oestrus. The iNOS mRNA concentration was significantly reduced in the isthmus at pro-oestrus (day 18) and oestrus (day 0) compared with persistently high levels in the ampulla. By in situ hybridisation, specific iNOS transcripts were additionally demonstrated in the oviduct epithelium. Immunoreactive iNOS protein was localised in secretory epithelial cells as well as in the lamina muscularis. The in vitro stimulation showed that both NOS were stimulated by progesterone, but not by oestradiol-17beta. The region-specific modulated expression of eNOS and iNOS provides evidence for an involvement of endogenously produced NO in the regulation of oviductal functions.
Abstract: Improvements of animal health, welfare and product quality are major goals of modern animal breeding. Thus, in addition to the classical production traits, functional traits such as disease resistance, fertility and longevity moved into the center of animal breeder's interests. Due to their low heritability, the improvement of functional traits using conventional approaches of phenotypic testing and quantitative genetics is difficult. A number of studies have been conducted worldwide in various species to map quantitative trait loci (QTLs) and to identify genetic markers for health traits. This has revealed a plethora of chromosome regions which may harbor genes with relevance for animal health. Functional genome research integrates holistic investigations at the level of the genome, at the level of gene activity (transcriptome, proteome) and at various levels of phenotypic expression. The integration of all these levels of information provides the basis for the functional dissection of complex traits. This review provides an overview of the most important strategies for holistic transcriptome and proteome analyses. The successful application of these techniques is exemplified by our studies of bovine reproductive biology.
Abstract: The glycan part endows cellular glycoconjugates with significant potential for biological recognition. N-Glycan branches often end with alpha2,3/alpha2,6-sialylation, posing the question whether and how placement of the sialic acid at 3 - or 6 -acceptor positions of galactose has cell biological relevance. As attractive model to study developmental regulation we monitored the expression of alpha2,3/alpha2,6-sialylated determinants in fetal and adult bovine testes and ovaries by lectin histochemistry. Distinct expression patterns were detected in both organ types. Oocyte staining, as a prominent example, was restricted to the presence of alpha2,6-sialylated glycans. Treatment with sialidase abolished binding and thus excluded sulfate esters as lectin targets. We added computer simulations to rationalize the observed evidence for non-random expression of the two closely related sialylgalactose isomers. Extensive molecular mechanics and molecular dynamics calculations reveal that the seemingly minor shift of the glycosidic bond from the alpha2,3 position to the alpha2,6 configuration causes significant shape and flexibility changes. They give each disaccharide its own characteristic meaning as signal in the sugar code.
Abstract: This study assessed the in vivo effects of recombinant growth hormone (rGH) administration on the expression of connexin-43 (Cx43) in bovine ovarian follicles. Two independent experiments were carried out using either estrous unsynchronized or synchronized multiparous Aberdeen Angus cows. rGH-treated animals were inoculated with a single dose of hormone (500 mg, intramuscular) while control animals were inoculated with hormone diluent. Five and 14 days after treatment (Experiments 1 and 2, respectively), ovarian Cx43 and apoptosis expression were assessed using immunohistochemistry. In both experiments primary, secondary, and tertiary follicles from rGH-treated and control groups distinctly expressed Cx43 protein. Primordial and atretic follicles were Cx43-negative. Interestingly, the number of Cx43 dots per granulosa cell did not show significant variation at different folliculogenesis stages neither in the rGH-treated nor in the control group. In unsynchronized animals, Cx43-positive follicles per total number of follicles ratio showed an interaction between stage of folliculogenesis and treatment due to significant differences between treatment groups in the early secondary follicle stage. In synchronized animals, there were significant differences between treatment groups and folliculogenesis stage. In both experiments, atretic follicles showed apoptosis-related DNA-fragmentation as determined by terminal uridin nick end labeling (TUNEL) assay. Tertiary follicles presented moderate TUNEL staining. Our results show significant increment in the number of ovarian follicles expressing the gap junction subunit Cx43 after in vivo rGH treatment. Therefore, we conclude that growth hormone can modulate in vivo gap junction assembly at early stages of folliculogenesis.
Abstract: Initiation and maintenance of pregnancy are critically dependent on an intact embryo-maternal communication in the preimplantation period. To get new insights into molecular mechanisms underlying this complex dialog, a holistic transcriptome study of endometrium samples from Day 18 pregnant vs. nonpregnant twin cows was performed. This genetically defined model system facilitated the identification of specific conceptus-induced changes of the endometrium transcriptome. Using a combination of subtracted cDNA libraries and cDNA array hybridization, 87 different genes were identified as upregulated in pregnant animals. Almost one half of these genes are known to be stimulated by type I interferons. For the ISG15ylation system, which is assumed to play an important role in interferon tau (IFNT) signaling, mRNAs of four potential components (IFITM1, IFITM3, HSXIAPAF1, and DTX3L) were found at increased levels in addition to ISG15 and UBE1L. These results were further substantiated by colocalization of these mRNAs in the endometrium of pregnant animals shown by in situ hybridization. A functional classification of the identified genes revealed several different biological processes involved in the preparation of the endometrium for the attachment and implantation of the embryo. Specifically, elevated transcript levels were found for genes involved in modulation of the maternal immune system, genes relevant for cell adhesion, and for remodeling of the endometrium. This first systematic study of maternal transcriptome changes in response to the presence of an embryo on Day 18 of pregnancy in cattle is an important step toward deciphering the embryo-maternal dialog using a systems biology approach.
Abstract: We established a short-term (24 h) culture system for bovine oviduct epithelial cells (BOECs), obtained on day 3.5 of the estrous cycle and evaluated the cells with respect to morphological criteria, marker gene expression, and hormone responsiveness. BOEC sheets were isolated mechanically from the ampulla with similar yields from oviducts ipsi- and contralateral to the ovulation site (57.9 +/- 4.6 and 56.4 +/- 8.0 x 10(6) cells). BOECs showed > 95% purity and cells cultured for 24 h maintained morphological characteristics present in vivo, as determined by light microscopy, scanning electron microscopy, and transmission electron microscopy. Both secretory cells with numerous secretory granules and ciliated cells with long, well-developed, and vigorously beating kinocilia were visible. Quantitative real-time PCR failed to detect significant differences in transcript levels between ipsi-and contralateral BOECs for the majority of marker genes (estrogen receptors alpha and beta (ESR1 and ESR2), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), oviductal glycoprotein 1 (OVGP1), progesterone receptor (PGR), and tumor rejection antigen 1 (TRA1)) throughout the 24 h culture period. However, the combined data of all time points for glutathione peroxidase 4 (GPX4), a gene previously shown to be expressed at higher levels in the ipsilateral oviduct in vivo, also indicated significantly different mRNA levels in vitro. The expression of marker genes remained stable after 6 h cell culture, indicating only a short adaptation period. Western blot analysis confirmed ESR1 and PGR protein expression throughout the culture period. In agreement with cyclic differences in vivo, estradiol-17beta stimulation increased PGR transcript abundance in BOECs. Our novel culture system provides functional BOECs in sufficient quantities for holistic transcriptome and proteome studies, e.g. for deciphering early embryo-maternal communication.
Abstract: Cell cycle progression is facilitated by cyclin dependent kinases (CDKs) that are activated by cyclins, including Cyclin D1 and inhibited by CDK inhibitors. Evidence of the involvement of cyclin gene alterations and over expression of various cyclins in human cancer is growing. The role of Cyclin D1 in malignant progression of papillary carcinomas of the thyroid has yet to be established. We therefore studied the expression of Cyclin D1 protein in thyroid carcinomas of young Kuwaiti patients (36 cases of conventional papillary thyroid carcinoma, 12 cases of its follicular variant, one case of tall cell thyroid carcinoma and one case of medullary carcinoma) using immunohistochemistry. In 23 patients (46%) circumscribed areas of cells were detected that showed a distinct to strong nuclear staining for immunoreactive Cyclin D1 whereas the remaining bulk of the carcinoma cells were negative or only showed a slight cytoplasmic staining. None of the tested clinical or path histological parameters showed a statistically significant correlation with the focal immunostaining. This does not rule out that the detected foci with positive nuclear Cyclin D1 immunostaining are areas where a progressive transformation to a more malignant phenotype occurs which eventually leading to lymph node and distant metastases.
Abstract: The endometrium plays a central role among the reproductive tissues in the context of early embryo-maternal communication and pregnancy. This study investigated transcriptome profiles of endometrium samples from day 18 pregnant vs non-pregnant heifers to get insight into the molecular mechanisms involved in conditioning the endometrium for embryo attachment and implantation. Using a combination of subtracted cDNA libraries and cDNA array hybridisation, 109 mRNAs with at least twofold higher abundance in endometrium of pregnant animals and 70 mRNAs with higher levels in the control group were identified. Among the mRNAs with higher abundance in pregnant animals, at least 41 are already described as induced by interferons. In addition, transcript levels of many new candidate genes involved in the regulation of transcription, cell adhesion, modulation of the maternal immune system and endometrial remodelling were found to be increased. The different expression level was confirmed with real-time PCR for nine genes. Localisation of mRNA expression in the endometrium was shown by in situ hybridisation for AGRN, LGALS3BP, LGALS9, USP18, PARP12 and BST2. A comparison with similar studies in humans, mice, and revealed species-specific and common molecular markers of uterine receptivity.
Abstract: Originally defined as a lymphokine inhibiting the random migration of macrophages, the macrophage migration inhibitory factor (MIF) is an important mediator of the host response to infection. Beyond its function as a classical cytokine, MIF is currently portrayed as a multifunctional protein with growth-regulating properties present in organ systems beyond immune cells. In previous studies, we detected substantial amounts of MIF in the rat epididymis and epididymal spermatozoa, where it appears to play a role during post-testicular sperm maturation and the acquisition of fertilization ability. To explore its presence in other species not yet examined in this respect, we extended the range of studies to the bull. Using a polyclonal antibody raised against MIF purified from bovine eye lenses, we detected MIF in the epithelium of the adult bovine epididymis with the basal cells representing a prominently stained cell type. A distinct accumulation of MIF at the apical cell pole of the epithelial cells and in membranous vesicles localized in the lumen of the epididymal duct was obvious. In the fetal bovine epididymis, we also detected MIF in the epithelium, whereas MIF accumulation was evident at the apical cell surface and in apical protrusions. By immunoelectron microscopy of the adult bovine epididymis, we localized MIF in apical protrusions of the epithelial cells and in luminal membrane-bound vesicles that were found in close proximity to sperm cells. Although the precise origin of the MIF-containing vesicles remains to be delineated, our morphological observations support the hypothesis that they become detached from the apical surface of the epididymal epithelial cells. Additionally, an association of MIF with the outer dense fibers of luminal spermatozoa was demonstrated. Data obtained in this study suggest MIF release by an apocrine secretion mode in the bovine epididymis. Furthermore, MIF localized in the basal cells of the epithelium and in the connective tissue could be responsible for regulating the migration of macrophages in order to avoid contact of immune cells with spermatozoa that carry a wide range of potent antigens.
Abstract: Growth factors are involved in development and function of the mammary gland. The aim of this study was the localization of fibroblast growth factor 1 (FGF-1) and its mRNA in the bovine mammary gland during different developmental and functional stages. Mammary tissue was obtained from German Brown Swiss cows (n = 23) during defined stages of mammogenesis (before and during pregnancy), lactogenesis, peak lactation and involution. The distribution of FGF-1 mRNA was studied using non-radioactive in situ hybridization, the corresponding FGF-protein was analysed using immunohistochemistry [avidin-biotin peroxidase complex (ABC)-method]. A moderate to distinct staining for FGF-mRNA was found in the epithelium of ducts and developing alveoli during mammogenesis. Post-partum at the same cellular locations, a considerable amount of FGF-1 mRNA, was seen that decreased during lactation. Also during early involution clear staining for FGF-mRNA could still be observed. Immunoreactive FGF-1 was found in considerable concentration in the epithelium of the mammary gland in heifers. The staining intensity generally decreased somewhat during mammogenesis and lactation, but could be always clearly demonstrated in the secretory epithelial cells of alveoli and glandular ducts. Also during the first day after the end of milking, the epithelium displayed a moderate to distinct epithelial immunostaining. Notably, After 4 weeks of involution, in many alveoli a shedding of the FGF-1 positive luminal cell layer was found. In our localization studies, no strict correlation between FGF-1 mRNA and its corresponding protein was found. The various reasons for this finding are discussed.
Abstract: The series of events associated with oocyte growth and maturation determines the oocyte's ability to undergo successful fertilization, cleavage and embryonic development. Among the molecules involved in these events, leptin has been identified as a modulator of oocyte function. Experiments were conducted to determine whether leptin treatment of oocytes during maturation affects their developmental capacity after fertilization and whether it has long-lasting effects on apoptosis and gene expression in the resulting blastocysts. Cumulus-oocyte complexes (COCs) were matured in serum-free medium containing 0 (control), 1, 10, or 100 ng/ml leptin or in medium supplemented with 10% (v/v) estrous cow serum (ECS). Addition of leptin during oocyte maturation had no effect on cleavage rate after fertilization. However, an increased proportion of oocytes that matured in the presence of 1 or 10 ng/ml leptin developed to blastocysts, which exhibited increased cell numbers. The proportion of apoptotic cells was reduced in blastocysts originating from leptin- or ECS-treated oocytes. Transcript levels of the genes encoding leptin receptor (LEPR), signal transducer and activator of transcription (STAT3), BCL2 associated X-protein (BAX), and baculoviral inhibitor of apoptosis protein repeat-containing 4 (BIRC4, also known as XIAP), were determined by reverse transcriptase-quantitative polymerase chain reaction analysis of expanded and hatched blastocysts. Depending on the dose used, leptin treatment of oocytes resulted in increased LEPR, STAT3, and BIRC4 mRNA levels and reduced BAX mRNA levels in blastocysts. In conclusion, leptin improved the ability of the oocyte to sustain embryonic development and had long-term effects on blastocyst apoptosis and transcript abundance of LEPR, STAT3, and apoptosis-associated genes.
Abstract: The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is regarded as the major molecular target of selenodeficiency in rodents, accounting for most of the histopathological and structural abnormalities of testicular tissue and male germ cells. PHGPx exists as a cytosolic form, mitochondrial form, and nuclear form (nPHGPx) predominantly expressed in late spermatids and spermatozoa. Here, we demonstrate that mice with a targeted deletion of the nPHGPx gene were, unlike mice with the full knockout (KO) of PHGPx, not only viable but also, surprisingly, fully fertile. While both morphological analysis of testis and epididymis and sperm parameter measurements did not show any apparent abnormality, toluidine blue and acridine orange stainings of spermatozoa indicated defective chromatin condensation in the KO sperm isolated from the caput epididymis. Furthermore, upon drying and hydrating, KO sperm exhibited a significant proportion of morphologically abnormal heads. Monobromobimane labeling and protein-free thiol titration revealed significantly less extensive oxidation in the cauda epididymis when compared to that in the wild type. We conclude that nPHGPx, by acting as a protein thiol peroxidase in vivo, contributes to the structural stability of sperm chromatin.
Abstract: The endometrium plays a central role among the reproductive tissues in the context of early embryo-maternal communication and pregnancy. It undergoes typical changes during the sexual/oestrous cycle, which are regulated by the ovarian hormones progesterone and oestrogen. To identify the underlying molecular mechanisms we have performed the first holistic screen of transcriptome changes in bovine intercaruncular endometrium at two stages of the cycle--end of day 0 (late oestrus, low progesterone) and day 12 (dioestrus, high progesterone). A combination of subtracted cDNA libraries and cDNA array hybridisation revealed 133 genes showing at least a 2-fold change of their mRNA abundance, 65 with higher levels at oestrus and 68 at dioestrus. Interestingly, genes were identified which showed differential expression between different uterine sections as well. The most prominent example was the UTMP (uterine milk protein) mRNA, which was markedly upregulated in the cranial part of the ipsilateral uterine horn at oestrus. A Gene Ontology classification of the genes with known function characterised the oestrus time by elevated expression of genes, for example related to cell adhesion, cell motility and extracellular matrix and the dioestrus time by higher expression of mRNAs encoding for a variety of enzymes and transport proteins, in particular ion channels. Searching in pathway databases and literature data-mining revealed physiological processes and signalling cascades, e.g. the transforming growth factor-beta signalling pathway and retinoic acid signalling, which are potentially involved in the regulation of changes of the endometrium during the oestrous cycle.
Abstract: It is now well established that normal development and function of testis are mediated by endocrine and paracrine pathways including hormones, growth factors and cytokines as well as by direct cell-to-cell contacts depending on tight, adhering and gap junctions. In the last two decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. Interestingly, most of these peptides are expressed not only in the adult mammalian testis during spermatogenesis but also during testicular morphogenesis in prenatal and postnatal life. Our study was launched to provide an overview of the expression, localization, and putative physiological roles of growth factors and their receptors in the mammalian testis. The growth factors considered in this part of our review are fibroblast growth factors and insulin-like growth factors. These factors are found in testicular cells in prenatal, postnatal, and adult animals and are implicated in the regulation of important testicular activities including testicular cord morphogenesis, modulation of testicular hormone secretion and control of spermatogenesis.
Abstract: Several fibroblast growth factors (FGFs) are implicated in proliferation and differentiation of both somatic and germ cells during testicular development, as well as in spermatogenesis of adult testis. The expression of FGF2 was studied in the adult bovine testis using quantitative RT-PCR, RNA in situ hybridization, and immunohistochemistry. Quantitative RT-PCR revealed consistent levels of FGF2 mRNA in parenchymal samples of the bovine testis. In situ hybridization localized FGF2 transcripts only in a constant fraction of Leydig and Sertoli cells as well as in modified Sertoli cells of the terminal segments. Immunohistochemistry revealed (a) no FGF2 protein in Sertoli cells (b) moderate cytoplasmic staining in Leydig cells and spermatogonia and (c) strong nuclear and faint cytoplasmic staining in myofibroblasts, in epithelial cells of straight tubules and rete testis and in blood vessels. These observations indicate a pleiotropic effect of FGF2 on the control of spermatogenesis in a paracrine and/or autocrine manner.
Abstract: Kuwait was chosen by the International Lymphoma Study Group (ILSG) as one of the sites attending in the project on "Clinical characteristics and pathological classification of non Hodgkin's lymphoma (NHL) in the developing countries". The Kuwait study involved 206 cases of NHL, diagnosed, staged and treated in the Kuwait Cancer Control Center (KCCC). All cases were reviewed and reclassified independently by the pathologists of KCCC and the International Lymphoma Study Group (ISLG) using the latest World Health Organization (WHO) classification of neoplastic disease of the hematopoietic and lymphoid tissues. Immunophenotyping as to B- or T-cell was documented in all cases. Three main pathological entities (diffuse large B-cell lymphoma, follicular lymphoma, peripheral T-cell lymphoma) were identified and studied thoroughly. The intense cooperation between experts of the ISLG and pathologists of the KCCC proved that the WHO classification was fully reproducible in Kuwait. The high incidence of extranodal lymphomas (53%) observed in the KCCC may not be due to special ethnic or environmental conditions in Kuwait but rather be due to a selection of patients coming to our center.
Abstract: The aim of this study was to investigate the possible participation of fibroblast growth factor (FGF) family members: FGF1, FGF2, and FGF7, and their receptor variants: FGFR, FGFR2IIIb, and FGFR2IIIc in theca interna (TI) and granulosa cell (GC) compartments of bovine follicles during final growth. A classification of follicles into five groups (<0.5; >0.5-5; >5-20; >20-180; >180 ng/ml, respectively) was performed according to the follicular fluid (FF) oestradiol-17beta (E) content. The mRNA expression and protein localization was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. FGF1 mRNA expression was relatively high in TI and lower in GC, and without any regulatory change for both tissue compartments during final follicular growth. The FGF1 protein could be predominantly localized in the cytoplasm of GC, in smooth muscle cells of blood vessels, in the rete ovarii, and at a lesser degree in theca cells. FGF2 mRNA in TI increased significantly in large follicles and was low and without any regulatory change in GC. FGF7 mRNA expression was relatively high in TI and very low in GC. For FGF7 in mature follicles a marked staining of the TI and the basal layers of the GC could be demonstrated. The mRNA signal for the FGFR in TI increased significantly with beginning of E production (E > 0.5-5 ng/ml FF) and was without any regulatory change in GC. The mRNA expression of FGFR2IIIb was relatively high in GC and increased significantly during final growth of follicles in contrast to the TI with very low expression. The FGFR2IIIc mRNA expression in TI and GC was relatively high but without any clear change. Our results suggest that FGF growth factor family members are involved in process of folliculogenesis and especially during final growth of the preovulatory (dominant) follicle by stimulation of angiogenesis and GC survival and proliferation.
Abstract: Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.
Abstract: A 3-year study was carried out to evaluate male and female effects on the efficiency of an in vitro fertilization (IVF) programme. The semen of different bulls used for artificial insemination was tested for the in vitro production of transferable blastocysts. The fertilization capacity was recorded for each bull. Bovine oocytes were matured in vitro, fertilized with frozen/thawed semen of 63 individual bulls and cultured during 8 days. The semen of one bull was used as control. The percentage of cleavage (36.3-93.4%) and blastocysts on day 7 (6.9-51.2%) varied from bull to bull. Despite high variability, blastocysts were produced with the semen of all bulls in the first trial. Moreover, oocytes fertilized with 85% of tested bulls reached a blastocyst rate not different to the control bull. The correlation coefficients of six bulls showed no significant male effect but an influence of oocytes on the cleavage rate (F-value 0.38, P > 0.05, and 12.4, P < 0.001, respectively). The development to blastocysts on day 7 was significantly influenced by sperms and also oocytes and session (P < 0.01), but no combined interaction was observed between female and male. It is concluded that transferable embryos can be produced in vitro in the first trial with frozen/thawed semen of 63 tested bulls. The results show different capacities of bulls to produce embryos and high male and female effects on the efficiency of an IVF programme.
Abstract: The emerging concept of the sugar code attributes functional significance to oligosaccharides of cellular glycoconjugates by protein (lectin)-carbohydrate interactions. Hence it follows that monitoring of glycan expression (glycomic profiling) is not only valuable to delineate characteristic (phenomenological) changes in the cell's glycosylation but will also come up with the localization of epitopes with potential in biorecognition. It is for this purpose that we have set up a panel of 16 markers (plant lectins and a carbohydrate-specific antibody). The selection met two criteria: a) to be able to detect the common constituents of natural glycans; and b) to place emphasis on detection of neutral carbohydrate units at the spatially accessible branch ends of glycan chains, which are known to be active as ligands for endogenous lectins in situ. Next, we incorporated recent insights into the importance of epitope clustering to turn less abundant oligosaccharides into potent ligands into our study design. To be able to focus on such high-affinity sites, we performed systematic titration studies aimed at defining the probe concentration at which carbohydrate-independent background staining is minimal while still yielding a clear signal. These requirements were met by marker concentrations of 1.25-2.5 microg/ml. Under these conditions, we defined cell-type- and differentiation-dependent changes in bovine testis. Sertoli cells lacked reactivity, whereas gonocytes were differentially reactive with the tested markers. The extent of staining intensity was subject to developmental changes, preferentially for Gal/GalNAc presentation and in this group most prominently with the galactoside-specific lectin from Viscum album L. (mistletoe). Of interest in this context, this lectin is known as a potent mitogen and signal inductor as well as haemagglutinin. The Gal/GalNAc-dependent signals decreased markedly in the course of development and staining was completely lost in the case of mistletoe lectin 12 weeks after gestation. Spermatids of adult testis presented respective glycan epitopes. In contrast to this developmental course of staining, endothelial cells either maintained a constant signal intensity or revealed a signal increase during development for Gal/GalNAc-specific lectins. Their binding of concanavalin A and the two phyto-haemagglutinins (PHA-E/L), which were not or only weakly reactive for gonocytes, served as inherent activity control. Based on lectin blot analysis with the mistletoe lectin as the marker which detected the most prominent change, the glycoprotein patterns from fetal and adult tissue specimens were qualitatively different, rendering changes in expression of the protein part of glycoproteins more likely than remodeling a glycoprotein's glycan chains. Methodologically, results of this procedure were compared to data obtained with lectin affinity chromatography and the combination of the two procedures. Differences in the profiles were discovered that can be assigned to the disparate ways to process the detergent extracts. When access to sample quantity is limited, as is possible in the case of fetal tissue, direct lectin blotting is recommended.
Abstract: A total of 935 patients with extranodal non-Hodgkin lymphoma (NHL) diagnosed in the period between January 1985 and December 2000 in Kuwait Cancer Center, serving the whole population of Kuwait, were used to describe the clinicopathological and epidemiological features of extranodal lymphomas in Kuwait. Extranodal lymphomas accounted for 45% of all NHL observed during this time. All NHL cases from Kuwait Cancer registry were analyzed and pathologically reclassified using the latest WHO (2000) classification. The most common lymphoma observed was diffuse large B-cell lymphoma (58.60%) followed by Burkitt s lymphoma (BL) (3.80%). In the pediatric group, BL comprises more than two thirds of all patients (77.20%). The most common extranodal sites were stomach (19.70%) and skin (17.80%) in the adult group, large intestine (29.80%) and small intestine (19.30%) in the pediatric age group. The majority (73.40%) of adult extranodal lymphomas was in stage IE-IIE and had a very good prognosis. On the contrary, the majority of pediatric extranodal lymphomas were found to be in stage III and IV. Variations in treatment policies (single agent or combined chemotherapy, radiotherapy, combined modality treatment) adopted and changed during the time period of 16 years of this retrospective study were documented.
Abstract: Oxygen radicals regulate many physiological processes, such as signaling, proliferation, and apoptosis, and thus play a pivotal role in pathophysiology and disease development. There are at least two thioredoxin reductase/thioredoxin/peroxiredoxin systems participating in the cellular defense against oxygen radicals. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. To begin to address this question, we generated and characterized mice lacking functional mitochondrial thioredoxin reductase (TrxR2). Ubiquitous Cre-mediated inactivation of TrxR2 is associated with embryonic death at embryonic day 13. TrxR2(TrxR2(-/-)minus;/TrxR2(-/-)minus;) embryos are smaller and severely anemic and show increased apoptosis in the liver. The size of hematopoietic colonies cultured ex vivo is dramatically reduced. TrxR2-deficient embryonic fibroblasts are highly sensitive to endogenous oxygen radicals when glutathione synthesis is inhibited. Besides the defect in hematopoiesis, the ventricular heart wall of TrxR2(TrxR2(-/-)minus;/TrxR2(-/-)minus;) embryos is thinned and proliferation of cardiomyocytes is decreased. Cardiac tissue-restricted ablation of TrxR2 results in fatal dilated cardiomyopathy, a condition reminiscent of that in Keshan disease and Friedreich's ataxia. We conclude that TrxR2 plays a pivotal role in both hematopoiesis and heart function.
Abstract: Pituitary LH and FSH are known to be the major regulators of ovarian function. In the last few years, however, there has been evidence that growth hormone (GH) is also involved in ovarian regulation. Therefore, the aim of our study was to elucidate the mechanisms of GH action during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs). As shown by detection of the nuclear cell proliferation-associated antigen Ki-67, COCs matured in vitro in the presence of GH revealed a significantly (P < 0.05) higher proportion of proliferating cumulus cells (12.6%) compared with the COCs matured in the control medium TCM 199 (9.9%). In contrast, the percentage of proliferating cells was not increased by supplementation of the medium with a combination of GH and insulin-like-growth factor I (IGF-I). Apoptosis as determined by TUNEL (terminal doxynucleotidyl transferase mediated dUTP nick-end labeling) was significantly (P < 0.05) reduced in the cumulus cells by GH treatment. COCs matured with a combination of GH and IGF-I revealed the lowest percentage of apoptotic cells (11%). The localization and quantification of the gap junction protein connexin 43 (Cx 43) demonstrated that GH induced a significant decrease in the synthesis of the Cx 43 protein in the cumulus cells. Our results imply that GH increases cumulus expansion by promotion of cell proliferation and inhibition of apoptosis. Whereas the increase in cell proliferation is a direct effect of GH, the antiapoptotic effects of GH during in vitro maturation are modulated by IGF-I. Stimulatory effects of GH on oocyte maturation are correlated with changes in the synthesis of gap junction proteins.
Abstract: Sperm binding and sperm penetration of the zona pellucida (zp) are regarded as species-specific. In this investigation, the interactions between bovine oocytes and porcine, respectively, equine spermatozoa have been studied under in vitro conditions and compared with the normal in vitro fertilization of bovine oocytes by bovine sperm. Surprisingly, many of the heterologous spermatozoa adhered firmly to the bovine oocytes and could not be removed by intense washing. On average, more than 100 boar or equine spermatozoa were bound to the zp of bovine oocytes. Electron microscopic studies clearly demonstrated that porcine sperm attached to the zona and underwent the acrosome reaction. Equine spermatozoa displayed a similar binding affinity, but unlike the porcine spermatozoa even penetrated the zp and were taken up into the oocyte after a longer period of co-incubation. Considering these new results the dogma of a strict species specificity of sperm zona interactions under in vitro conditions has to be reconsidered.
Abstract: Hyaluronan or hyaluronic acid (HA) is a normal component of mammalian follicular, oviduct, and uterine fluids. Granulosa and expanding cumulus cells secrete large amounts of HA, and when HA is added in maturation and culture media, it improves the developmental potential of oocytes and embryos. HA regulates gene expression, signaling, proliferation, motility, adhesion, and morphogenesis. Many of these biological activities of HA are mediated through binding to the receptor for HA-mediated motility/intracellular HA-binding protein (RHAMM/IHABP). We evaluated the presence and dynamics of RHAMM/IHABP mRNA and protein expression in different stages of in vitro-produced bovine embryos using quantitative reverse transcriptase-real time-polymerase chain reaction and immunohistochemistry. We also analyzed the effects of different culture systems on the relative abundance of RHAMM/IHABP transcripts. RHAMM/IHABP mRNA levels decreased from the 2-cell to the 16-cell stage, increased again at the morula stage, and reached their highest level at the expanded blastocyst stage. RHAMM/IHABP mRNA abundance was significantly (P < 0.05) lower in embryos recovered in serum-containing medium than in embryos from serum-free media. Immunohistochemistry revealed the presence of RHAMM/IHABP first in 8-cell stages. Whereas RHAMM staining in 8-cell and morula stages was intense, it was weaker in blastocysts. Embryonic secretion of HA increased from the 2-cell stage until the 8-cell stage and then decreased in 16-cell embryos. After this, HA secretion increased in expanded and hatched blastocyst stages. These data suggest that the positive effects of HA on in vitro-produced bovine embryos may be mediated at least in part by RHAMM/IHABP.
Abstract: It is now well established that oestrogen and progesterone are absolutely essential for mammary gland development. Lactation can be induced in non-pregnant animals by sex steroid hormone treatment. Most of the genomic actions of oestrogens are mediated by two oestrogen receptors (ER)-alpha and ERbeta, and for gestagens in ruminants by the progesterone receptor (PR). Our aim was the evaluation of mRNA expression and protein (localisation and Western blotting) during mammogenesis, lactogenesis, galactopoiesis (early, middle and late) and involution (8, 24, 28, 96-108 h and 14-28 days after the end of milking) in the bovine mammary gland (total no. 53). During these stages, the mRNA was assessed by means of real-time RT-PCR (LightCycler). The protein for ERalpha, ERbeta and PR was localised by immunohistochemistry and Western blotting. The mRNA expression results indicated the existence of ERalpha, ERbeta and PR in bovine mammary gland. Both ERalpha and PR are expressed in fg/ micro g total RNA range. The highest mRNA expression was found for ERalpha and PR in the tIssue of non-pregnant heifers, followed by a significant decrease to a lower level at the time of lactogenesis with low concentrations remaining during lactation and the first 4 weeks of involution. In contrast, the expression of ERbeta was about 1000-fold lower (ag/ micro g total RNA) and showed no clear difference during the stages examined, with a significant increase only 2-4 weeks after the end of milking. Immunolocalisation for ERalpha revealed a strong positive staining in nuclei of lactocytes in non-pregnant heifers, became undetectable during pregnancy, lactogenesis and lactation, and was again detectable 14-28 days after the end of milking. In contrast, PR was localised in the nuclei of epithelial cells in the mammary tIssue of non-pregnant heifers, in primigravid animals, and during late lactation and involution. During lactogenesis, peak and mid lactation, fewer nuclei of epithelial cells were positive, but increased staining of the cytoplasm of epithelial cells was obvious. ERalpha and ERbeta protein was found in all mammary gland stages examined by Western blotting. In contrast to mRNA expression, the protein signal for ERalpha was weaker in the tIssue of non-pregnant heifers and during involution (4 weeks). ERbeta protein showed a stronger signal (two isoform bands) in non-pregnant heifers and 4 weeks after the end of milking. This correlated with the mRNA expression data. Three isoforms of PR (A, B and C) were found by Western blotting in the tIssue of non-pregnant heifers, but only isoform B remained during the following stages (lactogenesis, galactopoiesis and involution). In conclusion, the mRNA expression and protein data for ER and PR showed clear regulatory changes, suggesting involvement of these receptors in bovine mammary gland development and involution.
Abstract: The morphology of canine cumulus-oocyte complexes (COCs) before puberty is still unknown. Therefore, the aim of our study was to elucidate the morphological characteristics of pre-pubertal oocytes and cumulus cells by light microscopy, scanning electron microscopy and transmission electron microscopy. The pre-pubertal oocyte was characterized by accumulation of lipid yolk droplets in the cytoplasm as well as high energy metabolism, low protein synthesis and high transcriptional activity of the cumulus cells. The cumulus cells, which revealed a prominent nucleus and few cytoplasm, communicated with each other by few short processes and exhibited merely a small amount of processes reaching the oocyte. Our studies imply that both the oocyte and the cumulus cells of canine COCs before puberty reveal characteristic morphological features which are correlated with changes in oocyte metabolism and cumulus cell communication.
Abstract: To determine the capacity of sewage treatment work effluents to disrupt the endocrine system under semifield conditions, two amphibian species, Xenopus laevis and Rana temporaria, were exposed to the effluent of a regional sewage treatment plant in South Bavaria during larval development until completion of metamorphosis. Exposure was carried out in river water (Würm) as a reference, and a 1:12-mixture sewage effluent representing the real situation on the spot, and in a higher concentration of sewage using a 1:2 mixture. An accidental impact of industrial wastewater into the reference and dilution medium, Würm, which was caused by a spate in the respective area during the sensitive period of sex differentiation of amphibian larvae, is assumed to be responsible for the relatively high percentage of females observed by histological analysis in all treatment groups. All of these values were higher than those determined in controls exposed to artificial tap water in laboratory experiments conducted in a comparable study design. Sex ratios between species, revealed by the semifield study with decreasing portions of females from control to 1:12 to 1:2, were strongly correlated. Determination of biomarker-mRNA-levels in Xenopus liver using semiquantitative RT-PCR at the end of the experimental phase, when exposure regime has turned into the initially expected situation with the highest load of potential estrogens in the effluent, followed by 1:2 and 1:12 mixture, resulted in a significant increase of Vitellogenin-mRNA in female juveniles exposed to the highest portion of sewage, whereas expression of both androgen and estrogen receptor-mRNA showed no clear differences. The results concerning the induction of estrogenic biomarkers are in accordance with our findings for estrogen receptor binding of sample extracts from the Würm and sewage taken in parallel at the end of the experiment, when sewage extracts possessed a much higher ability to displace [3H]estradiol from the estrogen receptor than the ones extracted from the mixtures.
Abstract: Early embryonic development, implantation and maintenance of a pregnancy are critically dependent on an intact embryo-maternal communication. So far, only few signals involved in this dialogue have been identified. In bovine and other ruminants, interferon tau is the predominant embryonic pregnancy recognition signal, exhibiting antiluteolytic activity. However, this is just one aspect of the complex process of embryo-maternal signalling, and a number of other systems are more likely to be involved. To gain a more comprehensive understanding of these important mechanisms, integrated projects involving specialists in embryology, reproductive biotechnology and functional genome research are necessary to perform a systematic analysis of interactions between pre-implantation stage embryos and oviduct or uterine epithelial cells, respectively. State-of-the-art transcriptomic and proteomic technologies will identify reciprocal signals between embryos and their maternal environment and the respective downstream reaction cascades. For in vivo studies, the use of monozygotic twins as recipient animals provides elegant model systems, thus eliminating genetic variability as a cause of differential gene expression. In addition, suitable systems for the co-culture of oviduct epithelial or endometrium cells with the respective embryonic stages need to be established for functional validation of candidate genes potentially involved in the dialogue between embryos and their maternal environment. The knowledge of these mechanisms should help to increase the pregnancy rate following embryo transfer and to avoid embryonic losses. Candidate genes involved in embryo-maternal communication will also be used to define new quality criteria for the selection of embryos for transfer to recipients. Another application is the supplementation of embryotrophic factors or components of embryo-maternal signalling in optimized formulations, such as bioartificial matrices. As a long-term goal, signalling mechanisms identified in bovine will also be functionally evaluated in other species, including the human.
Abstract: Growth hormone (GH) has recently been shown to exert distinct effects on the differentiation and metabolism of early embryos. Up to now, however, it is not clear whether GH is able to modulate apoptosis during early embryogenesis. Differential cell staining of 8-day-old bovine embryos cultured with 100 ng bovine recombinant GH (rbGH) per ml medium (synthetic oviduct fluid-polyvinylalcohol) demonstrated that GH significantly increased the number of inner cell mass (ICM) and trophectoderm cells in bovine expanded blastocysts. As shown by terminal deoxynucleotidyl transferase mediated dUTP labeling (TUNEL) supplementation of bGH decreased the percentage of 8-day-old embryos showing at least one apoptotic cell from 58 to 21%. The percentage of apoptotic cells in one blastocyst was significantly (P < 0.01) reduced from 4.6 to 1.1% by GH treatment. Incubation of the embryos with 150 mM vanillylnonanamide induced apoptosis in all embryos. Whereas in control embryos 14% of the embryonic cells were TUNEL-positive, the percentage of apoptotic cells declined to 2.7% in the GH treated embryos. Expression of immunoreactive bcl-2 in blastocysts was not affected by GH treatment. Synthesis of the bax protein which is known to promote apoptosis was reduced in embryos cultured with GH. Our results suggest that GH acts as survival factor during in vitro culture and reduces apoptosis by altering the bax to bcl-2 ratio during early embryogenesis.
Abstract: Spermadhesins, secretory proteins of the male genital tract, constitute the bulk of seminal plasma proteins in pig. The nucleotide sequences of spermadhesins AWN, AQN-1, and AQN-3 from the cDNAs, derived from seminal vesicles, were determined. The coding sequences of spermadhesins revealed an overall sequence similarity of 40% at nucleotide level. Expression of spermadhesins (AWN, AQN-1, AQN-3, PSP-I, and PSP-II) in porcine male and female reproductive tracts were studied by means of RT-PCR and immunological approaches. All spermadhesins are transcribed and translated in seminal vesicles and prostate. In caudal epididymis mRNA transcripts of all spermadhesins have been detected by RT-PCR. PSP-I showed additional signals in caput epididymis and rete testis. Translation can be detected only for AWN in tissue extracts by SDS-PAGE and Western blotting. Remarkably, AWN is the only spermadhesin that is also expressed in the uterus, the uterotubal junction, and the oviduct of the female genital tract as shown by RT-PCR, cDNA-sequencing, and immunological analysis. In sows at estrus and interestrus, in gilts and gilts 12 hr after insemination no obvious differences were noticed in the pattern of AWN-immunoreactivity in epithelial cells either of the uterotubal junction, isthmus, or ampulla. While strong staining was observed in the superficial uterine glands and in the glands of the uterotubal junction during estrus, in diestrus this declined distinctly. The role of spermadhesins in relation to their expression is discussed.
Abstract: Histochemical monitoring of developmental processes is presently centered on protein-protein interactions. However, oligosaccharides have the potential to store and transmit biological information. Carbohydrate chains of cellular glycoconjugates present determinants for binding of endogenous lectins. This interaction can be relevant for developmental processes. In fact, beta-galactosides and their derivatives serve as ligands for members of the lectin family of galectins. Since it is unclear to what extent functions of different galectins differ or overlap, hereby introducing redundancy into this system, monitoring of galectin presence during tissue maturation should include more than one type of galectin (galectin fingerprinting). Here, we focus on the two most frequently described ones, namely the homodimeric prototype galectin-1 and the chimera-type galectin-3, the latter one so far not characterized from bovine tissue. In the first step, we have detected its presence biochemically in addition to the abundant galectin-1 in bovine respiratory and digestive tracts during development. Evidently, diversification of the primitive foregut will not lead to an alteration of this property. Immunohistochemistry revealed clear differences in the galectins' localization profiles. Galectin-1 expression is strong in mesenchymal cells, especially smooth muscle cells, while epithelial lining harbors galectin-3. A gradual increase in staining intensity with development is especially observed in the case of galectin-3. Notably, this change is accompanied by a shift from primarily nuclear localization to the cytoplasm, an alteration not seen for galectin-1. However, nuclear presence of galectin-1 is encountered. Thus, the delineation of differences in expression of galectin-1 and -3 with respect to cell types and in the developmental course of subcellular localization argues in favor of mediation of nonoverlapping functions by these two homologous, endogenous lectins.
Abstract: Hyaluronic acid (HA) is the main glycosaminoglycan present in follicular, oviductal and uterine fluids. The main functions of HA include dynamic processes that are mediated through interaction with extracellular matrix components, regulation of gene expression, cell proliferation and cell differentiation. HA increases the viscosity of solutions and also has several physiological functions, including regulation of water distribution and water-binding capacity. The addition of 6 mg HA ml(-1) to synthetic oviduct fluid (SOF; SOF-HA) culture medium on day 5 (IVF = day 0) significantly (P < 0.001) increased the viscosity of the medium in comparison with SOF culture medium containing BSA (SOF-BSA). On day 8, rate of blastocyst development in SOF-HA culture medium was significantly (P < 0.05) higher than in SOF-BSA culture medium (38.2 versus 29.3%). The number of trophectoderm cells and the total number of cells of expanded blastocysts cultured in the presence of HA were significantly (P < 0.01) higher in comparison with expanded blastocysts cultured in the presence of BSA (88.9 +/- 7.3 versus 67.6 +/- 3.0 and 130.1 +/- 10.9 versus 104.8 +/- 2.5, respectively). After freezing and thawing, the percentage of day 8 embryos that re-expanded and hatched when cultured with SOF-HA was greater than that of embryos cultured with SOF-BSA (11.3 and 10.5% versus 75.5 and 36.8%, respectively). After thawing, the ATP contents of in vivo-derived, SOF-HA and SOF-BSA expanded blastocysts were similar. The embryos cultured with HA showed less ultrastructural deviation and de-differentiation after freezing and thawing than the embryos cultured with BSA. This study demonstrates that HA improves the developmental capacity of bovine embryos under in vitro conditions and is warranted as a supplement for in vitro production of bovine embryos, particularly if they are to be cryopreserved.
Abstract: The expression pattern of the intermediate filament protein cytokeratin 18 (CK 18) is described during pre- and post-natal development of the porcine lung using a monoclonal antibody against human CK 18. Lungs from 16 foetuses in pseudoglandular, canalicular, saccular and alveolar stages of lung development and lungs from 12 pigs ranging in age from birth to 49 days after birth were studied by immunohistochemistry. In the early pseudoglandular stage of development (day 70 of gestation) all the columnar epithelial cells lining the tubular endbuds strongly expressed CK 18 predominantly in the apical cell compartment. A modest staining was found in the more cuboidal cells of the canalicular stage (day 80 of gestation) where the labelling occurred as a distinct positive rim at the apical cell membrane in most of the cells lining the canaliculi. In 96- and 100-day-old foetuses, parts of the gas exchanging area were formed as terminal sacs by extreme attenuation of the epithelium. In this stage, CK 18 was clearly detectable in the flat type I as well as in the cuboidal type II alveolar epithelial cells. A marked change of the CK 18 expression pattern occurred during formation of the alveoli by septal outgrowth and maturation of the epithelium in 105- and 111-day-old foetuses. Differentiated type I cells no longer expressed CK 18, whereas type II cells were still labelled. Moreover, a specific change in the subcellular distribution pattern from the luminal periphery in immature porcine type II cells to a cytoplasmic localization in differentiated type II cells could be observed. Our investigation additionally demonstrated that the epithelium of bronchi, bronchioli and terminal bronchioli expressed CK 18 in all pre- and post-natal developmental stages. From the 96 days of gestation onwards the epithelial cells of developing bronchial glands were also labelled. Our results clearly show that during porcine lung development profound changes in the cellular expression pattern of CK 18 occur and that CK 18 can be regarded as a selective marker for differentiated porcine alveolar type II cells from the 105th day of gestation onwards. We also assume that the intermediate filament CK 18 could be of significance in the maturation process of the type II alveolar cells.
Abstract: This study evaluated the effects of bovine serum albumin (BSA; 4 mg/ml) and estrous cow serum (ECS; 10%) in North Carolina State University (NCSU) 23 medium on the development of in vitro-matured and in vitro-fertilized porcine oocytes. Early cleavage rate was significantly (P < 0.05) higher in NCSU/ECS (71.3 +/- 14.7%) vs. NCSU/BSA (60.6 +/- 4.7%). Cleavage beyond the four-cell stage was not different between the two culture media (43.5 +/- 9.5% and 41.4 +/- 17.7%, respectively). The proportion of development to blastocysts was--with borderline significance (P = 0.05)--higher in NCSU/BSA (28.0 +/- 4.4%) than in NCSU/ECS (20.4 +/- 7.3%). Blastocysts produced in NCSU/BSA had significantly (P < 0.001) higher cell numbers than those cultured in NCSU/ECS (29.5 +/- 20.1 vs. 16.9 +/- 10.8). The ultrastructure of in vitro-produced blastocysts from both culture systems was compared vs. in vivo-derived blastocysts. The latter showed a clear differentiation between trophectoderm (TE) and inner cell mass (ICM) cells. The TE cells were anchored to other TE cells or ICM cells by long, well-developed junctional complexes. The apical membrane of trophoblast cells was covered with numerous microvilli. Mitochondria were abundant, round to elongated in shape, and showed clear transverse cristae. The ultrastructure of blastocysts cultured in NCSU/BSA mimicked that of in vivo-derived embryos closely. In contrast, blastocysts from the NCSU/ECS culture system displayed an irregular ultrastructure with reduced numbers of organelles and numerous cytoplasmic inclusions, such as lipid-yolk-vacuoles and vacuoles with lipid content. In some sections of these embryos, cellular debris was detected in cytoplasm. The shape of mitochondria was more ovoid and cristae were not visible. In summary, our results demonstrate a beneficial influence of ECS in the culture medium on initial cleavage of in vitro-produced porcine embryos. Clearly negative effects of ECS in the subsequent culture period are associated with marked ultrastructural changes of embryonic cells.
Abstract: An in vitro model using co-culture of bovine in vitro-produced (IVP) embryos and bovine oviduct epithelial cells (bOECs) was established to study embryo-maternal interactions in the oviductal environment. In vitro conditions maintaining differentiated growth of oviductal cells were determined by evaluating several media supplemented with different sera at various concentrations. Morphological features were used as indicators of physiological growth, and it became obvious that synthetic oviduct fluid (SOF) supplemented with either oestrous cow serum (OCS) or dextran-coated charcoal-treated fetal calf serum (DCC-FCS) helped to prevent dedifferentiation of bOECs (Expt 1). RT-real-time-PCR analysis revealed an increased mRNA content of the oviduct-specific glycoprotein GP 85-97 when using lower serum concentrations (2 and 5% compared with 10%; Expt 2). In subsequent experiments in which cell-free cultured controls and co-cultured embryos were compared, co-cultured embryos showed an increased rate of cleavage (P < 0.05) after 3 days. Successive cell-free culture until day 8 resulted in a lower rate of blastocyst development (P < 0.05) and reduced ATP content (P < 0.05) of co-cultured versus control embryos (Expt 3). Long-term co-culture (8 days) in SOF with 5% OCS increased the expression of developmentally relevant genes (glucose transporter 1 (Glut-1) and heat shock protein (HSP 70)) in co-cultured versus control embryos (Expt 4). Higher embryonic Glut-1 mRNA expression after co-culture was obvious when using 10% DCC-FCS, but was not significant when culture medium was supplemented with 10% rather than 5% OCS (Expt 5). In conclusion, SOF with 5% OCS supports differentiated growth of bOECs. Co-culture under these conditions improves early cleavage rate, but not blastocyst development, and increases the expression of developmentally relevant genes influenced by type of serum and serum concentration.
Abstract: To study the involvement of the IGFs in mammary development and lactation of the cow, the temporal expressions of IGF-I and -II, its receptor type 1 (IGFR-1), IGF-binding proteins (IGFBPs)-1 to -6 and GH receptor (GHR) mRNA were examined. This was carried out for different stages of mammogenesis, lactogenesis, galactopoiesis and involution in the bovine mammary gland of 26 animals. Furthermore, IGF-I was localised by immunohistochemistry. The highest mRNA concentrations for IGF-I were detected in the mammary tissue of late pregnant heifers (days 255-272) and significantly lower expression was detected during lactogenesis and galactopoiesis. Immunohistochemistry of IGF-I revealed only a weak staining in the epithelium of the ducts during mammogenesis. The epithelium of the alveoli were negative during mammogenesis, lactogenesis and galactopoiesis but displayed distinct IGF-I activity during involution. In the stroma a distinct staining of the cytoplasm of adipocytes and of vascular smooth muscle cells was observed. A certain percentage of fibroblasts (usually 20-30%) were also immunopositive. In contrast, highest expression for IGFR-1 was detected during galactopoiesis and involution. The lowest mRNA concentration for IGFR-1 was found during pregnancy (days 194-213). In general, the expression of IGF-II was not regulated during mammogenesis and lactation, but decreased during involution. The mRNA for the six binding proteins was detected in the bovine mammary gland. The dominant binding proteins were IGFBP-3 and -5. The highest expression of IGFBP-3 was observed during mid-pregnancy and the lowest during late lactation, involution and in non-pregnant heifers. The mRNA for IGFBP-5 increased during late mammogenesis and lactogenesis followed by a decrease thereafter. In general, the mRNA concentrations for IGFBP-2, -4 and -6 were barely detectable during all stages. In contrast, the expression for IGFBP-1 was upregulated in the mammary gland of virgin heifers and increased around the onset of lactation. mRNA for GHR was found during all stages examined without outstanding fluctuations. In conclusion, locally produced IGF-I and -II may mediate mammogenesis. The high mammary IGFR-1 mRNA during lactation suggests a role for peripheral IGF-I in maintenance of lactation. The role of IGFBPs in the mammary gland needs further evaluation.
Abstract: The zona pellucida (ZP) is an extracellular matrix surrounding the oocyte and the early embryo that exerts several important functions during fertilization and early embryonic development. The ZP of most mammalian species is composed of three glycoproteins (ZPA, ZPB, ZPC), products of the gene families ZPA, ZPB and ZPC that have been found to be highly homologous within mammalian species. Most data on the structure and function of the ZP are obtained from studies in mouse. New data from pig and other domestic animals, however, indicate that the mouse model does not hold for all other species. Whereas in the mouse ZPB is the primary sperm receptor, in the pig ZPA has been shown to possess receptor activity. Contrary to the mouse, where the growing oocyte is the only source of zona glycoproteins, in domestic animals these proteins are expressed in both the oocyte and granulosa cells in a stage-specific pattern and may play also a role in granulosa cell differentiation. In several mammalian species, the epithelial secretory cells of the oviduct synthesize and secrete specific glycoproteins (oviductins) that become closely associated with the ZP of the ovulated oocyte. Once bound to the ZP, oviductin molecules could act as a protective layer around the oocyte and early embryo by virtue of their densely glycosylated mucin-type domains.
Abstract: The mutation of the p53 gene is a common phenomenon in numerous human tumors including breast cancer. It leads to an accumulation of nonfunctioning p53 protein in the cell nuclei, which can be detected by immunohistochemical techniques. In breast cancer overexpression of mutated p53 protein has been correlated to a poor prognosis. Our study is an immunohistochemical analysis of p53 in 82 cases of breast cancer in young (< or = 30 years old) Kuwaiti women, correlating it with histopathological grade, lymph node status, estrogen (ER) and progesterone receptor (PgR) content, tumor cell proliferation (immunostaining for Ki-67) and expression of c-erbB-2 oncoprotein. p53 immunostaining was found in 47 (57.32%) of the carcinomas. 65% of them displayed positive immunostaining for c-erbB-2. 63.7% of tumors with p53 overexpression were aneuploid. 64.8% of the p53 positive tumors were node positive. 93.5% of the p53 immunopositive carcinomas were ER-negative, and in 95.7% of this subclass of patients no PgR could be detected. The vast majority of p53 positive carcinomas were grade III (76.6%), 21.3% were grade II and 2.1% grade I, but neither tumor grade or tumor size showed a correlation with p53 expression. A significant negative correlation between ER- and PgR-content (p = 0.006) and immunostaining for p53 was observed. Our study provided evidence that the association of negative hormone receptor status and positivity for p53 immunostaining points to a greater tumor aggressiveness.
Abstract: During the last decade many experiments have been performed to study the effects of growth hormone (GH, somatotropin) on reproductive functions. Most of the studies found only slight or no effects of GH treatment, both on the oestrous cycle and on gonadotropin, progesterone. or oestrogen serum levels. In GH-treated animals, elevated levels of insulin-like growth factor I and GH in the serum could be correlated with an increased number of small (< 5 mm in diameter) ovarian follicles, possibly as a consequence of a reduction of apoptosis and follicular atresia. There is still controversy over the effects of GH on in vivo and in vitro embryo production and on the gestation period. Recent studies produced some evidence that GH-receptor is expressed in ovarian tissue, implying a direct role for GH in the ovary.
Abstract: Pituitary growth hormone (GH) stimulates postnatal growth and metabolism. The role of GH and its receptor (GHR) during prenatal development, however, is still controversial. As shown by reverse transcription polymerase chain reaction (RT-PCR), bovine in vitro fertilization embryos synthesized the transcript of GHR from Day 2 of embryonic life onwards. Real time RT-PCR revealed that synthesis of GHR mRNA was increased 5.9-fold in 6-day-old embryos compared with 2-day-old embryos. Using in situ hybridization, the mRNA encoding GHR was predominantly localized to the inner cell mass of blastocysts. The GHR protein was first visualized 3 days after fertilization. GH-specific transcripts were first detected in embryos on Day 8 of in vitro culture. As shown by transmission electron microscopy, GH treatment resulted in elimination of glycogen storage in 6- to 8-day-old embryos and an increase in exocytosis of lipid vesicles. These results suggest that a functional GHR able to modulate carbohydrate and lipid metabolism is synthesized during preimplantation development of the bovine embryo and that this GHR may be subject to activation by embryonic GH after Day 8.
Abstract: A 2-year comparative study was carried out to evaluate the effect of ovary size, follicle size and oocyte quality of 3-month-old Simmental calves and the efficiency of using calf ovaries in an in vitro fertilization (IVF) programme. We evaluated the effects of different concentrations of follicle-stimulating hormone (FSH) and oestradiol-17beta (E-17beta) in the maturation medium on the in vitro development of calf oocytes into morula and blastocysts. The proportion of recovered oocytes (62.1%; 42.8%; 25.3%) and the percentage of good quality cumulus oocyte complexes (84.2%: 59.8%; 45.9%) decreased significantly (P < 0.01) with decreasing ovary size (L, M and S). The rates of two or more cells on Day 2 and of blastocysts on Day 7 and Day 9 were significantly lower (P < 0.01) for calf oocytes (61.5%; 18.9%: 15.9%) compared with those from sexually matured females (70.1%: 32.3%; 22.2%). Calf oocytes. matured in medium supplemented with 20 microg/ml or 10 microg/ml FSH plus 2 microg/ml E-17beta had higher rates of cleavage on Day 2 (64.1% and 64.7%) and blastocysts on Day 7 (24.5% and 22.4%) than the control supplemented with 10 microg/ml FSH (55.6% and 19.2%, respectively). Groups supplemented with 20 microg/ml FSH plus 2 microg/ml E-17beta and 10 mg/ml plus 4 mg/ml E-17beta showed a significantly lower developmental rate of blastocysts on Day 7 (14.6% and 14.5%). High concentrations of E-17beta (4 microg/ml) resulted in a significantly lower development of blastocysts on Day 9 (8.1%) and hatched blastocysts on Day 13 (3.5%) (P < 0.01). We conclude that the proportion of calf oocytes obtained from immature animals and their suitability for IVF are lower than those of cows. Thus, the use of oocytcs from sexually immature females would decrease the relative efficiency of IVF programmes. Supplementation with high concentrations of FSH can improve the maturation and developmental capacity of oocytes from prepubertal calves.
Abstract: The zona pellucida (ZP) is an extracellular matrix surrounding the oocyte and the early embryo that exerts several important functions during fertilization and early embryonic development. The ZP of most mammalian species is composed of three major glycoproteins that show considerable heterogeneity due to extensive post-translational modifications. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of the ZP reveals three to four glycoproteins which have been nominated ZPI. ZP2, ZP3 and ZP4. As cloning and characterization of the ZP genes of a variety of mammalian species including domestic animals show a high homology, three classes of ZP genes, ZPA, ZPB and ZPC can be discerned. The corresponding proteins were named ZPA, ZPB and ZPC. Whereas in the mouse ZPB is the primary sperm receptor. the situation is more complicated in other species. For instance, in the pig ZPA has been shown to possess receptor activity. Interaction between gametes during fertilization is at least in part regulated by carbohydrate moieties of the ZP and carbohydrate-binding proteins of the sperm surface. In domestic animals zona proteins are expressed in both the oocyte and granulosa cells in a stage-specific pattern and may play a role in granulosa cell differentiation. The role of ZP glycoproteins in immunocontraception is briefly discussed.
Abstract: Fibroblast growth factor 2 (FGF-2, basic fibroblast growth factor) has been reported to be elevated in tissues from benign prostatic hyperplasia (BPH), the most frequent neoplastic disease in aging men. This suggests that FGF-2 may play a significant role in the development of BPH. In this study the cellular distribution pattern of FGF-2 in tissues from BPH has been investigated by immunohistochemical and molecular biological methods. Radioimmunoassay revealed high concentrations of FGF-2, ranging between 450 and 950 ng per g tissue. Immunoblots confirmed the presence of a 18 kDa FGF-2 in tissue extracts. By immunohistochemistry done with a polyclonal antibody to recombinant FGF-2 on paraffin sections, FGF-2 was localized in fibroblasts, endothelial cells and smooth muscle cells of tissue samples of BPH. Nuclei of these cells were labelled distinctly. Moreover the cytoplasm of smooth muscle cells was labelled moderately. No immunostaining was seen in prostatic epithelium. Non-radioactive in situ hybridization with digoxygenin-labelled oligonucleotides revealed the presence of mRNA for FGF-2 in smooth muscle cells of the prostatic stroma. These results provide evidence that FGF-2 may be produced locally in the human prostate as a stroma-specific mitogen and may play a causal role in the development of BPH.
Abstract: Our immunohistochemical results clearly demonstrated the occurrence of growth hormone receptors (GH-R) in the tumour cells of lipomas and liposarcomas. In liposarcomas staining intensity in the cytoplasm of tumour cells varied between weak and distinct but could not be correlated to the histological grade of the malignant tumours. These findings were corroborated to some extent by the RT-PCR results. RT-PCR analysis of human lipomas and liposarcomas revealed the amplified cDNA fragment of GH-R in 8 out of 12 lipomas but only in 3 out of 10 liposarcomas. The reduced number of GH-R positive tumours found with PCR may be explained by the extraction method of RNA from paraffin sections. An interesting finding was the distinct immunoreactivity of the endothelium of blood vessels in liposarcomas, which was especially pronounced in the newly forming capillaries. This points to an important role of GH-R in tumour angiogenesis which could significantly contribute to tumour growth in liposarcomas and may open the possibility for therapeutic intervention using antiangiogenic substances.
Abstract: Growth and differentiation of the mammary gland during development and lactation are controlled by complex hormonal mechanisms. Additionally growth factors are supposed to act as local mediators of the hormonally controlled developmental processes. Mammary tissue for this study was obtained from non pregnant control heifers, primigravid heifers (second part of pregnancy), around parturition, during lactation (early and late) and from dry cows. Using RT-PCR and ribonuclease protections assay (RPA) the expression of the following growth factors was studied in the different phases bovine mammary gland development: Insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), fibroblast growth factor 1 (FGF-I), fibroblast growth factor 2 (FGF-2), transforming growth factor alpha (TGF-alpha). Additionally the expression of fibroblast growth factor receptor (FGFR) and growth hormone receptor (GHR) was investigated. The cellular distribution pattern of several of these growth factors and GHR was obtained using Immunocytochemical techniques. The detailed expression and localization pattern of these growth factors are presented and their role in the local regulation of the bovine mammary gland is briefly discussed.
Abstract: Organogenesis and the establishment of the mature phenotype require an interplay between diverse recognition systems. Concerning protein-carbohydrate interactions, galectins are known to be involved in several extra- and intracellular functions. Due to the occurrence of two avian galectins in liver (chicken galectin-16 CG-16) and intestine (chicken galectin-14; CG-14) with different developmental regulation. the questions addressed are to what extent and where these galectins are present during chicken kidney development. Using Western blot analysis, the presence of both activities in tissue extracts was ascertained. A solid-phase assay showed peak levels at day 12 followed by a decline. A histochemical analysis was carried out in combination with routine staining. Epithelial cells of the mesonephric proximal tubules were immunoreactive in the cytoplasm for CG-14 from day 5 of incubation onwards. Additionally, epithelial cells of the metanephric collecting ducts were stained. For CG-16 a rather similar pattern of staining was seen, additional positivity in early glomerular podocytes being notable. At the electron microscopical level, a diffuse staining for CG-14 was seen in the cytoplasm, whereas immunoreactivity for CG-16 was observed mainly in mitochondria. These results demonstrate quantitative differences in the developmental regulation of the two avian galectins with obvious similarities in the cell-type pattern but with a disparate intracellular localisation profile.
Abstract: We have used immunohistochemistry and non-radioactive in situ hybridisation to localise the GH receptor and its transcript in the bovine mammary gland during mammogenesis, lactation and involution. We found a characteristic pattern of immunoreactive GH (irGH) receptor distribution in the epithelial and stromal compartments during the different stages of mammary gland development: The ductular epithelium showed a distinct staining for irGH receptor during most stages, whereas the alveolar epithelium contained a modest amount of GH receptor during pregnancy which increased during lactation and galactopoiesis. In dry cows, the immunostaining for GH receptors in the alveolar epithelium was very weak or negative. Curiously, the amount of GH receptor mRNA appeared relatively constant during mammogenesis and lactation. The epithelial cells of the alveoli and ducts as well as the endothelial cells showed a distinct signal in our in situ hy! bridisation studies. The predominant localisation of GH receptors in the epithelium of ducts and alveoli is supportive of a role for GH in epithelial differentiation and maintenance. Furthermore, the increased intensity of immunostaining in bovine mammary tissue post partum suggests a direct role for GH receptor in mediating the effect of GH in milk production and secretion.
Abstract: By reverse transcription-polymerase chain reaction the transcript of the growth hormone receptor (GHR) was proved in 21 human prostatic carcinomas and 19 benign prostatic hyperplasias. A new method for extracting RNA from paraffin-embedded tissue was established. Western immunoblotting using the monoclonal antibody mAb 263 revealed that the growth hormone receptor protein is equally expressed in prostatic carcinoma and hyperplasia. As shown by immunohistochemistry, the GHR protein is synthesized in the epithelial cells of the tumor acini. On the contrary, GHR was not found in normal prostatic epithelium. Our results imply that the growth hormone receptor may facilitate prostatic tumor cell growth.
Abstract: The oviduct is the physiological site for key events in reproduction, such as capacitation of spermatozoa, fertilization and early embryonic development. Interactions between oviduct epithelial cells and gametes or embryos cannot sufficiently be studied in vivo. Therefore, model systems are needed which mimic in vivo conditions most closely. In this study we optimised the method for isolating bovine oviduct cells and compared different cell support materials as well as two culture systems (perfusion vs static culture) for their ability to maintain characteristic morphological and functional features of oviduct cells. Out of nine different cell support materials tested, cellulose nitrate (0.45 micron pore size) was the most suitable to maintain cells in a manner similar to freshly isolated oviduct epithelial cells. Comparing static vs perfusion culture by electron microscopy, morphological differences of the cells were insignificant in the first days of culture, while they became more evident after 8 days. The cells in the static system lost typical characteristics such as columnar shape, cilia and secretory protrusions, while these features were still present in perfusion culture. In addition, intense ciliogenesis and cytoplasmic organelles for protein synthesis were found under perfusion conditions. These findings were underlined by differences in expression of the oviduct-specific oestrus-associated glycoprotein 85-97 kDa (GP 85-97) gene as revealed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The RNA levels of this specific gene were significantly higher in perfusion compared to the static culture system. Our data show clear advantages of perfusion vs static culture for primary bovine oviduct epithelial cells.
Abstract: The olfactory epithelium is unique in the mammalian nervous system as it is a site of continual neurogenesis. Constant turnover of primary sensory neurons in the periphery results in continuous remodeling of neuronal circuits and synapses in the olfactory bulb throughout life. Most of the specific mechanisms and factors that control and modulate this process are not known. Recent studies suggest that growth factors, and their receptors, may play a crucial role in the development and continuous regeneration of olfactory neurons, i.e. particularly in neuronal proliferation, neurite outgrowth, fasciculation and synapse formation of the olfactory system. The potential role of the following factors and their receptors in different species are reviewed: Nerve growth factor (NGF); insulin-like growth factors (IGFs); fibroblast growth factors (FGFs); epidermal growth factor (EGF); transforming growth factor alpha (TGF alpha); amphiregulin (AR) and transforming growth factors beta (TGFs beta).
Abstract: Immediately after ovulation a neovascular response occurs at the level of the theca interna. Pericytes and endothelial cells of post-capillary venules locally remodel the surrounding stroma, elongate and migrate into the avascular granulosa folds of the ruptured follicle. In order to examine the composition of the extracellular matrix as well as the growth characteristics of these newly formed vessels, we used immunohistochemical and electron microscopic methods. Initial sprouts were characterized by the appearance of a fibrillary network of fibronectin along the main axis of the sprout. Type IV collagen stained weakly and extracellular deposits of laminin were amorphous and patchy around immature capillary sprouts. In advanced maturational stages of the sprouts the capillaries were surrounded by increased deposits of fibronectin, whereas laminin and type IV collagen displayed a distinct and well-developed line around endothelial cells and pericytes. These observations indicate that the microvascular extracellular matrix undergoes a series of quantitative rather than qualitative changes during capillary development before achieving final maturation. Ultrastructural analyses showed that early capillary sprouts in the bovine corpus luteum were usually preceded by pericytes migrating at the tips of the sprouts. Endothelial cells comigrated in cohesive cylindrical projections, forming immediately a slit-like lumen which satisfies the criteria of the intercellular canalization type. Pericytes at the tips of endothelial sprouts exhibited a slender, bipolar morphology and were regularly surrounded by fragmented basal lamina, which is well-developed around pericytes in a more proximal position of the sprout. The regular association of pericytes with the leading front of the capillary sprouts suggests that these cell types may serve as guiding structures aiding outgrowth of endothelial cells in the bovine corpus luteum.
Abstract: Growth hormone (GH) exerts its regulatory functions in controlling metabolism, balanced growth and differentiated cell expression by acting on specific receptors, which trigger a phosphorylation cascade resulting in the modulation of numerous signalling pathways, and dictate gene expression. Immunohistochemical techniques were used to demonstrate the presence of growth hormone receptors in 126 formalin-fixed, paraffin-embedded melanocytic tumours comprising melanocytic naevi, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma and metastatic melanomas. The relative proportion of positive cells and intensity of staining was higher in neoplastic cells, compared to normal cutaneous cells. Of the 76 cases of common melanocytic naevi (CMN) studies, 46 were weakly reactive with MAb 263. Heterogeneity of immunoreactivity was found in primary melanoma lesions with a variable range of positive cells. Of 37 cases studied, 34 were moderately to strongly positive. Immunoreactivity showed subcellular localization of the GH-receptor in cell membranes, was predominantly cytoplasmic, but strong nuclear immunoreaction was also apparent in many instances. The nuclear localization of immunoreactivity is the result of nuclear GH-receptor/binding protein, identically to the cytosolic and plasma growth hormone binding protein. Intense immuno-reactivity was also observed in the cellular Golgi area of established cell lines and cultured tissue-derived cells in exponential growth phase, indicating cells are capable of GH-receptor synthesis. In the primary lesions, dermal tumour cells tended to be more immunoreactive relative to those seen in the dermal region. Metastatic lesions in various organs also expressed growth hormone receptors in secondary tumour cells and all of the metastatic cases were positive. The expression of GH-receptors in human melanoma cells means that these cells are directly responsive to GH action and that GH may stimulate local production of IGF-I, which then acts in an autocrine mechanism.
Abstract: The duplication of genes for recognition molecules and the ensuing diversification of the members of such families generate complex groups of homologous proteins. One example are galactoside-specific lectins whose sequences display constant features related to sugar binding, the galectins. Based on the inverse abundance of the chicken galectins CG-14 and CG-16 in adult intestine and liver, these two lectins represent a model to comparatively study expression of the related proteins and the galectin-reactive sites (glycoproteins and glycolipids) biochemically and histochemically. Functional overlap and/or acquisition of distinct functions would be reflected in qualitative and/or quantitative aspects of ligand display. Using five different stages of embryogenesis, differential regulation of the two galectins was detected in liver and intestine. The clear preference for one galectin (CG-14) was observed in intestine already at rather early stages, whereas equivalence for both proteins was noted in liver from day 12 to day 18 prior to hatching, as seen by ELISA assays and Western blot analysis. Presentation of galectin-reactive glycoproteins showed a tendency for gradual increase in both organs. Galectin-blotting analysis revealed primarily very similar patterns of positive bands at the different stages of development and only few quantitative and qualitative changes. The reactivity of glycolipids in a solid-phase assay was more variable, even surpassing the response of extracts of the adult organ at several embryonic stages. While the localization patterns of the galectins and galectin-reactive sites were nearly indistinguishable in the liver, intestinal tissue differed with respect to the placement and accessibility of binding sites. Thus, the results suggest a differential regulation of galectin activities in the two organs. As a sum they resemble the course of development of availability of glycoprotein ligands in vitro. These findings support the notion for a partial functional redundancy in this family. The described approach to employ galectin-specific antibodies and the labeled galectins as tools to assess presentation of ligands is suggested to be of general relevance to address the question of distinct vs. overlapping functions of related recognition molecules.
Abstract: Using in situ hybridization, mRNA encoding the growth hormone receptor (GHR) was localized in preimplantation embryos produced by in vitro fertilization (IVF) as well as in 30- to 70-day-old bovine embryos. In IVF embryos the transcript of GHR was demonstrated in the inner cell mass of 8-day-old blastocysts. In 30-day-old embryos, the mesonephros was the first organ to express the mRNA of GHR. In 40-day-old embryos, the transcript was found in the neurones of the spinal ganglions, the splanchnic nerves and the motoneurones of the spinal cord, in the vascular endothelium, and in the developing striated muscle tissue. Colocalization of the protein by immunohistochemistry showed an identical distribution pattern of GHR in 30- to 70-day-old embryos.
Abstract: Using nonradioactive in situ hybridization (ISH), the mRNA encoding the zona glycoprotein bZPC was localized in bovine ovaries, oocytes, and embryos. In the ovary, the distribution of the mRNA was correlated with the developmental stage of the follicle. Whereas in primordial and primary follicles the mRNA was predominantly seen in the oocyte, it was found in both the oocyte and the follicle cells of secondary and tertiary follicles. In 2-day-old embryos produced by in vitro fertilization (IVF), no mRNA encoding ZPC could be demonstrated. Immunoblotting using monospecific polyclonal antibodies against porcine ZPC revealed a distinct band at a molecular weight of 47 kD in the ovarian cortex of cows, calves, and fetuses as well as in bovine follicle cells. Immunohistochemistry using the ZPC antibody displayed a strong signal in the zona pellucida of bovine oocytes and 2- to 6-day-old embryos as well as in the follicle cells. Our results show that during follicular development bovine ZPC is synthesized by the oocyte of the primary follicle and by both the oocyte and the follicle cells of the secondary and tertiary follicle. After fertilization, the synthesis of the zona protein is finished.
Abstract: Growth hormone (GH) exerts its regulatory functions in controlling metabolism, balanced growth and differentiated cell expression by acting on specific receptors which trigger a phosphorylation cascade, resulting in the modulation of numerous signalling pathways dictating gene expression. A panel of five monoclonal antibodies was used in mapping the presence and somatic distribution of the GH receptor by immunohistochemistry in normal and neoplastic tissues and cultured cells of human, rat and rabbit origin. A wide distribution of the receptor was observed in many cell types. Not all cells expressing cytoplasmic GH receptors displayed nuclear immunoreactivity. In general, the relative proportion of positive cells and intensity of staining was higher in neoplastic cells than in normal tissue cells. Immunoreactivity showed subcellular localisation of the GH receptor in cell membranes and was predominantly cytoplasmic, but strong nuclear immunoreaction was also apparent in many instances. Intense immunoreactivity was also observed in the cellular Golgi area of established cell lines and cultured tissue-derived cells in exponential growth phase, indicating cells are capable of GH receptor synthesis. The presence of intracellular GH receptor, previously documented in normal tissues of mostly animal origin, is the result of endoplasmic reticulum and Golgi localisation. Heterogeneity of immunoreactivity was found in normal and neoplastic tissue with a variable range of positive cells. The nuclear localisation of immunoreactivity is the result of nuclear GH receptor/binding protein, identically to the cytosolic and plasma GH-binding protein, using a panel of five monoclonal antibodies against the GH receptor extracellular region. The expression of GH receptors, not only on small proliferating tumour cells such as lymphocytes, but also on well differentiated cells including keratinocytes, suggests that GH is necessary not only for differentiation of progenitor cells, but also for their subsequent clonal expansion, differentiation and maintenance.
Abstract: Growth hormone (GH) is known to play a key role in postnatal growth and differentiation. The role of GH and its receptor (GHR) in prenatal development, however, is still controversial. Using reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization (ISH) and immunohistochemistry we demonstrated the presence of GHR mRNA and protein in bovine mammary glands during fetal development. RT-PCR revealed GHR transcripts in fetal mammary glands from the third to the ninth month of pregnancy. By non-radioactive ISH, GHR mRNA was localized in the glandular epithelium, the surrounding mesenchymal cells, endothelial cells of vessels and in the stratum basale of the epidermis of fetal mammary glands. From the sixth month of fetal life onwards, GHR transcripts were also found in the cytoplasm of adipocytes. Immunohistochemical studies using the monoclonal antibody mAb 263 revealed the same distribution pattern as the mRNA. Our results imply that the growth hormone receptor is distinctly expressed in the immature mammary gland, suggesting that GH is involved in growth and differentiation of the fetal mammary gland.
Abstract: The purpose of this study was to investigate the expression of two zona pellucida gene families ZPB (ZP3 alpha) and ZPC (ZP3 beta). Sections of ovaries from bovine fetuses, calves and cows were labelled with polyclonal antibodies. Immunopositive labelling was found in both the follicle cells and the oocyte. Labelling was dependent on the stage of development. The specific sequence of immunopositive reactions suggests that in the bovine fetus both the ovary and the follicle cells contribute to the production of the zona pellucida during pre- and postnatal development.
Abstract: Spermadhesins are proteins with various functions in sperm capacitation and zona pellucida binding. In this study the cellular localization of the spermadhesin AWN-1 has been examined in the equine male genital tract. Results obtained by immunohistochemical methods reveal that in the horse AWN-1 is synthesized in spermatogonia, in the rete testis, the ductus epididymidis and the seminal vesicles. These findings indicate that the cellular origin of spermadhesins is species-specific.
Abstract: By reverse transcription-polymerase chain reaction, the transcript of the growth hormone receptor (GHR) was demonstrated in oocytes, follicular cells, and corpus luteum of the bovine ovary. Immunoblotting using the monoclonal antibody mAb 263 resulted in a distinct protein band at 120 kDa, confirming that translation of the mRNA takes place in the same cells. Nonradioactive in situ hybridization revealed that distribution of the mRNA encoding GHR was correlated with the developmental stage of the follicle. Whereas in primordial and primary follicles the oocyte showed distinct amounts of the transcript encoding GHR, in tertiary follicles the mRNA was predominantly localized in the cells of the cumulus oophorus. GHR mRNA was also expressed in the large granulosa lutein cells, in the germinal epithelium, and in the endothelial cells of ovarian vessels. Colocalization of the GHR protein showed a distribution pattern identical to that of the mRNA. In calves, oocyte and follicle cells changed GHR expression in the same way as in the adult ovary. During embryonic development of the ovary, distinct amounts of the mRNA encoding GHR were found in primordial follicles shortly before birth. Our results imply that the GHR is involved in ovarian ontogenesis, especially in early folliculogenesis.
Abstract: The goal of the study was to examine the expression and localization of members of the fibroblast growth factor family in the bovine mammary gland during different developmental and functional stages. Mammary tissue was obtained from German Brown Swiss cows (n = 23) during defined stages of mammogenesis (before and during pregnancy), lactogenesis, peak and late lactation, and involution. Extracted mRNA was analyzed by reverse transcription polymerase chain reaction and RNase protection assay. The tissue content of fibroblast growth factor-1 and fibroblast growth factor-2 was determined by radioimmunoassay, and the localization of fibroblast growth factor-2 was determined by immunohistochemistry. The highest mRNA concentration for fibroblast growth factor-1, -2, and -7 and their receptors was detected in the glands of virgin heifers or primigravid heifers during involution; less abundant fibroblast growth factor mRNA was detected during lactogenesis and galactopoiesis. Tissue protein concentrations of fibroblast growth factor-1 and fibroblast growth factor-2 showed similar tendencies. Immunoreactive fibroblast growth factor-2 was observed during mammogenesis and involution in endothelial cells, ductal epithelial cells, myoepithelial cells, and some alveolar cells. After positive staining, fibroblast growth factor-2 could only be observed in endothelial and myoepithelial cells during lactogenesis and could only be observed in myoepithelial cells during galactopoiesis. Expression, tissue concentration, and distinct localization suggest that fibroblast growth factors may be important in the local regulation of the bovine mammary gland.
Abstract: The olfactory system is a highly plastic region of the nervous system. Continuous remodeling of neuronal circuits in the olfactory bulb takes place throughout life as a result of constant turnover of primary sensory olfactory neurons in the periphery. Glycoconjugates are very important in olfactory development, regeneration and function. This article deals with different aspects of glycobiology relevant for the olfactory system. Various anatomical, developmental and functional subdivisions of the olfactory system have been labeled with exogenous lectins. The application of reverse lectin histochemistry resulted in the visualization of endogenous lectins, involved in fasciculation of olfactory axons. Numerous glycoproteins, among them members of the immunoglobulin superfamily, the cadherins and integrins as well as different glycolipids and proteoglycans can act as surface adhesion molecules in the olfactory system. The olfactory-specific form of the sialoglycoprotein neural cell adhesion molecule is implicated in olfactory neuronal and axonal guidance. Glycoconjugates including laminin, fibronectin and proteoglycans are abundant components of the olfactory extracellular matrix, influencing neurite outgrowth and cellular migration. Immunohistochemical labeling has revealed occurrence of the carbohydrate differentiation antigen, playing a role in neurulation and morphogenesis of the very early olfactory system. The synaptic vesicle glycoprotein, appearing also early in olfactory development, is used as a marker of olfactory tumors. Finally, membrane and transmembrane glycoconjugates as well as secreted glycoconjugates may act as olfactory receptor molecules.
Abstract: Interaction between gametes during fertilization is at least in part regulated by carbohydrate moieties of the zona pellucida (ZP) and carbohydrate binding proteins of the sperm surface. This review focuses on the protein-carbohydrate interactions during the primary binding of the sperm to the ZP in different species. Synthesis, structure and composition of the ZP are summarized. The functional significance of carbohydrate residues of the ZP as sperm receptor is discussed. Sperm surface proteins known to have specific ZP and carbohydrate-binding sites including the mouse beta1, 4-galactosyltransferase and sp56, the rabbit protein Sp17, a human mannose-binding protein and several members of the spermadhesin family are presented.
Abstract: By reverse transcription-polymerase chain reaction (RT-PCR) the transcript of the growth hormone receptor (GHR) was proved in bovine placentae of different gestational stages. Using non-radioactive in situ hybridization (ISH) the mRNA encoding GHR was localized in the uterine epithelium, glands, vessels and amniochorion of the placenta from the sixth week until the ninth month of gestation. Production of mRNA was low during the first trimester, significantly increased in the second trimester and peaked at the end of the sixth month of pregnancy. No transcript of GHR was found in the endometrium of non-pregnant uteri. The colocalization of the protein using a monoclonal antibody showed the same distribution pattern as the mRNA. Immunoblotting revealed a protein with a molecular weight of 120 kDa in the endometrium of pregnant cows which was not found in non-pregnant uteri. Our results imply that growth hormone is involved in placental metabolism and embryonic development from the early beginning of pregnancy until birth.
Abstract: Long-term culture of bone marrow derived stromal colony forming cells (S-CFC) in matrix and nutrient defined agar medium resulted in stromal cell colonies that pass sequentially through three distinct morphological stages: firstly, aggregated loose syncytium of round to avoid cells (stage I), a second developmental stage of large branching colonies in which the cells become enlarged, elongated with cytoplasmic projections forming a loosely anastomized network with adjacent cells (stage II), and finally cells become dissociated, loosing their long, thin cytoplasmic filaments and breaking their contacts with one another, but remain large and retain a bi-polar nature (stage III). Cells were also grown in liquid medium in a culture microenvironment closely resembling conditions of haemopoiesis in vitro. Using a panel of well defined monoclonal antibodies reactive against the rat, rabbit and human growth hormone receptors, this study found immunochemical evidence of the presence and localization of binding sites of growth hormone (GH) in the cell membrane and extra-nuclear Golgi area of long-term bone marrow derived human stromal cells in liquid and semi-solid nutrient agar mediums. GH-receptor immunoreactivity was present in small proliferating progenitor cells, myofibroblast-like cells, large reticular fibroblast cells, adipocytes and endothelial cells. Only MAb known to be reactive against human tissue resulted in strong immunoreactivity. The expression of GH-receptors not only on small proliferating, but also on the well differentiated cells, indicates a role for growth hormone on non-progenitor cells. GH-receptor immunoreactivity on differentiating and/or differentiated cells suggests that GH is also necessary for, or has a trophic function in differentiation. We propose that direct GH action is necessary not only for differentiation of progenitor cells as implied by the dual effector hypothesis, but also their subsequent clonal expansion, differentiation and maintenance.
Abstract: The display of cellular oligosaccharide chains is known to undergo marked developmental changes, as monitored histochemically with plant lectins. In conjunction with endogenous lectins respective ligand structures may have a functional role during fetal development. The assumption of a recognitive, functionally productive interplay prompts the study of the expression of a tissue lectin and of lectin-reactive glycoconjugates concomitantly. Focusing on common beta-galactosides as constituents of oligosaccharide chains and the predominant member of the family of galectins in mammals, namely galectin-1, the question therefore is addressed as to whether expression of lectin and lectin-reactive glycoconjugates exhibits alterations, assessed in three morphologically defined fetal stages and in adult bovine organs. Using a sandwich ELISA, the level of the rather ubiquitous galectin-1 is mostly increased in adult organs relative to respective fetal stages, except for the case of kidney. This developmental course is seen rather seldom, when the amounts of lectin-reactive glycoproteins or glycolipids are quantitated in solid-phase assays after tissue homogenization. Western blotting, combined with probing by labeled galectin-1, discloses primarily quantitative changes in the reactivity of individual glycoproteins. Performing the same assays on extract aliquots with a plant agglutinin, namely the galactoside-binding mistletoe lectin, whose fine specificity is different from galectin-1, its reduced extent of binding in solid-phase assays and the disparate profile of lectin-reactive glycoproteins reveal a non-uniform developmental alteration within the group of structural variants of beta-galactosides. Although sample preparation can affect ligand preservation and/or presentation and thus restricts the comparability of biochemical and histochemical results, especially for soluble reactants, the histochemical studies on frozen and paraffin-embedded sections of bovine heart, kidney and liver demonstrate that the localization of the galectin and of lectin-reactive epitopes can show a similar distribution, as seen in liver and heart, with organ-typical quantitative changes of a rather similar staining profile (heart, kidney) or notable changes in the spatial distribution (liver) in the course of development. This report emphasizes the potential value of combined monitoring of the lectin and its potential in vivo ligands to contribute to eventually unravel organ-related function(s) of a tissue lectin.
Abstract: It is now widely accepted that the mammary gland is under interconnected hormonal and local control. Growth factors are involved in the intercellular signalling of the gland. Our aim was the detection of transforming growth factors alpha (TGF-alpha) and beta 1 (TGF-beta 1) messenger RNA during mammogenesis, lactogenesis, galactopoiesis and involution in the bovine mammary gland (total n = 27). During these stages the RNA was assessed by means of ribonuclease protection assay and reverse transcription-polymerase chain reaction (RT-PCR). To study possible influences of oestrogen, progesterone and prolactin on growth factor expression, mammary RNA was obtained from heifers after induced mammogenesis and lactogenesis, with and without additional prolactin inhibition (total n = 20). Very low levels of TGF-alpha and TGF-beta 1 expression were detected during lactogenesis and galactopoiesis, increasing levels during mammogenesis of primigravid heifers, and highest levels during mammogenesis of virgin heifers and during involution. TGF-alpha expression after induced mammogenesis was greater than after induced lactogenesis or physiological mammogenesis during pregnancy. Furthermore, TGF-alpha mRNA contents increased after prolactin inhibition. TGF-beta 1 expression was almost equal after induced mammogenesis and lactogenesis, but greater than during the physiological mammogenesis and lactogenesis. In conclusion, it can be assumed that growth promoting TGF-alpha and growth inhibiting TGF-beta 1 are co-expressed in the bovine mammary gland. Higher mRNA contents of both factors during mammogenesis and involution may indicate autocrine or paracrine functions for these growth factors during proliferation and reorganisation of the mammary tissue.
Abstract: The purpose of the present study was to investigate potential modulations of endothelial cells of the bovine corpus luteum (CL) during pregnancy. Luteal endothelia of pregnant and non-pregnant cows were isolated and purity of cultures was verified by flow cytometric quantification of three independent endothelial markers (von Willebrand factor, angiotensin converting enzyme, Bandeiraea simplicifolia agglutinin I ligands). Different cellular parameters including light and electron microscopical investigation of morphology and growth characteristics as well as quantification of cellular lectin binding sites were compared. Extensive heterogeneity between luteal endothelial cells in pregnant and non-pregnant animals could be demonstrated, reflected in functional attributes like angiogenic activity, ultrastructural characteristics and the quantitative expression of cellular carbohydrates. Two different morphological types of cells ("cob-blestone growth pattern' and "arcuate growth pattern') were isolated from the CL of pregnancy as well as from the cyclic CL. Spontaneous angiogenic activities, including cellular migration in band-like structures and formation of ring-like structures, were observed in endothelial cells isolated from the CL of pregnant cows exclusively. This strongly suggests that microvascular luteal endothelium of pregnant animals, in contrast to the one of non-pregnant animals, is able to produce quantitatively and/or qualitatively specific angiogenesis factor(s). Heterogeneity between luteal endothelial cells in the pregnant and non-pregnant animal could also be demonstrated by quantification of lectin (Bandeiraea simplicifolia agglutinin I, concanavalin A, Dolichos biflorus agglutinin, Ulex europaeus agglutinin I, wheat germ agglutinin) binding sites: quantitative expression of specific endothelial cell surface carbohydrates could be correlated to the status of pregnancy, thus emphasizing the actual need of quantification of lectin binding.
Abstract: The mRNA of the zona pellucida glycoprotein ZP3 alpha was localized in frozen sections of pig ovaries, isolated oocytes and early embryos by in situ hybridization using biotinylated oligonucleotide probes. In follicles, the distribution of mRNA for ZP3 alpha was correlated with the developmental stage: in primordial and primary follicles, the mRNA was shown to be predominantly localized in the oocyte. In secondary follicles, mRNA was found in both the oocyte and follicle cells. In tertiary and preovulatory follicles, the follicle cells showed distinct staining, whereas the oocyte was labelled weakly. In the early embryo, i.e. 2 days after fertilization, mRNA for ZP3 alpha could not be demonstrated. Our results suggest that, in the pig, the zona pellucida protein ZP3 alpha is synthesized by the oocyte and the follicle cells in sequence. After fertilization, synthesis of ZP3 alpha is terminated.
Abstract: The purpose of this study was to isolate endothelial cells from different organs of porcine fetuses and to examine the binding of endothelial markers including lectins. Endothelial cells were isolated from the aorta, cerebral cortex, myocardium, ovary and testis. Binding of the antibodies to von Willebrand factor (vWF) and angiotensin converting enzyme (ACE), the presence of Weibel Palade bodies (WPB), uptake of acetylated low density lipoprotein (acLDL), and labelling with the lectins Bandeiraea simplicifolia agglutinin I (BS I), Peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA) and Ulex europaeus agglutinin I (UEA I) were examined. Cell preparations displayed cobblestone-like morphology with the exception of testicular endothelium, which formed arcuate structures. Endothelium isolated from the brain labelled more strongly than any other cell line with the lectin PNA, but it did not express ACE. In contrast to other cell preparations, myocardial endothelium showed very low binding of anti-vWF. Ovarian endothelium was able to perform in vitro angiogenesis. Moreover, these endothelial cells possessed the largest number of WPB. Testicular endothelium displayed highest binding of vWF. Endothelium isolated from the aorta, in contrast to all other endothelial cells, did not take up acLDL. These results demonstrate that organ- and tissue-specific heterogeneity is already expressed in fetal endothelium.
Abstract: AP-2 has been characterized previously as a unique 52 x 10(3) M(r) transcription activator encoded by a single gene that is expressed in a restricted pattern during embryonic morphogenesis of the peripheral nervous system, face, skin and nephric tissues. Here we report the isolation of genomic and cDNA clones encoding for a second AP-2 related transcription factor, designated AP-2 beta. AP-2 beta binds specifically to a series of well-characterized AP-2 binding sites, consensus to the sequence G/CCCN3GGC, and transactivates transcription from a reporter plasmid under the control of an AP-2-dependent promoter. A C-terminal domain known to mediate homodimerization of the previously cloned AP-2 alpha transcription activator is highly conserved and sufficient to mediate interaction between the two proteins. Northern blot and in situ hybridizations revealed that the two genes are expressed in murine embryos between days 9.5 and 19.5 p.c. Coexpression of both mRNAs was detected in many tissues at day 13.5 and 15.5 of embryogenesis but some regions of the developing brain and face including the primordium of midbrain and the facial mesenchyme differed in their expression pattern of AP-2 genes. AP-2 alpha and AP-2 beta signals in the central and peripheral nervous system overlapped with regions of developing sensory neurons. In adult tissues AP-2 alpha expression was found mainly in the skin, eye and prostate and AP-2 beta expression in the kidney. In summary, our analyses of embryonic and adult mice demonstrate that two different AP-2 transcription factors are specifically expressed during differentiation of many neural, epidermal and urogenital tissues.
Abstract: A panel of monoclonal antibodies to the growth hormone (GH) receptor/binding protein was used to demonstrate the existence and detail the expression of GH receptors in ductal and alveolar epithelial cells from rat and rabbit mammary glands by immunohistochemistry. Intense immunoreactivity was present in membrane, cytoplasm and some nuclei of epithelial cells during proliferation and lactation. Receptor expression decreased during weaning and was absent or weak in regressive mammary glands. Immunoreactivity was weak in ductal epithelial cells from virgin adult animals. Pronounced expression of GH receptor/binding protein was observed with two monoclonal antibodies and lesser reactivity was seen with others, paralleling their affinities for the receptor. The cytoplasmic presence of this putatively plasma membrane located GH receptor is accounted for by the existence of a soluble form on the GH receptor, namely the growth hormone binding protein derived from the membrane receptor by cleavage. Primary localization of the receptor in proliferating and lactating epithelial cells suggests that the rat and rabbit mammary gland is a GH target tissue. This finding is in contradiction to both classical GH action and the somatomedin hypothesis and challenges the widely held view that GH has no direct influence on mammary growth and function.
Abstract: Literature on the effect of steroid hormones (androgens, estrogens, and other steroids), of peptide hormones (e.g., prolactin), and growth factors (e.g., EGF, FGF, TGF-beta), on the effect of castration and of experimental hormone application on the prostate is reviewed. Androgens have inductive, repressive, and interactive effects. They counterbalance an agonistic effect on proliferation and an antagonistic effect on cell death; they may influence DNA synthesis and induce the synthesis of substances with mitogenic effects on the prostate. Estrogens exert direct and indirect effects on the prostate. They suppress the secretion of gonatropins, thus repressing testicular androgen secretion. They stimulate the fibromuscular stroma and induce squamous metaplasia of the epithelium. Estrogens may also be involved in the onset of benign prostatic hyperplasia. Prolactin is preferentially bound in the diseased human prostate. An abundance of information has been gained on EGF, FGF, TGF-beta, and other growth factors. They may be involved in the development of prostatic hyperplasia. Castration leads to a striking reduction in prostatic size in a short period of time due to autophagic and heterophagic processes. In castrated individuals, the prostate is enriched in androgen-independent cells. Experimental hormone application involves the substitution of androgens as well as anti-androgens, long-term application of different hormones, and application of combinations of drugs. The results of several studies are described. Further directions in the field of prostate research should concentrate on the role of growth factors in prostate development and pathology and on the effect of certain lectins on prostate diseases. We think that the investigation of interactions between steroid hormones and growth factors in normal and pathological neovascularization of the prostate is important.
Abstract: In the course of maintaining a cloned murine myocardium-derived endothelial cell line (mouse heart endothelial cell clone 5; MHEC5) a spontaneously transformed variant has been identified (clone MHEC5-T). On injection into histocompatible mice, clone MHEC5-T uniformly generated epithelioid haemangioendotheliomas. Clone MHEC5-T underwent significant additional alterations in addition to the acquisition of tumour-forming potential in vivo along with the diagnostic correlate of loss of cellular contact inhibition in vitro. Whereas the transformed cells maintained lectin-binding properties characteristic of endothelial cells, they lost the cell surface receptor(s) for acetylated low density lipoprotein and no longer bound antibodies to either angiotensin converting enzyme or von Willebrand factor-associated antigen. Vascular cell adhesion molecule-1 (VCAM-1), expressed constitutively on the parent clone, was down-regulated in the transformed cell line. The transformed cells acquired immunoreactivity to antibodies directed against cytokeratin, and they showed a markedly increased response to migration-inducing factors in vitro. The cell line described in this report demonstrates that the in vitro transformation of myocardium-derived endothelial cells can lead through transitional stages of differentiation to a new stable phenotype characterized by endothelial--to--epithelioid transition. The study of MHEC5-T cells, in addition to providing insight into the biology of cardiac neoplasms, may help to elucidate regulatory mechanisms involved in endothelial cell activation, transition and transformation.
Abstract: Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 alpha and ZP3 beta. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 alpha and anti-ZP3 beta. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 alpha and ZP3 beta. In secondary follicles, distinct labelling with anti-ZP3 beta and weak labelling with anti-ZP3 alpha could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 alpha and ZP3 beta. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 alpha and anti-ZP3 beta. Some granulosa cells showed staining for ZP3 alpha and ZP3 beta. The ZP displayed strong immunoreactivity for ZP3 beta and ZP3 alpha. Cells of the corona radiata were strongly immunopositive for ZP3 alpha and ZP3 beta. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 alpha and ZP3 beta strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.
Abstract: The present study was undertaken to define the temporal pattern and distribution of cells positive for chromogranin A (CgA) and tyrosine hydroxylase (TH) in various developmental stages of fetal bovine adrenal gland. CgA is an acidic protein, co-stored and co-released with amines and a variety of peptide hormones and neurotransmitters in dense core vesicles of neural and endocrine cells and can be used as a marker for these cells and their malignant counterparts. TH is the rate-limiting enzyme in catecholamine biosynthesis and reflects noradrenergic differentiation. The expression of CgA and TH was examined by immunohistochemistry. CgA immunoreactivity appears first in 35-day-old bovine fetuses. By the end of the second month, CgA-labelled cells are scattered throughout the entire primordium of the adrenal gland, and at a fetal age of 85-91 days most of these cells concentrate in the developing adrenal medulla. From this stage onwards, immunoreactive cells of the marginal zone of the medulla exhibit significantly stronger CgA immunoreaction than the central area. TH immunoreactivity appeared in the adrenal primordium for the first time at the end of the second month of gestation. The distribution pattern of TH-positive cells was similar to that described for CgA, and no significant differences in topographical arrangement between TH- and CgA-positive cells can be detected. The results show that bovine adrenal chromaffin cells express CgA already during their earliest stages of development and prior to TH. The stronger immunoreaction of marginal adrenal medullary cells suggests an adrenalcortical effect of glucocorticoids on the expression of CgA.
Abstract: Compared with normal prostatic tissue, the level of basic fibroblast growth factor (bFGF) is elevated in prostatic tumors. This suggests that bFGF may play a role in the development of prostatic neoplasms. The current study was undertaken to identify the cellular distribution of bFGF in benign prostatic hyperplasia (BPH) and prostatic carcinoma using a polyclonal antiserum against recombinant bFGF. In paraffin sections of prostatic tumors immunoreactive bFGF was found in fibroblasts, smooth muscle cells, and endothelial cells. Distinct staining was seen in most nuclei of these cells and a less intense immunoreaction occurred in the cytoplasm of smooth muscle cells. No immunostaining was seen in prostatic epithelial cells of prostatic tumors whether benign or malignant. With digoxigenin-labeled oligonucleotides in nonradioactive in situ hybridization, the presence of mRNA for bFGF was shown in smooth muscle cells of the stroma, suggesting that these cells are the main source of bFGF in BPH. Because no immunostaining for bFGF was obtained in the carcinoma cells, a specific role for bFGF cannot be seen for the development of malignant prostatic tumors.
Abstract: Carrier-immobilized carbohydrates are valuable tools for assessing the glycoligand-binding capacity of cell surfaces. A panel of 10 types of fluorescent neoglycoproteins has been synthesized to determine the extent of their specific binding to endothelial cells in vitro that have been obtained from porcine brain, ovary, and testis. Different sugar moieties revealed a nonuniform capacity to bind to the endothelial cells as determined by flow cytometric analysis, the histogenetic origin of the preparations being an important factor. Binding of mannose, xylose, and glucuronic acid was especially pronounced, but the extent of cell-associated fluorescence was significantly different dependent on the source of the endothelial cells. These results clearly reveal that endothelial cells in vitro display the capacity to specifically recognize defined carbohydrate moieties. Moreover, endothelial cells of different tissue origin can exhibit variability in this property, potentially endowing this cell type with site-dependent molecular properties.
Abstract: Galactosyltransferase (GalTase) has been discovered on a variety of cells where it is believed to be involved in cell-cell adhesion and cell-substratum adhesion as well as in metastasis of carcinoma cells. This immunohistochemical study was undertaken to identify the topography and the cellular distribution of GalTase in normal prostatic tissue, benign prostatic hyperplasia (11 cases), and prostatic carcinoma (26 cases). Immunoreactive GalTase was found to be exclusively associated with carcinoma cells and with premalignant epithelial cells in prostatic hyperplasia. In highly differentiated carcinomas, most of the carcinoma cells are positive for GalTase, whereas in poorly differentiated tumors, GalTase immunoreactivity was restricted to a subset of carcinoma cells with obviously invasive behavior. At the cellular level, GalTase was localized in the cytoplasm and at the cell membrane. In sections of normal prostatic tissue, as well as in unaltered acini of prostatic hyperplasic tissue, GalTase was not expressed. Fibroblasts, smooth muscle cells, and endothelial cells of the prostatic stroma were also consistently negative. With the use of immunoblots, we could confirm the presence of GalTase with a molecular mass of 45 kDa in the extracts of benign prostatic hyperplasic tissue and in prostatic carcinoma tissue but not in normal prostatic tissue. The results of our immunohistochemical study suggest that GalTase is a valuable marker to diagnose neoplastic transformation in prostatic tissue.
Abstract: Boar spermadhesin (AWN) is a 14-kDa multifunctional protein, attached to the surface of the spermatozoa and involved in sperm capacitation and zona pellucida binding. The cellular origin of AWN was previously unknown. Moreover, the region of the male genital tract in which AWN becomes attached to the surface of spermatozoa was also uncertain. By using monospecific polyclonal antibodies against AWN, the immunohistochemical distribution pattern of AWN epitopes has been investigated in tissue sections of the porcine male genital tract. Our study has revealed that AWN is synthesized in the rete testis and in the epithelium of the seminal vesicles. The latter are also the major contributors of seminal plasma AWN. In addition, immunoblotting analysis has shown that AWN is present on epididymal spermatozoa. Our results indicate that the cellular origin of spermadhesins is species-specific. The attachment of AWN to epididymal spermatozoa is probably important in developing the capacity for fertilization.
Abstract: We used several biotinylated neoglycoproteins as tumor markers to detect and localize endogenous carbohydrate-binding proteins in cultured hepatoblastoma, melanoma, and bladder carcinoma tumor cells. The neoglycoproteins used consisted of cellobiose, fucose, N-acetyl-galactosamine, N-acetyl-glucosamine, lactose, maltose, mannose, melibiose, and xylose. In addition, naturally occurring asialofetuin that was chemically disialylated was also used. Binding to the cultured tumor cells was made visible with the avidin-peroxidase technique. Depending on the type of neoglycoprotein used, markedly different expression of cytoplasmic and nuclear receptors for sugars (endogenous lectins) was obtained from rat hepatoblastoma, human melanoma, and bladder carcinoma tumor cells. The most pronounced staining differences were documented for asialofetuin and the neoglycoproteins containing fucose, N-acetyl-galactosamine, and lactose.
Abstract: alpha-Galactosyl residues in the carbohydrate part of cellular glycoconjugates can serve as cell type-associated markers and are implicated in intercellular adhesion and biosignaling. This biological significance explains the interest to characterize probes with respective specificity as the Griffonia simplicifolia I-isolectin B4. Due to the documented occurrence of an alpha-galactoside-binding immunoglobulin G fraction in human serum we compared the extent of binding and its pattern for the lectin and the antibody using surface-immobilized extract proteins and fixed sections of bovine testicular tissue with known lectin reactivity. The antibody fractions were obtained either solely from affinity chromatography isolation on immobilized melibiose or after an additional step to deplete this fraction of galactoside-binding activities without pronounced specificity to the alpha-anomeric linkage. They yielded a rather indistinguishable reactivity in comparison to that of the lectin, when an indirect approach was used. Labeling of the antibodies with a hydrazide derivative of biotin did not affect the pattern of binding. However, significant differences were noted, when conjugation of label was targeted to amino groups via N-hydroxy-succinimide esters of biotin and digoxigenin despite performance of the modification under activity-preserving conditions. Notably, the apparent strong staining of Leydig cells and nuclei of primary spermatocytes, respectively, was not inhibitable by sugar. These differences were corroborated by a nonidentical response of the various probes in solid-phase assays with extract proteins. Thus, care should be exercised in the interpretation of histochemical data, obtained with this type of modified antibody. When these precautions are fulfilled, this immunoglobulin fraction from human serum has the potential as an alpha-galactosyl-specific histochemical tool.
Abstract: This investigation for the first time has collected evidence of a specific glycoconjugate contribution of the acinar cells from accessory lacrimal glands to human tears. Amongst group III lectin binding glycoconjugates, monosaccharides seem to be more prominent than disaccharides. alpha (and less obvious beta) Galactose sugar moieties appear to be specifically important. A need for further differentiating investigations is outlined.
Abstract: Growth factors are known to take part in the regulation of reproduction. Here we present evidence for the expression of insulin-like growth factor 1 (IGF-1) in the bovine oviduct. Two IGF-1 specific mRNA-transcripts of 1.5 and 4.4 kb were detected during the whole oestrous cycle, and showed increased expression after ovulation. Complete homology to the known IGF-1 sequence was achieved by specific RT-PCR (reverse transcription-polymerase chain reaction) amplification followed by dideoxysequencing of this 210 bp fragment. IGF-1 protein was localized in the secretory cells of the oviduct epithelium using immunohistochemical techniques. We suggest possible effects of IGF-1 during ovulation either on the oviductal cells or on the early embryo.
Abstract: The present lectin histochemical comparison of cauda epididymal and ampullary bovine sperm was conducted to investigate whether ampullary secretions are involved in altering the plasma-membrane glycoconjugates of sperm. A marked redistribution of glycoconjugates between sperm from these two regions was indeed revealed on the basis of changes in binding patterns of the following fluoroscein-isothiocyanate (FITC)-labelled lectins: wheat-germ agglutinin (WGA), Maclura pomifera agglutinin (MPA), Griffonia simplicifolia I agglutinin (GS I) and Bauhinea purpurea agglutinin (BPA). This was evidenced in the first three cases by a relative reversal of staining intensity between the acrosomal and post-acrosomal regions, and by a pronounced increase in the staining of the midpiece. Changes in the distribution of BPA binding sites were limited to the latter phenomenon. The results are compared with previous findings, discussed in the context of the hypermotility characteristic of ampullary sperm and related to previously reported differences in the lectin-binding patterns of the luminal and glandular epithelia.
Abstract: The cellular distribution of insulin-like growth factors I and II (IGF-I and -II) was examined in bovine corpora lutea at different stages of the luteal phase using specific antibodies and the avidin-biotin immunoperoxidase technique. At the cellular level, intense immunostaining for IGF-I was exclusively observed in large and small luteal cells and in a limited number of endothelial cells. Positive IGF-I immunoreactivity in luteal cells was thereby distributed in a distinct topographical, lobule-specific manner. Immunoreactivity in central areas of luteal lobules was most abundant in large luteal cells, whereas in peripheral zones significantly (P < 0.05) more small luteal cells exhibited IGF-I immunoreactivity. This distribution pattern was evident from day 4 of the cycle onwards and occurred at all the stages investigated. The percentage of positive small (SLC) and large (LLC) luteal cells revealed by semiquantitative analysis depended on the stage of the cycle as follows: days 4-7: 34% LLC versus 21.3% SLC in central areas and 25.1% LLC versus 32.7% SLC in peripheral zones; days 8-12: 42.9% LLC versus 19.9% SLC in central areas and 23.5% LLC versus 35.2% SLC in peripheral zones; days 13-16: 47.7% LLC versus 19.4% SLC in central areas and 19.2% LLC versus 41.4% SLC in peripheral zones. In contrast to IGF-I, no expression of IGF-II immunoreactivity was seen in large or small luteal cells. Positive immunoreactivity was restricted to the perivascular fibroblasts of large blood vessels and to the pericytes of capillaries.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Endothelins (ETs) are important regulators of growth and function in many endocrine tissues. This study was designed to verify the expression of ETs in a series of normal human pituitaries and pituitary adenomas. We examined 13 normal pituitaries and 58 pituitary adenomas for the presence of immunoreactive (ir) ET-1 and ET-3. No ir-ET-1 was detected in any of the 13 normal pituitaries, whereas ir-ET-3 was observed in 4 of 13 (31%) cases. In contrast, 48% (28 of 58) of pituitary adenomas display immunoreactivity for ET-1, whereas 31% (18 of 58) show immunoreactivity for ET-3. With respect to the type of tumors, staining was as follows: nonfunctioning adenomas: ET-1, 14 of 33; ET-3, 9 of 33; somatotropinomas: ET-1, 8 of 16; ET-3, 6 of 16; corticotropinomas: ET-1, 5 of 5; ET-3, 2 of 5; and prolactinomas; ET-1, 1 of 4; ET-3, 1 of 4. Using double immunostaining, we found the colocalization of ET-3 in normal pituitaries and of ET-1 and ET-3 in pituitaries adenomas in each hormone-secreting cell. In Cushing adenomas, ET-1 was coexpressed in corticotropic cells in all 5 cases (100%). In the same tumors, by reverse transcriptase polymerase chain reaction, we investigated the presence of ET-1 and ET-3 messenger ribonucleic acids and found that they are expressed, respectively, in 18 of 21 and 7 of 11 tumors examined. Our findings demonstrate that pituitary adenomas frequently display ET-1 as well as ET-3 immunoreactivity, in contrast to normal pituitaries, in which only ET-3 was found.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: A panel of monoclonal antibodies to the growth hormone (GH) receptor/binding protein was used to demonstrate the existence and detail the expression of GH receptors in the cerebellum of 2, 10, 28 days old postnatal and adult rats and 10, 20 days old and adult rabbits by immunohistochemistry to define potential targets for endogenous GH action in the cerebellum. Receptors were localized in membrane and cytoplasmic components of neurons and glial cells and expression decreased with age. Intense immunoreactivity was observed in the cytoplasm and dendrites of Purkinje cells and in cells of the cerebellar nuclei. Glial cells also showed receptor expression. Strong immunoreactivity was observed with two monoclonal antibodies and lesser reactivity was seen with others, paralleling their affinities for the receptor. The cytoplasmic presence of this putatively plasma membrane located GH receptor is accounted for by the high receptor content of endoplasmic reticulum and the existence of a soluble form of the GH receptor, namely the GH binding protein (BP) derived from the membrane receptor by cleavage, and receptor localization reported here correlate well with the distribution of insulin-like growth factor 1 (IGF-1) mRNA and immunoreactivity in cerebellar Purkinje cells and glial cells. Primary localization of the receptor in the cerebellum is in direct contradiction to both classical GH action and the somatomedin hypothesis and supports and extends the theory of genetically regulated macroneuronal maturation.
Abstract: The leukocyte beta 2-integrin Mac-1 (CD11b/CD18) and its endothelial ligand intercellular adhesion molecule 1 (ICAM-1) are involved in leukocyte adhesion to and macromolecular leakage from postcapillary venules during inflammatory reactions. Both events are also encountered after ischemia-reperfusion of striated muscle, suggesting a central role of both adhesion proteins in reperfusion injury. Using intravital fluorescence microscopy and a microcirculation model in awake BALB/C mice, we investigated the effects of monoclonal antibodies (MAb) and Fab fragments to Mac-1 and MAb to ICAM-1 on leukocyte-endothelium interaction and macromolecular leakage of fluorescein isothiocyanate-dextran (1.5 x 10(5) mol wt) in striated skin muscle after 3 h of ischemia followed by reperfusion. We demonstrated that administration of MAb and Fab to Mac-1 before reperfusion was as effective as administration of MAb to ICAM-1, which was found to be significantly upregulated in the postischemic tissue by immunohistochemical analysis, in preventing postischemic leukocyte adhesion to and macromolecular leakage from postcapillary venules, whereas postischemic leukocyte rolling was not affected after MAb administration. Postischemic capillary perfusion was efficiently preserved in animals treated with anti-Mac-1 and anti-ICAM-1 MAb compared with animals receiving the isotype-matched control antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The present study deals with the immunohistochemical localization of S-100 protein in the testes of seven mammalian species including rat, cat, dog, pig, sheep, cattle and horse. Significant differences are demonstrated in the cellular distribution and intensity of immunoreaction for the protein. In bull, ram, boar and cat testes S-100 protein was localized in the cytoplasm and nuclei of Sertoli cells. A particularly intense staining was seen in the modified Sertoli cells of the terminal tubular segment. With the exception of the cat and horse S-100 protein immunoreactivity was additionally found in epithelial cells of the straight testicular tubules and in the epithelial cells of the rete testis. Endothelial cells of capillaries, veins and lymphatic vessels are regularly S-100 immunoreactive in ruminants. Leydig cells were found to be strongly positive for S-100 protein in the cat and rat testes and to a lower degree in pig and horse testes. Finally a distinct immunostaining of peritubular cells was restricted to the testis of dogs and rats. The remarkable species-specific variations of immunoreactivity for S-100 protein in different cell types of the testis support the hypothesis that S-100 protein is multifunctional protein and may have a different function in testicular physiology.
Abstract: cDNA probes derived from genes expressed specifically in the human epididymis were used to examine gene expression in the epididymides of boar, bull and stallion by Northern hybridization. Two probes for the HE1 and HE4 gene products were found to recognize tissue-specific transcripts in all three species, with a regionally differential distribution within the epididymis. Additionally, antibodies recognizing the HE4 protein were shown to react specifically in the epididymis of the boar and bull. An extensive study of the boar showed that, whereas mRNA for the HE1-homologue was up-regulated markedly only at puberty, the HE4-homologue was already present at moderate levels prepubertally. The distribution of the HE1-homologue changed at sexual maturity from a maximum in the cauda epididymis in the 3-4-week-old pig, to a maximum in the corpus/caput region in the adult, while the shift was in the opposite direction for the HE4-homologue. Evidently, gene expression is not fixed regionally through epididymal development in this species. The abdominal epididymis of a hemicryptorchid pig also showed a differential change in expression for the two gene products by comparison with the scrotal testis from the same animal. The results suggest that the HE1 and HE4 gene homologues may be sensitive markers for physiological changes within the mammalian epididymis, and that the boar could prove a useful model to examine the regulation of these human epididymal transcripts.
Abstract: Plant and invertebrate lectins are valuable cyto- and histological tools for the localization of defined carbohydrate determinants. The well-documented ubiquitous occurrence of sugar receptors encourages functional considerations. Undoubtedly, analysis of the presence of vertebrate lectins in tissues and cells is required to answer the pertinent and tempting question on the physiological relevance of protein (lectin)-carbohydrate recognition in situ. Carrier-immobilized glycoligands, derived from custom-made chemical synthesis, enable the visualization of respective binding sites. Histochemically inert proteins or synthetic polymers with appropriate functional groups are suitable carrier molecules for essential incorporation of ligand and label. The resulting neoglycoconjugates can track down tissue receptors that are neither impaired by fixation procedures nor blocked by endogenous high-affinity ligands. Lectins, especially the receptors of the tissue under investigation (endogenous lectins), and appropriately tailored immobilized glycoligands or lectin-specific antibodies (when available) are complementary tools to test the attractive hypothesis that diverse, functionally relevant glycobiological processes within or between cells are operative. Concomitant evaluation of both sides of lectin histochemistry, namely lectins as tools and lectins as functionally important molecules in situ, will indubitably render desired progress amenable in our often still fragmentary understanding of the importance of tissue lectin and glycoconjugate expression and its regulation.
Abstract: The concentration of the new bioactive acetic seminal fluid protein (aSFP) was determined in body fluids of bulls. An aSFP-specific radioimmunoassay was elaborated and established for routine measurement. No crossreaction of the aSFP antibody was found with other seminal plasma proteins and known growth factors. The 14 kDa aSFP was detected in secretions of ampulla and seminal vesicles by immunoblot analysis, but not in testis, epididymis or blood. Large quantities of aSFP were measured by radioimmunoassay in the fluid from the ampulla (2.6 +/- 0.3 mg ml-1) and seminal vesicles (3.0 +/- 0.4 mg ml-1). Immunohistochemistry techniques demonstrated that aSFP was localized mainly in the secretory epithelium of ampulla and seminal vesicles. Large amounts of aSFP (4.0 +/- 0.4 mg ml-1) were present in seminal plasma of bulls within a range of 1-7 mg ml-1, but aSFP could not be found on membranes of spermatozoa. Concentrations vary considerably among individuals, suggesting that aSFP may have a role in bovine reproduction.
Abstract: Monocytes/macrophages play a critical role in the pathogenesis of HIV infection, both as targets for virus replication and as sources of production of multifunctional cytokines. Endothelins, peptides with potent vasoconstricting activities originally isolated from endothelial cells, are also produced and secreted by macrophages in a manner similar to that of other cytokines. In an attempt to explore the potential role of endothelins in HIV-infection, we investigated the effect of the HIV-1 envelope glycoprotein, glycoprotein 120, on monocytic endothelin-1 production. This glycoprotein has been identified as a potent stimulator of monokines such as TNF-alpha and IL-6, which have been implicated as potential mediators of HIV-encephalopathy. We found that glycoprotein 120, similar to LPS, stimulates the secretion of endothelin-1, as well as TNF-alpha, from macrophages in a concentration-dependent manner. Using reverse transcriptase polymerase chain reaction, we found that circulating monocytes in HIV-infected individuals show a distinct expression of the endothelin-1 gene that is not detectable in healthy controls, indicating chronic activation of this gene in HIV-infection. In addition, cerebral macrophages in patients with HIV-encephalopathy were strongly positive for endothelin. Thus, monocytic endothelins appear to be stimulated during HIV infection. Their potent vasoactive properties render them potential candidates for mediating alterations in the cerebral perfusion pattern associated with the AIDS dementia complex.
Abstract: The present study deals with immunohistochemical localization of S-100 protein in the bovine testis. Immunoreactivity for the protein was seen in Sertoli cells of the seminiferous tubules and as a particularly intense staining in the terminal tubular segment (transitional region, middle portion, and terminal plug), which is mainly composed of modified Sertoli cells. Immunoreactivity was also found in epithelial cells of the straight testicular tubules and rete testis, and in the endothelium of capillaries, veins and lymphatic vessels. Although the functional significance of S-100 protein immunoreactivity in the Sertoli cells remains unclear, the present results suggest that it may be involved in the microtubule assembly-disassembly system. The specificity of the immunolabelling observed should enable the antigen and/or antibody to S-100 to be used as an investigative and diagnostic tool in the study of bovine Sertoli cell function.
Abstract: The histological affinity pattern of Dolichos biflorus agglutinin (DBA) in kidneys from mice (NMRI, Balb/c, CBA) and rats (Wistar) fixed by perfusion with formalin, Bouin, or HgCl2 was investigated with a horseradish peroxidase conjugate. The animals were examined from fetal stage to adulthood. Adult female NMRI mice exhibited constant DBA labelling, with DBA binding to cells of the proximal and collecting tubules. Moreover the vascular endothelium of the renal papilla was found to be DBA-positive in 50% of adult female animals. In contrast, there was only very little DBA binding in the kidneys of male adult NMRI mice. There was no sexual dimorphism in lectin labelling in kidneys from other strains of mice or from rats.
Abstract: The present study investigated the prophylactic effects of vitamin D metabolites and vitamin D metabolite combinations on static and dynamic, tetracycline-based, histomorphometric parameters in the axial skeleton of ovariectomized rats. Forty-three Fischer-344 rats (10 weeks old, 130 g each body weight, BW) were either bilaterally ovariectomized (OVX) or sham-operated (SHAM). The rats were allocated into the following groups: SHAM; OVX; OVX + 7.5 ng 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]/rat/day; OVX + 15 ng 1 alpha,24R,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3]/rat/day; OVX + 75 ng 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3]/rat/day; OVX + 7.5 ng 1,25(OH)2D3/rat/day + 15 ng 1,24,25(OH)3D3/rat/day; OVX + 7.5 ng 1,25(OH)2D3/rat/day + 75 ng 24,25(OH)2D3/rat/day. The vitamin D metabolites were fed orally starting 4 weeks after surgery. Urine and blood samples were collected 12 and 16 weeks postovariectomy, respectively. Sixteen weeks after surgery, all rats were sacrificed, and the first lumbar vertebrae were processed undecalcified for histomorphometric analysis. Ovariectomy induced a highly significant reduction (P less than 0.001) of cancellous bone mass in the secondary spongiosa of the lumbar vertebral body. The bone loss in OVX rats was accompanied by a distinct elevation of all histomorphometric parameters of bone formation and resorption. 1,25(OH)2D3 and both vitamin D metabolite combinations significantly raised serum calcium levels and prevented the bone loss by inhibiting the increased bone resorption in OVX rats. In the applied dosage, 1,24,25(OH)3D3 and 24,25(OH)2D3 alone were ineffective in preserving the cancellous bone of the lumbar vertebra in OVX rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The vascular endothelium has long been considered to be a homogeneous cell type. It is only recently that its remarkable heterogeneity has been shown. The selective and organ-specific adhesion of cells of the immune system, of metastasizing tumor cells and of microorganisms underscores the central position of the endothelium between the circulation and each organ. The use of antibodies and lectins led to the recognition of species-, organ-, and age-specific endothelial cell surface receptors. Identification of the endothelial adhesion molecules and -mechanisms, which are part of the "endothelial organ- and tissue address" would lead to the possibility of blocking the adhesion of cells, such as e.g. tumor cells.
Abstract: AWN is a boar protein which originates in secretions of the male accessory glands and which becomes sperm surface-associated upon ejaculation. It is one of the components thought to mediate sperm adhesion to the egg's zona pellucida through a carbohydrate-recognition mechanism. AWN may, thus, participate in the initial events of fertilization in the pig. In this report we describe its complete primary structure by combination of protein-chemical and mass spectrometric methods. AWN exists as two isoforms, AWN-1 and AWN-2, which differ in that AWN-2 is N-terminally acetylated. The amino acid sequence of AWN contains 133 amino acid residues and two disulphide bridges between nearest-neighbour cysteine residues. Analysis of the amino acid sequence of the AWN proteins showed significant similarity only to AQN-1 and AQN-3, two other boar spermadhesins.
Abstract: In the present study we report on the histotopographical distribution of lectin binding sites in the trophoblasts of day 18 to day 40 bovine embryos, using the FITC-labeled lectins BPA, Con A, DBA, GS I, GS II, MPA, PNA, SBA, UEA I and WGA. Lectin binding sites localized in giant binucleate cells differ from those localized in uninucleate cells, indicating changes in the biochemical structure of cell surfaces taking place during differentiation. In the trophoblast of the day 40 embryo, a distinct staining of uninucleate cells was seen after incubation with GS I, Con A and MPA, demonstrating N-acetylgalactosamine (GS I), Mannose (Con A) and Galactose (MPA) moieties, whereas giant binucleate cells showed intense reactions after incubation with DBA and WGA, indicating presence of N-acetylgalactosamine (DBA) and N-acetylglucosamine (WGA). GS II (specific for N-acetylglucosamine), SBA (specific for N-acetylgalactosamine) and UEA I (specific for L-Fucose) showed no affinity toward any of the examined tissues. We assume, that carbohydrate moieties in trophoblast cells play an important role in fetomaternal cell-cell adhesion and cell migration during implantation and placentation period.
Abstract: The blood supply and the microvascularization of the bovine penis (Bos taurus) were demonstrated using the scanning electron microscope. The Corpus cavernosum penis of the bull consists of a surprisingly well-developed mesh of small to intermediate vessels. The architecture of the cavernous body is largely determined by trabeculae of connective tissue, the extent of which varies between penis segments. The Corpus cavernosum is arranged primarily as a ring around the trabeculae of connective tissue. Chambers of large bore are found in the central portions of the Corpus cavernosum penis, whereas a relatively fine vascular network predominates in the periphery. Vessels in the cavernous body of the urethra, on the other hand, show strictly parallel orientation to the urethra, which they surround like a sleeve. They also stand in close association with vessels in the outer layer of the Tunica albuginea. The vascular systems of the Corpus cavernosum penis and the Corpus spongiosum penis are not connected with one another. The vascular architecture of the Glans penis is characterized by the inclusion of well-developed vascular arcades at regular intervals. These course through a connective tissue matrix rich in glycoproteins and stretch to just under the skin of the penis, where they are intimately associated with a subpapillary network of arteries and veins. The venous legs of the vascular arcades are supplied by this network of veins. The Glans penis of the bull is considered to represent a specialization of the penile integument.
Abstract: Yolk sac development was investigated in 69 ovine and 10 bovine embryos from the blastocyst stage to the 7th week of gestation. Light and electron microscopical findings are reported. The yolk sac in sheep and cattle is composed of an enlarged sac-like portion lying below the embryo and two ends which follow the elongated course of the trophoblast. In sheep, an open connection exists between the intestines and the yolk sac up to a crown-rump length (CRL) of 9 mm. It is closed by 12 mm CRL. The wall of the yolk sac is especially well vascularized in the enlarged, sac-like portion. Primary erythropoiesis occurs within the blood capillaries. In the blastocyst, the yolk sac entoderm is made up of elongated, flat cells. It becomes cuboidal in the 3 mm embryo (ovine) and later columnar. The up to 20 microns tall cells stain darkly and contain numerous light-colored vesicles. At 4.5 mm CRL light cells appear between the dark ones. Both cells are rich in rough endoplasmic reticulum (rER). The increased staining of the darker cells is due to an osmophilic cytoplasm and numerous, often parallel lamella of rER. The rER of the light cells is enlarged to irregularly-shaped cisternae, which nearly fill the entire cytoplasm and give them a rounded appearance. The dark cells contain polygonal nuclei, whereas those in the light cells are round with one or two nucleoli. The oval mitochondria have only a few peripheral cristae. Golgi fields are not very common. Cells of the entoderm are connected to one another over zonulae occludentes. They possess microvilli on the luminal surface and are supported by a basement membrane. From 5 mm CRL onwards (ovine), the yolk sac entoderm folds itself between the capillaries, thereby becoming stratified. The intercellular space between the cells expands as projections between neighboring cells interlock. Canaliculi arise between adjacent epithelia. The wall of the yolk sac thickens as a result of this infolding and the densely packed capillaries. Infoldings are especially predominant in the sac-like portion of the yolk sac, and only suggested in the ends. Involution of the yolk sac begins in the peripheral end segments and proceeds centripetally. Numerous glycogen particles appear in the yolk sac entoderm cells of the ovine fetus at a CRL of 36 mm, and by a CRL of 42 mm, the sac-like portion has also begun to show signs of degeneration. Mesenchyme is very sparse within the wall of the yolk sac throughout the entire period of development.(ABSTRACT TRUNCATED AT 400 WORDS)
Abstract: Previous studies with the electrically stimulated longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum suggested that opioid control of adenylate cyclase is confined to nerve somata. No indication was found for an opioid effect on the enzyme at nerve terminals of the neuro-muscular junction. The aim of the present investigation was to directly study the effect of opioids on cAMP generation in nerve fragments associated with somata or terminals of the myenteric plexus. Employing the ultracentrifugation technique an enrichment of cell organelles in fractions relating to either somata or terminals was achieved. Opioid binding studies revealed specific mu-receptors which in both fractions were regulated by GTP. Challenge of these fractions with forskolin and prostaglandin E1, respectively, resulted in an increased production of cAMP regardless of their neuroanatomical assignment. Examining the response of neuronal material to the selective mu-opioid DAMGO ([D-Ala2, MePhe4, Gly-ol5]enkephalin) revealed an inhibitory action on cAMP synthesis in somata-enriched fractions. No effect of DAMGO was observed in material linked to nerve terminals, although the presence of mu-opioid receptors and adenylate cyclase has been demonstrated. We conclude that opioid control of adenylate cyclase in the myenteric plexus of the guinea pig is confined to nerve somata.
Abstract: The Dunning R3327 rat carcinoma is an important model for human prostate adenocarcinoma. In the present study this tumor was further characterized by immunohistochemical demonstration of receptors for growth hormone (GH-R). Weak GH-R immunoreactivity was present in the secretory epithelial cells of the tumor acini. Large epithelial cells which were localized at the periphery of the acini and large cells in the stroma, which are probably derived from the epithelium ("Large neoplastic epithelial cells"), displayed a strong staining with one of the monoclonal antibodies (Mab 263) to GH-R. The presence of GH-R receptors in proliferating prostatic tumor cells supports the concept that GH reacts directly on prostate target tissue to facilitate tumor cell growth.
Abstract: The arrangement, origin, course and opening of the ductuli efferents testis of the bull (Bos taurus) were visualized using scanning electron microscopy. The corresponding capillary structure was also described and documented with the help of corrosive microanatomical casts. The number of the ductuli efferents testis of the bull varies narrowly between 12 to 13. Ductuli removed from the surrounding tissue have a length of up to 78 cm. The duct system is composed of linear as well as tortuous parts, permitting division into three distinct segments. Blind ending ductules are very short. We therefore do not see these structures as being associated with spermiostasis. The capillary density as well as the capillary architecture show regional peculiarities. All segments of the ductuli efferents testis are composed of capillaries of the continuous type. The findings indicate that the increased capillary density in the voluminous initial section of the ductuli efferents testis represents the basis for resorptive as well as secretory activities in the bull.
Abstract: In the present study we report on the histotopographical distribution of carbohydrate-binding proteins in the prostate and seminal vesicle of sexually mature NMRI mice using a panel of fluorescein-isothiocyanate labelled neoglycoproteins and asialoglycoproteins. Additionally, biochemical analysis using affinity chromatography and SDS-gel electrophoresis was performed to purify and characterize the respective proteins from the tissue. Our histochemical results clearly demonstrate the presence of endogenous receptors for the carbohydrate part of glycoconjugates in both glands. In the prostate a distinct staining was seen after incubation with melibiose-BSA-FTC, glucuronic acid-BSA-FTC and asialofetuin-FTC (only in the ventral prostate). In the epithelium of the seminal vesicle a weak staining occurred after incubation with asialofetuin-FTC and maltose-FTC. In the stroma of both accessory sex glands a distinct binding of several (neo)glycoproteins specific for beta-galactoside-binding proteins was observed which could be attributed to a beta-galactoside-binding lectin. Indeed biochemical analysis ascertained presence of such a histochemically detectable activity. We assume that the carbohydrate-binding proteins of the stroma, which were obviously linked to the elastic fibers, could play a role in the organisation of the extracellular matrix in the interstitium of the glands.
Abstract: Forty Fischer-344 rats (10 weeks old, 130 g BW) were either bilaterally ovariectomized (OVX) or sham-operated (SHAM). The rats were allocated to the following groups: SHAM; OVX; OVX + 15 ng 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]/rat/d; OVX + 30 ng 1 alpha,24R,25-trihydroxyvitamin D3 [1,24,25(OH)3D3]/rat/d; OVX + 15 ng 1,25(OH)2D3/rat/d + 30 ng 1,24,25(OH)3D3/rat/d. The vitamin D metabolites were fed orally starting 4 weeks after surgery. Urine and blood samples were taken at several time points during the experiment. Twenty-one weeks after surgery all rats were sacrificed, and the proximal tibiae and the first lumbar vertebrae were processed undecalcified for static bone histomorphometry. Ovariectomy induced a 40% reduction in vertebral cancellous bone area, and a 69% reduction in tibial cancellous bone area. This bone loss in OVX rats was associated with moderately increased biochemical and histomorphometric indices of bone formation and resorption as compared to values in sham-operated animals. Through inhibition of bone resorption, treatment of OVX rats with 1,25(OH)2D3, 1,24,25(OH)3D3, and the metabolite combination prevented the ovariectomy-induced osteopenia in the lumbar vertebra, and partially prevented cancellous bone osteopenia in the tibial metaphysis. However, OVX rats receiving 1,25(OH)2D3 alone or in combination with 1,24,25(OH)3D3 exhibited hypercalcemia, hyperphosphatemia, hypercalciuria, and impaired bone mineralization. Treatment of OVX rats with 1,24,25(OH)3D3 alone, on the other hand, only slightly increased serum calcium levels and did not impair bone mineralization. Furthermore, the inclusion of 1,24,25(OH)3D3 with 1,25(OH)2D3 partially antagonized the untoward effects of 1,25(OH)2D3 on bone mineralization. These data suggest that the actions of 1,24,25(OH)3D3 on bone metabolism might differ from that of 1,25(OH)2D3, and that 1,25(OH)2D3 and, particularly, 1,24,25(OH)3D3 may be potentially effective agents for the prophylaxis of postmenopausal osteoporosis.
Abstract: An important animal model for human prostatic adenocarcinoma is the Dunning R3327 rat carcinoma. In the present study this tumor was further characterized by analyzing the expression of endogenous sugar-binding proteins using glycohistochemistry and immunocytochemistry as well as affinity chromatography and gel electrophoresis. Our glycohistochemical and glycobiochemical results provide evidence for the presence of specific receptors for various carbohydrate moieties. Remarkably, basal cells of the Dunning tumor contain an endogenous lectin with specificity for beta-galactosides that is not found in basal cells of the normal rat prostate. This finding was corroborated using polyclonal antibodies against an immunologically related beta-galactoside-specific lectin from bovine heart. Basal cells of prostatic carcinoma may therefore behave different from normal basal cells. This difference could have a significant impact on the development of prostatic cancer.
Abstract: Structures characteristic of the omasum are already present by the third month of development. The fetal omasal laminae exhibit their typical arrangement (1434243414). Muscle fibers from the lamina circularis also extend into the first through third order laminae at this stage. Conical projections and small secondary papillae begin to appear on the laminae as the fetus ages. The lamina muscularis mucosae can be found in all of the laminae by the fourth to fifth month of development. The cuboidal to columnar epithelium of the stratum basale remains largely unaltered throughout the course of fetal development, whereas the stratum superficiale is subjected to a continuous series of changes. Superficial cell layers increase and the glycogen content of the epithelial cells fluctuates significantly.
Abstract: The human gastrointestinal tract, important for body salt and water balance, was investigated by endoscopic biopsy for the presence of atrial natriuretic peptide (ANP). Using immunohistochemistry, ANP-immunoreactive cells were identified in the lamina epithelialis mucosae of stomach, duodenum, jejunum, colon, and rectum. The findings indicate that ANP plays a role in intestinal salt and water regulation in man. ANP measurements in tissue specimens reached by endoscopic biopsy may be of major interest for future investigations on (patho-)physiological and pharmacological aspects of ANP.
Abstract: Changes in the vasculature of the canine kidney following four weeks obstruction of the ureter via double ligature is described on the basis of SEM investigation. Three significant alterations were observed: 1) A two-thirds reduction in the depth of the renal cortex as compared to controls. 2) Rarification of the entire cortical capillary bed. 3) Reduction in both the number and diameter of the glomeruli. The rarification of the post-glomerular capillaries is interpreted to be a pressure atrophy, whereas the reduction in the number of renal glomeruli and the concommitant diminuation of their capillary loops is thought to represent a functional atrophy.
Abstract: The goal of this study was to demonstrate the distribution of glycoproteins in the various segments of the Japanese quail nephron, using lectins labeled with HRP or FITC. Each one of the six labeled lectins had a characteristic distribution pattern along the nephron. The study shows that lectins are useful markers for certain nephron segments or for cell types in certain segments of the renal tubules. PNA marks the thin portion of the medullary loop, DBA marks the thick portion; it is thus possible to differentiate the nephron segments in the medullary cone of the kidney. Con A binds selectively with the epithelioid, granular cells of the tunica media of the vasa efferentia. The histochemical technic using labeled lectins makes it possible to identify certain renal structures that could not, or only with difficulty, be differentiated using conventional histology. Therefore, lectins as specific markers are gaining in importance for further studies of the morphology and physiology of the kidney.
Abstract: [1,2-Bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]dichloro-platinum (II), (C), a platinum complex with endocrine activity and a specific effect on hormone-dependent mammary tumors, was tested for its tumor-inhibitory activity in the hormone-sensitive R 3327 and Nb prostate carcinoma models of the rat and for its endocrine activities in comparison to the ligand L and diethylstilbestrol (DES). Established tumors of the R 3327 prostate tumor were strongly inhibited by C. Its effect equaled that of DES and was significantly better than that of L. Accessory sex organ weights and testosterone levels were strongly reduced by C as well as L. This antigonadotrophic effect, which is almost comparable to DES, was confirmed in 10 day experiments with intact, mature mice and rats, whereas a direct antiandrogenic activity was not given. A part of the antitumor action of C is therefore due to this antigonadotrophic activity. Affinities to estrogen, progesterone, and androgen receptors, however, were very low. The hormone-sensitive Noble Nb-R prostatic carcinoma was almost completely inhibited by C, whereas L had only a weak effect. As C has no significant effect on the hormone-independent R 3327 HI prostate tumor and as its effect on hormone-dependent tumors is significantly better than that of the ligand L in spite of their similar endocrine properties, an apparently specific antiproliferative effect of C only on hormone-dependent prostate tumors is obvious. This was further shown in a long-term experiment with the R 3327 prostate carcinoma. Whereas tumors in the castration group relapsed from androgen ablation and exerted a progressive tumor growth, therapy with C almost completely prevented this relapse phenomenon. After 25 weeks of treatment, C inhibited tumor growth by 90% compared to castration. Owing to these results, this new endocrine active platinum complex with an apparently specific effect on hormone-dependent prostate tumors can be of value for the therapy of the prostatic carcinoma.
Abstract: Five Fluorescein-isothiocyanate (FITC)-labelled lectins were used to study the postnatal development of carbohydrate constituents in the rat ventral prostate: Concanavalin A (Con A), wheat germ agglutinin (WGA), peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA) and Ricinus communis agglutinin I (RCA-I). With all the lectins tested, except RCA-I, specific binding sites could be shown for every stage of differentiation in the glandular epithelium. Binding sites for Con A, WGA, PNA and DBA were found from day 10 to 13 post partum onwards. Each lectin showed a characteristic localization. Binding sites for the lectins used changed to different extents during the following two weeks. After the 24th day post partum no further changes in the lectin binding pattern could be found. The development of the lectin binding properties showed that the changes in carbohydrate-containing constituents of the prostate correlate with the beginning of prostatic secretion and to prostatic epithelial differentiation. In the periacinar stroma the development of the lectin binding pattern was similar to that in the glandular epithelium. The changes of stromal binding sites for Con A and WGA during epithelial differentiation may reflect the changes of epithelial-stromal interactions in the prostate.
Abstract: In the present communication, ultrastructural and cytochemical aspects of mature bovine Sertoli cells and their relationship to the different stages of germ cell development are described. As in other mammalian species, different types of junctional specializations exist between Sertoli and germ cells in the bovine seminiferous epithelium, including desmosome-like junctions, Sertoli cell ectoplasmic specializations and tubulobulbar complexes. The functional significance of the morphological results and the interactions of Sertoli and germ cells during spermatogenesis are discussed.
Abstract: In the present study, the topographical distribution of carbohydrate binding sites on the plasma membrane of bovine epididymal spermatozoa was investigated using 15 different fluorescent neoglycoproteins and asialoglycoproteins. With mannose-bovine serum albumin (BSA)-fluoresceinthiocarbamyl (FTC), mannose-6-phosphate-BSA-FTC, lactose-BSA-FTC, maltose-BSA-FTC, asialolactoferrin-FTC and asialotransferrin-FTC a marked fluorescence was observed in the postacrosomal area. These results further substantiate the concept that carbohydrate binding sites of the spermatozoan plasma membrane and corresponding carbohydrates of the zona pellucida play a significant role in gamete interactions.
Abstract: Acidic extracts of guinea pig jejunum and colon contain atrial natriuretic peptide-like material (IR-ANP) detected by radioimmunoassay after purification by Sephadex G-50 gel filtration. Immunohistochemical analysis of guinea pig colon also revealed IR-ANP to be located directly beneath the lamina muscularis mucosae. High performance gel permeation chromatography (HP-GPC) and reverse phase high performance chromatography (RP-HPLC) of the IR-ANP showed correspondence to the 15 kD ANP precursor molecule (pro-ANP). No low molecular weight forms of ANP were detected. The extracted pro-ANP could be converted to alpha-ANP-like material by incubation with serum or supernatant of colonic homogenate. These data indicate the intestine to be a further site of ANP synthesis.
Abstract: A new polyester resin of low viscosity was used to study the three-dimensional structure of capillary sprouts of mature follicles and of corpora lutea. Vascular microcorrosion casts were obtained from ovaries of immature golden hamsters between days 4 and 7 after stimulation with pregnant mares' serum gonadotrophin (PMSG). On days 4 and 5 after PMSG, a nodular appearance of the organ was due to mature follicles and corpora lutea. Capillary sprouts were seen as dead-ending structures originating from the thecal capillary network. The sprouts were either concentrated around (follicle type 1) or towards (follicle type 2) the center of the antrum. Increased vascular permeability resulted in resin leakage only in type 2. Intact corpora lutea were characterized by radially grown capillary sprouts. Regressing corpora lutea showed a marked reduction of the vascular bed as displayed by filling defects in casts on day 6 and 7 after PMSG. It is assumed that: 1. Dead-ending capillary structures, resin leakage, and filling defects are not always artifacts of the microcorrosion cast technique, but can express functional microcirculatory changes. 2. The concentric growth of thecal capillary sprouts may be related to the presence of an intact follicular basal membrane, and the radial growth of sprouts be taken as a sign of a digested basal membrane.
Abstract: Resistance of hormone-dependent mammary carcinoma to cisplatin as a potent antitumor agent led to the synthesis of other estrophilic platinum complexes. In this investigation, the effects of a newly synthesized estrogen-receptor affine platinum complex on the mouse uterus were studied using light and electron-microscopy. The results have been compared with Tamoxifen, cisplatin and the estrophilic ligand. Both estrophilic ligand and estrophilic platinum complex produced strong estrogenic effects as well as features characteristic of the uterine epithelial cell in the luteal phase of the cycle, corresponding to a massive stimulation of the surface and glandular epithelial cells. The uteri showed large glandular lumina. An increase in the number of multivesicular and residual bodies, accompanied by a proliferation of eosinophilic granulocytes, was also seen. The appearance of inter- and intracellular lumina and the activation of smooth muscle cells represent further characteristic effects of the estrophilic ligand and estrophilic platinum complex. Anticipated increases in the incidence of cell death and/or deviant cyto-nuclear architecture in the uteri treated with cisplatin or platinum complex, were not observed.
Abstract: Two antiestrogenic phenylindoles (D 16726 and D 15413) were tested for their prostatic tumor-inhibiting activity. Both compounds exerted a strong inhibitory effect on prostate and seminal vesicle weight of intact rats and mice comparable to that of diethylstilbestrol. Their estrogenic properties, however, are much lower than those of DES. Therefore, there is no direct correlation between estrogenic potency and inhibition of accessory sex organ weights. The tumor-inhibiting activity of D 16726 and D 15413 on the androgen-dependent R 3327 Dunning prostatic carcinoma and the human prostatic tumor PC 82 implanted in nude mice equals that of castration or of diethylstilbestrol. Both 2-phenylindoles had good affinities for estrogen receptors from calf uterine and R 3327 tumor cytosol, but no affinities for androgen and progesterone receptors. As these 2-phenylindoles have much lower estrogenic properties than diethylstilbestrol, they may also have low side-effects, and can therefore be of interest for the therapy of the prostatic carcinoma.
Abstract: Antiestrogens and partial estrogens of the stilbene (1 and 4), triphenylbutene (2) and diphenylethane (3) series were tested for their potential prostatic tumor inhibiting activity. Compounds 1 and 2 exerted a strong inhibitory activity on prostate and seminal vesicle weight of intact rats and mice, whereas the strong antiestrogen 3 and compound 4 had no or only a slight effect. The tumor inhibiting activity of 1 and 2 on the hormone-dependent R 3327 Dunning prostatic carcinoma of the rat was strong and comparable to that of castration or administration of the potent estrogen DES. Compounds 1-4 had no direct antiandrogenic effect in castrated, testosterone-substituted rats and mice, and no affinity for the androgen or progesterone receptor. To the estrogen receptor from prostatic tumor cytosol, however, 1-4 had good receptor affinities. As the partial antiestrogen 1 and the partial estrogen 2 have much lower estrogenic properties than DES, but still have strong prostatic tumor inhibiting properties, they may offer a suitable alternative to conventional therapy of prostate carcinoma because of their possibly low estrogenic side effects.
Abstract: The ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase) in bovine Leydig cells has been studied and compared with the pattern of thiamine pyrophosphatase (TPPase) and acid phosphatase distribution in these cells. Using beta-nicotinamide adenine dinucleotide phosphate (beta-NADP+) as substrate, a marked staining is observed in the intermediate Golgi saccules with some focal extension to the trans aspect. Cisternae on the cis side and associated vesicles yielded only slightly positive reactions. The pattern of NADPase localization is clearly different from that of TPPase which consistently stains only the trans Golgi elements. The specificity of NADPase for its substrate, beta-NADP+, was clearly demonstrated by using substrates modified in either the nicotinamide region e.g. alpha-nicotinamide adenine dinucleotide phosphate (alpha-NADP+), beta-thionicotinamide adenine dinucleotide phosphate (Thio-NADP+), in the attachment site of the monoester phosphate group to the molecule (e.g. 2' monophospho-adenosine 5'-diphosphoribose (ATP-ribose) or adenosine-5-monophosphate (5'AMP). With these substrates only weak or negative reactions were obtained in the Golgi apparatus of the bovine Leydig cell.
Abstract: Within the uterine glands, the following enzymes were demonstrated by histochemical methods after 30, 58, 80, 100, and 110 d of pregnancy, respectively: beta-N-acetyl-hexosaminidase, beta-galactosidase, beta-glucuronidase, alpha-mannosidase, acid phosphatase, alkaline phosphatase, esterases, cytochrome oxidase, 5-nucleotidase, leucine aminopeptidase, adenosine triphosphatase, diaphorases (NADH, NADPH), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, succinate dehydrogenase, isocitrate dehydrogenase (NAD, NADP), beta-hydroxybutyrate dehydrogenase, glycero-3-phosphate dehydrogenase, NAD-glycero-3-phosphate dehydrogenase, glutamate dehydrogenase (NAD, NADP), lactate dehydrogenase. The results show that the activities of G-6-PDH, 6-PGDH, and cytochrome oxidase increase within secreting cells during the 2nd half of pregnancy. The activities of the other enzymes remained almost unchanged during the period of investigation. The description of our results distinguishes between gland neck, middle, and distal part of the secretory unit, respectively. In general, the enzyme activities are similar within the middle and distal gland segments, but lower in the epithelia of the neck region. The activity of dehydrogenases was medium to intensive within the middle and distal gland segments, but only low to medium within the neck portion. Of the hydrolases, the acid phosphatase, ATPase, leucine aminopeptidase, and beta-galactosidase demonstrated an intensive activity within activity secreting cells. The enzyme activities of the gland epithelia are compared with these of the uterine surface epithelia and the histochemical results are discussed in context with their significance in histiotrophic nutrition.
Abstract: The fine structure of bovine Sertoli cells was studied from the 4th to the 40th week post natum in order to correlate the progressive acquisition of normal adult morphology with functional development. The considerable increase in tubular size during the first 20 weeks is due to the proliferation of both presumptive Sertoli and germ cells. Aside from this, the presumptive Sertoli cells are seen to expand radially and lengthen considerably. From then on however, the observed increase in tubular diameter during the later period of postnatal development is solely due to the great increase in the number of germ cells. Presumptive Sertoli cells undergo morphological differentiation to mature Sertoli cells during the first 28 weeks of proliferative development. The maturation process includes distinct changes in cell shape, nucleus and cellular organelles, as well as an increase in and differentiation of Sertoli cell surface specializations. At 24 weeks the development of inter-Sertoli cell junctions has reached a point of differentiation where, in our opinion, a functional blood-testis barrier can be expected. During the first 8 weeks an extensive development of rough endoplasmic reticulum and a well-developed Golgi apparatus can be observed, which suggests a high secretory activity in the presumptive Sertoli cells at this time. We speculate that these secretory activities may play a role in the formation of the basal lamina which is extremely well developed during early postnatal life. The subsequent reduction of the basal lamina correlates well with diminished secretory activity in the Sertoli cells.
Abstract: In order to study the acrosome reaction in boar, spermatozoa were incubated in a calcium-containing medium in the presence of the calcium ionophore A23187. The time course of the acrosome reaction was assessed by phase-contrast microscopy and correlated with the movement characteristics of the spermatozoa determined by means of multiple-exposure photography (MEP). Different stages of the acrosome reaction could be observed by indirect immunofluorescence using an antibody fraction raised in rabbits against the isolated outer acrosomal membrane (OAM). At the start of the acrosome reaction, a bright fluorescence located exclusively at the acrosomal cap of the sperm head could be observed, whereas after 60-120 min, the fluorescence vanished, indicating the complete loss of the OAM. However, to gain more insight into the stages of the plasma membrane and OAM during the acrosome reaction, immunoelectron-microscopical studies were performed using anti-OAM antibodies detected by the protein-A gold method. Ultrathin sections and total preparations in combination with transmission electron microscopy (TEM) confirmed, that boar spermatozoa start their acrosome reaction by a vesiculation of the plasma membrane, thus exposing the heavily labelled OAM, which is then lost as sheets or large vesicles. The newly exposed inner acrosomal membrane did not show any labelling with gold, thereby indicating clear differences in the antigenicity of both acrosomal membranes.
Abstract: In porcine areolar placental epithelia, the following enzymes were demonstrated by histochemical methods after 30, 58, 80, 100, and 110 d of pregnancy, respectively: beta-N-acetyl-hexosaminidase, beta-galactosidase, beta-glucuronidase, alpha-mannosidase, acid phosphatase, alkaline phosphatase, nonspecific esterases, cytochrome oxidase, 5-nucleotidase, leucine aminopeptidase, adenosine triphosphatase, diaphorases (NADH, NADPH), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, succinate dehydrogenase, isocitrate dehydrogenase (NAD, NADP), beta-hydroxybutyrate dehydrogenase, glycero-3-phosphate dehydrogenase, NAD-glycero-3-phosphate dehydrogenase, glutamate dehydrogenase (NAD, NADP), lactate dehydrogenase. The results show that the enzyme activities remained almost unchanged during the period of investigation. Of the dehydrogenases, the diaphorases as well as succinate and lactate dehydrogenase demonstrated generally an intensive activity within the epithelia. The activity of the other dehydrogenases was only low. The activity of unspecific esterase was very intensive within the uterine epithelia but remarkably low within chorionic epithelia. Contrarily, the reaction of adenosine triphosphatase was more intensive within chorionic than uterine epithelia. All investigated glucosidases reacted distinctly positive within chorionic epithelia, but only beta-N-acetyl-hexosaminidase and beta-galactosidase in uterine epithelia. The high activity of acid phosphatase, especially within the chorionic epithelium, seems to be connected with uteroferrin, an iron-binding protein. The histochemical results are discussed in context with the function of the areolae in histiotrophic nutrition and iron transport.
Abstract: Modifications in bull sperm plasmamembrane during epididymal passage were investigated by the use of four different lectins: Concanavalin A (Con A); Ricinus communis I (RCA1); Wheat germ agglutinin (WGA); Ulex europaeus agglutinin I (UEA1). During sperm passage from caput to cauda epididymidis agglutination by RCA1 and WGA distinctly increased. Similar but somewhat less pronounced difference in the agglutinability was found for Con A. No agglutination was observed with UEA1. Ultrastructural examination of Con A binding sites on sperm plasma membrane with a Con A-horseradish peroxidase-gold technique (Con A-HRP-G) revealed a significant increase in the number of gold granules on the sperm tails during the epididymal passage of spermatozoa. No change in WGA-binding sites was observed between caput and cauda spermatozoa using a WGA-peroxidase method.
Abstract: Binding sites for Con A and WGA were detected on bovine spermatozoa during epididymal maturation. We used colloidal gold as an EM-marker. The spermatozoa were treated according to a two-step method for lectin and colloidal gold, then adsorbed to lysine-coated nickel grids and subsequently examined by TEM in toto. Using this method we rapidly got information about the topographic distribution of lectin-binding sites. Major differences exist for WGA between caput and cauda spermatozoa. Conceding that cell-thickness poses some limitation, we consider this method to be practical and especially useful in studies concerning topographic distribution of cell surface components in single cell systems.
Abstract: Using electron microscopic techniques the prostate glands of male Beagle dogs were studied 3 days after castration. At this time marked differences in the extent of alterations of the glandular epithelium were observed: Whereas several acini showed only minor changes with reduction of epithelial height and diminution of secretory granules, many acini were severely affected with pronounced alteration of cellular structure and accumulation of large lipid droplets. A constant feature was the stimulation of the basal cells of the grandular epithelium. Additionally, in some areas of the gland aggregations of stimulated basal cells forming an acinus-like structure with a slit-like lumen were found. Our study shows that castration leads to marked alterations of prostatic epithelium within a short time. Androgen deprivation causes regressive changes of secretory epithelial cells, but clearly stimulates the basal cell population.
Abstract: In porcine interareolar placental epithelia, the following enzymes were demonstrated by histochemical methods after 30, 58, 80, 100, and 110 d of pregnancy, respectively: beta-N-acetylhexosaminidase, beta-galactosidase, beta-glucuronidase, alpha-mannosidase, acid phosphatase, alkaline phosphatase, nonspecific esterases, cytochrome oxidase, 5-nucleotidase, leucine aminopeptidase, adenosine triphosphatase, diaphorases (NADH, NADPH), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, succinate dehydrogenase, isocitrate dehydrogenase (NAD, NADP), beta-hydroxybutyrate dehydrogenase, glycero-3-phosphate dehydrogenase, NAD-glycero-3-phosphate dehydrogenase, glutamate dehydrogenase (NAD, NADP), lactate dehydrogenase. The results show that most of the enzyme activities remained almost unchanged during the period of investigation. Only G-6-PDH and 6-PGDH activities increased within the uterine epithelium and nonspecific esterase activity within uterine as well as chorionic epithelia during the 2nd half of pregnancy. Within chorionic and uterine epithelia, hydrolases but not dehydrogenases demonstrated a higher activity at the bases of chorionic villi as compared to the apices and flanks of the latter. The action and influence of the demonstrated enzymes on metabolism, energy transfer, secretory, and resorptive activities of chorionic and uterine epithelia are discussed.
Abstract: The ultrastructure of the porcine uterine glands is described from material taken from 11 pregnant pigs at exactly known stages of gestation (day 30; 58; 80; 100; 110). Fixation was performed by perfusion via a branch of the uterine artery and the tissue was routinely processed for electron microscopy. Additionally, cytochemical studies (phosphotungstic acid reaction for glycoproteins, according to Rambourg 1967; acid phosphatase reaction; ultrastructural localization of cellular iron, according to Parmley et al. 1978) were performed. On day 30 of pregnancy the uterine glands are coiled, simple tubular glands with a narrow lumen. The epithelial lining is simple columnar and consists basically of two cell types, ciliated cells and secretory cells. The secretory activity of the glandular epithelium is low; only a few secretory granules are present in the supranuclear cytoplasm. At midpregnancy the ultrastructure of the glands has significantly changed and the cells now show all the characteristics of high secretory activity: numerous parallel cisternae of rough endoplasmic reticulum, an extensively developed Golgi apparatus and many secretory granules which give a positive reaction for acid phosphatase and glycoproteins. The lumina of the glands are significantly enlarged and filled with a great amount of a granular, acid phosphatase-positive material. In the last third of pregnancy, only minor changes in the ultrastructure of the uterine glands are observed. The secretory activity is still high. The amount of rough endoplasmic reticulum has further increased and parallel arrays of cisternae occupy a considerable part of the supranuclear cytoplasm. The importance of the uterine secretion for embryonic nutrition and development is only partly understood. One of the secreted glycoproteins, uteroferrin, is believed to play an important role in the iron transfer from mother to fetus. From midpregnancy onward, a special cell type, the "granule laden cell" is found scattered between normal secretory cells of the uterine glands. Contrary to the opinion of Perry and Cromby (1982), we could demonstrate that these cells frequently extend to the lumen of the gland; hence the term "basal cell" seems inappropriate for this cell type.
Abstract: Mammalian spermatozoa that leave the testis are neither motile nor fertile. Maturation of spermatozoa occurs during epididymal transport. Among the changes during epididymal passage alterations of the surface properties of spermatozoa appear especially interesting. In this report we describe ultrastructural localization of Con A and WGA-binding sites on bovine spermatozoa from caput and cauda epididymides using a indirect lectin-horse-radish-peroxidase gold technique. With Con A the plasma-membrane covering the sperm head was heavily labelled with gold granules in caput as well as in cauda epididymal spermatozoa. A different distribution was observed for WGA-binding sites. The acrosomal region of caput and cauda spermatozoa was heavily labelled with gold, both in caput and cauda spermatozoa. The postacrosomal region was only sparsely marked in caput sperm whereas in sperm cells originating from the cauda a calix like membrane area displayed intense labelling. Some differences in the number of binding sites were also seen on sperm tails: those of caput spermatozoa show generally more Con A binding sites that those from cauda epididymal spermatozoa. No changes in the number of WGA-binding sites on sperm tails was observed during epididymal passage. The technical aspects of the indirect lectin-horse-radish-peroxidase gold technique are briefly discussed.
Abstract: Mesangial cells derived from isolated glomeruli of rat kidney were grown as homogeneous cell lines in culture. They released, into the culture medium, erythropoietin that had free terminal galactosyl residues and was therefore not active in vivo. The production of erythropoietin by these cells was significantly enhanced by either lowering the PO2 in the incubation atmosphere or by adding cobalt chloride to the culture medium. Therefore, mesangial cells in culture may be considered as an in vitro system in which the regulation of erythropoietin production can be studied under well-defined conditions.
Abstract: Some of the critical steps in the qualitative histochemical localization of glucose-6-phosphate dehydrogenase (freezing procedures, incubation techniques and the influence of intermediate electron carriers, respiratory chain inhibitors and different tetrazolium salts) were evaluated in sections of bovine testis as a prerequisite for the microdensitometric estimation of the activity of the enzyme in bovine Leydig cells in situ. A modification of the gel incubation method of Rieder et al. (1978) gave the best results and was used for the quantitative investigations. Quantitative data for the dehydrogenase activity gained from microdensitometry of the formazan final reaction products in Leydig cells in situ were compared with the results of assays of the activity in homogenates of testis. The following apparent kinetic properties of glucose-6-phosphate dehydrogenase were obtained for the enzyme in Leydig cells in situ: Vmax = 0.11 absorbance units/min, Km = 0.37 mM. The quantitative characterization of glucose-6-phosphate activity in Leydig cells in situ appears to be suitable for combined morphological and functional diagnoses of small tissue samples such as testicular biopsies. This would give valuable information of the functional status of Leydig cells in normal and diseased testicular tissue.
Abstract: The intratesticular excurrent duct system of the bull is composed of rete testis, tubuli recti, and the terminal segment of the seminiferous tubules. Each terminal segment is surrounded by a vascular plexus and may be subdivided into a transitional region, middle portion, and terminal plug. The modified supporting cells of the middle portion and the terminal plug no longer display the typical Sertoli-Sertoli junctions seen in the transitional region and the seminiferous tubule proper. In the region of the terminal plug a distinct central lumen is generally not observed: spermatozoa and tubular fluid must pass through an intricate system of communicating clefts between the apices of the closely attached modified supporting cells. Vacuoles in the supranuclear region of the cells in the middle portion indicate strong transepithelial fluid transport. In analogy to the epithelium of rete testis and tubuli recti, the supporting cells of the terminal segment are capable of phagocytosing spermatozoa. The vascular plexus investing the terminal segment serves a dual purpose: it is a regulatory device for fluid and sperm transport, as well as an area of increased diapedesis for white blood cells.
Abstract: A prostate carcinoma arising spontaneously in a Fischer rat was transplanted by cell suspension to male and female Fischer rats. Tumour growth was rapid in both subcutaneous and intraprostatic sites of inoculation in males and subcutaneously in females. The growth rate was greater in male than in female hosts and the tumour developed as a solid form in both groups. It had a heterogeneous cell population with ultrastructural characteristics similar to basal cells of the normal prostate and to tumour cells seen in human prostate adenocarcinoma. Later stages of tumour growth transformed it to a cornifying squamous cell carcinoma. The morphological description of tumours produced by transplantation provides the basis for further studies using this method as a model for investigating the response of prostate carcinoma to endocrine manipulation.
Abstract: In the present study the development of the bovine acrosome was investigated using conventional electron-microscopical techniques as well as the phosphotungstic-acid (PTA) technique (Rambourg 1967) including enzymatic digestion experiments. As in other species and in accordance with previous light-microscopical studies (Clermont and Leblond 1955) four phases of acrosomal differentiation can be discerned; the Golgi-phase, cap-phase, acrosome-phase, and maturation-phase. In the bull no internal pattern of the acrosomal content can be observed, either with conventional uranyl acetate-lead citrate staining or with the PTA-techniques. Our results support the observation in other species (Fawcett et al. 1971) that no intrinsic polymerization or crystallization process of the acrosomal content is responsible for acrosomal shaping. Some of our results suggest the influence of external forces on acrosomal development in the bull. During the cap-phase and the acrosome-phase accumulation of smooth endoplasmic reticulum and a layer of fine filaments can be observed in the Sertoli-cell cytoplasm, immediately adjacent to the developing acrosome. A temporary influence of these structures on acrosomal development seems possible. The PTA-positive staining of the developing bovine acrosome is probably due to the presence of acrosomal glycoproteins; however, our results do not exclude the possibility that molecules other than glycoproteins contribute to the positive PTA-staining of the developing acrosome.
Abstract: The ultrastructure of the epididymal duct and ductuli efferentes in the dog has been studied by electron microscopy. The epididymidis can be separated into the classical divisions of caput, corpus and cauda epididymidis on the basis of general morphology and ultrastructure. The ductuli efferentes have a low epithelium with pronounced cilia at the apices of cells and appear to provide primarily a transport role for spermatozoa. In the epididymis proper the caput region is characterized by an extremely large Golgi apparatus with large numbers of lysosomes and nuclear inclusions. Secretory activity appears to be most common in the corpus region. Absorption and secretion are most active in the first two segments while in the cauda epididymidis the long-term storage of spermatozoa in the lumen is associated with many dense crystalline bodies formed in the epithelial cells within the Golgi apparatus and possibly deriving from absorbed macromolecular material from the lumen. The theory of whole sperm cell resorption by the epididymal duct is not supported by this study.
Abstract: The ultrastructure of the areolae in the porcine placenta is described. The areolae occur on day 30 of pregnancy as dome-shaped formation over the openings of the uterine glands. The lumen of the areolae is filled with the secretions of the uterine glands, the so-called histiotroph. The areolae lining epithelium is high collumnar, possessing long microvilli, a well-developed apical tubular system and numerous coated vesicles. This indicates that the epithelium has a high absorptive capacity. Our histochemical investigations reveal a high content of glycoproteins within the areolar lumen. The importance of one of the glycoprotein components of the histiotroph, uteroferrin, is discussed in connection with iron transfer from mother to the fetus.
Abstract: The intertubular stroma of the bovine testis is composed of narrow strands between two adjacent tubules and larger tri- and quadrangular interstices between three to four tubules. The latter contain the majority of Leydig cells, larger blood vessels and testicular lymph vessels. Leydig cells occur in groups or cords, not every cell being in close contact to a capillary, lymph vessel or venule. Between adjacent Leydig cells intercellular canaliculi and gap junctions are frequently encountered. Bovine Leydig cells are further characterized by an abundance of ribosome-associated endoplasmic reticulum, by mitochondria often containing crystalloid structures and displaying both tubular and lamelliform cristae, as well as by a relative paucity of lipid droplets and lysosomes. Independent of the size of intertubular lymph vessels their walls consist only of an endothelium of varying thickness, no typical basal lamina or associated musculature being present. The interstitial surface of the endothelium sends anchoring cytoplasmic pedicles into the subjacent ground substance and collagen fibrils. Among occasional plasma cells, mast cells and mononuclear leucocytes, a regular constituent of the intertubular region studied is a population of electron-lucid, irregularly shaped cells (light intercalated cells = LIC) with slender, pleomorphic processes. These cells are believed to be involved in testicular androgen storage and distribution.
Abstract: The finestructural changes of the interareolar porcine placenta during pregnancy are described. After perfusion fixation of the placenta the change in the thickness of the placental barrier from day 30 to day 110 of gestation is much more evident than after immerson fixation as has been used by all former authors. The alterations are due to the indentation of both the trophoblast and uterine epithelium by their corresponding capillary-network. This indentation is limited to the lateral wall and the summit of the chorionic ridges, while at the base the trophoblast as well as the uterine epithelium remains high columnar. This indicates that in the interareolar porcine placenta, which is represented by the chorionic ridges and the corresponding endometrial folds, at least two different areas with different structure and function may be discerned. 1) The lateral side and the top of the chorionic ridges seem to be predestinated for gaseous exchange. The placental barrier in this area is often less than 2 micrometers. 2) The transport of blood-borne nutrients takes place at the base of the chorionic ridges. This transport seems to be facilitated by an intercellular channel system between the uterine epithelial cells.
Abstract: During pregnancy and lactation marked changes are observed in the fine structure of the secretory cells in the Beagle mammary gland: especially pronounced are differences in cellular height, shape and size of the nuclei and distribution of mitochondria. In later stages of pregnancy a proceeding development of those cellular organelles involved in synthesis and extrusion of secretory material (i.e. rough endoplasmic reticulum, Golgi apparatus) can be observed. Myoepithelial cells which can be first discerned from secretory cells by ultrastructural features from day 40 on show only minor variations of their ultrastructure during pregnancy and lactation.
Abstract: Localization and activity of five hydrolases (alkaline phosphatase, adenosine triphosphatase, acid phosphatase, nonspecific esterase and leucylamino-peptidase) were evaluated histochemically in the epididymides of mature dogs. In the ductuli efferentes, cilia and apical parts of the epithelial cells displayed high activity of alkaline phosphatase and adenosine triphosphatase. Strong activity of acid phosphatase, nonspecific esterase and leucylamino-peptidase was present in the basal and supranuclear zones of the epithelium of the ductuli efferentes. Stereocilia of all three segments of the ductus epididymidis showed a high activity of alkaline phosphatase. Positive adenosine triphosphatase reaction was confined to the stereocilia of the initial segment. A complex pattern of acid phosphatase activity was observed in the middle segment. The subdivision of the middle segment in four subsegments was therefore suggested. In the epithelium of the initial segment only a few nonspecific esterase-positive cells were seen. The infranuclear and basal areas of the epithelium in the middle segment and the supranuclear zone of the terminal segment displayed distinct nonspecific esterase activity. The possible contribution of the hydrolases to the function of the epididymis is discussed.
Abstract: The histochemical localization of five glycosidases was studied in the epididymis of mature dogs. beta-Galactosidase showed a distinct to strong reaction in the epithelium of the ductuli efferentes and throughout the whole length of the ductus epididymidis. beta-N-Acetylglucosaminidase reactivity was weak in the initial segment, but increased significantly in the middle and terminal segment. The maximum beta-glucuronidase activity was found in the ductuli efferentes and in the initial segment. The alpha-mannosidase reaction was weak in all segments except the middle segment where a distinct activity was seen. With the method employed, no alpha-fucosidase activity could be detected. The physiological role of the glycosidases in the epididymis is discussed briefly.
Abstract: The histochemical localization of some oxidoreductases was investigated in the epididymides of adult tomcats. Succinate dehydrogenase, lactate dehydrogenase, beta-hydroxybutyrate-dehydrogenase revealed their highest activity in corpus and cauda epididymidis whereas glucose-6-phosphate-dehydrogenase was strongest in the caput. The activity of the diaphorases and of cytochrome oxidase in the epididymal epithelium increased from caput towards the cauda epididymidis. The reaction for isocitrate dehydrogenase was distinct throughout the whole length of the ductus epididymidis. Our findings were compared with the biochemical results of other authors and the functions of the oxidoreductases in the epididymal epithelium were briefly discussed.
Abstract: The boundary tissue of bovine testicular seminiferous tubules exhibits remarkable regional differences at the level of the seminiferous tubule proper, as compared with its terminal segment. The basal lamina of the seminiferous tubule proper is multilayered and possesses knob-like protrusions. At the level of the terminal segment the basal lamina is highly specialized; in the region of the terminal plug candelabrum-like projections of the tubular basal lamina invade the bases of the modified supporting cells up to a depth of 3.5 microns. The adjoining surface of these supporting cells is densely studded with hemidesmosomes. The elongated peritubular cells are arranged in 3--5 concentric layers around the tubulus seminiferus proper but form a loose association at the level of the terminal segment. Where the terminal segment joins the testicular straight tubule, peritubular cells may assemble to constitute a contractile spiral. Elastic tissue is situated mainly subjacent to the tubular basal lamina and to a lesser degree between the peritubular cell layers. A peritubular space lined by endothelium-like cells may surround the seminiferous tubule proper and also the transitional zone of the terminal segment.
Abstract: Ultrastructural examination showed that the epithelium of the bovine rete testis and the tubuli recti could phagocytose spermatozoa. Macrophages were regularly found in the basal parts of the epithelial cells and could be involved in the removal of degraded sperm material.
Abstract: In benign hyperplasia marked beta-glucuronidase and N-acetyl-beta-glucosaminidase activity was seen in the prostatic epithelium whereas the reactions for 5 other glycosidases were only slight or negative. The intensity of the reaction for the glycosidases in prostatic carcinoma was dependent upon the degree of the differentiation. The possible importance of glycosidases for the invasiveness of prostatic carcinoma is briefly discussed.
Abstract: The histochemical distribution of six glycosidases (N-acetyl-beta-glucosaminidase, beta-galactosidase, beta-glucuronidase, alpha-galactosidase, alpha-mannosidase and alpha-fucosidase) was investigated in the prostate, glandula vesicularis and glandula bulbourethralis of castrated and non-castrated adult boars. The functions of the glycosidases in the male accessory sex glands of the boar and their androgen dependence are discussed briefly.
Abstract: Ultrastructural and ultrahistochemical studies were performed on the uterovaginal sperm host glands of the quail (Coturnix coturnix japonica). The proximal parts of the glandular necks are lined by a pseudostratified epithelium, consisting of high columnar ciliated cells and small, irregular shaped, basal cells. The true glandular epithelium is composed only of columnar cells with microvilli on their luminal end. A characteristic luminal feature is a large lipid droplet in the perinuclear region. In the subplasmalemmal region numberous tubular profiles are seen which could represent a cellular resorption system. To evaluate the absorptive capacity of the uterovaginal sperm host glands, tracer studies with HRP, ferritin, lanthanum and ruthenium red were undertaken. Since between 5 min and 3 h after injection no absorption could be found with the techniques mentioned, it is suggested that phagocytosis of spermatozoa by the glandular epithelium is not likely to occur.
Abstract: The histochemical localization of 6 lysosomal enzymes was studied in the epididymis of adult tomcats. A weak to distinct reaction for acid phosphatase, leucyl-amino-peptidase and non--specific esterase could be observed in the epithelium of the ductus epididymidis in all three segments. Among the glycosidases, N-acetyl-beta-glucosaminidase displayed the strongest activity. alpha-man and alpha-fuc could not be demonstrated. For N-Acetyl-beta-glucosaminidase, beta-Galactosidase, acid phosphatase and non-specific esterase, an increase of enzyme activity from the initial segment towards the terminal segment was seen. Intracellularly, the maximum of enzyme activity of those four enzymes was supranuclear. This histochemically enstablished pattern of enzyme activity in the epididymis of the tomcat was compared with those of other mammals. The possible functions of enzymes in the epididymis was briefly discussed.
Abstract: Explants of canine prostate were cultured in a defined medium for periods of up to 5 days with and without added steroids. Testosterone and 5 alpha-dihydrotestosterone failed to maintain their histological integrity and induced a greatly increased formation of stromal elements. Epitestosterone and 5 alpha-dihydroepitestosterone were partially successful in maintaining epithelial height although secretory activity was not preserved. The only steroid that sustained epithelial height and secretory activity whilst keeping stromal growth at a minimum was 5 alpha-androstane-3 alpha, 17 alpha-diol. The three other epimeric androstanediols were ineffective.
Abstract: The activities of 6 glycosidases (n-acetyl-beta-glucosaminidase, beta-galactosidase, beta-glucuronidase, alpha-galactosidase, alpha-fucosidase and alpha-mannosidase) in the oviduct of the quail (Coturnix coturnix japonica) were studied with histochemical methods. Alpha-galactosidase and alpha-fucosidase showed a weak to moderate activity in the surface epithelium and in most of the glands of the oviduct. A Distinct reactivity of beta-glucuronidase was observed in the surface epithelium of the whole oviduct and in the glands of the uterovaginal-region. A moderate to distinct reactivity of n-acetyl-beta-glucosaminidase cound be demonstrated in the epithelium and in the glands of all regions of the oviduct. The comparatively highest activity of this enzyme was found in the glands of the magnum and in the surface epithelium of the uterus. The possible functions of the glycosidases in the oviduct are discussed briefly.
Abstract: X-ray microanalysis of zinc in ultrathin sections of dog prostate was performed by electron microscope microanalysis using the potassium pyroantimonate method of preparation. Prostates of both mature and immature dogs were examined and the metal was found to be localised primarily in the nucleolus, nuclear chromatin and secretory granules of epithelial cells. Differences in zinc concentrations were observed between mature and immature tissues, particularly in the nuclear chromatin. The metal was also incorporated into epithelial secretions, lysosomes and fibromuscular stroma. Variable binding of zinc to tissue components was revealed by a combination of histochemical precipitation and subcellular analysis.
Abstract: The surface epithelium of vagina, uterovaginal region and uterus as well as the uterine and uterovaginal glands of 18 mature female quails were studied with histochemical methods. As in other avian species also in the quail a storage of spermatozoa in the lightly coiled uterovaginal glands takes place. The functional specialization of these glands is underlined by their distinct enzyme pattern. A strong reactivity of enzymes from oxidative pathways and of adenosine triphosphatase between epithelium and glandular luminal content. Alkaline phosphatase in the glandular epithelium was observed only when an egg is transported through the uterovaginal region. As in other vertebrate sperm storing sites also in the uterovaginal region of the quail the presence of a strong steroid dehydrogenase activity is registered.
Abstract: The histochemical distribution of 4 glycosidases (alpha-Mannosidase, beta-Glucuronidase, N-Acetyl-beta-glucosaminidase and beta-Galactosidase) has been studied in the accessory sex glands of bulls. All glcosidases displayed the highest activity in the distal part of the caput epididymidis and in the cauda epididymidis. A distinct activity of N-Acetyl-beta-glucosaminidase and beta-galactosidase was observed in the seminal vesicle, the ampulla ductus deferentis and in the prostatic gland. In the epithelium of the bulbourethral gland only a weak content of beta-Galactosidase could be demonstrated histochemically. The functional role of the glycosidases in the accessory sex organs is discussed briefly.
Abstract: The ultrastructure of the basal cells of rat lateral and ventral prostate and of dog prostate has been studied. Basal cells from both species appear as undifferentiated cells, characterised by a lack of cytoplasmic organelles and a poorly developed Golgi complex and endoplasmic reticulum. The presence of cytoplasmic filaments and micropinocytosis is not considered to be sufficient evidence to assume any similarity to myoepithelium, as has been previously suggested. Basal cells are instead considered to be precursors of secretory epithelial cells.
