Abstract: The zona pellucida (ZP) is a unique extracellular coat surrounding the maturing oocyte, during ovulation, fertilization, and early embryo development. It is formed by three/four glycoproteins. Ultrastructural data obtained with transmission (TEM) and scanning electron microscopy (SEM) were compared with molecular data on the glycoproteins network from ovulation to blastocyst formation. Molecular models are quite different to the morphology obtained with TEM, which shows a microfibrillar architecture, or with SEM, which shows a spongy or smooth surface. The saponin-ruthenium red-osmium tetroxide-thiocarbohydrazide technique allows to show the ZP real microfilamentous structure and the related functional changes. These results support an ultrastructural supramolecular model, more similar and comparable to molecular models related with the glycoprotein network. A detailed mapping of single mammalian ZP proteins and their relationship within the supramolecular architecture of the zona matrix would clearly supply insights into the molecular basis of sperm-egg recognition. Differences in ZP glycoproteins among mammals do not affect structural morphology; further studies are needed to clarify the relationships between ultrastructural and molecular organizations.

Abstract: Supernumerary ossicles (or Wormian bones) of the cranial vault are formations associated with insufficient rate of suture closure, and regarded as "epigenetic" and "hypostotic" traits. These bones rest along sutures and/or fill fontanelles of the neonatal skull. In this autoptic report of a 66-year-old Caucasian woman, a peculiar supernumerary bone is described, unusual size and shape, filling completely the bregmatic fontanelle. The skull was CT-scanned through coronal sections at 80 kV and 60 mA, with a slice thickness of 1.0 mm and a resolution of 0.35 mm/pixel. Segmentation and 3D rendering were computed using MIMICS 7.0 (digital endocast). The bone was pentagonal and remarkably large, more on the exocranial surface than on the endocranial one, involving both tables and diploe of the vault. This feature might represent a wedge to completion of the vault architecture. Considering the functional and structural matrix of cranial morphogenesis, this case displays the possibility of discrete diversification of the ossification centres, as well as the relative stability of the structural skull matrix in response to discrete changes.

Abstract: In order to elucidate the ultrastructural dynamics of endometrium differentiation, uterine samples of fetuses aged 14 to 22 weeks of gestation (WG) were analyzed. Samples were processed for light (LM), transmission (TEM) and field-emission scanning electron microscopy (FE-SEM). Initial stratification of the uterine wall occurred at 14 WG: endometrial, myometrial, and perimetrial primordia were identified. At this age, the endometrial epithelium was simple columnar to pseudostratified and consisted of microvillous cells. Blood capillaries developed mainly in the stroma and between the myometrium and perimetrium primordia. At 18-20 WG the endometrial epithelium became clearly pseudostratified, with active ciliogenesis and a predominance of microvillous cells. Primordia of tubular glands were present at 20 WG. Microvillous cells still predominated in the endometrial epithelium at 21-22 WG and showed morphological features of apoptosis. The endometrial stroma at this stage was organizing into a thick lamina propria provided with subepithelial capillary plexuses. However, the stroma was formed by still undifferentiated mesenchymal cells during the whole period of study. Our data showed that the epithelial differentiation and distribution in the uterus occur in the human fetus in a similar way as in the adult. The above events are likely the expression of an early developmental patterning and related to future reproductive processes, such as the regulation of gamete passage and blastocyst implantation. Because the structure of the adult uterus is determined by the degree of paramesonephric duct fusion, septum absorption, and differentiation of the uterine primordial layers, our study may contribute toward clarifying to normal urogenital development.

Abstract: After dissection of the abdominal cavity of a 59-year-old male, a long artery, extending from the hepatic artery to the transverse colon, and comprised proximally within the neck of the pancreas and distally within the transverse mesocolon, has been detected. This "pancreato-colic" artery (P-C A) was 13 cm in length and 4 mm in diameter at the origin. Its mesocolic part (2.5 mm in diameter) contributed to the formation of the marginal arcade. No middle colic artery from the superior mesenteric was observed. Thus, the transverse colon was supplied by the distal part of the P-C A. Considering both the P-C A caliber and topography, attention should be paid during pancreatic resections and in the interposition of the transverse colon for esophageal replacement in order to avoid serious bleeding and necrosis. Embryologically, the proximal part of the P-C A might be regarded as an intrapancreatic variant of the tract of the longitudinal anastomosis between the ventral segmental arteries, persisting in the adult as dorsal pancreatic artery. Different from its usual retropancreatic location, this part might be entrapped inside the gland by the developing primitive pancreatic anlages. The distal, mesocolic, part of the P-C A might be regarded as a replacing middle colic artery into the dorsal mesentery during midgut rotation.

Abstract: Female reproductive aging is associated with several morphological changes of the genital tract with a subsequent decline in fertility; however, ultrastructural changes occurring after menopause have still not been well illustrated. Our aim was to characterize the three-dimensional microanatomy of the luminal surface of the human fallopian tube in perimenopause and postmenopause. Twenty bioptic samples of fallopian tubes were obtained after surgery under the informed consent of the patients. Samples were processed for transmission electron microscopy (TEM) and field-emission scanning electron microscopy (FE-SEM). As age increases the surface epithelium of the fallopian tube appeared somewhat flattened. Correlated TEM/FE-SEM observations showed gradual shortening of microvilli and deciliation. The most interesting finding was the gradual formation of microplicae-like structures on the surface epithelium, particularly from late perimenopause to postmenopause. Microplicae-like structures, associated with other regressive changes, represent an important adaptation of the epithelium of the fallopian tube; these are likely induced by the physiological process of aging, thus better withstanding hormonal changes associated with the advent of the menopause.

Abstract: This study describes the updated, fine structure of human gametes, the human fertilization process, and human embryos, mainly derived from assisted reproductive technology (ART). As clearly shown, the ultrastructure of human reproduction is a peculiar multistep process, which differs in part from that of other mammalian models, having some unique features. Particular attention has been devoted to the (1) sperm ultrastructure, likely "Tygerberg (Kruger) strict morphology criteria"; (2) mature oocyte, in which the MII spindle is barrel shaped, anastral, and lacking centrioles; (3) three-dimensional microarchitecture of the zona pellucida with its unique supramolecular filamentous organization; (4) sperm-egg interactions with the peculiarity of the sperm centrosome that activates the egg and organizes the sperm aster and mitotic spindles of the embryo; and (5) presence of viable cumulus cells whose metabolic activity is closely related to egg and embryo behavior in in vitro as well as in vivo conditions, in a sort of extraovarian "microfollicular unit." Even if the ultrastructural morphodynamic features of human fertilization are well understood, our knowledge about in vivo fertilization is still very limited and the complex sequence of in vivo biological steps involved in human reproduction is only partially reproduced in current ART procedures.

Abstract: The use of somatic cells for cocultures during in vitro fertilization (IVF) is currently finalized to obtain a higher number of healthy and viable embryos with a high potential of implantation. Among the different cell lines that can be used as feeder cells for cocultures, granulosa cells (GCs) are autologous cells, safe and easy to recover. The aim of the present study was to analyze the fine structure of human GCs used in a coculture system to evaluate, from a morphodynamic point of view, their role in supporting embryo development. GCs were collected during oocyte pick-up, 36 h after human chorionic gonadotropin administration, from patients undergoing IVF procedures, who had given their informed consent to be included in this protocol. After coculture, GCs were fixed and processed for light microscopy (LM) and transmission electron microscopy (TEM). By LM, GCs appeared as clusters of loosely packed cells, irregularly rounded or polyhedral in shape, varying in diameter from 18 to 25 microm. Mitotic cells, as well as regressing elements (with pyknotic nuclei or dense cytoplasm) and cell fragments were occasionally observed. By TEM, the plasma membrane was irregular due to the presence of cytoplasmic evaginations. Linear and annular gap junctions between neighboring GCs were found. GC nuclei, rounded and eccentrically located, contained finely dispersed chromatin, one (often two) prominent nucleoli and, infrequently, peripheral patches of heterochromatin. Numerous organelles populated the GC cytoplasm, among them, mitochondria were rod-shaped or elongated, usually provided with tubular-vesicular cristae but occasionally showing atypical, longitudinally oriented cristae. Membranes of smooth endoplasmic reticulum, Golgi stacks and vesicles, secretory-like granules, cisternae of rough endoplasmic reticulum (RER), free ribosomes and polysomes, lysosomal-like bodies, microfilaments, and lipid droplets were also seen in the GC cytoplasm. In most cells, RER was scarcely represented and numerous lipid droplets filled the perinuclear space. On the contrary, some GCs contained an abundant RER and rare lipid droplets scattered in the cytoplasm. In conclusion, our data demonstrated the presence, in a coculture system, of GCs provided with ultrastructural characteristics typical of healthy, metabolically active, mostly steroidogenic cells. Protein-synthetic cells have also been detected. These data, evaluated at the light of biochemical and clinical studies, sustain the capability of human GCs cocultures to positively affect early embryo development in vitro by the secretion of steroids and proteins, putative "embryotrophic" factors.

Abstract: The mammalian zona pellucida (ZP) is an extracellular matrix surrounding oocytes and early embryos, which is critical for normal fertilization and preimplantation development. It is made up of three/four glycoproteins arranged in a delicate filamentous matrix. Scanning electron microscopy (SEM) studies have shown that ZP has a porous, net-like structure and/or nearly smooth and compact aspect. In this study, the fine 3-D structure of the human and mouse ZP is reviewed with the aim to integrate ultrastructural and molecular data, considering that the mouse is still used as a good model for human fertilization. By conventional SEM observations, numerous evidences support that the spongy ZP appearance well correlates with mature oocytes. When observed through more sophisticated techniques at high resolution SEM, ZP showed a delicate meshwork of thin interconnected filaments, in a regular alternating pattern of wide and tight meshes. In mature oocytes, the wide meshes correspond to "pores" of the "spongy" ZP, whereas the tight meshes correspond to the compact parts of the ZP surrounding the pores. In conclusion, the traditional "spongy" or "compact" appearance of the ZP at conventional SEM appears to be only the consequence of a prevalence of different arrangements of microfilament networks, according to the maturation stage of the oocyte, and in agreement with the modern supramolecular model of the ZP at the basis of egg-sperm recognition. Despite great differences in molecular characterization of ZP glycoproteins between human and mouse ZP, there are no differences in the 3-D organization of glycoproteic microfilaments in these species.

Abstract: BACKGROUND: Primary varicose veins are commonly considered a progressive disease starting from the saphenous junctions and extending to tributaries in a retrograde fashion along the saphenous trunks. This theory has been criticized by studies indicating different patterns of development and progression of varicose veins. To contribute to the understanding of the pathogenesis of the disease, the anatomy of the venous bed was comparatively evaluated by duplex sonography in patients with varicose veins with a marked difference in age. METHODS: The study included 100 varicose limbs in 82 patients aged < 30 years and 238 limbs in 183 patients aged > 60 years. Veins were designated as saphenous veins (SVs), tributaries of the SVs (STVs), and veins not connected with the SVs (NSVs). Four main anatomic patterns were comparatively evaluated: (1) varicose changes only along SVs, (2) varicose changes along SVs and STVs, (3) varicose changes only in STVs, and (4) varicose changes only in NSVs. RESULTS: SVs were normal in 44% of varicose limbs. In most limbs from young subjects, varicose changes afflicted only SVTs (25%) and NSVs (36%). Varicose SVs were more frequent in the older group (62%) than in younger one (39%) owing to a higher prevalence of limbs with combined SV and STV varicosities (respectively, 59% and 37%). In the older group, varicosities in the STVs were more frequently observed in association with incompetence of the SV trunks. CONCLUSION: The frequent occurrence of normal SVs in varicose limbs of all patients does not support the crucial role commonly credited to SVs in the pathogenesis of primary varicosities. Moreover, the SV trunks were normal in most varicose limbs from young patients. These findings suggest that varicose disease may progressively extend in an antegrade fashion, spreading from the STVs to the SVs. This hypothesis suggests that the saphenous trunks could be spared in the treatment of a relevant number of varicose legs. Prospective longitudinal studies with serial duplex evaluations of large series of extremities are necessary to confirm this hypothesis.

Abstract: In order to understand the 3-D distribution of collagen in relation to vascularization, chorionic villi of human placentae, belonging to normal pregnancies at term, were studied by scanning electron microscopy (SEM) after alkali maceration techniques, and by transmission electron microscopy (TEM). The villous tree appeared made of an uninterrupted structure of collagen fibres. The collagen fibres connected the chorionic villi axis with their basal plates and organised differently according to the various levels of villous branching. The collagen of stem villi showed copious fibres. The external fibres (facing the villous surface) were arranged mainly longitudinally. The central core of the villi (inner fibres) were arranged concentrically around the wall of the fetal vessels. Both external and internal fibres formed stratified lamellae or small parallel bundles. The inner core of stem villi showed small holes housing capillary spaces. Mature intermediate and terminal villi showed a scarce amount of collagen arranged in thin concentric layer within the villous core, surrounding numerous dilated capillary and sinusoid spaces.These observations demonstrated that the extracellular matrix of human chorionic villi is highly compartmentalised and shows a variable structural 3-D distribution depending on the branching level of the villous tree, such a distribution ensures the most favourable microenvironment for feto-maternal exchanges and it is likely able to provide a modulated support to the developing chorionic fetal vessels and trophoblastic layer as well.

Abstract: Menopause signs a hallmark in women's life. When compared to traditional two-dimensional techniques, scanning electron microscopy (SEM) allows to discover the impressive microcosm of the human body and offers original views of the real three-dimensional ultrastructure of reproduction. On the basis of Prof. Motta's last and original book, herein we show a selection of microscopic images that characterize the 3D changes taking place on the surface epithelia of the aging woman's reproductive tract. Biopsies of ovaries, tubes, uteruses, cervices and vaginas were obtained from women (45-72 years old) during surgical or endoscopical procedures and after the informed consent of the patients. Samples were processed for SEM as reported elsewhere. Reproductive aging is associated to epithelial flatness, higher stromal density, absence of ovarian follicles, thickening of vascular walls and lack of glandular secretion. The uterus and the tube show a morphodynamic sequence of gradual microvilli shortening, patchy to complete deciliation and formation of microplicae like structures. The formation of microplicae like structures is interpreted as an adaptive mechanical response of the genital epithelia to the new microenvironment. We can propose that these structures may represent the initial stage of a physiologic metaplasia. In conclusion, imaging of menopause through the use of SEM allows a better understanding of the morphological and physiopathological bases of female aging and represents a perfect blend of art and aesthetics: a further confirmation of the endless beauty of our inner microscopic anatomy.

Abstract: A viscous elastic matrix secreted by the cumulus oophorus cells represent the "extracellular matrix" surrounding the human mature oocyte obtained from assisted reproductive technology (ART). The cumulus matrix is involved in several reproductive processes, including the pick-up of the oocyte-cumulus complex by the oviduct, the final maturation of the ovum and sperm-egg interaction. As showed by some Authors, the cumulus matrix is rich in hyaluronan, as well as in other proteins including inter-alpha-trypsin inhibitor, a dermatan sulfate proteoglycan, and a pentraxin-3. Proteins and hyaluronan are linked together to form a meshwork comprised of granules and filaments. We found in human cumulus oocyte complexes the presence of specialized cells still capable of producing fibronectin and tenascin-c in the post-ovulatory period. Moreover, fibronectin and tenascin-c are present within the hyaluronan matrix at fertilization during the tubal sojourn of the ovum and the embryo. Since pentraxin3 is important in matrix stabilization and gamete interactions, a possible role for fibronectin and tenascin-c may be postulated in the final maturation of the ovum, in the tubal pick-up and in the complex dialogue with the tubal epithelium.

Abstract: OBJECTIVE: To evaluate the microanatomical dynamics of the ovary during postreproductive life. DESIGN: Retrospective, observational research study.SETTING: Institutional (university). PATIENT(S): Eleven (43- to 72-year-old) women in perimenopause, menopause, or postmenopause. INTERVENTION(S): Biopsies of ovaries obtained from patients undergoing gynecologic surgery or diagnostic procedures. MAIN OUTCOME MEASURE(S): Analysis of the 3-dimensional microanatomy of the ovary by transmission and high-resolution scanning electron microscopy. RESULT(S): The surface epithelium gets gradually flatter and is always present, even in advanced age. The surface appears smoother because of a smaller number of papillae and crypts as well as a decreased number and shortening of microvilli on surface epithelial cells. Signs of atrophy and fibrosis are evident. Primordial follicles are usually absent in postmenopause, whereas corpora atretica, hemorrhagica, and albicantia, scar tissue, and simple follicular cysts are common after menopause. Apoptotic and necrotic cells appear frequently within the surface epithelium. Major common features are a marked reduction in number and caliber of blood vessels with thickening of the vascular walls and changes in endothelial cells. CONCLUSION(S): Scanning electron microscopy studies are a useful complement to ordinary gynecologic diagnostic methods. Variations among patients of the same age range or functional status should be considered.

Abstract: The spatial organisation of the extracellular fibrillar matrix of normal human placental villi at term can be directly visualised by scanning electron microscopy after 2N-NaOH maceration technique. By these methods, the extracellular fibrillar matrix of placental villi appears as a continuous network of isolated collagen fibrils and/or small fibrillar bundles interwoven each other. This sort of "collagenous fibrillar skeleton" forms the axis of chorionic villi and connects them with the basal plates running through the whole villous system of the placenta. Significant variations in the spatial arrangement as well as in the quantity of the extracellular matrix is observed at different levels of the villous ramification. Within the stem villi, the fibrillar extracellular matrix are abundant and, whereas the fibrils near the villous surface run parallel to the longitudinal axis of the villous (outer fibrils), those located in the inner core of the villous are arranged circularly around the wall of the fetal vessels (inner fibrils). In mature intermediate and terminal villi, viceversa, the extracellular fibrillar matrix is significandy reduced and the fibrils are mainly organised in a thin circular layer around the capillaries and sinusoids. The present study demonstrated the existence of a diverse spatial architecture of the extracellular matrix that results to be peculiar to the various levels of the ramification of the villous tree. Therefore, these morphological data strongly suggest a "compartmentalisation" of the villous tree as suggested by previous immunohistochemical study. Such a highly organised "collagenous fibrillar skeleton" stresses the important mechanical role of the extracellular matrix in sustaining the chorionic fetal vessels and the trophoblastic layer. Furthermore, the fine reticular-meshed network observed within the terminal villi suggests that at this level an additional role ensuring a favourable milieu for active feto-maternal exchanges may exist.

Abstract: Our study reviews and ultrastructurally characterises human pre-Sertoli cells between the 6th and the 20th week of gestation by means of integrated light microscopy, transmission electron microscopy and high resolution scanning electron microscopy (standard or following ODO maceration). The morphofunctional differentiation of Sertoli cells defines testicular differentiation. These somatic cells are mostly of mesonephric origin and can be first morphologically recognised in 7 week-old embryos altogether with the formation of testicular cords. The latter organise as primordial germ cells surrounded by pre-Sertoli cells. Due to the great synthetic activity of pre-Sertoli cells the rough endoplasmic reticulum develops. The basal lamina of the cords becomes distinguishable at 7 to 8 weeks of gestation. Both prespermatogonia and pre-Sertoli cells actively proliferate but the latter greatly outnumber prespermatogonia. Many interdigitations and cytoplasmic processes are observed between neighbouring pre-Sertoli cells. Due to cell proliferation a sort of compartmentalisation is established inside the cords in which pre-Sertoli cells tend to localise closer to the basal membrane embracing prespermatogonia with long and thin cytoplasmic processes. One of the main typical features of differentiating pre-Sertoli cells is the irregular nucleus and the prominent nucleolus. When the embryo is 14 to 20 weeks-old pre-Sertoli cells maintain their general morphology whereas the most significant change is the maximum development of Leydig cells. Testicular cords do not show any lumen at all so they cannot be termed "tubules".

Abstract: The aim of this study has been to observe, by electron microscopy, the morphological changes affecting mitochondria and associated organelles in the human female germ cell during oogenesis, maturation and fertilization. In the primordial germ cell (PGC), rounded mitochondria with a pale matrix and small vesicular cristae are disposed near the nucleus and significantly increase in number during PGC migration and settlement in the gonadal ridge, where they differentiate into oogonia. In these early stages of mammalian oogenesis, aggregates of mitochondria are typically clustered around or in close relationship with the nuage. In oocytes at early prophase stage, mitochondria proliferate while aligned along the outer surface of the nuclear membrane, contain a more dense matrix than before, and have lamellar cristae. Oocytes of primordial and primary follicles mostly contain round or irregular mitochondria whose matrix has become very light. These mitochondria show typical parallel, arched cristae, and are clustered near the nucleus with other organelles forming the Balbiani's vitelline body. When follicles grow, the mitochondria of the oocytes become even more numerous and are dispersed in the ooplasm. Both paranuclear accumulation and subsequent dispersion of mitochondria in the cytoplasm are likely to be regulated by microtubules. By ovulation, mitochondria are the most prominent organelles in the ooplasm. They form voluminous aggregates with smooth endoplasmic reticulum (SER) tubules and vesicles. These mitochondrial-SER aggregates (M-SER) and the mitochondrial-vesicle complexes (MV) could be involved in the production of a reservoir of substances or membranes anticipating subsequent fertilization and early embryogenesis. Just after fertilization, the mitochondria of the oocyte undergo a further substantial change in size, shape, and microtopography. In the pronuclear zygote, mitochondria concentrate around the pronuclei. During the first embryonic cleavage divisions, round or oval mitochondria with a dense matrix and few arched cristae are gradually replaced by elongated ones with a less dense matrix and numerous transverse cristae. A progressive reduction in size and number of M-SER aggregates and MV complexes also occurs. In summary, oocyte mitochondria show dynamic morphological changes as they increase in number and populate different cell domains within the oocyte. They form complex relationships with other cell organelles, according to the different energetic -metabolic needs of the cell during differentiation, maturation, and fertilization, and are ultimately inherited by the developing embryo, where they eventually assume a more typical somatic cell form.

Abstract: The three-dimensional organisation of the extracellular fibrillar matrix in normal human placental villi at term is investigated by scanning electron microscopic after 2N-NaOH-maceration technique. The latter method consists of a chemical digestion of cellular components of the villi that allows the visualisation of the extracellular fibrillar network in a real three-dimensional fashion by scanning electron microscope. By this approach, the extracellular fibrillar matrix, mainly composed by collagen fibrils, forms a continuous skeleton within the whole villous system of the placenta. This sort of collagenous fibrillar skeleton forms the axis of chorionic villi and connects them with the basal plates. Significant differences in quantity and arrangement of the extracellular matrix are observed at the various levels of the villous ramification. In fact, in the stem villi, the fibrillar extracellular matrix is considerable and the fibres are arranged longitudinally at the villous surface (outer fibrils) and circularly in the inner core of the villous around the wall of the fetal vessels (inner fibrils). On the contrary, in mature intermediate and terminal vili, the extracellular fibrillar matrix is significantly reduced and the fibrils are mainly arranged in a thin circular layer around the capillaries and sinusoids. The present study confirms morphologically the existence of a diverse organization of the extracellular matrix specific for the various levels of the villous ramification as suggested by previous immunohistochemical studies. Moreover, the presence of a highly organized collagenous fibrillar skeleton as observed in our study, stress the important mechanical role of the extracellular matrix in sustaining the chorionic fetal vessels and the trophoblastic layer. Furthermore, the fine reticular-meshed network of this skeleton observed in the terminal villi suggests an additional role of the extracellular matrix to ensure a favorable milieu for active feto-maternal exchanges at this level of the villous tree.

Abstract: When the embryo is 6-week-old the gonad is composed mostly of migrating primordial germ cells, surface coelomic mesothelium and mesenchymal cells. At 7 weeks of gestation (wg) testicular cords consist of prespermatogonia, larger, with a more regular outline and higher mitotic activity than primordial germ cells, embraced by somatic pre-Sertoli cells. The morphofunctional development of Sertoli cells defines testicular differentiation. Towards 7.5 wg the gonad is finally in its differentiated stage; the basal lamina of the cords becomes distinguishable, testicular cords radially branch and elongate, and Leydig cells are clearly recognized. Primordial germ cells are large and spherical, with rounded and eosinophilic nuclei and large nucleoli. Pre-Sertoli cells, in turn, show round or columnar nuclei and rough endopasmic reticulum. Prespermatogonia and mostly pre-Sertoli cells actively proliferate. Many interdigitations and cytoplasmic processes are observed between neighboring pre-Sertoli cells and between pre-Sertoli cells and prespermatogonia. A sort of com partmentalization is established inside the cords in which pre-Sertoli cells tend to localize closer to the basal membrane embracing the prespermatogonia with long and thin cytoplasmic processes. Between 14 and 16 wg the most significant changes besides maximum development of Leydig cells are differentiation of mesenchymal cells around the cords into future peritubular cells and maturational changes of pre-Sertoli cells. These likely reveal a peak coinciding in time with reported increases in either testosterone production and Müllerian Inhibiting Substance secretion. During the period herein considered testicular cords show no lumen neither any sign of canalization so they cannot be termed "tubules".

Abstract: The ultrastructural organization of mouse submandibular gland from 15 days gestation to 180 days of age was studied by field emission scanning electron microscopy. At 15 days of gestation several groups of acini, intercalated and striated ducts were present. They consisted of numerous pyramidal and/or polyhedral cells with spherical or elongated nuclei and short microvilli. At 3 days after birth different shapes of acinar structures covered by a network of fine collagen bundles were observed. The acinar portions corresponded to the terminal tubules and showed the acinar cells containing immature secretory granules. Fractured specimens of ducts revealed a flat surface with large central nuclei. At 14 days after birth acinar terminal portions possessed a round shape; branches of either intercalated and striated ducts were also observed. Gap junctions and interdigitations were numerous at the base of acinar cells. At 30 days after birth the acinar terminal portions and striated ducts revealed a large number of secretory granules. Acinar cells showed a pyramidal shape and basal nuclei. Freeze-cracked surfaces of the striated ductal cells evidenced also a pyramidal shape. Secretory granules ranged from 0.3 to 1.2 microns in size and were clearly observed by infoldings of the basement membranes. At 90 days after birth the ultrastructural features were more differentiated when compared with the previous ages. The freeze-cracked specimens showed very numerous secretory granules in all acinar and striated ductal cells. The granules occupied almost all the apical region of the cells. The outer surface of the basement membrane of acinar and myoepithelial cells was constituted by a spongy-like material covered by fine collagen fibrils. At 180 days after birth numerous secretory granules were seen either in acinar and ductal cells. A morphofunctional polarization of the cell was finally clearly observed in that the cytoplasmic organelles were concentrated in the basal portion whereas the secretory granules were located in the apical region of the cells.

Abstract: Testes from adult cats were studied by means of parallel transmission and scanning electron microscopy (SEM) after NaOH digestion technique, which selectively removed connective tissues or cells. The testis is covered by a dense fibroconnective tunica albuginea that partially divides the organ in lobules by sending septa into the parenchyma. The lamina propria of the seminiferous tubules consisted of one or two rows of cells. The interstitium was made up of randomly arranged collagen bundles. The most significant feature was the numerous Leydig cells rich in lipid droplets and displaying epithelioid features. Following alkali digestion and SEM these cells showed a cord-like arrangement. The cords were formed by one or two closely apposed cells, in between which some labyrinthine or canalicular-like spaces were left that in some areas opened in wide perivascular spaces. This particular arrangement of Leydig cells and the labyrinthine intercellular spaces is very likely designed to improve cell secretion of hormones, facilitating their transport into the blood, as well as the traffic of fluids and metabolites. The present techniques allowed the visualization of a real three-dimensional testicular microarchitecture and microtopography, not detectable with other methods. Such a study may help to better highlight the testicular morphophysiology.

Abstract: The organization of the collagen fibrils in the human umbilical cord at term is directly visualized by means of a scanning electron microscopy cell maceration method. This technique clearly reveals that there is a much more extensive collagen fibrillar architecture within the umbilical cord than that reported in the classical histological descriptions. The Wharton's jelly, in fact, appears as a spongy network of interlacing collagen fibres and small woven bundles apparently arranged at random and forming a continuous soft skeleton that encases the umbilical vessels. The collagen fibrillar network shows the presence of a wide system of interconnected cavities consisting of canalicular-like structures as well as cavernous and perivascular spaces. This system of cavities might play a mechanical role allowing the storing of the ground substance of the jelly and its diffusion during twisting or compression. Furthermore, it may have an important role facilitating the diffusion throughout the jelly of diffused materials (i.e. water and trophic metabolites) either from or to the umbilical vessels and the amniotic cavity, thus overcoming the lace of a proper vasculature with the jelly.

Abstract: Testes from adult cats were studied by means of scanning electron microscopy (SEM) after 2N-NaOH maceration method which selectively digests cells and tissue compounds. As seen by this technique, the testis appears covered by a dense fibroconnective tunica albuginea that divides partially the organ by sending septa into the parenchyma. The interstitium is made up by a rich connective tissue composed by randomly-arranged fine collagen bundles that clearly outline the spaces occupied by the seminiferous tubules. The basement membrane of the seminiferous tubules consists in two rough layers of collagen fibers that in some places leave small fenestrations. The three-dimensional arrangement of collagen fibers in the interstitium outlines a system of cavities likely to be lacunar spaces for Leydig cells and vascular imprints. Especially, the above chemical technique followed by deep observations under the SEM allowed a clear and complete view of the real three-dimensional microarchitecture of the connective tissue of the testis. Therefore, the collagen component was revealed to actually form a unique and complex skeleton for the whole organ. This three-dimensional figure closely follows indeed the classical histological compartmentalization of the testis with a better insight of its spatial microtopographical features. Moreover, this structural pattern is also likely to give rise to very fine morphofunctional subcompartmentalization, especially regarding the basement membrane of the seminiferous tubules and the lacunar spaces for Leydig cells including satellite vessels.

Abstract: This paper describes the spatial organization of the collagen fibrillar skeleton in pregnant rabbit endometrium at term, employing an alkali/low temperature/maceration technique followed by scanning electron microscopic (SEM) observations. Parallel light microscopic (LM) and transmission electron microscopic (TEM) investigations were made to identify the location and possible changes in the endometrial collagen network. Two different types of NaOH maceration were applied, demonstrating separately: 1) the collagen structures (2N-NaOH maceration removes the cells and basal laminae); and 2) the cellular elements (6N-NaOH maceration removes the collagen fibrils). After 2N-NaOH maceration, the collagen network of the endometrium is seen in a superficial compartment around the glands and a deep compartment situated near the endometrial-myometrial junction. Significant changes are observed only in the superficial compartment. The luminal mucosal surface is characterized by numerous thin projections reducing the uterine cavity which, as a consequence, further appears very irregular and highly convoluted. The subepithelial collagen network is composed of densely packed fibrils with a woven course. It contains many tubular or channel-like invaginations (100-150 microns in width and 200-300 microns in length) where endometrial glands are located. These invaginations, corresponding to the glandular impressions, are extremely dilated, enlarged and variable in shape. The collagen fibrils are arranged concentrically around the glandular orifices without forming bundles. At the bottom of the spaces between the mucosal projections, small fenestrations (4-8 microns in diameter each) are present. They form small groups of about 10-20 in number and are due to the endometrial blood capillaries. The deep compartment of the endometrial collagen network is little altered, preserving its general lamellar arrangement. The changes in the endometrial collagen skeleton are due to a variety of complex mechanical and hormonal stimuli affecting the uterus during pregnancy. These may be significant for correct implantation, placentation and delivery.

Abstract: The structure of the palatine salivary glands of rabbits was studied employing scanning electron microscopic methods. The fractured surface of the palatine salivary glands revealed that they were formed of small lobuli separated by interlobular spaces containing numerous bundles of collagen fibers. The NaOH treated specimens showed the organization of the collagen fibrils in their original location. In the interstitial stroma compartment, the collagen fibrils revealed a complex three-dimensional arrangement forming channel-like structures for supporting nerve fibers, small vessels and capillaries. Single groups of acinar cells were clearly demonstrated, and each acinus was enveloped by a characteristic basal lamina showing a sponge-like structure made up of several interconnected meshes of collagen fibrils. Furthermore, taking advantage of the ODO method and field emission scanning electron microscopy, within the cytoplasm of secretory acinar cells numerous organelles were clearly observed in a three-dimensional way such as Golgi complex lamellae, rough endoplasmic reticulum, mitochondria and secretory granules. All these cytoplasmic components revealed their real and microtopographical arrangement in some areas of the acinar cytoplasm clearly suggesting their direct involvement in energetic as well as secretory activities.

Abstract: The ultrastructure of human Leydig cells at different stages of the testicular prenatal development is described by means of transmission and scanning electron microscopy. Between 5 and 7 weeks of gestation (w.g.) the interstitial tissue of the gonad is filled with small undifferentiated mesenchymal cells, migrating primordial germ cells and blood vessels. When the embryo is 7 to 8 weeks-old Leydig cells (LC) appear in basically two morphological patterns, light and dark cells. Their most significative feature is the development of the smooth endoplasmic reticulum (SER) as a dense tubulo-vesicular network and the presence of numerous pleomorphic mitochondria with mainly lamellar cristae. At 14 and 16 w.g. the testicular interstitium reaches the maximum development; the cytoplasm of the LC shows a widespread network of anastomosing tubules of the SER and mitochondria with tubular cristae. Fetal LC show a partial cell coat, lack the crystals of Reinke, have few lipid droplets and show no signs of massive cell degeneration, at least until 16 w.g. These ultrastructural modifications in fetal LC are in accordance with the changes in both steroidogenic activity and hCG levels reported by the literature to occur at this stage of development. Junctional complexes were often observed among LC from 7 to 8 w.g. onwards.

Abstract: In the present study, a cell maceration/scanning electron microscopy method is employed in order to reveal the three-dimensional organization of the collagen fibrils in the Wharton's jelly of the human umbilical cord at term. By this method the jelly appears as a three-dimensional network of collagen fibrils that runs continuously from the amniotic membrane to the umbilical vessels. A diverse architecture of the collagen fibrillar network in the inner and outer region of the cord is observed suggesting an anatomical as well as a functional compartmentalization. In fact, the network is soft in the inner part and it is characterized by canalicular structures whereas in the outer region the collagen is dense and the network is characteristically composed by a sponge-like system of cavernous spaces. It is suggested that these cavities might store the ground substance and allow its diffusion during twisting or compression of the cord. Furthermore they may facilitate the flow of metabolites throughout the jelly from and to the umbilical vessels and the amniotic cavity.