Abstract: Uncoordinated-5 homologs 1-4 (UNC5H1-4) transmembrane netrin receptors are reported to control a number of cellular processes, including axonal guidance, angiogenesis and cell proliferation. These receptors are known as "dependence receptors" because they are able to induce apoptosis in the absence of their ligand, netrin. We have recently reported the localization of netrin-1 and its uncoordinated-5-B (UNC5B) receptor in both villous and extravillous cytotrophoblasts in the human placenta. However, the roles that netrin-1 and UNC5B play in the development of the placenta, as well as the regulation of their expression during the early stages of placental development, remain unexplored. Placental explants were used to demonstrate a proliferative effect of netrin-1 on cytotrophoblasts, as assessed by Ki67 staining. Primary cytotrophoblasts collected at different gestational ages during the first trimester of pregnancy indicated that netrin-1 mRNA expression decreased after 6 weeks of gestation (wg), whereas UNC5B expression increased gradually up to 13-14 wg. The BeWo cell line was used to evaluate the effect of hypoxia on the expression of netrin-1 and UNC5B. Primary cytotrophoblast and BeWo cells cultured under hypoxic conditions exhibited a decrease in the expression of UNC5B both at the mRNA and protein levels; in contrast, hypoxia induced no change in the levels of netrin-1. When hypoxia-inducible factor 1α (HIF-1α) was knocked down by siRNA, we found a significant increase in UNC5B expression, indicating that the HIF-1 pathway is involved in hypoxia-induced UNC5B transcriptional down-regulation. Altogether, these results demonstrate the role of netrin-1 as a new mitogenic factor for cytotrophoblastic cells, report the pattern of expression of netrin-1 and its receptor, UNC5B, in the human placenta during the first trimester of pregnancy, and bring insights into the direct control of the expression of UNC5B by HIF-1.
Abstract: Netrins are a family of proteins that mediate axonal guidance in the central nervous system (CNS). In addition to the CNS, netrins are involved in cell adhesion, motility, proliferation, differentiation, and survival. Because these processes occur in the placenta, we raised the question of whether netrin-1 is expressed by placental cells during development. In the present study, we analyzed the spatial and temporal distribution of netrin-1 and its two receptors, DCC (deleted in colorectal cancer) and UNC5B (uncoordinated-5 homolog) in human placenta using RT-PCR, Western blotting, and immunohistochemistry analysis. We demonstrated the presence of the proteins and transcripts of netrin-1 and its receptors in placenta and cytotrophoblasts. Furthermore, using immunohistochemistry, we localized endogenous netrin-1 protein staining to villous and extravillous cytotrophoblasts, and secreted netrin-1 outside the syncytiotrophoblasts. The DCC receptor was localized to syncytiotrophoblasts and invasive extravillous cytotrophoblasts during the first trimester and at term. On the other hand, the UNC5B receptor was localized to villous and extravillous cytotrophoblasts proximal to anchoring areas during the first trimester. At term, UNC5B was observed in decidual cells and weakly in extravillous cells. The discrete pattern of netrin-1 and netrin-1 receptor distribution suggested that netrin-1 protein functions might vary with its localization in the placenta and probably with time of gestation.
Abstract: OBJECTIVE: To evaluate the influence of aging on the achondroplasia mutation rate in the male germline. DESIGN: Studies in sperm and testis biopsy DNA according to donor's age. SETTING: University teaching hospital. PATIENT(S): Seventeen donors aged 30 to 65 years for sperm collection and 14 deceased donors aged 53 to 95 years for testis biopsies, all with normal stature. INTERVENTION(S): Testes were obtained from 14 deceased donors, and sperm was obtained from 17 patients who requested ART. MAIN OUTCOME MEASURE(S): Real-time polymerase chain reaction quantification of the G1138A mutation in sperm and testis biopsies. RESULT(S): The rate of G1138A mutation did not significantly vary with age in sperm, whereas in testis biopsies it increased markedly past the age of 70 years. Moreover, and for the first time, a mosaic for this mutation was detected in the testis of three subjects who were >80 years of age. CONCLUSION(S): These findings could contribute to providing a molecular explanation for the increased incidence of achondroplastic offspring with advanced paternal age.
Abstract: The effect of maternal age on the risk of meiotic abnormality is well documented. In contrast little is known about the effect of the paternal age. The question of the risk related to paternal age is raised because of the increased demand of Assisted Reproduction Techniques for older men. This review focuses on the alterations of male semen parameters, testis histology and genetic risks related to age. The motility, vitality and morphology of spermatozoa and semen volume are found decreasing with age. Histomorphometric studies reveal various alterations including a thickening of the basal membrane when spermatogenesis is arrested. The number of germinal and Sertoli cells decreases with increased age. Up to 95 years old, we could find subjects with complete spermatogenesis. Chromosomal analyses in different studies have provided controversial results. Our investigation on subjects aged from 29 to 102 showed that the rate of aneuploidy in the group of aged subjects with preserved spermatogenesis was not statistically different from the young control group. However the incidence of postmeiotic aneuploidy was increased when spermiogenesis had stopped. On the other hand from epidemiological studies, autosomal dominant diseases are known to be associated with paternal age. However, in the case of achondroplasia and Apert syndrome, direct DNA sperm analysis did not reveal significant increase in the mutation frequency with paternal age.
Abstract: OBJECTIVE: To study the chromosomal content of spermatozoa that could be selected for intracytoplasmic sperm injection (ICSI) in cases of macrocephalic sperm head syndrome. DESIGN: Case report. SETTING: Obstetrics, gynecology, urology, and reproductive biology departments. PATIENT(S): Two infertile patient candidates for ICSI presenting with total teratozoospermia (100%) with mainly large-headed spermatozoa (91% and 82%, respectively). INTERVENTION(S): Fluorescence in situ hybridization with X, Y, 18 centromeric probes on unselected spermatozoa (all migrated spermatozoa) and specifically on selected spermatozoa with normal-sized heads. MAIN OUTCOME MEASURE(S): Percentage of polyploid, diploid, aneuploid, and normal haploid spermatozoa, according to X, Y, 18 chromosome centromeric probes on selected spermatozoa (head size compatible with ICSI). RESULT(S): All the nonselected spermatozoa were abnormal, diploid, or polyploid. The rate of normal ploidy (haploid cells) among the selected sperm population was 1 per 28 for patient 1 and 5 per 51 for patient 2. CONCLUSION(S): This very low proportion of normal haploid spermatozoa among selected spermatozoa contraindicated ICSI for the two patients. We suggest performance of this selection and analysis before including (or not), in an ICSI program, patients with macrocephalic sperm head syndrome, associated or not with preimplantation genetic diagnosis.
Abstract: OBJECTIVE: To determine whether the size of CAG repeat in exon 1 of the androgen receptor (AR) gene is related to impaired spermatogenesis in older men. DESIGN: Study of two groups of older men: one with preserved spermatogenesis and the other with arrested spermatogenesis. SETTING: University teaching hospital. PATIENT(S): Twenty-eight men aged from 53 to 102 years. INTERVENTION(S): The DNA fragment encoding the AR polyglutamine tract was amplified from DNA of testis tissue. MAIN OUTCOME MEASURE(S): The size of the CAG repeat was evaluated by using fluorescent-labeled polymerase chain reaction performed on an ABI Prism 377 DNA sequencer followed by automated analysis with Genscan 3.1.2 software. RESULT(S): Mean CAG repeat length was 22.76 +/- 3 in the group of 13 aged men with preserved spermatogenesis and 21.86 +/- 2.23 in the group of 15 aged men with arrested spermatogenesis. CONCLUSION(S): Impaired spermatogenesis in elderly men does not seem to be correlated with the AR gene CAG repeat length, which therefore does not appear to be a risk factor for impaired spermatogenesis in older men.
Abstract: OBJECTIVE: The increase of frequency of Assisted Reproductive Techniques (ART) for elder men raises the question of the genetic risk for the offspring. Our aim was to evaluate the influence of ageing on the testicular histology, the aneuploidy rate in testis postmeiotic cells and the DNA fragmentation in sperm. PATIENTS AND METHODS: We performed a histomorphometric study of 36 men aged from 61 to 102 years and 10 young men from 29 to 40 years. The aneuploidy rate was evaluated by fluorescent in situ hybridation (FISH X, Y, 18) and DNA fragmentation in spermatozoa was evaluated by TUNEL. RESULTS: Histomorphometry showed various alterations of testicular histology with age including thickening of the basal membrane when spermatogenesis was arrested. The number of germinal cells and Sertoli cells decreased with age with important individual variations. Nevertheless spermatogenesis could be possible until 95 years. The rate of aneuploidy was not influenced by age when spermatogenesis was complete. However, we observed an increased aneuploidy rate in postmeiotic cells when spermiogenesis was arrested. On the other hand apoptosis was not increased with age. DISCUSSION AND CONCLUSION: Our study confirms that spermatogenesis is possible until a very advanced age (95 years) without any specific chromosome risk. The question of mutagenesis remains to be solved.
Abstract: OBJECTIVE: To evaluate the influence of aging on testicular histology and the aneuploidy rate in testis postmeiotic cells. DESIGN: Comparison between older men and younger men. SETTING: Deceased donors and patients who requested assisted reproductive technology (ART). PATIENT(S): Thirty-six older men (61-102 years old) and 10 younger men (29-40 years old). INTERVENTION(S): Testes were obtained from 35 deceased donors, and testicular biopsies were obtained from 11 patients who requested ART. MAIN OUTCOME MEASURE(S): Histomorphometry of testis and fluorescent in situ hybridization (FISH), with a three-probe set X, Y, and 18. RESULT(S): The histomorphometric study showed a thickening of the basal membrane when spermatogenesis was arrested. The number of germinal and Sertoli cells decreased as age increased. The rate of aneuploidy of postmeiotic cells was 1.1% for the control group, 1.29% for older subjects with preserved spermatogenesis, and 14.28% for the subjects with an arrested spermiogenesis. Only this last figure was higher than the control group. CONCLUSION(S): The rate of aneuploidy in older subjects (61-95 years old) with preserved spermatogenesis was not statistically different from that found in the control group; it was increased in older subjects with arrested spermatogenesis.
