Germ Cell Maturation Research Articles

Protein expression of the transcription factors DMRT1, TCLF5, and OCT4 in selected germ cell neoplasms of the testis

In the present study, we investigated protein expression of the transcription factors mammalian doublesex and mab-3 related transcription factor 1 (DMRT1), basic helix-loop-helix transcription factor-like 5 (TCLF5), and octamer-binding transcription factor 4 (OCT4) in normal human spermatogenesis, testicular mixed germ cell-sex cord stromal tumor (MGC-SCST), spermatocytic tumor, and seminoma. In normal human spermatogenesis, DMRT1 is expressed in the nuclei of spermatogonia but not in those of more mature germ cells. By way of contrast, TCLF5 is expressed in the nuclei of some clusters of primary spermatocytes that have entered meiosis 1, in secondary spermatocytes, and in round (early) spermatids in the seminiferous tubules of adults during the reproductive years. OCT4 is expressed in primordial germ cells but not in the seminiferous tubules of the normal adult testis during the reproductive years. DMRT1 is expressed in the germ cells of both testicular MGC-SCST and spermatocytic tumor, whereas TCLF5 is not expressed in either neoplasm. These low-grade neoplasms, however, differ histologically in that all the germ cell nuclei of testicular MGC-SCST resemble spermatogonia, whereas in spermatocytic tumor, the nuclei of the medium-sized and large cells resemble those of primary spermatocytes. Both neoplasms lack expression of OCT4. By way of contrast, in seminoma, a fully malignant testicular germ cell tumor, the germ cell nuclei express OCT4 but do not express either DMRT1 or TCLF5.

Small RNAs Are Trafficked from the Epididymis to Developing Mammalian Sperm

The biogenesis of the RNA payload of mature spermis of great interest, because RNAs delivered to the zygote at fertilization can affect early development. Here, we tested the hypothesis that small RNAs are trafficked to mammalian sperm during the process of post-testicular maturation in the epididymis. By characterizing small RNA dynamics during germ cell maturation in mice, we confirm and extend prior observations that sperm undergo a dramatic switch in the RNA payload from piRNAs to tRNA fragments (tRFs) upon exiting the testis and entering the epididymis. Small RNA delivery to sperm could be recapitulated invitro by incubating testicular spermatozoa with caput epididymosomes. Finally, tissue-specific metabolic labeling of RNAs in intact mice definitively shows that mature sperm carry RNAs that were originally synthesized in the epididymal epithelium. These data demonstrate that soma-germline RNA transfer occurs in male mammals, most likely via vesicular transport from the epididymis to maturing sperm.

Aging and chromatoid body assembly: Are these two physiological events linked?

The chromatoid body is a cytoplasmic male germ cell structure that plays a role in the regulation of mRNA transcription during spermatogenesis. A proteomic analysis of this structure has identified the presence of its classic molecular markers (MVH and MIWI), as well as a significant number of transient proteins. Circadian locomotor output cycles protein kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), which are molecular components of the circadian clock, are likely located in the chromatoid body in a transient fashion. This study sought to determine whether aging produces morphological changes in the chromatoid bodies of round spermatids similar to those previously observed in BMAL1 knockout mice. A sample of 30 male mice was divided into three groups: juvenile mice (45 days old), adult mice (120 days old), and old mice (+180 days old). Aging was confirmed by viability and sperm count analyses and testosterone dosage. Squash slides prepared with fragments of seminiferous tubules were immunostained for MVH, MIWI, BMAL1, and CLOCK detection. In juvenile and adult specimens, single round chromatoid bodies were observed using MVH/BMAL1 and MIWI/CLOCK immunostaining. In old specimens, many chromatoid bodies displayed changes in number and morphology, as well as an increase in the interactions between MVH and BMAL1; MIWI and CLOCK. Changes in chromatoid body morphology increased interactions between the proteins analyzed herein, and decreased amounts of these proteins in seminiferous tubules of older mice may indicate that aging influences the assembly and physiology of chromatoid bodies, which may, in turn, affect fertility. Impact statement The results discussed in this paper indicate that aging compromises the structure and physiology of chromatoid bodies (CBs) in post-meiotic male cells. Since CB is a fundamental structure for the differentiation of the mature male germ cell it is possible that this imbalance in CB physiology may play a role in the reduction of fertility in older men. It is important to note that not only the classic CB markers (such as the MIWI and MVH proteins) were used to showcase the structural changes in the CBs but also the main components of circadian cycle control (the CLOCK and BMAL1 proteins), indicating that the reduction of circadian control in aged males may contribute to these changes in CBs as well. Therefore, it is intriguing to evaluate the hypothesis that controlling these physiological/structural changes in CBs may be a way of delaying the effects of aging in males.

Mare and Stallion Effects on Blastocyst Production in a Commercial OPU-ICSI Program

Development of precise and reproducible culture system for in vitro differentiation of embryonic stem (ES) cells into germ cells counts as a major leap forward for understanding not only the remarkable process of gametogenesis, otherwise obscured by limited availability of precursor primordial germ cells (PGCs), but in finally treating the catastrophic infertility. Taking into account the significant role of retinoic acid (RA) during in vivo gametogenesis, we designed the present study to investigate the effects of its stimulation on directing the differentiation of ES cells into germ cells. The effects of RA were analyzed across dose-and-time upon various stages of gametogenesis like PGC induction, meiosis initiation and completion, haploid cell formation and development of the final gamete (oocyte and spermatozoa). Out of the series of RA doses (2, 4, 8, 16, 20 and 30 μM), 16 μM RA for 8 day culture interval was found to induce highest expression of PGC- and meiosis-associated genes like DAZL, VASA, SYCP3, MLH1, TNP1/2 and PRM2, while mature germ cell genes like BOULE and TEKT1 (Spermatocyte markers), GDF9 and ZP2 (Oocyte markers) showed higher expression at 2 μM RA dose, suggesting functional concentration-gradient of RA activity. Immunocytochemistry revealed expression of germ lineage-specific markers like: c-KIT, DAZL and VASA (PGC-markers); SYCP3, MLH1 and PROTAMINE1 (Meiotic-markers); ACROSIN and HAPRIN (Spermatocyte-markers); and GDF9 and ZP4 (Oocyte-markers) in optimally differentiated embryoid bodies (EBs) and adherent cultures. We observed significantly reduced (p < 0.05) concentration of 5-methyl-2-deoxycytidine in RA-differentiated EBs which is suggestive of the occurrence of methylation erasure. FACS analysis of optimally differentiated cultures detected 3.07% haploid cell population, indicating completion of meiosis. Oocyte-like structures (OLS) were obtained in adherent differentiated cultures. They had a big nucleus and a zona pellucida (ZP4) coat. They showed progression through 2-cell, 4-cell, 8-cell, morula and blastocyst-like structures upon extended culture beyond 14 days.

Selecting Infants With Cryptorchidism and High Risk of Infertility for Optional Adjuvant Hormonal Therapy and Cryopreservation of Germ Cells: Experience From a Pilot Study.

Orchiopexy for congenital cryptorchid testes is recommended between ½ and 1 year of age to preserve testicular germ cell maturation. Early operation is not enough to preserve fertility in 22 and 36% of cases. Aim of this study was to set up a protocol for optional adjuvant hormonal therapy after orchiopexy and thereafter cryopreservation of testicular biopsies from infants with bilateral cryptorchidism and high infertility risk. We included 17 boys with bilateral cryptorchidism, normal FSH, and impaired germ cell number per tubular transverse section (G/T) in testicular biopsies at orchiopexy, 7 months to 3½ years old. Postoperatively, optional adjuvant LHRH (kryptocur®) 0.2 mg/0.1 mL 2× every second day in 16 weeks were offered. Ten boys were applicable for age matching according to parent's choice of treatment regime and G/T. Five of them had kryptocur®, and five were controls. Repeat bilateral testicular biopsy evaluation and cryopreservation were offered to all boys 12 months after primary orchiopexy. For cryopreservation, tissue pieces were incubated with a cryoprotectant with a slow program freezing. Two out of five kryptorcur®-treated boys normalized both the average G/T and the number of adult dark spermatogonia (Ad-S). Another kryptocur®-treated boy with initial low G/T and no Ad-S increased the G/T and achieved normal number of Ad-S at time of cryopreservation. In the control group, two patients reached only normal lower range regarding the G/T and the number of Ad-S. None of boys with less than average 0.2 G/T improved significantly, whether they were kryptocur®-treated or not. Based on literature and the present results, we recommend adjuvant LHRH treatment to boys with cryptorchidism and insufficient genuine gonadotropin stimulation at time of surgery, as these patients have high infertility risk. Cryopreservation should be an option in case of treatment failure of adjuvant LHRH. However, to avoid repeat surgery with biopsy, some parents may choose biopsy for cryopreservation at time of the initial bilateral orchiopexy, well informed that the procedure may only be truly indicated in 22 and 36% of the cases.

The Expression of Markers for Intratubular Germ Cell Neoplasia in Normal Infantile Testes

Positive immunohistochemical expression of testicular cancer markers is often reported beyond 12 months of age in cryptorchid testes, which is assumed to indicate delayed maturation of the fetal germ cells, or neoplastic changes. These findings allowed for questions as to the extent of positive reaction in normal testes. The aim of the study was to clarify the expression of these markers in a normal material up to 2 years. Testicular material from 69 boys aged 1-690 days, who died of causes with no association of testicular pathology. Histology sections were incubated with primary antibodies including anti-placental-like alkaline phosphatase (PLAP), anti-C-Kit, anti-D2-40, and anti-Oct3/4. The mean germ cell number per tubular transverse section (G/T) was calculated based on the G/T of both testes of every boy. The mean G/T declined through the 690 days. PLAP appeared stably expressed throughout the ages studied. The likelihood of a positive reaction for C-Kit waned with increasing age within the study period. Positive staining for D2-40 and Oct3/4 was demonstrated up to 6 and 9 months respectively. Up to 1 or 2 years of age, normal infantile testes contain germ cells positive for the immunohistochemical markers commonly utilized to aid in the detection of testicular cancer. This finding supports the concept of germ cells undergoing a continuous maturational process in a heterogeneous fashion, and that this process is not complete by 2 years of age.

Preservation of future fertility in pediatric patients with cancer

The cure rate for childhood and adolescent patients with cancer has currently reached almost 80% and protecting future fertility and thereby promoting quality of life have become a major challenge in the care of these patients (Bioethics Law, 2004). Age, sex and associated treatments influence the risk of future subfertility. Certain chemotherapies (particularly alkylating agents) and radiotherapy fields that include the gonads or hypothalamopituitary axis may negatively impact the future fertility of patients. Evaluation of the gonadotoxic potential of therapeutic measures and the utilization of appropriate methods to preserve fertility require the combined efforts of a multidisciplinary team that includes pediatric oncologists, radiotherapists, surgeons, reproductive physicians and biologists and psychologists. Techniques for fertility preservation vary depending on the age of the child and range from surgical transposition of the gonads for pelvic radiotherapy to cryopreservation of the ovary or testicle in case of sterilizing chemotherapy. While scientists still do not yet fully understand the maturation of immature germ cells, these children will be seeking the assistance of Medically Assisted Procreation (MAP) in 20-30 years. In the meanwhile, it is to be hoped that many more advances will be achieved in the utilization of harvested germinal tissue.

Ancestral TCDD Exposure Induces Multigenerational Histologic and Transcriptomic Alterations in Gonads of Male Zebrafish.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the classic aryl hydrocarbon receptor (AhR) agonist, is a potent environmental toxicant and endocrine-disrupting chemical (EDC) with known developmental toxicity in humans, rodents, and fish. Early life exposure to some EDCs, including TCDD, is linked to the occurrence of adult-onset and multigenerational disease. Previous work exposing juvenile F0 zebrafish (Danio rerio) to 50ppt (parts per trillion) TCDD during reproductive development has shown male-mediated transgenerational decreases in fertility (F0-F2) and histologic and transcriptomic alterations in F0 testes. Here, we analyzed male germline alterations in F1 and F2 adult fish, looking for changes in testicular histology and gene expression inherited through the male lineage that could account for decreased reproductive capacity. Testes of TCDD-lineage F1 fish displayed an increase in spermatogonia (immature germ cells) and decrease in spermatozoa (mature germ cells). No histological changes were present in F2 fish. Transcriptomic analysis of exposed F1 and F2 testes revealed alterations in lipid and glucose metabolism, oxidation, xenobiotic response, and sperm cell development and maintenance genes, all of which are implicated in fertility outcomes. Overall, we found that differential expression of reproductive genes and reduced capacity of sperm cells to mature could account for the reproductive defects previously seen in TCDD-exposed male zebrafish and their descendants, providing insight into the distinct multigenerational effects of toxicant exposure.

Exposure to tris(1,3-dichloro-2-propyl) phosphate for Two generations decreases fecundity of zebrafish at environmentally relevant concentrations

Previous studies reported that exposure to environmentally relevant concentrations of TDCIPP significantly decreased the number of cumulative eggs in zebrafish, but effects on the quantity of eggs and sperms remained unknown. Therefore, in this study, effects of TDCIPP on yolk diameter, surface morphology of eggs, sperm density and total motility were evaluated. First generation (F0) zebrafish larvae (Danio rerio) were exposed to 0, 50, 500 or 5000 ng/L tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) from 14 days post fertilization (dpf) to 120 dpf. The F0 generation of zebrafish were paired and F1 generation of embryos were collected and continuously exposed to the same concentrations of TDCIPP until 150 dpf. TDCIPP bioconcentration in the whole body as well as effects on survival and fecundity were evaluated in F1 generation. Exposure to TDCIPP resulted in an accumulation of the chemical and decreased survival of F1 generation of zebrafish. TDCIPP decreased cumulative production and changed surface morphology of eggs in females. In males, TDCIPP decreased total motility of sperm but did not affect sperm density. These effects on quality of egg and sperm might be responsible for the decreased hatching rates observed in cross mating experiments. Furthermore, TDCIPP exposure resulted in down-regulated gene expression related to gonadal development and maturation of germ cells in females or/and males, and the down-regulation was correlated to decreased fecundity. Taken together, the results suggested that exposure to TDCIPP could decrease the quantity of eggs and sperms by down-regulating the expression of genes related to gonadal development and maturation of germ cells in zebrafish.

NEW EXPERIMENTAL PATHOGENETICALLY GROUNDED VARICOCELE MODE

Aim . The main aim of the study was to develop a pathogenetically grounded varicocele model and to compare it with the well-known one. Materials and methods. 30 male mongrel laboratory rats were divided in 3 equal groups, in the 1st group Saypol technique was performed, in the 2nd – one-time partial ligation of both spermatic cords with following testicles abdominisation. The 3rd group was the control one. There was a gonadectomy with following histological investigation on the 7th, 15th and 30th days. Results. Group 1 was presented with adhesions in abdominal cavity and hydronephrosis in all rats. The changes in the spermatic veins and testicles were reached both in the 1st and 2nd groups with more signifi ones in the 2nd group by the 15th day. There were phlebectasia, tunica media thinning, infl infi of the testicles with spermatogenous epithelium and mature germ cells necrobiosis, in some cases also arterial vessels proliferation and even spermatogenesis recovery. Conclusion. Thereby, the use of the suggested technique is more grounded from the technical, economical and pathogenetic points of view.

Effect of hCG and Ovaprim™ on reproductive characteristics of male Levantine scraper, Capoeta damascina (Valenciennes, 1842)

Species richness and abundance within the genus Capoeta has been depleted. As such, there is great need for developing assisted reproductive technologies for controlling reproduction in captivity. Here, we conducted in vivo studies with single administrations of human chorionic gonadotropin (hCG) and Ovaprim™ [(D-Arg6, Pro9NEt)-sGnRH + domperidone] in wild-caught Levantine scraper, Capoeta damascina and then evaluated milt characteristics, fertilization success, serum sex steroids, and spermatogenesis via histological testicular development. Spermiation responses were significantly stronger for Ovaprim injected fish than those injected with hCG or saline. hCG had a negative effect on milt quality by reducing the percentage of motile sperm and fertilization success at 12–48 h post injection (hpi), which was not observed after treatment with Ovaprim or the saline injection. Hormonal therapy resulted in higher sperm densities and spermatocrit, although sperm longevity was not impacted. Sex steroids were not impacted by hCG or saline injection, but Ovaprim effectively induced androgen and progestin release, as evident by higher serum levels of testosterone, and 17α,20β-dihydroxy-4-pregnen-3-one. Consequently, their levels peaked at 12 hpi, which coincided with maximal milt production. Histological analysis of the testes and quantification of germ cell types revealed that Ovaprim significantly stimulated spermiogenesis, as a higher number of accumulated spermatozoa were observed at 12 h and 24 hpi. Testes from saline and hCG-injected fish remained unchanged through the experiment, and contained all stages of germ cells, predominantly spermatocytes with few spermatozoa. In conclusion, Ovaprim treatment successfully induced steroidogenesis and maturation of spermatogenic germ cells, leading to spermiation and milt production without having any negative impacts on sperm quality and fertility in wild-caught C. damascina.

Immunohistochemical analysis of RHAMM expression in normal and neoplastic human tissues: a cell cycle protein with distinctive expression in mitotic cells and testicular germ cells.

Expression of Receptor for Hyaluronic Acid Mediated Motility (RHAMM) increases cellular motility and RHAMM overexpression promotes invasive phenotype and metastasis of cancer cells. RHAMM has been suggested as a biomarker for poor prognosis in several tumor types, including lung, breast, colorectal, gastric, pancreatic ductal, and ovarian cancers. RNA studies showed restricted RHAMM expression in normal tissues, but its protein expression data in tissues were limited. In light of its potential as a prognostic marker and a therapeutic target, we performed immunohistochemical analysis to systematically characterize RHAMM expression in normal and neoplastic human tissues. Among 29 normal adult tissues, RHAMM protein showed restricted expression and was observed in the thymus, lymph node/tonsil, small intestine, colon, skin, bone marrow, placenta, and testis. The cellular distribution patterns of RHAMM in these normal tissues were consistent with RHAMM being a G2/M cell cycle protein, and this was further supported in comparison to the expression of cyclin B2, another G2/M protein. However, unlike the subcellular localization of cyclin B2, RHAMM decorated mitotic spindles in both anaphase and metaphase. RHAMM expression in tumor tissues is variable; and higher RHAMM protein expression is associated with histologically higher-grade tumors in general. Distinct from its expression in somatic tissues, RHAMM showed diffuse, strong, stage-specific expression in the spermatocyte stage of germ cells in adult testis. The neoplastic counterpart, spermatocytic tumor, also showed strong RHAMM expression. This unique expression in testis suggests that RHAMM may function during normal testicular germ cell maturation.

Expression profiling of c-kit and its impact after esiRNA silencing during gonadal development in catfish

Receptor, c-Kit is a member of a family of growth factor receptors that have tyrosine kinase activity, and are involved in the transduction of growth regulatory signals across plasma membrane by activation of its ligand, kitl/scf. The present study analyzed mRNA and protein expression profiles of c-kit in the gonads of catfish, Clarias gariepinus, using real time PCR, in situ hybridization and immunohistochemistry. Tissue distribution analysis revealed higher expression mainly in the catfish gonads. Ontogeny studies showed minimal expression during early developmental stages and highest during 50–75 days post hatch, and the dimorphic expression in gonads decreased gradually till adulthood, which might suggest an important role for this gene around later stages of sex differentiation and gonadal development. Expression of c-kit was analyzed at various phases of gonadal cycle in both male and female, which showed minimal expression during the resting phase, and higher expression during the pre-spawning phase in male compared to females. In vitro and in vivo induction using human chorionic gonadotropin elevated the expression of c-kit indicating the regulatory influence of hypothalamo-hypophyseal axis. In vivo transient gene silencing using c-kit-esiRNA in adult catfish during gonadal recrudescence showed a decrease in c-kit expression, which affected the expression levels of germ cell meiotic marker sycp3, as well as several factors and steroidogenic enzyme genes that are involved in germ cell development. Decrease in the levels of 11-ketotestosterone and testosterone in serum were also observed after esiRNA silencing. The findings suggests that c-kit has an important role in the process of germ cell proliferation, development and maturation during gonadal development and recrudescence in catfish.

Age-related and photoperiodic variation of the DAZ gene family in the testis of the Syrian hamster (Mesocricetus auratus).

SummaryThe Deleted in AZoospermia (DAZ) gene family regulates the development, maturation and maintenance of germ cells and spermatogenesis in mammals. The DAZ family consists of two autosomal genes, Boule and Dazl (Daz-like), and the Daz gene on chromosome Y. The aim of this study was to analyze the localization of DAZL and BOULE during testicular ontogeny of the seasonal-breeding Syrian hamster, Mesocricetus auratus. We also evaluated the testicular expression of DAZ family genes under short- or long-photoperiod conditions. In the pre-pubertal and adult testis, DAZL protein was found mainly in spermatogonia. BOULE was found in the spermatogonia from 20 days of age and during the pre-pubertal and adult period it was also detected in spermatocytes and round spermatids. DAZL and BOULE expression in spermatogonia was strictly nuclear only in 20-day-old hamsters. We also detected the novel mRNA and protein expression of BOULE in Leydig cells. In adult hamsters, Dazl expression was increased in regressed testis compared with non-regressed testis and DAZL protein expression was restricted to primary spermatocytes in regressed testis. These results show that DAZL and BOULE are expressed in spermatogonia at early stages in the Syrian hamster, then both proteins translocate to the cytoplasm when meiosis starts. In the adult regressed testis, the absence of DAZL in spermatogonia might be related to the decrease in germ cell number, suggesting that DAZ gene family expression is involved in changes in seminiferous epithelium during photoregression.

Correlation between testicular mast cell count and spermatogenic epithelium in non-obstructive azoospermia.

Although there is emerging evidence that mast cells are involved in infertility, their exact role has not been elucidated clearly. Here we carried out a retrospective case-control study to find out whether there is a correlation between mast cell (MC) count and proliferation (Ki67 index) of the spermatogenic epithelium as well as of the Sertoli cells (vimentin-positive) in non-obstructive azoospermia (NOA). We assessed MCs, Ki67 and vimentin expression in Sertoli cells in testicular biopsies of germ cell aplasia (GCA, n=14) and maturation arrest (MA, n=14) vs. normal spermatogenesis (n=14) cases. There was a significant decrease in the spermatogonial Ki67 index (1.25±0.91, 4.21±1.81 vs. 39.57±3.92) and Johnsen score (2.48±0.65, 4.89±1.05 vs. 9.75±0.30) as well as a significant increase (P<0.001) in MC count (29.00±4.11, 7.57±1.95 vs. 3.00±1.30) in seminiferous tubules of infertile cases with GCA and MA vs. controls. On the other hand, the percentage of vimentin-expressing Sertoli cells was significantly decreased (P<0.001) in biopsies of cases with MA (35.50±15.62) compared to those of cases with GCA and controls (72.64±10.67 and 98.57±1.45 respectively). Additionally, a significant negative correlation was detected between MC count and Ki67 index as well as Johnsen score in the MA group which became more significant in the GCA group. The significant increase in MC count in the GCA group and to a lesser extent in the MA group indicates their possible role in NOA particularly at the spermatogonial proliferation level and this is supported by the significant negative correlation with the Ki67 index.

Differential expression profiles of conserved Snail transcription factors in the mouse testis.

Snail transcription factors are key regulators of cellular transitions during embryonic development and tumorigenesis. The closely related SNAI1 and SNAI2 proteins induce epithelial-mesenchymal transitions (EMTs), acting predominantly as transcriptional repressors, while the functions of SNAI3 are unknown. An initial examination of Snai2-deficient mice provided evidence of deficient spermatogenesis. To address the hypothesis that Snail proteins are important for male fertility, this study provides the first comprehensive cellular expression profiles of all three mammalian Snail genes in the post-natal mouse testis. To evaluate Snail transcript expression profiles, droplet digital (dd) PCR and insitu hybridization were employed. Snai1, 2 and 3 transcripts are readily detected at 7, 14, 28days post-partum (dpp) and 7weeks (adult). Unique cellular expression was demonstrated for each by insitu hybridization and immunohistochemistry using Western blot-validated antibodies. SNAI1 and SNAI2 are in the nucleus of the most mature germ cell types at post-natal ages 10, 15 and 26. SNAI3 is only detected from 15 dpp onwards and is localized in the Sertoli cell cytoplasm. In the adult testis, Snai1 and Snai2 transcripts are detected in spermatogonia and spermatocytes, while Snai3 is in both germ and Sertoli cells. SNAI1 protein is evident in nuclei of spermatogonia, spermatocytes, round spermatids and elongated spermatids (Stages IX-XII). SNAI2 is present in the nuclei of spermatogonia and spermatocytes, with a faint signal detected in round spermatids. SNAI3 was detected only in Sertoli cell cytoplasm, as in juvenile testes. Additionally, colocalization of SNAI1 and SNAI2 with previously identified key binding partners, LSD1 and PRC2 complex components, provides strong evidence that these important functional interactions are conserved during spermatogenesis to control gene activity. These distinct expression profiles suggest that each Snail family member has unique functions during spermatogenesis.

Analysis of CDK2 mutations in Chinese men with non-obstructive azoospermia who underwent testis biopsy.

To examine whether mutations of the CDK2 gene exist in Chinese men with non-obstructive azoospermia (NOA) with different histopathology, we recruited 175 Chinese men with idiopathic NOA who underwent testis biopsy, including hypospermatogenesis, germ cell maturation arrest and Sertoli cell only syndrome. Genomic DNA was extracted from peripheral blood samples. Subsequently, the seven exons of the CDK2 gene were amplified using polymerase chain reaction with specific primers, respectively. The polymerase chain reaction products were sequenced on an automated sequencer. We identified four known single nucleotide polymorphisms: c.324G>A in exon 1; c.363T>C in exon 2; c.*570G>A; and c.*1160G>C in the 3' UTR of the CDK2 gene. Comparison of the genotype and allele frequencies showed no significant differences between NOA cases and controls for the four single nucleotide polymorphisms. Furthermore, no significant differences were found between each pathological group and control group, respectively. The results indicate that mutations in the coding sequence of the CDK2 gene may not be responsible for idiopathic NOA in Chinese men. Future studies in large cohorts of different ethnic populations are warranted to establish whether associations exist between the CDK2 gene and NOA.

Utf1 contributes to intergenerational epigenetic inheritance of pluripotency

Undifferentiated embryonic cell transcription factor 1 (Utf1) is expressed in pluripotent embryonic stem cells (ESCs) and primordial germ cells (PGCs). Utf1 expression is directly controlled by pluripotency factors Oct4 and Sox2, which form a ternary complex with the Utf1 enhancer. The Utf1 protein plays a role in chromatin organization and epigenetic control of bivalent gene expression in ESCs in vitro, where it promotes effective cell differentiation during exit from pluripotency. The function of Utf1 in PGCs in vivo, however, is not known. Here, we report that proper development of Utf1 null embryos almost entirely depends on the presence of functional Utf1 alleles in the parental germline. This indicates that Utf1’s proposed epigenetic role in ESC pluripotency in vitro may be linked to intergenerational epigenetic inheritance in vivo. One component - or at least facilitator - of the relevant epigenetic mark appears to be Utf1 itself, since Utf1-driven tomato reporter and Utf1 are detected in mature germ cells. We also provide initial evidence for a reduced adult testis size in Utf1 null mice. Our findings thus point at unexpected functional links between the core ESC pluripotency factor network and epigenetic inheritance of pluripotency.

Identification, cloning and expression profile of sycp3 during gonadal cycle and after siRNA silencing in catfish

Molecular mechanisms underlying germ cell development, proliferation and maturation in teleosts revealed the importance of transcription factors through functional genomic approaches which is often comparable to mammals. In the present study, a proteomic approach was employed to find novel molecules with reference to germ cell development in teleosts. Based on this, current work utilized two-dimensional (2D) gel electrophoresis combined with matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry in an attempt to identify and compare the proteins which are expressed differentially in the male gonads of catfish during juvenile and adult stages. The 2D gel analysis identified the Sycp3 protein which was found to be up regulated in juvenile testis when compared to adult stages, and then the full length cDNA was cloned using the peptide sequence data from catfish testis. The spatio-temporal expression pattern of sycp3 was explored during the critical time period of gonadal development as well as at different phases of recrudescence in catfish using real-time PCR, followed by localization analysis by in situ hybridization and immunohistochemistry. In addition, in vivo transient knockdown using PEI mediated sycp3-esiRNA in adult catfish during recrudescence showed a decrease in sycp3 expression, which also affected the expression level of various testis-related genes. Present findings suggest that Sycp3 might have a potential role in the development and maintenance of testicular function in catfish.

A novel mutation in HAUS7 results in severe oligozoospermia in two brothers

Severe oligozoospermia (SO) is a common disease resulting in male infertility; however, its pathophysiology remains unclear. Here, we report two brothers with SO. Whole-exome sequencing (WES) identified a hemizygous variant in HAUS7 (c.G386T:p.G129V), an X-linked gene. HAUS7 has been reported to play a role in the meiotic maturation and chromosome alignment of germ cells. The two patients inherited this variant from their mother, and this variant was considered to be a highly pathogenic mutation by in silico analysis. Moreover, in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) was carried out in both the proband's wife and the brother's wife, but they failed to become pregnant after the embryo transfers. Therefore, this novel mutation in HAUS7 gene may be associated with severe oligozoospermia.