Germ Cells In Testis Research Articles

Antibody selection for immunocytochemical characterization of the male reproductive system in Psittaciformes

The success of breeding programs is limited by the sparse knowledge about endocrine regulation and biochemical reactions in the psittacine male tract. The immunocytochemical analysis of parrots' testicular tissues provides an insight into their reproductive system but is often hampered by the lack of reliable antibodies. In the present study, we tested a large panel of antibodies raised against steroid receptors, steroidogenic enzymes, relaxin peptides including their receptors, and proliferation markers on paraffin sections of testicular tissue from eight psittacine genera representing three continents. All investigated species displayed the tested markers in somatic and germ cells of testis and epididymis, even though cell distribution was partly heterogenous and in species-specific patterns. The 17β-hydroxysteroid-dehydrogenase-2, 3β-hydroxysteroid-dehydrogenase, and smooth muscle actin allowed the cross-species differentiation between active and nonactive gonads. The remaining steroidogenic enzymes, steroid receptors, relaxin peptides, and Ki67 proved to be suitable to define reproductive activity depending on the parrot species. Adapting immunocytochemical methods to different psittacines was successful, though various cellular expression patterns do not allow the transfer of results among different parrot species. However, the availability of a reliable repertory of sexual markers is important to examine reproductive biology of psittacine birds.

Effect of crude cottonseed oil containing gossypol on fertility of male and estrous cycle of female Bandicota bengalensis Gray and Hardwicke

Crude cottonseed oil (CCSO) containing 0.01% gossypol was evaluated in the present studies to record its effect on fertility of male and estrous cycle of female lesser bandicoot rat, Bandicota bengalensis Gray and Hardwicke. Laboratory feeding of bait (cracked wheat and powdered sugar in 98:2) containing 5 and 10% CCSO to female B. bengalensis for 13 days in no-choice feeding test, had no significant effect on the duration of the estrous cycle. However, laboratory feeding of bait containing 10% CCSO to male B. bengalensis for 15 and 30 days duration in a no-choice feeding test, significantly (P≤0.05) reduced sperm motility (%) and viability (%) in cauda epididymal fluid of treated rats compared to untreated ones. The weight of seminal vesicles of rats treated for 30 days was found to be significantly less (P≤0.05) than that for rats treated for 15 days. No significant effect of treatment was observed on the breeding success of rats, diameter of seminiferous tubules, cell count and association of various germ cells in testis. Further studies may be conducted by increasing the duration of treatment or by using CCSO containing a higher concentration of gossypol.

Apurinic/apyrimidinic endonuclease immunoreactivity in germ cells of experimental varicocele-induced rat testes

Increased germ cell apoptosis is related to oxidative DNA damage; therefore, we investigated whether there was a significant change in apurinic/apyrimidinic endonuclease (APE) in varicoceles. Experimental varicoceles were created by partial ligation of the left renal vein of adult male Sprague-Dawley rats, which were sacrificed at 1, 3 and 6 weeks after varicocele creation. Testicular tissues were sampled for TUNEL, Western blotting and immunohistochemistry. There was a significant increase in apoptotic germ cells in the ipsilateral testes 6 weeks after varicocele creation. Increased activation of p53, Bax and cleaved caspase-3 in the left testes was also noted. APE increased activation until 3 weeks after varicocele creation, and then decreased at 6 weeks after varicocele surgery. The spermatocytes were immunostained for both 8-hydroxy-2′-deoxyguanosine and APE, but the spermatogonia revealed only APE immunopositivity in the defective tubules. These results suggest that repression of APE is an underlying mechanism of augmented p53-dependent apoptosis in varicocele-induced rat testes and that remaining APE in the spermatogonia plays a decisive role in regaining testicular spermatogenic function after varicocelectomy.

The chemokine CXCL12 and its receptor CXCR4 are implicated in human seminoma metastasis

Seminoma and non-seminoma tumours increasingly occur within the western population. These tumours originate from carcinoma in situ (CIS) cells, which arise from dysfunctional gonocytes. CXCL12 and its receptors, CXCR4 and CXCR7, have been implicated in migration, proliferation and survival of gonocytes and their precursors and progeny, primordial germ cells and spermatogonial stem cells respectively. We previously found evidence that several miRNA molecules predicted to modulate CXCR4 signalling are differentially expressed during the differentiation of gonocytes into spermatogonia in mice. Bioinformatic analysis predicted these miRNA to modulate CXCR4 signalling, leading us to hypothesize that CXCL12-mediated CXCR4 signalling is involved in the disrupted differentiation of gonocytes that underpins CIS formation. Indeed, we detected CXCL12 in Sertoli cells of normal human testis, and relatively high expression in tumour stroma with concomitant weak staining in dispersed tumour cells. In contrast, CXCR4 was expressed in spermatogonial and meiotic germ cells of normal testis and in the majority of tumour cells. Quantitative RT-PCR identified elevated CXCR4 transcript levels in seminoma compared with normal testis and to non-seminoma, potentially reflecting the higher proportion of dysfunctional germ cells within seminomas. In the normal testis, expression of CXCR4 downstream signalling molecules phospho-MEK1/2 and phospho-ERK1/2 correlated with CXCR4/CXCL12 expression. Strikingly, this correlation was absent in seminoma and non-seminoma samples, suggesting that CXCL12 signalling is disrupted. Proliferation rate and cell survival were not altered by CXCL12 in either seminoma (TCam-2) or non-seminoma (833ke) cell lines. However, CXCL12 exposure induced TCam-2 cell invasion though simulated basement membrane, while in contrast, we provide the novel evidence that CXCR4-expressing non-seminoma cell lines 833ke and NTera2/D1 do not invade in response to CXCL12. These findings indicate that CXCL12 expression in the human testis may selectively influence seminoma migration and metastasis, correlating with its importance in gonocyte and spermatogonial stem cell biology.

The RHOX homeobox gene cluster is selectively expressed in human oocytes and male germ cells

What human tissues and cell types express the X-linked reproductive homeobox (RHOX) gene cluster? The RHOX homeobox genes and proteins are selectively expressed in germ cells in both the ovary and testis. The RHOX homeobox transcription factors are encoded by an X-linked gene cluster whose members are selectively expressed in the male and female reproductive tract of mice and rats. The Rhox genes have undergone strong selection pressure to rapidly evolve, making it uncertain whether they maintain their reproductive tissue-centric expression pattern in humans, an issue we address in this report. We examined the expression of all members of the human RHOX gene cluster in 11 fetal and 8 adult tissues. The focus of our analysis was on fetal testes, where we evaluated 16 different samples from 8 to 20 weeks gestation. We also analyzed fixed sections from fetal testes, adult testes and adult ovaries to determine the cell type-specific expression pattern of the proteins encoded by RHOX genes. We used quantitative reverse transcription-polymerase chain reaction analysis to assay human RHOX gene expression. We generated antisera against RHOX proteins and used them for western blotting, immunohistochemical and immunofluorescence analyses of RHOXF1 and RHOXF2/2B protein expression. We found that the RHOXF1 and RHOXF2/2B genes are highly expressed in the testis and exhibit low or undetectable expression in most other organs. Using RHOXF1- and RHOXF2/2B-specific antiserum, we found that both RHOXF1 and RHOXF2/2B are primarily expressed in germ cells in the adult testis. Early stage germ cells (spermatogonia and early spermatocytes) express RHOXF2/2B, while later stage germ cells (pachytene spermatocytes and round spermatids) express RHOXF1. Both RHOXF1 and RHOXF2/2B are expressed in prespermatogonia in human fetal testes. Consistent with this, RHOXF1 and RHOXF2/2B mRNA expression increases in the second trimester during fetal testes development when gonocytes differentiate into prespermatogonia. In the human adult ovary, we found that RHOXF1 and RHOXF2/2B are primarily expressed in oocytes. While the average level of expression of RHOX genes was low or undetectable in all 19 human tissues other than testes, it is still possible that RHOX genes are highly expressed in a small subset of cells in some of these non-testicular tissues. As a case in point, we found that RHOX proteins are highly expressed in oocytes within the human ovary, despite low levels of RHOX mRNA in the whole ovary. The cell type-specific and developmentally regulated expression pattern of the RHOX transcription factors suggests that they perform regulatory functions during human fetal germ cell development, spermatogenesis and oogenesis. Our results also raise the possibility that modulation of RHOX gene levels could correct some cases of human infertility and that their encoded proteins are candidate targets for contraceptive drug design.

The Relation between Adult Dark Spermatogonia and Other Parameters of Fertility Potential in Cryptorchid Testes

The fertility potential of boys with cryptorchidism may be related to the number of adult dark spermatogonia per tubular transverse section in testicular biopsies taken at orchiopexy. Placental-like alkaline phosphatase positive gonocytes in testes within year 1 of life indicate preserved ability for germ cell transformation. We related these parameters to the total number of tubular germ cells and other factors associated with fertility potential. The study comprised 89 boys 0.7 to 3 years old (median age 1.8) who underwent bilateral testicular biopsy at bilateral orchiopexy and provided blood samples for gonadotropins and inhibin B. Of 76 boys with adult dark spermatogonia 44 (58%) had a normal mean number of spermatogonia per tubular transverse section compared to 2 of 13 (15%) without adult dark spermatogonia (p <0.05). In the 30 boys with good fertility potential, including a normal mean number of tubular germ cells, and normal gonadotropins and inhibin B, the mean number of adult dark tubular germ cells was 0.081 vs 0.031 in the 38 with low fertility potential, including impaired tubular germ cells and/or low inhibin B but no reactive increase in gonadotropins (p <0.05). In the 21 patients with increased gonadotropins the mean number of adult dark spermatogonia per tubular transverse section was 0.063. Of the 20 boys with normal mean adult dark spermatogonia per tubular transverse section 12 (60%) had good fertility potential, including a normal mean number of tubular germ cells, normal gonadotropins and normal inhibin B, compared to only 18 of 69 (26%) with an impaired mean number of adult dark spermatogonia per tubular transverse section (p <0.05). Of 46 boys with a normal mean number of tubular germ cells 26 (57%) had placental-like alkaline phosphatase positive cells compared to 14 of 43 (33%) with a decreased mean number of tubular germ cells (p <0.05). The number of placental-like alkaline phosphatase positive gonocytes and adult dark spermatogonia per tubular transverse section are important parameters related to the fertility potential of boys with cryptorchid testes.

Mutations of NANOS1, a human homologue of the Drosophila morphogen, are associated with a lack of germ cells in testes or severe oligo-astheno-teratozoospermia

BackgroundThe Nanos gene is a key translational regulator of specific mRNAs involved in Drosophila germ cell development. Disruption of mammalian homologues, Nanos2 or Nanos3, causes male infertility in mice. In...

Very Small Embryonic-Like Stem Cells: Implications in Reproductive Biology

The most primitive germ cells in adult mammalian testis are the spermatogonial stem cells (SSCs) whereas primordial follicles (PFs) are considered the fundamental functional unit in ovary. However, this central dogma has recently been modified with the identification of a novel population of very small embryonic-like stem cells (VSELs) in the adult mammalian gonads. These stem cells are more primitive to SSCs and are also implicated during postnatal ovarian neo-oogenesis and primordial follicle assembly. VSELs are pluripotent in nature and characterized by nuclear Oct-4A, cell surface SSEA-4, and other pluripotent markers like Nanog, Sox2, and TERT. VSELs are considered to be the descendants of epiblast stem cells and possibly the primordial germ cells that persist into adulthood and undergo asymmetric cell division to replenish the gonadal germ cells throughout life. Elucidation of their role during infertility, endometrial repair, superovulation, and pathogenesis of various reproductive diseases like PCOS, endometriosis, cancer, and so on needs to be addressed. Hence, a detailed review of current understanding of VSEL biology is pertinent, which will hopefully open up new avenues for research to better understand various reproductive processes and cancers. It will also be relevant for future regenerative medicine, translational research, and clinical applications in human reproduction.

Induction of spermatogenic synchrony by retinoic acid in neonatal mice

Retinoic acid (RA) is required for the successful differentiation and meiotic entry of germ cells in the murine testis. The availability of RA to undifferentiated germ cells begins in a variable, uneven pattern during the first few days after birth and establishes the asynchronous pattern of germ cell differentiation in adulthood. It has been shown that synchronous spermatogenesis can be induced in 2 d postpartum mice, but not in adult mice, by treating vitamin A sufficient males with RA. In this study, neonatal males were treated at different ages with a single dose of RA and spermatogenesis was examined after recovery to adulthood. The failure of exogenous RA to alter asynchrony correlates with the appearance of meiotic preleptotene spermatocytes within the seminiferous epithelium.

Green fluorescence protein driven by the Na,K-ATPase α4 isoform promoter is expressed only in male germ cells of mouse testis

Expression of the Na,K-ATPase α4 isoform is required for sperm motility and fertility and is controlled by the Atp1a4 promoter. Here, we have investigated the specific tissue, cell type and developmental regulation of expression mediated by the Atp1a4 promoter. We have inserted the green fluorescent protein (GFP), downstream of the endogenous Atp1a4 promoter, in place of the Na,K-ATPase α4 gene, and used it as a marker for α4 expression in mice (Atp1a4 ( null(GFP) ) mice). Replacement of α4 by GFP completely disrupted α4 expression and activity, produced sperm morphological and functional abnormalities, and caused infertility of Atp1a4 ( null(GFP) ) male mice. Immunoblot analysis of Atp1a4 ( null(GFP) ) mouse tissues showed GFP expression in testis. This particular expression pattern was found in adult, but not in mouse embryos or in 7, 18day old mice. In agreement with expression of GFP, adult Atp1a4 ( null(GFP) ) mouse testis displayed the typical fluorescence of GFP. Immunocytochemistry of testis identified GFP in more differentiated male germ cells, but not in spermatogonia, Leydig or Sertoli cells. Further analysis, using immunoblot of fluorescently sorted testis cells with cell specific markers, detected GFP only in spermatocytes, spermatids and spermatozoa. While epididymis showed GFP expression, this was confined to the spermatozoa within the epididymal tubules. Our results show that the Atp1a4 promoter drives GFP expression exclusively in male germ cells of the testis, where it restricts it to post-meiotic stages of spermatogenesis. These findings highlight the exquisite spatial and temporal control of expression exerted by the Atp1a4 promoter on Na,K-ATPase α4, which is particularly well suited to fulfill the special functions of spermatozoa.

Germ line specific expression of a vasa homologue gene in turbot (Scophthalmus maximus): Evidence for vasa localization at cleavage furrows in euteleostei

Specification of primordial germ cells during early embryogenesis is a critical biological issue in reproduction and development. Yet, little is known in marine economic fish species. Vasa, a component of germ plasm, is the most-documented germ cell marker in teleosts. We isolated a full-length vasa cDNA (Smvas) from turbot (Scophthalmus maximus), a marine Euteleostei species, and investigated its expression patterns by RT-PCR and in situ hybridization during embryogenesis and gametogenesis to identify the germ cell lineage in this species. The deduced amino acid sequence of the isolated cDNA shared typical characteristics of Vasa protein and high identity to Vasa homologues in medaka (76.9%) and zebrafish (68.5%). The Smvas transcripts were exclusively detected in germ cells of testis and ovary, and exhibited an interesting dynamic localization pattern during oogenesis. The distribution pattern of Smvas during embyogenesis in this Euteleostei closely resembled the pattern observed in zebrafish (belonging to Osteriophysans) rather than medaka (belonging to Euteleostei). Thus, it is concluded that Smvas isolated in this study is a germ cell specific molecular marker in turbot. Furthermore, we hypothesize that Euteleostei could localize vasa mRNA by a special mode. The results not only facilitate the germ cell manipulation of the turbot, but also improve our understanding of germline development and evolution of vasa localization in teleost.

Intercellular adhesion molecule-2 is involved in apical ectoplasmic specialization dynamics during spermatogenesis in the rat

In this study, we investigated the role of intercellular adhesion molecule-2 (ICAM2) in the testis. ICAM2 is a cell adhesion protein having important roles in cell migration, especially during inflammation when leukocytes cross the endothelium. Herein, we showed ICAM2 to be expressed by germ and Sertoli cells in the rat testis. When a monospecific antibody was used for immunolocalization experiments, ICAM2 was found to surround the heads of elongating/elongated spermatids in all stages of the seminiferous epithelial cycle. To determine whether ICAM2 is a constituent of apical ectoplasmic specialization (ES), co-immunoprecipitation and dual immunofluorescence staining were performed. Interestingly, ICAM2 was found to associate with β1-integrin, nectin-3, afadin, Src, proline-rich tyrosine kinase 2, annexin II, and actin. Following CdCl2 treatment, ICAM2 was found to be upregulated during restructuring of the seminiferous epithelium, with round spermatids becoming increasingly immunoreactive for ICAM2 by 6–16 h. Interestingly, there was a loss in the binding of ICAM2 to actin during CdCl2-induced germ cell loss, suggesting that a loss of ICAM2–actin interactions might have facilitated junction restructuring. Taken collectively, these results illustrate that ICAM2 plays an important role in apical ES dynamics during spermatogenesis.

Inactivation of the Celf1 Gene that Encodes an RNA-Binding Protein Delays the First Wave of Spermatogenesis in Mice

BackgroundThe first wave of spermatogenesis in mammals is characterized by a sequential and synchronous appearance of germ cells in the prepubertal testis. Post-transcriptional controls of gene expression play important roles in this process but the molecular actors that underlie them are poorly known.Methodology/principal findingsWe evaluated the requirement for the RNA-binding protein CELF1 during the first wave of spermatogenesis in mice. Mice inactivated for Celf1 gene were not viable on pure genetic backgrounds. On a mixed background, we observed by histology and gene profiling by RT-qPCR that the testes of inactivated prepubertal mice were characterized by several features. (i) Spermiogenesis (differentiation of post-meiotic cells) was blocked in a subset of prepubertal inactivated mice. (ii) The appearance of the different stages of germ cell development was delayed by several days. (iii) The expression of markers of Leydig cells functions was similarly delayed.Conclusions/significance Celf1 disruption is responsible for a blockage of spermiogenesis both in adults and in prepubertal males. Hence, the spermiogenesis defects found in Celf1-inactivated adults appear from the first wave of spermiogenesis. The disruption of Celf1 gene is also responsible for a fully penetrant delayed first wave of spermatogenesis, and a delay of steroidogenesis may be the cause for the delay of germ cells differentiation.

Rodent Thyroid, Liver, and Fetal Testis Toxicity of the Monoester Metabolite of Bis-(2-ethylhexyl) Tetrabromophthalate (TBPH), a Novel Brominated Flame Retardant Present in Indoor Dust

Background: Bis-(2-ethylhexyl) tetrabromophthalate (TBPH) is widely used as a replacement for polybrominated diphenyl ethers (PBDEs) in commercial flame retardant mixtures such as Firemaster 550. It is also used in a commercial mixture called DP 45. Mono-(2-ethyhexyl) tetrabromophthalate (TBMEHP) is a potentially toxic metabolite.Objectives: We used in vitro and rodent in vivo models to evaluate human exposure and the potential metabolism and toxicity of TBPH.Methods: Dust collected from homes, offices, and cars was measured for TBPH by gas chromatography followed by mass spectrometry. Pregnant rats were gavaged with TBMEHP (200 or 500 mg/kg) or corn oil on gestational days 18 and 19, and dams and fetuses were evaluated histologically for toxicity. We also assessed TBMEHP for deiodinase inhibition using rat liver microsomes and for peroxisome proliferator-activated receptor (PPAR) α and γ activation using murine FAO cells and NIH 3T3 L1 cells.Results: TBPH concentrations in dust from office buildings (median, 410 ng/g) were higher than in main living areas in homes (median, 150 ng/g). TBPH was metabolized by purified porcine esterases to TBMEHP. Two days of TBMEHP exposure in the rat produced maternal hypothyroidism with markedly decreased serum T3 (3,3´,5-triiodo-l-thyronine), maternal hepatotoxicity, and increased multinucleated germ cells (MNGs) in fetal testes without antiandrogenic effects. In vitro, TBMEHP inhibited deiodinase activity, induced adipocyte differentiation in NIH 3T3 L1 cells, and activated PPARα- and PPARγ-mediated gene transcription in NIH 3T3 L1 cells and FAO cells, respectively.Conclusions: TBPH a) is present in dust from indoor environments (implying human exposure) and b) can be metabolized by porcine esterases to TBMEHP, which c) elicited maternal thyrotoxic and hepatotoxic effects and d) induced MNGs in the fetal testes in a rat model. In mouse NIH 3T3 L1 preadipocyte cells, TBMEHP inhibited rat hepatic microsome deiodinase activity and was an agonist for PPARs in murine FAO and NIH 3T3 L1 cells.

ブタ精巣におけるリラキシン関連因子（RLF）の発現・分泌動態と生殖細胞に対するパラクリン因子としての機能

ブタ精巣におけるリラキシン関連因子（RLF）の発現・分泌動態と生殖細胞に対するパラクリン因子としての機能

The high‐mobility group A1‐estrogen receptor β nuclear interaction is impaired in human testicular seminomas

It is well established that estrogens participate in the control of normal spermatogenesis and endogenous or environmental estrogens are involved in pathological germ cell proliferation including testicular germ cell tumors. The effects of estrogen are now known to be mediated by estrogen receptor-α (ERα) and ERβ subtypes, but only ERβ has been found in human germ cells of normal testis. However, its expression was markedly diminished in human testicular seminomas. The expression and the possible interaction of ERβ and HMGA1 were studied in normal germ cells and in human testicular seminomas. GC1 and TCam-2 germ cell lines, were used; in addition, a tissue micro-array (TMA) was built using the most representative areas from 35 cases of human testicular seminomas. The expression and the interaction of ERβ and HMGA1 were observed by using immunoprecipitation and Western blot analyses in combination with immunocytochemistry and immunofluorescence analyses. Here, we show that ERβ interacts with HMGA1 in normal germ cells, while down regulation of ERβ associates with transcriptional co-regulator HMGA1 over-expression and cytoplasmic localization both in human testicular seminomas and in TCam-2 cell line. In addition, we show that 17β-oestradiol induces an HMGA1 increased cytoplasmic expression associated to an ERβ down-regulation in TCam-2 cell line. Taken together, our results suggest that exposure to estrogens or estrogen-mimics, in some as of yet undefined manner, diminishes the ERβ-mediated growth restraint in human testicular seminoma, probably due to the HMGA1 cytoplasmic delocalization associated with ERβ down-regulation.

Bilateral Undescended Testes Classified According to Preoperative and Postoperative Status of Gonadotropins and Inhibin B in Relation to Testicular Histopathology at Bilateral Orchiopexy in Infant Boys

In recent series of boys with cryptorchidism gonadotropin levels have been higher and serum inhibin B levels have been lower than normal. To some extent the serum values of inhibin B reflect the state of germinative epithelium in cryptorchid testes. We evaluated whether blood samples of gonadotropins and inhibin B as well as histopathology could be used to classify undescended testes. A total of 69 boys (median age 2 years) who underwent surgery for bilateral cryptorchidism had blood samples taken preoperatively and 3 months to 2.1 years postoperatively. Testicular biopsies were performed bilaterally at orchiopexy. The average germ cell number per tubular transverse tubule was measured. Group 1 included 17 patients with increased follicle-stimulating hormone levels. Serum follicle-stimulating hormone and luteinizing hormone decreased significantly after surgery. In 77% of patients (13 of 17) follicle-stimulating hormone levels were normalized. Of these boys 35% (6 of 17) had a low postoperative serum inhibin B. Group 2 consisted of 27 patients with a decreased germ cell number and/or low preoperative inhibin B, but not increased serum follicle-stimulating hormone or luteinizing hormone. There were no significant postoperative changes in follicle-stimulating hormone and luteinizing hormone. Of these boys 22% (6 of 27) had a low serum inhibin B postoperatively. In group 3 there were 25 patients with a normal germ cell number, normal preoperative serum inhibin B and normal gonadotropins. There were no significant changes in luteinizing hormone and follicle-stimulating hormone postoperatively. Only 1 boy in this group had a low postoperative serum inhibin B. Patients with increased gonadotropin levels may have testicular dysgenesis and some may benefit from early surgery. Patients with normal gonadotropin levels and a decreased germ cell number have transient hypothalamus-pituitary-gonadal hypofunction and a poor fertility prognosis. These patients may benefit from gonadotropin treatment after orchiopexy. Patients with normal gonadotropins, inhibin B and germ cell number have a good fertility prognosis after surgery.

Genetic variants in the ETV5 gene in fertile and infertile men with nonobstructive azoospermia associated with Sertoli cell–only syndrome

To assess the association between genetic variants in the ETV5 gene with nonobstructive azoospermia (NOA) associated with Sertoli cell-only (SCO) syndrome. Genetic association study. University. Australian men (65 SCO, 53 NOA, and 242 fertile men) and American men (86 SCO and 54 fertile men). Paraffin-embedded human testicular tissue was sectioned and processed for immunofluorescence. Direct DNA sequencing and polymerase chain reaction-based SNP detection were performed to define genetic variants in the ETV5 gene. The localization of ETV5 in the human testis and the presence of ETV5 genetic variants in fertile and infertile men. ETV5 is localized to the cytoplasm and nucleus of Sertoli and germ cells in adult human testes. We identified six previously reported and six new genetic variants in the ETV5 gene. Of these, the allele frequency of the homozygous +48845 G>T (TT allele) variant was significantly higher in the SCO and NOA Australian men compared with fertile men. The homozygous +48845 G>T (TT allele) variant confers a higher risk for male infertility associated with NOA and SCO in Australian men.

Demethylation of the Coding Region Triggers the Activation of the Human Testis-Specific PDHA2 Gene in Somatic Tissues

Human PDHA2 is a testis-specific gene that codes for the E1α subunit of Pyruvate Dehydrogenase Complex (PDC), a crucial enzyme system in cell energy metabolism. Since activation of the PDHA2 gene in somatic cells could be a new therapeutic approach for PDC deficiency, we aimed to identify the regulatory mechanisms underlying the human PDHA2 gene expression. Functional deletion studies revealed that the −122 to −6 promoter region is indispensable for basal expression of this TATA-less promoter, and suggested a role of an epigenetic program in the control of PDHA2 gene expression. Indeed, treatment of SH-SY5Y cells with the hypomethylating agent 5-Aza-2′-deoxycytidine (DAC) promoted the reactivation of the PDHA2 gene, by inducing the recruitment of the RNA polymerase II to the proximal promoter region and the consequent increase in PDHA2 mRNA levels. Bisulfite sequencing analysis revealed that DAC treatment induced a significant demethylation of the CpG island II (nucleotides +197 to +460) in PDHA2 coding region, while the promoter region remained highly methylated. Taken together with our previous results that show an in vivo correlation between PDHA2 expression and the demethylation of the CpG island II in testis germ cells, the present results show that internal methylation of the PDHA2 gene plays a part in its repression in somatic cells. In conclusion, our data support the novel finding that methylation of the PDHA2 coding region can inhibit gene transcription. This represents a key mechanism for absence of PDHA2 expression in somatic cells and a target for PDC therapy.

Inactivation of Dicer1 has a severe cumulative impact on the formation of mature germ cells in mouse testes

Dicer1, an RNase III endonuclease, is indispensable for the maturation of miRNA and siRNA, which control gene expression through the RNAi pathway. The diverse functions of miRNA involving multiple developmental processes have been elucidated, but the role of Dicer1 in spermatogenesis is just beginning to be revealed. Mice lacking Dicer1 were reported to be embryonic lethal at E7.5. In the present study, mice with a Dicer1 conditional allele were crossed with Vasa-cre transgenic mice to delete Dicer1 as early as the prospermatogonia stage (at E15). At P40, seminiferous tubules of Dicer1 deficient mice showed several aberrant phenotypes. A large number of apoptotic germ cells were detected by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, but several events in meiosis of spermatocytes appeared unaffected. The mutant mice were found to be sterile, likely due to the extensive decrease in number and morphological abnormalities of mature sperm in the epididymis, which, together with the numerous haploid cells in the testis, indicated a severely affected transition from round to functional elongated spermatozoa. Additionally, we found milder phenotypes when Dicer1 was inactivated in later stages of spermatogenesis in Stra8-cre and Pgk2-cre transgenic mice. In conclusion, our findings suggest that the loss of Dicer1 has a continuous and cumulative effect on the process of spermatogenesis and blocks the germ cells in the stage of round spermatids to a large extent, ultimately leading to the generation of abnormal sperm.