Gonocyte Differentiation Research Articles

Molecular characterization, expression patterns and cellular localization of BCAS2 gene in male Hezuo pig.

Breast carcinoma amplified sequence 2 (BCAS2) participates in pre-mRNA splicing and DNA damage response, which is implicated in spermatogenesis and meiosis initiation in mouse. Nevertheless, the physiological roles of BCAS2 in the testes of large mammals especially boars remain largely unknown. In this study, testes were collected from Hezuo pig at three development stages including 30 days old (30 d), 120 days old (120 d), and 240 days old (240 d). BCAS2 CDS region was firstly cloned using RT-PCR method, and its molecular characteristics were identified using relevant bioinformatics software. Additionally, the expression patterns and cellular localization of BCAS2 were analyzed by quantitative real-time PCR (qRT-PCR), Western blot, immunohistochemistry and immunofluorescence. The cloning and sequence analysis indicated that the Hezuo pig BCAS2 CDS fragment encompassed 678 bp open reading frame (ORF) capable of encoding 225 amino acid residues, and possessed high identities with some other mammals. The results of qRT-PCR and Western blot displayed that BCAS2 levels both mRNA and protein were age-dependent increased (p < 0.01). Additionally, immunohistochemistry and immunofluorescence results revealed that BCAS2 protein was mainly observed in nucleus of gonocytes at 30 d testes as well as nucleus of spermatogonia and Sertoli cells at 120 and 240 d testes. Accordingly, we conclude that BCAS2 is critical for testicular development and spermatogenesis of Hezuo pig, perhaps by regulating proliferation or differentiation of gonocytes, pre-mRNA splicing of spermatogonia and functional maintenance of Sertoli cells, but specific mechanism still requires be further investigated.

Cloning, expression analysis and localization of DAZL gene implicated in germ cell development of male Hezuo pig

Deleted in azoospermia-like (DAZL) is essential for mammalian testicular function and spermatogenesis. To explore the molecular characterization, expression patterns, and cellular localization of the DAZL in Hezuo pig testes, testicular tissue was isolated from Hezuo pig at five development stages including 30 days old (30 d), 90 days old (90 d), 120 days old (120 d), 180 days old (180 d), and 240 days old (240 d). DAZL cDNA was first cloned using the RT-PCR method, and its molecular characterization was analyzed using relevant bioinformatics software. Subsequently, the expression patterns and cellular localization of DAZL were evaluated using quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry. The cloning and sequence analysis showed that the Hezuo pig DAZL cDNA fragment contained 888 bp open reading frame (ORF) capable of encoding 295 amino acid residues and exhibited high identities with some other mammals. The qRT-PCR and Western blot results indicated that DAZL was specifically expressed in Hezuo pig testes, and DAZL levels of both mRNA and protein were expressed at all five reproductive stages of Hezuo pig testes, with extremely significant higher expression levels in 90 d, 120 d, 180 d, and 240 d than those in 30 d (p < 0.01). Additionally, immunohistochemistry results revealed that DAZL protein was mainly localized in gonocytes at 30 d testes, primary spermatocytes, and spermatozoon at other developmental stages, and Leydig cells throughout five development stages. Together, these results suggested that DAZL may play an important role by regulating the proliferation or differentiation of gonocytes, development of primary spermatocytes and spermatozoon, and functional maintenance of Leydig cells in testicular development and spermatogenesis of Hezuo pig. Nevertheless, the specific regulatory mechanisms underlying these phenomena still requires further investigated and verified.

The Role of microRNAs in the Gonocyte Theory as Target of Malignancy: Looking for Potential Diagnostic Biomarkers.

Some pediatric patients with cryptorchidism preserve cells with gonocyte characteristics beyond their differentiation period, which could support the theory of the gonocyte as a target for malignancy in the development of testicular neoplasia. One of the key molecules in gonocyte malignancy is represented by microRNAs (miRNAs). The goal of this review is to give an overview of miRNAs, a class of small non-coding RNAs that participate in the regulation of gene expression. We also aim to review the crucial role of several miRNAs that have been further described in the regulation of gonocyte differentiation to spermatogonia, which, when transformed, could give rise to germ cell neoplasia in situ, a precursor lesion to testicular germ cell tumors. Finally, the potential use of miRNAs as diagnostic and prognostic biomarkers in testicular neoplasia is addressed, due to their specificity and sensitivity compared to conventional markers, as well as their applications in therapeutics.

Testicular cancer in mice: interplay between stem cells and endocrine insults.

BackgroundIncidence of type II germ cell tumors (T2GCT) has increased in young men possibly due to fetal/perinatal exposure to estrogenic compounds. Three-fold increased incidence of T2GCT was reported in men exposed in utero to diethylstilbestrol (DES). T2GCT is a development-related disease arising due to blocked differentiation of gonocytes into spermatogonia in fetal testes which survive as germ cell neoplasia in situ (GCNIS) and initiate T2GCT. In our earlier study, T2GCT-like features were observed in 9 out of 10 adult, 100-day-old mice testes upon neonatal exposure to DES (2 μg/pup/day on days 1–5). Neonatal DES exposure affected testicular very small embryonic-like stem cells (VSELs) and spermatogonial stem cells and resulted in infertility, reduced sperm counts and tumor-like changes leading to our postulate that testicular dysgenesis syndrome possibly has a stem cell basis. The present study was undertaken to further characterize testicular tumor in mice testes.MethodsDES-exposed mice pups (n = 70) were studied on D100 and after 12 months to understand how T2GCT progresses. Besides histological studies, a carefully selected panel of markers were studied by immuno-fluorescence and qRT-PCR.ResultsDES resulted in either atrophied or highly vascularized, big-sized testes and extra-testicular growth was also observed. GCNIS-like cells with big, vacuolated cytoplasm and increased expression of OCT-4, SSEA-1, SCA-1 and CD166 (cancer stem cells marker) along with reduced c-KIT, MVH and PTEN were evident. Global hypomethylation was found associated with altered expression of Dnmts, Igf2-H19 and Dlk-Meg3 imprinted genes along with reduced expression of Ezh2, cell cycle regulator p57KIP2 and Meg3; however, Pten remained unaltered. Increased expression of PCNA and Ki67 was observed in concert with complete lack of SOX-9 suggesting Sertoli cells independent proliferation.ConclusionsMouse model for T2GCT is described which will have immense potential to understand cancer initiation, cancer stem cells and also to develop effective therapies in future. T2GCT initiates from tissue-resident, pluripotent VSELs due to their altered epigenome. Neonatal exposure to DES blocks differentiation (spermatogenesis) and VSELs get transformed into CD166 positive cancer stem cells that undergo excessive self-renewal and initiate cancer in adult life challenging existing concept of fetal origin of T2GCT.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-022-02784-5.

A cooperative mechanism of target RNA selection via germ-cell-specific RNA-binding proteins NANOS2 and DND1.

The germ-cell-specific RNA-binding protein (RBP) NANOS2 plays a pivotal role in male gonocyte differentiation and spermatogonial stem cell maintenance. Although NANOS2 interacts with the CNOT deadenylation complex and Dead end 1 (DND1) to repress target RNAs, the molecular mechanisms underlying target mRNA selection remain unclear because of the limited cell resource invivo. Here, we demonstrate that exogenous NANOS2-DND1 suppresses target mRNAs in somatic cells. Using this somatic cell system, we find that NANOS2 interacts with RNA-bound DND1 and recruits the CNOT complex to the mRNAs. However, a fusion construct composed of the CNOT1-binding site of NANOS2 (NIM) and DND1 fails to repress the target gene expression. Therefore, NANOS2 is required not only for recruitment of the CNOT complex but also for selecting the target mRNA with DND1. This study reveals that NANOS2 functions as a second-layer RBP for the target recognition and functional adaptation of DND1.

Role of the Ubiquitin Ligase RNF149 in the Development of Rat Neonatal Gonocytes.

Male reproductive function depends on the formation of spermatogonial stem cells from their neonatal precursors, the gonocytes. Previously, we identified several UPS enzymes dynamically altered during gonocyte differentiation. The present work focuses on understanding the role of the RING finger protein 149 (RNF149), an E3 ligase that we found to be strongly expressed in gonocytes and downregulated in spermatogonia. The quantification of RNF149 mRNA from postnatal day (PND) 2 to 35 (puberty) in rat testis, brain, liver, kidney, and heart indicated that its highest levels are found in the testis. RNF149 knock-down in PND3 rat gonocytes was performed to better understand its role in gonocyte development. While a proliferative cocktail of PDGF-BB and 17β-estradiol (P+E) increased both the expression levels of the cell proliferation marker PCNA and RNF149 in mock cells, the effects of P+E on both genes were reduced in cells treated with RNF149 siRNA, suggesting that RNF149 expression is regulated during gonocyte proliferation and that there might be a functional link between RNF149 and PCNA. To examine RNF149 subcellular localization, EGFP-tagged RNF149 vectors were constructed, after determining the rat testis RNF149 mRNA sequence. Surprisingly, two variant transcripts were expressed in rat tissues, predicting truncated proteins, one containing the PA and the other the RING functional domains. Transfection in mouse F9 embryonal carcinoma cells and C18-4 spermatogonial cell lines showed differential subcellular profiles of the two truncated proteins. Overall, the results of this study support a role for RNF149 in gonocyte proliferation and suggest its transcription to variant mRNAs resulting in two proteins with different functional domains. Future studies will examine the respective roles of these variant proteins in the cell lines and isolated gonocytes.

Differentiation of mouse embryonic stem cells into cells with spermatogonia-like morphology with chemical intervention-dependent increased gene expression of LIM homeobox 1 (Lhx1)

Spermatogonial stem cells (SSCs) originate from gonocytes that differentiate from primordial germ cells (PGCs). In the developing mouse testis, expression of the gene LIM homeobox 1 (Lhx1) marks the most undifferentiated SSCs, which has not yet been reported for spermatogonia-like cells generated in vitro. Previously, it was shown that a chemical intervention in male mouse embryonic stem (ES) cells in serum culture, including Sirtuin 1 (SIRT1) inhibitor Ex-527, DNA methyltransferase (DNMT) inhibitor RG-108 and electrophilic redox cycling compound tert-butylhydroquinone (tBHQ), was associated with molecular markers of PGC to gonocyte differentiation. Here, we report the in vitro differentiation of male mouse ES cells, cultured under dual chemical inhibition of GSK3β and MEK (2i) with leukemia inhibitory factor (LIF) (2iL) and serum, into cells with spermatogonia-like morphology (CSMs) and population-averaged expression of spermatogonia-specific genes by removal of 2iL and a specific schedule of twice daily partial medium replacement. Combination of this new protocol with the previously reported chemical intervention increased population-averaged gene expression of Lhx1 in the resulting CSMs. Furthermore, we detected single CSMs with strong nuclear LHX1/5 protein signal only in the chemical intervention group. We propose that further investigation of CSMs may provide new insights into male germline development.

A0639 - Temperature is not critical for the differentiation of gonocytes into ad spermatogonia and fertility outcome in cryptorchid boys

A0639 - Temperature is not critical for the differentiation of gonocytes into ad spermatogonia and fertility outcome in cryptorchid boys

Etomoxir regulates the differentiation of male germ cells by specifically reducing H3K27ac level

BackgroundFatty acid oxidation plays an important role in a variety of developing and mature organ systems. However, the role of this metabolic pathway in different stages of testis development remains unknown. Here, we elucidate the mechanisms by which fatty acid oxidation regulates the maintenance and differentiation of gonocytes and spermatogonial stem cells.ResultsDuring E13.5-E15.5, male germ cells gradually enter the mitotic arrest phase, while the expression of CPT1A, a rate-limiting enzyme for fatty acid oxidation, gradually increases. Therefore, we treated pregnant mice (E13.5 to E15.5) with etomoxir, which is an inhibitor of CPT1A. Etomoxir-treated mice showed no difference in embryonic morphology; however, etomoxir-treated male gonocytes exited mitotic arrest, and cells of the gonad underwent apoptosis. In addition, etomoxir-treated mice at P7 displayed impaired homing of spermatogonia and increased cell apoptosis. We further demonstrated that inhibition of fatty acid oxidation in gonads was associated with gonocyte differentiation events and the histone modification H3K27ac.ConclusionsInhibiting fatty acid oxidation can specifically reduce the level of H3K27ac in the reproductive crest, which may be the cause of the down-regulation of male differentiation-specific gene expression, which ultimately leads to the male primordial germ cells exited from mitotic arrest. Our work uncovers metabolic reprogramming during male gonadal development, revealing that it plays an important role in the maintenance of gonocytes in a differentiated and quiescent state during foetal testis development.

Disorders in the function of Sertoli cells and arrest in the differentiation of gonocytes in patients with cryptochidism and microlithiasis

PurposeThis study aimed to correlate the presence of microlithiasis (ML) in cryptorchidism (CO) patients with the functionality of Sertoli cells and the arrest of gonocyte differentiation. MethodsTesticular biopsies were obtained from 21 inguinal CO pediatric patients and were classified in two groups as follows: patients with ML and those without ML. In both groups, the number of Sertoli cells/seminiferous cords and their functionality were determined, considering the concentrations of inhibin B. In addition, the area and the histological alterations of seminiferous epithelium were evaluated. The arrest of gonocyte differentiation was determined by immunoreactivity to SALL4, AP2ɣ, PLAP and POU5F1. ResultsWe found a statistical correlation between the presence of ML with the alterations in the functionality of Sertoli cells without reflecting in the differentiation of the gonocytes. ConclusionThe study of this population suggests that the association between CO and ML shows a malfunction of the Sertoli cells without necessarily causing arrest in the differentiation of gonocytes in these patients.

A Cooperative Mechanism of Target RNA Selection via Germ Cell-Specific RNA-Binding Proteins, NANOS2 and DND1

The germ cell-specific RNA-binding protein, NANOS2, plays a pivotal role in male gonocyte differentiation and spermatogonial stem cell maintenance. Although NANOS2 interacts with the CNOT deadenylation complex and Dead end 1 (DND1) to repress target RNAs, the molecular mechanisms underlying target mRNA selection remain unclear because of the limited cell resource in vivo. We succeeded in reproducing the NANOS2-DND1 function in somatic cells. Using this system, we found that NANOS2 recruits the CNOT complex to the mRNAs that are recognized and bound by DND1 through its own RNA-binding domain. In addition, by analyzing gonocytes expressing domain-swapped NANOS2 with the NANOS2-zinc finger (ZnF) domain substituted by the DND1-RNA recognition motifs (RRMs), we found that the NANOS2-ZnF domain is required for target mRNA selection and DND1 interaction. This study unveiled the mechanisms of the target RNA recognition underlying the partnership of two RNA-binding proteins, which leads to male germ cell development.

Evidence that activin A directly modulates early human male germline differentiation status.

Disrupted fetal germline development underpins testicular germ cell neoplasia, which is increasing worldwide. The complex signaling milieu during normal testis development includes TGFβ superfamily ligands; this study tests the hypothesis that, activin A, a TGFβ superfamily member, can influence gonocyte development. The human seminoma-derived cell line, TCam-2, a model of fetal gonocytes, was cultured with activin A (1.25-25 ng/mL) for 48 h, or with 5 ng/mL activin A for short- (6, 24, and 48 h) and long-term (13 days) exposures, and downstream targets measured by qRT-PCR. Transcripts that exhibited significant dose-dependent responses to activin A included the early germ cell markers KIT, NODAL, and CRIPTO (NODALl co-receptor and activin inhibitor) which all increased and the differentiation marker DNMT3L which decreased. After 48 h, KIT, NODAL, and CRIPTO levels were significantly higher, while the differentiation marker NANOS2 was significantly lower. Interestingly, activin A exposure also significantly reduced both transcript and protein levels of the PIWI/piRNA pathway component DNMT3L. Because TCam-2 cells produce the activin inhibitor CRIPTO, CRIPTO was reduced using siRNA prior to activin A exposure. This selectively increased KIT in response to activin A. Other ligands present in the fetal testis (BMP4, FGF9, TGFβ1, and TGFβ2) induced distinct effects on germline marker expression. This study showed that activin A can directly modulate germline markers in this human gonocyte-like cell, promoting a less-differentiated phenotype. Additional findings indicate evidence of signaling crosstalk between activin A and NODAL, leading to target-specific effects on gonocyte differentiation.

Cloning, Molecular Characterization and Expression Patterns of DMRTC2 Implicated in Germ Cell Development of Male Tibetan Sheep.

The double sex and mab-3-related transcription factors like family C2 (DMRTC2) gene is indispensable for mammalian testicular function and spermatogenesis. Despite its importance, what expression and roles of DMRTC2 possesses and how it regulates the testicular development and spermatogenesis in sheep, especially in Tibetan sheep, remains largely unknown. In this study, DMRTC2 cDNA from testes of Tibetan sheep was firstly cloned by the RT-PCR method, and its molecular characterization was identified. Subsequently, the expression and localization patterns of DMRTC2 were evaluated by quantitative real-time PCR (qPCR), Western blot, and immunofluorescence. The cloning and sequence analysis showed that the Tibetan sheep DMRTC2 cDNA fragment contained 1113 bp open reading frame (ORF) capable of encoding 370 amino acids, and displayed high identities with some other mammals, which shared an identical DM domain sequence of 47 amino acids ranged from residues 38 to 84. qPCR and Western blot results showed that DMRTC2 was expressed in testes throughout the development stages while not in epididymides (caput, corpus, and cauda), with higher mRNA and protein abundance in Tibetan sheep testes of one- and three-year-old (post-puberty) compared with that of three-month-old (pre-puberty). Immunofluorescence results revealed that immune staining for DMRTC2 protein was observed in spermatids and spermatogonia from post-puberty Tibetan sheep testes, and gonocytes from pre-puberty Tibetan sheep testes. Together, these results demonstrated, for the first time, in sheep, that DMRTC2, as a highly conserved gene in mammals, is essential for sheep spermatogenesis by regulating the proliferation or differentiation of gonocytes and development of spermatids in ram testes at different stages of maturity.

Protective Role of Peroxiredoxins against Reactive Oxygen Species in Neonatal Rat Testicular Gonocytes

Peroxiredoxins (PRDXs) are antioxidant enzymes that protect cells from oxidative stress and play a role in reactive oxygen species (ROS)-mediated signaling. We reported that PRDXs are critical for human fertility by maintaining sperm viability and regulating ROS levels during capacitation. Moreover, studies on Prdx6−/− mice revealed the essential role of PRDX6 in the viability, motility, and fertility competence of spermatozoa. Although PRDXs are abundant in the testis and spermatozoa, their potential role at different phases of spermatogenesis and in perinatal germ cells is unknown. Here, we examined the expression and role of PRDXs in isolated rat neonatal gonocytes, the precursors of spermatogonia, including spermatogonial stem cells. Gene array, qPCR analyses showed that PRDX1, 2, 3, 5, and 6 transcripts are among the most abundant antioxidant genes in postnatal day (PND) 3 gonocytes, while immunofluorescence confirmed the expression of PRDX1, 2, and 6 proteins. The role of PRDXs in gonocyte viability was examined using PRDX inhibitors, revealing that the 2-Cys PRDXs and PRDX6 peroxidases activities are critical for gonocytes viability in basal condition, likely preventing an excessive accumulation of endogenous ROS in the cells. In contrast to its crucial role in spermatozoa, PRDX6 independent phospholipase A2 (iPLA2) activity was not critical in gonocytes in basal conditions. However, under conditions of H2O2-induced oxidative stress, all these enzymatic activities were critical to maintain gonocyte viability. The inhibition of PRDXs promoted a two-fold increase in lipid peroxidation and prevented gonocyte differentiation. These results suggest that ROS are produced in neonatal gonocytes, where they are maintained by PRDXs at levels that are non-toxic and permissive for cell differentiation. These findings show that PRDXs play a major role in the antioxidant machinery of gonocytes, to maintain cell viability and allow for differentiation.

Cyclooxygenase 2 (COX2) expression and prostaglandin synthesis in neonatal rat testicular germ cells: Effects of acetaminophen and ibuprofen.

In infants, fever is often treated with acetaminophen or ibuprofen, two antipyretic and analgesic drugs inhibiting cyclooxygenases (COXs), enzymes catalyzing prostaglandin production. Infancy represents a critical developmental period when neonatal germ cells/gonocytes differentiate to spermatogonial stem cells required for spermatogenesis. (a) Determine the expression of Cox2 and associated genes in postnatal day (PND)3 rat gonocytes compared to spermatogonia. (b) Examine whether acetaminophen or ibuprofen disrupts neonatal gonocyte functions. (c) Determine whether neonatal gonocytes produce prostaglandins and whether this process is altered by acetaminophen and ibuprofen. The expression of Cox2 and related genes was determined by gene arrays and qPCR. Cox2 protein levels were determined by immunocyto/histochemistry and immunoblots. The effects of acetaminophen and ibuprofen on PND3 gonocyte viability, apoptosis, proliferation, and differentiation were examined alone and with a proliferation cocktail or differentiation factor. Prostaglandins were examined by immunocyto/histochemistry and LC-MS. Cox2 and related genes are highly expressed in gonocytes and spermatogonia. Acetaminophen and ibuprofen did not affect gonocyte survival or apoptosis, but they increased gonocyte proliferation. Ibuprofen significantly reduced RA-induced Stra8 expression, indicating an inhibitory effect on differentiation. Ibuprofen combined with RA decreased Cox2 mRNA and protein expression. PGE2 and PGF2α were produced by neonatal gonocytes and decreased by acetaminophen and ibuprofen. The concomitant decrease of Stra8 expression, Cox2 expression, and PGE2 and PGF2a production in gonocytes co-treated with RA suggests that Cox2 plays a role in PND3 gonocyte differentiation. The effects of acetaminophen and ibuprofen on proliferation suggest a negative relationship between Cox2 and proliferation. Treating neonates with acetaminophen or ibuprofen could disrupt gonocyte development, leading to adverse reproductive effects. Understanding COX2 role in neonatal gonocytes and the potential risk of acetaminophen and ibuprofen treatment of infants may help prevent male reproductive pathologies.

Role of Nodal signalling in testis development and initiation of testicular cancer.

Testicular development from the initially bipotential gonad is a tightly regulated process involving a complex signalling cascade to ensure proper sequential expression of signalling factors and secretion of steroid hormones. Initially, Sertoli cell specification facilitates differentiation of the steroidogenic fetal Leydig cells and establishment of the somatic niche, which is critical in supporting the germ cell population. Impairment of the somatic niche during fetal life may lead to development of male reproductive disorders, including arrest of gonocyte differentiation, which is considered the first step in the testicular cancer pathogenesis. In this review, we will outline the signalling pathways involved in fetal testis development focusing on the Nodal pathway, which has recently been implicated in several aspects of testicular differentiation in both mouse and human studies. Nodal signalling plays important roles in germ cell development, including regulation of pluripotency factor expression, proliferation and survival. Moreover, the Nodal pathway is involved in establishment of the somatic niche, including formation of seminiferous cords, steroidogenesis and Sertoli cell function. In our outline of fetal testis development, important differences between human and mouse models will be highlighted to emphasise that information obtained from mouse studies cannot always be directly translated to humans. Finally, the implications of dysregulated Nodal signalling in development of the testicular cancer precursor, germ cell neoplasia in situ, and testicular dysgenesis will be discussed - none of which arise in rodents, emphasising the importance of human models in the effort to increase our understanding of origin and early development of these disorders.

Sexual precocity in male microminipigs evaluated immunohistologically using spermatogonial stem cell markers

Microminipigs are one of the smallest miniature pigs characterized as sexually precocious; the males achieve sexual maturity at around 3–4.5 months of age. However, the physiology of this sexual precocity is still unclear. To understand sexual precocity in male microminipigs, we analyzed their testes at five developmental stages: neonatal (<7 days), 30-day-old, 45-day-old, 80-day-old, and adult (>24 months) stages. We used 4 pigs in each of the stages. To analyze testicular development histologically, the seminiferous tubule diameter (SD) was measured, and the presence or absence of the seminiferous lumen was confirmed. Changes in the expression of pluripotency markers, DBA, UCHL1, ZBTB16, and vimentin, were evaluated immunohistologically. For the analyses, cells positive for DBA, UCHL1, and ZBTB16 per 150 round seminiferous tubules in cross sections from each testis were counted to evaluate the total number of positive cells. The number of positive cells per 100 Sertoli cells (DBA+/Sertoli, UCHL1+/Sertoli, and ZBTB16+/Sertoli) was calculated to compare the five developmental stages. Histologically, SDs became larger with piglet growth, and precocity was confirmed; seminiferous lumens were observed from the 30-day-old stage. Immunohistologically, the number of DBA+/Sertoli, which indicates the number of gonocytes, decreased rapidly to an undetectable level by the 45-day-old stage. In the same period, the number of UCHL1+/Sertoli, which indicates total SSCs, increased significantly, suggesting that the proliferation of SSCs was accelerated before 30 days of age. Consequently, our study clarified that differentiation of SSCs in microminipigs started during the fetal period, the differentiation of gonocytes and proliferation of SSCs was then accelerated before 30 days of age, and the early phase of spermatogenesis was finally completed at around 45 days after birth. Consequently, sexual precocity in male microminipigs was characterized by a shorter duration of the early phase of spermatogenesis.

CDK2 kinase activity is a regulator of male germ cell fate.

The ability of men to remain fertile throughout their lives depends upon establishment of a spermatogonial stem cell (SSC) pool from gonocyte progenitors, and thereafter balancing SSC renewal versus terminal differentiation. Here, we report that precise regulation of the cell cycle is crucial for this balance. Whereas cyclin-dependent kinase 2 (Cdk2) is not necessary for mouse viability or gametogenesis stages prior to meiotic prophase I, mice bearing a deregulated allele (Cdk2Y15S ) are severely deficient in spermatogonial differentiation. This allele disrupts an inhibitory phosphorylation site (Tyr15) for the kinase WEE1. Remarkably, Cdk2Y15S/Y15S mice possess abnormal clusters of mitotically active SSC-like cells, but these are eventually removed by apoptosis after failing to differentiate properly. Analyses of lineage markers, germ cell proliferation over time, and single cell RNA-seq data revealed delayed and defective differentiation of gonocytes into SSCs. Biochemical and genetic data demonstrated that Cdk2Y15S is a gain-of-function allele causing elevated kinase activity, which underlies these differentiation defects. Our results demonstrate that precise regulation of CDK2 kinase activity in male germ cell development is crucial for the gonocyte-to-spermatogonia transition and long-term spermatogenic homeostasis.

Nodal Signaling Regulates Germ Cell Development and Establishment of Seminiferous Cords in the Human Fetal Testis

Disruption of human fetal testis development is widely accepted to underlie testicular germ cell cancer (TGCC) origin and additional disorders within testicular dysgenesis syndrome (TDS). However, the mechanisms for the development of testicular dysgenesis in humans are unclear. We used exvivo culture and xenograft approaches to investigate the importance of Nodal and Activin signaling in human fetal testis development. Inhibition of Nodal, and to some extent Activin, signaling disrupted seminiferous cord formation, abolished AMH expression, reduced androgen secretion, and decreased gonocyte numbers. Subsequent xenografting of testicular tissue rescued the disruptive effects on seminiferous cords and somatic cells but not germ cell effects. Stimulation of Nodal signaling increased the number of germ cells expressing pluripotency factors, and these persisted after xenografting. Our findings suggest a key role for Nodal signaling in the regulation of gonocyte differentiation and early human testis development with implications for the understanding of TGCC and TDS origin.

C-MET receptor as potential biomarker and target molecule for malignant testicular germ cell tumors.

Type II testicular germ cell tumors (TGCTs) represent the most frequent malignancy in Caucasian males (20–40 years). Even if diagnosed with disseminated disease, >80% of patients are cured; however, a small percentage of cases progress and result in death. It is commonly accepted that these cancers arise from a disturbed testicular embryonic niche that leads to the block of gonocyte differentiation. The subsequent development of the invasive seminomas and non-seminomas is due to a combination of genetic, epigenetic and microenvironment-based alterations (genvironment). Hepatocyte growth factor (HGF) is present in the testicular microenvironment, together with its receptor c-MET, from early embryonic development to an adult stage. In addition, c-MET is a well-known proto-oncogene involved in the onset and progression of various human cancers. Herein, we have investigated the expression and availability of HGF and c-MET in TCam-2, NCCIT and NT2D1 cells, which are type II (T)GCT representative cell lines, and the effect of c-MET activation/repression on the regulation of cancerous biological processes. We found that NT2D1 cells increase their proliferation, polarized migration, and invasion in response to HGF administration. NCCIT cells respond to HGF stimulation only partially, whereas TCam-2 cells do not respond to HGF, at least according to the investigated parameters. Interestingly, the immunohistochemical study of c-MET distribution in TGCTs confirm its presence in both seminoma and non-seminoma lesions with different patterns. Notably, we found the highest c-MET immunoreactivity in the epithelial elements of the various components of TGCTs: teratoma, yolk sac tumor and choriocarcinoma.