Spermatogonial Stem Cell Population Research Articles

Adult Human, but Not Rodent, Spermatogonial Stem Cells Retain States with a Foetal-like Signature.

Spermatogenesis involves a complex process of cellular differentiation maintained by spermatogonial stem cells (SSCs). Being critical to male reproduction, it is generally assumed that spermatogenesis starts and ends in equivalent transcriptional states in related species. Based on single-cell gene expression profiling, it has been proposed that undifferentiated human spermatogonia can be subclassified into four heterogenous subtypes, termed states 0, 0A, 0B, and 1. To increase the resolution of the undifferentiated compartment and trace the origin of the spermatogenic trajectory, we re-analysed the single-cell (sc) RNA-sequencing libraries of 34 post-pubescent human testes to generate an integrated atlas of germ cell differentiation. We then used this atlas to perform comparative analyses of the putative SSC transcriptome both across human development (using 28 foetal and pre-pubertal scRNA-seq libraries) and across species (including data from sheep, pig, buffalo, rhesus and cynomolgus macaque, rat, and mouse). Alongside its detailed characterisation, we show that the transcriptional heterogeneity of the undifferentiated spermatogonial cell compartment varies not only between species but across development. Our findings associate 'state 0B' with a suppressive transcriptomic programme that, in adult humans, acts to functionally oppose proliferation and maintain cells in a ready-to-react state. Consistent with this conclusion, we show that human foetal germ cells-which are mitotically arrested-can be characterised solely as state 0B. While germ cells with a state 0B signature are also present in foetal mice (and are likely conserved at this stage throughout mammals), they are not maintained into adulthood. We conjecture that in rodents, the foetal-like state 0B differentiates at birth into the renewing SSC population, whereas in humans it is maintained as a reserve population, supporting testicular homeostasis over a longer reproductive lifespan while reducing mutagenic load. Together, these results suggest that SSCs adopt differing evolutionary strategies across species to ensure fertility and genome integrity over vastly differing life histories and reproductive timeframes.

Targeting nuclear receptor corepressors for reversible male contraception

Despite numerous female contraceptive options, nearly half of all pregnancies are unintended. Family planning choices for men are currently limited to unreliable condoms and invasive vasectomies with questionable reversibility. Here, we report the development of an oral contraceptive approach based on transcriptional disruption of cyclical gene expression patterns during spermatogenesis. Spermatogenesis involves a continuous series of self-renewal and differentiation programs of spermatogonial stem cells (SSCs) that is regulated by retinoic acid (RA)-dependent activation of receptors (RARs), which control target gene expression through association with corepressor proteins. We have found that the interaction between RAR and the corepressor silencing mediator of retinoid and thyroid hormone receptors (SMRT) is essential for spermatogenesis. In a genetically engineered mouse model that negates SMRT-RAR binding (SMRTmRID mice), the synchronized, cyclic expression of RAR-dependent genes along the seminiferous tubules is disrupted. Notably, the presence of an RA-resistant SSC population that survives RAR de-repression suggests that the infertility attributed to the loss of SMRT-mediated repression is reversible. Supporting this notion, we show that inhibiting the action of the SMRT complex with chronic, low-dose oral administration of a histone deacetylase inhibitor reversibly blocks spermatogenesis and fertility without affecting libido. This demonstration validates pharmacologic targeting of the SMRT repressor complex for non-hormonal male contraception.

A Study of Dual Function and Expression Pattern of Klf4 between Two Different Processes of Differentiation and Maintenance of Pluripotency in Spermatogonial Stem Cells

Krüppel-like factor 4 (Klf4) is a transcription factor involved in proliferation, self-renewal, differentiation, apoptosis, and somatic cell reprogramming; thus, it has a critical role in accurate spermatogenesis. In this study, we examine the different protein localizations of Klf4 in the mouse testis and in two populations of spermatogonial stem cells (SSCs), including differentiated and undifferentiated cell types. We show that in contrast to its function during spermatogenesis in the adult testis, in vitro Klf4 is a factor in maintaining stemness in different SSC populations. Furthermore, we suggest that its functions could be age-dependent. Bioinformatics analysis in the Cytoscape platform was conducted to highlight pathways and networks of Klf4 interactions. Our results support the different functions of Klf4 dependent on the age and differentiation state of SSCs.

SPRY4-dependent ERK negative feedback demarcates functional adult stem cells in the male mouse germline†.

Niche-derived growth factors support self-renewal of mouse spermatogonial stem and progenitor cells through ERK MAPK signaling and other pathways. At the same time, dysregulated growth factor-dependent signaling has been associated with loss of stem cell activity and aberrant differentiation. We hypothesized that growth factor signaling through the ERK MAPK pathway in spermatogonial stem cells is tightly regulated within a narrow range through distinct intracellular negative feedback regulators. Evaluation of candidate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)-responsive genes known to dampen downstream signaling revealed robust induction of specific negative feedback regulators, including Spry4, in cultured mouse spermatogonial stem cells in response to glial cell line-derived neurotrophic factor or fibroblast growth factor 2. Undifferentiated spermatogonia in vivo exhibited high levels of Spry4 mRNA. Quantitative single-cell analysis of ERK MAPK signaling in spermatogonial stem cell cultures revealed both dynamic signaling patterns in response to growth factors and disruption of such effects when Spry4 was ablated, due to dysregulation of ERK MAPK downstream of RAS. Whereas negative feedback regulator expression decreased during differentiation, loss of Spry4 shifted cell fate toward early differentiation with concomitant loss of stem cell activity. Finally, a mouse Spry4 reporter line revealed that the adult spermatogonial stem cell population in vivo is demarcated by strong Spry4 promoter activity. Collectively, our data suggest that negative feedback-dependent regulation of ERK MAPK is critical for preservation of spermatogonial stem cell fate within the mammalian testis.

Differential responsiveness of spermatogonia to retinoic acid dictates precocious differentiation but not meiotic entry during steady-state spermatogenesis†.

The foundation of mammalian spermatogenesis is provided by undifferentiated spermatogonia, which comprise of spermatogonial stem cells (SSCs) and transit-amplifying progenitors that differentiate in response to retinoic acid (RA) and are committed to enter meiosis. Our laboratory recently reported that the foundational populations of SSCs, undifferentiated progenitors, and differentiating spermatogonia are formed in the neonatal testis in part based on their differential responsiveness to RA. Here, we expand on those findings to define the extent to which RA responsiveness during steady-state spermatogenesis in the adult testis regulates the spermatogonial fate. Our results reveal that both progenitor and differentiating spermatogonia throughout the testis are capable of responding to exogenous RA, but their resulting fates were quite distinct-undifferentiated progenitors precociously differentiated and proceeded into meiosis on a normal timeline, while differentiating spermatogonia were unable to hasten their entry into meiosis. This reveals that the spermatogonia responding to RA must still complete the 8.6day differentiation program prior to their entry into meiosis. Addition of exogenous RA enriched testes with preleptotene and pachytene spermatocytes one and two seminiferous cycles later, respectively, supporting recent clinical studies reporting increased sperm production and enhanced fertility in subfertile men on long-term RA analog treatment. Collectively, our results reveal that a well-buffered system exists within mammalian testes to regulate spermatogonial RA exposure, that exposed undifferentiated progenitors can precociously differentiate, but must complete a normal-length differentiation program prior to entering meiosis, and that daily RA treatments increased the numbers of advanced germ cells by directing undifferentiated progenitors to continuously differentiate.

RNA Interference as a Method of Gene Knockdown in Cultured Spermatogonia.

Maintenance and self-renewal of the spermatogonial stem cell (SSC) population in the testis are dictated by the expression of a unique suite of genes. In manipulating gene expression through loss-of-function approaches, we can identify important regulatory mechanisms that dictate spermatogonial fate decisions. One such approach is RNA interference (RNAi), which uses natural cellular responses to small interfering RNAs to decrease levels of a targeted transcript. RNAi is performed in primary cultures of undifferentiated spermatogonia, and can be paired with techniques such as spermatogonial transplantation to assess the functional consequences of downregulated expression of the target gene on stem cell maintenance. This approach provides an alternative or complementary strategy to the generation of knockout mouse lines / cell lines. Here, we describe the methodology of RNAi in undifferentiated spermatogonia, and outline its inherent advantages and disadvantages over other technologies in the study of gene regulation in these cells.

In vitro propagation of XXY human Klinefelter spermatogonial stem cells: A step towards new fertility opportunities.

Klinefelter Syndrome (KS) is characterized by a masculine phenotype, supernumerary sex chromosomes (47, XXY), and impaired fertility due to loss of spermatogonial stem cells (SSCs). Early testicular cryopreservation could be an option for future fertility treatments in these patients, including SSCs transplantation or in vitro spermatogenesis. It is critically essential to adapt current in vitro SSCs propagation systems as a fertility option for KS patients. KS human testicular samples (13,15- and 17-year-old non-mosaic KS boys) were donated by patients enrolled in an experimental testicular tissue banking program. Testicular cells were isolated from cryopreserved tissue and propagated in long-term culture for 110 days. Cell-specific gene expression confirmed the presence of all four main cell types found in testes: Spermatogonia, Sertoli, Leydig, and Peritubular cells. A population of ZBTB16+ undifferentiated spermatogonia was identified throughout the culture using digital PCR. Flow cytometric analysis also detected an HLA-/CD9+/CD49f+ population, indicating maintenance of a stem cell subpopulation among the spermatogonial cells. FISH staining for chromosomes X and Y showed most cells containing an XXY karyotype with a smaller number containing either XY or XX. Both XY and XX populations were able to be enriched by magnetic sorting for CD9 as a spermatogonia marker. Molecular karyotyping demonstrated genomic stability of the cultured cells, over time. Finally, single-cell RNAseq analysis confirmed transcription of ID4, TCN2, and NANOS 3 within a population of putative SSCs population. This is the first study showing successful isolation and long-term in vitro propagation of human KS testicular cells. These findings could inform the development of therapeutic fertility options for KS patients, either through in vitro spermatogenesis or transplantation of SSC, in vivo.

Culture of Kenyan Goat (Capra hircus) Undifferentiated Spermatogonia in Feeder-Free Conditions.

The undifferentiated spermatogonial population in mammalian testes contains a spermatogonial stem cell (SSC) population that can regenerate continual spermatogenesis following transplantation. This capacity has the potential to be exploited as a surrogate sires breeding tool to achieve widespread dissemination of desirable genetics in livestock production. Because SSCs are relatively rare in testicular tissue, the ability to expand a population in vitro would be advantageous to provide large numbers for transplantation into surrogate recipient males. Here, we evaluated conditions that would support long-term in-vitro maintenance of undifferentiated spermatogonia from a goat breed that is endemic to Kenyan livestock production. Single-cell suspensions enriched for undifferentiated spermatogonia from pre-pubertal bucks were seeded on laminin-coated tissue culture plates and maintained in a commercial media based on serum-free composition. The serum-free media was conditioned on goat fetal fibroblasts and supplemented with a growth factor cocktail that included glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), stromal cell-derived factor (SDF), and fibroblast growth factor (FGF) before use. Over 45 days, the primary cultures developed a cluster morphology indicative of in-vitro grown undifferentiated spermatogonia from other species and expressed the germ cell marker VASA, as well as the previously defined spermatogonial marker such as promyelocytic leukemia zinc finger (PLZF). Taken together, these findings provide a methodology for isolating the SSC containing undifferentiated spermatogonial population from goat testes and long-term maintenance in defined culture conditions.

LncRNA HOTAIR promotes proliferation and suppresses apoptosis of mouse spermatogonium GC-1 cells by sponging miR-761 to modulate NANOS2 expression.

LncRNA HOX antisense intergenic RNA (HOTAIR) can regulate cancer-related gene expression and promote stem cell and tumor cell proliferation via mechanisms including the competing endogenous RNA (ceRNA) mechanism. HOTAIR is abundantly expressed in the genital tubercle of E11.5, E12.5, and E13.5 embryos, whereas it became barely detectable at E13.5 and expressed again in adult mouse testis. However, the underlying function and mechanism of HOTAIR in spermatogenesis have not been elucidated. Interestingly, other researchers reported that the function of gene Nanos C2HC-Type Zinc Finger 2 (nanos2) includes the maintenance of both the primordial germ cells (PGCs) and germline stem cells, and Nanos2 protein and transcripts (NANOS2) were detected only in PGCs from day E11.5 and undifferentiated spermatogonia in spermatogenesis. We therefore investigated the relationship between HOTAIR and NANOS2 in maintaining spermatogonial stem cell population. We found that, compared to the adult mouse, the expression levels of HOTAIR and NANOS2 in embryo mouse were significantly higher and miR-761expression level was lower. In mouse GC-1 spermatogonia cells, overexpression of miRNA-761 significantly inhibited the expression of NANOS2 and HOTAIR, suppressed the proliferation, and promotes apoptosis of cells. Knock down and overexpression of HOTAIR indicated that HOTAIR expression was positively correlated with NANOS2 expression; overexpressed HOTAIR could promote proliferation and suppresses apoptosis of GC-1 cells. By a rescue experiment and dual luciferase reporter assay, miR-761 was identified as a direct target of HOTAIR, and NANOS2 was identified as the direct target of miR-761. The above results indicate that HOTAIR promotes proliferation and suppresses apoptosis of mouse spermatogonium GC-1 cells by sponging miR-761 to modulate NANOS2 expression. Our findings elucidate one of possible mechanisms and importance of HOTAIR in maintaining spermatogonial stem cell population, and provide new candidate genes and possible pathogenesis for male infertility.

Hormonal, Histological, and Comparative Study of the Effect of Pure Ginseng on Testicular Function in the Breeding/Non-Breeding Season of Rams in Basrah.

This study aimed to investigate the effect of the administration of powdered Panax ginseng as a dietary supplement on testosterone concentration, spermatogenesis stimulating hormone, interstitial cell-stimulating hormone levels, sperms morphological characteristics, testis histological traits, and testicular size in the breeding and non-breeding season in adult rams. In total, 20 adult rams were included and randomly divided into three groups. The first group of adult rams (n=8) was subdivided into two subgroups of four rams (Sub-G1-B and Sub-G2-B). TheSub-G1-B and Sub-G2-B were fed 2 and 5 g of P. ginseng once a day, respectively, for 90 days during the breeding season. The second group of adult rams (n=8) was subdivided into two subgroups of four rams (Sub-G1-NB and Sub-G2-NB). The Sub-G1-NB and Sub-G2-NB were fed 2 and 5 g of P. ginseng once a day, respectively, for 90 days during the non-breeding season. The third group of adult rams (n=4) was considered the control group two times (in and out of the season). The results showed that the testosterone concentration and gonad protective and interstitial cell-stimulating hormone levels increased significantly (P<0.05) in both the experimental groups that received ginseng supplementation, compared to the control group in and out of the breeding season. The evaluation of sperm morphometric parameters, such as total sperm count, total motility, and progressive motility, showed superiority in improving the above-mentioned parameters. However, the total immotile sperms and non-progressive sperms underwent a significant decrease (P<0.05) in both experimental groups of ginseng supplemented, compared to the control group in and out of season. The angiogenesis of the seminiferous tubules increased significantly (P<0.05) in both experimental groups. Through a microscopic examination, the recorded data showed a significant increase in the population of spermatogonial stem cells as well as primary and secondary spermatocytes in both experimental groups. Values of testicular diameter showed a significant increase (P<0.05) after a period of 75 and 90 days following the initiation of treatments in both experimental groups, compared to the control group in and out of the season. It can be concluded that P. ginseng has some beneficial effects on the antioxidant status of the semen, the morphometric parameters, and other critical traits of sperm and testicles which are the important factors in male fertility.

Analysis of the Reversible Impact of the Chemodrug Busulfan on Mouse Testes.

Spermatogenesis is a process within the testis that leads to the production of spermatozoa. It is based on a population of spermatogonial stem cells, which have the capacity to self-renew and to differentiate throughout life to ensure the functions of reproduction are maintained. Male fertility disorders are responsible for half of the cases of infertility in couples worldwide. It is well known that cancer treatments are associated with reversible or irreversible fertility disorders. Busulfan (Bu) is an alkylating agent that significantly inhibits spermatogenesis. The present study relied on a combination of in vivo and in vitro approaches as well as RNAseq analysis to characterize the effects of Bu, in which mouse testes were used as a model. An in silico analysis revealed that many of the Bu-modulated genes are potentially regulated by the SIN3 Transcription Regulator Family Member A (SIN3A) and E2F Transcription Factor (E2F) families of transcription factors. The results demonstrate that the deregulated genes function in processes related to the cell cycle, DNA repair, and cell death mechanisms, including the Tumor Protein 53 (TP53) pathway. This reinforces the role of the TP53 signaling pathway as a major player in Bu effects. In addition, Bu altered the patterns of mRNA accumulation for various genes in undifferentiated spermatogonia. This work provides significant insight into the kinetics and impacts of busulfan, which could pave the way for developing strategies to minimize the impact of chemodrugs and, thus, could lead to germ cell lineage regeneration following anticancer treatments.

O-189 Male fertility restoration by direct transplantation of human infant testicular cells into infertile recipient mouse testis

Abstract Study question Is colonization of human gonocytes and spermatogonial stem cells (SSCs) directly transplanted to seminiferous tubules of busulfan sterilised mice testis during an 8-week period feasible? Summary answer Gonocytes and SSCs from infant boys can settle on the basal membrane and form germline stem cell colonies in the seminiferous tubules of recipient mice. What is known already The neonatal or immature animal provides higher populations of gonocytes and/or SSCs than adults, and the number of transplanted donor SSCs directly affects the colonization rate of the recipient testes. Along with SSC transplantation restoring the recipient’s spermatogenesis, donor gonocyte was also reported to be capable of establishing spermatogenesis in rodents. Study design, size, duration Transplantation of human testicular cells including gonocytes and SSCs into seminiferous tubules of infertile recipient mice. We included 10 infant testis biopsies from which single-cell suspension was transplanted individually into the seminiferous tubules of 10 immunodeficient mice. The immunodeficient mouse testes were injected with busulfan to deplete germ cells. Four weeks later, we did the xenotransplantation. Then after eight weeks, we collected all mouse testes to do further analysis. Participants/materials, setting, methods Testis biopsies were obtained from cryptorchid boys undergoing orchidopexy. After enzymatic digestion of the testis biopsies, dissociated single-cell suspensions were pre-labeled with a green fluorescent dye. Then the single-cell suspensions were transplanted into seminiferous tubules of the infertile recipient mice. Eight weeks later, the presence of gonocytes and SSCs was determined by immunohistochemistry and whole-mount immunofluorescence. Main results and the role of chance Without in vitro propagation, naturally enriched human germline stem cells settled on the basal membrane of seminiferous tubules and survived in the mouse testes at least for two months demonstrating that human gonocytes and SSCs were capable of colonizing the recipient mouse seminiferous tubules. Limitations, reasons for caution The study samples were from infant boys with undescended testes that were more likely to contain gonocytes. It was not possible to determine which germ-cell type at transplantation resulted in the detected gonocytes and SSC colonies after xenotransplantation. Transplantation of gonocytes may include the potential risk of stem cell-related malignancy. Wider implications of the findings Without in vitro propagation, male germline stem cell-based transplantation could provide a relatively safe therapeutic treatment for prepubertal boys with cryptorchidism and boys diagnosed with cancer. This method could also facilitate clinical translation. Trial registration number not applicable

A regulatory role for CHD4 in maintenance of the spermatogonial stem cell pool.

SummaryMaintenance and self-renewal of the spermatogonial stem cell (SSC) population is the cornerstone of male fertility. Here, we have identified a key role for the nucleosome remodeling protein CHD4 in regulating SSC function. Gene expression analyses revealed that CHD4 expression is highly enriched in the SSC population in the mouse testis. Using spermatogonial transplantation techniques it was established that loss of Chd4 expression significantly impairs SSC regenerative capacity, causing a ∼50% reduction in colonization of recipient testes. An scRNA-seq comparison revealed reduced expression of “self-renewal” genes following Chd4 knockdown, along with increased expression of signature progenitor genes. Co-immunoprecipitation analyses demonstrated that CHD4 regulates gene expression in spermatogonia not only through its traditional association with the remodeling complex NuRD, but also via interaction with the GDNF-responsive transcription factor SALL4. Cumulatively, the results of this study depict a previously unappreciated role for CHD4 in controlling fate decisions in the spermatogonial pool.

Long-term follow-up of boys who have undergone a testicular biopsy for fertility preservation.

Long-term follow-up of boys who have undergone a testicular biopsy for fertility preservation.

Silk Nanofibrous Electrospun Scaffold Amplifies Proliferation and Stemness Profile of Mouse Spermatogonial Stem Cells

Electrospun nanofibrous scaffolds provide a structural microenvironment similar to the native extracellular matrix, which leads to the maintenance and propagation of the cultured cells under in vitro conditions. Furthermore, we used the sertoli cells to improve SSC proliferation due to its secretory role in growth factors. In this study, a silk electrospun nanofibrous scaffold has been used as a microenvironment for guiding the proliferation of spermatogonial stem cells (SSCs). SSCs were seeded on the prepared scaffolds and then cultured in vitro for 2 weeks. The attachment of SSCs was examined by scanning electron microscopy (SEM). Cell attachment and viability were evaluated via 4′, 6-diamidino-2-phenylindole staining and 3-(4, 5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide assessment, respectively. The expression of the specific SSC genes including stimulated by retinoic acid gene 8 (Stra8), deleted in azoospermia-like (DAZL) and piwil-like RNA-mediated gene silencing 2 (Piwil2), was quantified using real-time-PCR and confirmed by immunofluorescent staining. The data indicated that the prepared scaffold guided more effective SSC attachment and proliferation in comparison to the monolayer control. However, a significant upregulation of DAZL, Stra8, and piwill2 genes was observed in the cells grown on the silk scaffold. Also, the expression of Srta8 and Oct4 in the cultured cells was confirmed by immunofluorescence staining 2 weeks post-culture. Silk scaffold increases the in vitro viability of SSCs and amplifies their proliferation during the culture period. It is concluded that silk scaffold can serve as an appropriate substrate with broad implications for SSC proliferation under in vitro culture medium. We used a silk scaffold for guiding the proliferation of spermatogonial stem cell (SSC) population. Spermatogonia stem cells were seeded and cultured on silk scaffolds for 2 weeks. The attachment of SSCs was examined via SEM and viability was evaluated by DAPI staining and MTT assessments. The expression of specific spermatogonial stem cell markers including Stra8 gene, DAZL, and Piwill2 was quantified via Q-PCR and immunofluorescent staining. Scaffold guides firm attachment of SSCs and proliferates more than a monolayer layer. The significant upregulation of DAZL, Stra8, piwill2, and Oct4 in the experiment was confirmed by the real-time PCR and immunofluorescence staining 2 weeks after culture. Silk scaffold increases the in vitro viability of SSCs and amplifies their proliferation during the culture period. Silk scaffold can serve as an appropriate substrate with wide implications for SSC proliferation under in vitro condition.

The Non-hormonal Male Contraceptive Adjudin Exerts its Effects via MAPs and Signaling Proteins mTORC1/rpS6 and FAK-Y407.

Adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (formerly called AF-2364), is a nonhormonal male contraceptive, since it effectively induces reversible male infertility without perturbing the serum concentrations of follicle stimulating hormone (FSH), testosterone, and inhibin B based on studies in rats and rabbits. Adjudin was shown to exert its effects preferentially by perturbing the testis-specific actin-rich adherens junction (AJ) at the Sertoli-spermatid interface known as apical ectoplasmic specialization (apical ES), thereby effectively inducing spermatid exfoliation. Adjudin did not perturb germ cell development nor germ cell function. Also, it had no effects on Sertoli cell-cell AJ called basal ectoplasmic specialization (basal ES), which, together with tight junction constitute the blood-testis barrier (BTB), unless an acute dose of adjudin was used. Adjudin also did not perturb the population of spermatogonial stem cells nor Sertoli cells in the testis. However, the downstream signaling protein(s) utilized by adjudin to induce transient male infertility remains unexplored. Herein, using adult rats treated with adjudin and monitored changes in the phenotypes across the seminiferous epithelium between 6 and 96 h in parallel with the steady-state protein levels of an array of signaling and cytoskeletal regulatory proteins, recently shown to be involved in apical ES, basal ES and BTB function. It was shown that adjudin exerts its contraceptive effects through changes in microtubule associated proteins (MAPs) and signaling proteins mTORC1/rpS6 and p-FAK-Y407. These findings are important to not only study adjudin-mediated male infertility but also the biology of spermatogenesis.

WTAP Function in Sertoli Cells Is Essential for Sustaining the Spermatogonial Stem Cell Niche.

SummarySertoli cells are the major component of the spermatogonial stem cell (SSC) niche; however, regulatory mechanisms in Sertoli cells that dictate SSC fate decisions remain largely unknown. Here we revealed features of the N6-methyladenosine (m6A) mRNA modification in Sertoli cells and demonstrated the functions of WTAP, the key subunit of the m6A methyltransferase complex in spermatogenesis. m6A-sequencing analysis identified 21,909 m6A sites from 15,365 putative m6A-enriched transcripts within 6,122 genes, including many Sertoli cell-specific genes. Conditional deletion of Wtap in Sertoli cells resulted in sterility and the progressive loss of the SSC population. RNA sequencing and ribosome nascent-chain complex-bound mRNA sequencing analyses suggested that alternative splicing events of transcripts encoding SSC niche factors were sharply altered and translation of these transcripts were severely dysregulated by Wtap deletion. Collectively, this study uncovers a novel regulatory mechanism of the SSC niche and provide insights into molecular interactions between stem cells and their cognate niches in mammals.

Developmental underpinnings of spermatogonial stem cell establishment.

The germline serves as a conduit for transmission of genetic and epigenetic information from one generation to the next. In males, spermatozoa are the final carriers of inheritance and their continual production is supported by a foundational population of spermatogonial stem cells (SSCs) that forms from prospermatogonial precursors during the early stages of neonatal development. In mammals, the timing for which SSCs are specified and the underlying mechanisms guiding this process remain to be completely understood. To propose an evolving concept for how the foundational SSC population is established. This review summarizes recent and historical findings from peer-reviewed publications made primarily with mouse models while incorporating limited studies from humans and livestock. Establishment of the SSC population appears to follow a biphasic pattern involving a period of fate programming followed by an establishment phase that culminates in formation of the SSC population. This model for establishment of the foundational SSC population from precursors is anticipated to extend across mammalian species and include humans and livestock, albeit on different timescales.

CD2 is a surface marker for mouse and rat spermatogonial stem cells.

The spermatogonial stem cell (SSC) population in testis is small, and the lack of SSC markers has severely handicapped research on these cells. During our attempt to identify genes involved in SSC aging, we found that CD2 is expressed in cultured SSCs. Flow cytometric analysis and spermatogonial transplantation experiments showed that CD2 is expressed in SSCs from mature adult mouse testes. Cultured SSCs transfected with short hairpin RNAs (shRNAs) against CD2 proliferated poorly and showed an increased frequency of apoptosis. Moreover, functional analysis of transfected cells revealed impairment of SSC activity. Fluorescence activated cell sorting and spermatogonial transplantation experiments showed that CD2 is expressed not only in mouse but also in rat SSCs. The results indicate that CD2 is a novel SSC surface marker conserved between mouse and rat SSCs.

Genome-wide DNA-binding profile of SRY-box transcription factor 3 (SOX3) in mouse testes.

Spermatogenesis is the male version of gametogenesis, where germ cells are transformed into haploid spermatozoa through a tightly controlled series of mitosis, meiosis and differentiation. This process is reliant on precisely timed changes in gene expression controlled by several different hormonal and transcriptional mechanisms. One important transcription factor is SRY-box transcription factor 3 (SOX3), which is transiently expressed within the uncommitted spermatogonial stem cell population. Sox3-null mouse testes exhibit a block in spermatogenesis, leading to infertility or subfertility. However, the molecular role of SOX3 during spermatogonial differentiation remains poorly understood because the genomic regions targeted by this transcription factor have not been identified. In this study we used chromatin immunoprecipitation sequencing to identify and characterise the endogenous genome-wide binding profile of SOX3 in mouse testes at Postnatal Day 7. We show that neurogenin3 (Neurog3 or Ngn3) is directly targeted by SOX3 in spermatogonial stem cells via a novel testes-specific binding site. We also implicate SOX3, for the first time, in direct regulation of histone gene expression and demonstrate that this function is shared by both neural progenitors and testes, and with another important transcription factor required for spermatogenesis, namely promyelocytic leukaemia zinc-finger (PLZF). Together, these data provide new insights into the function of SOX3 in different stem cell contexts.