Mouse Spermatogonial Stem Cells Research Articles

Correction to: Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells

Correction to: Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells

The effect of epididymosomes on the development of frozen-thawed mouse spermatogonial stem cells after culture in a decellularized testicular scaffold and transplantation into azoospermic mice.

This study examined SSC proliferation on an epididymosome-enriched decellularized testicular matrix (DTM) hydrogel and spermatogenesis induction in azoospermic mice. Epididymosomes were extracted and characterized using SEM and western blotting. After cryopreservation, thawed SSCs were cultured in a hydrogel-based three-dimensional (3D) culture containing 10 ng/mL GDNF or 20µg/mL epididymosomes. SSCs were assessed using the MTT assay, flow cytometry, and qRT-PCR after two weeks of culture. The isolated SSCs were microinjected into the efferent ducts of busulfan-treated mice. DiI-labeled SSCs were followed, and cell homing was assessed after two weeks. After 8 weeks, the testes were evaluated using morphometric studies and immunohistochemistry. The expression of PLZF, TGF-β, and miR-10b did not increase statistically significantly in the 3D + GDNF and 3D + epididymosome groups compared to the 3D group. Among the groups, the GDNF-treated group exhibited the highest expression of miR-21 (*P < 0.05). Caspase-3 expression was lower in the epididymosome-treated group than in the other groups (***P < 0.001). Compared to the 3D and negative control groups, the 3D + epididymosomes and 3D + GDNF groups showed an increase in spermatogenic cells. Immunohistochemical results confirmed the growth and differentiation of spermatogonial cells into spermatids in the treatment groups. The DTM hydrogel containing 20µg/mL epididymosomes or 10 ng/mL GDNF is a novel and safe culture system that can support SSC proliferation in vitro to obtain adequate SSCs for transplantation success. It could be a novel therapeutic agent that could recover deregulated SSCs in azoospermic patients.

In vitro proliferation and differentiation of mouse spermatogonial stem cells in decellularized human placenta matrix.

Utilizing natural scaffold production derived from extracellular matrix components presents a promising strategy for advancing in vitro spermatogenesis. In this study, we employed decellularized human placental tissue as a scaffold, upon which neonatal mouse spermatogonial cells (SCs) were cultured three-dimensional (3D) configuration. To assess cellular proliferation, we examined the expression of key markers (Id4 and Gfrα1) at both 1 and 14 days into the culture. Our quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed a notable increase in Gfrα1 gene expression, with the 3D culture group exhibiting the highest levels. Furthermore, the relative frequency of Gfrα1-positive cells significantly rose from 38.1% in isolated SCs to 46.13% and 76.93% in the two-dimensional (2D) and 3D culture systems, respectively. Moving forward to days 14 and 35 of the culture period, we evaluated the expression of differentiating markers (Sycp3, acrosin, and Protamine 1). Sycp3 and Prm1 gene expression levels were upregulated in both 2D and 3D cultures, with the 3D group displaying the highest expression. Additionally, acrosin gene expression increased notably within the 3D culture. Notably, at the 35-day mark, the percentage of Prm1-positive cells in the 3D group (36.4%) significantly surpassed that in the 2D group (10.96%). This study suggests that the utilization of placental scaffolds holds significant promise as a bio-scaffold for enhancing mouse in vitro spermatogenesis.

Insulin-like growth factor-1 promotes the testicular sperm production by improving germ cell survival and proliferation in high-fat diet-treated male mice.

A decrease in semen volume among men is comparable to the rising prevalence of obesity worldwide. The anabolic hormone insulin-like growth factor-1 (IGF-1) can promote proliferation and differentiation in cultured mouse spermatogonial stem cells and alleviate abnormal in vitro spermatogenesis. Additionally, serum IGF-1 level is negatively correlated with body mass index. Whereas the role of IGF-1 in the sperm production in obese men remains unclear. To investigate the therapeutic effect and potential mechanism of IGF-1 on spermatogenesis of high-fat diet (HFD)-induced obesity mice. An HFD-induced obesity mouse model was established. Alterations in testicular morphology, sperm count, proliferation, and apoptosis were observed by H&E staining,immunohistochemistry, immunofluorescence, and Western blotting. Exogenous recombinant IGF-1 was administered to obese mice to investigate the correlations between altered testicular IGF-1 levels and sperm production. The sperm count was reduced, the testicular structure was disordered, and sex hormone levels were abnormal in HFD-fed mice compared with normal diet-fed mice. The expression of proliferation-related antigens such as proliferating cell nuclear antigen (PCNA) and Ki-67 was decreased, while that of proapoptotic proteins such as c-caspase3 was increased in testes from HFD-fed mice. Most importantly, the phosphorylation of insulin-like growth factor-1 receptor (IGF-1R) in testes was decreased due to reductions in IGF-1 from hepatocytes and Sertoli cells. Recombinant IGF-1 alleviated these functional impairments by promoting IGF-1R, Akt, and Erk1/2 phosphorylation in the testes. Insufficient IGF-1/IGF-1R signaling is intimately linked to damaged sperm production in obese male mice. Exogenous IGF-1 can improve survival and proliferation as well as sperm production. This study provides a novel theoretical basis and a target for the treatment of obese men with oligozoospermia.

Postnatal development of mouse spermatogonial stem cells as determined by immunophenotype, regenerative capacity, and long-term culture-initiating ability: a model for practical applications

Spermatogonial stem cells (SSCs) are the foundation of life-long spermatogenesis. While SSC research has advanced greatly over the past two decades, characterization of SSCs during postnatal development has not been well documented. Using the mouse as a model, in this study, we defined the immunophenotypic profiles of testis cells during the course of postnatal development using multi-parameter flow cytometry with up to five cell-surface antigens. We found that the profiles progress over time in a manner specific to developmental stages. We then isolated multiple cell fractions at different developmental stages using fluorescent-activated cell sorting (FACS) and identified specific cell populations with prominent capacities to regenerate spermatogenesis upon transplantation and to initiate long-term SSC culture. The data indicated that the cell fraction with the highest level of regeneration capacity exhibited the most prominent potential to initiate SSC culture, regardless of age. Interestingly, refinement of cell fractionation using GFRA1 and KIT did not lead to further enrichment of regenerative and culture-initiating stem cells, suggesting that when a high degree of SSC enrichment is achieved, standard markers of SSC self-renewal or commitment may lose their effectiveness to distinguish cells at the stem cell state from committed progenitors. This study provides a significant information resource for future studies and practical applications of mammalian SSCs.

Splicing factor SRSF1 is essential for homing of precursor spermatogonial stem cells in mice.

Spermatogonial stem cells (SSCs) are essential for continuous spermatogenesis and male fertility. The underlying mechanisms of alternative splicing (AS) in mouse SSCs are still largely unclear. We demonstrated that SRSF1 is essential for gene expression and splicing in mouse SSCs. Crosslinking immunoprecipitation and sequencing data revealed that spermatogonia-related genes (e.g. Plzf, Id4, Setdb1, Stra8, Tial1/Tiar, Bcas2, Ddx5, Srsf10, Uhrf1, and Bud31) were bound by SRSF1 in the mouse testes. Specific deletion of Srsf1 in mouse germ cells impairs homing of precursor SSCs leading to male infertility. Whole-mount staining data showed the absence of germ cells in the testes of adult conditional knockout (cKO) mice, which indicates Sertoli cell-only syndrome in cKO mice. The expression of spermatogonia-related genes (e.g. Gfra1, Pou5f1, Plzf, Dnd1, Stra8, and Taf4b) was significantly reduced in the testes of cKO mice. Moreover, multiomics analysis suggests that SRSF1 may affect survival of spermatogonia by directly binding and regulating Tial1/Tiar expression through AS. In addition, immunoprecipitation mass spectrometry and co-immunoprecipitation data showed that SRSF1 interacts with RNA splicing-related proteins (e.g. SART1, RBM15, and SRSF10). Collectively, our data reveal the critical role of SRSF1 in spermatogonia survival, which may provide a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying homing of precursor SSCs.

Splicing factor SRSF1 is essential for homing of precursor spermatogonial stem cells in mice

Spermatogonial stem cells (SSCs) are essential for continuous spermatogenesis and male fertility. The underlying mechanisms of alternative splicing (AS) in mouse SSCs are still largely unclear. We demonstrated that SRSF1 is essential for gene expression and splicing in mouse SSCs. Crosslinking immunoprecipitation and sequencing data revealed that spermatogonia-related genes (e.g. Plzf, Id4, Setdb1, Stra8, Tial1/Tiar, Bcas2, Ddx5, Srsf10, Uhrf1, and Bud31) were bound by SRSF1 in the mouse testes. Specific deletion of Srsf1 in mouse germ cells impairs homing of precursor SSCs leading to male infertility. Whole-mount staining data showed the absence of germ cells in the testes of adult conditional knockout (cKO) mice, which indicates Sertoli cell-only syndrome in cKO mice. The expression of spermatogonia-related genes (e.g. Gfra1, Pou5f1, Plzf, Dnd1, Stra8, and Taf4b) was significantly reduced in the testes of cKO mice. Moreover, multiomics analysis suggests that SRSF1 may affect survival of spermatogonia by directly binding and regulating Tial1/Tiar expression through AS. In addition, immunoprecipitation mass spectrometry and co-immunoprecipitation data showed that SRSF1 interacts with RNA splicing-related proteins (e.g. SART1, RBM15, and SRSF10). Collectively, our data reveal the critical role of SRSF1 in spermatogonia survival, which may provide a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying homing of precursor SSCs.

Cytoplasmatic Localization of Six1 in Male Testis and Spermatogonial Stem Cells.

Sine oculis homeobox 1 (Six1) is an important factor for embryonic development and carcinoma malignancy. However, the localization of Six1 varies due to protein size and cell types in different organs. In this study, we focus on the expression and localization of Six1 in male reproductive organ via bioinformatics analysis and immunofluorescent detection. The potential interacted proteins with Six1 were also predicted by protein-protein interactions (PPIs) and Enrichr analysis. Bioinformatic data from The Cancer Genome Atlas and Genotype-Tissue Expression project databases showed that SIX1 was highly expressed in normal human testis, but low expressed in the testicular germ cell tumor sample. Human Protein Atlas examination verified that SIX1 level was higher in normal than that in cancer samples. The sub-localization of SIX1 in different reproductive tissues varies but specifically in the cytoplasm and membrane in testicular cells. In mouse cells, single cell RNA-sequencing data analysis indicated that Six1 expression level was higher in mouse spermatogonial stem cells (mSSCs) and differentiating spermatogonial than in other somatic cells. Immunofluorescence staining showed the cytoplasmic localization of Six1 in mouse testis and mSSCs. Further PPIs and Enrichr examination showed the potential interaction of Six1 with bone morphogenetic protein 4 (Bmp4) and catenin Beta-1 (CtnnB1) and stem cell signal pathways. Cytoplasmic localization of Six1 in male testis and mSSCs was probably associated with stem cell related proteins Bmp4 and CtnnB1 for stem cell development.

Induction of Meiotic Initiation in Long-Term Mouse Spermatogonial Stem Cells Under Retinoid Acid and Nutrient Restriction Conditions.

Spermatogonial stem cells (SSCs) produce haploid sperm via mitosis and meiosis in vivo. Although the technique to culture mouse SSCs has been well established, induction of meiosis in vitro has remained a challenge. Retinoic acid (RA) is required for meiosis in vivo; however, RA alone is not sufficient to induce meiosis in vitro. Here, we describe a method in which nutrient restriction and RA synergistically induce meiotic initiation into meiotic prophase I in cultured mouse SSCs.

Ultrastructural study: in vitro and in vivo differentiation of mice spermatogonial stem cells.

Mouse testicular tissue is composed of seminiferous tubules and interstitial tissue. Mammalian spermatogenesis is divided into three stages: spermatocytogenesis (mitotic divisions) in which spermatogonial stem cells (SSCs) turn into spermatocytes, followed by two consecutive meiotic divisions in which spermatocytes form spermatids. Spermatids differentiate into spermatozoa during spermiogenesis. Various factors affect the process of spermatogenesis and the organization of cells in the testis. Any disorder in different stages of spermatogenesis will have negative effects on male fertility. The aim of the current study was to compare the in vitro and in vivo spermatogenesis processes before and after transplantation to azoospermic mice using ultrastructural techniques. In this study, mice were irradiated with single doses of 14 Gy 60Co radiation. SSCs isolated from neonatal mice were cultured in vitro for 1 week and were injected into the seminiferous tubule recipient's mice. Testicular cells of neonatal mice were cultured in the four groups on extracellular matrix-based 3D printing scaffolds. The transplanted testes (8 weeks after transplantation) and cultured testicular cells in vitro (after 3 weeks) were then processed for transmission electron microscopy studies. Our study's findings revealed that the morphology and ultrastructure of testicular cells after transplantation and in vitro culture are similar to those of in vivo spermatogenesis, indicating that spermatogenic cell nature is unaltered in vitro.

The Effect of Chitosan/Alginate/Graphene Oxide Nanocomposites on Proliferation of Mouse Spermatogonial Stem Cells.

Male survivors of childhood cancer have been known to be afflicted with azoospermia. To combat this, the isolation and purification of spermatogonial stem cells (SSCs) are crucial. Implementing scaffolds that emulate the extracellular matrix environment is vital for promoting the regeneration and proliferation of SSCs. This research aimed to evaluate the efficiency of nanocomposite scaffolds based on alginate, chitosan, and graphene oxide (GO) in facilitating SSCs proliferation. To analyze the cytotoxicity of the scaffolds, an MTT assay was conducted at 1, 3, and 7 days, and the sample containing 30 µg/mL of GO (ALGCS/GO30) exhibited the most favorable results, indicating its optimal performance. The identity of the cells was confirmed using flow cytometry with C-Kit and GFRα1 markers. The scaffolds were subjected to various analyses to characterize their properties. FTIR was employed to assess the chemical structure, XRD to examine crystallinity, and SEM to visualize the morphology of the scaffolds. To evaluate the proliferation of SSCs, qRT-PCR was used. The study's results demonstrated that the ALGCS/GO30 nanocomposite scaffold exhibited biocompatibility and facilitated the attachment and proliferation of SSCs. Notably, the scaffold displayed a significant increase in proliferation markers compared to the control group, indicating its ability to support SSC growth. The expression level of the PLZF protein was assessed using the Immunocytochemistry method. The observations confirmed the qRT-PCR results, which indicated that the nanocomposite scaffolds had higher levels of PLZF protein expression than scaffolds without GO. The biocompatible ALGCS/GO30 is a promising alternative for promoting SSC proliferation in in vitro applications.

Dysregulation of MTFR2, ATP5IF1 and BAK1 in Sertoli cells relates to idiopathic non-obstructive azoospermia via inhibiting mitochondrial fission and inducing mitochondrial dysfunction†.

Non-obstructive azoospermia affects more than 10% of infertile men with over 70% patients are idiopathic with uncharacterized molecular mechanisms, which is referred as idiopathic non-obstructive azoospermia. In this study, we checked the morphology of Sertoli cell mitochondria in testis biopsies from patients with idiopathic non-obstructive azoospermia and patients with obstructive azoospermia who have normal spermiogenesis. The expression of 104 genes controlling mitochondria fission and fusion were analyzed in three gene expression datasets including a total of 60 patients with non-obstructive azoospermia. The levels of 7 candidate genes were detected in testis biopsies from 38 patients with idiopathic non-obstructive azoospermia and 24 patients with obstructive azoospermia who have normal spermatogenesis by RT-qPCR. Cell viability, apoptosis, mitochondria membrane potential, adenosine triphosphate production, oxygen consumption, and mitochondria morphology were examined in primary human Sertoli cells. Mouse spermatogonial stem cells were used to detect the cell supporting capacity of Sertoli cells. We observed that patients with idiopathic non-obstructive azoospermia had elongated mitochondria. MTFR2 and ATP5IF1 were downregulated, whereas BAK1 was upregulated in idiopathic non-obstructive azoospermia testis and Sertoli cells. Sertoli cells from patients with idiopathic non-obstructive azoospermia had reduced viability, mitochondria membrane potential, adenosine triphosphate production, oxygen consumption rate, glycolysis and increased apoptosis. Knockdown MTFR2 in Sertoli cells increased the mitochondria size. Knockdown ATP5IF1 did not change mitochondrial morphology but increased adenosine triphosphate hydrolysis. Overexpression of BAK1 reduced membrane potential and upregulated cell apoptosis. The dysregulation of all these three genes contributed to the dysfunction of Sertoli cells, which provides a clue for idiopathic non-obstructive azoospermia treatment.

Loss of RBFOX2 inhibits meiotic initiation in male mice

Meiotic initiation is a critical step in gametogenesis. Recently, some genes required for meiotic initiation have been identified. However, meiosis-initiating factors and the underlying mechanisms are far from being fully understood. We have established a long-term culture system of spermatogonial stem cells (SSCs) and an in vitro model of meiotic initiation using mouse SSCs. Our previous study revealed that the RNA-binding protein RBFOX2 may regulate meiotic initiation, but the role and the mechanism need to be further elucidated. In this study, we constructed RBFOX2 knockdown SSC lines by using lentivirus-mediated gene delivery method, and found that the knockdown SSCs underwent normal self-renewal, mitosis and differentiation. However, they were unable to initiate meiosis when treated with retinoic acid, and they underwent apoptosis. These results indicate that RBFOX2 plays an essential role in meiotic initiation of spermatogonia. This work provides new clues for understanding the functions of RNA-binding proteins in meiotic initiation.

Hyaluronic acid/silicon nanoparticle scaffold induces proliferation and differentiation of mouse spermatogonial stem cells transplanted to epididymal adipose tissue.

Spermatogonia stem cells (SSCs) are a unique cell population maintaining male spermatogenesis during life, through their potential for proliferation and differentiation. The application of silicon nanoparticles (SNs) and hyaluronic acid (HA) to induce the differentiation of SSCs seems promising. Herein, we investigate the effect of SN and HA scaffolds on the progression of SSCs spermatogenesis in mice. Initially SSCs were isolated from healthy immature mice and cultured on prepared scaffolds (HA, SN, and HA/SN) in a 3D culture system. Then viability of SSCs cultured on scaffolds was examined using MTT assay and Acridine Orange staining. Then SSCs cultured on scaffolds were transplanted into epididymal adipose tissue (EAT) in mature mice and the result was studied by H&E and IHC staining 8weeks after transplantation. MTT and Acridine Orange analysis revealed that among three different scaffolds HA/SN based scaffold causes considerable toxicity on SSCs (P < 0.05) while H&E staining showed that culture of SSCs on HA, SN, and HA/SN scaffolds has a positive effect on the progression of SSCs spermatogenesis after transplantation into EAT. IHC staining identified TP1, TEKT1, and PLZF as crucial biomarkers in the spermatogenesis development of SSCs transplanted to EAT. According to the presence of these biomarkers in different experimental groups, we found the most spermatogenesis development in SSCs cultured on HA/SN scaffold (PLZF, P < 0.01) (TEKT1, P < 0.01) (TP1, P < 0.001). Our study showed that, although the cytotoxic effect of the HA/SN scaffold decreases the viability rate of SSCs; however, SSCs that survive on HA/SN scaffold showed more ability to progress in spermatogenesis after transplantation into EAT.

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.

Expression of Phospholipase D Family Member 6 in Bovine Testes and Its Molecular Characteristics.

Spermatogonial stem cells (SSCs) are the only primitive spermatogonial cells in males that can naturally transmit genetic information to their offspring and replicate throughout their lives. Phospholipase D family member 6 (PLD6) has recently been found to be a surface marker for SSCs in mice and boars; however, it has not been validated in cattle. The results of reversed transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) found that the relative expression of the PLD6 gene in the testicular tissues of two-year-old Simmental calves was significantly higher than that of six-month-old calves. Immunofluorescent staining further verified the expression of PLD6 protein in bovine spermatogenic cells like germ cell marker DEAD box helicase 4 (DDX4, also known as VASA). Based on multiple bioinformatic databases, PLD6 is a conservative protein which has high homology with mouse Q5SWZ9 protein. It is closely involved in the normal functioning of the reproductive system. Molecular dynamics simulation analyzed the binding of PLD6 as a phospholipase to cardiolipin (CL), and the PLD6-CL complex showed high stability. The protein interaction network analysis showed that there is a significant relationship between PLD6 and piwi-interacting RNA (piRNA) binding protein. PLD6 acts as an endonuclease and participates in piRNA production. In addition, PLD6 in bovine and mouse testes has a similar expression pattern with the spermatogonium-related genes VASA and piwi like RNA-mediated gene silencing 2 (PIWIL2). In conclusion, these analyses imply that PLD6 has a relatively high expression in bovine testes and could be used as a biomarker for spermatogenic cells including SSCs.

Dissecting the spermatogonial stem cell niche using spatial transcriptomics

Spermatogonial stem cells (SSCs) in the testis support the lifelong production of sperm. SSCs reside within specialized microenvironments called "niches," which are essential for SSC self-renewal and differentiation. However, our understanding of the molecular and cellular interactions between SSCs and niches remains incomplete. Here, we combine spatial transcriptomics, computational analyses, and functional assays to systematically dissect the molecular, cellular, and spatial composition of SSC niches. This allows us to spatially map the ligand-receptor (LR) interaction landscape in both mouse and human testes. Our data demonstrate that pleiotrophin regulates mouse SSC functions through syndecan receptors. We also identify ephrin-A1 as a potential niche factor that influences human SSC functions. Furthermore, we show that the spatial re-distribution of inflammation-related LR interactions underlies diabetes-induced testicular injury. Together, our study demonstrates a systems approach to dissect the complex organization of the stem cell microenvironment in health and disease.

Alpha-tocopherol enhances spermatogonial stem cell proliferation and restores mouse spermatogenesis by up-regulating BMI1.

Spermatogonial stem cells (SSCs) are essential for maintaining reproductive function in males. B-lymphoma Mo-MLV insertion region 1 (BMI1) is a vital transcription repressor that regulates cell proliferation and differentiation. However, little is known about the role of BMI1 in mediating the fate of mammalian SSCs and in male reproduction. This study investigated whether BMI1 is essential for male reproduction and the role of alpha-tocopherol (α-tocopherol), a protective agent for male fertility, as a modulator of BMI1 both in vitro and in vivo. Methyl thiazolyl tetrazolium (MTT) and 5-ethynyl-2'-deoxyuridine (EDU) assays were used to assess the effect of BMI1 on the proliferative ability of the mouse SSC line C18-4. Real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence were applied to investigate changes in the mRNA and protein expression levels of BMI1. Male mice were used to investigate the effect of α-tocopherol and a BMI1 inhibitor on reproduction-associated functionality in vivo. Analysis revealed that BMI1 was expressed at high levels in testicular tissues and spermatogonia in mice. The silencing of BMI1 inhibited the proliferation of SSCs and DNA synthesis and enhanced the levels of γ-H2AX. α-tocopherol enhanced the proliferation and DNA synthesis of C18-4 cells, and increased the levels of BMI1. Notably, α-tocopherol rescued the inhibition of cell proliferation and DNA damage in C18-4 cells caused by the silencing of BMI1. Furthermore, α-tocopherol restored sperm count (Ctrl vs. PTC-209, p = 0.0034; Ctrl vs. PTC-209 + α-tocopherol, p = 0.7293) and normalized sperm malformation such as broken heads, irregular heads, lost and curled tails in vivo, as demonstrated by its antagonism with the BMI1 inhibitor PTC-209. Analysis demonstrated that α-tocopherol is a potent in vitro and in vivo modulator of BMI1, a transcription factor that plays an important role in in SSC proliferation and spermatogenesis. Our findings identify a new target and strategy for treating male infertility that deserves further pre-clinical investigation.

Lovastatin promotes the self-renewal of murine and primate spermatogonial stem cells.

The spermatogonial stem cell (SSC) niche is critical for SSC maintenance and subsequent spermatogenesis. Numerous reproductive hazards impair the SSC niche, thereby resulting in aberrant SSC self-renewal and male infertility. However, promising agents targeting the impaired SSC niche to promote SSC self-renewal are still limited. Here, we screen out and assess the effects of Lovastatin on the self-renewal of mouse SSCs (mSSCs). Mechanistically, Lovastatin promotes the self-renewal of mSSCs and inhibits its inflammation and apoptosis through the regulation of isoprenoid intermediates. Remarkably, treatment by Lovastatin could promote the proliferation of undifferentiated spermatogonia in the male gonadotoxicity model generated by busulfan injection. Of note, we demonstrate that Lovastatin could enhance the proliferation of primate undifferentiated spermatogonia. Collectively, our findings uncover that lovastatin could promote the self-renewal of both murine and primate SSCs and have implications for the treatment of certain types of male infertility using small compounds.

Glutamine protects mouse spermatogonial stem cells against NOX1-derived ROS for sustaining self-renewal division in vitro.

Reactive oxygen species (ROS) are generated from NADPH oxidases and mitochondria; they are generally harmful for stem cells. Spermatogonial stem cells (SSCs) are unique among tissue-stem cells because they undergo ROS-dependent self-renewal via NOX1 activation. However, the mechanism by which SSCs are protected from ROS remains unknown. Here, we demonstrate a crucial role for Gln in ROS protection using cultured SSCs derived from immature testes. Measurements of amino acids required for SSC cultures revealed the indispensable role of Gln in SSC survival. Gln induced Myc expression to drive SSC self-renewal in vitro, whereas Gln deprivation triggered Trp53-dependent apoptosis and impaired SSC activity. However, apoptosis was attenuated in cultured SSCs that lacked NOX1. In contrast, cultured SSCs lacking Top1mt mitochondria-specific topoisomerase exhibited poor mitochondrial ROS production and underwent apoptosis. Gln deprivation reduced glutathione production; supra-molar Asn supplementation allowed offspring production from SSCs cultured without Gln. Therefore, Gln ensures ROS-dependent SSC-self-renewal by providing protection against NOX1 and inducing Myc.