[Effects and mechanism of p53 gene deletion on energy metabolism during the pluripotent transformation of spermatogonial stem cells].

Previous studies have shown that long-term spermatogonial stem cells (SSCs) have the potential to spontaneously transform into pluripotent stem cells, which is speculated to be related to the tumorigenesis of testicular germ cells, especially when p53 is deficient in SSCs which shows a significant increase in the spontaneous transformation efficiency. Energy metabolism has been proved to be strongly associated with the maintenance and acquisition of pluripotency. Recently, we compared the difference in chromatin accessibility and gene expression profiles between wild-type (p53+/+) and p53 deficient (p53-/-) mouse SSCs using the Assay for Targeting Accessible-Chromatin with high-throughput sequencing (ATAC-seq) and transcriptome sequencing (RNA-seq) techniques, and revealed that SMAD3 is a key transcription factor in the transformation of SSCs into pluripotent cells. In addition, we also observed significant changes in the expression levels of many genes related to energy metabolism after p53 deletion. To further reveal the role of p53 in the regulation of pluripotency and energy metabolism, this paper explored the effects and mechanism of p53 deletion on energy metabolism during the pluripotent transformation of SSCs. The results of ATAC-seq and RNA-seq from p53+/+ and p53-/- SSCs revealed that gene chromatin accessibility related to positive regulation of glycolysis and electron transfer and ATP synthesis was increased, and the transcription levels of genes encoding key glycolytic enzymes and regulating electron transport-related enzymes were markedly increased. Furthermore, transcription factors SMAD3 and SMAD4 promoted glycolysis and energy homeostasis by binding to the chromatin of the Prkag2 gene which encodes the AMPK subunit. These results suggest that p53 deficiency activates the key enzyme genes of glycolysis in SSCs and enhances the chromatin accessibility of genes associated with glycolysis activation to improve glycolysis activity and promote transformation to pluripotency. Moreover, SMAD3/SMAD4-mediated transcription of the Prkag2 gene ensures the energy demand of cells in the process of pluripotency transformation and maintains cell energy homeostasis by promoting AMPK activity. These results shed light on the importance of the crosstalk between energy metabolism and stem cell pluripotency transformation, which might be helpful for clinical research of gonadal tumors.

FOXP4 promotes proliferation of human spermatogonial stem cells.

Continuous self-renewal and differentiation of spermatogonial stem cells (SSCs) is vital for maintenance of adult spermatogenesis. Although several spermatogonial stem cell regulators have been extensively investigated in rodents, regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established. We analyzed single-cell sequencing data from the human testis and found that forkhead box P4 (FOXP4) expression gradually increased with development of SSCs. Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential. Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis. FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis. These findings imply that FOXP4 is involved in human SSC proliferation, which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.

The molecular evolution of spermatogenesis across mammals.

The testis produces gametes through spermatogenesis and evolves rapidly at both the morphological and molecular level in mammals1-6, probably owing to the evolutionary pressure on males to be reproductively successful7. However, the molecular evolution of individual spermatogenic cell types across mammals remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from 11 species that cover the three main mammalian lineages (eutherians, marsupials and monotremes) and birds (the evolutionary outgroup), and include seven primates. We find that the rapid evolution of the testis was driven by accelerated fixation rates of gene expression changes, amino acid substitutions and new genes in late spermatogenic stages, probably facilitated by reduced pleiotropic constraints, haploid selection and transcriptionally permissive chromatin. We identify temporal expression changes of individual genes across species and conserved expression programs controlling ancestral spermatogenic processes. Genes predominantly expressed in spermatogonia (germ cells fuelling spermatogenesis) and Sertoli (somatic support) cells accumulated on X chromosomes during evolution, presumably owing to male-beneficial selective forces. Further work identified transcriptomal differences between X- and Y-bearing spermatids and uncovered that meiotic sex-chromosome inactivation (MSCI) also occurs in monotremes and hence is common to mammalian sex-chromosome systems. Thus, the mechanism of meiotic silencing of unsynapsed chromatin, which underlies MSCI, is an ancestral mammalian feature. Our study illuminates the molecular evolution of spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals.

Effects and mechanism of p53 gene deletion on energy metabolism during the pluripotent transformation of spermatogonial stem cells / 生理学报

Previous studies have shown that long-term spermatogonial stem cells (SSCs) have the potential to spontaneously transform into pluripotent stem cells, which is speculated to be related to the tumorigenesis of testicular germ cells, especially when p53 is deficient in SSCs which shows a significant increase in the spontaneous transformation efficiency. Energy metabolism has been proved to be strongly associated with the maintenance and acquisition of pluripotency. Recently, we compared the difference in chromatin accessibility and gene expression profiles between wild-type (p53+/+) and p53 deficient (p53-/-) mouse SSCs using the Assay for Targeting Accessible-Chromatin with high-throughput sequencing (ATAC-seq) and transcriptome sequencing (RNA-seq) techniques, and revealed that SMAD3 is a key transcription factor in the transformation of SSCs into pluripotent cells. In addition, we also observed significant changes in the expression levels of many genes related to energy metabolism after p53 deletion. To further reveal the role of p53 in the regulation of pluripotency and energy metabolism, this paper explored the effects and mechanism of p53 deletion on energy metabolism during the pluripotent transformation of SSCs. The results of ATAC-seq and RNA-seq from p53+/+ and p53-/- SSCs revealed that gene chromatin accessibility related to positive regulation of glycolysis and electron transfer and ATP synthesis was increased, and the transcription levels of genes encoding key glycolytic enzymes and regulating electron transport-related enzymes were markedly increased. Furthermore, transcription factors SMAD3 and SMAD4 promoted glycolysis and energy homeostasis by binding to the chromatin of the Prkag2 gene which encodes the AMPK subunit. These results suggest that p53 deficiency activates the key enzyme genes of glycolysis in SSCs and enhances the chromatin accessibility of genes associated with glycolysis activation to improve glycolysis activity and promote transformation to pluripotency. Moreover, SMAD3/SMAD4-mediated transcription of the Prkag2 gene ensures the energy demand of cells in the process of pluripotency transformation and maintains cell energy homeostasis by promoting AMPK activity. These results shed light on the importance of the crosstalk between energy metabolism and stem cell pluripotency transformation, which might be helpful for clinical research of gonadal tumors.

Histone methyltransferase DOT1L is essential for self-renewal of germline stem cells.

Self-renewal of spermatogonial stem cells is vital to lifelong production of male gametes and thus fertility. However, the underlying mechanisms remain enigmatic. Here, we show that DOT1L, the sole H3K79 methyltransferase, is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L fail to maintain spermatogonial stem cells, characterized by a sequential loss of germ cells from spermatogonia to spermatids and ultimately a Sertoli cell only syndrome. Inhibition of DOT1L reduces the stem cell activity after transplantation. DOT1L promotes expression of the fate-determining HoxC transcription factors in spermatogonial stem cells. Furthermore, H3K79me2 accumulates at HoxC9 and HoxC10 genes. Our findings identify an essential function for DOT1L in adult stem cells and provide an epigenetic paradigm for regulation of spermatogonial stem cells.

Hypobaric hypoxia exposure alters transcriptome in mouse testis and impairs spermatogenesis in offspring.

Male fertility relies on continual and robust spermatogenesis. Environmental hypoxia adversely affects reproductive health in humans and animal studies provide compelling evidences that hypoxia impairs spermatogenesis in directly exposed individuals. However, a detail examination of hypoxia induced changes in testicular gene expression is still lacking and spermatogenesis in offspring of hypoxia exposed animals of awaits investigation. In this study, a hypobaric hypoxic chamber was used to simulate hypoxic conditions in mice and effects of hypoxia on spermatogenesis, fertility and testicular gene expression were evaluated. The results showed that hypoxia exposure reduced the number of undifferentiated spermatogonia but did not change the regenerative capacity of spermatogonial stem cells (SSCs) after transplantation. Hypoxia significantly increased the percent of abnormal sperm and these defects were recovered 2 months after returning to the normoxia. Transcriptome analysis of testicular tissues from control and hypoxia treated animals revealed that 766 genes were up-regulated and 965 genes were down-regulated. Surprisingly, expressions of genes that regulate epigenetic modifications were altered, indicating hypoxia-induced damage to spermatogenesis may be intergenerational. Indeed, animals that were sired by hypoxia exposed males exhibited impaired spermatogenesis. Together, these findings suggest that hypoxia exposure alters testicular gene expression and causes long-lasting damage to spermatogenesis.

Unraveling three-dimensional chromatin structural dynamics during spermatogonial differentiation.

Spermatogonial stem cells (SSCs) are able to undergo both self-renewal and differentiation. Unlike self-renewal, which replenishes the SSC and progenitor pool, differentiation is an irreversible process committing cells to meiosis. Although the preparations for meiotic events in differentiating spermatogonia (Di-SG) are likely to be accompanied by alterations in chromatin structure, the three-dimensional chromatin architectural differences between SSCs and Di-SG, and the higher-order chromatin dynamics during spermatogonial differentiation, have not been systematically investigated. Here, we performed in situ high-throughput chromosome conformation capture, RNA-seq, and chromatin immunoprecipitation-sequencing analyses on porcine undifferentiated spermatogonia (which consist of SSCs and progenitors) and Di-SG. We identified that Di-SG exhibited less compact chromatin structural organization, weakened compartmentalization, and diminished topologically associating domains in comparison with undifferentiated spermatogonia, suggesting that diminished higher-order chromatin architecture in meiotic cells, as shown by recent reports, might be preprogrammed in Di-SG. Our data also revealed that A/B compartments, representing open or closed chromatin regions respectively, and topologically associating domains were related to dynamic gene expression during spermatogonial differentiation. Furthermore, we unraveled the contribution of promoter-enhancer interactions to premeiotic transcriptional regulation, which has not been accomplished in previous studies due to limited cell input and resolution. Together, our study uncovered the three-dimensional chromatin structure of SSCs/progenitors and Di-SG, as well as the interplay between higher-order chromatin architecture and dynamic gene expression during spermatogonial differentiation. These findings provide novel insights into the mechanisms for SSC self-renewal and differentiation and have implications for diagnosis and treatment of male sub-/infertility.

Pyroglutamylated RFamide peptide (QRFP): Role in early testicular development in relation to Sertoli cell maturation in prepubertal mice.

QRFP, an orexigenic neuropeptide, binds to its cognate receptor GPR103 and regulates various biological functions. We have recently shown that QRFP and its receptor are present in mice testes and that their expression is high during early postnatal period. The present study aimed to investigate the effect of sustained high level of QRFP on Sertoli cells proliferation and differentiation and to relate these events with germ cell differentiation and lumen formation in the seminiferous tubules in mice testes during prepubertal period. QRFP was injected intraperitoneally to male mice from postnatal day 5 to 16. Morphometric analysis and various markers related to Sertoli cell maturation (WT1, p27kip1, AMH, AR and CYP19A1) and germ cell proliferation and differentiation (PCNA, GDNF and c-Kit) were evaluated. QRFP administration caused an early lumen formation in the seminiferous tubules in testis of treated mice. Further, there was a significant increase in p27kip1 expression and a marked decrease in AMH expression in QRFP-treated mice compared to controls. However, no appreciable change was noted in AR expression in treated mice. QRFP treatment also caused an increase in c-Kit expression in treated mice compared to controls, suggesting an accelerated spermatogonial differentiation in testis of QRFP-treated mice. Taken together, the present results suggest that the prolonged high level of QRFP increases Sertoli cell maturation, which, in turn, plays a contributory role in increasing the pace of germ cell differentiation and formation of lumen in the seminiferous tubules.

Autophagy modulation alleviates cryoinjury in murine spermatogonial stem cell cryopreservation.

BACKGROUND: Cryopreservation can expand the usefulness of spermatogonial stem cells (SSCs) in various fields. However, previous investigations that have attempted to modulate cryoinjury-induced mechanisms to increase cryoprotective efficiency have mainly focused on apoptosis and necrosis. OBJECTIVES: This study aimed to establish an effective molecular-based cryoprotectant for SSC cryopreservation via autophagy modulation. MATERIALS AND METHODS: To determine the efficacy of autophagy modulation, we assessed the recovery rate and relative proliferation rate and performed western blotting for the determination of autophagy flux, immunocytochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) for SSC characterization, and spermatogonial transplantation for in vivo SSC functional activity. RESULTS: The results showed that a basal level of autophagy caused a higher relative proliferation rate (pifithrin-µ 0.01 µM, 184.2 ± 11.2%; 3-methyladenine 0.01 µM, 175.3 ± 10.3%; pifithrin-µ 0.01 µM + 3-methyladenine 0.01 µM, P3, 224.6 ± 22.3%) than the DMSO control (100 ± 6.2%). All treatment groups exhibited normal characteristics, suggesting that these modulators could be used as effective cryoprotectants without changing the properties of the undifferentiated germ cells. According to the results of the in vivo spermatogonial transplantation assay, the colonies per total number of cultured SSCs was significantly higher in the pifithrin-µ 0.01 µM (1596.7 ± 172.5 colonies), 3-methyladenine 0.01 µM (1522.1 ± 179.2 colonies), and P3 (1727.5 ± 196.5 colonies) treatment groups than in the DMSO control (842.8 ± 110.08 colonies), which was comparable to that of the fresh control (1882.1 ± 132.1 colonies). DISCUSSION: A basal level of autophagy is more essential for resilience in frozen SSCs after thawing, rather than the excessive activation or inhibition of autophagy. CONCLUSION: A basal level of autophagy plays a critical role in the pro-survival response of frozen SSCs after thawing; herein, a new approach by which SSC cryoprotective efficiency can be improved was identified.

Genomic stability of mouse spermatogonial stem cells in vitro.

Germline mutations underlie genetic diversity and species evolution. Previous studies have assessed the theoretical mutation rates and spectra in germ cells mostly by analyzing genetic markers and reporter genes in populations and pedigrees. This study reported the direct measurement of germline mutations by whole-genome sequencing of cultured spermatogonial stem cells in mice, namely germline stem (GS) cells, together with multipotent GS (mGS) cells that spontaneously dedifferentiated from GS cells. GS cells produce functional sperm that can generate offspring by transplantation into seminiferous tubules, whereas mGS cells contribute to germline chimeras by microinjection into blastocysts in a manner similar to embryonic stem cells. The estimated mutation rate of GS and mGS cells was approximately 0.22 × 10-9 and 1.0 × 10-9 per base per cell population doubling, respectively, indicating that GS cells have a lower mutation rate compared to mGS cells. GS and mGS cells also showed distinct mutation patterns, with C-to-T transition as the most frequent in GS cells and C-to-A transversion as the most predominant in mGS cells. By karyotype analysis, GS cells showed recurrent trisomy of chromosomes 15 and 16, whereas mGS cells frequently exhibited chromosomes 1, 6, 8, and 11 amplifications, suggesting that distinct chromosomal abnormalities confer a selective growth advantage for each cell type in vitro. These data provide the basis for studying germline mutations and a foundation for the future utilization of GS cells for reproductive technology and clinical applications.

Differential Effects of Low and High Radiation Dose Rates on Mouse Spermatogenesis.

The adverse effects of radiation are proportional to the total dose and dose rate. We aimed to investigate the effects of radiation dose rate on different organs in mice. The mice were subjected to low dose rate (LDR, ~3.4 mGy/h) and high dose rate (HDR, ~51 Gy/h) radiation. LDR radiation caused severe tissue toxicity, as observed in the histological analysis of testis. It adversely influenced sperm production, including sperm count and motility, and induced greater sperm abnormalities. The expression of markers of early stage spermatogonial stem cells, such as Plzf, c-Kit, and Oct4, decreased significantly after LDR irradiation, compared to that following exposure of HDR radiation, in qPCR analysis. The compositional ratios of all stages of spermatogonia and meiotic cells, except round spermatid, were considerably reduced by LDR in FACS analysis. Therefore, LDR radiation caused more adverse testicular damage than that by HDR radiation, contrary to the response observed in other organs. Therefore, the dose rate of radiation may have differential effects, depending on the organ; it is necessary to evaluate the effect of radiation in terms of radiation dose, dose rate, organ type, and other conditions.

Three-dimensional morphological analysis of spermatogenesis in aged mouse testes.

Spermatogenesis, which is a continuous process from undifferentiated spermatogonia to spermatozoa in the seminiferous tubules, declines with age. To investigate changes in spermatogenesis with aging, we reconstructed the seminiferous tubules of 12 mice aged 12 to 30 months from serial sections and examined age-related and region-specific alterations in the seminiferous epithelium and spermatogenic waves in three dimensions. The basic structure of the seminiferous tubules, including the numbers of tubules, terminating points, branching points, and total tubule length, did not change with age. Age-related alterations in spermatogenesis, primarily assessed by the formation of vacuoles in Sertoli cells, were detected in the seminiferous tubules at 12 months. The proportion of altered tubule segments with impaired spermatogenesis further increased by 24 months, but remained unchanged thereafter. Altered tubule segments were preferentially distributed in tubule areas close to the rete testis and those in the center of the testis. Spermatogenic waves became shorter in length with age. These results provide a basis for examining the decline of spermatogenesis not only with aging, but also in male infertility.

Direct reprogramming of human Sertoli cells into male germline stem cells with the self-renewal and differentiation potentials via overexpressing DAZL/DAZ2/BOULE genes.

We propose a new concept that human somatic cells can be converted to become male germline stem cells by the defined factors. Here, we demonstrated that the overexpression of DAZL, DAZ2, and BOULE could directly reprogram human Sertoli cells into cells with the characteristics of human spermatogonial stem cells (SSCs), as shown by their similar transcriptomes and proteomics with human SSCs. Significantly, human SSCs derived from human Sertoli cells colonized and proliferated in vivo, and they could differentiate into spermatocytes and haploid spermatids in vitro. Human Sertoli cell-derived SSCs excluded Y chromosome microdeletions and assumed normal chromosomes. Collectively, human somatic cells could be converted directly to human SSCs with the self-renewal and differentiation potentials and high safety. This study is of unusual significance, because it provides an effective approach for reprogramming human somatic cells into male germ cells and offers invaluable male gametes for treating male infertility.

Current scenario and challenges ahead in application of spermatogonial stem cell technology in livestock.

PURPOSE: Spermatogonial stem cells (SSCs) are the source for the mature male gamete. SSC technology in humans is mainly focusing on preserving fertility in cancer patients. Whereas in livestock, it is used for mining the factors associated with male fertility. The review discusses the present status of SSC biology, methodologies developed for in vitro culture, and challenges ahead in establishing SSC technology for the propagation of superior germplasm with special reference to livestock. METHOD: Published literatures from PubMed and Google Scholar on topics of SSCs isolation, purification, characterization, short and long-term culture of SSCs, stemness maintenance, epigenetic modifications of SSCs, growth factors, and SSC cryopreservation and transplantation were used for the study. RESULT: The fine-tuning of SSC isolation and culture conditions with special reference to feeder cells, growth factors, and additives need to be refined for livestock. An insight into the molecular mechanisms involved in maintaining stemness and proliferation of SSCs could facilitate the dissemination of superior germplasm through transplantation and transgenesis. The epigenetic influence on the composition and expression of the biomolecules during in vitro differentiation of cultured cells is essential for sustaining fertility. The development of surrogate males through gene-editing will be historic achievement for the foothold of the SSCs technology. CONCLUSION: Detailed studies on the species-specific factors regulating the stemness and differentiation of the SSCs are required for the development of a long-term culture system and in vitro spermatogenesis in livestock. Epigenetic changes in the SSCs during in vitro culture have to be elucidated for the successful application of SSCs for improving the productivity of the animals.

Actions and Roles of FSH in Germinative Cells.

Follicle stimulating hormone (FSH) is produced by the pituitary gland in a coordinated hypothalamic-pituitary-gonadal (HPG) axis event, plays important roles in reproduction and germ cell development during different phases of reproductive development (fetal, neonatal, puberty, and adult life), and is consequently essential for fertility. FSH is a heterodimeric glycoprotein hormone of two dissociable subunits, α and ß. The FSH ß-subunit (FSHß) function starts upon coupling to its specific receptor: follicle-stimulating hormone receptor (FSHR). FSHRs are localized mainly on the surface of target cells on the testis and ovary (granulosa and Sertoli cells) and have recently been found in testicular stem cells and extra-gonadal tissue. Several reproduction disorders are associated with absent or low FSH secretion, with mutation of the FSH ß-subunit or the FSH receptor, and/or its signaling pathways. However, the influence of FSH on germ cells is still poorly understood; some studies have suggested that this hormone also plays a determinant role in the self-renewal of germinative cells and acts to increase undifferentiated spermatogonia proliferation. In addition, in vitro, together with other factors, it assists the process of differentiation of primordial germ cells (PGCLCs) into gametes (oocyte-like and SSCLCs). In this review, we describe relevant research on the influence of FSH on spermatogenesis and folliculogenesis, mainly in the germ cell of humans and other species. The possible roles of FSH in germ cell generation in vitro are also presented.

Mammalian in vitro gametogenesis.

Germ cells differentiate into sexually dimorphic gametes, oocytes, and spermatozoa, which unite to form new individuals. Accordingly, germ cell development entails intricate regulations of genome functions for genetic and epigenetic inheritance. The past decade has seen considerable advances in in vitro gametogenesis (IVG), which aims to recreate germ cell development from pluripotent stem cells (PSCs) in culture. Mouse PSCs can be induced into functional oocytes and spermatozoa, whereas human PSCs can be induced into early oocytes and prospermatogonia, promoting mechanistic understanding of mammalian germ cell development. The prospect for inducing human gametes with appropriate functions has been heightened, and such advances will create possibilities in reproductive medicine, including modeling infertility to explore remedies. The use of IVG-derived gametes for human reproduction will require careful legal and ethical discussions.

Cryopreservation of Germ Cells of Banana Shrimp (Fenneropenaeus merguiensis) and Black Tiger Shrimp (Penaeus monodon).

Germ cell cryopreservation has been used to preserve many fish species. However, this method has not been established for crustaceans; thus, we attempted to do this herein. The efficiency of slow freezing was compared to vitrification methods for germ cell cryopreservation in two types of marine shrimp, Fenneropenaeus merguiensis and Penaeus monodon. In situ hybridization with a vasa probe was used to identify germ cells. The effects of three cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (GLY), and magnesium chloride (MgCl2), on germ cell viability and recovery rate were compared at three concentrations (5%, 10%, and 15%). The effects of thawing temperature, including 10 and 27 °C, were also investigated. We discovered that 10% DMSO with the vitrification is suitable for preserving the germ cells of F. merguiensis for a long time, whereas 10% GLY with vitrification is suitable for P. monodon. Moreover, the most suitable thawing temperature was 10 °C for both species. This is the first report of germ cell cryopreservation in crustaceans. Thus, we provide evidence that crustacean germ cells can be preserved long-term in liquid nitrogen; this is the first step in the sustainable preservation of crustaceans, especially shrimp.

Insights into differentiation and function of the transition region between the seminiferous tubule and rete testis.

Seminiferous tubules physically connect to the rete testis through short segments called the transition region (TR). During fetal development, this specialized junction is considered the initial site where testis cords begin to form and to grow in length well beyond birth and into adulthood and form convoluted tubular cores. Mitotic activity of the Sertoli cell, the somatic cell of the epithelium, ceases before puberty, but modified Sertoli cells in the TR remain immature and capable of proliferation. This review presents what is known about this specialized region of the testis, with an emphasis on the morphological, molecular and physiological features, which support the hypothesis that this short region of epithelial transition serves as a specialized niche for undifferentiated Sertoli cells and spermatogonial stem cells. Also, the region is populated by an elevated number of immune cells, suggesting an important activity in monitoring and responding to any leakage of autoantigens, as sperm enter the rete testis. Several structure/function characteristics of the transition region are discussed and compared across species.

RHOX10 drives mouse spermatogonial stem cell establishment through a transcription factor signaling cascade.

Spermatogonial stem cells (SSCs) are essential for male fertility. Here, we report that mouse SSC generation is driven by a transcription factor (TF) cascade controlled by the homeobox protein, RHOX10, which acts by driving the differentiation of SSC precursors called pro-spermatogonia (ProSG). We identify genes regulated by RHOX10 in ProSG in vivo and define direct RHOX10-target genes using several approaches, including a rapid temporal induction assay: iSLAMseq. Together, these approaches identify temporal waves of RHOX10 direct targets, as well as RHOX10 secondary-target genes. Many of the RHOX10-regulated genes encode proteins with known roles in SSCs. Using an in vitro ProSG differentiation assay, we find that RHOX10 promotes mouse ProSG differentiation through a conserved transcriptional cascade involving the key germ-cell TFs DMRT1 and ZBTB16. Our study gives important insights into germ cell development and provides a blueprint for how to define TF cascades.

Spermatogonial stem cell transplantation into nonablated mouse recipient testes.

Spermatogonial transplantation has been used as a standard assay for spermatogonial stem cells (SSCs). After transplantation into the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) between Sertoli cells and settle in a niche. Unlike in the repair of other self-renewing systems, SSC transplantation is generally performed after complete destruction of endogenous spermatogenesis. Here, we examined the impacts of recipient conditioning on SSC homing. Germ cell ablation downregulated the expression of glial cell line-derived neurotrophic factor, which has been shown to attract SSCs to niches, implying that nonablated niches would attract SSCs more efficiently. As expected, SSCs colonized nonablated testes when transplanted into recipients with the same genetic background. Moreover, although spermatogenesis was arrested at the spermatocyte stage in Cldn11-deficient mice without a BTB, transplantation not only enhanced donor colonization but also restored normal spermatogenesis. The results show promise for the development of a new transplantation strategy to overcome male infertility.