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Summary Histone H3 lysine 9 (H3K9) methylation is dynamically regulated by methyltransferases and demethylases. In spermatogenesis, prospermatogonia differentiate into differentiating or undifferentiated spermatogonia after birth. However, the epigenetic regulation of prospermatogonia to spermatogonia transition is largely unknown. We found that perinatal prospermatogonia have extremely low levels of di-methylated H3K9 (H3K9me2) and that H3K9 demethylases, JMJD1A and JMJD1B, catalyze H3K9me2 demethylation in perinatal prospermatogonia. Depletion of JMJD1A and JMJD1B in the embryonic germline resulted in complete loss of male germ cells after puberty, indicating that H3K9me2 demethylation is essential for male germline maintenance. JMJD1A/JMJD1B-depleted germ cells were unable to differentiate into functional spermatogonia. JMJD1 isozymes contributed to activation of several spermatogonial stem cell maintenance genes through H3K9 demethylation during the prospermatogonia to spermatogonia transition, which we propose is key for spermatogonia development. In summary, JMJD1A/JMJD1B-mediated H3K9me2 demethylation promotes prospermatogonia to differentiate into functional spermatogonia by establishing proper gene expression profiles., Highlights • JMJD1A/JMJD1B catalyze H3K9 demethylation in prospermatogonia • JMJD1A/JMJD1B are required for prospermatogonia to spermatogonia transition • JMJD1A/JMJD1B activate genes required for spermatogonial stem cell maintenance, In this article, Kuroki et al. show that the H3K9 demethylases, JMJD1A and JMJD1B, are essential for differentiation and maintenance of male germ cells in mice. JMJD1A/JMJD1B-mediated H3K9 demethylation regulates prospermatogonia to spermatogonia transition by ensuring proper gene expression.

SUMMARY 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., Graphical abstract, In brief Using a battery of genome-wide approaches, Tan et al. identify genes regulated by the RHOX10 transcription factor in mouse pro-spermatogonia. Temporal waves of RHOX10 targets, including both direct and indirect targets, are defined. Among these genes are those that participate in a conserved transcription factor signaling cascade driving pro-spermatogonia differentiation.

Mammalian male germ cell development takes place in the testis under the influence of a variety of somatic cells and an incompletely defined paracrine and endocrine influences. Since it is not recapitulated well in vitro, researchers studying spermatogenesis often manipulate the germline by creating transgenic or knockout mice or by administering pharmaceutical agonists/antagonists or inhibitors. The effects of these types of manipulations on germline development can often be determined following microscopic imaging, both of stained and immunostained testis sections. Here, we describe approaches for microscopic analysis of the developing male germline, provide detailed protocols for a variety of immunostaining approaches, and discuss transgenic fluorescent reporter lines for studying the early stages of spermatogenesis. Keywords: Prospermatogonia, Spermatogonia, Testis, Immunofluorescence, Immunohistochemistry

This review focuses on those mouse mutations that cause an effect on the morphology, viability, and/or behavior of primordial germ cells (PGCs) and gonocytes at specific steps of their fetal development up to the start of spermatogenesis, a few days after birth. To restrict the area covered, mice with mutations that cause abnormal hormone levels or mutations of genes not expressed in germ cells that secondarily cause spermatogenic problems are not discussed. To make our literature search as comprehensive as possible, Pubmed was searched for "(primordial germ cells OR prospermatogonia OR prespermatogonia OR gonocytes OR spermatogonia or meiosis or spermiogenesis or spermatogenesis) AND mouse AND (knockout or mutant or transgenic)." This search started at 2003 as mutants created earlier were already retrieved for a previous review. The resulting citations were then further selected for complete or partial arrests at the level of PGCs and/or gonocytes. Fifty-nine protein coding genes and two miRNA coding genes were found that arrest the development of PGCs and gonocytes at specific steps providing a better insight into the regulation of the development of these cells. As to be expected, often problems in fetal germ cell development have an effect on the fertility of the mice at adulthood.

SummaryThe developmental origins of most adult stem cells are poorly understood. Here, we report the identification of a transcription factor—RHOX10—critical for the initial establishment of spermatogonial stem cells (SSCs). Conditional loss of the entire 33-gene X-linked homeobox gene cluster that includes Rhox10 causes progressive spermatogenic decline, a phenotype indistinguishable from that caused by loss of only Rhox10. We demonstrate that this phenotype results from dramatically reduced SSC generation. By using a battery of approaches, including single-cell-RNA sequencing (scRNA-seq) analysis, we show that Rhox10 drives SSC generation by promoting pro-spermatogonia differentiation. Rhox10 also regulates batteries of migration genes and promotes the migration of pro-spermatogonia into the SSC niche. The identification of an X-linked homeobox gene that drives the initial generation of SSCs has implications for the evolution of X-linked gene clusters and sheds light on regulatory mechanisms influencing adult stem cell generation in general.

This review focuses on those mouse mutations that cause an effect on the morphology, viability, and/or behavior of primordial germ cells (PGCs) and gonocytes at specific steps of their fetal development up to the start of spermatogenesis, a few days after birth. To restrict the area covered, mice with mutations that cause abnormal hormone levels or mutations of genes not expressed in germ cells that secondarily cause spermatogenic problems are not discussed. To make our literature search as comprehensive as possible, Pubmed was searched for "(primordial germ cells OR prospermatogonia OR prespermatogonia OR gonocytes OR spermatogonia ormeiosis or spermiogenesis or spermatogenesis) AND mouse AND (knockout or mutant or transgenic)." This search started at 2003 as mutants created earlier were already retrieved for a previous review. The resulting citations were then further selected for complete or partial arrests at the level of PGCs and/or gonocytes. Fifty-nine protein coding genes and two miRNA coding genes were found that arrest the development of PGCs and gonocytes at specific steps providing a better insight into the regulation of the development of these cells. As to be expected, often problems in fetal germ cell development have an effect on the fertility of the mice at adulthood. Summary Sentence: Many gene mutations cause an arrest at specific developmental steps of mouse primordial germ cells and gonocytes, also called prospermatogonia providing valuable clues for the regulation of their development.

The stem cell properties of gonocytes and prospermatogonia at prepubertal stages are still largely unknown: it is not clear whether gonocytes and prospermatogonia are a special cell type or similar to adult undifferentiated spermatogonia. To characterize these cells, we have established transgenic mice carrying EGFP (enhanced green fluorescence protein) cDNA under control of an Oct4 18-kb genomic fragment containing the minimal promoter and proximal and distal enhancers; Oct4 is reported to be expressed in undifferentiated spermatogonia at prepubertal stages. Generation of transgenic mice enabled us to purify gonocytes and prospermatogonia from the somatic cells of the testis. Transplantation studies of testicular cells so far have been done with a mixture of germ cells and somatic cells. This is the first report that establishes how to purify germ cells from total testicular cells, enabling evaluation of cell-autonomous repopulating activity of a subpopulation of prospermatogonia. We show that prospermatogonia differ markedly from adult spermatogonia in both the size of the KIT-negative population and cell cycle characteristics. The GFP+ KIT− fraction of prospermatogonia has much higher repopulating activity than does the GFP+KIT+ population in the adult environment. Interestingly, the GFP+KIT+ population still exhibits repopulating activity, unlike adult KIT-positive spermatogonia. We also show that ALCAM, activated leukocyte cell adhesion molecule, is expressed transiently in gonocytes. Sertoli cells and myoid cells also express ALCAM at the same stage, suggesting that ALCAM may contribute to gonocyte–Sertoli cell adhesion and migration of gonoyctes toward the basement membrane.

Expression of PCNA has been described at various stages of human foetal development

Background In the male germline, neonatal prospermatogonia give rise to spermatogonia, which include stem cell population (undifferentiated spermatogonia) that supports continuous spermatogenesis in adults. Although the levels of DNA methyltransferases change dynamically in the neonatal and early postnatal male germ cells, detailed genome-wide DNA methylation profiles of these cells during the stem cell formation and differentiation have not been reported. Results To understand the regulation of spermatogonial stem cell formation and differentiation, we examined the DNA methylation and gene expression dynamics of male mouse germ cells at the critical stages: neonatal prospermatogonia, and early postntal (day 7) undifferentiated and differentiating spermatogonia. We found large partially methylated domains similar to those found in cancer cells and placenta in all these germ cells, and high levels of non-CG methylation and 5-hydroxymethylcytosines in neonatal prospermatogonia. Although the global CG methylation levels were stable in early postnatal male germ cells, and despite the reported scarcity of differential methylation in the adult spermatogonial stem cells, we identified many regions showing stage-specific differential methylation in and around genes important for stem cell function and spermatogenesis. These regions contained binding sites for specific transcription factors including the SOX family members. Conclusions Our findings show a distinctive and dynamic regulation of DNA methylation during spermatogonial stem cell formation and differentiation in the neonatal and early postnatal testes. Furthermore, we revealed a unique accumulation and distribution of non-CG methylation and 5hmC marks in neonatal prospermatogonia. These findings contrast with the reported scarcity of differential methylation in adult spermatogonial stem cell differentiation and represent a unique phase of male germ cell development. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1833-5) contains supplementary material, which is available to authorized users.