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Spermatogenesis is sustained by homeostatic balance between the self-renewal and differentiation of spermatogonial stem cells, which is dependent on the strict regulation of transcription factor and chromatin modulator gene expression. Chromodomain helicase DNA-binding protein 4 is highly expressed in spermatogonial stem cells but roles in mouse spermatogenesis are not fully understood. Here, we report that the germ-cell-specific deletion of chromodomain helicase DNA-binding protein 4 resulted in complete infertility in male mice, with rapid loss of spermatogonial stem cells and excessive germ cell apoptosis. Chromodomain helicase DNA-binding protein 4-knockdown in cultured spermatogonial stem cells also promoted the expression of apoptosis-related genes and thereby activated the tumor necrosis factor signaling pathway. Mechanistically, chromodomain helicase DNA-binding protein 4 occupies the genomic regulatory region of key apoptosis-related genes, including Jun and Nfkb1. Together, our findings reveal the determinant role of chromodomain helicase DNA-binding protein 4 in spermatogonial stem cells survival in vivo, which will offer insight into the pathogenesis of male sterility and potential novel therapeutic targets., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

More than a decade ago, the ENCODE and NIH Epigenomics Roadmap consortia organized large multilaboratory efforts to profile the epigenomes of >110 different mammalian somatic cell types. This generated valuable publicly accessible datasets that are being mined to reveal genome-wide patterns of a variety of different epigenetic parameters. This consortia approach facilitated the powerful and comprehensive multiparametric integrative analysis of the epigenomes in each cell type. However, no germ cell types were included among the cell types characterized by either of these consortia. Thus, comprehensive epigenetic profiling data are not generally available for the most evolutionarily important cells, male and female germ cells. We discuss the need for reproductive biologists to generate similar multiparametric epigenomic profiling datasets for both male and female germ cells at different developmental stages and summarize our recent effort to derive such data for mammalian spermatogonial stem cells and progenitor spermatogonia., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Although reactive oxygen species (ROS) are required for spermatogonial stem cell (SSC) self-renewal, they induce DNA damage and are harmful to SSCs. However, little is known about how SSCs protect their genome during self-renewal. Here, we report that Ogg1 is essential for SSC protection against ROS. While cultured SSCs exhibited homologous recombination-based DNA double-strand break repair at levels comparable with those in pluripotent stem cells, they were significantly more resistant to hydrogen peroxide than pluripotent stem cells or mouse embryonic fibroblasts, suggesting that they exhibit high levels of base excision repair (BER) activity. Consistent with this observation, cultured SSCs showed significantly lower levels of point mutations than somatic cells, and showed strong expression of BER-related genes. Functional screening revealed that Ogg1 depletion significantly impairs survival of cultured SSCs upon hydrogen peroxide exposure. Thus, our results suggest increased expression of BER-related genes, including Ogg1, protects SSCs from ROS-induced damage., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Humanin (HN) is a mitochondrial-derived peptide that protects many cells/tissues from damage. We previously demonstrated that HN reduces stress-induced male germ cell apoptosis in rodents. HN action in neuronal cells is mediated through its binding to a trimeric cell membrane receptor composed of glycoprotein 130 (gp130), IL-27 receptor subunit (IL-27R, also known as WSX-1/TCCR), and ciliary neurotrophic factor receptor subunit (CNTFR). The mechanisms of HN action in testis remain unclear. We demonstrated in ex-vivo seminiferous tubules culture that HN prevented heat-induced germ cell apoptosis was blocked by specific anti-IL-27R, anti-gp130, and anti-EBI-3, but not by anti-CNTFR antibodies significantly. The cytoprotective action of HN was studied by using groups of il-27r-/- or ebi-3-/- mice administered the following treatment: (1) vehicle; (2) a single intraperitoneal (IP) injection of HN peptide; (3) testicular hyperthermia; and (4) testicular hyperthermia plus HN. We demonstrated that HN inhibited heat-induced germ cell apoptosis in wildtype but not in il-27r-/- or ebi-3-/- mice. HN restored heat-suppressed STAT3 phosphorylation in wildtype but not il-27r-/- or ebi-3-/- mice. Dot blot analyses showed the direct interaction of HN with IL-27R or EBI-3 peptide. Immunofluorescence staining showed the co-localization of IL-27R with HN and gp130 in Leydig cells and germ cells. We conclude that the anti-apoptotic effects of HN in mouse testes are mediated through interaction with EBI-3, IL-27R, and activation of gp130, whereas the role of CNTFR needs further studies. This suggests a multicomponent tissue-specific receptor for HN in the testis and links HN action with the IL-12/IL-27 family of cytokines., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Spermatogonial stem cells (SSCs) undergo continuous self-renewal division in response to self-renewal factors. The present study identified ephrin type-A receptor 2 (EPHA2) on mouse SSCs and showed that supplementation of glial cell-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), which are both SSC self-renewal factors, induced EPHA2 expression in cultured SSCs. Spermatogonial transplantation combined with magnetic-activated cell sorting or fluorescence-activated cell sorting also revealed that EPHA2 was expressed in SSCs. Additionally, ret proto-oncogene (RET) phosphorylation levels decreased following the knockdown (KD) of Epha2 expression via short hairpin ribonucleic acid (RNA). Although the present immunoprecipitation experiments did not reveal an association between RET with EPHA2, RET interacted with FGFR2. The Epha2 KD decreased the proliferation of cultured SSCs and inhibited the binding of cultured SSCs to laminin-coated plates. The Epha2 KD also significantly reduced the colonization of testis cells by spermatogonial transplantation. EPHA2 was also expressed in human GDNF family receptor alpha 1-positive spermatogonia. The present results indicate that SSCs express EPHA2 and suggest that it is a critical modifier of self-renewal signals in SSCs., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The self-renewal, proliferation, and differentiation of the spermatogonial populations must be finely coordinated in the mammalian testis, as dysregulation of these processes can lead to subfertility, infertility, or the formation of tumors. There are wide gaps in our understanding of how these spermatogonial populations are formed and maintained, and our laboratory has focused on identifying the molecular and cellular pathways that direct their development. Others and we have shown, using a combination of pharmacologic inhibitors and genetic models, that activation of mTOR complex 1 (mTORC1) is important for spermatogonial differentiation in vivo. Here, we extend those studies to directly test the germ cell-autonomous requirement for mTORC1 in spermatogonial differentiation. We created germ cell conditional knockout mice for "regulatory associated protein of MTOR, complex 1" (Rptor), which encodes an essential component of mTORC1. While germ cell KO mice were viable and healthy, they had smaller testes than littermate controls, and no sperm were present in their cauda epididymides. We found that an initial cohort of Rptor KO spermatogonia proliferated, differentiated, and entered meiosis (which they were unable to complete). However, no self-renewing spermatogonia were formed, and thus the entire germline was lost by adulthood, resulting in Sertoli cell-only testes. These results reveal the cell autonomous requirement for RPTOR in the formation or maintenance of the foundational self-renewing spermatogonial stem cell pool in the mouse testis and underscore complex roles for mTORC1 and its constituent proteins in male germ cell development., (© The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

miRNAs play important roles during mammalian spermatogenesis. However, the function of most miRNAs in spermatogenesis and the underlying mechanisms remain unknown. Here, we report that miR-202 is highly expressed in mouse spermatogonial stem cells (SSCs), and is oppositely regulated by Glial cell-Derived Neurotrophic Factor (GDNF) and retinoic acid (RA), two key factors for SSC self-renewal and differentiation. We used inducible CRISPR-Cas9 to knockout miR-202 in cultured SSCs, and found that the knockout SSCs initiated premature differentiation accompanied by reduced stem cell activity and increased mitosis and apoptosis. Target genes were identified with iTRAQ-based proteomic analysis and RNA sequencing, and are enriched with cell cycle regulators and RNA-binding proteins. Rbfox2 and Cpeb1 were found to be direct targets of miR-202 and Rbfox2 but not Cpeb1, is essential for the differentiation of SSCs into meiotic cells. Accordingly, an SSC fate-regulatory network composed of signaling molecules of GDNF and RA, miR-202 and diverse downstream effectors has been identified., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Precise separation of spermatogonial stem cells (SSCs) from progenitor spermatogonia that lack stem cell activity and are committed to differentiation remains a challenge. To distinguish between these spermatogonial subtypes, we identified genes that exhibited bimodal mRNA levels at the single-cell level among undifferentiated spermatogonia from Postnatal Day 6 mouse testes, including Tspan8, Epha2, and Pvr, each of which encode cell surface proteins useful for cell selection. Transplantation studies provided definitive evidence that a TSPAN8-high subpopulation is enriched for SSCs. RNA-seq analyses identified genes differentially expressed between TSPAN8-high and -low subpopulations that clustered into multiple biological pathways potentially involved in SSC renewal or differentiation, respectively. Methyl-seq analysis identified hypomethylated domains in the promoters of these genes in both subpopulations that colocalized with peaks of histone modifications defined by ChIP-seq analysis. Taken together, these results demonstrate functional heterogeneity among mouse undifferentiated spermatogonia and point to key biological characteristics that distinguish SSCs from progenitor spermatogonia., (© 2016 by the Society for the Study of Reproduction, Inc.)

In many mammalian species, surface markers have been used to obtain enriched populations of spermatogonial stem cells (SSCs) for assisted reproduction and other applications; however, little is known about the expression patterns of feline SSCs. In this study, we assessed expression of the SSC surface markers commonly used in other species, KIT, ITGA6, CD9, GFRalpha1, ADGRA3, and THY1, in addition to the less frequently used pluripotent markers TRA-1-60, TRA-1-81, SSEA-1, and SSEA-4 in SSCs of both prepubertal and adult domestic cats (Felis catus). To further characterize cat SSCs, we sorted cells using SSC-specific markers and evaluated the expression of the pluripotent transcription factors NANOG, POU5F1, and SOX2 and the proto-oncogene MYC within these populations. We concluded that SSC surface markers used in other mammalian species were not specific for identifying cat SSCs. However, the pluripotent markers we evaluated were more specific to cat spermatogonia, and the presence of SSEA-1 and SSEA-4 in fewer and primarily individual cells suggests that these two markers may be used for enrichment of cat SSCs. The expression of pluripotent transcription factors at mRNA level by single-stained cells positive for SSEA-4 and by dual-stained cells positive for both GFRalpha1 and SSEA-4 reflects the undifferentiated stage of cat SSCs. The absence of transcription factors in double-stained cells positive for only one marker implies the loss of the stem cell-like identity with the loss of either GFRalpha1 or SSEA-4. Further investigation is warranted to elucidate the biological characteristics of these spermatogonial subpopulations., (© 2016 by the Society for the Study of Reproduction, Inc.)

The F-box and leucine-rich repeat protein 10 (Fbxl10) gene encodes a protein that catalyzes demethylation of H3K4 and H3K36. In this study, we show the important roles of FBXL10 as a histone demethylase in sustainable sperm production using mice in which the JmjC domain of Fbxl10 was deleted (Fbxl10(DeltaJ/DeltaJ)). In histological analysis, testis sections from 10-wk-old Fbxl10(DeltaJ/DeltaJ) mice appeared normal. On the other hand, testes from 7-mo-old Fbxl10(DeltaJ/DeltaJ) mice contained a greater ratio of seminiferous tubules exhibiting degeneration of spermatogenesis. Further analysis using an in vitro spermatogonia culture system, that is, germline stem cells (GSCs), revealed that Fbxl10(DeltaJ/DeltaJ) GSCs expressed a significantly higher level of P21 and P19 mRNA, cyclin-dependent kinase inhibitors and also known as cellular senescence markers, than wild-type (WT) GSCs. Furthermore, the ratio of Fbxl10(DeltaJ/DeltaJ) GSCs in G0/G1 phase was higher and the ratios in S and G2/M phases were lower than the corresponding ratios of WT GSCs, and the doubling speed of Fbxl10(DeltaJ/DeltaJ) GSCs was significantly slower than that of WT GSCs. In addition to these in vitro results, an in vivo study indicated that recovery of spermatogenesis after a transient reduction in the number of testicular germ cells by busulfan treatment was significantly slower in Fbxl10(DeltaJ/DeltaJ) mice than in WT mice. These data suggest that Fbxl10 plays important roles in long-term sustainable spermatogenesis via regulating cell cycle., (© 2016 by the Society for the Study of Reproduction, Inc.)

Stem cell niche is a specialized microenvironment crucial to self-renewal. The testis in Drosophila contains two different types of stem cells, the germline stem cells and the somatic cyst stem cells that are sustained by their respective niche signals, thus is a good system for studying the interaction between the stem cells and their hosting niche. The JAK-STAT and BMP pathways are known to play critical roles in the self-renewal of different kinds of stem cells, but the roles of several other pathways have emerged recently in a complex signaling network in the testis niche. Reports of independent observations from three research groups have uncovered an important role of Hedgehog (Hh) in the Drosophila testis niche. In this review, we summarize these recent findings and discuss the interplay between the Hh signaling mechanisms and those of the JAK-STAT and BMP pathways. We also discuss directions for further investigation.