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

ABSTRACT: 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.

SUBMITTER: Tan K

PROVIDER: S-EPMC8357189 | biostudies-literature |

REPOSITORIES: biostudies-literature

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RHOX10 Drives Mouse Spermatogonial Stem Cell Establishment Through A Transcription Factor Signaling Cascade

Project description: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 stimulating 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 we design: iSLAMseq. Together, these approaches identify temporal waves of direct targets, as well as secondary-target genes. Many of the RHOX10-regulated genes encode proteins with known roles in SSCs. Using an in vitro ProSG differentiation assay that we develop, 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.

2021-06-18 | GSE165372 | GEO

The Homeobox Transcription Factor RHOX10 Drives Mouse Spermatogonial Stem Cell Establishment.

Project description:The 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.

| S-EPMC5063083 | biostudies-literature

RHOX10 Drives Mouse Spermatogonial Stem Cell Establishment Through A Transcription Factor Signaling Cascade

Project description:RHOX10 Drives Mouse Spermatogonial Stem Cell Establishment Through A Transcription Factor Signaling Cascade

| PRJNA694173 | ENA

ROS amplification drives mouse spermatogonial stem cell self-renewal.

Project description:Reactive oxygen species (ROS) play critical roles in self-renewal division for various stem cell types. However, it remains unclear how ROS signals are integrated with self-renewal machinery. Here, we report that the MAPK14/MAPK7/BCL6B pathway creates a positive feedback loop to drive spermatogonial stem cell (SSC) self-renewal via ROS amplification. The activation of MAPK14 induced MAPK7 phosphorylation in cultured SSCs, and targeted deletion of Mapk14 or Mapk7 resulted in significant SSC deficiency after spermatogonial transplantation. The activation of this signaling pathway not only induced Nox1 but also increased ROS levels. Chemical screening of MAPK7 targets revealed many ROS-dependent spermatogonial transcription factors, of which BCL6B was found to initiate ROS production by increasing Nox1 expression via ETV5-induced nuclear translocation. Because hydrogen peroxide or Nox1 transfection also induced BCL6B nuclear translocation, our results suggest that BCL6B initiates and amplifies ROS signals to activate ROS-dependent spermatogonial transcription factors by forming a positive feedback loop.

| S-EPMC6448598 | biostudies-literature

Identification of a Transcription Factor that Promotes Spermatogonial Stem Cell Establishment

Project description:Spermatogonial stem cells (SSCs) are critical for maintaining spermatogenesis throughout adult life. Little is known about how SSCs are first generated. Here, we report the identification of a transcription factor—RHOX10—that promotes the initial establishment of SSCs. We were led to this discovery because we found that conditional loss of a large X-linked gene cluster comprised of 33 related homeobox genes, including Rhox10, causes defects predicted if SSCs fail to be generated or maintained. Remarkably, KO of only Rhox10 elicits SSC-related defects indistinguishable from KO of the entire gene cluster. Using a battery of approaches, including single cell-RNAseq analysis, we determined that loss of Rhox10 causes accumulation of undifferentiated germ cells—Pro-spermatogonia (ProSG)—at a time when they normally would form SSCs. The identification of a transcription factor that drives the initial generation of SSCs has potential therapeutic applications for infertility.

2016-09-27 | GSE82174 | GEO

Establishment of a proteome profile and identification of molecular markers for mouse spermatogonial stem cells.

Project description:Spermatogonial stem cells (SSCs) are undifferentiated cells that are required to maintain spermatogenesis throughout the reproductive life of mammals. Although SSC transplantation and culture provide a powerful tool to identify the mechanisms regulating SSC function, the precise signalling mechanisms governing SSC self-renewal and specific surface markers for purifying SSCs remain to be clearly determined. In the present study, we established a steady SSC culture according to the method described by Shinohara's lab. Fertile progeny was produced after transplantation of cultured SSCs into infertile mouse testis, and the red fluorescence exhibited by the culture cell membranes was stably and continuously transmitted to the offspring. Next, via advanced mass spectrometry and an optimized proteomics platform, we constructed the proteome profile, with 682 proteins expressed in SSCs. Furthermore bioinformatics analysis showed that the list contained several known molecules that are regulated in SSCs. Several nucleoproteins and membrane proteins were chosen for further exploration using immunofluorescence and RT-PCR. The results showed that SALL1, EZH2, and RCOR2 are possibly involved in the self-renewal mechanism of SSCs. Furthermore, the results of tissue-specific expression analysis showed that Gpat2 and Pld6 were uniquely and highly expressed in mouse testes and cultured SSCs. The cellular localization of PLD6 was further explored and the results showed it was primarily expressed in the spermatogonial membrane of mouse testes and cultured SSCs. The proteins identified in this study form the basis for further exploring the molecular mechanism of self-renewal in SSCs and for identifying specific surface markers of SSCs.

| S-EPMC4369810 | biostudies-literature

Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat.

Project description:Self-renewal of spermatogonial stem cells (SSCs) is the foundation for maintenance of spermatogenesis throughout life in males and for continuation of a species. The molecular mechanism underlying stem cell self-renewal is a fundamental question in stem cell biology. Recently, we identified growth factors necessary for self-renewal of mouse SSCs and established a serum-free culture system for their proliferation in vitro. To determine whether the stimulatory signals for SSC replication are conserved among different species, we extended the culture system to rat SSCs. Initially, a method to assess in vitro expansion of SSCs was developed by using flow cytometric analysis, and, subsequently, we found that a combination of glial cell line-derived neurotrophic factor, soluble glial cell line-derived neurotrophic factor-family receptor alpha-1 and basic fibroblast growth factor supports proliferation of rat SSCs. When cultured with the three factors, stem cells proliferated continuously for >7 months, and transplantation of the cultured SSCs to recipient rats generated donor stem cell-derived progeny, demonstrating that the cultured stem cells are normal. The growth factor requirement for replication of rat SSCs is identical to that of mouse; therefore, the signaling factors for SSC self-renewal are conserved in these two species. Because SSCs from many mammals, including human, can replicate in mouse seminiferous tubules after transplantation, the growth factors required for SSC self-renewal may be conserved among many different species. Furthermore, development of a long-term culture system for rat SSCs has established a foundation for germ-line modification of the rat by gene targeting technology.

| S-EPMC1242328 | biostudies-literature

Establishment of cell lines with porcine spermatogonial stem cell properties.

Project description:BackgroundSpermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to mature functional spermatozoa, being the only adult stem cells in the males that can transmit genetic information to the next generation. Porcine SSCs hold great value in transgenic pig production and in establishment of porcine models for regenerative medicine. However, studies and applications of porcine SSCs have been greatly hampered by the low number of SSCs in the testis as well as the lack of an ideal stable long-term culture system to propagate porcine SSCs perpetually.ResultsIn the present study, by lentiviral transduction of plasmids expressing the simian virus 40 (SV40) large T antigen into porcine primary SSCs, we developed two immortalized cell lines with porcine SSC attributes. The established cell lines, with the expression of porcine SSC and germ cell markers UCHL1, PLZF, THY1, VASA and DAZL, could respond to retinoic acid (RA), and could colonize the recipient mouse testis without tumor formation after transplantation. The cell lines displayed infinite proliferation potential, and have now been cultured for more than 7 months and passaged for over 35 times without morphological abnormalities.ConclusionsWe have for the first time established porcine SSC lines that could provide abundant cell sources for mechanistic studies on porcine SSC self-renewal and differentiation, thereby facilitating development of an optimal long-term culture system for porcine primary SSCs and their application to animal husbandry and medicine.

| S-EPMC7146966 | biostudies-literature

CXCL12-CXCR4 signaling is required for the maintenance of mouse spermatogonial stem cells.

Project description:Continual spermatogenesis relies on the activities of a tissue-specific stem cell population referred to as spermatogonial stem cells (SSCs). Fate decisions of stem cells are influenced by their niche environments, a major component of which is soluble factors secreted by support cells. At present, the factors that constitute the SSC niche are undefined. We explored the role of chemokine (C-X-C motif) ligand 12 (CXCL12) signaling via its receptor C-X-C chemokine receptor type 4 (CXCR4) in regulation of mouse SSC fate decisions. Immunofluorescent staining for CXCL12 protein in cross sections of testes from both pup and adult mice revealed its localization at the basement membrane of seminiferous tubules. Within the undifferentiated spermatogonial population of mouse testes, a fraction of cells were found to express CXCR4 and possess stem cell capacity. Inhibition of CXCR4 signaling in primary cultures of mouse undifferentiated spermatogonia resulted in SSC loss, in part by reducing proliferation and increasing the transition to a progenitor state primed for differentiation upon stimulation by retinoic acid. In addition, CXCL12-CXCR4 signaling in mouse SSCs was found to be important for colonization of recipient testes following transplantation, possibly by influencing homing to establish stem-cell niches. Furthermore, inhibition of CXCR4 signaling in testes of adult mice impaired SSC maintenance, leading to loss of the germline. Collectively, these findings indicate that CXCL12 is an important component of the growth factor milieu of stem cells in mammalian testes and that it signals via the CXCR4 to regulate maintenance of the SSC pool.

| S-EPMC4074255 | biostudies-literature

Determination phase at transition of gonocytes to spermatogonial stem cells improves establishment efficiency of spermatogonial stem cells in domestic cats.

Project description:The development of germ cells has not been entirely documented in the cat especially the transition phase of the gonocyte to the spermatogonial stem cell (G/SSC). The aims of study were to examine testicular development and to identify the G/SSC transition in order to isolate and culture SSCs in vitro. Testes were divided into 3 groups according to donor age (I, < 4 months; II, 4-6 months; and III, > 6 months). In Exp. 1, we studied testicular development by histology, transmission electron microscopy and immunohistochemistry. In Exp. 2, we determined the expression of GFR?-1, DDX-4 and c-kit and performed flow cytometry. The SSCs isolated from groups II and III were characterized by RT-PCR and TEM (Exp. 3). Chronological changes in the G/SSC transition were demonstrated. The size, morphology and ultrastructure of SSCs were distinguishable from those of gonocytes. The results demonstrated that group II contained the highest numbers of SSCs per seminiferous cord/tubule (17.66 ± 2.20%) and GFR?-1(+) cells (14.89 ± 5.66%) compared with the other groups. The findings coincided with an increased efficiency of SSC derivation in group II compared with group III (74.33 ± 2.64% vs. 23.33 ± 2.23%). The colonies expressed mRNA for GFRA1, ZBTB16, RET and POU5F1. Our study found that the G/SSC transition occurs at 4-6 months of age. This period is useful for isolation and improves the establishment efficiency of cat SSCs in vitro.

| S-EPMC4685225 | biostudies-literature

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