Crucial genes and pathways in chicken germ stem cell differentiation
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ABSTRACT: The male germ cell differentiation is a subtle and complex regulation processes, currently its regulatory mechanism is not fully understood. In our experiment, we performed the first comprehensive genome wide analyses of the crucial genes in three kinds of crucial cells (ESCs, PGCs and SSCs) associated with the male germ cells differentiation. We identified thousands of differentially expressed genes (DEGs) in this process andwe choosed 173 candidate genes involved in the differentiation and metabolic processes, like GAL9, AMH, PLK1, PSMD7 and so on. Further exploration found that the candidate genes express patterns were the same between in vitro induction experiments and transcriptome results. Our results clues to the mechanistic basis of male germ cell differentiation and provides an important reference for future studies.
Project description:The male germ cell differentiation is a subtle and complex regulation processes, currently its regulatory mechanism is not fully understood. In our experiment, we performed the first comprehensive genome wide analyses of the crucial genes in three kinds of crucial cells (ESCs, PGCs and SSCs) associated with the male germ cells differentiation. We identified thousands of differentially expressed genes (DEGs) in this process andwe choosed 173 candidate genes involved in the differentiation and metabolic processes, like GAL9, AMH, PLK1, PSMD7 and so on. Further exploration found that the candidate genes express patterns were the same between in vitro induction experiments and transcriptome results. Our results clues to the mechanistic basis of male germ cell differentiation and provides an important reference for future studies. Gene expression in chicken germ stem cells was measured at different stages of development. Three independent experiments were performed at each stage (ESCs, PGCs and SSCs).
Project description:Male spermatogenesis is sustained by homeostatic balance between the self-renewal and differentiation of spermatogonial stem cells (SSCs), which is dependent on the strict regulation of transcriptional factor and chromatin modulator gene expression. Chromodomain helicase DNA-binding protein 4 (CHD4) is highly expressed in SSCs but roles in mouse spermatogenesis are unexplored. Here, we report that the germ-cell-specific deletion of Chd4 resulted in complete infertility in male mice, with rapid loss of SSCs and excessive germ cell apoptosis. Chd4-knockdown in cultured SSCs also promoted the expression of apoptosis-related genes and thereby activated the tumor necrosis factor signaling pathway. Mechanistically, CHD4 occupies the genomic regulatory region of key apoptosis-related genes including Jun and Nfkb1. Together, our findings reveal the determinant role of CHD4 in SSCs survival in vivo, which will offer insight into the pathogenesis of male sterility and potential novel therapeutic targets.
Project description:Male spermatogenesis is sustained by homeostatic balance between the self-renewal and differentiation of spermatogonial stem cells (SSCs), which is dependent on the strict regulation of transcriptional factor and chromatin modulator gene expression. Chromodomain helicase DNA-binding protein 4 (CHD4) is highly expressed in SSCs but roles in mouse spermatogenesis are unexplored. Here, we report that the germ-cell-specific deletion of Chd4 resulted in complete infertility in male mice, with rapid loss of SSCs and excessive germ cell apoptosis. Chd4-knockdown in cultured SSCs also promoted the expression of apoptosis-related genes and thereby activated the tumor necrosis factor signaling pathway. Mechanistically, CHD4 occupies the genomic regulatory region of key apoptosis-related genes including Jun and Nfkb1. Together, our findings reveal the determinant role of CHD4 in SSCs survival in vivo, which will offer insight into the pathogenesis of male sterility and potential novel therapeutic targets.
Project description:Protein phosphatase 6 (PP6) is a member of the PP2A-like subfamily, which plays significant roles in numerous fundamental biological activities. We found that PPP6C plays important roles in male germ cells recently. Spermatogenesis is supported by the Sertoli cells in seminiferous epithelium. In this study, we crossed Ppp6cF/F mice with AMH-Cre mice to gain mutant mice with specific depletion of the Ppp6c gene in the Sertoli cells. We discovered that the PPP6C cKO male mice were absolutely infertile and germ cells were largely lost during spermatogenesis. By combing phosphoproteome with bioinformatics analysis, we showed that the phosphorylation status of β-catenin at S552 (a marker of adherens junctions) was significantly upregulated in mutant mice. Abnormal β-catenin accumulation resulted in impaired testicular junction integrity, thus led to abnormal structure and functions of BTB. Taken together, our study reveals a novel function for PPP6C in male germ cell survival and differentiation by regulating the cell-cell communication through dephosphorylating β-catenin at S552.
Project description:Spermatogonial stem cells (SSCs) provide a continuous spermatogenesis and male fertility. However, 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. Specific deletion of Srsf1 in mouse germ cells impairs self-renewal and homing of SSCs leading to male infertility. Whole-mount staining data showed the absence of germ cells in the testes of adult cKO mice, which indicates severe non-obstructive azoospermia (NOA) in cKO mice. Expression of SSC-related genes (Gfra1, Pou5f1, Plzf, Dnd1, Stra8, and Taf4b) was significantly reduced in the testes of conditional knockout (cKO) mice. CLIP-seq data found that SSC-related genes (Plzf, Id4, Setdb1, Stra8, Tial1, Bcas2, Ddx5, Srsf10, Uhrf1, and Bud31) were bound by SRSF1 in the mouse testes. Moreover, multi-omics analysis suggests that SRSF1 directly binds and regulates the expression of Tial1 via AS to implement SSCs self-renewal and differentiation. Collectively, our data reveal the critical role of an SRSF1-mediated AS in SSCs self-renewal and differentiation, which may provide a framework to elucidate the molecular mechanisms of the post-transcriptional network underlying SSCs.
Project description:In this study, we have demonstrated, for the first time, that overexpression of three genes of DAZ family members, namely DAZL, DAZ2, and BOULE, could directly reprogram human Sertoli cells to the cells with the biochemical phenotypes, the self-renewal and differentiation capacities of human SSCs. This study thus offers invaluable male gametes for treating the infertility of azoospermia patients. We propose a new concept that human somatic cells can be converted to become male germline stem cells by the defined factors. Here we have demonstrated that the overexpression of DAZL, DAZ2, and BOULE could directly reprogram human Sertoli cells into the cells with the characteristics of human spermatogonial stem cells (SSCs), as evidenced 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 spermatocyte and haploid spermatids in vitro. Human Sertoli cells-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 & 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.
Project description:Fsh-mediated regulation of zebrafish spermatogenesis includes modulating the expression of testicular growth factors. Here, we study if and how two Sertoli cell-derived Fsh-responsive growth factors, anti-Müllerian hormone (Amh; inhibiting steroidogenesis and germ cell differentiation) and insulin-like growth factor 3 (Igf3; stimulating germ cell differentiation), cooperate in regulating spermatogonial development. In dose response and time course experiments with primary testis tissue cultures, Fsh upregulated igf3 transcript levels and down-regulated amh transcript levels; igf3 transcript levels were more rapidly up-regulated and responded to lower Fsh concentrations than were required to decrease amh mRNA levels. Quantification of immunoreactive Amh and Igf3 on testis sections showed that Fsh increased slightly Igf3 staining but decreased clearly Amh staining. Studying the direct interaction of the two growth factors showed that Amh compromised Igf3-stimulated proliferation of type A (both undifferentiated [Aund] and differentiating [Adiff]) spermatogonia. Also the proliferation of those Sertoli cells associated with Aund spermatogonia was reduced by Amh. To gain more insight into how Amh inhibits germ cell development, we examined Amh-induced changes in testicular gene expression by RNA sequencing. The majority (69%) of the differentially expressed genes was down-regulated by Amh, including several stimulators of spermatogenesis, such as igf3 and steroidogenesis-related genes. At the same time, Amh increased the expression of inhibitory signals, such as inha and id3, or facilitated prostaglandin E2 (PGE2) signaling. Evaluating one of the potentially inhibitory signals, we indeed found in tissue culture experiments that PGE2 promoted the accumulation of Aund at the expense of Adiff and B spermatogonia. Our data suggest that an important aspect of Fsh bioactivity in stimulating spermatogenesis is implemented by restricting the different inhibitory effects of Amh and by counterbalancing them with stimulatory signals, such as Igf3
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.
Project description:Mammalian spermatogenesis and male fertility are sustained by a small population of spermatogonial stem cells (SSCs) in the testis. In particular, germ cells are highly sensitive to chemotherapies and radiation, placing male cancer patients at a higher risks of treatment-induced infertility. While SSCs surive the treatment may restore the germline and ferility, it remains unknown how SSCs mediate the regenerative responses to re-establish the germline. In this study, we used a mouse model of chemotherapy-induced germline damage and recovery to identify differentially expressed genes in homeostatic (from untreated control mice) versus regenerative SSCs (busulfan-treated mice) and potential effectors driving the initiation of regeneration.