Nanos2-regulated mRNAs in the cultured spermatogonial stem cells (SSCs)
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ABSTRACT: Spermatogonial stem cells are foundation of spermatogenesis. In our previous study, we observed a rapid degeneration of spermatogenesis by conditional knockout (cKO) of Nanos2 in adult spermatogonia, but the underlying mechanisms are largely unknown. While, conditional overexpression (cOE) of Nanos2 also inhibited germ cell development and stayed in undifferentiation status. Here we used cultured germline stem cells (GSCs) to study the mechanisms of Nanos2 in spermatogonia. We compared Nanos2 expression in cultured GSCs with in vivo isolated Nanos2 positive cells, and found the level of Nanos2 is significantly lower in GSCs as compared to Nanos2 positive cells. Thus we generated Nano2 cOE GSCs lines. By conditional inducing expression of Nanos2 we compared gene expression profile to control GSCs. These data provide the first evidence of Nanos2 regulating genes in spermatogonial stem cells.
Project description:Little is known of the fundamental processes governed by epigenetic mechanisms in the supplier cells of spermatogenesis, the spermatogonial stem cells (SSCs). The histone H3 lysine demethylase KDM1A is expressed in spermatogonia. We hypothesized that KDM1A serves in transcriptional regulation of SSCs and fertility. Using a conditional deletion of Kdm1a [conditional knockout (cKO)] in mouse spermatogonia, we determined that Kdm1a is essential for spermatogenesis as adult cKO males completely lack germ cells. Analysis of postnatal testis development revealed that undifferentiated and differentiating spermatogonial populations form in Kdm1a-cKO animals, yet the majority fail to enter meiosis. Loss of germ cells in the cKO was rapid with none remaining by postnatal day (PND) 21. To gain insight into the mechanistic implications of Kdm1a ablation, we isolated PND 6 spermatogonia enriched for SSCs and analyzed their transcriptome by RNA sequencing. Loss of Kdm1a was associated with altered transcription of 1206 genes. Importantly, differentially expressed genes between control and Kdm1a-cKO animals included those that are essential for SSC and progenitor maintenance and spermatogonial differentiation. The complete loss of fertility and failure to establish spermatogenesis indicate that Kdm1a is a master controller of gene transcription in spermatogonia and is required for SSC and progenitor maintenance and differentiation.
Project description:Here, gene profiles in rat spermatogonial stem cell lines are compared to publicly available mouse, monkey and human spermatogonial gene profiles. Interestingly, rat spermatogonia expressed metabolic control factors Foxa1, Foxa2 and Foxa3. Germline Foxa2 was enriched in Gfra1Hi and Gfra1Low undifferentiated A-single spermatogonia. Foxa2-bound loci in spermatogonial chromatin were over-represented by conserved stemness genes (Dusp6, Gfra1, Etv5, Rest, Nanos2, Foxp1) that intersect bioinformatically with conserved glutathione/pentose phosphate metabolism genes (Tkt, Gss, Gclc, Gclm, Gpx1, Gpx4, Fth), marking elevated spermatogonial GSH:GSSG. Cystine-uptake and intracellular conversion to cysteine typically couple glutathione biosynthesis to pentose phosphate metabolism. Rat spermatogonia, curiously, displayed poor germline stem cell viability in cystine-containing media, and, like primate spermatogonia, exhibited reduced transsulfuration pathway markers. Exogenous cysteine, cysteine-like mercaptans, somatic testis cells and ferroptosis inhibitors counteracted the cysteine starvation-induced spermatogonial death and stimulated spermatogonial growth factor activity in vitro.
Project description:Spermatogonial differentiation is a developmental process that is essential for spermatogenesis, but the molecular and cellular changes that germ cells must undergo to transition from undifferentiated spermatogonia to differentiating spermatogonia remain largely undefined. Retinoic acid (RA) is necessary and sufficient for spermatogonial differentiation. Using the postnatal mouse testis, we examine the transcriptome changes that accompany spermatogonial differentiation. Spermatogenesis was synchronized by administration of potent and selective RA synthesis inhibitor; as a result, testes contained only undifferentiated spermatogonia. Then, the inhibitor was discontinued, and mice were given a single dose of exogenous RA to initiate spermatogonial differentiation. We measured transcriptomes in FACS-enriched germ cells either before RA administration, when the cells correspond to Aal spermatogonia (and a minor contribution of spermatogonial stem cells) or at two points after RA administration, when the cells correspond to A1 or A3 differentiating spermatogonia. The results of this study reveal the full transcriptome changes accompanying spermatogonial differentiation in the mouse.
Project description:To identify Nanos2-associated mRNAs, we performed microarray analysis of mRNAs coprecipitated with FLAG-tagged NANOS2 with postnatal testis from Tg-Nanos2 enhancer-3xFLAG-Nanos2-3'UTR at P7.
Project description:To identify Nanos2-associated mRNAs, we performed microarray analysis of mRNAs coprecipitated with FLAG-tagged NANOS2 with male gonad from Tg-Nanos2 enhancer-3xFLAG-Nanos2-3'UTR at E14.5.
Project description:To investigate the biochemical function of NANOS2, we performed expression microarray analysis of the embyornic male gonad of Nanos2 hetero, Nanos2 KO, and Nanos2 KO_Tg 3×FLAG-tagged Nanos2-ΔN10, which is truncated form of Nanos2 in the N-terminal region, transgenic mice.
Project description:To identify Nanos2-associated mRNAs, we performed microarray analysis of mRNAs coprecipitated with FLAG-tagged NANOS2 with male gonad from Tg-Nanos2 enhancer-3xFLAG-Nanos2-3'UTR at E14.5. Biological duplicates were examined at each sample.
Project description:To investigate the biochemical function of NANOS2, we performed expression microarray analysis of the embyornic male gonad of Nanos2 hetero, Nanos2 KO, and Nanos2 KO_Tg 3M-CM-^WFLAG-tagged Nanos2-M-NM-^TN10, which is truncated form of Nanos2 in the N-terminal region, transgenic mice. 3M-CM-^WFLAG-tagged Nanos2-M-NM-^TN10 protein function was validated by rescue experiment in the Nanos2-null male gonad of mouse embryo at E14.5. Biological duplicates were examined at each genotype, Nanos2 hetero, Nanos2 KO, and Nanos2 KO_Tg 3M-CM-^WFLAG-tagged Nanos2-M-NM-^TN10 for each experiment.
Project description:The in vitro derivation and propagation of spermatogonial stem cells (SSCs) from pluripotent stem cells (PSCs) is a key goal in reproductive science. We show here that when aggregated with embryonic testicular somatic cells (reconstituted testes), primordial germ cell-like cells (PGCLCs) induced from mouse embryonic stem cells differentiate into spermatogonia-like cells in vitro and are expandable as cells that resemble germline stem cells (GSCs), a primary cell line with SSC activity. Remarkably, GSC-like cells (GSCLCs), but not PGCLCs, colonize adult testes and, albeit less effectively than GSCs, contribute to spermatogenesis and fertile offspring. Whole-genome analyses reveal that GSCLCs exhibit aberrant methylation at vulnerable regulatory elements, including those critical for spermatogenesis, which may restrain their spermatogenic potential. Our study establishes a strategy for the in vitro derivation of SSC activity from PSCs, which, we propose, relies on faithful epigenomic regulation.
Project description:The in vitro derivation and propagation of spermatogonial stem cells (SSCs) from pluripotent stem cells (PSCs) is a key goal in reproductive science. We show here that when aggregated with embryonic testicular somatic cells (reconstituted testes), primordial germ cell-like cells (PGCLCs) induced from mouse embryonic stem cells differentiate into spermatogonia-like cells in vitro and are expandable as cells that resemble germline stem cells (GSCs), a primary cell line with SSC activity. Remarkably, GSC-like cells (GSCLCs), but not PGCLCs, colonize adult testes and, albeit less effectively than GSCs, contribute to spermatogenesis and fertile offspring. Whole-genome analyses reveal that GSCLCs exhibit aberrant methylation at vulnerable regulatory elements, including those critical for spermatogenesis, which may restrain their spermatogenic potential. Our study establishes a strategy for the in vitro derivation of SSC activity from PSCs, which, we propose, relies on faithful epigenomic regulation.