Project description:Spermatogonial stem cells are quiescent, undergo self-renewal or differentiating divisions, thereby forming the cellular basis of spermatogenesis. This cellular development is orchestrated by follicle-stimulating hormone (FSH), through the production of Sertoli cell-derived factors, and by Leydig cell-released androgens. Here, we investigate the transcriptional events induced by Fsh in a steroid-independent manner on the restart of zebrafish (Danio rerio) spermatogenesis ex vivo, using testis from adult males where type A spermatogonia were enriched by estrogen treatment in vivo. Under these conditions, RNA sequencing preferentially detected differentially expressed genes in somatic/Sertoli cells. Fsh-stimulated spermatogonial proliferation was accompanied by modulating several signaling systems (i.e. Tgf-β, Hedgehog, Wnt and Notch pathways). In silico protein-protein interaction analysis indicated a role for Hedgehog family members potentially integrating signals from different pathways during fish spermatogenesis. Moreover, Fsh had a marked impact on metabolic genes, such as lactate and fatty acid metabolism, or on Sertoli cell barrier components. Fish Leydig cells express the Fsh receptor and one of the most robust Fsh-responsive genes was insulin-like 3 (insl3), a Leydig cell-derived growth factor. Follow-up work showed that recombinant zebrafish Insl3 mediated pro-differentiation effects of Fsh on spermatogonia in an androgen-independent manner. Our experimental approach allowed focusing on testicular somatic genes in zebrafish and showed that the activity of signaling systems known to be relevant in stem cell systems was modulated by Fsh, providing promising leads for future work, as exemplified by the studies on Insl3.
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:MafB is a transcription factor acts as an important regulator of the development and differentiation of various organs and tissues. MafB is expressed in sertoli, leydig, and germ cells in adult mice testis. However, analysis of MafB cKO mice testis showed that MAFB is required for the initiation of the first spermatogenic wave but not the subsequent waves. We examined the transcriptome profile of MafB-cKO Sertoli cells during the first spermatogenic wave. Results of the down-regulated genes revealed that some genes are direct targets of MAFB since the presence of MARE sites in their promoters. Thus, they are candidates of maintaining MAFB function in sertoli cells.
Project description:In this study, we aim to figure out if Sertoli cells released exosomes can regulate the survival and steroidogenesis of Leydig cells. We found Sertoli cells released exosomes can cross the blood-testis barrier, deliver their contents into the Leydig cells, and promote the survival of Leydig cells through CCL20.
Project description:Spermatogenesis requires the presence of functional somatic Sertoli cells in the seminiferous tubules of the testis. Sertoli cells provide support and factors necessary for the successful progression of germ cells into spermatozoa. Sertoli cells are regulated to a large degree by the glycoprotein hormone FSH, which is required for the testis to acquire full size and spermatogenic capacity. Signaling events initiated by the binding of FSH to its receptor lead to an alteration of Sertoli cell gene expression. To characterize the changes in gene expression in FSH-treated Sertoli cells, we used the mRNA from these cells to screen Affymetrix U34A rat GeneChip oligonucleotide microarrays. Sertoli cells from 20-d-old rats were cultured in the presence of 25 ng/ml ovine FSH. At 0, 2, 4, 8, and 24 h after the addition of FSH, total RNA was purified and used to prepare biotinylated target, which was hybridized to the U34A rat microarray containing approximately 9000 rat genes. Analysis identified 100-300 transcripts at each time point that were up-regulated or down-regulated by 2-fold or greater. Genes previously reported to be FSH or cAMP regulated in rat Sertoli cells were identified, in addition to numerous genes not reported to be expressed or FSH regulated in Sertoli cells. The expression patterns of five of these genes, encoding nerve growth factor inducible gene B, PRL-1, PC3 nerve growth factor-inducible antiproliferative putative secreted protein, diacylglycerol acyltransferase, and an expressed sequence tag, in FSH- and N,O'-dibutyryl cAMP-treated rat Sertoli cells were confirmed and characterized by Northern blot analysis. Thus, we have begun to define the transcriptome induced and repressed by FSH in rat Sertoli cells, and we have generated datasets of genes available for further analysis in regard to spermatogenesis and Sertoli cell signaling.
Project description:MafB is a transcription factor acts as an important regulator of the development and differentiation of various organs and tissues. MafB is expressed in sertoli, leydig, and germ cells in adult mice testis. However, analysis of MafB cKO mice testis showed no dissruption of spermatogenesis or obvious abnormal structure. Thus, the exact function is unclear. We examined any change in the transcriptome profile of MafB-cKO Sertoli cells. Results of signaling pathway analysis of the downregulated genes was related to immune-function but not spermatogenesis.
Project description:BACKGROUND: Phthalates are manmade industrial additives used mostly as plasticizers. In addition to their deleterious effects on male genital development, population studies have recently documented correlations between phthalates exposure and subtle impacts on reproductive tract development and on the metabolic syndrome in male adults. In mature rodents liver di-(2-ethylhexyl)-phthalate (DEHP) activates the peroxisome proliferators-activated receptor (PPARalpha), a member of the nuclear receptor (NR) superfamily. OBJECTIVES: Using a systems biology approach, we aimed at defining potential mechanisms underlying the impacts of DEHP on adult mouse liver and testis. METHODS: Thus, we performed a parallel analysis of transcript and metabolic profiles in the liver from adult mice exposed to varying DEHP doses. Moreover, we obtained pangenomic mRNA profiles of laser-captured Leydig and Sertoli cells from mature animals exposed to DEHP. RESULTS: Hepatic genes modulated by DEHP are predominantly PPARalpha targets. However, the induction of some prototypic cytochrome P450 genes strongly supports the activation of additional NR pathways. Integration of hepatic transcriptomic and metabonomic profiles further revealed a correlation between the impacts of DEHP on a cluster of genes and metabolites linked to heme synthesis and on a cluster of Rev-erbalpha target genes related to metabolic and circadian clock pathways. Cell-specific effects of DEHP were investigated in the adult testis and a noticeable impact of DEHP was observed on Leydig cells transcriptome. CONCLUSIONS: We report a detailed analysis of DEHP interference with hepatic Constitutive Androstane Receptor (CAR) and Rev-erbalpha pathways and a novel transcriptional impact of DEHP on adult endocrine cells of the testis. Experiment Overall Design: two condition experiment, Sertoli cells-rich areas from DEHP-treated mice vs. Sertoli cells-rich areas from vehicle-treated mice. Biological replicates: 4 DEHP-treated samples and 4 vehicle-treated samples. Dye-swap design. N=4 DEHP-treated vs vehicle-treated x 2 microarrays (dye-swap)=8 microarrays