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Discovery of FSH-regulated and estrus stage-specific transcriptional networks in mouse ovaries

ABSTRACT: Follicle-stimulating hormone (FSH) acts by binding to FSHRs on ovarian granulosa cells to produce estradiol. FSH is essential for female fertility because mice lacking FSH (Fshb KO) are anestrus, and infertile. Although several in vitro and ex vivo cell culture approaches combined with pharmacological hormone treatments were used to identify FSH-regulated genes, how FSH orchestrates ovarian gene networks in vivo has not been investigated. Similarly, whether FSH-regulated genes display estrus stage-specific expression changes has also not been studied. Here, we functionally rescued Fshb null mice with a well-established gonadotrope-targeted HFSHB transgene-mediated in vivo genetic rescue approach and performed RNA-Seq analysis on ovarian RNAs obtained from FSH-intact (WT), FSH-deficient (Fshb KO) and FSH-rescue (HFSHB+ rescue) mice. By comparing WT vs Fshb KO and Fshb KO vs Fshb KO HFSHB+ ovarian gene expression datasets, we identified bona-fide FSH-responsive genes in vivo in mouse ovaries. Cross interrogation of WT vs Fshb KO and Fshb KO vs Fshb KO HFSHB+ data sets further allowed us to identify several transcription factors and RNA-binding proteins specifically in FSH - regulated genes. In an independent set of experiments, we performed RNA-Seq analysis on ovarian RNAs obtained from mice in diestrus (DE), proestrus (PE) and estrus (E) stages and identified estrus stage-specific ovarian gene expression patterns. Interestingly, many of the FSH-regulated transcription factors themselves were estrus-stage specifically expressed. We found that ESR2 and GATA6, two known FSH-responsive granulosa cell-specific transcription factors and their target genes are reciprocally regulated with distinct patterns of expression in estrus stages. Thus, our in vivo genetic models and RNA-Seq analyses identify FSH-regulated ovarian genes in specific estrus stages that are under transcriptional and post-transcriptional control.

ORGANISM(S): Mus musculus

PROVIDER: GSE268258 | GEO | 2025/01/22

REPOSITORIES: GEO

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Project description:The forkhead box transcription factor FOXO1 is highly expressed in granulosa cells of growing follicles but is down-regulated by FSH in culture or by LH-induced luteinization in vivo. To analyze the function of FOXO1, we infected rat and mouse granulosa cells with adenoviral vectors expressing two FOXO1 mutants: a gain-of-function mutant FOXOA3 that has two serine residues and one threonine residue mutated to alanines rendering this protein constitutively active and nuclear, and a FOXOA3-mutant DNA-binding domain (mDBD) in which the DBD is mutated. The infected cells were then treated with vehicle or FSH for specific time intervals. Infection of the granulosa cells was highly efficient, caused only minimal apoptosis, and maintained FOXO1 protein at levels of the endogenous protein observed in cells before exposure to FSH. RNA was prepared from control and adenoviral infected cells exposed to vehicle or FSH for 12 and 24 h. Affymetrix microarray and database analyses identified, and real time RT-PCR verified, that genes within the lipid, sterol, and steroidogenic biosynthetic pathways (Hmgcs1, Hmgcr, Mvk, Sqle, Lss, Cyp51, Tm7sf2, Dhcr24 and Star, Cyp11a1, and Cyp19), including two key transcriptional regulators Srebf1 and Srebf2 of cholesterol biosynthesis and steroidogenesis (Nr5a1, Nr5a2), were major targets induced by FSH and suppressed by FOXOA3 and FOXOA3-mDBD in the cultured granulosa cells. By contrast, FOXOA3 and FOXOA3- mDBD induced expression of Cyp27a1 mRNA that encodes an enzyme involved in cholesterol catabolism to oxysterols. The genes up-regulated by FSH in cultured granulosa cells were also induced in granulosa cells of preovulatory follicles and corpora lutea collected from immature mice primed with FSH (equine choriogonadotropin) and LH (human choriogonadotropin), respectively. Conversely, Foxo1 and Cyp27a1 mRNAs were reduced by these same treatments. Collectively, these data provide novel evidence that FOXO1 may play a key role in granulosa cells to modulate lipid and sterol biosynthesis, thereby preventing elevated steroidogenesis during early stages of follicle development. Experiment Overall Design: Immature rat granulosa cells were affected with either control (GFP) or FOXO1 mutant adenovirus (FOXOA3 and FOXOA3-mDBD) for 4 hours. Then the cells were treated with either FSH or vehicle for 24 hours. The gene expression profiles for the four treatment samples (control, control +FSH, FOXOA3 + FSH, FOXOA3-mDBD + FSH) were compared using Rat Genome 230.2 Arrays with one array per sample.

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Discovery of FSH-regulated and estrus stage-specific transcriptional networks in mouse ovaries

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