Identification of FSH-regulated and estrous stage-specific transcriptional networks in mouse ovaries

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. Overall design: RNA-seq samples from WT, Fshb (+/-), Fshb (-/-), Fshb (-/-) HFshb (+/+) and WT cycling mice staged in DE, P, and E stages of estrus cycle.