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

Abstract

Follicle-stimulating hormone (FSH) acts by binding to FSHRs expressed on ovarian granulosa cells and produces estradiol. FSH is essential for female fertility because mice lacking FSH (<i>Fshb KO</i>) are anestrous and infertile. Although several in vitro cell culture and ex vivo approaches combined with pharmacological hormone treatment were used to identify FSH-regulated genes, how FSH orchestrates ovarian gene networks in vivo has not been investigated. Whether FSH-regulated genes display estrous stage-specific expression changes has also not been studied. Here, we functionally rescued <i>Fshb</i> null mice with a gonadotrope-targeted <i>HFSHB</i> transgene and performed RNA-Seq analysis on ovarian RNAs obtained from FSH-intact (<i>WT</i>), FSH-deficient (<i>Fshb KO</i>), and FSH-rescue (<i>HFSHB⁺</i> rescue) mice. By comparing <i>WT</i> vs. <i>Fshb KO</i> and <i>Fshb KO</i> vs. <i>HFSHB⁺</i> rescue ovarian gene expression datasets, we identified FSH-responsive genes in vivo. Cross interrogation of these datasets further allowed us to identify several transcription factors (TFs) and RNA-binding proteins specific to FSH-regulated genes. In an independent set of experiments, we performed RNA-Seq analysis on ovarian RNAs from mice in diestrous (DE), proestrous (PE), and estrous (E) and identified estrous stage-specific ovarian gene expression patterns. Interestingly, many of the FSH-regulated TFs themselves were estrous-stage specifically expressed. We found that ESR2 and GATA6, two known FSH-responsive TFs, and their target genes are reciprocally regulated with distinct patterns of expression in estrous stages. Together, our in vivo models and RNA-Seq analyses identify FSH-regulated ovarian genes in specific estrous stages that are under transcriptional and posttranscriptional control.