Differential susceptibility of male and female germ cells to glucocorticoid-mediated signaling [TAGseq_KO_ALL]
While physiologic stress has long been known to impair mammalian reproductive capacity through hormonal dysregulation, mounting evidence now suggests that stress experienced prior to or during gestation may also negatively impact the health of future offspring. Rodent models of gestational physiologic stress can induce neurologic and behavioral phenotypes that persist for up to three generations, suggesting that stress signals can induce lasting epigenetic changes in the germline. Treatment with glucocorticoid stress hormones is sufficient to recapitulate the transgenerational phenotypes seen in physiologic stress models. These hormones are known to bind and activate the glucocorticoid receptor (GR), a ligand-inducible transcription factor, thus implicating GR-mediated signaling as a potential contributor to the transgenerational inheritance of stress-induced phenotypes. Here we demonstrate dynamic spatiotemporal regulation of GR expression in the mouse germline, showing expression in the fetal oocyte as well as the perinatal and adult spermatogonia. Functionally, we find that fetal oocytes are intrinsically buffered against changes in GR signaling, as neither genetic deletion of GR nor GR agonism with dexamethasone altered the transcriptional landscape or the progression of fetal oocytes through meiosis. In contrast, our studies revealed that the male germline is susceptible to glucocorticoid-mediated signaling, specifically by regulating RNA splicing within the spermatogonia, although this does not abrogate fertility. Together, our work suggests a sexually dimorphic function for GR in the germline, and represents an important step towards understanding the mechanisms by which stress can modulate the transmission of genetic information through the germline. Overall design: To investigate the role of glucocorticoid signaling in female germ cells, we performed RNA-seq on germ cells of the ovary following genetic deletion of the glucocorticoid receptor (GR). To generate embryos homozygous for the GR knockout allele (GR KO/KO), heterozygous females (GR KO/+) were crossed to heterozygous males also carrying an Oct4-GFP transgene (GR KO/+ ; Tg:Oct4 GFP/GFP) to facilitate FACS sorting of germ cells.  To generate embryos with a conditional deletion of GR (GR cKO/cKO), females homozygous for an exon 3 floxed GR allele (GR flox/flox) were crossed to similar males that were also heterozygous for the germ cell specific Oct4CreERT2 allele (GR flox/flox ; Oct4CreERT2+ ; Tg:Oct4 GFP/GFP). Pregnant dams were injected at E10.5 with 125 µg / g tamoxifen to induce recombination and deletion of GR exon 3 specifically in the germ cells. For both full body knockout and GR conditional deletion models, pregnant dams were dissected the morning of E17.5, genotyped, and Oct4 GFP+ germ cells were FACS sorted from individual embryos prior to performing low input 3' Tag-Seq.