Srd5a1is Differentially Regulated and Methylated During Prepubertal Development in the Ovary and Hypothalamus

Abstract

5α-reductase-1 catalyzes production of various steroids, including neurosteroids. We reported previously that expression of its encoding gene, Srd5a1, drops in murine ovaries and hypothalamic pre-optic area (POA) after early-life immune stress, seemingly contributing to delayed puberty and ovarian follicle depletion, while in their ovaries the first intron was more methylated at two CpGs. Here we hypothesized that this CpG-containing locus comprises a methylation-sensitive transcriptional enhancer for Srd5a1. We found that ovarian Srd5a1 mRNA increased 8-fold and methylation of the same two CpGs decreased upto 75% between post-natal days (PND) 10-30. Estradiol (E2) levels rise during this pre-pubertal stage, and exposure of ovarian cells to E2 increased Srd5a1 expression. Chromatin immunoprecipitation (ChIP) in an ovarian cell line confirmed ESR1 binding to this differentially-methylated genomic region, and also enrichment of the enhancer modification, H3K4me1. Targeting dCas9-DNMT3 to this locus increased CpG2 methylation 2.5-fold and abolished the Srd5a1 response to E2. In the POA, Srd5a1 mRNA levels decreased 70% between PND 7-10 and then remained constant without correlation to CpG methylation levels. Srd5a1 mRNA levels did not respond to E2 in hypothalamic GT1-7 cells, even after dCas9-TET1 reduced CpG1 methylation by 50%. The neonatal drop in POA Srd5a1 expression occurs at a time of increasing glucocorticoids, and treatment of GT1-7 cells with dexamethasone reduced Srd5a1 mRNA levels, while ChIP confirmed GR binding at the enhancer. Our findings on the tissue-specific regulation of Srd5a1 and its methylation-sensitive control by E2 in the ovaries illuminate epigenetic mechanisms underlying reproductive phenotypic variation which impact life-long health.