Genome-wide identification of estrogen receptor binding sites reveals novel estrogen-responsive pathways in adult male germ cells

Spermatogenesis occurs in the seminiferous epithelium that shows presence of estrogen receptors alpha (ERα) and beta (ERβ), both of which regulate gene transcription by binding to the DNA. Hormone responsive phases of spermatogenesis are well documented; yet, the genes regulated remain inexplicit. To study the regulation of genes by estrogen in male germ cells, we performed chromatin immunoprecipitation (ChIP) sequencing for ERα and ERβ under normal physiological conditions. We observed a total of 27,221 peaks in ERα and 20,926 peaks in ERβ. Majority of the peaks were present in the intronic regions and located 20kb upstream or downstream from the transcription start site (TSS). Upon pathway analysis, the genes enriched showed involvement in several biological pathways. Genes involved in pathways whose role in spermatogenesis is unexplored were validated; these included prolactin, GnRH and oxytocin signalling. All the genes showed presence of estrogen response elements (EREs) and majority of them showed significant enrichment by ChIP-qPCR. Functional validation using seminiferous tubule culture after treatment with receptor specific agonist and antagonist confirmed the regulation of these genes by estrogen through its receptors. This study provides new insights about the estrogen regulated genes during spermatogenesis. Examination of 2 different nuclear receptors (estrogen receptor alpha and beta) in germ cells of adult rat testes