Estrogen Receptor Signaling Alters Sperm DNA Methylation Landscape in Adult Male Rats

Estrogen through its receptors, ERα and ERβ, regulate various aspects of spermatogenesis and male fertility. Because the sperm epigenome is an important contributing factor to male fertility, we evaluated the effects of estrogen signaling activation through the ERs on sperm DNA methylome in adult rats. Whole genome-bisulfite sequencing in caudal sperm DNA was performed. The differentially methylated CpG (DMC) sites were validated by pyrosequencing, and the expression of differentially methylated genes (DMGs) was evaluated in testis by quantitative RT-PCR. Activation of ERα signaling brought about large-scale changes in the sperm DNA methylome compared to ERβ. There were 28074 DMCs and 5189 DMGs obtained after ERα agonist 4,4',4''-(4-Propyl-[1H] pyrazole-1,3,5-triyl) (PPT) treatment, whereas 1492 DMCs and 336 DMGs for ERβ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN). In genic regions, most of the DMCs were intronic, followed by promoter and upstream regions. DMCs were distributed around the transcription start site and in transcription factor-binding regions, implicating their plausible role in gene expression regulation. Genes important for spermatogenesis were identified and validated which showed a similar trend of differential methylation as obtained by whole genome-bisulfite sequencing. The expression of the DMGs was also found to be altered in the testis. There was a considerable overlap (14% to 50%) of PPT DMGs with the DMGs reported to be affected in clinical conditions of male infertility. This study highlights the role of ERs in shaping the sperm epigenome and that aberrant estrogen signaling could be a contributing factor in clinical conditions of male infertility.