Luteinizing hormone promotes functional remodeling of immature porcine Leydig cells associated with DNA methylation changes.

Luteinizing hormone (LH) is a central regulator of testicular development, while the epigenetic mechanisms underlying LH-mediated regulation of immature Leydig cell (iLC) development and function warrant further investigation. In the present study, graded administration of a gonadotropin-releasing hormone agonist (GnRH-a) established differential serum LH levels in Shaziling boars. Reduced LH levels were associated with delayed testicular development and increased body weight, body length, and chest circumference. In vitro, LH treatment promoted G1-to-S phase progression and cell proliferation and increased adenosine triphosphate (ATP) content and total-protein-normalized insulin-like peptide 3 (INSL3) concentrations. Both in vivo and in vitro analyses showed that DNA methyltransferase 3 alpha (DNMT3A) expression was inversely associated with LH levels. Integrated analysis of reduced representation bisulfite sequencing (RRBS) and RNA sequencing (RNA-seq) identified 220 genes exhibiting both differential methylation and differential expression. These genes were enriched in pathways related to Leydig cell function, particularly the cyclic adenosine monophosphate (cAMP) signaling pathway. Functional validation showed that knockdown of the hub genes SLIT-ROBO Rho GTPase activating protein 3 (SRGAP3) and hyaluronan synthase 1 (HAS1) reduced ATP content, while HAS1 knockdown also inhibited cell proliferation. Collectively, these findings identify DNA methylation-associated transcriptional regulation as a potential epigenetic mechanism linking altered LH levels to iLC function.