PRM1 modulates proliferation, apoptosis, and testosterone synthesis in bovine Leydig cells

Testicular development is crucial for spermatogenesis and reproductive capacity of bulls. The synthesis and secretion of testosterone by Leydig cells influence testicular physiological functions. The protamine 1 (PRM1) gene is highly expressed in adult bull testes; however, its effects on bovine Leydig cells remain unclear. In this study, bovine Leydig cells were isolated and cultured, followed by overexpression and knockdown of PRM1. The effects of PRM1 on cell proliferation, apoptosis, and testosterone synthesis were assessed by quantitative real-time PCR, western blotting, Cell Counting Kit-8, EdU staining, flow cytometry, and ELISA. Overexpression of PRM1 enhanced cell viability, increased the proportion of cells in the S phase, and upregulated the expression of proliferation-related genes proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinase 2 (CDK2) (P < 0.01); reduced the number of apoptotic cells and downregulated the expression of apoptosis-related genes BAX and Caspase3 (P < 0.05); and promoted testosterone secretion as well as the expression of testosterone synthesis-related genes cytochrome P450 family 17 subfamily A member 1 (CYP17A1), 17β-hydroxysteroid dehydrogenase 3 (HSD17B3), and steroidogenic acute regulatory protein (STAR) (P < 0.05). Conversely, PRM1 knockdown decreased cell viability, reduced the proportion of cells in the S phase, and downregulated the expression of PCNA and CDK2 (P < 0.01); increased the number of apoptotic cells and upregulated the expression of BAX and Caspase3 (P < 0.05); suppressed testosterone secretion along with the expression of CYP17A1, HSD17B3, and STAR (P < 0.05). Overall, PRM1 promotes cell proliferation and inhibits cell apoptosis in bovine Leydig cells, while enhancing testosterone synthesis and secretion. This study provides a theoretical foundation and potential applications for improving semen quality in bulls.