Novel circNSD2 promotes proliferation and testosterone synthesis while inhibiting apoptosis in bovine Leydig cells

Leydig cells (LCs) are the primary cells responsible for testosterone synthesis in male animals, and their steroidogenic function is crucial for testicular development and spermatogenesis in bulls. Circular RNAs (circRNAs) play important roles in various biological processes and reproductive development; however, their functions in bovine LCs remain largely unexplored. Our previous whole-transcriptome sequencing of calf and adult bovine testes identified a differentially expressed circRNA, circ_0027599 (designated circNSD2 after its host gene NSD2), that was highly expressed in adult testes. To investigate its function, circNSD2 was overexpressed in bovine LCs, and its effects on cell proliferation, apoptosis, and testosterone synthesis were assessed. The results showed that circNSD2 overexpression significantly 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 CDK2). Concurrently, circNSD2 overexpression significantly reduced the number of apoptotic cells, downregulated the expression of pro-apoptotic genes (BAX and Caspase3), and upregulated the anti-apoptotic gene (Bcl2). Furthermore, circNSD2 overexpression promoted testosterone secretion and increased the expression of key steroidogenic genes (STAR, CYP11A1, CYP17A1, and HSD17B3). In conclusion, circNSD2 promotes proliferation, inhibits apoptosis, and enhances testosterone synthesis and secretion in bovine LCs. These findings reveal a potential regulatory role of circNSD2 in bovine testicular development and provide a theoretical foundation and molecular target for improving reproductive performance in breeding bulls.