MiR-27b-3p suppresses proliferation and testosterone synthesis in goat Leydig cells by activating the AMPK pathway...

Leydig cells (LCs) are the primary sites for testosterone synthesis in male animals, with their proliferation and steroidogenic capacity directly affecting reproductive system development. Understanding the molecular regulation of testicular development in male goats supports semen quality improvement and treatment of testicular dysfunction, while also informing strategies to enhance reproductive efficiency in goat breeding. Although microRNAs (miRNAs) are known to influence LC function by targeting key genes, the specific role of miR-27b-3p remains unclear. Our study in Qianbei Ma goats identified a critical regulatory axis during testicular development, where miR-27b-3p expression decreased while PPARG and AMPK signaling increased. Functionally, overexpressing miR-27b-3p significantly inhibited LC proliferation-shown by reduced Cyclin B1/E2 levels (P < 0.05)-and testosterone synthesis, indicated by lower HSD3B1 and STAR protein levels (P < 0.05), while also downregulating PPARG (P < 0.05). Inhibiting miR-27b-3p produced opposite effects. Further mechanistic studies confirmed that PPARG acts upstream of AMPK signaling to regulate LC proliferation and steroidogenesis (P < 0.05-0.005). AMPK activation via AICAR partially rescued the defects caused by PPARG silencing (P < 0.05). These findings demonstrate that miR-27b-3p suppresses Leydig cell proliferation and testosterone production by targeting PPARG, thereby modulating AMPK signaling. This regulatory mechanism offers new insights for developing therapeutic strategies targeting male goat reproductive dysfunction.