miR-182-5p regulates proliferation and senescence in chicken Leydig cells via the Wnt/β-catenin pathway by targeting FOXN3.

The reproductive performance of roosters is an important factor influencing production efficiency and genetic progress in the poultry industry. Leydig cells (LCs) support male reproductive function through testosterone synthesis and secretion; however, the post-transcriptional mechanisms regulating chicken LCs function remain incompletely understood. Previous studies have shown that testicular miR-182-5p expression is higher in 200-day-old than in 500-day-old roosters, suggesting a possible association with age-related changes in testicular function. This study aims to investigate the role of miR-182-5p in vitro. miR-182-5p overexpression promoted proliferation, inhibited senescence, and elevated testosterone levels in chicken LCs, whereas miR-182-5p inhibition produced the opposite effects. A dual-luciferase reporter assay performed in the chicken fibroblast cell line (DF-1 cells) demonstrated that miR-182-5p targets the 3'untranslated region (3'UTR) of FOXN3. FOXN3 knockdown produced effects consistent with those of miR-182-5p overexpression, supporting FOXN3 as a functional downstream mediator of miR-182-5p. Moreover, miR-182-5p enhanced Wnt/β-catenin signaling, and co-transfection with FOXN3 overexpression, which inhibited this pathway, attenuated the miR-182-5p-induced increases in proliferation and testosterone levels as well as the decrease in senescence. These findings support a miR-182-5p/FOXN3/Wnt/β-catenin regulatory axis that modulates proliferation, senescence, and testosterone levels in chicken LCs in vitro. Further age-comparative and in vivo studies are warranted to determine whether this mechanism contributes to natural reproductive aging or influences reproductive performance in roosters.