Synergistic roles of follicle-stimulating hormone and testosterone in Sertoli cell proliferation and testicular function in roosters

Spermatogenesis relies on the intricate interactions between testicular somatic cells and germ cells. While follicle-stimulating hormone (FSH) and testosterone (T) are considered pivotal regulatory factors in this process, the mechanisms by which these hormones regulate the spermatogonial stem cell (SSC) microenvironment via Sertoli cells during testicular development in roosters remain unclear. Here, we examined how FSH and T regulate Sertoli-cell proliferation and the expression of glial cell-derived neurotrophic factor (GDNF) during rooster testicular development. We observed that at 4 weeks of age, the seminiferous tubules of roosters were fully developed, accompanied by the migration of spermatocytes into the lumen. At this stage, both serum and testicular levels of FSH and T were elevated in parallel. In highly purified Sertoli cells cultures, FSH treatment (25-100 ng/mL) significantly promoted proliferation and cell-cycle progression, and induced GDNF protein expression in a time- and dose-dependent manner, with the strongest response observed at 75 ng/mL. Although T did not significantly affect Sertoli cell proliferation, it markedly upregulated GDNF protein expression via the androgen receptor (AR) signaling pathway. Specifically, T (0.1-0.4 ng/mL) increased GDNF without altering proliferation, and this effect was blunted by pharmacological AR blockade (enzalutamide), indicating AR dependence. Furthermore, combined treatment with FSH and T demonstrated a synergistic effect, amplifying the expression of GDNF protein. In vivo experiments revealed that subcutaneous FSH administration increased Sertoli-cell proliferative activity (PCNA) and testicular growth, whereas testosterone administration enhanced testicular GDNF expression. Notably, combined FSH+T treatment produced the highest testicular GDNF level among groups, consistent with a synergistic effect in vivo. These findings suggest that FSH drives the expansion of Sertoli cell numbers by promoting their proliferation and cell cycle progression, while testosterone enhances the functional maturation of Sertoli cells through the regulation of GDNF expression. The synergistic actions of these two hormones together optimize the spermatogenic microenvironment, providing novel insights into the hormonal regulation of male reproduction.