AMH drives chicken ovarian germline stem cell proliferation through the canonical Smad pathway in the avian ovary

The OSCs population in postnatal chickens is a potential reservoir for follicular renewal, however, the signals that regulate OSC maintenance and expansion are not well understood. Here, we identify anti-Müllerian hormone (AMH) as a key mitogen for chicken OSCs and define its signaling mechanism. Following the successful isolation and characterization of OSCs from adult hens, we found that these cells exhibit a distinctive hormonal receptor profile: they highly express the AMH type II receptor (AMHR2) while showing negligible expression of canonical gonadotropin receptors (FSHR, LHR) and estrogen receptors (ESR1, ESR2). Functionally, AMH robustly stimulated OSCs proliferation in both culture and ex vivo ovarian explants. Transcriptomic analysis revealed that AMH treatment alters the expression of 305 genes, coordinating the activation of major pro-proliferative pathways-including ErbB, mTOR, and Wnt signaling-and concurrently suppressing apoptotic and necroptotic pathways. This effect is mediated through phosphorylation of Smad1/5/8 without affecting their mRNA or total protein levels. Critically, pharmacological inhibition of Smad1/5/8 phosphorylation completely blocked AMH-induced proliferation and reversed the AMH-driven upregulation of proliferative genes (MYC, EGR4, CDK1, BLK) as well as the downregulation of the pro-apoptotic gene FASLG. Our results establish a novel, pro-proliferative role for AMH in avian ovarian biology, operating through a conserved AMH expand the OSCs pool by dual regulation of cell cycle and survival pathways. This work reframes AMH's physiological role beyond its classical function in follicle recruitment and offers new insights into the hormonal control of OSCs, with implications for enhancing reproductive longevity in poultry.