ERα-NOS/NO/cGMP/PKG-mTOR/KITL/FOXO3a signaling cascade mediates estrogen-induced primordial follicle activation in goats.

With increasing consumer demand for goat milk, accelerating the breeding of elite dairy goats through livestock embryo engineering has become imperative. Elucidating estrogen's molecular mechanism in primordial follicle activation is crucial for improving in vitro embryo production to enhance breeding efficiency. This study aimed to elucidate the molecular mechanisms by which estrogen regulates the activation of primordial follicles in goats. By supplementing the ovarian cortical fragment culture system with estrogen and small molecular reagents, and utilizing experimental techniques including hematoxylin and eosin staining, immunohistochemical staining, immunofluorescence staining, reverse transcription quantitative PCR, western blot analysis, and ELISA, we demonstrated that the protein levels of phosphorylated (p) mechanistic target of rapamycin kinase (mTOR) mTOR, p-ribosomal protein S6 (rpS6), and KIT ligand (KITL) were significantly higher in granulosa cells of growing follicles than in those of primordial follicles in goat ovaries. Supplementing 17β-estradiol (E2) in cultured goat ovarian cortex fragments markedly increased activated follicle proportion. Mechanistically, E2 activated the mTOR/KITL/FOXO3a signaling cascade, wherein mTOR/KITL mediated E2-induced FOXO3a phosphorylation and primordial follicle activation. Furthermore, nitric oxide synthase (NOS) mRNA and protein were detected in goat ovaries, and inhibition of NOS/nitric oxide (NO) signaling reversed E2-driven follicle activation. We found that E2 upregulated protein expression and phosphorylation of endothelial NOS (eNOS) and neuronal NOS (nNOS) through the nuclear estrogen receptor pathway, triggering NO/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signaling to induce mTOR/KITL/FOXO3a activation. Specific activation of estrogen receptor α (ERα) by the agonist propyl pyrazole triol (PPT) induced NO/cGMP/PKG signaling, which mediated PPT promotion of mTOR/KITL/forkhead box O3a (FOXO3a) activation and follicle development. Collectively, estrogen activates primordial follicles in dairy goats through ERα-dependent upregulation of NOS, initiating NO/cGMP/PKG → mTOR/KITL/FOXO3a signaling cascades. This study deciphers estrogen's regulatory mechanism in follicular activation, offering novel insights for increasing oocyte yield via in vitro culture in goat breeding.