In vivo evidence for FOXO3-mediated suppression of primordial follicle activation in chickens

Primordial follicle activation is a critical determinant of female reproductive lifespan. Although forkhead box O3 (FOXO3) conservatively regulates primordial follicle dormancy in mammals, its role in avian species remains unknown. Using the herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV)-mediated germ cell ablation system, we generated germline chimeric chickens carrying FOXO3-overexpressing (OE-FOXO3) primordial germ cells (PGCs). Overexpression was confirmed by RT-qPCR and Western blot. Compared with WT, OE-FOXO3 PGCs showed inhibited proliferation but retained germline identity and migration. In vivo, OE-FOXO3 chimeras exhibited delayed primordial follicle activation. Quantitative follicle staging at 3, 4, 7, and 21 days post-hatch (dph) revealed that OE-FOXO3 ovaries consistently maintained a higher proportion of primordial follicles and fewer growing follicles. By 21 dph, this difference was most evident: OE-FOXO3 follicles remained dormant with sustained nuclear/perinuclear FOXO3, whereas control follicles progressed, showing cytoplasmic or peripheral granulosa cell FOXO3 staining. At 3 dph, OE-FOXO3 ovaries contained fewer germ cell clusters and reduced apoptosis. By 4 dph, coinciding with the onset of primordial follicle activation, Western blot analysis revealed that the PI3K/AKT signaling was significantly activated in the control ovaries, whereas this activation was suppressed in the OE-FOXO3 ovaries. Collectively, these findings provide the first in vivo evidence that FOXO3 functions as a negative regulator of primordial follicle activation in chickens. Notably, this suppressed activation state was associated with reduced PI3K/AKT signaling. This study extends the functional conservation of FOXO3 beyond mammals, offering new insights into avian ovarian reserve establishment.