GDNF sustains female germline stem cell homeostasis during ovarian aging in laying hens.

Ovarian aging is a major factor contributing to the decline in egg production in laying hens, particularly during the late laying period. Female germline stem cells (FGSCs), which possess self-renewal and differentiation capacities, are considered important for replenishing the oocyte pool and maintaining ovarian function. However, the mechanisms regulating FGSC function during ovarian aging remain largely unknown. In this study, we investigated the role of glial cell line-derived neurotrophic factor (GDNF) in FGSC proliferation. We found that aged hens with high laying performance exhibited significantly greater FGSC abundance and higher ovarian expression of GDNF and its receptors, GFRα1 and RET, than low-laying hens. In vitro experiments further showed that GDNF significantly promoted FGSC proliferation. Transcriptome analysis revealed that GDNF treatment was associated with enhanced PI3K-Akt and mTOR signaling and reduced FoxO- and p53-related signaling, indicating a transcriptional shift toward cell proliferation and survival. Consistently, GDNF increased the expression of multiple proliferation-associated genes and significantly expanded endogenous FGSC populations in ovarian organ cultures. Collectively, these findings demonstrate that exogenous GDNF promotes FGSC proliferation, while higher GDNF expression is associated with greater FGSC abundance in high-laying aged hens. These results suggest that GDNF may contribute to FGSC maintenance by modulating signaling networks related to cell proliferation and survival, providing new insights into the cellular and molecular mechanisms underlying ovarian aging and reproductive decline in laying hens.