Abstract / Summary
Zinc oxide nanoparticles (ZnO NPs) are increasingly used as feed additives and antimicrobial alternatives in livestock production, but their potential reproductive toxicity remains insufficiently understood. Autophagy and apoptosis are central regulators of follicular atresia and granulosa cell fate, but whether ZnO NPs disrupt these processes in granulosa cells and how they interact mechanistically remain unclear. In this study, we tested whether ZnO NPs couple autophagy to apoptosis in mouse granulosa cells (GCs) through Notch3 signaling. Primary GCs were exposed to ZnO NPs and analyzed via viability assays, RT-qPCR, immunoblotting, and RNA-seq, with pharmacological perturbation of autophagy, apoptosis, and Notch signaling. ZnO NPs reduced viability and induced autophagic and apoptotic responses. The autophagy inhibitor 3-methyladenine (3-MA) partially attenuated ZnO NPs-induced autophagy-associated alterations, improved viability, and attenuated apoptosis. Conversely, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) reduced apoptosis and partially modulated autophagy-associated markers but did not fully reverse the ZnO NPs-induced autophagic response. Transcriptomics identified altered Notch signaling, and ZnO NPs reduced NOTCH3, the NOTCH3 intracellular domain, and HES1. 3-MA partially restored this axis, linking autophagy-associated alterations to Notch3 signaling suppression. Consistent with a protective role for Notch signaling, the γ-secretase inhibitor DAPT aggravated ZnO NPs-induced viability loss and apoptosis. Together, these pharmacological interventions support a model in which ZnO NPs-induced autophagic alterations contribute to apoptosis through suppression of Notch3 signaling, while apoptosis inhibition only partially modulates the autophagic response. This autophagy–Notch3–apoptosis regulatory network provides a framework for evaluating the ovarian toxicity of ZnO NPs.