OONO<sup>−</sup> reduces the expression of steroidogenic genes and proteins to inhibit estrogen secretion in porcine ovarian granulosa cells

Granulosa cells (GCs), as critical somatic components of ovarian follicles, maintain female reproductive physiology through steroid hormone regulation and follicular development. To investigate the effects of the peroxynitrite anion (OONO⁻) on GCs' function and estrogen (E₂) secretion in sows, GCs were treated with 3-morpholinosydnonimine (SIN-1, an OONO⁻ donor), with subsequent validation using carboxy-PTIO (cPTIO, a NO scavenger) to confirm OONO⁻-specific effects. RNA sequencing (RNA-seq) revealed that OONO⁻-induced transcriptional alterations in GCs, with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealing significant pathway disruptions, including estrogen metabolism and cellular proliferation. The mechanism of the effect of OONO⁻ on GCs' E₂ secretion was further explored. The experimental data showed that OONO⁻ inhibited E₂ secretion by significantly downregulating key steroidogenic regulators (CYP19A1, 3<italic>β</italic>-HSD, and follicle-stimulating hormone receptor [FSHR]). In conclusion, this study indicated that OONO⁻ impairs GCs physiological functions via molecular network dysregulation, leading to inhibited E₂ secretion and affecting follicular development.