C-type natriuretic peptide mitigates apoptosis in ovarian granulosa cells through the cGMP pathway independent of PKG signaling

Background Mammalian follicular development is a highly coordinated process governed by hormonal and paracrine cues. Apoptosis of granulosa cells is a central driver of follicular atresia, triggered by insufficient anti-apoptotic signals or activation of pro-apoptotic pathways. C-type natriuretic peptide (CNP), a naturally occurring peptide present in follicular fluid, has been widely recognized as a oocyte meiosis arrester. However, the regulation of CNP on apoptosis of ovarian granulosa cells remains poorly defined. Methods RNA sequencing (RNA-seq) was performed on bovine granulosa cells treated with or without CNP to characterize transcriptomic changes and identify differentially regulated pathways. For functional assays, in vitro culture models of bovine mural granulosa cells, cumulus-oocyte complexes (COCs), and oocytectomized complexes (OOXs) were established to evaluate the effects of CNP on cell survival. Cultured granulosa cells were pretreated with pharmacologic inhibitors to identify the signaling pathways involved in apoptosis regulation by CNP. Apoptosis was assessed by TUNEL assay, while apoptosis-related gene and protein expression levels were analyzed by RT-qPCR and western blotting, respectively. In vitro maturation (IVM) of oocytes was performed to evaluate functional significance of cGMP-dependent protein kinase (PKG) inhibition. Results Transcriptomic profiling revealed that CNP administration significantly downregulated multiple apoptosis-related pathways, including the IL17 signaling pathway, TNF signaling, and NF-κBpathways. Functionally, CNP suppressed apoptosis in both mural granulosa cells and cumulus cells, independent of oocyte presence. Notably, PKG inhibition by KT5823 also reduced granulosa cell apoptosis, and the anti-apoptotic effects of CNP were preserved despite PKG blockade. Conversely, ectopic PKG overexpression enhanced apoptosis, demonstrating a pro-apoptotic role for PKG in this context. Incorporation of KT5823 into the IVM system attenuated cumulus cell apoptosis and improved bovine and ovine oocyte developmental competence. Conclusions These findings reveal that the anti-apoptotic effect of CNP on granulosa cells is independent of PKG signaling. CNP exerts transcriptional suppression of apoptosis-related pathways, while pharmacological PKG inhibition represents a promising approach to enhance the quality of in vitro matured oocytes.