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

Abstract

Background: Mammalian follicular development is regulated by endocrine and paracrine factors, and apoptosis of granulosa cells represents a major cause of follicular atresia. C-type natriuretic peptide (CNP), a paracrine factor in follicular fluid, maintains meiotic arrest of oocytes through activation of the NPR2–cGMP pathway. Protein kinase G (PKG) is a principal downstream effector of cGMP, but its function in granulosa cell apoptosis has not been fully characterized. Methods: Bovine granulosa cells were treated with CNP, cGMP or the PKG inhibitor KT5823. Transcriptomic analysis was performed to identify differentially expressed genes and signaling pathways. The effects of CNP on apoptosis were further evaluated in mural granulosa cells, cumulus–oocyte complexes (COCs), and oocytectomized complexes (OOXs). Apoptosis was examined by TUNEL assay, and expression of apoptosis-related genes and proteins was determined by RT-qPCR and Western blotting. The influence of PKG inhibition on oocyte developmental competence was assessed during in vitro maturation (IVM). Results: CNP treatment downregulated multiple apoptosis-related pathways, including TNF, IL-17, and NF-κB signaling. CNP reduced apoptosis in both granulosa and cumulus cells, and this effect was not affected by PKG inhibition. Overexpression of PKG increased apoptosis, suggesting a pro-apoptotic role of PKG in granulosa cells. Addition of KT5823 during IVM decreased apoptosis in cumulus cells and improved blastocyst development and ICM cell numbers in bovine and ovine oocytes. Conclusions: CNP suppresses apoptosis of granulosa cells through a PKG-independent mechanism involving transcriptional downregulation of apoptosis-related pathways. PKG appears to promote, rather than mediate, apoptosis in this system. Inhibition of PKG during IVM may improve oocyte quality and provide a useful reference for optimization of in vitro maturation systems.