Screening of key regulatory factors in apoptosis of granulosa cells in healthy and atretic pig follicles based on RNA-seq

Abstract

The molecular mechanism underlying abnormal follicular atresia remains to be elucidated. Research conducted to date has indicated that ovarian granulosa cell (GC) apoptosis is a significant contributor to follicular atresia. Abnormalities in follicular atresia can result in decreased reproductive efficiency in pigs. It is evident that comparative studies focusing on healthy, early atresia and progressively atresia follicles, particularly from the perspective of follicular atresia induced by ovarian GC apoptosis, have not yet been reported. Specifically, the use of RNA-seq technology to systematically analyse differentially expressed genes and associated signaling pathways in GCs at different stages of atresia remains unexplored. The research was divided into three distinct groups: control, early atresia, and progressively atresia. Key genes and signaling pathways were elucidated through RNA-seq. A Venn diagram revealed 86 overlapping genes that were upregulated during early atresia and downregulated during progressively atresia. Additionally, another 47 overlapping genes were found to be downregulated during early atresia and upregulated during progressively atresia. These 133 overlapping genes were significantly enriched in multiple KEGG pathways. Additionally, in conjunction with the key gene-related network diagram, 6 signaling pathways related to ovarian GC apoptosis were further screened out. These pathways include ovarian steroidogenesis, progesterone-mediated oocyte maturation, gonadotropin-releasing hormone signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, and chemokine signaling pathway. Altogether, 12 key genes were identified: SCARB1, IGF1, PRKACA, ADCY6, LDLR, PLD1, CXCL10, IRF7, ISG15, RNase L, OAS2, and STAT1. Six of these genes were randomly selected for qRT-PCR verification, and their expression levels were found to be consistent with the sequencing results. Consequently, the identification of key regulatory factors involved in the apoptotic process provides a theoretical foundation for investigating the mechanisms underlying abnormal follicular atresia in pigs.