2,5-Hexanedione Affects Ovarian Granulosa Cells in Swine by Regulating the CDKN1A Gene: A Transcriptome Analysis

SIMPLE SUMMARY: Porcine ovarian granulosa cells (pGCs), the main somatic cells in the follicular microenvironment, are close to oocytes. Under the action of various regulatory factors and exogenous substances, their abnormal proliferation, apoptosis and oxidative stress directly affect the quality of oocytes and embryonic development. Although 2,5-hexanedione (2,5-HD), a metabolite of n-hexane, has been linked to animal fertility in recent studies, the underlying mechanisms are still not clearly known. In this research, we aim to investigate the effects of different concentrations of 2,5-HD on cell morphology and apoptosis in pGCs. RNA-seq analyses revealed 4817 differentially expressed genes (DEGs) following 2,5-HD treatment. GO enrichment and KEGG pathway analysis suggested that the DEG, CDKN1A, was significantly enriched in the p53 signaling pathway that had the functions of apoptosis, growth inhibition and inhibition of cell cycle progression. After the interference of the CDKN1A gene, we found that it decreased pGC apoptosis, with lower cells in the G1 phase and higher cells in the S phase. Through a 2,5-HD cytotoxicity study and transcriptome bioinformatics analysis, these results deepen our understanding of the toxicological effects of 2,5-HD on porcine ovarian granulosa cells (pGCs), which is expected to help to understand the toxic mechanism of 2,5-HD in the female animal reproductive system. ABSTRACT: N-hexane, a common industrial organic solvent, causes multiple organ damage owing to its metabolite, 2,5-hexanedione (2,5-HD). To identify and evaluate the effects of 2,5-HD on sows’ reproductive performance, we used porcine ovarian granulosa cells (pGCs) as a vehicle and carried out cell morphology and transcriptome analyses. 2,5-HD has the potential to inhibit the proliferation of pGCs and induce morphological changes and apoptosis depending on the dose. RNA-seq analyses identified 4817 differentially expressed genes (DEGs), with 2394 down-regulated and 2423 up-regulated following 2,5-HD exposure treatment. The DEG, cyclin-dependent kinase inhibitor 1A (CDKN1A), according to the Kyoto Encyclopedia of Genes and Genomes enrichment analysis, was significantly enriched in the p53 signaling pathway. Thus, we evaluated its function in pGC apoptosis in vitro. Then, we knocked down the CDKN1A gene in the pGCs to identify its effects on pGCs. Its knockdown decreased pGC apoptosis, with significantly fewer cells in the G1 phase (p < 0.05) and very significantly more cells in the S phase (p < 0.01). Herein, we revealed novel candidate genes that influence pGCs apoptosis and cell cycle and provided new insights into the role of CDKN1A in pGCs during apoptosis and cell cycle arrest.