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Adequate regulation of the speed of follicular development has been reported to prolong the reproductive life of the ovary. The aim of the present study was to assess the potential effects and mechanism of the Ca2+/calmodulin‑dependent protein kinase II (CaMKII) pathway on the development of ovarian follicle. In the present study, the expression of CaMKII was measured in the ovary of mice at different developmental stages by immunofluorescence, confirming that CaMKII has a role in follicular development. Subsequently, the 17.5 days post‑coitus (dpc) embryonic ovaries were collected and cultured with KN93 for 4 days in vitro. It was revealed that KN93 inhibited the development of follicles, where it reduced the expression levels of oocyte and granulosa cell markers DEAD‑box helicase 4 (DDX4) and forkhead box L2 (FOXL2). These results suggested that KN93 could delay follicular development. Proteomics technology was then used to find that 262 proteins of KN93 treated 17.5 dpc embryonic ovaries were significantly altered after in vitro culture. Bioinformatics analysis was used to analyze these altered proteins. In total, four important Kyoto Encyclopedia of Genes and Genome pathways, namely steroid biosynthesis, p53 signaling pathway and retinol metabolism and metabolic pathways, were particularly enriched. Further analysis revealed that the upregulated proteins NADP‑dependent steroid dehydrogenase‑like (Nsdhl), lanosterol synthase (Lss), farnesyl‑diphosphate farnesyltransferase 1 (Fdft1), cytochrome P450 family 51 family A member 1 (Cyp51a1), hydroxymethylglutaryl‑CoA synthase 1 (Hmgcs1), fatty acid synthase (Fasn) and dimethylallyltranstransferase (Fdps) were directly interacting with each other in the four enriched pathways. In summary, the potential mechanism of KN93 in slowing down follicular development most likely lies in its inhibitory effects on CaMKII, which upregulated the expression of Nsdhl, Lss, Fdft1, Cyp51a1, Hmgcs1, Fasn and Fdps. This downregulated the expression of oocyte and granulosa cell markers DDX4 and FOXL2 in the follicles, thereby delaying follicular development. Overall, these results provide novel insight into the potential mechanism by which KN93 and CaMKII can delay follicular development.
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