Effects and potential mechanism of Ca2+/calmodulin‑dependent protein kinase II pathway inhibitor KN93 on the development of ovarian follicle

Abstract

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.

Keywords: KN93; calmodulin‑dependent protein kinase II; mouse; primary follicle; primordial follicle.

Figure 1

Expression pattern of CaMKII in the embryonic and neonatal mouse ovaries. The embryonic and neonatal mouse ovaries were stained for CaMKII (red) and the oocyte specific marker DDX4 (green) at the indicated time points. The nuclei were counter-stained using Hoechest (blue). CaMKII was mainly localized to the cytoplasm of granulosa cells in either primordial follicles or primary follicles. The thin arrows indicated primordial follicles, and the thick arrows indicated primary follicles. Scale bar, 40 µm (the first four columns) and 100 µm (the last column). CaMKII, calmodulin-dependent protein kinase II; DDX4, DEAD-box helicase 4; dpc, days post-coitus; dpp, days post-partum.

Figure 2

Western blotting for the 17.5 dpc ovaries that were cultured with or without KN93 for 4 days in vitro. The results revealed that KN93 significantly reduced the expression of CaMKII and the oocyte, granulosa cells specific marker DDX4 and FOXL2 in ovarian follicle. GAPDH was used as a loading control. The data are presented as the mean ± SD (n=3). ^**P<0.01, ^***P<0.001. dpc, days post-coitus; CaMKII, calmodulin-dependent protein kinase II; DDX4, DEAD-box helicase 4; FOXL2, forkhead box L2.

Figure 3

Immunofluorescence analysis for the 17.5 dpc ovaries that were in vitro cultured with or without KN93 for 4 days. Immunofluorescence detection results demonstrated that the development of follicles in KN93 treatment group was markedly inhibited compared with those in the respective controls. The ovaries were stained for the oocyte specific marker DDX4 (green) and the granulosa cells specific marker FOXL2 (red). The nuclei were counter-stained by Hoechest (blue). The thin arrows indicated primordial follicles, and the thick arrows indicated primary follicles. Scale bar, 20 µm (the first four columns) and 40 µm (the last column). dpc, days post-coitus; DDX4, DEAD-box helicase 4; FOXL2, forkhead box L2.

Figure 4

Proteomic techniques revealed differential proteins influenced by KN93. (A) Principal component analysis of the identified proteins in the KN93 treatment group and control group. Red and blue dots represent the KN93 treatment group and control group, respectively. (B) Volcano plot indicated the differential proteins between KN93 treatment group and control group. Red dots indicated significantly upregulated proteins with adjusted P-values <0.01, blue dots indicated significantly down-regulated proteins with adjusted P-values <0.01 and gray dots indicated non-significantly expressed proteins. (C) Differential proteins were verified by western blotting, which was then (D) quantified. GAPDH was used as a loading control. The data are presented as the mean ± SD (n=3). ^**P<0.01,^***P<0.001. NS, no significant difference; CYP51A1, cytochrome P450 family 51 family A member 1; FADD, fas-associated death domain; NCAM1, neural cell adhesion molecule 1; Cyct, cytochrome c, testis; IGFBP3, insulin-like growth factor binding protein 3; PLS1, plastin-1; PDZK1, PDZ domain-containing 1; ZP2, zona pellicida sperm-binding protein; YBX2, Y-box binding protein 2; BNIP3L, Bcl-2-interacting protein 3-like.

Figure 5

GO and KEGG enrichment analysis of differential proteins affected by KN93. (A) Enriched the top 20 GO terms in the (A) molecular function, (B) cell components and (C) biological process categories for the differential proteins. (D) Enriched the top 20 KEGG information for the differential proteins. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 6

Analysis of potential mechanism for KN93 retarding ovarian follicle development. (A) Interaction analysis of differential proteins in four key KEGG enriched pathways. Nsdhl, Lss, Fdft1, Cyp51a1, Hmgcs1, Fasn and Fdps have direct interactions, which was marked through the pale green elliptic region. (B) Model of KN93 retarding follicular development. After CaMKII was inhibited by the inhibitor KN93, seven interacting proteins were upregulated (Nsdhl, Lss, Fdft1, Cyp51a1, Hmgcs1, Fasn and Fdps), then downregulated oocyte marker DDX4 and granulosa cell marker FOXL2 in follicles, ultimately slowing down primordial follicles formation and activation, thereby leaded to the delay of the ovarian follicle development. Nsdhl, NADP-dependent steroid dehydrogenase-like; Lss, lanosterol synthase; Fdft1, farnesyl-diphosphate farnesyltransferase; Cyp51a1, cytochrome P450 family 51 family A member 1; Hmgcs1, hydroxymethylglutaryl-CoA synthase 1; Fasn, fatty acid synthase; Fdps, dimethylallyltranstransferase; CaMKII, calmodulin-dependent protein kinase II; DDX4, DEAD-box helicase 4; FOXL2, forkhead box L2.