The Regulatory Effect And Mechanism Of ID3 On Progesterone Synthesis In Bovine Cumulus Cells

Progesterone is a biologically active steroid hormone secreted by the ovary and plays an important role in embryo attachment and pregnancy maintenance.It has been found that the steroid hormone in the COC is mainly derived from the cumulus cells(CCs)and regulates oocyte quality by altering the production capacity.ID3 plays a key role as a DNA binding repressor for maintaining CCs proliferation and inhibiting differentiation,and also regulates mammalian ovarian function.The group’s previous study found that the physiological state and function of CCs were altered after adding BMP15 and GDF9 to bovine CCs and the ID3 gene showed significant differential expression in this process,however,its specific role and mechanism are still unclear.To investigate the effect of ID3 on the function of bovine CCs,we inhibited ID3 expression in bovine CCs by siRNA and construct-based differential transcriptome gene expression profiles to reveal the ID3-mediated regulatory network.The results showed that there were 197 differentially expressed genes,including 110 up-regulated genes and 87 down-regulated genes,after knockdown of ID3 compared with the control group.GO enrichment analysis showed that the differentially expressed genes were involved in biological processes such as cholesterol transport and meiosis,with a total of 412 pathways.KEGG analysis showed that the differentially expressed genes were mainly involved in 156 pathways including Gn RH secretion,MAPK signaling pathway,p53 signaling pathway,and progesterone-mediated oocyte maturation.Subsequently,to clarify the effect of ID3 on progesterone synthesis and the potential mechanism of action,ELISA and qRT-PCR,Western blot were used to detect changes in the levels of progesterone and key regulatory genes and proteins of its biological synthesis,respectively.The results showed that the mRNA levels of St AR,CYP11A1 and HSD3B1 were down-regulated to 0.69 ± 0.04,0.45 ± 0.05 and 0.58 ±0.09 times of the control group,respectively,after knocking down ID3(P<0.01);the protein levels were down-regulated to 0.68 ± 0.11,0.71 ± 0.13 and 0.64 ± 0.06 times of the control group,respectively(P<0.05)and 0.64 ± 0.06-fold(P<0.01),respectively.After ID3 inhibition,ATP levels significantly decreased to 0.78 ± 0.05-fold of the control(P<0.01),Parkin and DRP1 protein levels significantly increased to 1.43 ± 0.20-fold(P<0.05)and 3.08 ± 0.22-fold(P<0.01)of the control,while MFN1 and MFN2 protein levels significantly decreased to 0.53 ± 0.14-fold of the control,respectively.0.53 ± 0.14-fold and 0.31 ± 0.07-fold,respectively(P<0.01).Meanwhile,inhibition of ID3 expression significantly increased the apoptosis rate and decreased the ERK1/2phosphorylation level in CCs.Finally,the effects of knocking down ID3 on oocyte development were clarified by detecting the changes in oocyte first polar body extrusion rate,oocyte maturationpromoting factors BMP15 and GDF9,as well as the changes in the expression levels of CCs expansion factors PTX3 and HAS2 mRNA.The results showed that inhibition of ID3 expression levels in oocytes in the COC reduced the oocyte first polar body extrusion rate to 0.79 ± 0.09 times that of the control group(P<0.01),down-regulated oocyte mass-related factors BMP15 and GDF9 to 0.70 ± 0.07 and 0.70 ± 0.04 times that of the control group,respectively(P<0.01),and oocyte PTX3 and HAS2 were also down-regulated to 0.50 ± 0.03 and 0.67 ± 0.06-fold of the control group,respectively(P<0.01).To investigate the effect of ID3 inhibition on oocyte quality,we examined changes in ATP levels and changes in mitochondrial function-associated proteins in oocytes.The results showed that inhibition of ID3 significantly decreased intracellular ATP levels to 0.66 ± 0.02-fold(P<0.01),increased Parkin and DRP1 protein expression to 1.17 ± 0.16-fold(P<0.05)and 1.27 ± 0.05-fold(P<0.01)of the control,and decreased OPA1 protein levels to 0.89 ± 0.07-fold of the control(P<0.05).To further investigate the effect of ID3 inhibition on oocyte quality,we determined its effect on the antioxidant capacity of oocytes by measuring the intracellular levels of ROS,GSH,CAT and SOD2.The results showed that the inhibition of ID3 increased the accumulation of ROS and decreased the accumulation of GSH;in addition,the mRNA expression levels of CAT and SOD2 decreased to 0.64 ± 0.09(P<0.01)and 0.74 ± 0.14(P<0.05)times,respectively,of the control group,indicating that the antioxidant capacity of oocytes was somewhat reduced.In summary,this study shows that ID3 can influence oocyte development and quality by regulating the level of progesterone synthesis in bovine CCs through regulating the oxidative stress balance and mitochondrial function.The findings will provide a new basis for understanding the biological role of ID3 as well as oocytes.