The Effect Of Kidney-tonifying Treatment On Cumulus Expansion During Oocyte Maturation In Controlled Ovarian Hyperstimulation Mice And Its Mechanism Of Regulating Mapks Signaling Pathway

Objective: The purpose of this study was to observe and analyze the effects of sequential administration of Bushen Tiaojing Fang Ⅱ and Ⅲ on cumulus expansion grading,cumulus expansion-related factors,FSH,and MAPKs signal pathways,and to reveal the role and mechanism of kidney-tonifying method to improve the extracellular matrix of cumulus,promote cumulus expansion,oocyte development and maturity,and enrich the theory of "kidney dominates reproduction".Methods: In total,360 female Kunming mice with normal estrous cycle were randomly divided into normal group,control group and Bushen Tianjing Fang(BSTJF)group(n =120 mice per group).The control and BSTJF group were intraperitoneally injected with Gn RHa on the 1st and 9th days.On day 9,PMSG was administered simultaneously,followed by h CG 48 h later.Mice in the normal group were injected with an equal volume of 0.9% saline.Mice in the BSTJD group received oral Bushen Tiaojing Fang Ⅱ daily by gavage on days 1 to 9 followed by Bushen Tiaojing Fang Ⅲ on days 10 and 11.Mice in the normal and control group were given the same volume of distilled water.At 6 h,9 h,and 12 h after injection of normal saline or h CG,mice in each group were decapitated,blood was centrifuged,and serum was collected.The cumulus expansion grade of COCs was observed and counted under a microscope,and then COCs were collected.Radioimmunoassay for detecting serum FSH content in mice;Quantitative real-time polymerase chain reaction for detecting cumulus expansion-related factors HAS2,TNFAIP6,PTX3,PTGS2 m RNA expression in COCs of mice;Immunofluorescence staining for detecting cumulus expansion-related factors HAS2,TNFAIP6,PTX3,PTGS2 protein expression in COCs of mice;Western blot was used to detect the expression levels of cumulus expansion related factors HAS2,TNFAIP6,PTX3,PTGS2 in COCs of mice;Western blot was used to detect MAPK14 and MAPK3/1 protein expression and phosphorylation levels in COCs of mice.Result: 1 Morphology and structure of COCs at different grades of cumulus expansion Grade 0: The cumulus expansion cannot be seen,but the oocytes begin to attach to the culture plate and begin to form a monolayer.Grade 1: The smallest degree of cumulus expansion can be observed.At this time,the shape of the COCs is radial.Grade 2: The outer cumulus cells of COCs begin to expand.Grade 3: Except for the radiating canopy cumulus cells that are close to the oocytes,the other cumulus cells have expanded.Grade 4: All cumulus cells have expanded,reaching maximum cumulus expansion.2 Grading of cumulus expansion in COCs at different times in each group of mice.Compared with the normal group,there were no significant differences in the proportions of the expansion grades of the COCs in the control group(P >0.05);compared with the normal group,the proportions of COCs in the BSTJF group decreased at 6 h of grade 1,9 h,and 12 h of grades 1 and 2(P <0.05),and the proportions of grades 2,3,and 4 at 6 h,and grades 3 and 12 at 9 h increased(P <0.05);compared with the control group,the proportions of COCs in the BSTJF group decreased at 6 h of grade 1,and grades 1 and 2 at 9 h,and grades 0,1 and 2 at 12 h(P <0.05),the proportions of grades 2 and 4 at 6 h,grades 3 at 9 h,and grade 4 at 12 h increased(P <0.05).3 Comparisons of serum FSH content of mice in different groups at different times.Compared with the normal group,there were no statistically significant differences in serum FSH content at 6 h,9 h,and 12 h after h CG injection in control mice(P >0.05);compared with the control group,the serum FSH content in the BSTJF group increased(P <0.05).Compared with the normal group,the serum FSH content at 6 and 12 h after h CG injection in the BSTJF group mice increased(P <0.05).4 Comparisons of the m RNA expression of HAS2,TNFAIP6,PTX3,PTGS2 in COCs of mice in different groups at different times6 h: Compared with the normal group,the expression of HAS2,TNFAIP6,PTX3,PTGS2 m RNA in the control group were not statistically significant(P >0.05);compared with the normal group,the expression of HAS2 m RNA in the BSTJF group was not statistically significant(P >0.05),the expression of TNFAIP6,PTX3,PTGS2 m RNA increased(P <0.05);compared with the control group,the expression of HAS2,PTGS2 m RNA in the BSTJF group were not statistically significant(P >0.05),the expression of TNFAIP6 and PTX3 m RNA increased(P <0.05).9 h: Compared with the normal group,the expression of HAS2,TNFAIP6,PTX3,and PTGS2 m RNA in the control group were not statistically significant(P >0.05);compared with the normal group,the expression of PTGS2 m RNA in the BSTJF group was not statistically significant(P >0.05),the m RNA expression of HAS2,TNFAIP6,and PTX3 increased(P <0.05);compared with the control group,the expression of HAS2,TNFAIP6,PTX3,and PTGS2 m RNA increased in the BSTJF group(P <0.05).12 h: Compared with the normal group,the expression of HAS2,TNFAIP6,PTX3,and PTGS2 m RNA in the control group were not statistically significant(P >0.05);compared with the normal group,the expression of HAS2,TNFAIP6,PTX3,and PTGS2 m RNA in the BSTJF group increased(P <0.05).Compared with the control group,the expression of HAS2 m RNA in the BSTJF group was not statistically significant(P >0.05),and the expression of TNFAIP6,PTX3,and PTGS2 m RNA increased(P <0.05).5 Comparisons of the expression of HAS2,TNFAIP6,PTX3,and PTGS2 proteins in COCs at different times in each group of mice5.1 Immunofluorescence detection of HAS2,TNFAIP6,PTX3,PTGS2 protein expression in COCs at different times in each group of miceHAS2,TNFAIP6,PTX3,and PTGS2 proteins were expressed in cumulus cells and oocytes.5.2 Western blot to detect the expression levels of HAS2,TNFAIP6,PTX3,and PTGS2 proteins in COCs at different times in each group of mice6 h: Compared with the normal group,the expression of HAS2,TNFAIP6,PTX3,and PTGS2 proteins in the control group were not statistically significant(P >0.05).Compared with the normal group,the expression of HAS2 protein in the BSTJF group was not statistically significant(P >0.05);the expression of TNFAIP6,PTX3,and PTGS2 proteins in the BSTJF group increased(P <0.05).Compared with the control group,the expression of HAS2 and PTX3 proteins in the BSTJF group were not statistically significant(P >0.05);the expressions of TNFAIP6 and PTGS2 proteins in the BSTJF group increased(P <0.05).9 h: Compared with the normal group,the expression of HAS2,TNFAIP6,PTX3,and PTGS2 proteins in the control group were not statistically significant(P >0.05).Compared with the normal group,the expression of HAS2,TNFAIP6,PTX3,and PTGS2 proteins in the BSTJF group increased(P <0.05).Compared with the control group,the expression of HAS2,TNFAIP6,PTX3,and PTGS2 proteins in the BSTJF group increased(P <0.05).12 h: Compared with the normal group,the expression of HAS2,TNFAIP6,PTX3,and PTGS2 proteins in the control group were not statistically significant(P >0.05).Compared with the normal group,the expression of HAS2,TNFAIP6,PTX3,and PTGS2 proteins in the BSTJF group increased(P <0.05).Compared with the control group,the expression of HAS2,TNFAIP6,PTX3,and PTGS2 proteins in the BSTJF group increased(P <0.05).6 Comparison of MAPK14,MAPK3/1 proteins and their phosphorylation levels in COCs at different times in each group of miceAt 6 h,9 h,and 12 h after h CG injection,compared with the normal group,the phosphorylation levels of MAPK14 and MAPK3/1 in the control group were not statistically significant(P >0.05);compared with the normal group,the phosphorylation levels of MAPK14 and MAPK3/1 in the BSTJF group increased(P <0.05);compared with the control group,the phosphorylation levels of MAPK14 and MAPK3/1 in the BSTJF group increased(P <0.05).Conclusion:1.Bushen Tiaojing Fang Ⅱ and Ⅲ can promote cumulus expansion in mice and thus promote oocyte development and maturity.2.Bushen Tiaojing Fang Ⅱ and Ⅲ can up-regulate the expression of cumulus expansion-related factors HAS2,TNFAIP6,PTX3,PTGS2 m RNAs and proteins in mice COCs,thereby promoting cumulus expansion.Among them,HAS2,TNFAIP6,and PTX3 are important components of the extracellular matrix of the cumulus cells.Therefore,Bushen Tiaojing Fang Ⅱ and Ⅲ can improve the extracellular matrix of the cumulus cells.3.The mechanism of Bushen Tiaojing Fang Ⅱ and Ⅲ in promoting the expression of cumulus expansion-related factors in COCs of mice may be partly achieved by activating the MAPK14 and MAPK3/1 signaling pathways by regulating the serum hormone FSH expression levels in mice.