Effect And Mechanism Of High-Fat Diet Induced Obesity On Ovarian Function In Mice During Early Pregnancy

ObjectiveRecent years,the prevalence of obesity has increased dramatically around the world.Obesity contributes to an increased prevalence of obesity-related metabolic dysfunction,cardiovascular diseases,diabetes and some cancers.In addition,obesity exerts a negative influence on female fertility,and it also exerts adverse effects both on gravidas and on fetuses.Obese pregnant women have a higher risk of adverse pregnancy outcomes and impaired fertility.As the incidence of obesity increases these years,it is of great significance to study the ovarian insufficiency caused by obesity.During early pregnancy,the follicles stop developing,and the corpus luteum plays endocrine function in the ovary to maintain pregnancy.It is worth further study whether obesity affects ovaries in early pregnancy.Main function of ovary in early pregnancy is to secret hormone.To date,research about impact of obesity on ovarian corpus luteum function at early pregnancy is less.During early pregnancy,corpus luteum formation,hormone secretion and cell proliferation differentiation,require a lot of energy,the role of glucose metabolism and lipid metabolism in obese pregnant mice ovary remains unknown,this study try to explore obesity’effects on ovarian function from perspective of glucose and lipid metabolism during early pregnancy.MethodsAnimal experiments in vivo to explore the effects of high-fat diet on ovarian function in mice during early pregnancy and its possible mechanism:1.Animal model establishment and sample collection: 4 weeks of C57BL/6J female mice,fed chow diet to adapt for a week,randomly divided into two groups,using Research DIETS food company formula feeding,namely the control group given controlled diet,high-fat diet group was given 60 kcal % fat high-fat diet,after 12 weeks,with the same strain chow diet male mice which have sexual maturation in one cage at 18:00PM.The next morning to check vaginal plug and record for the first day of pregnancy(D1).At the day 7 of pregnancy(D7),take the blood and ovary from the mice.Verification of the animal model:(1)The mice were weighed every week,and the body weight curve of the mice was obtained for 12 weeks.(2)After 12 weeks of feeding,glucose tolerance test(GTT)was measured,and serum triglyceride(TG)and total cholesterol(TC)levels of the pregnant mice were measured to verify the model.2.Effects of obesity induced by high-fat diet on ovarian function in mice during early pregnancy:(1)The histological morphology of the ovary at D7 was observed by H&E staining,and the number of luteum was counted.From the perspective of morphology,the changes in the ovary of pregnant mice under the condition of obesity were analyzed.(2)The levels of Estradiol(E2)and Progesterone(P4)in serum were detected to observe whether obesity affected ovarian hormone synthesis.(3)Western blot analysis and immunohistochemistry were used to detect the expressions of ovarian function marker CYP19A1,CYP11A1 and St AR in the ovary,and to observe whether the ovarian function of pregnant mice changed under the condition of obesity from the molecular level.3.Study on the mechanism of glucose metabolism involved in obesity induced by high-fat diet on abnormal ovarian function in mice during early pregnancy:(1)RT-qPCR was used to detect the expression of glucose metabolism pathway related genes Slc2a1,Slc2a4,Hk2,Pfkl,Pgk1,Pkm2,Ldha,Pdha1 and Pdk1 in the ovary of pregnant mice during early pregnancy,and to observe whether glucose metabolism pathway changed in the ovary of pregnant mice under the condition of obesity from the perspective of molecular level.(2)Western-blot analysis and immunohistochemical methods were used to detect the expression of HIF1-α,and to observe whether the hypoxic environment of ovarian tissue in early pregnancy was changed in the condition of obesity from the perspective of molecular level.(3)Western-blot analysis and immunohistochemical methods were used to detect the expressions of molecules PDP1,PDK1 and PDHA1 related to glycolytic pathway,and to observe whether the oxidative pathway of ovarian tissue was changed in early pregnancy under the condition of obesity from the perspective of molecular level.(4)The lactic acid kit was used to detect the lactic acid levels in the ovarian tissues,and to observe whether the glycolysis of the ovarian tissues had been changed in the early pregnancy under the condition of obesity from the perspective of the metabolic small molecule level.4.Study on the mechanism of lipid metabolism involved in obesity induced by high-fat diet on abnormal ovarian function in mice during early pregnancy:(1)Tissue TG detection kit,oil red O staining,and transmission electron microscopy were used to detect whether lipid deposition occurred in ovarian tissue under the condition of obesity.(2)The expression of genes related to lipid metabolism pathway Acsl4,Elovl5,Cd36,Slc27a4,Hadha and Cpt1 a in the ovary during early pregnancy was detected by RT-qPCR,and the changes of lipid metabolism pathway were observed from the perspective of molecular level under the condition of obesity.(3)The expression of fatty acid β-oxidation related molecule CPT1 A in lipid metabolism was detected by Western blotting were observed at the molecular level under the condition of obesity during early pregnancy.(4)ATP detection kit was used to detect the ATP levels of ovarian tissue.The energy level of ovarian tissue in early pregnancy was observed under the condition of obesity.(5)RT-qPCR was used to detect the expression of genes related to tricarboxylate cycle pathway Cs,Idh2,Suclg1,Sdh2,Mdh2,Ndufb6,Cox10 and Atp5b in the ovary during early pregnancy,and to observe whether the tricarboxylate cycle pathway was changed in the ovary of pregnant mice under the condition of obesity at the molecular level.Cell experiment in vitro to explore the effect of high-fat diet on ovarian function in early pregnancy mice and its possible mechanism:1.Establishment of cell model: Human granulosa-like tumor cell line(KGN)was cultured with Oleate acid(OA)and Palmitic acid(PA)to establish high-fat cell model,and luteinization was induced by h CG in vitro.2.Effects of OA+PA culture and h CG induced luteinization on the function of KGN cells:(1)CCK-8 was used to detect the effect of OA+PA and h CG on KGN cell viability.(2)The levels of E2 and P4 in the culture medium of KGN cells were detected by ELISA.(3)The expressions of CYP19A1,CYP11A1 and STAR in KGN cells were detected by Western blotting.3.Study on the mechanism of whether glucose metabolism is involved in the dysfunction of KGN cells:(1)The expressions of glucose metabolism pathway related genes SLC2A1,PFK2,PGK1,LDHA and PDHA1 in luteinized KGN cells cultured in OA+PA and induced by h CG were detected by RT-qPCR.(2)The expression of HIF1-α was detected by western blotting to observe whether hypoxia occurred in KGN cell.(3)The expressions of glycolytic oxidation pathway related proteins PDP1,PDK1 and PDHA1 were detected by Western blotting,and whether the aerobic oxidation pathway of KGN cells was changed.(4)The lactic acid level in the medium of KGN cells was detected to observe whether the glycolysis was changed.4.Study on whether lipid metabolism is involved in the dysfunction of KGN cells:(1)TG content in KGN cells was detected;The distribution of lipid droplets in KGN cells was observed by oil red O staining.(2)The expressions of genes related to lipid metabolism pathway ACSL4,ELOVL5,CD36,SLC27A4,HADHA and CPT1 A in KGN cells were detected by RT-qPCR to observe whether the lipid metabolism pathway was changed in vitro.(3)The expression of CPT1 A in KGN cells was detected by western blotting to observe whether the fatty acid β-oxidation was changed.(4)The ATP level of KGN cells was detected to observe whether the energy level changed.5.Etomoxir,a specific inhibitor of CPT1 A,was used to intervene fatty acid β-oxidation in KGN cells to observe whether fatty acid β-oxidation was involved in the dysfunction of KGN cells.(1)CCK-8 was used to detect the effects of different concentrations of etomoxir on the activity of KGN cells cultured with OA+PA and induced luteinization by h CG.Western-blot was used to detect the inhibitory effect of different concentrations of etomoxir on CPT1 A.(2)Oil red O staining and transmission electron microscopy were used to detect the effect of etomoxir intervention on the distribution of lipid droplets in KGN cells.(3)The expression of tricarboxylic acid cycle pathway and electron transport chain related genes CS,IDH2,SUCLG1,SDH2,MDH2,NDUFB6,COX10 and ATP5 b in KGN cells by RT-qPCR to observe whether the tricarboxylic acid cycle pathway was reversed after inhibiting the oxidation of fatty acid β-oxidation.(4)The ATP level of KGN cells was detected to observe whether the ATP level returned to normal level.(5)The levels of E2 and P4 in the supernatant of KGN cells were detected to observe whether the levels of E2 and P4 returned to normal level.Results1.Compared with the control group,the body weight of mice in the HFD group increased significantly and glucose tolerance was abnormal after 12 weeks of high-fat feeding.2.Compared with the control group,the number of ovarian corpus luteum in HFD group increased at D7;The levels of E2 and P4 in serum were significantly increased;Protein expressions of ovarian function marker CYP19A1,CYP11A1 and STAR were also increased.3.Compared with the control group,the expression of Hk2 gene related to glucose metabolism pathway in ovary tissue of mice in HFD group decreased at D7,while the expression of most other genes showed no significant difference.The protein expression of HIF1-α,PDK1,PDP1 and PDHA1 had no significant difference.There was no significant change in lactic acid production.4.Oil red O staining,transmission electron microscopy and TG results showed that lipid accumulation in ovary of HFD group was significantly increased at D7;the expression of fatty acid synthesis related genes(ACSL4 and ELOVL5)and genes involved in fatty acid uptake and transport(SLC27A4)and fatty acid β-oxidation related gene(CPT1A)were increased.CPT1 A protein expression increased;ATP level increased;Some genes(IDH2,NDUFB6,COX10)of the tricarboxylic acid cycle and electron transport chain were expressed abnormally.5.In vitro results showed that cultured with OA+PA and luteinization induced by h CG had no effect on the viability of KGN cells;Compared with the control group,the E2 and P4 level in KGN cell medium in OA+PA group were significantly increased.The protein expressions of CYP19A1,CYP11A1 and STAR were also increased.6.Compared with the control group,the expression of PFK2 gene in KGN cells of OA+PA group was decreased,and the expression of other genes related to glucose metabolism pathway were not significantly changed;Protein expression of HIF1-α,PDK1,PDP1 and PDHA1 had no significant difference.There was no significant change in lactic acid production in cell medium.7.The results of oil red O staining and TG test showed that lipid deposition occurred in KGN cells in OA+PA group compared with the control group;fatty acid β-oxidation related gene(CPT1A)expression increased;CPT1A protein expression increased;ATP levels increased.8.After inhibited the β-oxidation of fatty acid by etomoxir,oil red O staining and transmission electron microscopy showed that the lipid deposition in the inhibitor group was deeper than that in the OA+PA group;however,the expression of some genes(Cs,IDH2,ATP5b)of the tricarboxylic acid cycle and electron transport chain and the level of ATP were decreased.The level of E2 and P4 and the expression of ovarian hormone secretion related proteins CYP19A1,CYP11A1 and STAR were also decreased.ConclusionsIn early pregnancy,obesity induced by high-fat diet impaired mice ovarian function.High-fat diet through fatty acid β-oxidation rather than glycolysis,that impaired ovarian function.