The Regulation Of High Insulin Levels On Ovary Apoptosis In Early Pregnant Mice

Objective : Metabolic syndrome has become a health problem worldwide.Metabolic syndrome and its comorbidities,characterized by hyperglycemia,obesity,insulin resistance and hypertension,are a serious threat to the health of the population.Insulin resistance and the associated metabolic syndrome,such as PCOS,are associated with reproductive problems.Uterine decidualization is important to the development of embryo.An abnormal decidualization could lead to embryo can not be normal nutrition,resulting in abnormal development of embryo.Our previous indicated that high levels of insulin has effect on uterine decidualization in early pregnant mice,but the mechanism remains elusive.Hormones estradiol(E2)and progesterone(P4)are secreted by the ovary is the critical regulator for a successful endometrial decidualization process.An increasing body of both experimental and clinical evidence suggests that high levels insulin could lead to ovarian dysfunction.Although increased high levels insulin have been linked to subfertility and early pregnancy loss,whether high levels of insulin result in ovarian dysfunction during early pregnancy,especially during endometrial decidualization process,has not been explored.Studies show that insulin could regulates apoptosis through a series of signaling pathways,and the balance of apoptosis of ovarian cells is essential to normal decidualization.Here,we studied the role of high insulin levels on ovarian cells apoptosis during early pregnancy,and further explored whether the the possible mechanism.Methods:1.Establishment of animal model and sample collection: 8-week-old KUNMING mice obtained from the Laboratory Animal Centre of Chongqing Medical University.The high insulin-exposed group and control group were established.Adult female mice from two group were mated with fertile(or vasectomized)males to induce pregnancy(or pseudopregnancy).The day in which a vaginal plug was found after mating was considered the first day of pregnancy(D1/PD1).Serum and ovarian tissues on D7/PD7 and D8/PD8 was collected on ice for further analyses.2.Ovarian cells apoptosis is impaired in early pregnancy mice with high insulin treatment: TUNEL was used to detect apoptosis of the ovarian cells.Immunohistochemistry was used to detect the protein expression of Bax and Bcl2 in D8.Western blot was used to detect the expression of apoptosis related proteins in D7,D8,PD7 and PD8.3.Alters of PI3K/Akt signaling pathway related proteins under high insulin levels treatment: Western blot was used to detect the expression of PI3K/Akt pathway related protein in D7,D8,PD7 and PD8.PI3K/Akt pathway specific inhibitor LY294002 was injected to two groups and further analysis of the effect of high levels of insulin on apoptosis of ovarian tissues.Results:1.Hormone secretion from the ovary is altered in high insulin-exposed mice: ELISA test showed that a marked increase in the serum level of insulin after high insulin treatment.In addition,there was a marked decrease in the serum levels of oestrogen and progesterone.2.Ovarian cells apoptosis is impaired with high insulin treatment: TUNEL detection showed that high insulin-exposed group had less apoptosis cells.Immunohistochemistry studies showed that Bax and Bcl2 proteins were widely distributed in the cytoplasm of the ovaries,and the number of Bax-positive cells was significantly less in the high insulin-exposed mice,while the number of Bcl2-positive cells was obviously increased.Western blot indicated that high insulin-exposed group expressed less Bax,cleaved-Casepase3,cleaved PARP while expression of Bcl2 was increased.3.PI3K/AKT signalling pathway is involved in apoptosis inhibited by high insulin levels: Western blot indicated that the expression of p-Akt was significantly upregulated after a high insulin treatment.Furthermore,LY294002 was injected into the lumen of one uterine horn adjacent to the ovary in two group.Western blot showed that LY294002 eliminated the effect of the high insulin level on the upregulation of Akt phosphorylation and the downregulation of apoptosis in the high insulin-exposed group.Conclusion: The apoptosis of ovarian was inhibited with high levels of insulin treatment in early pregnancy.Meanwhile,expression of related proteins in PI3K/Akt signaling pathway from the insulin group have significantly differences.All of these results showed that ovarian apoptosis is imbalanced under the role of insulin and then impaired the process of decidualization,the PI3K/AKT signalling pathway might participate in this process.