The regulation of high insulin levels on ovary apoptosis in early pregnant mice

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Highlights

  • We found that hormone secretion from the ovary is altered after insulin treatment.

  • Ovary Apoptosis is reduced in early pregnancy mice with high insulin treatment.

  • PI3K/AKT signalling pathway is involved in apoptosis inhibited by high insulin levels.

Abstract

Studies have shown that metabolic syndrome is associated with reproductive problems. Women with metabolic syndrome, characterized by hyperinsulinemia, have common ovarian dysfunction, but the mechanism remains elusive. The aim of this research is to explore the effects of high levels of insulin on ovary function during early pregnancy. Mice in the high insulin-exposed group were given a subcutaneous injection of human recombinant insulin. After insulin treatment, changes in various hormones were tested using ELISA kits which showed hormones secreted by the ovary were significantly altered in the insulin group. TUNEL staining showed less TUNEL-positive cells in the insulin group. A significant decrease in Bax and an increase in Bcl2 in the ovary were found in the insulin group by immunohistochemical studies. Western blotting showed the expressions of apoptosis related proteins in the ovaries from the insulin group were obviously altered. In addition, expression of p-Akt proteins in the ovaries from the insulin group was significantly upregulated. Moreover, the Akt inhibitor LY294002 reversed the anti-apoptotic effects of high insulin in the ovary tissues in early pregnancy mice. All of these results showed that insulin impaired ovarian function during early pregnancy and ovarian apoptosis is imbalanced under the role of insulin. The PI3K/AKT signalling pathway might participate in this process.

Introduction

Metabolic syndrome has become a health problem worldwide [1]. Metabolic syndrome and its comorbidities, characterized by hyperglycemia, obesity, insulin resistance and hypertension, are a serious threat to the health of the population [1], [2]. Insulin resistance and the associated metabolic syndrome are especially problematic in reproductive-age women because they are associated with reproductive problems [3], [4].

Uterine decidualization is a critical event during mammalian pregnancy. In humans, decidualization is initiated at every menstrual cycle when ovulation occurs and when the corpus luteum produces progesterone (P4). In mice, decidualization occurs with blastocyst attachment to the uterine lining, which peaks on day 7 and 8 of pregnancy. Aberrant decidualization has been linked to numerous pregnancy complications, including infertility, recurrent miscarriage, preeclampsia, intrauterine growth restriction, and preterm delivery [5], [6], [7].

Hormones estradiol (E2) and progesterone (P4) are secreted by the ovary, which is the critical regulator for a successful endometrial decidualization process [8] and responsible for the function of normal mammalian ovary [9], [10]. An increasing body of both experimental and clinical evidence suggests that high levels insulin could lead to ovarian dysfunction both in human and in mice [11], [12], [13]. Although increased high levels insulin have been linked to subfertility and early pregnancy loss [14], whether high levels of insulin result in ovarian dysfunction during early pregnancy, especially during endometrial decidualization process, has not been explored.

It ensures the selection of the dominant follicle [10], [15], the demise of excessive follicles and the degeneration of old corpora lutea, which are essential processes in maintaining the normal cyclicity of ovarian steroidogenesis. A balance between cellular proliferation and apoptosis is essential for the normality of ovarian function. Studies show that after exposure to a high concentration of insulin, blastocyst become insulin resistant and the balance between proliferation and apoptosis is broken. It is similar to what occurs in other cell systems in response to chronic insulin stimulation [16], [17]. However, it is still not known whether ovary apoptosis is an effect of high insulin levels during early pregnancy.

Here, we studied the role of high insulin levels on ovary function during early pregnancy, and then, we further explored whether the impaired apoptosis was responsible for the dysfunctional ovaries treated with high insulin levels.

Section snippets

Animals and tissue collection

Eight-week-old KUNMING mice, approved for experimental use by the Laboratory Animal Centre of Chongqing Medical University (NO. 20110016), were allowed regular food and water and were kept at room temperature (21–23 °C) on a 12L: 12D cycle. The high insulin-exposed mice model was established according to the method described in a previous report [18]. Briefly, female mice were randomly divided into two groups (n = 40 in each group). The control group was injected i.h. with normal saline, and

Hormone secretion from the ovary is altered in high insulin-exposed mice

Control and high insulin-exposed female mice were mated with fertile male mice, and the serum samples were detected using ELISA in early pregnancy (D7 and D8). We found that a marked increase (P < 0.001) in the serum level of insulin after high insulin treatment (Fig. 1A) in early pregnancy (D7 and D8). To study whether high insulin levels in female mice affects circulating hormones levels in early pregnancy mice, an ELISA test was further conducted. The results showed that both serum oestrogen

Discussion

Increasing experimental and clinical evidence suggests that maternal metabolic syndrome is associated with poor reproductive outcomes [14], [22], [23]. Although high levels of insulin are linked to subfertility and early pregnancy loss [24], its effects on ovarian function during early pregnancy have not been explored. Here, we addressed this question by injecting mice with a high level of insulin. First, the high insulin-exposed mice significantly altered the secretion of hormones of the ovary

Conflict of interest

The authors declared that they have no conflicts of interest to this work.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81300486, No 31271546), the Natural Science Foundation of Chongqing (No. cstc2015jcyjA10013), Scientific Research Program of Science and Technology Commission of Yuzhong District of Chongqing (No.20150104), the Excellent Young Scholars of Chongqing Medical University (No. CYYQ201508).

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  • 1

    These authors contributed equally to this work.

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