Effect And Mechanism Of Prenatal Androgen Excess On Polycystic Ovary Syndrome In Pubertal Female Rats

Polycystic ovary syndrome(PCOS) is a heterogeneous reproductive and metabolic disorder characterized by anovulation, hyperandrogenism and polycystic ovaries, and the early pathophysiological mechanisms of PCOS remain unclear. It has been proposed that PCOS is one of fetal origin of adult disease. The abnormal endocrine and metabolism in mothers and fetus, including prenatal androgen exposure, mother obesity, fetal growth pattern and low birth weight, reprogram the developmental trajectory of multiple organ systems in the fetus, reduce hypothalamic sensitivity to steroid negative feedback,and derange the function of pancreas, which increase the later risk of PCOS in adolescents and adult women.The clinical manifestations of PCOS often emerge before or during puberty. Peripubertal metabolic dysfunction is one of the first phenotypic traits observed in adolescent girls with PCOS. Insulin resistance and hyperinsulinemia play a pivotal role in the pathogenesis of PCOS in adolescents. The hyperinsulinemia elevates the level of androgen in ovaries, resulting in an increased number of atresia follicles and deranging the development of follicles and ovulation. In addition, insulin resistance is thought to be the metabolic abnormality most closely linked to an increased risk of obesity, type 2 diabetes mellitus, metabolic syndrome and cardiovascular disease. Due to the significant overlap between PCOS-related symptoms and the normal physiologic changes associated with puberty, PCOS is possibly being under-evaluated in the adolescent population. Therefore, an improved understanding of the pathogenesis of the metabolic derangements in adolescents with PCOS may be a key to minimizing the development of symptoms and preventing the onset of the long-term complications associated with this syndrome.In this study, we administrated pregnant rats with 5α-dihydrotestosterone(DHT) and investigated the role of prenatal androgen excess in the development of reproductive and metabolic derangements in female offspring rats at the peripubertal stage. We also determined whether the prenatally androgenized(PNA) rats manifested impaired glucose tolerance(IGT) and insulin resistance during the pubertal development stage and whether the resulting metabolic derangements were mediated by peripheral insulin signaling. In addition, we elucidated the role of Kisspeptin/Neurokinin B/Dynorphin(KNDy) in the regulation of gonadotrophin releasing hormone(Gn RH) and luteinizing hormone(LH) in the hypothalamus of PCOS rats at the peripubertal stage.Part Ⅰ Effect of Prenatal Androgen Excess on Reproductive and Metabolic Derangements in Pubertal Female RatsObjective To determine the effect of prenatal androgen excess on developmental, reproductive and metabolic derangements in female offspring rats during pubertal development.Methods On days 16–19 of gestation, the pregnant rats were treated with daily subcutaneous injections of either 0.5 ml DHT(10mg/ml) or a vehicle as a control. The body weight, ovarian weight, ovarian morphology, serum steroid hormones(testosterone and estradiol) and fasting plasma biochemical profiles of the female offspring were determined at the age of 4 to 8 weeks.Results Prenatal DHT treatment resulted in markedly elevated body weight in PNA female offspring from postnatal 5 to 8 weeks(P < 0.01). No differences were observed in the ovarian weight and ovary/body weight ratio between the two groups during puberty. Ovaries from the prenatally androgenized(PNA) rats had an increased number of total, cystic and atretic follicles, compared with the number in control(P < 0.01). From postnatal 5 to 8 weeks, the plasma levels of total testosterone and 17β-estradiol were not different between two groups(P > 0.05). Fasting glucose, insulin, leptin levels and homeostatic model assessment for insulin resistance were elevated in the PNA rats at the age of 5 to 8 weeks(P < 0.01 or P < 0.05). Frompostnatal 4 to 7 weeks, the serum total cholesterol, triglyceride and free fatty acids levels were unaltered in both the experimental and control groups; however, 8-week-old PNA rats had increased free fatty acids levels compared with control(P < 0.05).Part Ⅱ Mechanism of Prenatal Androgen Excess on Insulin Resistance in Pubertal Female Rats with PCOSObjective To determine whether prenatal androgen excess induced impaired glucose tolerance and insulin resistance in pubertal female offspring and whether the resulting insulin resistance were mediated by peripheral insulin signaling during the pubertal development stage.Methods To establish pubertal rats model with PCOS mentioned above. From postnatal 5 to 8 weeks, intraperitoneal glucose tolerance tests(IPGTTs) were performed weekly in the female offspring. Blood samples were obtained 0, 15, 30, 60 and 120 min after the glucose load and the levels of glucose and insulin were measured. The intraperitoneal insulin tolerance tests(IPITTs) were performed weekly in the PNA rats and control from postnatal 4 to 8 weeks. Glucose levels were measured 0, 15, 30, 60 and 120 min after insulin stimulation. In addition, in order to explore insulin signaling, half of the animals in the experimental and control groups at the age of 6 weeks were intraperitoneally injected with 2 U/kg of regular human insulin, and half were injected with a vehicle, 10 min before death. The protein and phosphorylation of different components of the IRS-AKT pathway, including IRS1, IRS2, AKT and GSK-3, were investigated in the soleus muscles, liver and parametrial fat of 6-week-old PCOS rats.Results Following IPITTs, serum glucose and insulin levels did not significantly differ between the two groups at the age of 5 to 8 weeks(P > 0.05). Although no difference was detected in the glucose levels at 15, 30 and 120 min after insulin administration between the two groups, the 60-min glucose levels were significantly higher in the PNA group than in the control group at postnatal 5 and 6 weeks(P <0.01 or P < 0.05). Moreover, prenatal DHT treatment induced significantly elevated glucose levels at 30 and 60 min after IPITTs in 7- and 8-week-old PNA rats(P < 0.05). The abundance of IRS1 and IRS2 was significantly decreased in the skeletal muscles and liver of 6-week-old PCOS rats after stimulation with insulin(P < 0.01). Prenatal DHT treatment remarkably decreased insulin-stimulated phosphorylation of AKT in the skeletal muscles(P < 0.01), whereas phosphorylation of AKT were unaltered in the liver of PNA rats. In the adipose tissue of 6-week-old PCOS rats, no decreases were detected in the phosphorylation of insulin signaling proteins, including IRS1, IRS2, AKT and GSK-3(P > 0.05).Part Ⅲ Effect of Prenatal Androgen Excess on Kisspeptin/Neurokinin B/Dynorphin(KNDy) in hypothalamus of Pubertal Female Rats with PCOSObjective To determine the effect of prenatal androgen excess on Kisspeptin/Neurokinin B/Dynorphin(KNDy) neurons in hypothalamus of pubertal female rats with PCOS, and elucidated the role of KNDy in the regulation of Gn RH and LH in the hypothalamus of PCOS rats at the peripubertal stage.Methods To establish pubertal rats model with PCOS mentioned above. At the age of 4, 6 and 8 weeks, we measured the levels of serum LH and evaluated Kiss1, kiss1 r, NKB, NKB3 R, NPY, NPYR, Ob Rb, Gn RH, Ag RP and POMC m RNA levels in the hypothalamic arcuate nucleus of PCOS rats. The location of Kisspeptin, NKB and Ob Rb in the arcuate nucleus of 6-week-old PCOS rats was determined using immunofluorescence. Kisspeptin, Senktide(NKB receptor agonist) or Leptin were intracerebrally injected into the lateral cerebral ventricle of 6-week-old PCOS rats. The serum LH were measured 0, 15, 30 and 60 min after administration and Kiss1, NKB and Ob Rb m RNA levels were evaluated in the hypothalamic arcuate nucleus of PCOS rats.Results The serum LH levels did not significantly differ between the PCOS rats and control groups at the age of 4, 6 and 8 weeks; however, the frequencys of serum LHpulse in 6-week-old PCOS rats were higher than control(P < 0.01). Prenatal DHT treatment resulted in markedly elevated Kiss1 m RNA levels in the arcuate nucleus of 6- and 8-week-old PCOS rats(P < 0.01), whereas no differences were observed in the levels of Kiss1 m RNA at the age of 4 weeks between the two groups. Prenatal DHT administration remarkably elevated NKB m RNA levels in the arcuate nucleus than control at the age of 4, 6 and 8 weeks(P < 0.05 or P < 0.01) and increased NKB3 R m RNA levels at the age of 4 weeks(P < 0.05). The 4- and 6-week-old PCOS rats had significantly increased Ob Rb m RNA levels in the arcuate nucleus compared with the control(P < 0.05). However, the levels of kiss1 r, NPY, NPYR, Gn RH, Ag RP and POMC m RNA were not different between two groups(P > 0.05). A large number of Kisspeptin and NKB neurons were detected in the arcuate nucleus of 6-week-old PCOS rats and the expression of Ob Rb were observed in these neurons. After Kisspeptin, Senktide or Leptin administration, the 15, 30 and 60-min LH levels in 6-week-old PCOS rats were significantly higher than vehicle(P < 0.05 or P < 0.01). In addition, the Kiss1 m RNA levels in the arcuate nucleus were significantly increased after leptin treatment(P < 0.01), whereas the levels of NKB m RNA did not differ between the two groups.Conclusion1. In this study, we administrated pregnant rats with DHT and established purbtal rat modle with PCOS. The prenatally androgenized rats exhibited PCOS-related reproductive and metabolic derangements during puberty, including polycystic ovaries, elevated body weight, hyperglycemia, hyperinsulinemia, hyperleptinemia and insulin resistance, similar to the development of PCOS in adolescents.2. We investigated the contribution of prenatal androgen excess to glucose tolerance and insulin sensitivity in pubertal PCOS rats. Prenatal androgen excess resulted in impaired insulin sensitivity in the PCOS rats during pubertal development and a trend toward more severe insulin resistance during and after puberty. We found the impaired insulin signaling through IRS and AKT may result in the peripheral insulin resistance during pubertal development.3. Exposure to excessive androgens in utero resulted in high frequency of serum LH pulses and enhanced expression of Kisspeptin, NKB and Ob Rb m RNA in the hypothalamic arcuate nucleus of pubertal PCOS rats. Kisspeptin, NKB receptor agonists and Leptin stimulated the release of LH in pubertal PCOS rats. Therefore, the Kisspeptin, NKB neurons and Leptin may play a role in the regulation of Gn RH and LH in the hypothalamus of PCOS rats at the peripubertal stage.4. These findings validate the contribution of prenatal androgen excess to developmental, reproductive and metabolic derangements of PCOS in pubertal female rats. In this study, we elucidated that the early pathophysiological mechanisms of PCOS as a fetal origin of adult disease in pubertal rat modle, which could be valuable for minimizing the development of symptoms and preventing the onset of the long-term complications associated with this syndrome in adolescents with PCOS.