The Expression Of The HTRA3 In The Ovary Of PCOS Rat And Its Impact On Follicular Development

Part IThe construction of rat model with polycystic ovarian syndrome(PCOS) and the expression of HTRA3 in the granulosa cells (GCs) and the serum of rats with PCOSObjective:1,To establish the polycystic ovarian syndrome (PCOS) rat model with the juvenile female Wistar rats by dehydroepiandrosterone (DHEA), and to observe the changes of ovarian morphology, sexual hormone and the insulin resistance.2,To compare the expression of HtrA3 mRNA and proteins in GCs and ovary , to test the levels of HtrA3 in serum and supernatant fluid of GCs from the rats with PCOS and the rats with functional ovary. To investigate the effect of HtrA3 gene on the pathogenesis involved in PCOS. Methods:1,Female Wistar rats of 21 days old were divided into two groups with 20 numbers each randomly, establishing the PCOS rat model by subcutaneous injecting DHEA6mg/100g weights with 0.2ml oils for 20 days, and the control group rats were injected the same volume oil. Prove the model with the methods as follow: Detect Testosterone, Estradiol, Luteinizing hormone levels, fasting blood glucose(FBG)and fasting insulin (FINS) levers by radioimmunoassay (RIA) and calculate the insulin resistant index (HOMA1-IR), view ovarian morphological changes by HE stain, and measure the ovarian weights and body weights.2,Select 20 rats from two groups, anesthetize them and take out of the two ovaries of each rat, slice and extract GCs for culturing, and collect serum. The expression of HtrA3 mRNA in GCs of PCOS group and control group were measured and evaluated by reverse transcription polymerase chain reaction (RT-PCR) and gel image analysis system.The expression of HtrA3 proteins in PCOS group and control group were tested by SABC immunohistochemical technique. And the serum and supernatant fluid levers of HtrA3 in the two groups were detected by enzyme linked immunosorbent assay (ELISA).Results:1,The serum level of testosterone , fasting insulin, fasting blood glucose concentration were significantly higher in model group than those in control group(P<0.05),and so as the insulin resistance index (P<0.05). The body weight in two groups showed no significant differences (P>0.05). In consideration of body weight influence, the ovarian weight in model group were higher than the control group (P<0.05). The HE stain of ovarian slices showed that in the model group, the number of follicular cysts were increased, While the developing follicles, corpus lutea (CL) and GCs were significantly diminished.There are many CL and developing follicles in different stages in the control group.2,Compared with the control group,the gel image analysis system demonstrated that the transcription of HtrA3mRNA were decreased significantly in PCOS group (P<0.01). The HtrA3 proteins were detected in all kinds of follicles, and the expression of them in model group were lower than in control group (P<0.05). Compared with the control group, the HtrA3 levers in the serum and supernatant fluid of model group were decreased (P<0.05, P<0.01).Conclusion:1,The changes of the ovarian morphology and sexual hormone in the PCOS rat model induced by DHEA were proved to be similar with that of the PCOS patients, and the rat model also has insulin resistance.2,HtrA3 gene is expressed in all stages of follicles, especially in the endochylema and nucleus of the oocytes and GCs.3,In the PCOS group, the expression of HtrA3mRNA and proteins in ovary and the level of HtrA3 in serum and supernatant fluid of GCs are all lower than in control group, which indicate HtrA3 gene may involved in the pathogenesis and development of PCOS.Part IIThe research of the relationship between HTRA3 and TGF-β1 level in PCOS ratsObjective: 1,To compare the levels of HtrA3 and TGF-β1 in serum and supernatant fluid of GCs from the rats with PCOS and the rats with functional ovary, and to analyze the roles of the two factors in PCOS.2,To analyze the relationship between HtrA3 and TGF-β1 level,for exploring the way HtrA3 involved in polycystic ovarian syndrome. Methods:The levels of HtrA3 and TGF-β1 in the two groups were detected by enzyme linked immunosorbent assay (ELISA), and analyze relationship between the two factors.Results:Compared with the control groups, the serum and supernatant fluid level of HtrA3 was decreased in the model group (P<0.05, P<0.01), but the level of TGF-β1 was increased (P<0.01, P<0.05). To analyze the relationship between HtrA3 and TGF-β1 concentration in serum and supernatant fluid of GCs, we can see the two factors have a negative correlation(r= -0.4327, P<0.01; r= -0.3997, P<0.05).Conclusion:The levels of HtrA3 and TGF-β1 in serum and in supernatant fluid of GCs in two groups are all have significant difference, and the two factors was negatively correlated both in serum and in supernatant fluid of GCs, which indicated that HtrA3 may involved in the pathogenesis of PCOS by regulating TGF-β1 signal pathway.