The Local Role Of Oocyte, GDF-9 And TGF-β₁ In The PCOS Rat

Objective:Using the PCOS rat model induced by Dehydroepiandrosterone(DHEA).To study the local role of oocyte, GDF-9 and TGF-β1 in the formation of cystic follicles, stroma fibrosis, capsular thickening, no dominant follicle, and the change of hormone values in PCOS.Methods:1. Established PCOS rat model by subcutaneous injection DHEA. Estradiol (E2), Testosterone (T), Follicular stimulating hormone (FSH), Luteinizing hormone (LH), LH/FSH and fasting insulin(FINS) hormone values of the model were tested by microparticle enzyme immunoassay, the pathohistology of PCOS rats ovarian structure was observed by HE staining, and its ultra- structure was tested by transmission electron microscope (TEM).2. The ultra-structure of oocyte in PCOS groups (w=8), and control groups (n=8) was investigated by transmission electron microscope.3. The expression of GDF-9, TGF-β1 protein in PCOS groups (n=20),and control groups( n =20) were detected and evaluated by immunohistochemistry and image analysis system.4. The transcription of BMPR-Ⅱ mRNA, a receptor of GDF-9, TβR Ⅰ and TβR Ⅱ mRNA,the receptors of TGF-β1, in PCOS groups (n=8) and control groups (n=8), were measured and evaluated by semi-quantitative RT-PCR (reverse transcription polymerase chain reaction) and gel image analysis system.5. The expression of Smad4 protein in PCOS groups (n=20) and control groups (n =20) were measured by immunohistochemistry, and the transcription of Smad4 and Smad7 mRNA in PCOS groups (n=8), and control groups (n=8), were measured and evaluated by semi-quantitative RT-PCR (reverse transcription polymerase chain reaction) and gel image analysis system.Results:1. The values of T, E2 and FINS in PCOS groups were significantly higher than those of the control groups (P<0.05), the pathological structure showed that there were more cystic follicles and atresia follicles in PCOS, the layers of granulose cell became thinner, but the theca layers were thickened, corpora lutea(CL) were only occasionally observed. The ultrastructure showed that collagen was increased in stroma, and abundant of mitochondria, smooth endoplasmic reticulum (SER) and lipid droplet was seen in the theca-interstitial cells (TIC).2. The changes of cell organelles, and microvilli(MV) of oocytes and the degeneration or necrosis of grannulosa cell(GC) around the oocyte were found in PCOS oocytes of preantral and antral follicles. The number of abnormal oocytes of preantral and antral follicles in PCOS groups was significantly more than that in the control groups (p<0.01).3. There was no significant difference of GDF-9 expression intensity between PCOS groups and control groups (P>0.05), but the expression intensity of TGF-β1 in the theca cells of antral follicles and the stromal cells of PCO groups were markedly higher than those of the control groups respectively (P<0.05).4. Compared with the control groups, the transcription of GDF-9 receptor BMPR-Ⅱ mRNA was increased signiffcantly in PCOS groups (P<0.05), and TGF-β1 receptorsTβR Ⅰ , TβR Ⅱ mRNA were also higher in PCOS groups (P<0.05).5. Compared with the control groups, the expression intensity of Smad4 protein in the stromal cells of PCOS groups was markedly higher (P<0.05), and the transcription of Smad4 mRNA and Smad7 mRNA was also higher in PCOS groups (P<0.05).Conclusions:1. The results of serum E2, T, LH/FSH, FINS, and the pathological-histology, the ultrastructure of ovary show that the model is established successfully.2. The change of oocyte structure causes the PCOS specific morphology; the apoptosis of oocyte might cause a follicle atresia and the necrosis of oocyte is the basis for cyst formation.3. GDF-9 plays an important role in regulating the follicle development and maintaining the growth of theca- interstitial cells and androgen secretion in PCOS.4. The increasing expression of GDF-9 receptor BMPR- II and signal transduction proteinSmad4 and Smad7 in PCOS might promote androgen secretion, and influence the function of GDF-9 in regulating ovary development and ovulation.5. The abnormal expression of TGF-β1 in PCOS is the main causes of capsular thickening, stroma fibrosis; TGF-β1 also has effects in regulating follicle growth, atresia and disturbing steroidogenesis.6. The increasing expression of TGF-β1 receptors, signal transduction protein Smad4 and Smad7 in PCOS enhance the function of TGF-β1 in inducing stroma fibrosis, and influence the function of TGF-β1 in regulating follicles development.