Study On The Role And Modulation Mechanism Of Tumor Necosis Factor-related Apoptosis-inducing Ligand Signaling Pathway During Follicle Development In Polycystic Ovary Syndrome Rats

Part 1 Establishing a rat model of polycystic ovary syndromeObjective: To establish an animal model of polycystic ovary syndrome(PCOS)．Methods: 23-day-old female Sprague-Dawley rats were choosed in this research．The ratsin experiment group were injected daily S.C. (Subcutaneous injection) with SodiumPrasterone Sulfate 9mg/100g for 20 days．The rats in control group were injected withwater for injection．The ovarian aspect,weight,morphology were observed,the ovarianultrastructure were examined with transmission electron microscope(TEM),and the serumtestosterone(T),estradiol(E2),progesterone(P),prolactin(PRL) levels were assessedbetween each group．Results:1.In experiment group rats,the ovarian weight were significantly higher than that incontrol group(P＜0.01),and there were many cystic follicles on the surface of theovary．2. Haematoxylin eosin staining of light microscope showed there were many cystic folliclesin the experiment group rats．The granular cell layer get attenuated,theca cellsproliferated and there were less corepus luteum forming．3. In experiment group rats,TEM showed that the granulosa cells were fusiform shape,nucleus were irregularly and intranuclear chromatin condensed．The fiber between cellincremented,theca cells layer increased,and there were lipid droplet in the theca cells．4. The serum T and E2 levels of rats in experiment group were significantly higher thanthose of control group(P＜0.05)．The serum P and PRL levels of rats in experiment group were similar to those of control group(P＞0.05)．Conclusion: The morphologic and endocrinological changes in the ovarian of the SodiumPrasterone Sulfate induced PCOS rat model were similar to those of PCOS patients．It wasproved to be an ideal PCOS animal model．Part 2 The relationship between follicle development arrested andgranulosa cells apoptosis in polycystic ovary syndrome ratsObjective:To investigate the relationship between follicle development arrested andgranulosa cells apoptosis in PCOS rats．Methods: Using Sodium Prasterone Sulfate induced PCOS rat model,the apoptosis of thegranulose cells were observed by TEM and TdT-mediated dUTP-biotin nickend-labeling(TUNEL)．Results:1. The ultrastructure of the granulose cells in PCOS rats displayed apoptoticcharacteristics:karyotheca of nucleus shrinkaged,karyotype aligned irregularly,intranuclear chromatin condensed,mitochondria swelled,mitochondrial cristae broke ordisappeared,and there were representative crescent-shaped apoptotic bodies in thegranulosa cells on the later stage of apoptosis．2. The apoptosis was detected by TUNEL．The incidence of apoptosis in granulosa cells ofantral follicles was significantly higher in PCOS rats than that in control group (P<0.01).The incidence of apoptosis in granulosa cells of preantral follicles was similar betweenthe two groups (P>0.05).There were no signs of apoptosis in granulosa cells ofprimordial follicles in either group．Conclusion: The incidence of apoptosis in granulosa cells of antral follicles wassignificantly higher in the PCOS rats．This may be one of the reason that induced arrested growth of antral follicles in the PCOS．Part 3 The role of tumor necosis factor-related apoptosis-inducingligand,death receptor 4 and decoy receptor 1 during follicle developmentin polycystic ovary syndrome ratsObjective:To investigate the role of necosis factor-related apoptosis-inducingligand(TRAIL),death receptor 4(DR4) and decoy receptor 1(DcR1) during follicledevelopment in polycystic ovary syndrome rats．Methods:Using Sodium Prasterone Sulfate induced PCOS rat model,detected theexpression of TRAIL,DR 4 and DcR1 in granulosa cells within polycystic ovary syndromerats through the immunhistochemical staining,reverse transcription polymerase chainreaction (RT-PCR) analysis and western blot analysis．Results:1. Immunohistochemical analyses showed,the expression of TRAIL protein in granulosacells were significantly stronger in antral follicles from the PCOS rats than in thosefrom the control rats(P<0.05),the expression of TRAIL protein in granulosa cells werenot significantly difference in preantral follicles between the PCOS rats and the controlrats(P>0.05),there were no positive staining of TRAIL protein in granulosa cells ofprimordial follicles in either group．RT-PCR and western blot analysis showed,theexpression of TRAIL mRNA and TRAIL protein were significantly increased ingranulosa cells from the PCOS rats than in those from the control rats(P<0.01)．2. Immunohistochemical analyses showed,the expression of DR4 protein in granulosacells were not significantly difference in preantral and antral follicles between thePCOS rats and the control rats (P>0.05),there were no positive staining of DR4 protein in granulosa cells of primordial follicles in either group．RT-PCR and western blotanalysis showed,the expression of DR4 mRNA and DR4 protein were not significantlydifference in granulosa cells between the PCOS rats and the control rats(P>0.05)．3. Immunohistochemical analyses showed,the expression of DcR1 protein in granulosacells were significantly weaker in antral follicles from the PCOS rats than in those fromthe control rats(P<0.01),the expression of DcR1 protein in granulosa cells were notsignificantly difference in preantral follicles between the PCOS rats and the controlrats(P>0.05),there were no positive staining of DcR1 protein in granulosa cells ofprimordial follicles in either group．RT-PCR and western blot analysis showed,theexpression of DcR1 mRNA and DcR1 protein were significantly decreased in granulosacells from the PCOS rats than in those from the control rats(P<0.01)．Conclusion:TRAIL,DR4 and DcR1 played a role in regulation the apoptosis of granulosecells during follicle development in PCOS rats．The abnormal expression of TRAIL,DR4and DcR1 in granulosa cells may be one of the modulation mechanism that induced thefailure during follicle development in PCOS．Part 4 The expression of caspase3 in granulosa cells within polycysticovary syndrome ratsObjective:To investigate the finally effective stage of TRAIL signaling pathway duringapoptosis in granulosa cells within polycystic ovary syndrome rats．Methods:Using Sodium Prasterone Sulfate induced PCOS rat model,detected theexpression of caspase3 in granulosa cells within polycystic ovary syndrome rats through thewestern blot analysis and reverse transcription polymerase chain reaction (RT-PCR) analysis．Results:1. Western blot analysis showed,the expression of actived caspase3 protein weresignificantly increased in granulosa cells from the PCOS rats than in those from thecontrol rats(P<0.01).2. RT-PCR analysis showed,the expression of caspase3 mRNA were significantlyincreased in granulosa cells from the PCOS rats than in those from the controlrats(P<0.01).Conclusion:TRAIL signaling pathway may be pass the caspase cascade finally during theapoptosis of granulosa cells in PCOS rats,it induced apoptosis by activating caspase3．...