The Relevant Study Between PCOS Granulosa Cell Apoptosis And MTOR Expression

Polycystic ovary syndrome（PCOS）is a kind of complex female endocrine and metabolism disorder with an incidence of 5-10%.At present,the mechanism of PCOS has not been fully elucidated.Mammalian Target of Rapamycin（mTOR）is a classic highly conserved silk/threonine protein kinases,the study found that mTOR can accept and integrate the stimulation of various signaling pathways inside and outside the cell,such as hormones,growth factors,nutrition,energy metabolism and stress,and then translate proteins.Physiological processes such as cell growth,cell proliferation,cell survival,autophagy and metabolism of cells are regulated.And mTOR is also involved in the whole process of follicular growth and development.So mTOR is also very important in reproductive field.The abnormal follicular atresia of PCOS may be also due to the disorder of mTOR and granulosa cell apoptosis.In this study,we aimed to analyze the change of ovulation induction on mTOR and granulosa cell apoptosis of PCOS patients and PCOS rats,and to discover the correlationship between mTOR and granulosa cell apoptosis of PCOS.Part one The relevant study between PCOS granulosa cell apoptosis and mTOR expression in IVF-ETObjecttve:To investigate the mechanism of abnormal follicular development in PCOS patients,and to find the correlation by analyzing methods between mTOR and granulosa cell apoptosis in PCOS patients of IVF-ET.Methods:A total of 120 patients with ICSI treatment because of male moderate or severe asthenosperm and azoospermatism were and enrolled in the Reproductive Medicine department from January 2015 to December 2016.They were devided into the PCOS group and the control group,each group with 60 patients.Befor the ovarian stimulation,blood was collected during the2-3 menstrual cycle.The levels of serum follicle stimulating hormone（FSH）,luteinizing hormone（LH）,estrogen（E₂）and testosterone（T）were test using chemiluminescence.After eggs retrieval,granulosa cells were separated and extracted.The expressions of mTOR,Akt,S6K1,caspase-3,bcl-2 and bax in the two groups were detected by RT-PCR and Western blot,apoptosis rates were calculated by TUNEL.Statistics methods were used to anlysis the correlationship between mTOR and granulosa cell apoptosis of PCOS patients.Results:The levels of LH and T in the serum of PCOS group were significantly higher than those of the control group（P<0.05）,the level of E₂was significantly lower than that of the control group（P<0.05）,and the levels of FSH was not different with those of the control group（P>0.05）.In the PCOS group,the levels of mTOR,Akt,S6K1 m RNA and relative protein expression were significantly higher than those in the control group（P<0.05）.In the PCOS group,the levels of caspase-3,bax m RNA,relative protein expression and apoptosis rate were significantly higher than those in the control group（P<0.05）,while the m RNA and relative protein expression of bcl-2 was lower than control group（P<0.05）.Conclusion:During IVF-ET treatment of PCOS,mTOR was actived and granulosa cell apoptosis was more increasing,there were certain correlations between the maturity of oocyt,early abortion rate and mTOR and granulosa cell apoptosis.Part two Effect of ovulation induction on PCOS rats granulosa cell apoptosis and mTOR expressionObjecttve:To explore the mechanism of granulosa cell apoptosis mediated by mTOR in abnormal follicular development in PCOS,and the effect of ovulation induction drugs on the expression of mTOR and granulosa cell apoptosis,so as to provide experimental basis for the treatment of PCOS.Methods:Thirty healthy SD female rats aged 6 weeks with an average weight of 200g were divided into control group（n=10）,PCOS model group（n=10）,PCOS ovulation induction group.Rats were injected 0.2ml DHEA（6mg/100g/d）on the neck and back for 21 days.During the period of model building,vaginal smears changes in each group rats were continuous observated.Except the the control group rats,vaginal smear results of all other group rats indicated that regular ovulation disappeared,this phenomenon could be judged as PCOS animal model was builded successfully.After PCOS rats models were built by DHEA,serum follicle stimulating hormone（FSH）,luteinizing hormone（LH）,estrogen（E₂）,testosterone（T）levels,fasting blood-glucose（FBG）,fasting insulin（FINS）and HOMA index were tested about insulin resistance.Then PCOS ovulation induction group rats were continuedly injected400U/kg pregnant horse serum（PMSG）.Then 48h later,they had intraperitoneal injection of 1000U/kg HCG.Body weight and ovary weight of rats were compared among three groups.Ovarian histology morphology were observed by HE staining,immunohistochemical staining were used to found positive expression and localization of mTOR.Rats follicular granulosa cells were separated and cultured in vitro,and finally RT-PCR,Western blot and TUNEL were taken to detect expressions of mTOR,Akt,S6K1 in mTOR signaling pathways,caspase-3,bal-2,bax and apoptosis rates.Statistics methods were used to anlysis the correlationship between mTOR and granulosa cell apoptosis of PCOS rats before and after ovulation induction treatment.Results:The average body weights and ovary weights of three group rats were significant different by statistics analysing（P<0.05）.After PCOS animal model building with DHEA,serum hormon levels of three groups indicated that LH and T in PCOS group and PCOS ovulation induction group were significantly higher than control group（P<0.05）,E₂was significantly reduced,and significantly lower than the control group（P<0.05）,FSH was same among three groups.Serum FBG and FINS levels,HOMA index were significantly higher in PCOS group and PCOS ovulation induction group than in control group（P<0.05）.These changes suggestted that insulin resistance existed in the PCOS rat model after DHEA injection that were more coincident with PCOS characteristics.In PCOS group,typical polycystic change of rats ovaries occured,and cystic expansion and more small follicles and large follicles were observed with microscope,without luteum forming especially in the inner of granular cell.On the contrary,in the control group and PCOS ovulation induction group,luteum and different period follicles emerged,such as preantral follicles and mature follicels.mTOR was widely expressed in ovarian tissue,follicular fluid,inner theca cells and granulosa cells,but it was relatively low expressed in follicular fluid.In granular cells,the positive expression rate of mTOR was 58.7%in the control group,it was 86.7%in PCOS group,and it was 60.1%in the PCOS ovulation induction group.There was statistically difference（P<0.05）between the PCOS group and the control group or PCOS ovulation induction group,while there was no statistically difference between the control group and PCOS ovulation induction group（P>0.05）.The levels of mTOR,Akt,S6K1 m RNA and relative protein expression in the PCOS group were significantly higher than those in the control group and PCOS ovulation induction group（P<0.05）.The levels of caspase-3,bax m RNA,relative protein expression and apoptosis rate in the PCOS group were significantly higher than those in the control group and PCOS ovulation induction group（P<0.05）.While the m RNA and relative protein expression of bcl-2 in the PCOS group were significantly lower than those in the control group and PCOS ovulation induction group（P<0.05）.Conclusion:mTOR can adjust the follicle development,maturation and apoptosis.As the treatment of PCOS ovulation obstacle,ovulation induction drugs can reduce follicular granulosa cell apoptosis and promote the follicular development of PCOS rats,possibly through effect on mTOR signaling pathway function.Thus mTOR is expected to become the new targets for PCOS treatment.