Altered Protein Expression Profiles In PCOS Ovary And Lysosomal Cathepsin D Related Mechanisms Of Follicle Development

Polycystic ovary syndrome(PCOS),is a common disorder,affecting approximately 6～10%of women during their childbearing years.It is characterized with chronic anovulation,menstrual irregularites and infertile;evidence of hyperandrogenism(either clinical,manifested as hirsutism,ache,or biochemical,manifested by elevated serum adrenal/ovarian androgen concentration),and is also associated with obesity,insulin resistance and hyperinsulinaemia.Women with PCOS are also at increased risk of cardiovascular disease and endometrial cancer.The condition is now well recognized as having a major effect throughout life on the reproductive,metabolic,and cardiovascular health of affected women.Over the years,a variety of theories have been proposed to explain the development of PCOS,although its underlying etiology remains unknown and the pathophysiology is unclear.Arrest of follicle maturation in polycystic ovary is a characteristic feature of PCOS.A complete understanding of protein expression profiles of female ovary,a main gonad with responsibility for producing oocyte,could provide underlying mechanism of folliclogenesis.Proteomic analysis has proved itself to be a sophisticated technique, which enabled us to generate a protein expression profile in any given tissue.In present study,two-dimensional electrophoresis(2-DE) combining with matrix assisted laser desorption/ionization-time-of-flight mass spectrometry(MALDI-TOF-MS) was undertaken to characterize differences in protein expression patterns in ovarian tissues obtained from PCOS patients and normal controls in order to detect ovarian proteins regarding follicular development,and to reveal underlying pathogenesis of PCOS.Cathepsin D(cath-D) is an aspartic endo-protease that is ubiquitously distributed in lysosomes.Cath-D may promote apoptosis as a key mediator of induced-apoptosis, although its function in apoptosis is not yet fully understood.In the present study,we have detected that cath-D level decreased significantly in PCOS patient ovaries by using 2-DE combined with MALDI-TOF-MS.Further studies of cath-D expression in cultured immature rat follicles and granulose cells stimulated by over-dose dehydroepiandrosterone indicated a correlation of follicular/cell apoptosis with cath-D levels.Cath-D gene silence in rat granulose cells approved its role in cell apoptosis upon stimulation with over-dose dehydroepiandrosterone which mimic hyperandrogenism of PCOS.Meanwhile,we found follicle-stimulating hormone had an adverse effect on cath-D level of immature follciles and granulose cells.These findings may contribute to a better understanding of molecular mechanism of follicular development and provide clues for insight into the pathogenesis and treatment of PCOS. Part OneAltered Protein Expression Profiles and Lower Lysosomal Cathepsin D level in PCOS OvaryObjective:To investigate the changes of protein expression profile of PCOS ovaries in order to detect ovarian proteins regarding folliculogenesis.Method:Two-dimensional electrophoresis(2-DE) combining with matrix assisted laser desorption/ionization-time-of-flight mass spectrometry(MALDI-TOF-MS) was performed to describ protein expression profiles in human ovaries comparing PCOS patients vs.healthy individuals.Changes of cath-D and annexin 6 expressions were further validated in by using western blot analysis.Results:Image analysis of silver-stained 2-dimensional gels identified total of 23 proteins that were up- or downregulated in PCOS ovaries,18 proteins were identified by MALDI-TOF MS.These proteins are involved in Ca2+ signaling,apoptosis,toxicity, and lipoprotein metabolism,and so on.Conclusion:Altered protein expression in PCOS ovary,including lower level of apoptosis associated cath-D,may involve in folliclogenesis. Part Two Effects of Follicle-Stimulating Hormone and Dehydroepiandrosterone on the Morphology and Expression of Cathepsin D of Immature Rat Follicles in CultureObjective:To investigate the effects of gonadotrophic hormone and steroids on the morphology and expression of cath-D of immature rat follicles in cultureMethod:A histological analysis was performed after culturing immature follicles for 4～6 days with FSH,17β-estradiol,dehydroepiandrosterone or alone(control).Cath-D expression was detected by using western blot analysis.Results:When immature rat follicles were cultured in the presence of the 10ng/ml FSH, the diameter of follicles increased significantly compared with control.Meanwhile,the level of cath-D decreased following the treatment with FSH.10^-6 M. dehydroepiandrosterone had an adverse effect on immature rat follicle proliferation compared with FSH.No obvious change of cath-D expression in follicles was observed with treatment of over-dose dehydroepiandrosterone.Both 10^-8M 17β-estradiol and 10^-8M dehydroepiandrosterone had no effect on follicle morphology and cath-D expression.Conclusion:1.Over-dose dehydroepiandrosterone induced apoptosis of immature rat follicles in culture.2.FSH promoted immature rat follicle proliferation and down-regulated cath-D levels. Part Three Role of Follicle-Stimulating Hormone and Steroids on Expression of Cathepsin D of Cultured Rat Granulose Cells and Its Underlying Mechanism Regarding Over-dose Dehydroepiandrosterone Induced ApoptosisObjective:To investigate the role of gonadotrophic hormone and steroids on cath-D expression of cultured rat granulose cells and its underlying mechanism regarding over-dose dehydroepiandrosterone induced apoptosis.Method:Cath-D level of in vitro rat granulose cells cultured for 48h with FSH, 17β-estradiol,dehydroepiandrosterone or alone(control).The mitotic and apoptotic indexes of cath-D gene silenced granulose cells were evaluated by alamar blue analysis and flow cytometry.Location and expression of caspase-3,a cath-D dependent apoptotic inducer,were detected by using immunofluorescence technique.Results:Both 10ng/ml and 100ng/ml FSH had promoted granulose cell proliferation, and decreased the level of cath-D of granulose cells.On the opposite,10^-6 M dehydroepiandrosterone induced granulose cell increased the level of cath-D.The rate of cath-D knockdown granulose cell apoptosis decreased after treated with 10^-6 M dehydroepiandrosterone.Expression of caspase-3 also decreased in cath-D knockdown granulose cell cultured with over-dose dehydroepiandrosterone.Conclusion:1.Over-dose dehydroepiandrosterone up-regulated level of cath-D of rat granulose cells in culture.2.FSH down-regulated cath-D expression of rat granulose cell. 3.10^-8 M estradiol and 10^-8 M dehydroepiandrosterone had no effect on cath-D expression.4.Caspase-3 dependent cath- D pathway,may participate the mechanism of rat granulose cell apoptosis induced by over-dose dehydroepiandrosterone.