PCOS卵巢组织蛋白表达的改变及组织蛋白酶D与卵泡发育调节机制研究
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摘要
研究背景
多囊卵巢综合征(polycystic ovary syndrome,PCOS)是最常见的一种内分泌疾病,在育龄期妇女人群中发病率约6～10%。主要临床症状表现为:卵巢无排卵导致的月经稀发、闭经和不孕;高雄激素血症及多毛、唑疮等症状;肥胖、胰岛素抵抗和高胰岛素血症;并存在发生心血管疾病、子宫内膜癌等远期疾病风险。PCOS病程常从青春期开始,部分病人甚至终身受其影响。PCOS病因和病理机制至今尚未完全阐明,相应地也缺乏有效的治疗手段,严重威胁着女性健康,是目前妇产科急待解决的临床问题之一。
卵泡发育障碍是PCOS的一个特征性病理改变,卵巢做为卵子发生的唯一场所,其局部蛋白表达及功能异常将直接影响卵泡发育的结局。因此明确PCOS卵巢组织与正常卵巢组织蛋白表达的差异并找到参与调控卵泡发育机制的相关蛋白是研究PCOS卵泡发育机制的一个有效切入点,对最终阐明PCOS病理机制改变有着重要的意义。基于上述观点,本研究采用高通量高效率的蛋白组学技术筛查出PCOS卵巢组织中与正常卵巢组织相比差异表达的蛋白质,并挑选可能与卵泡发育异常机制相关的蛋白行进一步研究。
组织蛋白酶D(cathepsin D,cath-D)是凋亡机制途径中一种重要的参与因子。卵巢蛋白组学研究首次发现了cath-D在PCOS卵巢组织总蛋白中表达降低。由于卵巢组织细胞成分复杂,为进一步明确cath-D在卵泡发育过程中所参与的调节机制,分别在体外培养的大鼠未成熟卵泡和颗粒细胞体系中,使用卵泡刺激素(follicle-stimulating hormone,FSH)、17β雌二醇(17β-estradiol,17β-E_2)和脱氢表雄酮(dehydroepiandrosterone,DHEA)刺激干预,观察cath-D表达与卵泡及颗粒细胞增殖/凋亡的相关性,证实cath-D表达受到FSH和DHEA的调节。进一步对cath-D基因行RNA干扰(RNA imerference,RNAi)证明了cath-D参与DHEA诱导卵泡凋亡的调节机制。离体研究中模拟PCOS特征性高雄激素血症改变,采用超生理浓度DHEA干预卵泡和颗粒细胞生长,首次发现并证实了cath-D参与高雄激素导致卵泡发育障碍的调控机制,为深入明确PCOS病理生理机制研究提供了有价值的理论基础,并为PCOS临床治疗药物的开发研制提供有利线索。
目的:比较PCOS卵巢组织和正常卵巢组织差异表达的蛋白质,寻找可能与PCOS卵巢中卵泡发育机制相关的蛋白质。
方法:采用双向电泳方法分离PCOS组和正常对照组总蛋白,经PDQuest软件分析,分别找到表达上升/下降的蛋白点;利用基质辅助激光解吸附电离飞行时间质谱技术(matrix assisted laser desorption/ionization-time-of-flight mass spectrometry,MALDI-TOF MS)对差异蛋白进行鉴定,获得肽质量图谱,通过与蛋白数据库中的资料对比,得到目标蛋白,并用westem blot法进行鉴定。
结果:PCOS组和正常对照组卵巢组织双向电泳蛋白谱存在差异;通过软件分析,共找到23个表达水平差异的蛋白点;经过MALDI-TOF MS技术,共鉴定出18个蛋白点(17种蛋白质)。选择其中两个蛋白Cathepsin D和Annexin6为代表性蛋白进行验证,结果和双向电泳结果一致。
结论:
1.PCOS卵巢组织中部分蛋白质表达与正常卵巢组织相比存在差异,差异表达的蛋白质与多种生命活动机制相关。
2.包括Cathepsin D和Annexin6在内的多个蛋白质表达差异可能与PCOS病理机制相关。
目的:观察促性腺激素和性激素作用下,体外培养的大鼠未成熟卵泡形态学改变,以及相应的cath-D表达改变。
方法:体外培养的大鼠未成熟卵泡分别予以不同种类和剂量的激素干预4-6d,观察卵泡大体和微观形态学的改变,并以western blot法检测cath-D蛋白表达。
结果:10ng/mlFSH能促进体外培养大鼠未成熟卵泡的直径增大,同时western blot法检测发现FSH作用将减少卵泡细胞cath-D蛋白的表达。10~(-6)M DHEA诱发未成熟卵泡出现凋亡的形态学改变,但是卵泡cath-D蛋白表达水平无明显改变。10~(-8)M 17β-E_2和10~(-8)M DHEA条件作用下,体外培养的大鼠未成熟卵泡形态和cath-D表达无明显改变。
结论:1.超生理浓度的DHEA作用将诱使体外培养的大鼠未成熟卵泡发生凋亡。
2.生理浓度的FSH能促进体外培养大鼠未成熟卵泡生长。FSH对体外培养的卵泡中cath-D表达起降调作用。
第三部分卵泡刺激素和甾体激素作用下大鼠颗粒细胞cathepsin D表达及其参与高雄激素诱导细胞发生凋亡的机制研究
目的:观察卵泡刺激素和甾体激素作用下大鼠颗粒细胞中cath-D表达改变。探讨cath-D参与雄激素诱导的颗粒细胞发生凋亡的机制及后续途径。
方法:体外培养的大鼠颗粒细胞以不同种类和浓度激素干预,western blot法观察cath-D表达改变。Cath-D基因行RNAi后,采用高雄激素刺激后,alamar blue analysis测定细胞代谢率,流式细胞分析仪测定细胞凋亡情况,荧光免疫细胞化学法观察caspase-3表达定位。
结果:10ng/ml和100ng/ml FSH均能促进大鼠颗粒细胞生长,同时降低颗粒细胞中cath-D的表达;而10~(-6)M DHEA诱导颗粒细胞发生凋亡并使cath-D表达升高;10~(-8)M 17β-E_2和10~(-8)M DHEA作用对颗粒细胞cath-D表达无影响。颗粒细胞cath-D基因行RNAi后,采用同样的超生理浓度DHEA(10~(-6)M)作用,细胞代谢率提高先于未RNAi组,且流式细胞分析仪测定显示凋亡细胞比例减少,荧光免疫细胞化学法观察caspase-3荧光表达减弱。
结论:1.超生理浓度的DHEA上调颗粒细胞cath-D表达。
2.生理浓度的FSH对颗粒细胞cath-D表达起降调作用。
3.生理浓度的17β-E_2和DHEA对大鼠颗粒细胞cath-D表达无明显调节作用。
4.依赖于caspase-3途径的cath-D参与雄激素诱导颗粒细胞发生凋亡机制。
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 One
Altered Protein Expression Profiles and Lower Lysosomal Cathepsin D level in PCOS Ovary
Objective: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 Culture
Objective:To investigate the effects of gonadotrophic hormone and steroids on the morphology and expression of cath-D of immature rat follicles in culture
Method: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~(-8)M 17β-estradiol and 10~(-8)M 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 Apoptosis
Objective: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.
多囊卵巢综合征(polycystic ovary syndrome,PCOS)是最常见的一种内分泌疾病,在育龄期妇女人群中发病率约6～10%。主要临床症状表现为:卵巢无排卵导致的月经稀发、闭经和不孕;高雄激素血症及多毛、唑疮等症状;肥胖、胰岛素抵抗和高胰岛素血症;并存在发生心血管疾病、子宫内膜癌等远期疾病风险。PCOS病程常从青春期开始,部分病人甚至终身受其影响。PCOS病因和病理机制至今尚未完全阐明,相应地也缺乏有效的治疗手段,严重威胁着女性健康,是目前妇产科急待解决的临床问题之一。
卵泡发育障碍是PCOS的一个特征性病理改变,卵巢做为卵子发生的唯一场所,其局部蛋白表达及功能异常将直接影响卵泡发育的结局。因此明确PCOS卵巢组织与正常卵巢组织蛋白表达的差异并找到参与调控卵泡发育机制的相关蛋白是研究PCOS卵泡发育机制的一个有效切入点,对最终阐明PCOS病理机制改变有着重要的意义。基于上述观点,本研究采用高通量高效率的蛋白组学技术筛查出PCOS卵巢组织中与正常卵巢组织相比差异表达的蛋白质,并挑选可能与卵泡发育异常机制相关的蛋白行进一步研究。
组织蛋白酶D(cathepsin D,cath-D)是凋亡机制途径中一种重要的参与因子。卵巢蛋白组学研究首次发现了cath-D在PCOS卵巢组织总蛋白中表达降低。由于卵巢组织细胞成分复杂,为进一步明确cath-D在卵泡发育过程中所参与的调节机制,分别在体外培养的大鼠未成熟卵泡和颗粒细胞体系中,使用卵泡刺激素(follicle-stimulating hormone,FSH)、17β雌二醇(17β-estradiol,17β-E_2)和脱氢表雄酮(dehydroepiandrosterone,DHEA)刺激干预,观察cath-D表达与卵泡及颗粒细胞增殖/凋亡的相关性,证实cath-D表达受到FSH和DHEA的调节。进一步对cath-D基因行RNA干扰(RNA imerference,RNAi)证明了cath-D参与DHEA诱导卵泡凋亡的调节机制。离体研究中模拟PCOS特征性高雄激素血症改变,采用超生理浓度DHEA干预卵泡和颗粒细胞生长,首次发现并证实了cath-D参与高雄激素导致卵泡发育障碍的调控机制,为深入明确PCOS病理生理机制研究提供了有价值的理论基础,并为PCOS临床治疗药物的开发研制提供有利线索。
目的:比较PCOS卵巢组织和正常卵巢组织差异表达的蛋白质,寻找可能与PCOS卵巢中卵泡发育机制相关的蛋白质。
方法:采用双向电泳方法分离PCOS组和正常对照组总蛋白,经PDQuest软件分析,分别找到表达上升/下降的蛋白点;利用基质辅助激光解吸附电离飞行时间质谱技术(matrix assisted laser desorption/ionization-time-of-flight mass spectrometry,MALDI-TOF MS)对差异蛋白进行鉴定,获得肽质量图谱,通过与蛋白数据库中的资料对比,得到目标蛋白,并用westem blot法进行鉴定。
结果:PCOS组和正常对照组卵巢组织双向电泳蛋白谱存在差异;通过软件分析,共找到23个表达水平差异的蛋白点;经过MALDI-TOF MS技术,共鉴定出18个蛋白点(17种蛋白质)。选择其中两个蛋白Cathepsin D和Annexin6为代表性蛋白进行验证,结果和双向电泳结果一致。
结论:
1.PCOS卵巢组织中部分蛋白质表达与正常卵巢组织相比存在差异,差异表达的蛋白质与多种生命活动机制相关。
2.包括Cathepsin D和Annexin6在内的多个蛋白质表达差异可能与PCOS病理机制相关。
目的:观察促性腺激素和性激素作用下,体外培养的大鼠未成熟卵泡形态学改变,以及相应的cath-D表达改变。
方法:体外培养的大鼠未成熟卵泡分别予以不同种类和剂量的激素干预4-6d,观察卵泡大体和微观形态学的改变,并以western blot法检测cath-D蛋白表达。
结果:10ng/mlFSH能促进体外培养大鼠未成熟卵泡的直径增大,同时western blot法检测发现FSH作用将减少卵泡细胞cath-D蛋白的表达。10~(-6)M DHEA诱发未成熟卵泡出现凋亡的形态学改变,但是卵泡cath-D蛋白表达水平无明显改变。10~(-8)M 17β-E_2和10~(-8)M DHEA条件作用下,体外培养的大鼠未成熟卵泡形态和cath-D表达无明显改变。
结论:1.超生理浓度的DHEA作用将诱使体外培养的大鼠未成熟卵泡发生凋亡。
2.生理浓度的FSH能促进体外培养大鼠未成熟卵泡生长。FSH对体外培养的卵泡中cath-D表达起降调作用。
第三部分卵泡刺激素和甾体激素作用下大鼠颗粒细胞cathepsin D表达及其参与高雄激素诱导细胞发生凋亡的机制研究
目的:观察卵泡刺激素和甾体激素作用下大鼠颗粒细胞中cath-D表达改变。探讨cath-D参与雄激素诱导的颗粒细胞发生凋亡的机制及后续途径。
方法:体外培养的大鼠颗粒细胞以不同种类和浓度激素干预,western blot法观察cath-D表达改变。Cath-D基因行RNAi后,采用高雄激素刺激后,alamar blue analysis测定细胞代谢率,流式细胞分析仪测定细胞凋亡情况,荧光免疫细胞化学法观察caspase-3表达定位。
结果:10ng/ml和100ng/ml FSH均能促进大鼠颗粒细胞生长,同时降低颗粒细胞中cath-D的表达;而10~(-6)M DHEA诱导颗粒细胞发生凋亡并使cath-D表达升高;10~(-8)M 17β-E_2和10~(-8)M DHEA作用对颗粒细胞cath-D表达无影响。颗粒细胞cath-D基因行RNAi后,采用同样的超生理浓度DHEA(10~(-6)M)作用,细胞代谢率提高先于未RNAi组,且流式细胞分析仪测定显示凋亡细胞比例减少,荧光免疫细胞化学法观察caspase-3荧光表达减弱。
结论:1.超生理浓度的DHEA上调颗粒细胞cath-D表达。
2.生理浓度的FSH对颗粒细胞cath-D表达起降调作用。
3.生理浓度的17β-E_2和DHEA对大鼠颗粒细胞cath-D表达无明显调节作用。
4.依赖于caspase-3途径的cath-D参与雄激素诱导颗粒细胞发生凋亡机制。
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 One
Altered Protein Expression Profiles and Lower Lysosomal Cathepsin D level in PCOS Ovary
Objective: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 Culture
Objective:To investigate the effects of gonadotrophic hormone and steroids on the morphology and expression of cath-D of immature rat follicles in culture
Method: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~(-8)M 17β-estradiol and 10~(-8)M 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 Apoptosis
Objective: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.
引文
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[3] M.Bergendah,J.D.Veldhuis.Is there a physiological role for gonadotrophin oligosaccharide heterogeneity in humans? HumReprod 2001;6:1058-1064.
[4] Suhail A.R.D.,Philip A.T.,Christopher J.S.,Kamal A.S.PCOS:an ovarian disorder that leads to dysregulation in the hypothalamic-pituitary-adrenal axis?Euro J Obstet Gynecol Reprod Biol 2005;118:4-16.
[5] Aradhana M.V.,Andrea D.,Anne C.Insulin resistance in polycystic ovary syndrome:progress and paradoxes.Recent Prog Horm Res.2001;56:295-308.
[6] Franks S.,McCarthy M.I.,Hardy K.Development of polycystic ovary syndrome:involvement of genetic and environmental factors.Inter J Andro 2006;29:278-285.
[7] Gilling-Smith C,Story EH,Franks S:Evidence for a primary abnormality of thecal cell steroidogenesis in the polycystic ovary syndrome.Clin Endocrinol 1997;47:93-99.
[8] Pache TD,Hop WC,de Jong FH,Leerentveld RA,van Geldorp,H,Van de Kamp TM,Gooren LJ,Fauser BC:17 beta-Oestradiol,androstenedione and inhibin levels in fluid from individual follicles of normal and polycystic ovaries,and in ovaries from androgen treated female to male transsexuals.Clin Endocrinol(Oxf)1992;36:565-572.
[9] Hanrahan JP,Gregan SM,Mulsant P,Mullen M,Davis GH,Powell R,Galloway SM Mutations in the genes for oocyte-derived growth factors GDF9 and BMP 15 are associated with both increased ovulation rate and sterility in Cambridge and Belclare sheep(Ovis aries).Biol Reprod.2004;70:900-909.
[10]Lee WS,Yoon SJ,Yoon TK,Cha KY,Lee SH,Shimasaki S,Lee S,Lee KA.Effects of bone morphogenetic protein-7(BMP-7)on primordial follicular growth in the mouse ovary.Mol Reprod Dev.2004;69:159-163.
[11]Attisano L,Wrana JL Signal transduction by the TGF-beta superfamily.Science.2002;296:1646-1647.
[12]Fortune J.E.The early stages of follicular development:activation of primordial follicles and growth of preantral follicles.Animal Reprod Sci 2003;78:135-163.
[13]Marzieh S.Rafael B.V.,Arsalan S.,Alina G.,Leonid P.Pathogenesis of polycystic ovary syndrome:what is the role of obesity.Metabolism 2004;53:358-376.
[14]Makoto O,Sanae O,Jin-Yi J,Jesse G,Yifang W,Fumikazu K,Benjamin KT:Growth Differentiation Factor 9 Is Antiapoptotic during Follicular Development from Preantral to Early Antral Stage.Mol Endocrinol 2006;20:2456-2468.
[15]McNatty KP,Juengel JL,Reader KL,Lun S,Myllymaa S,Lawrence SB,Western A,Meerasahib MF,Mottershead DG,Groome NP,Ritvos O,Laitinen MP:Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants.Reproduction 2005;129:481-487.
[16]Wood J.R.,Nelson V.L.,Ho C,Jansen E.,Wanf CY.,Urbanek M.,Mcallister J.M.,Mosselman S.,Strauss J.F.The molecular phenotype of polycystic ovary syndrome(PCOS)theca cells and new candidate PCOS genes defined by microarray analysis.J Biol Chem 2003;278:26380-26390.
[17]Thompson W.E.,Powell J.,Thomas K.H.,Whittaker J.A.Immunolocaliztion and expression of the steroidogenic acute regulatory protein during the transitional stages of rat follicular differentiation.J Histochem Cytochem 1999,47:769-776.
[18]Liu AX,Zhu YM,Luo Q,Wu YT,Gao HJ,Zhu XM,Xu CM,Huang HF.Specific peptide patterns of follicular fluids at different growth stages analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.Biochim Biophys Acta.2007;1770:29-38.
[19]Guojie Song,Sian E.Harding,Michael R.Duchen,Richard Tunwell,Peter O'Gara,Tim E.Hawkins,and Stephen E.Moss Altered mechanical properties and intracellular calcium signaling in cardiomyocytes from annexin 6 null-mutant mice.FASEB J.2002;16:622-624.
[20]Gu SS,Shi N,Wu MP.The protective effect of ApolipoproteinA-I on myocardial ischemia-reperfusion injury in rats.Life Sci.2007;81:702-709.
[21]Emert-Sedlak L,Shangary S,Rabinovitz A,Miranda MB,Delach SM,Johnson DE.Involvement of cathepsin D in chemotherapy-induced cytochrome c release,caspase activation,and cell death.Mol Cancer Ther.2005;4:733-742.
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