【作者】 张蓓；

【导师】 崔毓桂；

【作者基本信息】 南京医科大学 ， 妇产科学， 2017， 硕士

【摘要】 研究背景与目的:睾丸中产生的成熟精子是男性生殖的前提。精子发生从精原细胞开始,是一个连续的过程,包括精原细胞的增殖,精母细胞的减数分裂以及精子形成三个阶段。在生理状态下,卵泡刺激素(Follicle-stimulating Hormone,FSH)促进精原细胞增殖并进入减数分裂;睾酮(Testosterone,T)维持减数分裂的完成以及精子形成,它们相互协同促进。除此之外,精子发生还受到其他激素以及睾丸局部调节因子的影响。对于睾丸局部调节机制,至今尚不完全清楚。精子形成是圆形精子细胞分化形成成熟精子,伴随着多余胞质的脱落以及核致密染色质的形成。在精子形成过程中,组蛋白H3、H4发生高度乙酰化使核染色质变得松散,组蛋白被过渡蛋白(Transition nuclear protein1/Transition nuclear protein2,Tnp1/Tnp2)替换下来,在睾丸特异性布罗莫结构域蛋白(bromodomaintestis-specific,BRDT)的诱导下被精子蛋白酶体或者其他蛋白水解机制所降解,过渡蛋白再被鱼精蛋白(Protamine1/Protamine2,Prm1/Prm 2)所替代,鱼精蛋白使得核染色质高度浓缩致密,从而避免精子头部的遗传物质被女性体内的有害物质所破坏,这是形成成熟精子非常关键的步骤。其中组蛋白乙酰化主要受组蛋白乙酰化酶(Histone acetyltransferases,HATs)以及组蛋白去乙酰化酶(Histone deacetylases,HDACs)的调节,两者受睾丸内局部因子的调节。研究精子发生和精子形成的调节,对于深入理解男性不育症的发病机制具有重要意义。研究表明,半数以上男性不育症患者表现为少精子症、严重少精子症、弱精子或死精子症、畸形精子症,病因也大多不明确,其核心问题是精子发生障碍以及精子形成障碍。SET蛋白是一个多功能蛋白,在调节细胞周期中起重要作用,包括DNA复制、核小体装配、染色体修饰、DNA转录、细胞增殖、细胞凋亡等。其也被称作乙酰化酶抑制剂(inhibitor of acetyltransferases,INHAT),是INHAT复合物中的分子之一,能明显抑制组蛋白乙酰化。本课题前期研究发现SET蛋白主要表达于精原细胞,精母细胞,在精子细胞中有少量表达,并且SET蛋白能促进精原细胞增殖。因此我们认为,SET蛋白作为睾丸局部因子,参与调节精子发生和精子形成。为了证明以上假说,深入研究SET蛋白在精子发生和精子形成中的作用及其机制,本文采用小鼠精母细胞系GC-2 spd为模型,研究SET蛋白在精母细胞中的表达与定位;通过干涉SET的表达研究SET蛋白调节精母细胞增殖;进一步研究SET蛋白调节精子组蛋白乙酰化的作用。材料与方法:(1)体外培养GC-2 spd细胞,通过免疫荧光对精母细胞标志物乳酸脱氢酶C(lactate dehydrogenase C,LDHC)和睾丸特异激酶 1(Testis-Specific Kinase 1,TESK1)进行鉴定。(2)构建空载腺病毒(AdH1-siRNA/NS)和SET干涉腺病毒(AdH1-siRNA/SET),通过免疫荧光技术、实时定量逆转录聚合酶链反应(Realtime RT-PCR)和蛋白质印迹(Western blot)等方法证明其干涉有效性。(3)分别将空载腺病毒(AdH1-siRNA/NS,对照组)和SET干涉腺病毒(AdH1-siRNA/SET,干涉组)转染至GC-2 spd细胞中,体外分别培养24h、48h、72h,用细胞计数的方法检测细胞增殖。(4)分别将腺病毒AdH1-siRNA/NS和AdH1-siRNA/SET转染至GC-2 spd细胞中,体外培养72h,用Western blot及RT-PCR方法检测组蛋白乙酰化水平、组蛋白乙酰化酶(HATs)以及组蛋白去乙酰化酶(HDACs)的表达水平,用HAT活性试剂盒以及HDAC活性试剂盒检测HAT以及HDAC酶活性。(5)从体外培养的GC-2 spd细胞中提取蛋白质,用琼脂糖珠免疫共沉淀试剂盒检测SET蛋白与组蛋白H3、H4的结合。(6)统计学方法:SPSS 16.0软件进行统计学处理,数据以均数±标准差(Means±SD)表示。P<0.05为统计学有显著性差异。结果:(1)免疫荧光显示精母细胞株GC-2 spd阳性表达LDHC和TESK1。因此,GC-2 spd细胞系能被当作精母细胞在本实验中使用。(2)SET蛋白表达于GC-2 spd细胞的胞核和胞质中。AdH1-siRNA/NS和AdH1-siRNA/SET前期在本实验室已经构建。干涉SET后,胞核以及胞质中的SET蛋白均明显减少,细胞中SET蛋白表达量明显降低70～80%(P<0.01),表明构建的AdH1-siRNA/SET腺病毒是高效的。(3)干涉SET后,GC-2 spd细胞数明显降低(P<0.05),细胞增殖明显受到抑制。(4)干涉SET后,GC-2 spd的组蛋白H3、H4乙酰化水平明显增高2～3倍(P<0.05),但组蛋白乙酰化酶(HATs)和去乙酰化酶(HDACs)的mRNA以及蛋白表达水平差异无统计学意义(P>0.05),它们的酶活性比值(HAT活性/HDAC活性)的差异也没有统计学意义(P>0.05)。(5)GC-2 spd细胞中的蛋白质免疫共沉淀结果提示GC-2 spd中SET蛋白与组蛋白H3、H4结合。结论:(1)SET蛋白在精母细胞核内和胞浆中均表达;敲除SET蛋白,抑制精母细胞增殖。提示SET蛋白通过促进精母细胞增殖参与调节精子发生。(2)SET蛋白抑制精子细胞组蛋白H3、H4乙酰化。SET蛋白通过结合组蛋白H3、H4,阻止其赖氨酸乙酰化位点的暴露,从而抑制H3、H4的乙酰化,进而可能抑制精子形成。(3)本研究证明,SET蛋白作为睾丸内的局部因子参与调节睾丸精子发生和精子形成。进一步阐明其作用机制,对于阐明男性不育症,尤其是男性少精症、弱精症、无精症、畸形精子症的病因机制具有重要的理论意义。更多还原

【Abstract】 Background and Objectives:The mature spermatozoon produced from the testis is the precondition of male fertility.The process from the spermatogonial stem cells to mature spermatozoa is constant,which can be divided into the mitotic,meiotic and postmeiotic(spermiogenic)phases.At the physiological status,FSH acts to stimulate spermatogonial proliferation and entry into meiosis and testosterone acts to ensure completion of meiosis and spermiogenesis,both play synergistic effects.Besides,the other hormones or local factors can also regulate spermatogenesis.The local regulating mechanism of spermatogenesis is still obscure.Spermiogenesis refers to the haploidic round spermatids undergo dramatic morphological changes,including cytoplasmic dropping and the form of compact nuclear chromatin,and transform into spermatozoa.During which,hyperacetylated nuclear histones are replaced by more basic transition proteins,and ultimately protamines to facilitate denser packaging of the paternal genome into the sperm head.The compated nuclear can protect the genome from the deleterious materials from female genital tract.BRDT(bromodomaintestis-specific)recognize the replaced hyperacetylated histones and induce its evicted,which would then be degraded through the action of spermatoproteasome or/and yet unknown proteolytic systems.The acetylized histone is mainly regulated by HATs and HDACs,local factors in addition.Observing the mechanism of spermatogenesis and spermiogenesis has important significance in the cause of male infertility.Since previous studies have demonstrated that more than half of male infertility patients showed oligospermia,asthenospermia,azoospermia and teratozoospermia.The cause is not clear and the core mechanism is abnormal spermatogenesis and spermiogenesis.SET is a multifunctional protein that plays an important role in the regulation of cell cycle,DNA replication,nucleosome assembly,chromatin modification,transcription,cell proliferation,apoptosis and so on.It is also called the inhibitor of acetyltransferase(INHAT)and is the component of the INHAT complex,which can inhibit histone acetylation.Our previous study showed that SET protein is mainly expressed in spermatogonia and spermatocytes,with low expression in spermatid.What’s more,SET protein promoted the spermatogonium proliferation.Therefore,we propose this hypothesis that SET,as a local factor,regulate spermatogenesis and spermiogenesis.To confirm this hypothesis,with further investigations of mechanisms of spermatogenesis and spermiogenesis,we applied GC-2 spd cell line to carry out some experiments.We observed the expression and subcellular location of SET protein in GC-2 spd;focused on the effects of SET protein in regulating the spermatocyte proliferation by knockdown the SET protein;further studied the potential mechanisms in SET regulating histone acetylation during spermiogenesis.Material and Methods:(1)The GC-2 spd cell was cultured in vitro,the immunofluorescence was used to identify two markers of spermatocytes,LDHC(lactate dehydrogenase C)and TESK1(Testis-Specific Kinase 1).(2)Constructed AdH1-siRNA/NS and AdH1-siRNA/SET adenovirus.Immunofluorescence,Western blot and Real time RT-PCR were used to prove the expression validity.(3)The cells were transfected with AdH1-siRNA/SET to knockdown the expression of SET protein,AdH1-siRNA/NS as control.Cells were in vitro cultured for 24h,48h,72h,cells number was used to detect the cell proliferation.(4)AdH1-siRNA/NS and AdH1-siRNA/SET adenovirus were transfected in GC-2 spd cells for 72h.Western blot and Real time RT-PCR was used to detect the expression levels of SET protein,Ac-histone,histone acetyltransferases and histone deacetylases,HAT Activity Assay Kit and HDAC Activity Assay Kit to assess the HAT and HDAC activity.(5)The combination of SET and H4 or SET and H3 were detected by an immunoprecipitation kit from the protein extracted from in vitro GC-2 spd cells.(6)Statistical methods.The SPSS 16.0 software was used for statistical analysis.Data were presented as mean±standard deviation(Means ± SD).P<0.05 was considered significant difference.Results:(1)Immunofluorescence confirmed the positive expression of Two markers of spermatocytes,LDHC and TESK1.Therefore,GC-2 spd cell line was used in our study as spermatocyte.(2)SET protein was expressed in both cytoplasm and nucleus of GC-2 spd cell.AdHl-siRNA/NS and AdH1-siRNA/SET adenovirus were successfully constructed in our laboratory.The expression of SET protein in the cytoplasm and nucleus of the cells transfected with AdHl-siRNA/SET was significantly decreased,the expression levels of SET was inhibited by 70～80%(P<0.05).So,these adenovirus vectors with high efficient were used our study.(3)In GC-2 spd treated with AdH1-siRNA/SET,knockdown of SET protein significantly decreased the cell number,inhibited the cell proliferation(P<0.05).(4)After knockdown of SET protein,the acetylation level of H3 and H4 was significantly increased(P<0.05)by 2-3 fold.However,there were no significantly differences in the mRNA and protein expressions of HATs and HDACs,the same with HAT activity/HDAC activity.(5)Immunoprecipitation showed that SET protein combined with H3 and H4 in the protein extracted from in vitro GC-2 spd cells.Conclusion:(1)SET protein was expressed in both cytoplasm and nucleus of spermatocytes.The knockdown the expression of SET protein inhibited the cell proliferation,which indicated that SET regulated spermatogenesis by promoting spermatocytes proliferation.(2)SET protein inhibited the acetylation of histone H3 and H4 by combining and masking the lysine acetylation sites in spermatid,thus possibly inhibiting the spermiogenesis.(3)This study demonstrates that SET,as a local factor,is involved in the regulation of spermatogenesis and spermiogenesis.This study enhance our knowledge on the regulating mechanisms of spermatogenesis and spermiogenesis and provide us a theoretical basis of cause for male infertility,especially in oligospermia,asthenospermia,azoospermia and teratozoospermia.更多还原