【作者】 李超；

【导师】 康现江；

【作者基本信息】 河北大学 ， 动物学， 2023， 博士

【摘要】 精子发生精密而复杂,精原细胞经减数分裂和精细胞变态分化为成熟精子。期间大多胞质和细胞器被抛却或整合,最终形成精子特有膜性细胞器-顶体。通常认为顶体来源于溶酶体,且与高尔基体密切相关,但也有报道发现部分线粒体和内质网蛋白也存在于顶体中。膜性细胞器相互作用及其在顶体形成过程中的功能仍需深入研究,线粒体相关膜(Mitochondria associated membrane,MAM)在细胞器互作领域研究最为深入,多集中于酵母或疾病相关的研究,而在雄性生殖及精子发生过程中的功能尚未见研究报道。中华绒螯蟹(Eriocheir sinensis)属于甲壳纲,十足目,爬行亚目,具有十分重要的经济价值。作为爬行亚目动物的代表,中华绒螯蟹精子具有非浓缩核,无鞭毛、不运动等特征,由于其顶体占比较大且结构独特,也可以作为研究顶体形成的理想材料。本论文围绕中华绒螯蟹精子发生过程中主要膜性细胞器参与精子发生和顶体形成的特征和功能,主要结果如下:1.中华绒螯蟹精巢发育规律及精子发生过程中细胞器动态首先,研究统计了全年各月份雄性中华绒螯蟹生殖系数(w/w),使用细胞涂片统计不同月份各时期生精细胞占比,以揭示其发育时间规律。研究发现6～9月份是不同时期精细胞的发育阶段。解剖精巢生精小管,利用组织切片和压片观察精巢生精小管中生精细胞分布的空间规律。结果发现中华绒螯蟹生精小管长约40 cm,有60个以上明暗相间的生殖周期,可根据明暗相间不同区段,取材不同时期生精细胞,如明区主要为精原和精母细胞器,暗区主要为精子,明暗相间区域则为不同时期生精细胞区段。使用透射电镜进一步观察精子发生过程中主要膜性细胞器动态,发现典型高尔基体及MAM结构,同时观察了顶体形成过程及关键事件,如前顶体囊外排,细胞核异质化现象等。2.中华绒螯蟹精子顶体释放蛋白与主要膜性细胞器蛋白为了分析主要膜性细胞器参与精子顶体形成的功能,比较分析钙离子载体A23187和Ionomycin诱导精子顶体反应率,结果显示二者诱导中华绒螯蟹精子顶体反应率分别为58.4%和95.8%,且Ionomycin诱导的顶体反应精子多数处于顶体反应的第3、4阶段。分离顶体反应释放蛋白,LC-MS/MS鉴定了945种精子顶体释放蛋白,其中部分蛋白来源于线粒体(166种)、内质网(15种)、高尔基体(2种)和溶酶体(17种)。免疫荧光观察发现线粒体蛋白柠檬酸合酶(citrate synthase)和内质网蛋白钙网蛋白(calreticulin)均参与精子发生和顶体形成并最终定位于成熟精子顶体上,而溶酶体蛋白组织蛋白酶D(cathepsin D)参与精子发生但并未定位在顶体上。3.中华绒螯蟹精子发生过程中线粒体特征和功能为了进一步研究中华绒螯蟹精子发生过程中线粒体的特征和功能,利用线粒体膜电位探针检测精子发生过程中线粒体动态,结果显示,线粒体持续存在直至精子成熟。使用免疫荧光结合不同区域线粒体蛋白抗体如VDAC2(voltage-dependent anion channel2),CC(cytochrome c),COX4和MDH(malate dehydrogenase)综合探究线粒体蛋白参与顶体形成的特征,结果发现VDAC2、细胞色素C和MDH均参与了精子顶体的形成,而COX4则一直定位于线粒体并未转移至顶体。4.VDAC1、GRP75和IP3R参与中华绒螯蟹精子顶体形成的特征和功能为了分析MAM参与中华绒螯蟹发生和顶体形成的特征和功能,利用分级分离和超速离心的方法分离了中华绒螯蟹精巢MAM结构并结合质谱获得了其蛋白质组学数据,共鉴定到3644种蛋白,分析了其中的VDAC1(voltage-dependent anion channel 1),GRP75(75 k Da glucose-regulated protein)和IP3R(inositol 1,4,5-trisphosphate receptor)三种蛋白。免疫荧光显示早期精细胞,GRP75和IP3R均定位于细胞质中,环核分布,而VDAC1则位于细胞的一端,毗邻细胞核;中期精细胞,VDAC1聚集于前顶体囊顶端,GRP75同样定位于前顶体囊前端但与VDAC1特征不同,IP3R则定位于前顶体囊膜和顶体管上。成熟精子中VDAC1和GRP75共定位于顶体头帽,IP3R则定位于顶体囊膜上。IP(immunoprecipitation)结果表明中华绒螯蟹精巢中IP3R与VDAC1存在相互作用。5.中华绒螯蟹精子亚顶体结构蛋白质组学为了更为全面的理解中华绒螯蟹精子顶体蛋白组以及主要膜性细胞器参与亚顶体结构的功能。通过差速离心和超速离心收集脱落的片层结构,借助顶体管+头帽复合结构与非浓缩细胞核的差异性结合超声破碎和差速离心分离顶体管和头帽复合结构,进一步利用质谱分析不同亚顶体结构蛋白质学。精子顶体片层结构蛋白组共鉴定到699种蛋白,其中部分蛋白来源于线粒体(154种)、内质网(13种)、高尔基体(2种)和溶酶体(4种)。顶体管+头帽复合结构蛋白组共鉴定到773种蛋白,其中部分蛋白来源于线粒体(168种)、内质网(9种),未发现高尔基体和溶酶体蛋白。综上,本研究揭示了中华绒螯蟹精巢特征以及生精小管中生精细胞分布的时空规律;比较分析了精子顶体反应释放蛋白、不同亚顶体结构蛋白和MAM结构的蛋白质组,发现MAM结构蛋白也是顶体蛋白的重要成员;阐述了主要膜性细胞器参与顶体形成动态和功能。研究结果为进一步探明中华绒螯蟹精子发生过程中主要膜性细胞器及其相关蛋白参与精子形成的功能提供参考,具有理论价值。同时也为十足目动物精子质量评价提供基础参考。更多还原

【Abstract】 The complex process of spermatogenesis is precisely regulated.Spermatogonia undergo meiosis and metamorphosis to produce mature sperm.During this process,most of the cytoplasm and organelles are integrated or discarded,and finally the acrosome,an unique membranous organelle,emerges.In general,the acrosome is thought to be derived from the lysosomes and it is closely related to the Golgi apparatus.But it has also been reported that proteins from mitochondria and endoplasmic reticulum transfer to the acrosome.The functions of membranous organelles interactions in the process of acrosome formation need further research.Mitochondria associated membrane(MAM)as one of the organelle interactions has been well studied,most reports focused on yeast or disease,while few researches on male reproduction and spermatogenesis.Eriocheir sinensis belongs to crustacea,decapod,reptilia,which has very important economic value.As a representative animal of reptilia,the sperm of E.sinensis has no flagella which leads the movement loss,and the nuclei is uncondensed.The large proportion of acrosome and unique structures makes it an ideal material for studying the acrosome formation.The research focuses on the characteristics and functions of the main membranous organelles participating in the acrosome formation during spermatogenesis in E.sinensis.The main results are as follows:1.Characteristics of testis development and organelles dynamics during spermatogenesis in E.sinensisFirstly,the reproductive coefficient(w/w)were counted in each month of the year in E.sinensis,and cell smearing were used to count the proportion of germ cells in different months to illustrate the developmental time line of spermatogenic cells.The results showed that from June to September is the developmental stage of different spermatogenic cells.The seminiferous tubules of the testis were dissected,and the spatial pattern of the distribution of spermatogenic cells in the seminiferous tubules was observed by tissue sections and slices.The results showed that the seminiferous tubule in E.sinensis testes is about 40 cm and it contains more than 60 light and dark reproductive cycles.The different periods of spermatogenic cells can be obtained according to the different light and dark regions,spermatogonia and spermatocytes are major in the light regions,while the dark regions contain sperm,the spermtids distribute between the light and dark regions.The transmission electron microscope was used to further observe the dynamics of the main membranous organelles during spermatogenesis,typical Golgi apparatus and MAM structures were found exist during spermatogenesis.The key events during acrosome formation were observed,such as the efflux of preacrosome vesicle and the heterogeneity of the nucleus.2.Sperm acrosomal released proteins and main membranous organelle proteins in E.sinensisTo analyze the functions of major membranous organelles involved in acrosome formation during spermatogenesis,the rate of acrosome reaction induced by calcium ionophore A23187 and Ionomycin were compared,the results showed that the acrosome rate of calcium ionophore Ionomycin is 95.8% and A23187 is 58.4%.Most sperm were at stage 3or 4 of acrosome reaction induced by Ionomycin.The sperm acrosomal released proteins were collected and analyzed by LC-MS/MS,945 proteins were identified,partial of these proteins were from mitochondria(166 kinds),endoplasmic reticulum(15 kinds),Golgi apparatus(2kinds)and lysosomes(17 kinds).The characteristic of membranous organelles proteins were analyzed using immunofluorescence(IF),the results showed that citrate synthase(mitochondria)and the calreticulin(endoplasmic reticulum)were involved in acrosome formation during spermatogenesis,and they finally transfer to the acrosome in sperm,while the cathepsin D(lysosome)exist in spermatogenesis but absent from the acrosome.3.Characteristics and functions of mitochondria during spermatogenesis in E.sinensisTo further study the characteristics and functions of mitochondria in the acrosome formation during spermatogenesis in E.sinensis,mitochondrial potential probe was used to detect mitochondrial dynamics during spermatogenesis.The results showed that mitochondria persist during spermatogenesis and exist in mature sperm.The dynamics of different mitochondrial regions proteins such as VDAC2(voltage-dependent anion channel 2),CC(cytochrome c),COX4 and MDH(malate dehydrogenase)were analyzed using IF to illustrate the characteristics in the acrosome formation during spermatogenesis,the results showed that mitochondrial proteins like VDAC2,CC and MDH are involved in acrosome formation.While COX4 settle on the mitochondria during spermatogenesis instead of transfer to the acrosome.4.The characteristics and functions of VDAC1,GRP75 and IP3 R in the acrosome formation in E.sinensis.To study the characteristics and functions of mitochondria associated membrane(MAM)in the acrosome formation during spermatogenesis in E.sinensis,the MAM structures from the testis of E.sinensis were separated by fractionation and ultracentrifugation,and the proteomic data was obtained using LC-MS/MS.A total of 3644 proteins were identified.The target proteins VDAC1(voltage-dependent anion channel 1),GRP75(75 k Da glucose-regulated protein)and IP3R(inositol 1,4,5-trisphosphate receptor)were analyzed,the IF results showed that in early spermatids,GRP75 and IP3 R were located in the cytoplasm and distributed around the nuclei,while VDAC1 was located at one side of the spermtids,adjacent to the nuclei.In middle spermatids,VDAC1 accumulated at the top of the preacrosomal vesicle,GRP75 was also located at the front of the preacrosomal capsule but presented different characteristics compared with VDAC1,while IP3 R was located on the preacrosomal capsule membrane and acrosomal tubule.In sperm,VDAC1 and GRP75co-localized on the acrosomal apical cap and IP3 R was localized on the acrosomal membrane.Results of IP showed that IP3 R interacted with VDAC1 in the testes of E.sinensis.5.Proteome of subacrosome structures of sperm in E.sinensisTo understand more comprehensively of the acrosomal proteome and the functions of main membranous organelles involved in the subacrosomal structures of E.sinensis.The lamellar structures exfoliated were collected using differential centrifugation and ultracentrifugation,the acrosomal tubule connected apical cap structures were separated using sonication and differential centrifugation based on the differences between the complex structures and the uncondensed nuclei.LC-MS/MS was used to obtain the proteme data of different subacrsomal structures.A total of 699 proteins were identified in the acrosomal lamellar structure(LS)proteome in E.sinensis,partial proteins are from mitochondria(154kinds),endoplasmic reticulum(13 kinds),Golgi apparatus(2 kinds)and lysosomes(4 kinds).A total of 773 proteins were identified in the acrosomal tubule connected apical cap(TC)proteome,some of which are derived from mitochondria(168 kinds),endoplasmic reticulum(9 kinds),no proteins were found from Golgi apparatus or lysosomes.In summary,the present researches revealed the testicular characteristics and the spatiotemporal patterns of germ cells distribution in seminiferous tubules in E.sinensis.The proteome of sperm acrosomal released proteins,different subacrosomal structures and MAM structures were analyzed comparatively,which illustrate that proteins from MAM structures are important members of the acrosomal proteins.The results provide a reference for further exploring the functions of the main membranous organelles and their related proteins involved in sperm formation during spermatogenesis in E.sinensis,which has important theoretical value.Besides,the article provides a basic reference for the evaluation of sperm quality in decapod animals.更多还原