【作者】 曲迪；

【导师】 王群；

【作者基本信息】 华东师范大学 ， 动物学， 2007， 硕士

【摘要】 本文根据目前我国中华绒螯蟹养殖经济中的繁育质量问题和国内外对动物生殖系统的研究情况，围绕精荚变成精子的过程，对该蟹雌体纳精囊，雄体副性腺以及精子顶体酶活力进行必要的研究。一方面从科研的角度来为进一步研究甲壳动物生殖系统提供资料；另一方面，也为我国淡水经济资源能得到更好的发展提供基本理论依据。1、纳精囊是中华绒螯蟹雌性生殖系统的重要组成部分，在精子储藏、排卵以及受精过程中起着十分重要的作用。利用组织学，组织化学以及透射电子显微镜技术，对中华绒螯蟹成熟雌体纳精囊进行初步研究，以了解纳精囊壁的结构及所含主要化学物质。纳精囊由囊壁和囊腔两大部分组成，囊壁由外至内分别为：结缔组织、肌肉层（局部有）、基膜和柱状上皮细胞（顶分泌型腺细胞）几个大的部分。但并非纳精囊壁的所有地方都按此顺序拥有所有的结构，有的只有一个或两个部分，中华绒螯蟹纳精囊壁属于典型的移行上皮。透射电镜结果表明：纳精囊壁的柱状上皮细胞向腔内伸出很多微绒毛，纳精囊腔内具有多种不同类型的分泌物，它们在生殖过程中起不同的作用。组织化学结果显示纳精囊壁分泌型细胞含有大量的糖类物质、一些蛋白质和脂类物质，其糖类物质分泌量明显多于其它的各种细胞。精子在进入纳精囊后其保持新陈代谢的能量由纳精囊上皮细胞分泌物提供。由此可推测，顶分泌细胞分泌的碳水化合物是精子在纳精囊中主要能量来源，而上皮细胞分泌物中的蛋白质和脂类物质可能参与精子自身的酶反应和其他一些反应。2、中华绒螯蟹精子从前输精管开始以精荚形式存在，经过输精管（vasa deferentia）、储精囊和射精管（部分精荚经过副性腺），随精液和雄性腺分泌液一起进入雌体纳精囊中。精荚进入到雌体纳精囊后，很快便破裂，释放出游离精子，以该形式存在直至与雌性卵结合，接着雌蟹抱卵。但是关于精荚的破裂机制至今都没有证据充足的结论。本实验旨在找到精荚破裂的场所和物质。基于此，取中华绒螯蟹精荚，人工海水冲洗，使精荚浓度为1500个／ml左右。取精液以及副性腺，卵巢和纳精囊三种生殖器官，分别匀浆，冷冻离心提取蛋白，考马斯亮蓝法检测并平衡蛋白浓度。在同一条件下设立空白对照组，精液组，副性腺蛋白组，卵巢蛋白组和纳精囊蛋白组。各组分别加入到精荚中。载玻片取30微升混合液，显微镜下计数剩余完整精荚数量，在第5min，10min，20min，40min，60min和90min分别计数。结果显示，对照组，精液，卵巢蛋白和纳精囊蛋白均对精荚破裂无影响，而副性腺蛋白可以使精荚显著破裂。由此可知：副性腺蛋白在精荚破裂中起重要作用。3、已经验证中华绒螯蟹副性腺蛋白对精荚破裂有促进作用。中华绒螯蟹雄体副性腺位于储精囊和射精管交界处，与储精囊和射精管相通，并且在蟹体两侧对称分布，白色，呈发达分枝状盲管。本试验在证明中华绒螯蟹副性腺蛋白有助于精荚破裂的基础上，通过扫描电子显微镜技术以及蛋白质电泳技术，进一步研究探讨副性腺中使精荚破裂的具体副性腺蛋白来源种类以及所处位置等。扫描电子显微镜样品消化精荚证明副性腺中含有能使精荚破裂的蛋白，但平时并不分泌到副性腺腔中；扫描电镜结果表明：副性腺自溶物与完整内容物相比，大泡变小，形状由规则变为不规则，小泡数量明显增多。蛋白质电泳结果表明，副性腺自溶物中的小泡蛋白条带数量明显比大泡和对照增多几条，则可以证明：能够使精荚破裂的就是副性腺自溶物蛋白中的小泡蛋白。4、精子顶体酶是精子顶体部一种特殊的酶，为丝氨酸蛋白酶，是受精过程中的一种重要的蛋白水解酶。此酶能水解卵细胞的透明带，使得精卵能相互融合。顶体酶还能够促进生殖系统中的激肽的释放，后者能够增强精子的活力和促进精子的运动。经离体实验证明：纳精囊蛋白不能够使精荚破裂，而副性腺分泌物能够使精荚在较短时间内破裂。本文采用Kennedy方法，适当加以调整，来检测副性腺蛋白消化精荚后得到的游离精子和雌蟹纳精囊内的精子的顶体酶活力区别，由此进一步分析副性腺蛋白的作用和中华绒螯蟹精子顶体酶活力与其它物种精子顶体酶活力的区别。经副性腺消化精荚所得的精子，顶体酶活力分别是3.216μIU·10^-6和2.232μIU·10^-6，显著低于雌蟹纳精囊中的精子顶体酶活力：239.090μIU·10^-6和219.297μIU·10^-6，证明：中华绒螯蟹副性腺蛋白尽管能使精荚破裂，但是和精子顶体酶活力增高关系不大。影响精子顶体酶活力的物质应该来自于雌蟹纳精囊内，实验中的扫描电子显微镜照片证明纳精囊内分泌物丰富，以及组织化学证明纳精囊分泌细胞分泌丰富物质，推测纳精囊内这些内容物可能会对精子顶体酶活力的提高起到作用。更多还原

【Abstract】 This paper was based on the propagate problems in breed aquatics and research status quo about the Chinese mitten-handed crabs （Eriocheir Sinensis） at home and abroad. The research, which circumfusing the changing process that from Spermatophores to sperms, worked on the spermatheca in female, accessory gland in male and other aspects hailed from these. On the one hand, it can contribute to natural science research by offering basic theoretics material and on the other hand, it can help to make the freshwater economic resourch of our country developed and utilized better.1. The spermatheca, which is an important organ in Eriocheir Sinensis female procreation system, plays vital function on sperm storage, ovulation and fecundation activities. Using histological, histochemical and transmission electron microscope（TEM） technology, basic research was operated on the spermatheca of the mature female Eriocheir Sinensis in order to reveal the main structure of the spermatheca wall and the chemical inclusion of spermatheca cells.The spermatheca is devided into two parts: vesicle wall and vesicle lumen. From the outside in, vesicle wall contains: Connective tissue（CT）, Layler of muscle（LM）, basement membrane cell（BM） and Columnar epithelium cell（CEC）. These parts are not existed in the same place and some position of the spermatheca own only one or two parts. So, vesicle wall of Eriocheir Sinensis spermatheca is typical transitional epidermal.Result of TEM showed: many microvilli（MV） existed on the end of CEC and several different kinds of lumen secretion（LS） existed in the vesicle lumen. The function of this structure is hypothesized playing dissimilar action in procreation process.Analysing the main structure and chemical inclusion of cells of spermatheca to make sure that the spermatheca does a contribution to depositing nutrition when crabs are mating and breeding, therefore we can speculate the source of nutrition of the sperms after mating. The apical excreting cell contains lots of carbohydrate , some protein and fat, especially, it excrete much more carbohydrate than any other cells. The spermathecal epithelium cells provide nurishment to sperms keeping metabolism in spermatheca, so it can be assumed the carbohydrate from apical excreting cells be the main nutrients of sperms in spermatheca, and the protein and fat excreted from epithelium cells may participate in enzymatic reaction of the sperm.2. Spermatophores of the Eriocheir sinensis, which appeared in Proximal vasa deferentia, throughing vasa deferentia, sperm bundles, accessory gland and ejaculatory duct, entered into spermatheca with semen and other male sex gland exudates. Short time after the Spermatophores entered the spermatheca, it dissoluted and existed as single sperm until combining with germ cell. The aim of this experiment is to find out more information about the process from spermatophore to single sperm.Using man-made seawater to wash and dilute Spermatophores of the Chinese mitten-handed crabs （Eriocheir sinensis） to made the concentration of which was 1500/ ml. Three important reproductive apparatus: accessory gland, ovary and spermatheca were made to homogenization, and four different protein solutions were distilled from these three homogenate liquids and spermatic in the condition of refrigerant centrifugal machine, separately.Method of Coomassie Brilliant Blue was adopted to detect and balance the concentration of proteins to the same concentration. Five protein groups: contrast, spermatic, accessory gland, ovary and spermatheca were set up in the same condition and combined with Spermatophores. Put 30ul mixed reactive solution to slide in each group and at fixed time and taken count of the survival number of integrated Spermatophores. Results showed that four proteins: contrast, spermatic, overy and spermatheca have no prominent impact on dissolution of Spermatophores but accessory gland protein has marked function to accelerate the Spermatophores dissolve. Thus we can concluded that accessory gland protein play a vital role in dissolution of spermatophora.3. The accessory gland of Eriocheir sinensis located between sperm bundles and ejaculatory duct, and these three organ are communicate. The accessory gland is white, distribute on both side of the crab symmetrically. It has been proved that accessorygland protein make function on spermatophore dissolution.On the foundation that accessory gland protein can dissolute the spermatophore, by using scanning electron microscopy（SEM） and protein electrophoresis technology, more information about which kind of protein it is and where it situates can be researched futher.Using SEM sample 2 and 3 digesting Spermatophores reveals that the protein that can dissolute spermatophore exists in the accessory gland, but it is not excreted into the vesicle lumen at peacetime. Rusult of SEM showed: compared to integrate inclusion, the big vesicles become small and losing its regular rotundity, and what’s more, the number of small vesicles is huge. The result of the protein electrophoresis showed: the number of proteins in small vesicles is remarkable more than that in big vesicles. It can be concluded that the protein in small vesicles of accessory gland play vital or full function on spermatophore dissolution.4. Acrosin, a sperm-specific arosomal proteinase, has an essential role in the fertilization process and is thought to be important in the acrosome reaction, sperm-zona binding and zona penetration. The spermatozoa acrosin activity and the accessory gland acrosin activity of Eriocheir sinensis are measured with a simplified clinical assay of total sperm acrosin activity. This method is based on the measurement of amidase activity of sperm suspensions. The acrosin activity assay has been used in many animals. Results of these studies indicated that use of this assay method could provide valuable information concerning semen quality.The accessory gland acrosin activity of Eriocheir sinensis is about 3.216μIU·10^-6 and 2.232μIU·10^-6,which is remarkable lower than the spermatheca gland acrosin activity: 239.090μIU·10^-6 and 219.297μIU·10^-6. It can be concluded that although the protein of accessory gland can dissolute the spermatophore, it have less relation to the high acrosin activity in spermatheca. The factor that effect the acrosin activity should come from spermatheca itself. Former experiment proves there are abundant secretion in spermatheca and these secretion maybe have some relation with improvement of the acrosin activity. This result indicates that the clinical assay of acrosin activity can be used for crab spermatozoa to evaluate the quality of crab semen.更多还原