中华绒螯蟹精子膜蛋白提取、抗体制备及其免疫定位研究
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摘要
中华绒螯蟹(Eriocheir sinensis)是我国特有的水产珍品，也是我国一种重要的经济蟹类，广泛存在于我国南北各地淡水水域中。中华绒螯蟹是我国名优淡水养殖业的主要品种。近年来，其养殖业在沿海各地蓬勃兴起，它以优良的生物学品质，极高的营养价值和独特的风味蜚声海内外。养殖业的发展，进一步促进了基础科学研究的发展，而基础科学的发展，又反过来加速了养殖业的蓬勃发展。
本文就中华绒螯蟹精子膜蛋白提取、抗体制备及其免疫定位进行了研究，为其受精生物学、发育生物学方面的深入研究提供参考资料。主要从以下几个方面进行了研究：
1．对中华绒螯蟹雄性生殖系统进行解剖，得到贮精囊，进而分离到精荚，利用玻璃匀浆器匀浆的方法，对中华绒螯蟹精荚进行破碎，得到精荚基质和精子；使用两种去污剂对中华绒螯蟹精子膜蛋白进行了提取，在变性聚丙烯酰胺凝胶电泳(SDS-PAGE)条件下，对其精子膜蛋白组分进行了分析，实验结果表明，在双去污剂作用下，提取得到的精子膜蛋白与脱膜精子部分蛋白谱带差异明显；并对提取的精子膜蛋白进行了分子量、等电点和糖蛋白检测，结果显示，所提取的12种精子膜蛋白，其分子量均在100ku以下，等电点范围在4.4-6.2之间，并均为糖蛋白，说明所提取的中华绒螯蟹精子膜蛋白是低分子量的酸性糖蛋白，这与其他一些哺乳动物精子膜蛋白的研究结果类似；
2．乳酸脱氢酶(LDH)尤其是精子特异性乳酸脱氢酶，定位于膜上，也为精子膜蛋白的一种。利用天然聚丙烯酰胺凝胶电泳(Native-PAGE)和特异性染色的方法，对中华绒螯蟹进行了乳酸脱氢酶同工酶电泳分析，实验结果表明，以两种不同底物(乳酸钠和α-羟基丁酸钠)分别作用于血清、鳃、心脏、肌肉、肝胰腺、成熟蟹精巢、未成熟蟹精巢和精子，实验结果显示，乳酸脱氢酶同工酶具有组织特异性，并且首次在中华绒螯蟹体内发现了精子特异性乳酸脱氢酶，这种酶在中华绒螯蟹精子成熟过程中均存在，并随着精子的成熟，其含量和酶活性逐渐增强，可能在精子成熟过程中起作用；
3．利用抽提的精子膜蛋白免疫雌性新西兰白兔，制备多克隆抗血清；利用琼脂糖双向免疫扩散分别对多克隆抗血清活性、效价进行了分析，实验结果表明，制备的多克隆抗血清发生抗原抗体沉淀反应，具有免疫活性，且其效价为1：16；在精子膜蛋白雌雄组织特异性检测中，其实验结果表明，制备的多克隆抗血清与精巢、输精管和贮精囊粗提
Eriocheir sinensis is an unique aquatic treasure as well as important economic crab, which extensively exists throughout South and North of freshwater in China. As one of the famous freshwater species for aquaculture, in recent years, its aquaculture in coastal areas is prosperous. It is having great renown for its excellent biological character, the extremely high nutritional value and the unique flavor all the world. The progress of aquaculture further promotes that of fundamental research, or otherwise, fundamental research accelerates aquaculture, too.
This paper is about extraction, antisera preparation and immunolocalization of the sperm membrane proteins in E. sinensis. It provides some references for the further researches on the Fertilization Biology and Developmental Biology. Our study is mainly from the following aspects:
1. Male reproductive system of E. sinensis was dissected, spermatophore was obtained from seminal vesicle and then matrix of spermatophores as well as sperms was separated from the spermatophores by using glass homogenizer. Next, sperm membrane proteins of the crab were extracted by two detergent solutions and subjected to SDS Polyacrylamide Gel Electrophoresis(SDS-PAGE). Our results indicated that under the function of double-detergent, the proteins we got had significant differences from those of the demembranated sperms. We found that the molecular weight of 12 extracted proteins was less than 100ku, isoelectric point(pI) ranged from 4.4-6.2, and all of them were glycoproteins. Thus demonstrate that sperm membrane proteins of E. sinensis are lower molecular acidic glycoproteins, which is similar with those in mammalian sperms.
2. Lactate dehydrogenase(LDH), is one of the membrane proteins, which is also found on sperm membrane, so we speculated that sperm membrane of E. sinensis may contain this protein. By Native-PAGE and specific staining, we researched on the sperm-specific LDH of E. sinensis, the experiments showed that in the presence of two substrates(sodium lactate and α-hyd roxybutyric acid sodium), we analyzed its serum, gill, heart, muscle, hepatopancreas,
本文就中华绒螯蟹精子膜蛋白提取、抗体制备及其免疫定位进行了研究，为其受精生物学、发育生物学方面的深入研究提供参考资料。主要从以下几个方面进行了研究：
1．对中华绒螯蟹雄性生殖系统进行解剖，得到贮精囊，进而分离到精荚，利用玻璃匀浆器匀浆的方法，对中华绒螯蟹精荚进行破碎，得到精荚基质和精子；使用两种去污剂对中华绒螯蟹精子膜蛋白进行了提取，在变性聚丙烯酰胺凝胶电泳(SDS-PAGE)条件下，对其精子膜蛋白组分进行了分析，实验结果表明，在双去污剂作用下，提取得到的精子膜蛋白与脱膜精子部分蛋白谱带差异明显；并对提取的精子膜蛋白进行了分子量、等电点和糖蛋白检测，结果显示，所提取的12种精子膜蛋白，其分子量均在100ku以下，等电点范围在4.4-6.2之间，并均为糖蛋白，说明所提取的中华绒螯蟹精子膜蛋白是低分子量的酸性糖蛋白，这与其他一些哺乳动物精子膜蛋白的研究结果类似；
2．乳酸脱氢酶(LDH)尤其是精子特异性乳酸脱氢酶，定位于膜上，也为精子膜蛋白的一种。利用天然聚丙烯酰胺凝胶电泳(Native-PAGE)和特异性染色的方法，对中华绒螯蟹进行了乳酸脱氢酶同工酶电泳分析，实验结果表明，以两种不同底物(乳酸钠和α-羟基丁酸钠)分别作用于血清、鳃、心脏、肌肉、肝胰腺、成熟蟹精巢、未成熟蟹精巢和精子，实验结果显示，乳酸脱氢酶同工酶具有组织特异性，并且首次在中华绒螯蟹体内发现了精子特异性乳酸脱氢酶，这种酶在中华绒螯蟹精子成熟过程中均存在，并随着精子的成熟，其含量和酶活性逐渐增强，可能在精子成熟过程中起作用；
3．利用抽提的精子膜蛋白免疫雌性新西兰白兔，制备多克隆抗血清；利用琼脂糖双向免疫扩散分别对多克隆抗血清活性、效价进行了分析，实验结果表明，制备的多克隆抗血清发生抗原抗体沉淀反应，具有免疫活性，且其效价为1：16；在精子膜蛋白雌雄组织特异性检测中，其实验结果表明，制备的多克隆抗血清与精巢、输精管和贮精囊粗提
Eriocheir sinensis is an unique aquatic treasure as well as important economic crab, which extensively exists throughout South and North of freshwater in China. As one of the famous freshwater species for aquaculture, in recent years, its aquaculture in coastal areas is prosperous. It is having great renown for its excellent biological character, the extremely high nutritional value and the unique flavor all the world. The progress of aquaculture further promotes that of fundamental research, or otherwise, fundamental research accelerates aquaculture, too.
This paper is about extraction, antisera preparation and immunolocalization of the sperm membrane proteins in E. sinensis. It provides some references for the further researches on the Fertilization Biology and Developmental Biology. Our study is mainly from the following aspects:
1. Male reproductive system of E. sinensis was dissected, spermatophore was obtained from seminal vesicle and then matrix of spermatophores as well as sperms was separated from the spermatophores by using glass homogenizer. Next, sperm membrane proteins of the crab were extracted by two detergent solutions and subjected to SDS Polyacrylamide Gel Electrophoresis(SDS-PAGE). Our results indicated that under the function of double-detergent, the proteins we got had significant differences from those of the demembranated sperms. We found that the molecular weight of 12 extracted proteins was less than 100ku, isoelectric point(pI) ranged from 4.4-6.2, and all of them were glycoproteins. Thus demonstrate that sperm membrane proteins of E. sinensis are lower molecular acidic glycoproteins, which is similar with those in mammalian sperms.
2. Lactate dehydrogenase(LDH), is one of the membrane proteins, which is also found on sperm membrane, so we speculated that sperm membrane of E. sinensis may contain this protein. By Native-PAGE and specific staining, we researched on the sperm-specific LDH of E. sinensis, the experiments showed that in the presence of two substrates(sodium lactate and α-hyd roxybutyric acid sodium), we analyzed its serum, gill, heart, muscle, hepatopancreas,
引文
[1] 王旻晨，杨亚安．人精子包被抗原的提取法研究．苏州医学院学报，1997，17(5)：848-850．
[2] 王桂兰，崔存德，夏振开等．人精子顶体膜蛋白的纯化及性质的研究．中国应用生理学杂志，2003，19(4)：376-378．
[3] 沙国柱，黄宇烽，徐建平．人精子膜抗原的提纯和分析．男科学报，1999，5(3)：138-140．
[4] 王浩飞，向祖琼，王益鑫．人精子膜蛋白的二维电泳实验研究．中华男科学，2003，9(7)：504-506．
[5] 魏曙光，王琳芳，缪时英．一种与受精有关的人精子膜蛋白(BS-17)的分离纯化．生物化学杂志，1994，10(2)：236-241．
[6] 林典梁．人精子甘露糖受体纯化及其对受精能力的影响．福建医科大学硕士学位论文(单行本)，2002．
[7] 缪时英，严缘昌，李月华等．编码人精子膜蛋白BS-84的cDNA研究．自然科学进展——国家重点实验室通讯，1995，5(2)：225-228．
[8] 丁之德，Hansen S，Thiel S，et al．人精子膜甘露糖结合蛋白的研究——蛋白的定位．中国男科学杂志，2000，14(1)：19-21．
[9] 丁之德，Hansen S，Thiel s，et al，人精子膜甘露糖结合蛋白的研究．生殖与避孕，2000，20(1)：20-25．
[10] 高爱武，何晓兵，严缘昌．一种人精子膜蛋白的基因定位及其抗体对小鼠体外受精的影响．生殖与避孕，2000，20(3)：142-146．
[11] 缪时英，李月华，周荔申等．一种人精子膜蛋白基因的克隆与鉴定．基础医学与临床，1990，10(6)：23-28．
[12] 魏曙光，缪时英，张梅林．一种Calpastatin具有部分同源结构的人精子膜蛋白的发现与研究．生物化学杂志，1994，10(2)：242-246．
[13] 夏欣一．人附睾精子蛋白P34H的克隆、表达及单克隆抗体制备．南京师范大学硕士学位论文(单行本)，2003．
[14] 徐崇，陈复，刘莉等．84kD精子膜蛋白与抗精子抗体．生殖与避孕，1990，10(2)：39-42．
[15] 杨宏毅．人精子甘露糖受体的表达及其与精子．透明带相互作用的关系．福建医科大学硕士学位论文(单行本)，2002．
[16] 江澍．人精子甘露糖受体对精卵融合的影响．福建医科大学硕士学位论文(单行本)，2002．
[17] 周红，曾仲奎，刘荣华．人精子膜上凝集素受体的研究．生殖与避孕，1992，12(2)：11-17．
[18] Yu J, Wang LF, Miao SY, et al. Antiidiotypic antibody related to the 84kD human sperm membrane protein. Cell. Res., 1990, 2: 163-172.
[19] Quill TA, Garbers DL. Sperad is a novel sperm-specific plasma membrane protein homologous to a family of cell adhesion proteins. J. Biol. Chem., 1996, 271(52): 33509-33514.
[20] Cheng A, Le T, Palacios M, et al. Sperm-egg recognition in the mouse: characterization of sp56, a sperm protein having specific affinity for ZP3. J. Cell, Biol., 1994, 125(4): 867-878.
[21] Zhuang DZ, Zhang TY, Chen DY. A protein extracted from mouse sperm that plays an important role in fertilization. Devel. Reprod Biol., 1994, 3(I): 1-9.
[22] 孙秉贵，周占祥，邓泽沛．SA-30抗原在小鼠睾丸、附睾、精子及早期胚的定位．解剖学报，2000，31(2)：167-171．
[23] 丁之德，王卓群，吴明章．大鼠精子在附睾成熟中精子膜变化的研究．生殖医学杂志，1994，3(2)：114-117．
[24] 李彦锋，靳风烁，江军．精子表面蛋白受精素β在精卵结合和融合中的作用研究．中国男科学杂志，2001，15(4)：225-228．
[25] 庄大中，宋祥芬，胡国俊等．精子抗原MSH27参与精卵融合过程．科学通报，1999，44(14)：1508-1513．
[26] 韩之明，庄大中，高绍荣等．小鼠精子在附睾成熟过程中质膜糖蛋白的变化．动物学报，1999，45(1)：93-98．
[27] 柏家林，苟克勉，安晓荣等．一种新的精子膜蛋白sp18基因的克隆及其在E．coli中的表达．农业生物技术学报，2003，11(3)：268-272．
[28] 何晓兵．与大鼠受精相关的两种受体分子-GABA-BR和大鼠sp56的研究．中国科学院研究生院(上海生命科学研究院)．2003．
[29] Hardy DM, Garbers DL. A sperm membrane protein that binds in a species-specific manner to the egg extracellular matrix is homologous to von willebrand factor. J Biol. Chem., 1995, 270(44): 26025-26028.
[30] Haden NP, Hickox JR, Whisnant CS, et al. Systematic characterization of sperm-specific membrane proteins in swine. Biol. Reprod, 2000, 63: 1839-1847.
[31] 赵明，费坚，孙册．猪精子凝集素的纯化、性质及其作用．生物化学与生物物理学报，1995，27(4)：346-354．
[32] 赵明，孙册．猪精子凝集素在精卵结合中的作用机制．生物化学与生物物理学报，1995，27(4)：375-383．
[33] 赵明，孙册．猪镜子中与卵透明带糖蛋白ZP3结合的蛋白质．生物化学与生物物理学报，1996，28(2)：145-152．
[34] 刘建喜，林爱星，杨毅等．家兔精子膜蛋白rSP10在大肠杆菌中的高效表达及其抗血清的制备． 生物工程学报，2001，17(3)：314-317．
[35] 梁植权，王琳芳，缪时英等．精子膜蛋白的研究Ⅰ．具有同种抗原性的兔精子膜蛋白A的分离． 中国医学科学院学报，1980，2(3)：147-151．
[36] 王琳芳，缪时英，赵敏顺等．精子膜蛋白的研究Ⅱ．具有同种抗原性的兔精子膜蛋白A的免疫活性及理化性质．中国医学科学院学报，1980，2(3)：152—155．
[37] 王琳芳，缪时英，曹淑兰等．精子膜蛋白的研究Ⅲ．兔精子膜蛋白“A”的定位及其酯酶活性的观察．中国医学科学院学报，1982，4(2)：82-85．
[38] Zahler WL, Doak GA. Isolation of the outer acrosomal membrane from bull sperm. Biochimica. et. Biophysica. Acta., 1975, 406: 479-488.
[39] Hardy DM, Garbers DL. Species-specific binding of sperm proteins to the extracellular matrix(zona pellucida)of the egg. I. Biol. Chem., 1994, 269(29): 19000-19004.
[40] 柳荣，李天俊，陶丙春等．奶牛精子膜的分离及纯化．畜牧兽医学报，1997，28(5)：409-415．
[41] 曾少举，桑建利，梁前进．一种牛精子膜蛋白的纯化及在生殖中的作用．北京师范大学学报(自然科学版)，2000，36(5)：683-686．
[42] 周占祥，邓泽沛．羊精子表面的凝集素标记特征．中国组织化学与细胞化学杂志，1994，3(3)：256-261．
[43] 周占祥，邓泽沛．羊精子在成熟和获能过程中表面的凝集素标记变化．解剖学报，1994，25(3)：276-280
[44] Sabeur K, Foristall K, Ball BA. Characterization of PH-20 in canine spermatozoa and testis. Theriogenology, 2002, 57(2): 977-987.
[45] 杨书婷，桂建芳．雌核发育银鲫和两性生殖彩鲫精子蛋白组份的比较研究．动物学报，2001，47(1)：79-84．
[46] 丁军，蒋一珪，单仕新等．异源精子质膜蛋白质在银鲫卵雌核发育初级控制中的作用．科学通报，1993，38(5)：455-458．
[47] Tian JD, Gong H, Gerald H, et al. Xenopus laevis sperm-egg adhesion is regulated by modifications in the sperm receptor and the egg vitelline envelope. Devel. Biol., 1997, 187: 143-153.
[48] 奚耕思，杨月红，郑哲民．蟋蟀精子表面LCA及ConA结合糖复合物的分布变化．动物学报，2002，48(1)：125-130．
[49] Cattaneo F, Ogiso M, Hoshi M, et al. Purification and characterization of the plasma membrane glycosidases of Drosophila melanogaster spermatozoa, lns. Bioch. Mol. Biol., 2002, 32:929-941.
[50] 王宏田，张培军．体外受精动物的精卵识别．海洋科学，2000，24(5)：21-23．
[51] Vacquier VD, Moy GM. Isolation of bindin: The protein responsible for adhesion of sperm to sea urchin eggs. Proc. Natl. Acad. Sci. USA, 1977, 74: 2456-2460.
[52] Ulrich AS, Otter M, Glabe CG, et al. Membrane fusion is induced by a distinct peptide sequence of the sea urchin fertilization protein bindin. J. Biol. Chem., 1998, 273(27): 16748-16755.
[53] Podell SB, Vacquier VD. Purification of the Mr 80,000 and Mr 210,000 proteins of the sea urchin sperm plasma membrane. J. Biol. Chem., 1985, 260(5): 2715-2718.
[54] 刘慧慧，李太武，苏秀榕等．鲍配子识别蛋白的研究．动物学杂志，2003，38(6)：104-109．
[55] 刘慧慧，李太武，苏秀榕．贝类配子细胞识别物的初步研究．水产科学，2004，23(5)：10-14．
[56] 程立均，康现江，穆淑梅等．中华绒螯蟹精荚分级分离及其蛋白组分分析．动物学杂志，2005，40(5)：95-98．
[57] Brown GG. Ultrastructural studies of sperm morphology and sperm-egg interaction in the decapod Callinectes sapidus. J. Ultrastruct. Res., 1966, 14(5): 425-40.
[58] 堵南山，赖伟，薛鲁征．中华绒螫蟹精子的研究Ⅰ．精子的形态及超微结构．海洋与湖沼，1987，18(2)：119-126．
[59] Yasuzumi G. Spermatogenesis in animals as revealed by electron microscopy. Ⅶ. Spermatid differentiation in crab, Eriocheir japonicus. J. Biophs. Biochem. Cytol., 1960, 7: 73-87.
[60] Jamison RGM. The ultrastructure of the spermatozoa of four species of xanthid crabs(Crustacea, Brachyura, Xanthidae). J. Submicrosc. Cytol. Pathol., 1989, 21(3): 579-584.
[61] Reger JF. Studies on the fine strrcture of spermatids and spermatozoa of the crab, Pinnixia sp. J. Morphol., 1970, 132: 89-100.
[62] 王兰，堵南山，赖伟．长江华溪蟹精子的超微结构．动物学报，2000，46(2)：227-229．
[63] Langreth SG. Spermiogenesis in Cancer crabs. J. Cell. Biol., 1969, 43: 575-603.
[64] 上官步敏，李少菁．锯缘青蟹精子超微结构的研究．动物学报，1994，40(1)：7-12．
[65] 李太武．三疣梭子蟹精子的发生及超微结构研究．动物学报，1995，41(1)：41-49．
[66] Hinsch GW. Sperm structure of Oxyrhyncha. Can. J. Zool., 1973, 51: 421-429.
[67] Geldziler BP, Kadandale A, Singson. Molecular genetic approaches to studying fertilization in model systems. Reproduction, 2004b, 127: 409-416.
[68] 丁蓓蓓，蒋俶，蒋文珏等．受精蛋白B在人精子表面的免疫组织化学定位．实验生物学报，2001，34(4)：283-289．
[69] 张军，李彦锋．精子抗原受精素B的研究进展．中华男科学，2004，10(1)：52-55．
[70] 吕年青，王兴海，黄宇烽．精子抗原作为候选免疫避孕疫苗的研究进展．男科学报，1999，5(3)：154-158．
[71] Myles DG, Primakoff P. Why did the sperm cross the cumulus? To get to the oocyte, Functions of the sperm surface proteins PH-20 and fertilin in arriving at, and fusing with, the egg. Bio. Reprod, 1997, 56: 320.
[72] Phelps BM, Primakoff P, Koppel DE, et al. Restricted lateral diffusion of PH-20, a PI-anchored sperm membrane protein. Science, 1988, 240: 1780.
[73] Richardson RT, Yamasaki N, O'rand MG. Sequence of a rabbit sperm zona pellucida binding protein and localization during the acrosome reacting. Devel. Biol., 1994, 165(2): 688-701.
[74] Kurth BE, Weston C, Reddi PP, et al. Oviductal antibody response to a defined recombinant sperm antigen in macques. Biol. Reprod., 1997, 57: 981-989.
[75] Santhanam R, Naz Rk. Novel human testis-specific cDNA: molecular cloning, expression and immunobiological effects of the recombinant protein. Mol. Reprod. Dev., 2001, 60(1): 1-12.
[76] Trivedi RN, Naz RK. Testis-specific antigen (TSA-1) is expressed in murine sperm and its antibodies inhibit fertilization. Am. J. Reprod. Immunol., 2002, 47(1): 38-45.
[77] Zhang XD, Miao SY, Wang LF, et al. Human sperm membrane protein (hSMP-1): a developmental testis-specific component during germ cell differentiation. Arch. Androl., 2000, 45(3): 239-346.
[78] Wang H, Miao S, Chen D, et al. Assignment of chromosomal locus and evidence for alternatively spliced mRNAs of a human sperm membrane protein (hSMP-1). Biochim. Biophys. Acta., 1999, 1447(1): 119-124.
[79] Calogero AE, Burrello N, Barone N, et al. Effects of progesterone on sperm function: mechanism of action. Hum. Reprod., 2000, 15(Suppl 1): 28-45.
[80] 贾悦，崔毓桂，狄福松．孕激素与男性生殖．中华男科学，2001，7(2)：117-120．
[81] Blackmore PF, Lattanzio FA. Cell surface localization of a novel non-genomic progesterone receptor on the head of human sperm. Biochem. Biophys. Res. Commun.,1991,181(1): 331-336.
[82] Blackmore PF, Neulen J, Lattanzio FA, et al. Cell surface binding sites for progesterone mediate calcium uptake in human sperm. J. Biol. Chem.,1991,266(28): 8655-8659.
[83] Yang J, Serres C, Philibert D, et al. Progesterone and RU486: opposing effect on human sperm.. Proc. Natl. Acad. Sci. USA.,1994,91(2): 529-533.
[84] He XB, Hu J H, Wu Q, et al. Indentification of GABA(B) receptor in rat testis and sperm. Biochem. Biophys. Res. Commun., 2001,27(283): 243-247.
[85] 王春年，刘海卫，袁玉英等．γ-氨基丁酸诱发人精子顶体反应及其对若干离子转运影响的研究．生殖与避孕，1996，16(2)：118-121．
[86] 边淑玲，张玮，朱辉等．γ-氨基丁酸对精子顶体酶活性的影响．中华男科学，2002，5(8)：326-328．
[87] Spat A. Astaturable receptor for P-inositol-1,4,5-trisphosphate in hepatocytes and neutrophils. Nature,1986, 319:514.
[88] Vinson Gp, Saridogan E, Puddefoot JR, et al. Tissue rennin-angiotensin systems and reproduction. Human. Reprod., 1997, 12: 651-662.
[89] Naz RK. Application of sperm antigens in immunocontraception. Front. Biosci., 1996, 1: 87-95.
[90] Naz RK. Effects of antisperm antibodies on early cleavage of fertilized ova. Biol. Reprod., 1992, 46(1): 130-139.
[91] Boue F, Sullivan R. Cases of human infertility are associated with the absence of P34H an epididymal sperm antigen. Biol. Reprod., 1996, 54(5): 1018-1024.
[92] 黄勋彬．纤溶酶原激活因子／纤溶酶系统与精子之间的关系．国外医学计划生育分册，1996，4(15)：222-224．
[93] Leyton L, LeGuen P, Bunch D, et al. Regulation of mouse gamete interaction by a sperm tyrosine kinase. Pro. Natl. Acad. Sci. USA., 1992, 89: 11692-11695.
[94] Burks DJ, Carballada R, Moore HDM, et al. Interaction of tyrosine kinase from human sperm with the zona pellucida at fertilization. Science, 1995, 269: 83-86.
[95] Brewis IA, Clayton R, Martin M, et al. Tyrosine phosphprylation of 95kD protein and induction of the acrosome reaction in human spermatozoa in response to recombinant human ZP3. Mol. Hum. Reprod., 1998, 4(12): 1136-1144.
[96] Pukazhenthi BS, Wildt DE, Ottinger MA, et al. Compromised sperm protein phosphorylation after capacitation, swim-up, and zona pelucida exposure in teratospermic domestic cats. J. Androl., 1996, 17(4): 409-419.
[97] Wassarman PM, Jovine L, Litscher ES. A profile of fertilization in mammals. Nat. Cell. Biol., 2001, 3: E59-E64.
[98] Miller DJ, Shi XD, Burkin HB. Molecular basis of mammalian gamete binding. Recent. Prog. Horm. Res., 2002, 57: 37-73.
[99] Youakim A, Hathaway HJ, Miller DJ, et al. Overexpressing sperm surface β-1,4-galactosyltransferase in transgenic mice affects multiple aspects of sperm-egg interactions. J. Cell. Biol., 1994, 126(6): 1573-1583.
[100] Lu RIM, Shur K. Male mice deficient for germ-cell cyritestin are infertile.Biol. Reprod., 1999, 61: 1445-1451.
[101] Foster LH, Cheng A, Bleil JD. Tissue-and species-specific expression of sp56, a mouse sperm fertilization protein. Science, 1995, 269: 86-89.
[102] Kim KS, Cha MC, Gerton GL. Mouse sperm protein sp56 is a component of the acrosomal matrix. Biol. Reprod., 2001, 64: 36-43.
[103] Cohen N, Wassarman PM. Association of egg zona pellucida glycoprotein mZP3 with sperm protein sp56 during fertilization in mice. Int. J. Dev. Biol., 2001, 45: 569-576.
[104] Hardy CM, Mobbs KJ. Expression of recombinant mouse sperm protein sp56 and assessment of its potential for use as an antigen in an immunocontraceptive vaccine. Mol. Reprod. Dev., 1999, 52: 216-224.
[105] He XB, Yan YC, Li YP, et al. Cloning of rat sp56, the homologue of mouse sperm ZP3 receptor-sp56. Cell Res., 2003, 13(2): 121-129.
[106] Hickox JR, Bi M, Hardy DM. Heterogeneous processing and zona pellucida binding activity of pig zonadhesion. J. Biol. Chem., 2001, 276(44): 41502-41509.
[107] Lea IA, Sivashanmugam P, O'Rand MG. Zonadhesion: Characterization, localization,and zona pellucida binding. Biol. Reprod., 2001, 65: 1691-1700.
[108] Williams RM, Jones R. Specificity of binding of zona pellucida glycoroteins to sperm proacrosin and related proteins. J. Exp. Zool., 1993, 226(1): 65-73.
[109] Jansen S, Quigley M, Reik W, et al. Analysis of polysulfate-binding domains in porcine proacrosin, a putaive zona adhesin protein from mammalian spermatozoa, Int. J. Dev. Biol., 1995, 39(3): 501-510.
[110] 宛传丹，黄宇烽，许晓风．精子蛋白SP22研究进展．中华男科学杂志，2005，11(1)：56-59．
[111] Hao Z, Wolkowicz MJ, Shetty J, et al. SAMP32, a testis-specific, isoantigenic sperm acrosomal membrane-associated protein. Biol. Reprod., 2002, 66:735-744.
[112] Blobel CP. Proteolytic process of a protein involved in sperm-egg fusion correlates with acqutisition of fertilization competence. J. Cell. Biol., 1999, 111 (1): 69.
[113] 丁之德，吴明章．哺乳类动物精子在附睾内成熟过程中膜蛋白的变化．男性学杂志，1993，7(2)：119-121．
[114] Brown CR, von Glos KI, Jones R. Changes in plasma membrane glycoproteins of rat spermatozoa during maturation in the epididymis. J. Cell Biol., 1983, 96(1): 256-264.
[115] Gaunt SJ. A 28K-dalton cell surface autoantigen of spermatogenesis: characterization using a monoclonal antibody. Dev. Biol., 1982, 89(1): 92-100.
[116] Voglmayr JK, Fairbanks G, Lewis RG. Surface glycoprotein changes in ram spermatozoa during epididymal maturation. Bio. Reprod, 1983, 29(3): 767-775.
[117] Swanson WJ, Vacquier VD. Liposome fusion induced by a Mr. 18,000 protein localized to the acrosomal region of acrosome-reacted abalone spermatozoa. Biochemistry, 1995, 34(43): 14202-14208．
[118] 刘慧慧，李太武，苏秀榕．几种动物精卵识别机制．海洋湖沼通报，2004，1：16-21．
[119] Esponda P, Guerra R. Plasma-membrane glycoproteins changes during sperm iogenesis and in the sperm atozoa of some orthoptera. Cell. Tissue. Res., 1991, 264:507-513.
[120] Long G, Sottile R, Privitera R. Letin binding to the sperm surface before and after insemination in a grasshopper Eyprepocnomicsplorans( charp).Eur Arch. Biol.,1994, 105(1):35-40.
[121] Marti S, Cacho D., Jass A. Plasma membrane glycoproteins of mature and immature drone honey bee spermatozoa:Letin binding as seen by light and electronmicoscope. Theriogenology, 1996, 46(1):181-190.
[122] Cattaneo F, Pasini ME. Glycosidases are present on the surface of Drosophila melanogaster spermatozoa. Mol. Reprod, 1997, 48(2):276-281.
[123] 堵南山．中华绒螯蟹的受精．水产科技情报，1998，25(1)：9-13．
[124] Takizawa S, Sawada H, Someno T, et al. Effects of protease inbibitors on binding of sperm to the vitelline coat of Ascidian eggs: Implications for participation of a proteasome (Muticatalytic proteinase complex). J. Exp. Zool., 1993,267:86-91.
[125] Foltz KR, Lennarz WJ. The molecular basis of sea urchin gamete interactions at the egg plasma membrane. Devel. Biol., 1993, 158(1): 46-61.
[126] 杨万喜，姜乃澄，卢建平．十足类甲壳动物受精细胞学研究进展．东海海洋，1998，4：57-63．
[127] 范桢，周孝瑚，章仁安等．大鼠精子特异的LDH-C4的提纯及其免疫性质分析．上海医科大学学报，1989，16(4)：253-258．
[128] 方丁，房世荣主编．同工酶在医学上的应用．北京：人民卫生出版社，1982，66-94．
[129] Markert CL, Moiler, F. Multiple forms of enzymes: tissue, ontogenetic, and species specific patterns. Proc. Natl. Acad Sci. USA., 1959, 45: 753-763.
[130] 陈明，周念辉，李爱媛等．东方对虾精子中的乳酸脱氢酶C．科学通报，1994，39(24)：2272-2274．
[13I] 曾科文，郑成洋，潘雪峰．水貂组织乳酸脱氢酶同工酶电泳分析．东北林业大学学报，1987，15(6)：28-32．
[132] Blanco A, Zinkham WH. Lactate dehydrogenases in human testes. Science, 1963, 139: 601-602.
[133] 李明．精子乳酸脱氢酶同工酶的研究进展．临床检验杂志，2001，19(6)：375-378．
[134] Goldberg E. Lactic and malic dehydrogenase in human spermatozoa. Science, 1963, 139: 602.
[135] O'Flaherty CM, Beorlegui NB, Beconi MT. Lactate dehydrogenase-C4 is involved in heparin-and NADH-dependent bovine sperm capacitation. Andrologia, 2002, 34: 91-97.
[136] O'Hern PA, Liang ZG, Bambra CS, et al. Colinear synthesis of an antigen-specific B-cell epitope with a 'promiscuous' tetanus toxin T-cell epitope: a synthetic peptide immunocontraceptive. Vaccine, 1997, 15: 1761-1766.
[137] Goldberg E. The ontogeny of sperm-sperific lactate dehydrogenase in mice. J.. Exp. Zool., 1967, 164: 309.
[138] Zinkham WH, Blanco A, Kupchyk L. Lactate dehydrogenase in testis: dissociation and recombination of subunits. Science, 1963, 142: 1303-1304.
[139] Markert CL. Lactate dehydrogenase.: biochemistry and function of lactate dehydrogenase. Cell. Biochem. Funct., 1984, 2(3): 131-134.
[140] Baldwin K, Mortimer AP. Do ascidians possess the ancestral subunit type of vertebrate lactate dehydrogenase? J. Exp. Zoo., 1988, 246(2): 109-114.
[141] 张龙翔，张庭芳，李令媛主编．生化实验方法和技术．北京：高等教育出版社，1997，379-381．
[142] Faulk WP, Taylor GM. An immunocolloid method for the electron microscope, Immunochemistry, 1971, 8(11): 1081-1083.
[143] 堵南山．中华绒螯蟹的同属种类及其英文名称．水产科技情报，1998，25(3)：108-109．
[144] 胡自强，胡运瑾．河蟹生殖系统的形态学和组织结构．湖南师范大学自然科学学报，1997，20(3)：71-76．
[145] 堵南山，薛鲁征，赖伟．中华绒螯蟹(Eriocheir sinensis)雄性生殖系统的组织学研究．动物学报，1988，34(4)：329-335．
[146] 吴萍，楼允东，邱高峰．中华绒螯蟹性腺发育的形态学、组织学和组织化学变化．上海水产大学学报，2003，12(2)：106-112．
[147] 堵南山，赖伟，薛鲁征．中华绒螯蟹(Eriocheir sinensis)精子顶体反应的研究．动物学报，1987，33(1)：8-13．
[148] 王群，赵云龙，陈立侨．中华绒螯蟹雄性生殖系统生化组成及精子代谢．水产学报，2002，26(5)： 411-416．
[149] 康现江，米娅，刘彬彬等．摘除眼柄对中华绒螯蟹精巢发育及其氨基酸含量的影响．台湾海峡，2000，19(3)：360-364．
[150] 吴萍，邱高峰，楼允东．中华绒螯蟹促雄腺结构变化对精子发生的影响．水产学杂志，2002，15(1)：88-92．
[151] 堵南山，薛鲁征，赖伟．中华绒螯蟹精子的研究Ⅱ．精子发生．海洋与湖沼，1988，19(1)：71-78．
[152] 堵南山，赖伟，安婴等．中华绒螯蟹受精的细胞学研究．中国科学(B辑)，1992，3：260-265．
[153] 堵南山．中华绒螯蟹的受精生物学(一)．生物学通报，1998，33(12)：5-8．
[154] 堵南山．中华绒螯蟹的受精生物学(二)．生物学通报，1999，34(1)：10-12．
[155] 堵南山．中华绒螯蟹是体内还是体外受精?水产科技情报，2000，27(5)：217-218．
[156] 程立均．中华绒螯蟹精子膜蛋白的提取分离及部分生化性质研究．河北大学硕士学位论文(单行本)，2004．
[157] 汪家政，范明主编．蛋白质技术手册．北京：科学出版社，2002，42-47．
[158] 芮菊生，杜懋琴，陈海明等编著．组织切片技术．北京：人民教育出版社，1980，225-231．
[159] 北原文雄，玉井康胜，早野茂夫等．表面活性剂．北京：化学工业出版社，1984，529．
[160] 乔新美，曹维孝，邹世平．长江、瓯江中华绒螯蟹几种同工酶的分析比较．淡水渔业，1994，24(5)：10-13．
[161] 许加武，李思发．长江口中华绒螯蟹与其它几种同科蟹的同工酶比较．水产科技情报，1996，23(4)：159-162．
[162] 王丹，于伟君．辽河长江两水系中华绒螯蟹酯酶和乳酸脱氢酶的同工酶比较研究．辽宁大学学报(自然科学版)，1995，22(4)：79-81．
[163] 程家骅，王云龙，许加武等．苏北南部沿海几种蟹类蛋白和同工酶的比较研究．中国水产科学，1998，5(1)：10-17．
[164] 薛俊增．患“抖抖病”中华绒螯蟹可溶性蛋白质和同工酶分析．动物学杂志，2002，37(2)：17-21．
[165] 贾守菊，应雪萍，陈艳乐等．中华绒螯蟹不同发育阶段腹肢粘液腺同工酶的比较．海洋湖沼通报，2004，4：52-60．
[166] 雷焕宗，贾守菊，应雪萍等．中华绒螫蟹不同生理时期肌肉组织中同工酶的分析比较．河南科学，2004，22(4)：480-483．
[167] 王群，赵云龙，陈立侨．中华绒螯蟹雄性生殖系统生化组成及精子代谢．水产学报，2002，26(5)：411-416．
[168] 哈密斯BD，利克伍德D著，刘毓秀，程桂芳译．蛋白质的凝胶电泳——实践方法．北京：科学出版社，1986，159-160．
[169] 汪谦主编．现代医学实验方法．北京：人民卫生出版社，1997，712-715．
[170] 李建武，余瑞元，袁明秀等．生物化学实验原理和方法．北京：北京大学出版社，1994，381-386．
[2] 王桂兰，崔存德，夏振开等．人精子顶体膜蛋白的纯化及性质的研究．中国应用生理学杂志，2003，19(4)：376-378．
[3] 沙国柱，黄宇烽，徐建平．人精子膜抗原的提纯和分析．男科学报，1999，5(3)：138-140．
[4] 王浩飞，向祖琼，王益鑫．人精子膜蛋白的二维电泳实验研究．中华男科学，2003，9(7)：504-506．
[5] 魏曙光，王琳芳，缪时英．一种与受精有关的人精子膜蛋白(BS-17)的分离纯化．生物化学杂志，1994，10(2)：236-241．
[6] 林典梁．人精子甘露糖受体纯化及其对受精能力的影响．福建医科大学硕士学位论文(单行本)，2002．
[7] 缪时英，严缘昌，李月华等．编码人精子膜蛋白BS-84的cDNA研究．自然科学进展——国家重点实验室通讯，1995，5(2)：225-228．
[8] 丁之德，Hansen S，Thiel S，et al．人精子膜甘露糖结合蛋白的研究——蛋白的定位．中国男科学杂志，2000，14(1)：19-21．
[9] 丁之德，Hansen S，Thiel s，et al，人精子膜甘露糖结合蛋白的研究．生殖与避孕，2000，20(1)：20-25．
[10] 高爱武，何晓兵，严缘昌．一种人精子膜蛋白的基因定位及其抗体对小鼠体外受精的影响．生殖与避孕，2000，20(3)：142-146．
[11] 缪时英，李月华，周荔申等．一种人精子膜蛋白基因的克隆与鉴定．基础医学与临床，1990，10(6)：23-28．
[12] 魏曙光，缪时英，张梅林．一种Calpastatin具有部分同源结构的人精子膜蛋白的发现与研究．生物化学杂志，1994，10(2)：242-246．
[13] 夏欣一．人附睾精子蛋白P34H的克隆、表达及单克隆抗体制备．南京师范大学硕士学位论文(单行本)，2003．
[14] 徐崇，陈复，刘莉等．84kD精子膜蛋白与抗精子抗体．生殖与避孕，1990，10(2)：39-42．
[15] 杨宏毅．人精子甘露糖受体的表达及其与精子．透明带相互作用的关系．福建医科大学硕士学位论文(单行本)，2002．
[16] 江澍．人精子甘露糖受体对精卵融合的影响．福建医科大学硕士学位论文(单行本)，2002．
[17] 周红，曾仲奎，刘荣华．人精子膜上凝集素受体的研究．生殖与避孕，1992，12(2)：11-17．
[18] Yu J, Wang LF, Miao SY, et al. Antiidiotypic antibody related to the 84kD human sperm membrane protein. Cell. Res., 1990, 2: 163-172.
[19] Quill TA, Garbers DL. Sperad is a novel sperm-specific plasma membrane protein homologous to a family of cell adhesion proteins. J. Biol. Chem., 1996, 271(52): 33509-33514.
[20] Cheng A, Le T, Palacios M, et al. Sperm-egg recognition in the mouse: characterization of sp56, a sperm protein having specific affinity for ZP3. J. Cell, Biol., 1994, 125(4): 867-878.
[21] Zhuang DZ, Zhang TY, Chen DY. A protein extracted from mouse sperm that plays an important role in fertilization. Devel. Reprod Biol., 1994, 3(I): 1-9.
[22] 孙秉贵，周占祥，邓泽沛．SA-30抗原在小鼠睾丸、附睾、精子及早期胚的定位．解剖学报，2000，31(2)：167-171．
[23] 丁之德，王卓群，吴明章．大鼠精子在附睾成熟中精子膜变化的研究．生殖医学杂志，1994，3(2)：114-117．
[24] 李彦锋，靳风烁，江军．精子表面蛋白受精素β在精卵结合和融合中的作用研究．中国男科学杂志，2001，15(4)：225-228．
[25] 庄大中，宋祥芬，胡国俊等．精子抗原MSH27参与精卵融合过程．科学通报，1999，44(14)：1508-1513．
[26] 韩之明，庄大中，高绍荣等．小鼠精子在附睾成熟过程中质膜糖蛋白的变化．动物学报，1999，45(1)：93-98．
[27] 柏家林，苟克勉，安晓荣等．一种新的精子膜蛋白sp18基因的克隆及其在E．coli中的表达．农业生物技术学报，2003，11(3)：268-272．
[28] 何晓兵．与大鼠受精相关的两种受体分子-GABA-BR和大鼠sp56的研究．中国科学院研究生院(上海生命科学研究院)．2003．
[29] Hardy DM, Garbers DL. A sperm membrane protein that binds in a species-specific manner to the egg extracellular matrix is homologous to von willebrand factor. J Biol. Chem., 1995, 270(44): 26025-26028.
[30] Haden NP, Hickox JR, Whisnant CS, et al. Systematic characterization of sperm-specific membrane proteins in swine. Biol. Reprod, 2000, 63: 1839-1847.
[31] 赵明，费坚，孙册．猪精子凝集素的纯化、性质及其作用．生物化学与生物物理学报，1995，27(4)：346-354．
[32] 赵明，孙册．猪精子凝集素在精卵结合中的作用机制．生物化学与生物物理学报，1995，27(4)：375-383．
[33] 赵明，孙册．猪镜子中与卵透明带糖蛋白ZP3结合的蛋白质．生物化学与生物物理学报，1996，28(2)：145-152．
[34] 刘建喜，林爱星，杨毅等．家兔精子膜蛋白rSP10在大肠杆菌中的高效表达及其抗血清的制备． 生物工程学报，2001，17(3)：314-317．
[35] 梁植权，王琳芳，缪时英等．精子膜蛋白的研究Ⅰ．具有同种抗原性的兔精子膜蛋白A的分离． 中国医学科学院学报，1980，2(3)：147-151．
[36] 王琳芳，缪时英，赵敏顺等．精子膜蛋白的研究Ⅱ．具有同种抗原性的兔精子膜蛋白A的免疫活性及理化性质．中国医学科学院学报，1980，2(3)：152—155．
[37] 王琳芳，缪时英，曹淑兰等．精子膜蛋白的研究Ⅲ．兔精子膜蛋白“A”的定位及其酯酶活性的观察．中国医学科学院学报，1982，4(2)：82-85．
[38] Zahler WL, Doak GA. Isolation of the outer acrosomal membrane from bull sperm. Biochimica. et. Biophysica. Acta., 1975, 406: 479-488.
[39] Hardy DM, Garbers DL. Species-specific binding of sperm proteins to the extracellular matrix(zona pellucida)of the egg. I. Biol. Chem., 1994, 269(29): 19000-19004.
[40] 柳荣，李天俊，陶丙春等．奶牛精子膜的分离及纯化．畜牧兽医学报，1997，28(5)：409-415．
[41] 曾少举，桑建利，梁前进．一种牛精子膜蛋白的纯化及在生殖中的作用．北京师范大学学报(自然科学版)，2000，36(5)：683-686．
[42] 周占祥，邓泽沛．羊精子表面的凝集素标记特征．中国组织化学与细胞化学杂志，1994，3(3)：256-261．
[43] 周占祥，邓泽沛．羊精子在成熟和获能过程中表面的凝集素标记变化．解剖学报，1994，25(3)：276-280
[44] Sabeur K, Foristall K, Ball BA. Characterization of PH-20 in canine spermatozoa and testis. Theriogenology, 2002, 57(2): 977-987.
[45] 杨书婷，桂建芳．雌核发育银鲫和两性生殖彩鲫精子蛋白组份的比较研究．动物学报，2001，47(1)：79-84．
[46] 丁军，蒋一珪，单仕新等．异源精子质膜蛋白质在银鲫卵雌核发育初级控制中的作用．科学通报，1993，38(5)：455-458．
[47] Tian JD, Gong H, Gerald H, et al. Xenopus laevis sperm-egg adhesion is regulated by modifications in the sperm receptor and the egg vitelline envelope. Devel. Biol., 1997, 187: 143-153.
[48] 奚耕思，杨月红，郑哲民．蟋蟀精子表面LCA及ConA结合糖复合物的分布变化．动物学报，2002，48(1)：125-130．
[49] Cattaneo F, Ogiso M, Hoshi M, et al. Purification and characterization of the plasma membrane glycosidases of Drosophila melanogaster spermatozoa, lns. Bioch. Mol. Biol., 2002, 32:929-941.
[50] 王宏田，张培军．体外受精动物的精卵识别．海洋科学，2000，24(5)：21-23．
[51] Vacquier VD, Moy GM. Isolation of bindin: The protein responsible for adhesion of sperm to sea urchin eggs. Proc. Natl. Acad. Sci. USA, 1977, 74: 2456-2460.
[52] Ulrich AS, Otter M, Glabe CG, et al. Membrane fusion is induced by a distinct peptide sequence of the sea urchin fertilization protein bindin. J. Biol. Chem., 1998, 273(27): 16748-16755.
[53] Podell SB, Vacquier VD. Purification of the Mr 80,000 and Mr 210,000 proteins of the sea urchin sperm plasma membrane. J. Biol. Chem., 1985, 260(5): 2715-2718.
[54] 刘慧慧，李太武，苏秀榕等．鲍配子识别蛋白的研究．动物学杂志，2003，38(6)：104-109．
[55] 刘慧慧，李太武，苏秀榕．贝类配子细胞识别物的初步研究．水产科学，2004，23(5)：10-14．
[56] 程立均，康现江，穆淑梅等．中华绒螯蟹精荚分级分离及其蛋白组分分析．动物学杂志，2005，40(5)：95-98．
[57] Brown GG. Ultrastructural studies of sperm morphology and sperm-egg interaction in the decapod Callinectes sapidus. J. Ultrastruct. Res., 1966, 14(5): 425-40.
[58] 堵南山，赖伟，薛鲁征．中华绒螫蟹精子的研究Ⅰ．精子的形态及超微结构．海洋与湖沼，1987，18(2)：119-126．
[59] Yasuzumi G. Spermatogenesis in animals as revealed by electron microscopy. Ⅶ. Spermatid differentiation in crab, Eriocheir japonicus. J. Biophs. Biochem. Cytol., 1960, 7: 73-87.
[60] Jamison RGM. The ultrastructure of the spermatozoa of four species of xanthid crabs(Crustacea, Brachyura, Xanthidae). J. Submicrosc. Cytol. Pathol., 1989, 21(3): 579-584.
[61] Reger JF. Studies on the fine strrcture of spermatids and spermatozoa of the crab, Pinnixia sp. J. Morphol., 1970, 132: 89-100.
[62] 王兰，堵南山，赖伟．长江华溪蟹精子的超微结构．动物学报，2000，46(2)：227-229．
[63] Langreth SG. Spermiogenesis in Cancer crabs. J. Cell. Biol., 1969, 43: 575-603.
[64] 上官步敏，李少菁．锯缘青蟹精子超微结构的研究．动物学报，1994，40(1)：7-12．
[65] 李太武．三疣梭子蟹精子的发生及超微结构研究．动物学报，1995，41(1)：41-49．
[66] Hinsch GW. Sperm structure of Oxyrhyncha. Can. J. Zool., 1973, 51: 421-429.
[67] Geldziler BP, Kadandale A, Singson. Molecular genetic approaches to studying fertilization in model systems. Reproduction, 2004b, 127: 409-416.
[68] 丁蓓蓓，蒋俶，蒋文珏等．受精蛋白B在人精子表面的免疫组织化学定位．实验生物学报，2001，34(4)：283-289．
[69] 张军，李彦锋．精子抗原受精素B的研究进展．中华男科学，2004，10(1)：52-55．
[70] 吕年青，王兴海，黄宇烽．精子抗原作为候选免疫避孕疫苗的研究进展．男科学报，1999，5(3)：154-158．
[71] Myles DG, Primakoff P. Why did the sperm cross the cumulus? To get to the oocyte, Functions of the sperm surface proteins PH-20 and fertilin in arriving at, and fusing with, the egg. Bio. Reprod, 1997, 56: 320.
[72] Phelps BM, Primakoff P, Koppel DE, et al. Restricted lateral diffusion of PH-20, a PI-anchored sperm membrane protein. Science, 1988, 240: 1780.
[73] Richardson RT, Yamasaki N, O'rand MG. Sequence of a rabbit sperm zona pellucida binding protein and localization during the acrosome reacting. Devel. Biol., 1994, 165(2): 688-701.
[74] Kurth BE, Weston C, Reddi PP, et al. Oviductal antibody response to a defined recombinant sperm antigen in macques. Biol. Reprod., 1997, 57: 981-989.
[75] Santhanam R, Naz Rk. Novel human testis-specific cDNA: molecular cloning, expression and immunobiological effects of the recombinant protein. Mol. Reprod. Dev., 2001, 60(1): 1-12.
[76] Trivedi RN, Naz RK. Testis-specific antigen (TSA-1) is expressed in murine sperm and its antibodies inhibit fertilization. Am. J. Reprod. Immunol., 2002, 47(1): 38-45.
[77] Zhang XD, Miao SY, Wang LF, et al. Human sperm membrane protein (hSMP-1): a developmental testis-specific component during germ cell differentiation. Arch. Androl., 2000, 45(3): 239-346.
[78] Wang H, Miao S, Chen D, et al. Assignment of chromosomal locus and evidence for alternatively spliced mRNAs of a human sperm membrane protein (hSMP-1). Biochim. Biophys. Acta., 1999, 1447(1): 119-124.
[79] Calogero AE, Burrello N, Barone N, et al. Effects of progesterone on sperm function: mechanism of action. Hum. Reprod., 2000, 15(Suppl 1): 28-45.
[80] 贾悦，崔毓桂，狄福松．孕激素与男性生殖．中华男科学，2001，7(2)：117-120．
[81] Blackmore PF, Lattanzio FA. Cell surface localization of a novel non-genomic progesterone receptor on the head of human sperm. Biochem. Biophys. Res. Commun.,1991,181(1): 331-336.
[82] Blackmore PF, Neulen J, Lattanzio FA, et al. Cell surface binding sites for progesterone mediate calcium uptake in human sperm. J. Biol. Chem.,1991,266(28): 8655-8659.
[83] Yang J, Serres C, Philibert D, et al. Progesterone and RU486: opposing effect on human sperm.. Proc. Natl. Acad. Sci. USA.,1994,91(2): 529-533.
[84] He XB, Hu J H, Wu Q, et al. Indentification of GABA(B) receptor in rat testis and sperm. Biochem. Biophys. Res. Commun., 2001,27(283): 243-247.
[85] 王春年，刘海卫，袁玉英等．γ-氨基丁酸诱发人精子顶体反应及其对若干离子转运影响的研究．生殖与避孕，1996，16(2)：118-121．
[86] 边淑玲，张玮，朱辉等．γ-氨基丁酸对精子顶体酶活性的影响．中华男科学，2002，5(8)：326-328．
[87] Spat A. Astaturable receptor for P-inositol-1,4,5-trisphosphate in hepatocytes and neutrophils. Nature,1986, 319:514.
[88] Vinson Gp, Saridogan E, Puddefoot JR, et al. Tissue rennin-angiotensin systems and reproduction. Human. Reprod., 1997, 12: 651-662.
[89] Naz RK. Application of sperm antigens in immunocontraception. Front. Biosci., 1996, 1: 87-95.
[90] Naz RK. Effects of antisperm antibodies on early cleavage of fertilized ova. Biol. Reprod., 1992, 46(1): 130-139.
[91] Boue F, Sullivan R. Cases of human infertility are associated with the absence of P34H an epididymal sperm antigen. Biol. Reprod., 1996, 54(5): 1018-1024.
[92] 黄勋彬．纤溶酶原激活因子／纤溶酶系统与精子之间的关系．国外医学计划生育分册，1996，4(15)：222-224．
[93] Leyton L, LeGuen P, Bunch D, et al. Regulation of mouse gamete interaction by a sperm tyrosine kinase. Pro. Natl. Acad. Sci. USA., 1992, 89: 11692-11695.
[94] Burks DJ, Carballada R, Moore HDM, et al. Interaction of tyrosine kinase from human sperm with the zona pellucida at fertilization. Science, 1995, 269: 83-86.
[95] Brewis IA, Clayton R, Martin M, et al. Tyrosine phosphprylation of 95kD protein and induction of the acrosome reaction in human spermatozoa in response to recombinant human ZP3. Mol. Hum. Reprod., 1998, 4(12): 1136-1144.
[96] Pukazhenthi BS, Wildt DE, Ottinger MA, et al. Compromised sperm protein phosphorylation after capacitation, swim-up, and zona pelucida exposure in teratospermic domestic cats. J. Androl., 1996, 17(4): 409-419.
[97] Wassarman PM, Jovine L, Litscher ES. A profile of fertilization in mammals. Nat. Cell. Biol., 2001, 3: E59-E64.
[98] Miller DJ, Shi XD, Burkin HB. Molecular basis of mammalian gamete binding. Recent. Prog. Horm. Res., 2002, 57: 37-73.
[99] Youakim A, Hathaway HJ, Miller DJ, et al. Overexpressing sperm surface β-1,4-galactosyltransferase in transgenic mice affects multiple aspects of sperm-egg interactions. J. Cell. Biol., 1994, 126(6): 1573-1583.
[100] Lu RIM, Shur K. Male mice deficient for germ-cell cyritestin are infertile.Biol. Reprod., 1999, 61: 1445-1451.
[101] Foster LH, Cheng A, Bleil JD. Tissue-and species-specific expression of sp56, a mouse sperm fertilization protein. Science, 1995, 269: 86-89.
[102] Kim KS, Cha MC, Gerton GL. Mouse sperm protein sp56 is a component of the acrosomal matrix. Biol. Reprod., 2001, 64: 36-43.
[103] Cohen N, Wassarman PM. Association of egg zona pellucida glycoprotein mZP3 with sperm protein sp56 during fertilization in mice. Int. J. Dev. Biol., 2001, 45: 569-576.
[104] Hardy CM, Mobbs KJ. Expression of recombinant mouse sperm protein sp56 and assessment of its potential for use as an antigen in an immunocontraceptive vaccine. Mol. Reprod. Dev., 1999, 52: 216-224.
[105] He XB, Yan YC, Li YP, et al. Cloning of rat sp56, the homologue of mouse sperm ZP3 receptor-sp56. Cell Res., 2003, 13(2): 121-129.
[106] Hickox JR, Bi M, Hardy DM. Heterogeneous processing and zona pellucida binding activity of pig zonadhesion. J. Biol. Chem., 2001, 276(44): 41502-41509.
[107] Lea IA, Sivashanmugam P, O'Rand MG. Zonadhesion: Characterization, localization,and zona pellucida binding. Biol. Reprod., 2001, 65: 1691-1700.
[108] Williams RM, Jones R. Specificity of binding of zona pellucida glycoroteins to sperm proacrosin and related proteins. J. Exp. Zool., 1993, 226(1): 65-73.
[109] Jansen S, Quigley M, Reik W, et al. Analysis of polysulfate-binding domains in porcine proacrosin, a putaive zona adhesin protein from mammalian spermatozoa, Int. J. Dev. Biol., 1995, 39(3): 501-510.
[110] 宛传丹，黄宇烽，许晓风．精子蛋白SP22研究进展．中华男科学杂志，2005，11(1)：56-59．
[111] Hao Z, Wolkowicz MJ, Shetty J, et al. SAMP32, a testis-specific, isoantigenic sperm acrosomal membrane-associated protein. Biol. Reprod., 2002, 66:735-744.
[112] Blobel CP. Proteolytic process of a protein involved in sperm-egg fusion correlates with acqutisition of fertilization competence. J. Cell. Biol., 1999, 111 (1): 69.
[113] 丁之德，吴明章．哺乳类动物精子在附睾内成熟过程中膜蛋白的变化．男性学杂志，1993，7(2)：119-121．
[114] Brown CR, von Glos KI, Jones R. Changes in plasma membrane glycoproteins of rat spermatozoa during maturation in the epididymis. J. Cell Biol., 1983, 96(1): 256-264.
[115] Gaunt SJ. A 28K-dalton cell surface autoantigen of spermatogenesis: characterization using a monoclonal antibody. Dev. Biol., 1982, 89(1): 92-100.
[116] Voglmayr JK, Fairbanks G, Lewis RG. Surface glycoprotein changes in ram spermatozoa during epididymal maturation. Bio. Reprod, 1983, 29(3): 767-775.
[117] Swanson WJ, Vacquier VD. Liposome fusion induced by a Mr. 18,000 protein localized to the acrosomal region of acrosome-reacted abalone spermatozoa. Biochemistry, 1995, 34(43): 14202-14208．
[118] 刘慧慧，李太武，苏秀榕．几种动物精卵识别机制．海洋湖沼通报，2004，1：16-21．
[119] Esponda P, Guerra R. Plasma-membrane glycoproteins changes during sperm iogenesis and in the sperm atozoa of some orthoptera. Cell. Tissue. Res., 1991, 264:507-513.
[120] Long G, Sottile R, Privitera R. Letin binding to the sperm surface before and after insemination in a grasshopper Eyprepocnomicsplorans( charp).Eur Arch. Biol.,1994, 105(1):35-40.
[121] Marti S, Cacho D., Jass A. Plasma membrane glycoproteins of mature and immature drone honey bee spermatozoa:Letin binding as seen by light and electronmicoscope. Theriogenology, 1996, 46(1):181-190.
[122] Cattaneo F, Pasini ME. Glycosidases are present on the surface of Drosophila melanogaster spermatozoa. Mol. Reprod, 1997, 48(2):276-281.
[123] 堵南山．中华绒螯蟹的受精．水产科技情报，1998，25(1)：9-13．
[124] Takizawa S, Sawada H, Someno T, et al. Effects of protease inbibitors on binding of sperm to the vitelline coat of Ascidian eggs: Implications for participation of a proteasome (Muticatalytic proteinase complex). J. Exp. Zool., 1993,267:86-91.
[125] Foltz KR, Lennarz WJ. The molecular basis of sea urchin gamete interactions at the egg plasma membrane. Devel. Biol., 1993, 158(1): 46-61.
[126] 杨万喜，姜乃澄，卢建平．十足类甲壳动物受精细胞学研究进展．东海海洋，1998，4：57-63．
[127] 范桢，周孝瑚，章仁安等．大鼠精子特异的LDH-C4的提纯及其免疫性质分析．上海医科大学学报，1989，16(4)：253-258．
[128] 方丁，房世荣主编．同工酶在医学上的应用．北京：人民卫生出版社，1982，66-94．
[129] Markert CL, Moiler, F. Multiple forms of enzymes: tissue, ontogenetic, and species specific patterns. Proc. Natl. Acad Sci. USA., 1959, 45: 753-763.
[130] 陈明，周念辉，李爱媛等．东方对虾精子中的乳酸脱氢酶C．科学通报，1994，39(24)：2272-2274．
[13I] 曾科文，郑成洋，潘雪峰．水貂组织乳酸脱氢酶同工酶电泳分析．东北林业大学学报，1987，15(6)：28-32．
[132] Blanco A, Zinkham WH. Lactate dehydrogenases in human testes. Science, 1963, 139: 601-602.
[133] 李明．精子乳酸脱氢酶同工酶的研究进展．临床检验杂志，2001，19(6)：375-378．
[134] Goldberg E. Lactic and malic dehydrogenase in human spermatozoa. Science, 1963, 139: 602.
[135] O'Flaherty CM, Beorlegui NB, Beconi MT. Lactate dehydrogenase-C4 is involved in heparin-and NADH-dependent bovine sperm capacitation. Andrologia, 2002, 34: 91-97.
[136] O'Hern PA, Liang ZG, Bambra CS, et al. Colinear synthesis of an antigen-specific B-cell epitope with a 'promiscuous' tetanus toxin T-cell epitope: a synthetic peptide immunocontraceptive. Vaccine, 1997, 15: 1761-1766.
[137] Goldberg E. The ontogeny of sperm-sperific lactate dehydrogenase in mice. J.. Exp. Zool., 1967, 164: 309.
[138] Zinkham WH, Blanco A, Kupchyk L. Lactate dehydrogenase in testis: dissociation and recombination of subunits. Science, 1963, 142: 1303-1304.
[139] Markert CL. Lactate dehydrogenase.: biochemistry and function of lactate dehydrogenase. Cell. Biochem. Funct., 1984, 2(3): 131-134.
[140] Baldwin K, Mortimer AP. Do ascidians possess the ancestral subunit type of vertebrate lactate dehydrogenase? J. Exp. Zoo., 1988, 246(2): 109-114.
[141] 张龙翔，张庭芳，李令媛主编．生化实验方法和技术．北京：高等教育出版社，1997，379-381．
[142] Faulk WP, Taylor GM. An immunocolloid method for the electron microscope, Immunochemistry, 1971, 8(11): 1081-1083.
[143] 堵南山．中华绒螯蟹的同属种类及其英文名称．水产科技情报，1998，25(3)：108-109．
[144] 胡自强，胡运瑾．河蟹生殖系统的形态学和组织结构．湖南师范大学自然科学学报，1997，20(3)：71-76．
[145] 堵南山，薛鲁征，赖伟．中华绒螯蟹(Eriocheir sinensis)雄性生殖系统的组织学研究．动物学报，1988，34(4)：329-335．
[146] 吴萍，楼允东，邱高峰．中华绒螯蟹性腺发育的形态学、组织学和组织化学变化．上海水产大学学报，2003，12(2)：106-112．
[147] 堵南山，赖伟，薛鲁征．中华绒螯蟹(Eriocheir sinensis)精子顶体反应的研究．动物学报，1987，33(1)：8-13．
[148] 王群，赵云龙，陈立侨．中华绒螯蟹雄性生殖系统生化组成及精子代谢．水产学报，2002，26(5)： 411-416．
[149] 康现江，米娅，刘彬彬等．摘除眼柄对中华绒螯蟹精巢发育及其氨基酸含量的影响．台湾海峡，2000，19(3)：360-364．
[150] 吴萍，邱高峰，楼允东．中华绒螯蟹促雄腺结构变化对精子发生的影响．水产学杂志，2002，15(1)：88-92．
[151] 堵南山，薛鲁征，赖伟．中华绒螯蟹精子的研究Ⅱ．精子发生．海洋与湖沼，1988，19(1)：71-78．
[152] 堵南山，赖伟，安婴等．中华绒螯蟹受精的细胞学研究．中国科学(B辑)，1992，3：260-265．
[153] 堵南山．中华绒螯蟹的受精生物学(一)．生物学通报，1998，33(12)：5-8．
[154] 堵南山．中华绒螯蟹的受精生物学(二)．生物学通报，1999，34(1)：10-12．
[155] 堵南山．中华绒螯蟹是体内还是体外受精?水产科技情报，2000，27(5)：217-218．
[156] 程立均．中华绒螯蟹精子膜蛋白的提取分离及部分生化性质研究．河北大学硕士学位论文(单行本)，2004．
[157] 汪家政，范明主编．蛋白质技术手册．北京：科学出版社，2002，42-47．
[158] 芮菊生，杜懋琴，陈海明等编著．组织切片技术．北京：人民教育出版社，1980，225-231．
[159] 北原文雄，玉井康胜，早野茂夫等．表面活性剂．北京：化学工业出版社，1984，529．
[160] 乔新美，曹维孝，邹世平．长江、瓯江中华绒螯蟹几种同工酶的分析比较．淡水渔业，1994，24(5)：10-13．
[161] 许加武，李思发．长江口中华绒螯蟹与其它几种同科蟹的同工酶比较．水产科技情报，1996，23(4)：159-162．
[162] 王丹，于伟君．辽河长江两水系中华绒螯蟹酯酶和乳酸脱氢酶的同工酶比较研究．辽宁大学学报(自然科学版)，1995，22(4)：79-81．
[163] 程家骅，王云龙，许加武等．苏北南部沿海几种蟹类蛋白和同工酶的比较研究．中国水产科学，1998，5(1)：10-17．
[164] 薛俊增．患“抖抖病”中华绒螯蟹可溶性蛋白质和同工酶分析．动物学杂志，2002，37(2)：17-21．
[165] 贾守菊，应雪萍，陈艳乐等．中华绒螯蟹不同发育阶段腹肢粘液腺同工酶的比较．海洋湖沼通报，2004，4：52-60．
[166] 雷焕宗，贾守菊，应雪萍等．中华绒螫蟹不同生理时期肌肉组织中同工酶的分析比较．河南科学，2004，22(4)：480-483．
[167] 王群，赵云龙，陈立侨．中华绒螯蟹雄性生殖系统生化组成及精子代谢．水产学报，2002，26(5)：411-416．
[168] 哈密斯BD，利克伍德D著，刘毓秀，程桂芳译．蛋白质的凝胶电泳——实践方法．北京：科学出版社，1986，159-160．
[169] 汪谦主编．现代医学实验方法．北京：人民卫生出版社，1997，712-715．
[170] 李建武，余瑞元，袁明秀等．生物化学实验原理和方法．北京：北京大学出版社，1994，381-386．