EPPIN基因多态性及低表达对雄性生殖功能的影响
|
摘要
附睾蛋白酶抑制剂EPPIN(Epididymal Protease Inhibitor)作为一种极具发展潜力的男性避孕疫苗候选靶标在近年来引起众多研究者的关注。该蛋白特异性地表达于睾丸和附睾,研究表明它在精子功能以及男性生殖方面发挥着重要作用。本研究拟从分子流行病学和整体动物两个方面探讨EPPIN在雄性生殖中的作用,从而为男性不育的病因学研究提供新的线索,同时从新的角度阐明EPPIN发挥生物学功能的分子机制。
第一部分EPPIN基因多态性与原发性男性不育的相关性研究
研究表明,单核苷酸多态性(Single nucleotide polymorphisms,SNPs)作为基因突变中最为常见的变异类型,对基因的表达和功能产生着重要影响。对于EPPIN基因, NCBI数据库中已列出了其可能存在的众多SNPs(http://www.ncbi.nlm.nih.gov/projects/SNP/),但是,到目前为止,未见有关该基因SNPs与精液质量和男性不育的相关报道。因此,为阐明EPPIN基因多态性与精液质量及男性不育的关系,在本部分研究中,我们首先以473例确诊的男性不育病例为对象,分析了EPPIN基因四个标签SNPs(rs6124715, rs2231829, rs2227290和rs11594)与精液质量的关系。为进一步明确这些SNPs位点与原发性男性不育发病风险之间的关系,我们增加了由198例正常生育男性组成的对照组,分析了这些SNPs在病例组和对照各组间的分布差异并比较了SNPs对各组血清睾酮水平的影响,同时采用生物信息学方法探讨了阳性SNPs发挥生物学功能的可能机制。结果发现,位点rs2231829与精子数量之间以及位点rs6124715、rs11594与精子运动能力之间均存在显著相关性。在SNPs与发病风险的分析中,我们发现多态性位点rs2231829和rs11594的存在能显著改变原发性男性不育的发病风险,并且这种风险在精液质量异常的病例中更加明显,然而,这些标签SNPs各基因型对应的血清睾酮水平没有显著性差异。生物信息学分析发现,位点rs2231829可影响转录因子的结合、而位点rs11594可影响mRNA结构。因此,我们推测EPPIN基因多态性的存在可能通过影响转录因子的结合以及mRNA的结构而影响该基因的生物学功能,进而影响精子成熟过程和生育结局,但是由于EPPIN相关研究资料的有限性,这一假设还有待进一步研究证实。
第二部分EPPIN基因低表达对精子运动功能的影响
已有研究提示我们, EPPIN可能参与了精子发生和精子成熟的过程,但是,到目前为止还缺乏相关研究从整体动物角度对这些假说予以证实。在本部分研究中,我们以小鼠为对象,采用整体动物RNA干扰(RNAi)结合睾丸显微注射以及膜片钳技术,研究EPPIN基因低表达对小鼠精子运动功能的影响,并对其可能的作用机制进行探讨。结果发现,EPPIN基因低表达后小鼠精子运动能力明显下降,主要表现为活力(motility)、平均路径速度(VAP)、直线速度(VSL)和曲线速度(VCL)的显著下降。进一步研究发现,与精子运动能力变化相一致的是,小鼠睾丸生精细胞的T型钙电流也显著下降,T型钙通道的mRNA水平也明显降低。因此可以得出以下结论:EPPIN基因低表达导致睾丸生精细胞T型钙通道mRNA水平的降低和T型钙电流的下降,从而引起精子运动能力的下降。但是,精子运动能力的获得是一个非常复杂的过程,不仅与精子自身结构密切相关,而且精子所处的外环境对其亦有重要影响,虽然钙离子在精子运动中起着关键性作用,但是T型钙通道的改变如何影响精子运动能力还有待进一步研究。
As a potential candidate for male immunocontraception vaccine, EPPIN (Epididymal protease inhibitor) has attracted the attention of many researchers in the recent decade. This protein is expressed specifically in the testis and epididymis. Many studies have demonstrated its importance in sperm function and male fertility. In the present study, we sought to investigate the function of EPPIN in male fertility from two aspects of molecular epidemiology and animal models, which could provide some new clues for the etiology of male infertility and clarify the molecule mechanism of EPPIN’s biological function from new perspective.
Part I The association study between variants in the EPPIN gene and idiopathic male infertility
Single nucleotide polymorphisms (SNPs) represent the most abundant class of inheritable human gene mutations, and have profound influence on gene expression and function. For the EPPIN gene, although a number of SNPs were listed on the website http://www.ncbi.nlm.nih.gov/projects/SNP/, there is no related report to date on the roles of EPPIN genetic variants on semen quality and male infertility in the general male population. Therefore, in order to clarify the association between EPPIN variant and semen quality and male infertility, we first analyzed the association between four EPPIN tagSNPs (rs6124715, rs2231829, rs2227290 and rs11594) and semen quality in 473 males with definite idiopathic infertility. Then, to clarify the association between EPPIN genetic variants and idiopathic male infertility, we expanded the sample capacity by adding a control group consisting of 198 fertile men and analyzed the genotype distribution of each SNP, evaluated the influence of these genetic variants on serum testosterone level. Additionally, we explored that how the positive SNPs play the biological functions by bioinformatics methods. We have demonstrated significant associations between genetic variant rs2231829 and sperm number, and between variants rs6124715 and rs11594 and sperm motility characteristics. In the analysis of SNPs to male infertility risk, we found variants rs2231829 / rs11594 are associated with decreased / increased male infertility risks, while the patients with abnormal semen parameters might be at lower / higher risk. Almost similar serum testosterone levels among different EPPIN genotypes were observed for each group. The bioinformatics analysis found that variant rs2231829 could alter transcription factor binding site, variant rs11594 could change the mRNA structure, and in turn affect the sperm maturation process and male fertility. Whether this indeed is the case requires further investigation.
Part II The down-regulated expression of EPPIN gene on sperm motility
We know from existing research that EPPIN participates in the process of spermatogenesis and sperm maturation, but to date, there was no effective in vitro or animal model to confirm its function in these processes. In the present study, we investigated the effect of down-regulated expression of EPPIN gene to mouse sperm motility in a mouse model by the utilization of RNA interference in vivo with the combination of testis microinjection and patch clamp, and explored the possible mechanism. We found the sperm motility declined significantly when the EPPIN gene was down-regulated, which was mainly manifested as the decline of motility, VAP, VSL and VCL. Further studies demonstrated that, according with the decline of sperm motility, the T-type calcium current and the T-type calcium channel mRNA level of spermatogenic cells also declined significantly. Therefore, we concluded that the low expression of EPPIN gene induced the decline of T-type calcium current and the T-type calcium channel mRNA level, which in turn resulted in the decline of sperm motility. However as we know, the acquisition of sperm motility is an extremely complex process, which is associated with not only the sperm structure but also the external environment. Although the calcium ion plays a key role in sperm motility, we needs further study to elucidate how the alteration of T-type calcium channel affects the sperm motility.
第一部分EPPIN基因多态性与原发性男性不育的相关性研究
研究表明,单核苷酸多态性(Single nucleotide polymorphisms,SNPs)作为基因突变中最为常见的变异类型,对基因的表达和功能产生着重要影响。对于EPPIN基因, NCBI数据库中已列出了其可能存在的众多SNPs(http://www.ncbi.nlm.nih.gov/projects/SNP/),但是,到目前为止,未见有关该基因SNPs与精液质量和男性不育的相关报道。因此,为阐明EPPIN基因多态性与精液质量及男性不育的关系,在本部分研究中,我们首先以473例确诊的男性不育病例为对象,分析了EPPIN基因四个标签SNPs(rs6124715, rs2231829, rs2227290和rs11594)与精液质量的关系。为进一步明确这些SNPs位点与原发性男性不育发病风险之间的关系,我们增加了由198例正常生育男性组成的对照组,分析了这些SNPs在病例组和对照各组间的分布差异并比较了SNPs对各组血清睾酮水平的影响,同时采用生物信息学方法探讨了阳性SNPs发挥生物学功能的可能机制。结果发现,位点rs2231829与精子数量之间以及位点rs6124715、rs11594与精子运动能力之间均存在显著相关性。在SNPs与发病风险的分析中,我们发现多态性位点rs2231829和rs11594的存在能显著改变原发性男性不育的发病风险,并且这种风险在精液质量异常的病例中更加明显,然而,这些标签SNPs各基因型对应的血清睾酮水平没有显著性差异。生物信息学分析发现,位点rs2231829可影响转录因子的结合、而位点rs11594可影响mRNA结构。因此,我们推测EPPIN基因多态性的存在可能通过影响转录因子的结合以及mRNA的结构而影响该基因的生物学功能,进而影响精子成熟过程和生育结局,但是由于EPPIN相关研究资料的有限性,这一假设还有待进一步研究证实。
第二部分EPPIN基因低表达对精子运动功能的影响
已有研究提示我们, EPPIN可能参与了精子发生和精子成熟的过程,但是,到目前为止还缺乏相关研究从整体动物角度对这些假说予以证实。在本部分研究中,我们以小鼠为对象,采用整体动物RNA干扰(RNAi)结合睾丸显微注射以及膜片钳技术,研究EPPIN基因低表达对小鼠精子运动功能的影响,并对其可能的作用机制进行探讨。结果发现,EPPIN基因低表达后小鼠精子运动能力明显下降,主要表现为活力(motility)、平均路径速度(VAP)、直线速度(VSL)和曲线速度(VCL)的显著下降。进一步研究发现,与精子运动能力变化相一致的是,小鼠睾丸生精细胞的T型钙电流也显著下降,T型钙通道的mRNA水平也明显降低。因此可以得出以下结论:EPPIN基因低表达导致睾丸生精细胞T型钙通道mRNA水平的降低和T型钙电流的下降,从而引起精子运动能力的下降。但是,精子运动能力的获得是一个非常复杂的过程,不仅与精子自身结构密切相关,而且精子所处的外环境对其亦有重要影响,虽然钙离子在精子运动中起着关键性作用,但是T型钙通道的改变如何影响精子运动能力还有待进一步研究。
As a potential candidate for male immunocontraception vaccine, EPPIN (Epididymal protease inhibitor) has attracted the attention of many researchers in the recent decade. This protein is expressed specifically in the testis and epididymis. Many studies have demonstrated its importance in sperm function and male fertility. In the present study, we sought to investigate the function of EPPIN in male fertility from two aspects of molecular epidemiology and animal models, which could provide some new clues for the etiology of male infertility and clarify the molecule mechanism of EPPIN’s biological function from new perspective.
Part I The association study between variants in the EPPIN gene and idiopathic male infertility
Single nucleotide polymorphisms (SNPs) represent the most abundant class of inheritable human gene mutations, and have profound influence on gene expression and function. For the EPPIN gene, although a number of SNPs were listed on the website http://www.ncbi.nlm.nih.gov/projects/SNP/, there is no related report to date on the roles of EPPIN genetic variants on semen quality and male infertility in the general male population. Therefore, in order to clarify the association between EPPIN variant and semen quality and male infertility, we first analyzed the association between four EPPIN tagSNPs (rs6124715, rs2231829, rs2227290 and rs11594) and semen quality in 473 males with definite idiopathic infertility. Then, to clarify the association between EPPIN genetic variants and idiopathic male infertility, we expanded the sample capacity by adding a control group consisting of 198 fertile men and analyzed the genotype distribution of each SNP, evaluated the influence of these genetic variants on serum testosterone level. Additionally, we explored that how the positive SNPs play the biological functions by bioinformatics methods. We have demonstrated significant associations between genetic variant rs2231829 and sperm number, and between variants rs6124715 and rs11594 and sperm motility characteristics. In the analysis of SNPs to male infertility risk, we found variants rs2231829 / rs11594 are associated with decreased / increased male infertility risks, while the patients with abnormal semen parameters might be at lower / higher risk. Almost similar serum testosterone levels among different EPPIN genotypes were observed for each group. The bioinformatics analysis found that variant rs2231829 could alter transcription factor binding site, variant rs11594 could change the mRNA structure, and in turn affect the sperm maturation process and male fertility. Whether this indeed is the case requires further investigation.
Part II The down-regulated expression of EPPIN gene on sperm motility
We know from existing research that EPPIN participates in the process of spermatogenesis and sperm maturation, but to date, there was no effective in vitro or animal model to confirm its function in these processes. In the present study, we investigated the effect of down-regulated expression of EPPIN gene to mouse sperm motility in a mouse model by the utilization of RNA interference in vivo with the combination of testis microinjection and patch clamp, and explored the possible mechanism. We found the sperm motility declined significantly when the EPPIN gene was down-regulated, which was mainly manifested as the decline of motility, VAP, VSL and VCL. Further studies demonstrated that, according with the decline of sperm motility, the T-type calcium current and the T-type calcium channel mRNA level of spermatogenic cells also declined significantly. Therefore, we concluded that the low expression of EPPIN gene induced the decline of T-type calcium current and the T-type calcium channel mRNA level, which in turn resulted in the decline of sperm motility. However as we know, the acquisition of sperm motility is an extremely complex process, which is associated with not only the sperm structure but also the external environment. Although the calcium ion plays a key role in sperm motility, we needs further study to elucidate how the alteration of T-type calcium channel affects the sperm motility.
引文
[1] Richardson RT, Sivashanmugam P, Hall SH, Hamil KG, Moore PA, Ruben SM, French FS, O'Rand M. Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis. Gene, 2001, 270(1-2):93-102.
[2] Wang Z, Widgren EE, Sivashanmugam P, O'Rand MG, Richardson RT. Association of eppin with semenogelin on human spermatozoa. Biology of reproduction, 2005, 72(5):1064-70.
[3] Sivashanmugam P, Hall SH, Hamil KG, French FS, O'Rand MG, Richardson RT. Characterization of mouse Eppin and a gene cluster of similar protease inhibitors on mouse chromosome 2. Gene, 2003, 312:125-34.
[4] O'Rand MG, Widgren EE, Wang Z, Richardson RT. Eppin: an effective target for male contraception. Molecular and Cellular Endocrinology, 2006, 250(1-2):157-62.
[5] Wang Z, Widgren EE, Richardson RT, O'Rand MG. Characterization of an eppin protein complex from human semen and spermatozoa. Biology of reproduction, 2007, 77(3):476-84.
[6] O'rand MG, Widgren EE, Sivashanmugam P, Richardson RT, Hall SH, French FS, VandeVoort CA, Ramachandra SG, Ramesh V, Jagannadha Rao A. Reversible immunocontraception in male monkeys immunized with eppin. Science, 2004, 306(5699):1189-90.
[7] DubéE, Hermo L, Chan PT, Cyr DG. Alterations in gene expression in the caput epididymides of nonobstructive azoospermic men. Biol Reprod, 2008, 78:342-351.
[8] Robert M, Gagnon C. Semenogelin I: a coagulum forming, multifunctional seminal vesicle protein. Cell Mol Life Sci, 1999, 55:944–60.
[9] Mitra A, Richardson RT and O’Rand MG. Analysis of Recombinant Human Semenogelin as an Inhibitor of Human Sperm Motility. Biol Reprod, 2009, DOI:10.1095/biolreprod.109.081331
[10] O’Rand MG, Widgren EE, Beyler S and Richardson RT. Inhibition of human sperm motility by contraceptive anti-eppin antibodies from infertile male monkeys: effect on cyclic adenosine monophosphate. Biol Reprod, 2009, 80:279-85.
[11] Denolet E, De Gendt K, Allemeersch J, Engelen K, Marchal K, Van Hummelen P, Tan KA, Sharpe RM, Saunders PT, Swinnen JV, Verhoeven G. The effect of a sertoli cell-selective knockout of the androgen receptor on testicular gene expression in prepubertal mice. Mol Endocrinol, 2006, 20(2):321-34.
[12] Willems A, De Gendt K, Allemeersch J, Smith LB, Welsh M, Swinnen JV, Verhoeven G.. Early effects of Sertoli cell-selective androgen receptor ablation on testicular gene expression. Int J Androl. 2009 Apr 12. [Epub ahead of print].
[13] Lim P, Robson M, Spaliviero J, McTavish KJ, Jimenez M, Zajac JD, Handelsman DJ, Allan CM. Sertoli cell androgen receptor DNA binding domain is essential for the completion of spermatogenesis. Endocrinology, 2009, 150(10):4755-65.
[14] De Gendt K, McKinnell C, Willems A, Saunders PT, Sharpe RM, Atanassova N, Swinnen JV, Verhoeven G. Organotypic cultures of prepubertal mouse testes: a method to study androgen action in sertoli cells while preserving their natural environment. Biol Reprod, 2009, 81(6):1083-92.
[15] Schauwaers K, De Gendt K, Saunders PT, Atanassova N, Haelens A, Callewaert L, Moehren U, Swinnen JV, Verhoeven G, Verrijdt G, Claessens F. Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model. Proc Natl Acad Sci U S A, 2007, 104(12):4961-6.
[16] Yenugu S, Richardson RT, Sivashanmugam P, Wang Z, O'rand MG, French FS, Hall SH. Antimicrobial activity of human EPPIN, an androgen-regulated, sperm-bound protein with a whey acidic protein motif. Biology of reproduction, 2004, 71(5):1484-90.
[17] Krausz C, Forti G. Clinical aspects of male infertility. In: The Genetic Basis of Male Infertility, (ed McElreavey, K.), Springer-Verlag, Berlin, Heidelberg, Germany. 2000, pp. 1-21.
[18] Thonneau P, Marchand S, Tallec A, Ferial ML, Ducot B, Lansac J, Lopes P, Tabaste JM, Spira A. Incidence and main causes of infertility in a resident population (1,850,000) of three French regions (1988-1989). Hum Reprod, 1991, 6(6):811-6
[19] Skakkebaek NE, Giwercman A, de Kretser D. Pathogenesis and management of male infertility. Lancet, 1994, 343:1473-79.
[20] Huynh T, Mollard R, Trounson A. Selected genetic factors associated with male infertility. Human Reproduction Update, 2002, 8:183-98.
[21] Jamsai D, Reilly A, Smith SJ, Gibbs GM, Baker HW, McLachlan RI, de Kretser DM, O'Bryan MK. Polymorphisms in the human cysteine-rich secretory protein 2 (CRISP2) gene in Australian men. Hum Reprod, 2008, 23(9):2151-9.
[22] Guo Y, Jamison DC. The distribution of SNPs in human gene regulatory regions. BMC Genomics, 2005, 6:140.
[23] Cram DS, O’Bryan MK, de Kretser DM. Male infertility genetics-the future. Journal of Andrology, 2001, 22:738-46.
[24] Ferlin A, Raicu F, Gatta V, Zuccarello D, Palka G, Foresta C. Male infertility: role of genetic background. Reprod Biomed Online, 2007, 14(6):734-45.
[25] Shi YY and He L. SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci. Cell Res, 2005, 15:97-98.
[26] World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 4th ed, 1999.Cambridge University Press, Cambridge, UK.
[27] Sherins, R.J. Are semen quality and male fertility changing? N. Engl. J. Med., 1995, 332:327-28.
[28] Akre, O., Cnattingius, S., Bergstrom, R., Kvist, U., Trichopoulos, D., Ekbom, A. Human fertility does not decline: evidence from Sweden. Fertil. Steril, 1999, 71: 1066-69.
[29] Guzick DS, Overstreet JW, Factor-Litvak P. Sperm morphology, motility, and concentration in fertile and infertile men. N Engl J Med, 2001, 345:1388–93.
[30] Skakkeb?k NE, Rajpert-De Meyts E, Main KM. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod, 2001, 16:972–78.
[31] Stouffs K, Tournaye H, Liebaers I, Lissens W. Male infertility and theinvolvement of the X chromosome. Hum Reprod Update, 2009, 15(6):623-37.
[32] Le Magueresse-Battistoni B. Serine proteases and serine protease inhibitors in testicular physiology: the plasminogen activation system. Reproduction, 2007, 134: 721-29.
[33] Monsees TK, Schill WB, Miska W. Protease-protease inhibitor interactions in Sertoli cell-germ cell crosstalk. Advances in Experimental Medicine & Biology, 1997, 424:111-23.
[34] Sripriya S, Nirmaladevi J, George R, Hemamalini A, Baskaran M, Prema R, Ve Ramesh S, Karthiyayini T, Amali J, Job S et al. OPTN gene: profile of patients with glaucoma from India. Mol Vis, 2006, 12:816-20.
[35] Verrijzer CP and Van der Vliet PC. POU domain transcription factors. Biochim Biophys Acta, 1993, 1173:1-21.
[36] Ryan AK and Rosenfeld MG. POU domain family values: flexibility, partnerships, and developmental codes. Genes Dev, 1997, 11:1207-25.
[37] Gronostajski RM. Roles of the NFI/CTF gene family in transcription and development. Gene, 2000, 249:31-45.
[38] Gruber AR, Lorenz R, Bernhart SH, Neub?ck R and Hofacker IL. The Vienna RNA websuite. Nucleic Acids Res, 2008, 36 (Web Server issue):W70-74.
[39] Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature, 1985, 317(6034):230-4.
[40]刘建忠,敖敬,田为宇,马季骅.基因敲除技术研究进展.化学与生物工程,2006, 23(2):4-6.
[41] Lu PY, Xie F, Woodle MC. In vivo application of RNA interference: from functional genomics to therapeutics. Adv Genet, 2005, 54:117–42.
[42] Robert M, Gagnon C. Sperm motility inhibitor from human seminal plasma: association with semen coagulum. Human reproduction, 1995, 10:2192–97.
[43] Robert M, Gagnon C. Purification and characterization of the active precursor of a human sperm motility inhibitor secreted by the seminal vesicles: identity with semenogelin. Biology of reproduction, 1996, 55:813–21.
[44] Bourgeon F, Evrard B, Brillard-Bourdet M, Colleu D, Jégou B, Pineau C. Involvement of semenogelin-derived peptides in the antibacterial activity of human seminal plasma. Biology of reproduction, 2004, 70(3):768-74.
[45] Trevi?o CL, Felix R, Castellano LE, Gutiérrez C, Rodríguez D, Pacheco J, López-González I, Gomora JC, Tsutsumi V, Hernández-Cruz A, Fiordelisio T, Scaling AL, Darszon A. Expression and differential cell distribution of low-threshold Ca channels in mammalian male germ cells and sperm. FEBS Lett, 2004, 563:87-92. 2+
[46] Kirichok Y, Navarro B, Clapham DE. Whole-cell patch-clamp measurements of spermatozoa reveal an alkaline-activated Ca2 + channel. Nature, 2006, 439: 737-40.
[47] Darszon A, Labarca P, Nishigaki T, Espinosa F. Ion channels in sperm physiology. Physiological Reviews, 1999, 79: 481-510.
[1] Bongaarts, J. and Watkins, S. C. Social interactions and contemporary fertility transitions. Population and Development Review, 1996, 22: 639- 82.
[2] Bongaarts, J. The role of family planning programmes in contemporary fertility transitions. In: The Continuing Demographic Transition, Edited by Jones, G. W., Douglas, R. M., Caldwell, J. C. and D’Souza, R. M. 1997, Oxford: Clarendon Press, pp. 422–43.
[3] Blithe D. Male contraception: what is on the horizon? Contraception, 2008, 78:S23-7.
[4] Fu H, Darroch JE, Haas T, Ranjit N. Contraceptive failure rates: new estimates from the 1995 National Survey of Family Growth. Fam Plann Perspect, 1999, 31:56–63.
[5] Schlegel PN, Goldstein M. Vasectomy. In: Sharpe D, Haseltine FP, eds. Contraception. 1993, New York: Springer-Verlag; 181–191.
[6] Ansbacher R. Sperm-agglutinating and sperm-immobilizing antibodies in vasectomized men. Fertil Steril 1971, 22:629–32.
[7] Hoesl CE, Saad F, P?ppel M, Altwein JE. Reversible, non-barrier male contraception: status and prospects. Eur Urol, 2005, 48(5):712-22; discussion 722-3.
[8] Richardson RT, Sivashanmugam P, Hall SH, Hamil KG, Moore PA, Ruben SM, French FS, O'Rand M. Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis. Gene, 2001, 270(1-2):93-102.
[9] Wang Z, Widgren EE, Sivashanmugam P, O'Rand MG, Richardson RT. Association of eppin with semenogelin on human spermatozoa. Biology of reproduction, 2005, 72(5):1064-70.
[10] Sivashanmugam P, Hall SH, Hamil KG, French FS, O'Rand MG, Richardson RT. Characterization of mouse Eppin and a gene cluster of similar protease inhibitors on mouse chromosome 2. Gene, 2003, 312:125-34.
[11] O'Rand MG, Widgren EE, Wang Z, Richardson RT. Eppin: an effective target for male contraception. Molecular and Cellular Endocrinology, 2006, 250(1-2):157-62.
[12] Wang Z, Widgren EE, Richardson RT, O'Rand MG. Characterization of an eppin protein complex from human semen and spermatozoa. Biology of reproduction, 2007, 77(3):476-84.
[13] O'rand MG, Widgren EE, Sivashanmugam P, Richardson RT, Hall SH, French FS, VandeVoort CA, Ramachandra SG, Ramesh V, Jagannadha Rao A. Reversible immunocontraception in male monkeys immunized with eppin. Science, 2004, 306(5699):1189-90.
[14] DubéE, Hermo L, Chan PT, Cyr DG. Alterations in gene expression in the caput epididymides of nonobstructive azoospermic men. Biol Reprod 2008;78:342-351.
[15] Robert M, Gagnon C. Semenogelin I: a coagulum forming, multifunctional seminal vesicle protein. Cell Mol Life Sci 1999;55:944–960.
[16] Mitra A, Richardson RT and O’Rand MG. Analysis of Recombinant Human Semenogelin as an Inhibitor of Human Sperm Motility. Biol Reprod 2009; DOI:10.1095/biolreprod.109.081331.
[17] O’Rand MG, Widgren EE, Beyler S and Richardson RT. Inhibition of human sperm motility by contraceptive anti-eppin antibodies from infertile male monkeys: effect on cyclic adenosine monophosphate. Biol Reprod 2009;80:279-285.
[18] Zhang J, Ding X, Bian Z, Xia Y, Lu C, Wang S, Song L, Wang X. The effect of anti-eppin antibodies on ionophore A23187-induced calcium influx and acrosome reaction of human spermatozoa. Hum Reprod. 2010, 25(1):29-36.
[19] Denolet E, De Gendt K, Allemeersch J, Engelen K, Marchal K, Van Hummelen P, Tan KA, Sharpe RM, Saunders PT, Swinnen JV, Verhoeven G. The effect of a sertoli cell-selective knockout of the androgen receptor on testicular gene expression in prepubertal mice. Mol Endocrinol. 2006;20(2):321-34.
[20] Willems A, De Gendt K, Allemeersch J, Smith LB, Welsh M, Swinnen JV, Verhoeven G.. Early effects of Sertoli cell-selective androgen receptor ablation on testicular gene expression. Int J Androl. 2009 Apr 12. [Epub ahead of print].
[21] Lim P, Robson M, Spaliviero J, McTavish KJ, Jimenez M, Zajac JD, Handelsman DJ, Allan CM. Sertoli cell androgen receptor DNA binding domain is essential for the completion of spermatogenesis. Endocrinology.2009;150(10):4755-65.
[22] De Gendt K, McKinnell C, Willems A, Saunders PT, Sharpe RM, Atanassova N, Swinnen JV, Verhoeven G. Organotypic cultures of prepubertal mouse testes: a method to study androgen action in sertoli cells while preserving their natural environment. Biol Reprod. 2009; 81(6):1083-92.
[23] Schauwaers K, De Gendt K, Saunders PT, Atanassova N, Haelens A, Callewaert L, Moehren U, Swinnen JV, Verhoeven G, Verrijdt G, Claessens F. Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model. Proc Natl Acad Sci U S A. 2007;104(12):4961-6.
[24] Yenugu S, Richardson RT, Sivashanmugam P, Wang Z, O'rand MG, French FS, Hall SH. Antimicrobial activity of human EPPIN, an androgen-regulated, sperm-bound protein with a whey acidic protein motif. Biology of reproduction, 2004, 71(5):1484-90.
[25] McCrudden MT, Dafforn TR, Houston DF, Turkington PT, Timson DJ. Functional domains of the human epididymal protease inhibitor, eppin. The FEBS journal, 2008, 275(8):1742-50.
[26] Alexander NJ. Reprod Immunology Bronson RA(ed) 1996, Blackwell Science Inc Boston pp640-646.
[27]李彦锋,何畏,靳风烁.避孕疫苗研究现状.国外医学泌尿系统分册,2001,21(3):118-121.
[2] Wang Z, Widgren EE, Sivashanmugam P, O'Rand MG, Richardson RT. Association of eppin with semenogelin on human spermatozoa. Biology of reproduction, 2005, 72(5):1064-70.
[3] Sivashanmugam P, Hall SH, Hamil KG, French FS, O'Rand MG, Richardson RT. Characterization of mouse Eppin and a gene cluster of similar protease inhibitors on mouse chromosome 2. Gene, 2003, 312:125-34.
[4] O'Rand MG, Widgren EE, Wang Z, Richardson RT. Eppin: an effective target for male contraception. Molecular and Cellular Endocrinology, 2006, 250(1-2):157-62.
[5] Wang Z, Widgren EE, Richardson RT, O'Rand MG. Characterization of an eppin protein complex from human semen and spermatozoa. Biology of reproduction, 2007, 77(3):476-84.
[6] O'rand MG, Widgren EE, Sivashanmugam P, Richardson RT, Hall SH, French FS, VandeVoort CA, Ramachandra SG, Ramesh V, Jagannadha Rao A. Reversible immunocontraception in male monkeys immunized with eppin. Science, 2004, 306(5699):1189-90.
[7] DubéE, Hermo L, Chan PT, Cyr DG. Alterations in gene expression in the caput epididymides of nonobstructive azoospermic men. Biol Reprod, 2008, 78:342-351.
[8] Robert M, Gagnon C. Semenogelin I: a coagulum forming, multifunctional seminal vesicle protein. Cell Mol Life Sci, 1999, 55:944–60.
[9] Mitra A, Richardson RT and O’Rand MG. Analysis of Recombinant Human Semenogelin as an Inhibitor of Human Sperm Motility. Biol Reprod, 2009, DOI:10.1095/biolreprod.109.081331
[10] O’Rand MG, Widgren EE, Beyler S and Richardson RT. Inhibition of human sperm motility by contraceptive anti-eppin antibodies from infertile male monkeys: effect on cyclic adenosine monophosphate. Biol Reprod, 2009, 80:279-85.
[11] Denolet E, De Gendt K, Allemeersch J, Engelen K, Marchal K, Van Hummelen P, Tan KA, Sharpe RM, Saunders PT, Swinnen JV, Verhoeven G. The effect of a sertoli cell-selective knockout of the androgen receptor on testicular gene expression in prepubertal mice. Mol Endocrinol, 2006, 20(2):321-34.
[12] Willems A, De Gendt K, Allemeersch J, Smith LB, Welsh M, Swinnen JV, Verhoeven G.. Early effects of Sertoli cell-selective androgen receptor ablation on testicular gene expression. Int J Androl. 2009 Apr 12. [Epub ahead of print].
[13] Lim P, Robson M, Spaliviero J, McTavish KJ, Jimenez M, Zajac JD, Handelsman DJ, Allan CM. Sertoli cell androgen receptor DNA binding domain is essential for the completion of spermatogenesis. Endocrinology, 2009, 150(10):4755-65.
[14] De Gendt K, McKinnell C, Willems A, Saunders PT, Sharpe RM, Atanassova N, Swinnen JV, Verhoeven G. Organotypic cultures of prepubertal mouse testes: a method to study androgen action in sertoli cells while preserving their natural environment. Biol Reprod, 2009, 81(6):1083-92.
[15] Schauwaers K, De Gendt K, Saunders PT, Atanassova N, Haelens A, Callewaert L, Moehren U, Swinnen JV, Verhoeven G, Verrijdt G, Claessens F. Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model. Proc Natl Acad Sci U S A, 2007, 104(12):4961-6.
[16] Yenugu S, Richardson RT, Sivashanmugam P, Wang Z, O'rand MG, French FS, Hall SH. Antimicrobial activity of human EPPIN, an androgen-regulated, sperm-bound protein with a whey acidic protein motif. Biology of reproduction, 2004, 71(5):1484-90.
[17] Krausz C, Forti G. Clinical aspects of male infertility. In: The Genetic Basis of Male Infertility, (ed McElreavey, K.), Springer-Verlag, Berlin, Heidelberg, Germany. 2000, pp. 1-21.
[18] Thonneau P, Marchand S, Tallec A, Ferial ML, Ducot B, Lansac J, Lopes P, Tabaste JM, Spira A. Incidence and main causes of infertility in a resident population (1,850,000) of three French regions (1988-1989). Hum Reprod, 1991, 6(6):811-6
[19] Skakkebaek NE, Giwercman A, de Kretser D. Pathogenesis and management of male infertility. Lancet, 1994, 343:1473-79.
[20] Huynh T, Mollard R, Trounson A. Selected genetic factors associated with male infertility. Human Reproduction Update, 2002, 8:183-98.
[21] Jamsai D, Reilly A, Smith SJ, Gibbs GM, Baker HW, McLachlan RI, de Kretser DM, O'Bryan MK. Polymorphisms in the human cysteine-rich secretory protein 2 (CRISP2) gene in Australian men. Hum Reprod, 2008, 23(9):2151-9.
[22] Guo Y, Jamison DC. The distribution of SNPs in human gene regulatory regions. BMC Genomics, 2005, 6:140.
[23] Cram DS, O’Bryan MK, de Kretser DM. Male infertility genetics-the future. Journal of Andrology, 2001, 22:738-46.
[24] Ferlin A, Raicu F, Gatta V, Zuccarello D, Palka G, Foresta C. Male infertility: role of genetic background. Reprod Biomed Online, 2007, 14(6):734-45.
[25] Shi YY and He L. SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci. Cell Res, 2005, 15:97-98.
[26] World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 4th ed, 1999.Cambridge University Press, Cambridge, UK.
[27] Sherins, R.J. Are semen quality and male fertility changing? N. Engl. J. Med., 1995, 332:327-28.
[28] Akre, O., Cnattingius, S., Bergstrom, R., Kvist, U., Trichopoulos, D., Ekbom, A. Human fertility does not decline: evidence from Sweden. Fertil. Steril, 1999, 71: 1066-69.
[29] Guzick DS, Overstreet JW, Factor-Litvak P. Sperm morphology, motility, and concentration in fertile and infertile men. N Engl J Med, 2001, 345:1388–93.
[30] Skakkeb?k NE, Rajpert-De Meyts E, Main KM. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod, 2001, 16:972–78.
[31] Stouffs K, Tournaye H, Liebaers I, Lissens W. Male infertility and theinvolvement of the X chromosome. Hum Reprod Update, 2009, 15(6):623-37.
[32] Le Magueresse-Battistoni B. Serine proteases and serine protease inhibitors in testicular physiology: the plasminogen activation system. Reproduction, 2007, 134: 721-29.
[33] Monsees TK, Schill WB, Miska W. Protease-protease inhibitor interactions in Sertoli cell-germ cell crosstalk. Advances in Experimental Medicine & Biology, 1997, 424:111-23.
[34] Sripriya S, Nirmaladevi J, George R, Hemamalini A, Baskaran M, Prema R, Ve Ramesh S, Karthiyayini T, Amali J, Job S et al. OPTN gene: profile of patients with glaucoma from India. Mol Vis, 2006, 12:816-20.
[35] Verrijzer CP and Van der Vliet PC. POU domain transcription factors. Biochim Biophys Acta, 1993, 1173:1-21.
[36] Ryan AK and Rosenfeld MG. POU domain family values: flexibility, partnerships, and developmental codes. Genes Dev, 1997, 11:1207-25.
[37] Gronostajski RM. Roles of the NFI/CTF gene family in transcription and development. Gene, 2000, 249:31-45.
[38] Gruber AR, Lorenz R, Bernhart SH, Neub?ck R and Hofacker IL. The Vienna RNA websuite. Nucleic Acids Res, 2008, 36 (Web Server issue):W70-74.
[39] Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature, 1985, 317(6034):230-4.
[40]刘建忠,敖敬,田为宇,马季骅.基因敲除技术研究进展.化学与生物工程,2006, 23(2):4-6.
[41] Lu PY, Xie F, Woodle MC. In vivo application of RNA interference: from functional genomics to therapeutics. Adv Genet, 2005, 54:117–42.
[42] Robert M, Gagnon C. Sperm motility inhibitor from human seminal plasma: association with semen coagulum. Human reproduction, 1995, 10:2192–97.
[43] Robert M, Gagnon C. Purification and characterization of the active precursor of a human sperm motility inhibitor secreted by the seminal vesicles: identity with semenogelin. Biology of reproduction, 1996, 55:813–21.
[44] Bourgeon F, Evrard B, Brillard-Bourdet M, Colleu D, Jégou B, Pineau C. Involvement of semenogelin-derived peptides in the antibacterial activity of human seminal plasma. Biology of reproduction, 2004, 70(3):768-74.
[45] Trevi?o CL, Felix R, Castellano LE, Gutiérrez C, Rodríguez D, Pacheco J, López-González I, Gomora JC, Tsutsumi V, Hernández-Cruz A, Fiordelisio T, Scaling AL, Darszon A. Expression and differential cell distribution of low-threshold Ca channels in mammalian male germ cells and sperm. FEBS Lett, 2004, 563:87-92. 2+
[46] Kirichok Y, Navarro B, Clapham DE. Whole-cell patch-clamp measurements of spermatozoa reveal an alkaline-activated Ca2 + channel. Nature, 2006, 439: 737-40.
[47] Darszon A, Labarca P, Nishigaki T, Espinosa F. Ion channels in sperm physiology. Physiological Reviews, 1999, 79: 481-510.
[1] Bongaarts, J. and Watkins, S. C. Social interactions and contemporary fertility transitions. Population and Development Review, 1996, 22: 639- 82.
[2] Bongaarts, J. The role of family planning programmes in contemporary fertility transitions. In: The Continuing Demographic Transition, Edited by Jones, G. W., Douglas, R. M., Caldwell, J. C. and D’Souza, R. M. 1997, Oxford: Clarendon Press, pp. 422–43.
[3] Blithe D. Male contraception: what is on the horizon? Contraception, 2008, 78:S23-7.
[4] Fu H, Darroch JE, Haas T, Ranjit N. Contraceptive failure rates: new estimates from the 1995 National Survey of Family Growth. Fam Plann Perspect, 1999, 31:56–63.
[5] Schlegel PN, Goldstein M. Vasectomy. In: Sharpe D, Haseltine FP, eds. Contraception. 1993, New York: Springer-Verlag; 181–191.
[6] Ansbacher R. Sperm-agglutinating and sperm-immobilizing antibodies in vasectomized men. Fertil Steril 1971, 22:629–32.
[7] Hoesl CE, Saad F, P?ppel M, Altwein JE. Reversible, non-barrier male contraception: status and prospects. Eur Urol, 2005, 48(5):712-22; discussion 722-3.
[8] Richardson RT, Sivashanmugam P, Hall SH, Hamil KG, Moore PA, Ruben SM, French FS, O'Rand M. Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis. Gene, 2001, 270(1-2):93-102.
[9] Wang Z, Widgren EE, Sivashanmugam P, O'Rand MG, Richardson RT. Association of eppin with semenogelin on human spermatozoa. Biology of reproduction, 2005, 72(5):1064-70.
[10] Sivashanmugam P, Hall SH, Hamil KG, French FS, O'Rand MG, Richardson RT. Characterization of mouse Eppin and a gene cluster of similar protease inhibitors on mouse chromosome 2. Gene, 2003, 312:125-34.
[11] O'Rand MG, Widgren EE, Wang Z, Richardson RT. Eppin: an effective target for male contraception. Molecular and Cellular Endocrinology, 2006, 250(1-2):157-62.
[12] Wang Z, Widgren EE, Richardson RT, O'Rand MG. Characterization of an eppin protein complex from human semen and spermatozoa. Biology of reproduction, 2007, 77(3):476-84.
[13] O'rand MG, Widgren EE, Sivashanmugam P, Richardson RT, Hall SH, French FS, VandeVoort CA, Ramachandra SG, Ramesh V, Jagannadha Rao A. Reversible immunocontraception in male monkeys immunized with eppin. Science, 2004, 306(5699):1189-90.
[14] DubéE, Hermo L, Chan PT, Cyr DG. Alterations in gene expression in the caput epididymides of nonobstructive azoospermic men. Biol Reprod 2008;78:342-351.
[15] Robert M, Gagnon C. Semenogelin I: a coagulum forming, multifunctional seminal vesicle protein. Cell Mol Life Sci 1999;55:944–960.
[16] Mitra A, Richardson RT and O’Rand MG. Analysis of Recombinant Human Semenogelin as an Inhibitor of Human Sperm Motility. Biol Reprod 2009; DOI:10.1095/biolreprod.109.081331.
[17] O’Rand MG, Widgren EE, Beyler S and Richardson RT. Inhibition of human sperm motility by contraceptive anti-eppin antibodies from infertile male monkeys: effect on cyclic adenosine monophosphate. Biol Reprod 2009;80:279-285.
[18] Zhang J, Ding X, Bian Z, Xia Y, Lu C, Wang S, Song L, Wang X. The effect of anti-eppin antibodies on ionophore A23187-induced calcium influx and acrosome reaction of human spermatozoa. Hum Reprod. 2010, 25(1):29-36.
[19] Denolet E, De Gendt K, Allemeersch J, Engelen K, Marchal K, Van Hummelen P, Tan KA, Sharpe RM, Saunders PT, Swinnen JV, Verhoeven G. The effect of a sertoli cell-selective knockout of the androgen receptor on testicular gene expression in prepubertal mice. Mol Endocrinol. 2006;20(2):321-34.
[20] Willems A, De Gendt K, Allemeersch J, Smith LB, Welsh M, Swinnen JV, Verhoeven G.. Early effects of Sertoli cell-selective androgen receptor ablation on testicular gene expression. Int J Androl. 2009 Apr 12. [Epub ahead of print].
[21] Lim P, Robson M, Spaliviero J, McTavish KJ, Jimenez M, Zajac JD, Handelsman DJ, Allan CM. Sertoli cell androgen receptor DNA binding domain is essential for the completion of spermatogenesis. Endocrinology.2009;150(10):4755-65.
[22] De Gendt K, McKinnell C, Willems A, Saunders PT, Sharpe RM, Atanassova N, Swinnen JV, Verhoeven G. Organotypic cultures of prepubertal mouse testes: a method to study androgen action in sertoli cells while preserving their natural environment. Biol Reprod. 2009; 81(6):1083-92.
[23] Schauwaers K, De Gendt K, Saunders PT, Atanassova N, Haelens A, Callewaert L, Moehren U, Swinnen JV, Verhoeven G, Verrijdt G, Claessens F. Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model. Proc Natl Acad Sci U S A. 2007;104(12):4961-6.
[24] Yenugu S, Richardson RT, Sivashanmugam P, Wang Z, O'rand MG, French FS, Hall SH. Antimicrobial activity of human EPPIN, an androgen-regulated, sperm-bound protein with a whey acidic protein motif. Biology of reproduction, 2004, 71(5):1484-90.
[25] McCrudden MT, Dafforn TR, Houston DF, Turkington PT, Timson DJ. Functional domains of the human epididymal protease inhibitor, eppin. The FEBS journal, 2008, 275(8):1742-50.
[26] Alexander NJ. Reprod Immunology Bronson RA(ed) 1996, Blackwell Science Inc Boston pp640-646.
[27]李彦锋,何畏,靳风烁.避孕疫苗研究现状.国外医学泌尿系统分册,2001,21(3):118-121.