Systematic Mapping and Functional Analysis of a Family of Human Epididymal Secretory Sperm-Located Proteins

Abstract

The mammalian spermatozoon has many cellular compartments, such as head and tail, permitting it to interact with the female reproductive tract and fertilize the egg. It acquires this fertilizing potential during transit through the epididymis, which secretes proteins that coat different sperm domains. Optimal levels of these proteins provide the spermatozoon with its ability to move to, bind to, fuse with, and penetrate the egg; otherwise male infertility results. As few human epididymal proteins have been characterized, this work was performed to generate a database of human epididymal sperm-located proteins involved in maturation. Two-dimensional gel electrophoresis of epididymal tissue and luminal fluid proteins, followed by identification using MALDI-TOF/MS or MALDI-TOF/TOF, revealed over a thousand spots in gels comprising 745 abundant nonstructural proteins, 408 in luminal fluids, of which 207 were present on spermatozoa. Antibodies raised to 619 recombinant or synthetic peptides, used in Western blots, histological sections, and washed sperm preparations to confirm antibody quality and protein expression, indicated their regional location in the epididymal epithelium and highly specific locations on washed functional spermatozoa. Sperm function tests suggested the role of some proteins in motility and protection against oxidative attack. A large database of these proteins, characterized by size, pI, chromosomal location, and function, was given a unified terminology reflecting their sperm domain location. These novel, secreted human epididymal proteins are potential targets for a posttesticular contraceptive acting to provide rapid, reversible, functional sterility in men and they are also biomarkers that could be used in noninvasive assessments of male fertility. The mammalian spermatozoon has many cellular compartments, such as head and tail, permitting it to interact with the female reproductive tract and fertilize the egg. It acquires this fertilizing potential during transit through the epididymis, which secretes proteins that coat different sperm domains. Optimal levels of these proteins provide the spermatozoon with its ability to move to, bind to, fuse with, and penetrate the egg; otherwise male infertility results. As few human epididymal proteins have been characterized, this work was performed to generate a database of human epididymal sperm-located proteins involved in maturation. Two-dimensional gel electrophoresis of epididymal tissue and luminal fluid proteins, followed by identification using MALDI-TOF/MS or MALDI-TOF/TOF, revealed over a thousand spots in gels comprising 745 abundant nonstructural proteins, 408 in luminal fluids, of which 207 were present on spermatozoa. Antibodies raised to 619 recombinant or synthetic peptides, used in Western blots, histological sections, and washed sperm preparations to confirm antibody quality and protein expression, indicated their regional location in the epididymal epithelium and highly specific locations on washed functional spermatozoa. Sperm function tests suggested the role of some proteins in motility and protection against oxidative attack. A large database of these proteins, characterized by size, pI, chromosomal location, and function, was given a unified terminology reflecting their sperm domain location. These novel, secreted human epididymal proteins are potential targets for a posttesticular contraceptive acting to provide rapid, reversible, functional sterility in men and they are also biomarkers that could be used in noninvasive assessments of male fertility. There are two major challenges for population and reproductive health. First, rapid global population expansion, which will see the world population reach 9 billion by 2045 (http://www.census.gov/ipc/www/idb/worldpopgraph.php). Second, infertility is suffered by around 180 million couples, comprising about 10%–15% worldwide and over 25% in some countries, of which 40%–50% is related to the male (http://www.who.int/reproductivehealth/publications/infertility/progress_63/en/index.html). Natural fertilization requires tightly coordinated and sequential sperm functions (1Yanagimachi R. Mammalian fertilization Knobil E. Neill J.D. The Physiology of Reproduction. 2nd Ed. Raven, New York1994: 189Google Scholar), from maturation in the epididymis (2Cooper T.G. Yeung C.H. Sperm maturation in the human epididymis.in: De Jonge C Barratt C.L.R. The Sperm Cell: Production, Maturation, Fertilization, Regeneration. CUP, Cambridge, UK2006: 72Crossref Scopus (27) Google Scholar) and survival in the female tract to fusion with the ovum (3Suarez S.S. Pacey A.A. Sperm transport in the female reproductive tract.Hum Reprod Update. 2006; 12: 23-37Crossref PubMed Scopus (740) Google Scholar). Fertility and infertility are both sides of the same reproductive coin so that understanding one can provide clues to the other. On the one hand, the widespread application to infertile couples of artificial reproductive technology, which bypasses natural fertilization mechanisms, has diverted attention from understanding the biological causes of the infertility for its cure, to that of overcoming the symptom of childlessness. On the other, controlling population growth by contraceptive means is currently only available to large populations of women. Male reproductive research lags far behind that of other disciplines and cannot meet the social needs for family planning, although hormonal contraception for men has been successful in China (4Gu Y. Liang X. Wu W. Liu M. Song S. Cheng L. Bo L. Xiong C. Wang X. Liu X. Peng L. Yao K. Multicenter contraceptive efficacy trial of injectable testosterone undecanoate in Chinese men.J. Clin. Endocrinol. Metab. 2009; 94: 1910-1915Crossref PubMed Scopus (164) Google Scholar). An alternative to suppression of sperm production for male contraception is to produce dysfunctional spermatozoa. Spermatozoa produced by the testis are immature, and since the epididymis provides a special microenvironment for their maturation and storage before ejaculation (5Turner T.T. On the epididymis and its role in the development of the fertile ejaculate.J. Androl. 1995; 16: 292-298PubMed Google Scholar), it is a prime target for such attack. The concept of sperm maturation, first mooted by Young (6Young W.C. A study of the function of the epididymis. III. Functional changes undergone by spermatozoa during their transit through the epididymis and vas deferens in the guinea pig.J. Exp. Biol. 1931; 8: 109-210Google Scholar) as a time-dependent process, was challenged by Bedford (7Bedford J.M. Effects of duct ligation on the fertilizing ability of spermatozoa from different regions of the rabbit epididymis.J. Exp. Zool. 1967; 166: 271-281Crossref PubMed Scopus (108) Google Scholar) and Orgebin-Crist (8Orgebin-Crist M.C. Fertility in does inseminated with epididymal spermatozoa.J. Reprod. Fertil. 1967; 14: 346-347Crossref PubMed Google Scholar) who provided evidence for the role of the epididymis in this process. Subsequent studies characterized potentially important epididymal proteins. Rat epididymal secretory proteins B/C and D/E found in epididymal luminal fluids (9Brooks D.E. Higgins S.J. Characterization and androgen-dependence of proteins associated with luminal fluid and spermatozoa in the rat epididymis.J. Reprod. Fertil. 1980; 59: 363-375Crossref PubMed Scopus (149) Google Scholar), bound to different sperm domains (10Brooks D.E. Tiver K. Localization of epididymal secretory proteins on rat spermatozoa.J. Reprod. Fertil. 1983; 69: 651-657Crossref PubMed Scopus (68) Google Scholar) and currently thought to regulate the process of capacitation (11Roberts K.P. Johnston D.S. Nolan M.A. Wooters J.L. Waxmonsky N.C. Piehl L.B. Ensrud-Bowlin K.M. Hamilton D.W. Structure and function of epididymal protein cysteine-rich secretory protein-1.Asian J. Androl. 2007; 9: 508-514Crossref PubMed Scopus (47) Google Scholar), necessary for fertilization, have been cloned from a rat epididymal complementary DNA (cDNA) 1The abbreviations used are:cDNAcomplementary DNAWBWestern blottingIHCImmunohistochemistryRTroom temperatureMS/MStandem mass spectrometryCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidMALDImatrix-assisted laser desorption ionizationTOFtime of flightRT-PCRreverse transcription-PCRBSAbovine serum albuminPBSphosphate buffered saline. library (12Brooks D.E. Means Tiver of the for secreted by the rat J. PubMed Scopus Google Scholar). rat epididymal proteins, the An found in the male reproductive of PubMed Scopus Google Scholar) are involved in sperm R. Y. K. of spermatozoa in the epididymis of the Chinese J. PubMed Scopus Google Scholar) suggested that interact with spermatozoa the epididymal and this has been in the M. C. J.M. E. of and spermatozoa during epididymal Reprod. 2006; PubMed Scopus Google Scholar). epididymal studies the work and confirm functional changes in epididymal spermatozoa T.G. Yeung C.H. Physiology of sperm maturation and E. S. Male and Scopus Google Scholar) and specific proteins the and protein The of human epididymal from to the of to human epididymal S. K. R. C. proteins of epididymal Reprod. PubMed Scopus Google Scholar) that proteins with different functions C. in the PubMed Google Scholar). J.L. M. Y. epididymal and Endocrinol. 2006; PubMed Scopus Google Scholar) performed gel to the human epididymal and have been to human epididymal proteins to spermatozoa R. J. are involved in the of sperm proteins during epididymal J. Androl. 2007; 9: PubMed Scopus Google Scholar). complementary DNA Western mass laser desorption of serum buffered saline. the posttesticular rapid, and it requires with specific epididymal T.G. epididymal and spermatozoa.J. Reprod. Fertil. Google T.G. Yeung C.H. to epididymal Reprod. Update. PubMed Scopus Google Scholar), few such are The application of global has the epididymal of a fertile Wang Liu J. Liu Liu of a library from human PubMed Scopus Google Scholar), which found by from E. R. the human Reprod. 2007; PubMed Scopus Google E. C. R. Effects of on the human Reprod. PubMed Scopus Google Scholar) and in epididymal library from C. in the PubMed Google Scholar). is of the epididymal in the epididymis of have been J.L. J.L. of epididymal secretory proteins for spermatozoa PubMed Scopus Google J.L. C. R. M. X. J.L. Mammalian epididymal Endocrinol. 2009; PubMed Scopus Google Scholar), of which about interact with few have functions that could be for As can be to or male the proteins involved in and understanding the process of fertilization this this study to produce a database and a quality antibody library of human epididymal sperm proteins. The was to from to and luminal fluid from epididymal tissue of spermatozoa from both proteins were and for their and functional by two gel electrophoresis mass and Antibodies were raised to these proteins and used to confirm the of the proteins in epididymal tissue and epididymal fluid by Western and to on tissue by and on spermatozoa by that were found in tissue sections, in epididymal and present in spermatozoa were epididymal secretory sperm-located proteins and the role of some proteins in sperm maturation was by sperm function tests using or recombinant proteins. from who in were artificial for epididymis was and to and one epididymis was in protein the other epididymis was as for the of human and Cambridge, Scholar) from was by of and was used for a of sperm sperm or T.G. C. E. Effects of of on and sperm as as from and Reprod. 8: PubMed Scopus Google Scholar) and spermatozoa were by to the were by the of were of human epididymal fluid proteins on gel J. Androl. PubMed Scopus Google Scholar) from the epididymal which C.H. T.G. M. of in the human and the of their J. PubMed Scopus Google Scholar) luminal fluid was by the epididymis and it and were and by for and by to the were by for and the to produce for protein were washed washed and and for spermatozoa were found of the the were to in for in and for The and the luminal fluid were with of for and for were washed with in and protein were with the The of proteins using a PubMed Scopus Google Scholar) Two-dimensional gel electrophoresis was performed as W. electrophoresis for PubMed Scopus Google Scholar). the used for were in two with and with The were on gel and with were in to confirm the and were with a were with proteins in the fluid Western were with of the proteins have been of by the of the Western or to spots from two gels were with and followed by with in for was to a of in was to the gel which were for was and with the and S. R. 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The mass was in the with of a of and a were in was performed with the and protein mass the of laser from different were by The used were and by that from the or were mass was performed on a The were used to the to a mass mass were in and mass was used with laser A of with of were for An of was used for and were for of the and database were by the a for protein for was performed with against the protein database for was performed with which the against the database for The was to as the for one and as a of and as a protein mass mass was to database mass of and was to and were the was and for mass and for one protein was in a the protein with the was from the were by a and protein was in only one The proteins were first to their function in the and the of and database proteins that could be were in the database were also for protein domains by database and with and were as a of potential epididymal secretory and sperm-located proteins, against epididymal tissue and fluid proteins were two to were to in to available were by male New with recombinant proteins C. in the PubMed Google Scholar) or synthetic from which or of the a of the successful is present to the of the these and their will be available to were by with recombinant proteins the nonstructural proteins of the fluid proteins, were tissue proteins, to of nonstructural proteins were The quality of the against fluid and tissue proteins was by against their the 619 revealed only one to the one with a major to the and one and major which be and tissue of by were to and with for they were for with the antibody with with in buffered were for with a of in were using a from human was with the were to provided with the and for the of epididymal proteins were from on the of in and are in was performed with a and were on a by with and by a New for epididymal were by in other from and were in their from the and in were on with for and for with in buffered saline. were with and washed in in buffered saline. were for with serum in buffered and for with antibody in with buffered were for with a of in was revealed by a were with and for with serum and was used as and Wang Liu J. 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Androl. 2009; PubMed Scopus Google Scholar) as a for spermatozoa were to for the sperm was washed with and and the sperm to Sperm were for to were two was to and antibody was to the were for in were in Hamilton and motility was by sperm Hamilton Spermatozoa by as The sperm was in buffered and was to the of and were with and in the only was were for in was as The quality of the gels of protein of which were in the fluids and in tissue A of protein could be by MALDI-TOF/MS and A of proteins from protein spots were found in epididymal luminal fluid and proteins from protein spots in the epididymal tissue There were proteins to both epididymal fluid and that of the abundant luminal fluid proteins are also present as abundant proteins in the of abundant proteins in the tissue were as abundant proteins in the the human epididymal 745 proteins in fluid and tissue of which were proteins which were against luminal fluid proteins and against tissue were raised against nonstructural proteins and were by against their fluids or tissue and 619 were These were used to the proteins in tissue and luminal fluid preparations to the abundant fluid proteins with 180 proteins by raised against tissue proteins were in luminal fluids by that the fluid over different proteins. revealed that of the proteins were associated with the epididymal with or in different epididymal and many were on the although of these were in fluids by Western of by the regional in epididymal and also of some of these in other There were of the 207 sperm-located proteins also in epididymal that in There was and were some of the proteins in the epididymal regions by in to in only two for proteins in the and in the the the protein have been in the of the the tissue the the the was of of epididymal secretory sperm-located proteins location as in protein and of A protein in a The antibody library epididymal protein to be on spermatozoa of antibody location were found 207 sperm-located proteins were of identification and of the 207 proteins from 745 tissue and fluid proteins. The of proteins was by proteins to be and were raised to the proteins with the 619 Western revealed that 408 proteins were present in epididymal fluid and and proteins were sperm-located proteins found in epididymal and the proteins were associated with found in fluid or sperm sperm-located sperm sperm-located of epididymal luminal On the of and proteins were as tissue proteins, secretory proteins, and sperm-located proteins. tissue proteins were as present in the tissue in epididymal secretory proteins were as present in epididymal and sperm-located proteins were on washed spermatozoa. The terminology for tissue proteins was human epididymal tissue the specific and for secretory proteins human epididymal secretory on their location on sperm-located proteins were with in a to the location on the sperm-located proteins were for secretory and for sperm-located proteins could be sperm or their of proteins that are sperm domains. sperm-located protein that was with of the secreted and tissue proteins are in which a 619 epididymal proteins were used to the proteins by Western in epididymal luminal by in in regions of the epididymis, and by on different domains of spermatozoa. these proteins were secretory proteins secretory sperm-located proteins tissue sperm-located proteins and proteins of which were found on the of the of the sperm-located proteins have been by other as on the sperm The of the human epididymal proteins from to 9 and the of the human epididymal proteins were with and The chromosomal location of the epididymal the secretory proteins and sperm-located proteins were on the or on the on the The proteins as spots on gels and by mass were functional on the of the and PubMed The of proteins were related to other protein functions were related to different sperm the of 207 proteins found on different sperm domains that are associated with different The antibody to protein the of motility and of spermatozoa from in the of of The antibody to the of head from that of the of against secreted human epididymal proteins on the sperm on the of Antibodies against the sperm and in the the of spermatozoa with of head different from and spermatozoa in were in the or in motility motility in a of spermatozoa with in the of were spermatozoa. 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The of human spermatozoa has been by the of found in serum from infertile gels and fertile D.S. Wooters J. Y. K.P. of the human sperm PubMed Scopus Google Scholar), and gels and J. J.M. J.L. R. identification of human sperm 2006; PubMed Scopus Google Scholar). There is study of the epididymal to R. J. J.M. in the study of the sperm and Biol. Reprod. Scopus Google R. S. J.M. Sperm 2009; 9: PubMed Scopus Google Scholar) and was on proteins of epididymal they are necessary for sperm maturation and are potential posttesticular contraceptive The specific functions and locations of the proteins on specific sperm domains will a of functional the 619 raised to human epididymal proteins, the of 207 with washed spermatozoa that the of the human epididymal sperm-located protein family is this such human epididymal proteins been and by were found in different by different from the used which also of the proteins were epididymal secretory proteins from their in epididymal fluid the for some were by other as some epididymal protein were found in the it is that such proteins in the epididymal fluid from the testis or also from the epididymis. The location on spermatozoa of some proteins from this different or sperm and requires of the proteins were epididymal secretory proteins from their in epididymal fluid they are since were by other The of proteins in epididymal fluid is of epididymal since they could also have in the testis or have in both the testis and epididymis. the of of the proteins the was their epididymal On the other hand, epididymal sperm-located proteins could be as secreted they were in the luminal these be present the of the Western of proteins the sperm has been was to proteins on the from in the so the proteins in spermatozoa are that such proteins are of posttesticular of spermatozoa is of spermatozoa is are currently to human epididymal which spermatozoa. The of proteins in a spermatozoon is to be around M.A. and 2009; Google Scholar). The present work proteins, by epididymal tissue and present in luminal that were sperm-located and is a that these proteins are of epididymal The of in the epididymis, with of of the that luminal R. J. are involved in the of sperm proteins during epididymal J. Androl. 2007; 9: PubMed Scopus Google Scholar) proteins a can be secreted the epididymal The specific of proteins in in the to the sperm has been in R. J. are involved in the of sperm proteins during epididymal J. Androl. 2007; 9: PubMed Scopus Google Scholar). The of human of proteins, has been M. R. of human during a and Reprod. PubMed Scopus Google Scholar) and were with of the luminal proteins and with of the 207 sperm-located proteins in this the and sperm proteins important during sperm maturation M.A. R. L. L. and that during sperm maturation using in gel PubMed Scopus Google Scholar). of the epididymal secretory proteins found on the washed spermatozoa are also found in the of human and spermatozoa to proteins for sperm development or of the of proteins M.A. and 2009; Google Scholar) the and the of and of or were in this These the two proteins, and protein electrophoresis in is one of sperm proteins to from to M.A. R. L. L. and that during sperm maturation using in gel PubMed Scopus Google Scholar), of epididymal and by spermatozoa. the in the rat is highly in the of the epididymis T.T. E. Johnston D.S. The rat epididymal of in the rat and Reprod. 2007; PubMed Scopus Google Scholar). The present the epididymal proteins to the and the in with from the human epididymal Wang Liu J. Liu Liu of a library from human PubMed Scopus Google Wang Liu J. Wang Liu J. and identification of a sperm with and in the human Reprod. PubMed Scopus Google Scholar). Functional that epididymal proteins the luminal microenvironment and sperm maturation New epididymal and Reprod. Update. 2009; PubMed Scopus Google Scholar). These proteins be related to during maturation or fertilization T.G. Neill J.D. Knobil E. of Reproduction. Scholar), related to or transport The ability of against proteins the and of the to sperm only that the proteins are on the sperm that they be in some of the ability of to is by its function, this is from their identification as protein protein and the of they could as targets for of the proteins were involved in and the protein Wang Liu J. Wang Liu J. and identification of a sperm with and in the human Reprod. PubMed Scopus Google Scholar). The proteins in protection of epididymal spermatozoa in the male and female The ability of the protein to the in its identification as a and a function as the male the epididymal found on about were proteins that be important for sperm functions during the process of epididymal sperm transit and fertilization T.G. Neill J.D. Knobil E. of Reproduction. Scholar). The functional of proteins on different sperm domains revealed that proteins to the sperm to the sperm and The be related to the survival of spermatozoa as in the female tract and the to the sequential that the sperm head and fertilization, and fusion E. M. L. in human spermatozoa.J. Reprod. PubMed Scopus Google Scholar). work has the human epididymal and a large of sperm proteins a family by a that the of protein on spermatozoa. The epididymal secretory sperm-located proteins are important proteins in sperm maturation and the that proteins of different function are different sperm domains will a of functional that sperm functions are by against proteins from the sperm head and J. Wu J. R. Y. Y. X. Liu J. X. J. is a potential for human fertilization Reprod. 2007; PubMed Scopus Google C. S. C. C. Characterization and of a human sperm protein with a potential role in Reprod. PubMed Scopus (27) Google Y. X. S. S. S.S. Wang L. infertility of on the of female 2009; PubMed Scopus Google L. K. J. protein is a human involved in and Androl. PubMed Scopus Google Scholar). of the of sperm this database of sperm proteins as a of for research and The of the epididymal proteins have to be before they can be used to and for a this work will men to important role in family to the of for in gel spots with a for on and Yeung of and of for and of the of for and of the with