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Evaluation of Sperm Proteins in Infertile Men: A Proteomic Approach

2011; Elsevier BV; Volume: 95; Issue: 8 Linguagem: Inglês

10.1016/j.fertnstert.2011.03.112

1556-5653

Stetson Thacker, Satya Prakash Yadav, Rakesh Sharma, Anthony H. Kashou, Belinda Willard, Dongmei Zhang, Ashok Agarwal,

Iron Metabolism and Disorders

In this study, the sperm protein profile was compared between fertile and infertile men using 2-dimensional gel electrophoresis, liquid chromatography mass spectrometer analysis, and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry. Four unique proteins, semenogelin II precursor, prolactin-induced protein, clusterin isoform 1, and prostate-specific antigen isoform 1 preproprotein, were predominantly present in the semen of healthy men; however, semenogelin II precursor and clusterin isoform 1 were not seen in the semen of infertile men, suggesting unique differences in the spermatozoa protein profiles of fertile and infertile men. In this study, the sperm protein profile was compared between fertile and infertile men using 2-dimensional gel electrophoresis, liquid chromatography mass spectrometer analysis, and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry. Four unique proteins, semenogelin II precursor, prolactin-induced protein, clusterin isoform 1, and prostate-specific antigen isoform 1 preproprotein, were predominantly present in the semen of healthy men; however, semenogelin II precursor and clusterin isoform 1 were not seen in the semen of infertile men, suggesting unique differences in the spermatozoa protein profiles of fertile and infertile men. One in 10 couples are infertile, and male factor defects account for as many as 50% of all infertility cases (1Sharlip I.D. Jarow J.P. Belker A.M. Lipshultz L.I. Sigman M. Thomas A.J. et al.Best practice policies for male infertility.Fertil Steril. 2002; 77: 873-882Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Proteomics allows the differential expression of proteins. A large number of proteins such as semenogelin (SG) (2Lilja H. Olbring J. Rannevik G. Laurell C.-B. Seminal vesicle-secreted proteins and their reactions during gelation and liquefaction of human semen.J Clin Invest. 1987; 80: 281-285Crossref PubMed Scopus (329) Google Scholar, 3Jonsson M. Frohm B. Malm J. Binding of semenogelin I to intact human spermatozoa studied by flow cytometry and surface plasmon resonance.J Androl. 2010; 6: 560-565Crossref Scopus (8) Google Scholar), serine protease, prostate-specific antigen (PSA) (4Kise H. Nishioka J. Kawamura J. Suzuki K. Characterization of semenogelin II and its molecular interaction with prostate-specific antigen and protein C inhibitor.Eur J Biochem. 1996; 15: 88-96Crossref Scopus (47) Google Scholar), clusterin (5Fritz I.B. Burdzy K. Setchell B. Blaschuk O. Ram rete testis fluid contains a protein (clusterin) which influences cell-cell interaction in vitro.Biol Reprod. 1983; 28: 1173-1188Crossref PubMed Scopus (198) Google Scholar, 6Silkensen J.R. Skubitz K.M. Skubitz A.P. Chmielewski D.H. Manivel J.C. Dvergsten J.A. et al.Clusterin promotes the aggregation and adhesion of renal porcine epithelial cells.J Clin Invest. 1995; 96: 2646-2653Crossref PubMed Scopus (68) Google Scholar), fibronectin (7McGee R.S. Herr J.C. Human seminal vesicle-specific antigen is a substrate for prostate-specific antigen (or P-30).Biol Reprod. 1988; 39: 499-510Crossref PubMed Scopus (72) Google Scholar), prolactin-induced protein (PIP) (8Haagensen D.E. Mazoujian G. Biochemistry and immunohistochemistry of fluid proteins of the breast in gross cystic disease.in: Haagensen D.E. Diseases of the breast. W.B. Saunders Co., Philadelphia1986: 474-500Google Scholar), heat shock protein, and lactoferrin (9Lilja H. Abrahamsson P.A. Lundwall A. Semenogelin, the predominant protein in human semen. Primary structure and identification of closely related proteins in the male accessory sex glands and on the spermatozoa.J Biol Chem. 1989; 25: 1894-1900Google Scholar) have been identified and may play a role in male fertility. In this pilot study, the objective was to compare the protein profile from fertile and infertile men as a first step toward identifying unique seminal proteins that may be associated with male infertility using novel proteomic tools such as 2-dimensional gel electrophoresis (2-DGE), liquid chromatography-mass spectroscopy, and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Following institutional review board approval, semen samples were obtained from 1 healthy man of proven fertility and 3 infertile men (low count and motility) according to World Health Organization 1999 guidelines. Spermatozoa were solubilized in lysis buffer, isoelectrofocusing was performed, and 2-DGE was used for further resolution of proteins isolated from sperm cells. The uniquely identified spots were excised from the gels, washed, and destained in 50% acetonitrile containing 5% acetic acid. The dried gel pieces were digested overnight with trypsin (5 μL of 10 ng/μL) in 50 mM ammonium bicarbonate. The extracts were combined and evaporated to reduce the volume to 10 μL. The extracts were subsequently resuspended in 15% acetic acid to make a final volume of approximately 30 μL for liquid chromatography-mass spectroscopy. Ten microliters of the extracted peptide samples were injected on a C18 reverse-phase capillary chromatography column for separation before introduction into an online mass spectrometer. Peptides eluted from the column were analyzed by mass spectrometer to determine peptide molecular weights and product ion spectra to determine amino acid sequence. Data were analyzed by using all collisionally induced dissociation spectra collected in the experiment to search the National Center for Biotechnology Information nonredundant database with the search engine Mascot (Matrix Science). Additional searches using the programs Sequest and Blast were conducted as needed. An aliquot (15%) from the samples was dried and spotted on MALDI plates for MALDI-TOF/TOF analysis. All gels were run in duplicate under identical conditions. The resulting spot from each gel was superimposed, and distinct spots were identified. Only those spots that were unique (i.e., not superimposed) were subsequently studied. Ten spots from 1 Coomassie blue-stained gel from control (S1) and 4 unique spots from 3 Coomassie blue-stained gels from the infertile patients (S2–S4) were used for subsequent steps (Fig. 1). In the healthy control sample, 10 distinctive spots were used for liquid chromatography-mass spectroscopy analysis for protein identification. Spots 1, 4, and 6 were identified as an N-terminally truncated form of SGII precursor, a 65-kDa protein. The sequence coverage in spots 1, 4, and 6 was 5%, 6%, and 10% respectively. All of the peptides identified in this protein were derived from amino acids 484–582, indicating that these N-terminally truncated forms of SGII migrated to different spots during 2-DGE. Spots 2, 3, and 11 were identified as PIP, a 16-kDa protein. The protein coverage in spots 2 and 3 was 50% and 40%, respectively, mainly from amino acids 39–146, indicating truncated or modified PIP isoforms. A peptide, (118)ELGICPDDAAVIPIK(133), with masses of 1,611, 1,602, 1,627, and 1,643 Da, was identified in spots 2 and 11, corresponding to various modifications at Cys123 in the sequence. The peptide with a mass of 1,611 Da is the carbamidomethylated form of cysteine. We identified several additional forms of this peptide containing various cysteine modifications including cysteic acid and oxidized carbamidomethylated cysteine. One interesting observation was that the cysteic acid form of this peptide was only identified in spot 2 (Fig. 1). Spot 8 was identified as clusterin isoform 1, a 58-kDa protein, with coverage of 4% from amino acids 97–130 of the protein sequence. Spots 9 and 10 were identified as PSA isoform I preprotein (29 kDa) with 10% and 16% of peptide coverage, respectively. Two spots, spots 5 and 7, could not be identified when analyzed on both LCQ and LTQ linear ion trap mass spectrometer systems (Thermo Scientific). In the samples from infertile men, 2 unique proteins were identified in this set as PIP (16 kDa) and PSA isoform 1 preproprotein (29 kDa). PIP was identified in spots 11 and 12 by 7 peptides, each covering 44% and 53% of the protein sequence, respectively. PSA isoform 1 preproprotein had 6 peptides with 21% peptide coverage. Spot 13 could not be identified by both mass spectrometers. A peptide with a mass ranging between 1,627 and 1,643 Da was identified in spot 11, corresponding to peptide (119)ELGICPDDAAVIPIK(133) and showed various oxidized states of Cys123. In this pilot study, we identified the presence of 4 major proteins in human seminal plasma that were unique and different in fertile and infertile men. These were SGII precursor, PIP, clusterin isoform, and PSA isoform 1 preproprotein. Although all 4 were present in the sample from the healthy control, the samples from the infertile men lacked SGII precursor and clusterin isoform I. The major proteins of the human semen coagulum are SGI and, to a lesser extent, SGII (10Robert M. Gagnon C. Semenogelin I. A coagulum forming, multifunctional seminal vesicle protein.Cell Mol Life Sci. 1999; 55: 944-960Crossref PubMed Scopus (145) Google Scholar). SGI and SGII represent 20% to 40% of the seminal plasma proteins and share a 78% homology at the amino acid level; the difference in mass between SGI (52 kDa) and SGII (71 kDa) is related to the presence of an extended C-terminal in SGII (11Lilja H. Lundwall A. Molecular-cloning of epididymal and seminal vesicular transcripts encoding a semenogelin-related protein.Proc Natl Acad Sci U S A. 1992; 89: 4559-4563Crossref PubMed Scopus (88) Google Scholar). They are characterized by a basic isoelectric point (>9.5) and a high capacity for zinc binding (10Robert M. Gagnon C. Semenogelin I. A coagulum forming, multifunctional seminal vesicle protein.Cell Mol Life Sci. 1999; 55: 944-960Crossref PubMed Scopus (145) Google Scholar, 12Mandal A. Bhattacharyya A.K. Biochemical composition of washed seminal coagulum in comparison to sperm-free semen from the same donors.J Reprod Fertil. 1990; 88: 113-118Crossref PubMed Scopus (14) Google Scholar, 13Robert M. Gibbs B.F. Jacobson E. Gagnon C. Characterization of prostate-specific antigen proteolytic activity on its major physiological substrate, the sperm motility inhibitor precursor/semenogelin I.Biochemistry. 1997; 36: 3811-3819Crossref PubMed Scopus (158) Google Scholar). We identified SGII using 2-DGE only in the control samples; all of the peptides identified in this protein were derived from amino acids 484–582, suggesting N-terminally truncated forms of SGII that migrated to different spots during 2-DGE. We did not detect SGII in the samples from the infertile men, contrary to an earlier report (14Zhao C. Huo R. Wang F.Q. Lin M. Zhou Z.M. Sha J.H. Identification of several proteins involved in regulation of sperm motility by proteomic analysis.Fertil Steril. 2007; 87: 436-438Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Data suggest that SG, its degradation products, or both may be natural regulators of sperm capacitation that could prevent this process from occurring prematurely. PSA has been reported to be involved in degradation of SG (9Lilja H. Abrahamsson P.A. Lundwall A. Semenogelin, the predominant protein in human semen. Primary structure and identification of closely related proteins in the male accessory sex glands and on the spermatozoa.J Biol Chem. 1989; 25: 1894-1900Google Scholar, 15Robert M. Gagnon C. Purification and characterization of the active precursor of a human sperm motility inhibitor secreted by the seminal vesicles: identity with semenogelin.Biol Reprod. 1996; 55: 813-821Crossref PubMed Scopus (96) Google Scholar) and might therefore be expected to have a positive impact on sperm motility. PSA is a 33-kDa androgen-regulated serine protease that cleaves SGI and SGII in the seminal coagulum. We demonstrated the presence of PSA isoform I preprotein (29 kDa) in both the control and infertile specimens. In the control specimen, spots 9 and 10 were identified as PSA isoform I preprotein (29 kDa) with 10% and 16% of peptide coverage. In the infertile specimens, the PSA isoform 1 preproprotein had 6 peptides with 21% peptide coverage. Low levels of seminal PSA have been reported in patients with low sperm motility (16Ahlgren G. Rannevik G. Lilja H. Impaired secretory function of the prostate in men with oligo-asthenozoospermia.J Androl. 1995; 16: 491-498PubMed Google Scholar). Clusterin is a 70- to 80-kDa highly conserved heterodimeric glycoprotein (17O’Bryan M.K. Baker H.W. Saunders J.R. Kirszbaum L. Walker I.D. Hudson P. et al.Human seminal clusterin (SP-40). Isolation and characterization.J Clin Invest. 1990; 85: 1477-1486Crossref PubMed Scopus (106) Google Scholar). It has been implicated in a number of diverse biologic processes, including cell-cell interactions (5Fritz I.B. Burdzy K. Setchell B. Blaschuk O. Ram rete testis fluid contains a protein (clusterin) which influences cell-cell interaction in vitro.Biol Reprod. 1983; 28: 1173-1188Crossref PubMed Scopus (198) Google Scholar, 6Silkensen J.R. Skubitz K.M. Skubitz A.P. Chmielewski D.H. Manivel J.C. Dvergsten J.A. et al.Clusterin promotes the aggregation and adhesion of renal porcine epithelial cells.J Clin Invest. 1995; 96: 2646-2653Crossref PubMed Scopus (68) Google Scholar), sperm maturation (18Collard M.W. Griswold M.D. Biosynthesis and molecular cloning of sulfated glycoprotein 2 secreted by rat Sertoli cells.Biochemistry. 1987; 16: 3297-3303Crossref Scopus (271) Google Scholar, 19Sylvester S.R. Morales C. Oko R. Griswold M.D. Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat.Biol Reprod. 1991; 45: 195-207Crossref PubMed Scopus (139) Google Scholar), apoptosis (20Buttyan R. Olsson C.A. Pintar J. Chang C. Bandyk M. Ng P.Y. et al.Induction of the TRPM-2 gene in cells undergoing programmed death.Mol Cell Biol. 1989; 9: 3473-3481Crossref PubMed Scopus (421) Google Scholar), complement inhibition (21Murphy B.F. Kirszbaum L. Walker I.D. d’Apice A.J. SP-40, 40, a newly identified normal human serum protein found in the SC5b-9 complex of complement and in the immune deposits in glomerulonephritis.J Clin Invest. 1988; 81: 1858-1864Crossref PubMed Scopus (252) Google Scholar), lipid transport (22de Silva H.V. Stuart W.D. Duvic C.R. Wetterau J.R. Ray M.J. Ferguson D.G. A 70-kDa apolipoprotein designated apo J is a marker for subclasses of human plasma high density lipoproteins.J Biol Chem. 1990; 265: 13240-13247Abstract Full Text PDF PubMed Google Scholar, 23Jenne D.E. Tschopp J. Molecular structure and functional characterization of a human complement cytolysis inhibitor found in blood and seminal plasma: identity to sulfated glycoprotein 2, a constituent of rat testis fluid.Proc Natl Acad Sci U S A. 1989; 86: 7123-7127Crossref PubMed Scopus (338) Google Scholar), tissue remodeling, membrane recycling (24Danik M. Chabot J.G. Mercier C. Benabid A.L. Chauvin C. 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Clusterin, an abundant serum factor, is a possible negative regulator of MT6-MMP/MMP-25 produced by neutrophils.J Biol Chem. 2003; 278: 36350-36357Crossref PubMed Scopus (54) Google Scholar). Clusterin prevents damaging oxidative reactions (28Reyes-Moreno C. Boilard M. Sullivan R. Sirard M.A. Characterization and identification of epididymal factors that protect ejaculated bovine sperm during in vitro storage.Biol Reprod. 2002; 66: 159-166Crossref PubMed Scopus (47) Google Scholar), protein precipitation (29Ibrahim N.M. Gilbert G.R. Loseth K.L. Crabo B.G. Correlation between clusterin-positive spermatozoa determined by flow cytometry in bull semen and fertility.J Androl. 2000; 21: 887-894PubMed Google Scholar), and agglutination of abnormal spermatozoa (17O’Bryan M.K. Baker H.W. Saunders J.R. Kirszbaum L. Walker I.D. Hudson P. et al.Human seminal clusterin (SP-40). Isolation and characterization.J Clin Invest. 1990; 85: 1477-1486Crossref PubMed Scopus (106) Google Scholar, 30O’Bryan M.K. Murphy B.F. Liu D.Y. Clarke G.N. Baker H.W. The use of anticlusterin monoclonal antibodies for the combined assessment of human sperm morphology and acrosome integrity.Hum Reprod. 1994; 9: 1490-1496PubMed Google Scholar) and controls complement-induced sperm lysis (31Jenne D.E. Lowin B. Peitsch M.C. Böttcher A. Schmitz G. Tschopp J. Clusterin (complement lysis inhibitor) forms a high density lipoprotein complex with apolipoprotein A-I in human plasma.J Biol Chem. 1991; 266: 11030-11036Abstract Full Text PDF PubMed Google Scholar). In the control sample, spot 8 was identified as clusterin isoform 1, a 58-kDa protein, with coverage of 4% from amino acids 97–130 of the protein sequence. Both clusterin and its precursor are present only in abnormal human spermatozoa and in asthenozoospermic men (17O’Bryan M.K. Baker H.W. Saunders J.R. Kirszbaum L. Walker I.D. Hudson P. et al.Human seminal clusterin (SP-40). Isolation and characterization.J Clin Invest. 1990; 85: 1477-1486Crossref PubMed Scopus (106) Google Scholar, 29Ibrahim N.M. Gilbert G.R. Loseth K.L. Crabo B.G. Correlation between clusterin-positive spermatozoa determined by flow cytometry in bull semen and fertility.J Androl. 2000; 21: 887-894PubMed Google Scholar, 30O’Bryan M.K. Murphy B.F. Liu D.Y. Clarke G.N. Baker H.W. The use of anticlusterin monoclonal antibodies for the combined assessment of human sperm morphology and acrosome integrity.Hum Reprod. 1994; 9: 1490-1496PubMed Google Scholar, 32Clark A.M. Griswold M.D. Expression of clusterin/sulfated glycoprotein-2 under conditions of heat stress in rat Sertoli cells and a mouse Sertoli cell line.J Androl. 1997; 18: 257-263PubMed Google Scholar). PIP is an aspartyl proteinase with specificity to fibronectin, one of the major protein constituents of the seminal coagulum (9Lilja H. Abrahamsson P.A. Lundwall A. Semenogelin, the predominant protein in human semen. Primary structure and identification of closely related proteins in the male accessory sex glands and on the spermatozoa.J Biol Chem. 1989; 25: 1894-1900Google Scholar, 33Caputo E. Manco G. Mandrich L. Guardiola J. A novel aspartyl proteinase from apocrine epithelia and breast tumors.J Biol Chem. 2000; 275: 7935-7941Crossref PubMed Scopus (60) Google Scholar, 34Autiero M. Abrescia P. Guardiola J. Interaction of seminal plasma proteins with cell surface antigens: presence of a CD4-binding glycoprotein in human seminal plasma.Exp Cell Res. 1991; 197: 268-271Crossref PubMed Scopus (49) Google Scholar, 35Schaller J. Akiyama K. Kimura H. Hess D. Affolter M. Rickli E.E. Primary structure of a new actin-binding protein from human seminal plasma.Eur J Biochem. 1991; 196: 743-750Crossref PubMed Scopus (43) Google Scholar, 36Caputo E. Carratore V. Ciullo M. Tiberio C. Mani J.C. Piatier-Tonneau D. et al.Biosynthesis and immunobiochemical characterization of p17/GCDFP-15. A glycoprotein from seminal vesicles and from breast tumors, in HeLa cells and in Pichia pastoris yeast.Eur J Biochem. 1999; 265: 664-670Crossref PubMed Scopus (23) Google Scholar, 37Yoshida K. Yamasaki T. Yoshiike M. Takano S. Sato I. Iwamoto T. Quantification of seminal plasma motility inhibitor/semenogelin inhuman seminal plasma.J Androl. 2003; 24: 878-884PubMed Google Scholar). It is found in the postacrosomal zone and may have a role in fertilization (38Bergamo P. Balestrieri M. Cammarota G. Guardiola J. Abrescia P. CD4-mediated anchoring of the seminal antigen gp17 onto the spermatozoon surface.Hum Immunol. 1997; 58: 30-41Crossref PubMed Scopus (26) Google Scholar). A decrease in the amount of PIP and PIP precursors may lead to a more viscous seminal vesicular fluid and incomplete liquefaction of the ejaculate. We identified PIP 16-kDa proteins in both the control and patient samples. The protein coverage in spots 2, 3, and 11 was 50%, 40%, and 44%, respectively, mainly from amino acids 39–146, indicating truncated or modified isoforms of PIP. The condensed chromatin of mammalian spermatozoa is extensively stabilized by disulfide bridges (39Loir M. Lanneau M. Structural function of the basic nuclear proteins in ram spermatids.J Ultrastruct Res. 1984; 86: 262-272Crossref PubMed Scopus (38) Google Scholar, 40Hammadeh M.E. Hamad M.F. Montenarh M. Fischer-Hammadeh C. Protamine contents and P1/P2 ratio in human spermatozoa from smokers and non-smokers.Hum Reprod. 2010; 25: 2708-2720Crossref PubMed Scopus (78) Google Scholar). We observed various cysteine modifications; the cysteic acid form of this peptide/protein was found to be present only in the sperm sample of fertile men, whereas the cysteic acid-modified form was absent in the sample from infertile men. Although the study limitation was the small number of samples, this was a pilot study in which we used these novel approaches to identify the proteins that may have a key role in infertility. This study will be further expanded by increasing the number of samples taken from infertile individuals, whose sperm proteomes will be analyzed and compared with those who have fertile spermatozoa proteomes.