- Email: [email protected]
Evaluation of Sperm Proteins in Infertile Men: A Proteomic Approach
Evaluation of Sperm Proteins in Infertile Men: A Proteomic Approach 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. (Fertil Steril 2011;95:2745–8. 2011 by American Society for Reproductive Medicine.) Key Words: Sperm, semenogelin II precursor, prolactin-induced protein, clusterin, prostate-specific antigen
One in 10 couples are infertile, and male factor defects account for as many as 50% of all infertility cases (1). Proteomics allows the differential expression of proteins. A large number of proteins such as semenogelin (SG) (2, 3), serine protease, prostatespecific antigen (PSA) (4), clusterin (5, 6), fibronectin (7), prolactin-induced protein (PIP) (8), heat shock protein, and lactoferrin (9) 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).
a
Stetson Thacker Satya P. Yadav, Ph.D.a Rakesh K. Sharma, Ph.D.b Anthony Kashou, B.S.b Belinda Willard, Ph.D.c Dongmei Zhang, Ph.D.c Ashok Agarwal, Ph.D.b a Molecular Biotechnology Core Laboratory, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio b Center for Reproductive Medicine, Glickman Urological & Kidney Institute, Cleveland Clinic and Obstetrics and Gynecology and Women’s Health Institute, Cleveland, Ohio c Proteomics Core, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio Received January 24, 2011; revised March 15, 2011; accepted March 17, 2011; published online May 4, 2011. S.T. has nothing to disclose. S.P.Y. has nothing to disclose. R.K.S. has nothing to disclose. A.K. has nothing to disclose. B.W. has nothing to disclose. D.Z. has nothing to disclose. A.A. has nothing to disclose. S.T. is a summer high school student. Reprint requests: Ashok Agarwal, Ph.D., Center for Reproductive Medicine, Glickman Urological & Kidney Institute, Cleveland Clinic, 9500 Euclid Avenue, Desk A19.1, Cleveland, Ohio 44195 (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.03.112
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 mL of 10 ng/mL) in 50 mM ammonium bicarbonate. The extracts were combined and evaporated to reduce the volume to 10 mL. The extracts were subsequently resuspended in 15% acetic acid to make a final volume of approximately 30 mL 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.
Fertility and Sterility Vol. 95, No. 8, June 30, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
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FIGURE 1 Coomassie blue-stained 2-dimensional gel from 1 control and 3 infertile patients. Ten spots were obtained from 1 control and 4 unique spots from 3 infertile patients. All spots from the control and patients were superimposed. Only those spots that were not common in both but unique (11–14) were examined. S1 ¼ control; S2–S4 ¼ infertile patients.
Thacker. Correspondence. Fertil Steril 2011.
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.
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Correspondence
A peptide with a mass ranging between 1,627 and 1,643 Da was identified in spot 11, corresponding to peptide (119)ELGICPD DAAVIPIK(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 (10). 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 (11). They are characterized by a basic isoelectric point (>9.5) and a high capacity for zinc binding (10, 12, 13). 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 (14). 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 (9, 15) and might therefore be expected to have a positive impact on sperm motility. PSA is a 33-kDa androgen-regulated serine
Vol. 95, No. 8, June 30, 2011
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 (16). Clusterin is a 70- to 80-kDa highly conserved heterodimeric glycoprotein (17). It has been implicated in a number of diverse biologic processes, including cell-cell interactions (5, 6), sperm maturation (18, 19), apoptosis (20), complement inhibition (21), lipid transport (22, 23), tissue remodeling, membrane recycling (24, 25), clearance of cellular debris (26), and degradation of the extracellular matrix (27). Clusterin prevents damaging oxidative reactions (28), protein precipitation (29), and agglutination of abnormal spermatozoa (17, 30) and controls complement-induced sperm lysis (31). 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 (17, 29, 30, 32).
PIP is an aspartyl proteinase with specificity to fibronectin, one of the major protein constituents of the seminal coagulum (9, 33–37). It is found in the postacrosomal zone and may have a role in fertilization (38). 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 (39, 40). 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.
REFERENCES 1. Sharlip ID, Jarow JP, Belker AM, Lipshultz LI, Sigman M, Thomas AJ, et al. Best practice policies for male infertility. Fertil Steril 2002;77:873–82. 2. Lilja 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;80:281–5. 3. Jonsson M, Frohm B, Malm J. Binding of semenogelin I to intact human spermatozoa studied by flow cytometry and surface plasmon resonance. J Androl 2010;6:560–5. 4. Kise 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;15:88–96. 5. Fritz IB, Burdzy K, Setchell B, Blaschuk O. Ram rete testis fluid contains a protein (clusterin) which influences cell-cell interaction in vitro. Biol Reprod 1983;28:1173–88. 6. Silkensen JR, Skubitz KM, Skubitz AP, Chmielewski DH, Manivel JC, Dvergsten JA, et al. Clusterin promotes the aggregation and adhesion of renal porcine epithelial cells. J Clin Invest 1995;96:2646–53. 7. McGee RS, Herr JC. Human seminal vesiclespecific antigen is a substrate for prostate-specific antigen (or P-30). Biol Reprod 1988;39:499–510. 8. Haagensen DE, Mazoujian G. Biochemistry and immunohistochemistry of fluid proteins of the breast in gross cystic disease. In: Haagensen DE, ed. Diseases of the breast. Philadelphia: W.B. Saunders Co., 1986:474–500. 9. Lilja H, Abrahamsson PA, 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;25:1894–900. 10. Robert M, Gagnon C, Semenogelin I. A coagulum forming, multifunctional seminal vesicle protein. Cell Mol Life Sci 1999;55:944–60.
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11. Lilja H, Lundwall A. Molecular-cloning of epididymal and seminal vesicular transcripts encoding a semenogelin-related protein. Proc Natl Acad Sci U S A 1992;89:4559–63. 12. Mandal A, Bhattacharyya AK. Biochemical composition of washed seminal coagulum in comparison to sperm-free semen from the same donors. J Reprod Fertil 1990;88:113–8. 13. Robert M, Gibbs BF, 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;36:3811–9. 14. Zhao C, Huo R, Wang FQ, Lin M, Zhou ZM, Sha JH. Identification of several proteins involved in regulation of sperm motility by proteomic analysis. Fertil Steril 2007;87:436–8. 15. 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. Biol Reprod 1996;55:813–21. 16. Ahlgren G, Rannevik G, Lilja H. Impaired secretory function of the prostate in men with oligoasthenozoospermia. J Androl 1995;16:491–8. 17. O’Bryan MK, Baker HW, Saunders JR, Kirszbaum L, Walker ID, Hudson P, et al. Human seminal clusterin (SP-40). Isolation and characterization. J Clin Invest 1990;85:1477–86. 18. Collard MW, Griswold MD. Biosynthesis and molecular cloning of sulfated glycoprotein 2 secreted by rat Sertoli cells. Biochemistry 1987;16:3297–303. 19. Sylvester SR, Morales C, Oko R, Griswold MD. Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat. Biol Reprod 1991;45:195–207. 20. Buttyan R, Olsson CA, Pintar J, Chang C, Bandyk M, Ng PY, et al. Induction of the TRPM-2 gene in cells undergoing programmed death. Mol Cell Biol 1989;9:3473–81. 21. Murphy BF, Kirszbaum L, Walker ID, d’Apice AJ. SP-40, 40, a newly identified normal human serum
22.
23.
24.
25.
26.
27.
28.
protein found in the SC5b-9 complex of complement and in the immune deposits in glomerulonephritis. J Clin Invest 1988;81:1858–64. de Silva HV, Stuart WD, Duvic CR, Wetterau JR, Ray MJ, Ferguson DG. A 70-kDa apolipoprotein designated apo J is a marker for subclasses of human plasma high density lipoproteins. J Biol Chem 1990;265:13240–7. Jenne DE, 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;86:7123–7. Danik M, Chabot JG, Mercier C, Benabid AL, Chauvin C, Quirion R, et al. Human gliomas and epileptic foci express high levels of a mRNA related to rat testicular sulfated glycoprotein 2, a purported marker of cell death. Proc Natl Acad Sci U S A 1991;88:8577–81. Palmer DJ, Christie DL. Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H), and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules. J Biol Chem 1992;267:19806–12. Bartl MM, Luckenbach T, Bergner O, Ullrich O, Koch-Brandt C. Multiple receptors mediate apoJdependent clearance of cellular debris into nonprofessional phagocytes. Exp Cell Res 2001;15: 130–41. Matsuda A, Itoh Y, Koshikawa N, Akizawa T, Yana I, Seiki M. Clusterin, an abundant serum factor, is a possible negative regulator of MT6MMP/MMP-25 produced by neutrophils. J Biol Chem 2003;278:36350–7. Reyes-Moreno C, Boilard M, Sullivan R, Sirard MA. Characterization and identification of epididymal factors that protect ejaculated bovine sperm during in vitro storage. Biol Reprod 2002;66:159–66.
2747
29. Ibrahim NM, Gilbert GR, Loseth KL, Crabo BG. Correlation between clusterin-positive spermatozoa determined by flow cytometry in bull semen and fertility. J Androl 2000;21:887–94. 30. O’Bryan MK, Murphy BF, Liu DY, Clarke GN, Baker HW. The use of anticlusterin monoclonal antibodies for the combined assessment of human sperm morphology and acrosome integrity. Hum Reprod 1994;9:1490–6. 31. Jenne DE, Lowin B, Peitsch MC, B€ottcher 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;266:11030–6. 32. Clark AM, Griswold MD. Expression of clusterin/ sulfated glycoprotein-2 under conditions of heat stress in rat Sertoli cells and a mouse Sertoli cell line. J Androl 1997;18:257–63.
2748
Thacker et al.
33. Caputo E, Manco G, Mandrich L, Guardiola J. A novel aspartyl proteinase from apocrine epithelia and breast tumors. J Biol Chem 2000;275: 7935–41. 34. Autiero 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;197:268–71. 35. Schaller J, Akiyama K, Kimura H, Hess D, Affolter M, Rickli EE. Primary structure of a new actin-binding protein from human seminal plasma. Eur J Biochem 1991;196:743–50. 36. Caputo E, Carratore V, Ciullo M, Tiberio C, Mani JC, 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;265:664–70.
Correspondence
37. Yoshida K, Yamasaki T, Yoshiike M, Takano S, Sato I, Iwamoto T. Quantification of seminal plasma motility inhibitor/semenogelin inhuman seminal plasma. J Androl 2003;24:878–84. 38. Bergamo P, Balestrieri M, Cammarota G, Guardiola J, Abrescia P. CD4-mediated anchoring of the seminal antigen gp17 onto the spermatozoon surface. Hum Immunol 1997;58:30–41. 39. Loir M, Lanneau M. Structural function of the basic nuclear proteins in ram spermatids. J Ultrastruct Res 1984;86:262–72. 40. Hammadeh ME, Hamad MF, Montenarh M, Fischer-Hammadeh C. Protamine contents and P1/ P2 ratio in human spermatozoa from smokers and non-smokers. Hum Reprod 2010;25:2708–20.
Vol. 95, No. 8, June 30, 2011
One in 10 couples are infertile, and male factor defects account for as many as 50% of all infertility cases (1). Proteomics allows the differential expression of proteins. A large number of proteins such as semenogelin (SG) (2, 3), serine protease, prostatespecific antigen (PSA) (4), clusterin (5, 6), fibronectin (7), prolactin-induced protein (PIP) (8), heat shock protein, and lactoferrin (9) 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).
a
Stetson Thacker Satya P. Yadav, Ph.D.a Rakesh K. Sharma, Ph.D.b Anthony Kashou, B.S.b Belinda Willard, Ph.D.c Dongmei Zhang, Ph.D.c Ashok Agarwal, Ph.D.b a Molecular Biotechnology Core Laboratory, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio b Center for Reproductive Medicine, Glickman Urological & Kidney Institute, Cleveland Clinic and Obstetrics and Gynecology and Women’s Health Institute, Cleveland, Ohio c Proteomics Core, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio Received January 24, 2011; revised March 15, 2011; accepted March 17, 2011; published online May 4, 2011. S.T. has nothing to disclose. S.P.Y. has nothing to disclose. R.K.S. has nothing to disclose. A.K. has nothing to disclose. B.W. has nothing to disclose. D.Z. has nothing to disclose. A.A. has nothing to disclose. S.T. is a summer high school student. Reprint requests: Ashok Agarwal, Ph.D., Center for Reproductive Medicine, Glickman Urological & Kidney Institute, Cleveland Clinic, 9500 Euclid Avenue, Desk A19.1, Cleveland, Ohio 44195 (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.03.112
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 mL of 10 ng/mL) in 50 mM ammonium bicarbonate. The extracts were combined and evaporated to reduce the volume to 10 mL. The extracts were subsequently resuspended in 15% acetic acid to make a final volume of approximately 30 mL 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.
Fertility and Sterility Vol. 95, No. 8, June 30, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
2745
FIGURE 1 Coomassie blue-stained 2-dimensional gel from 1 control and 3 infertile patients. Ten spots were obtained from 1 control and 4 unique spots from 3 infertile patients. All spots from the control and patients were superimposed. Only those spots that were not common in both but unique (11–14) were examined. S1 ¼ control; S2–S4 ¼ infertile patients.
Thacker. Correspondence. Fertil Steril 2011.
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.
2746
Thacker et al.
Correspondence
A peptide with a mass ranging between 1,627 and 1,643 Da was identified in spot 11, corresponding to peptide (119)ELGICPD DAAVIPIK(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 (10). 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 (11). They are characterized by a basic isoelectric point (>9.5) and a high capacity for zinc binding (10, 12, 13). 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 (14). 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 (9, 15) and might therefore be expected to have a positive impact on sperm motility. PSA is a 33-kDa androgen-regulated serine
Vol. 95, No. 8, June 30, 2011
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 (16). Clusterin is a 70- to 80-kDa highly conserved heterodimeric glycoprotein (17). It has been implicated in a number of diverse biologic processes, including cell-cell interactions (5, 6), sperm maturation (18, 19), apoptosis (20), complement inhibition (21), lipid transport (22, 23), tissue remodeling, membrane recycling (24, 25), clearance of cellular debris (26), and degradation of the extracellular matrix (27). Clusterin prevents damaging oxidative reactions (28), protein precipitation (29), and agglutination of abnormal spermatozoa (17, 30) and controls complement-induced sperm lysis (31). 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 (17, 29, 30, 32).
PIP is an aspartyl proteinase with specificity to fibronectin, one of the major protein constituents of the seminal coagulum (9, 33–37). It is found in the postacrosomal zone and may have a role in fertilization (38). 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 (39, 40). 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.
REFERENCES 1. Sharlip ID, Jarow JP, Belker AM, Lipshultz LI, Sigman M, Thomas AJ, et al. Best practice policies for male infertility. Fertil Steril 2002;77:873–82. 2. Lilja 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;80:281–5. 3. Jonsson M, Frohm B, Malm J. Binding of semenogelin I to intact human spermatozoa studied by flow cytometry and surface plasmon resonance. J Androl 2010;6:560–5. 4. Kise 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;15:88–96. 5. Fritz IB, Burdzy K, Setchell B, Blaschuk O. Ram rete testis fluid contains a protein (clusterin) which influences cell-cell interaction in vitro. Biol Reprod 1983;28:1173–88. 6. Silkensen JR, Skubitz KM, Skubitz AP, Chmielewski DH, Manivel JC, Dvergsten JA, et al. Clusterin promotes the aggregation and adhesion of renal porcine epithelial cells. J Clin Invest 1995;96:2646–53. 7. McGee RS, Herr JC. Human seminal vesiclespecific antigen is a substrate for prostate-specific antigen (or P-30). Biol Reprod 1988;39:499–510. 8. Haagensen DE, Mazoujian G. Biochemistry and immunohistochemistry of fluid proteins of the breast in gross cystic disease. In: Haagensen DE, ed. Diseases of the breast. Philadelphia: W.B. Saunders Co., 1986:474–500. 9. Lilja H, Abrahamsson PA, 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;25:1894–900. 10. Robert M, Gagnon C, Semenogelin I. A coagulum forming, multifunctional seminal vesicle protein. Cell Mol Life Sci 1999;55:944–60.
Fertility and Sterility
11. Lilja H, Lundwall A. Molecular-cloning of epididymal and seminal vesicular transcripts encoding a semenogelin-related protein. Proc Natl Acad Sci U S A 1992;89:4559–63. 12. Mandal A, Bhattacharyya AK. Biochemical composition of washed seminal coagulum in comparison to sperm-free semen from the same donors. J Reprod Fertil 1990;88:113–8. 13. Robert M, Gibbs BF, 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;36:3811–9. 14. Zhao C, Huo R, Wang FQ, Lin M, Zhou ZM, Sha JH. Identification of several proteins involved in regulation of sperm motility by proteomic analysis. Fertil Steril 2007;87:436–8. 15. 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. Biol Reprod 1996;55:813–21. 16. Ahlgren G, Rannevik G, Lilja H. Impaired secretory function of the prostate in men with oligoasthenozoospermia. J Androl 1995;16:491–8. 17. O’Bryan MK, Baker HW, Saunders JR, Kirszbaum L, Walker ID, Hudson P, et al. Human seminal clusterin (SP-40). Isolation and characterization. J Clin Invest 1990;85:1477–86. 18. Collard MW, Griswold MD. Biosynthesis and molecular cloning of sulfated glycoprotein 2 secreted by rat Sertoli cells. Biochemistry 1987;16:3297–303. 19. Sylvester SR, Morales C, Oko R, Griswold MD. Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat. Biol Reprod 1991;45:195–207. 20. Buttyan R, Olsson CA, Pintar J, Chang C, Bandyk M, Ng PY, et al. Induction of the TRPM-2 gene in cells undergoing programmed death. Mol Cell Biol 1989;9:3473–81. 21. Murphy BF, Kirszbaum L, Walker ID, d’Apice AJ. SP-40, 40, a newly identified normal human serum
22.
23.
24.
25.
26.
27.
28.
protein found in the SC5b-9 complex of complement and in the immune deposits in glomerulonephritis. J Clin Invest 1988;81:1858–64. de Silva HV, Stuart WD, Duvic CR, Wetterau JR, Ray MJ, Ferguson DG. A 70-kDa apolipoprotein designated apo J is a marker for subclasses of human plasma high density lipoproteins. J Biol Chem 1990;265:13240–7. Jenne DE, 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;86:7123–7. Danik M, Chabot JG, Mercier C, Benabid AL, Chauvin C, Quirion R, et al. Human gliomas and epileptic foci express high levels of a mRNA related to rat testicular sulfated glycoprotein 2, a purported marker of cell death. Proc Natl Acad Sci U S A 1991;88:8577–81. Palmer DJ, Christie DL. Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H), and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules. J Biol Chem 1992;267:19806–12. Bartl MM, Luckenbach T, Bergner O, Ullrich O, Koch-Brandt C. Multiple receptors mediate apoJdependent clearance of cellular debris into nonprofessional phagocytes. Exp Cell Res 2001;15: 130–41. Matsuda A, Itoh Y, Koshikawa N, Akizawa T, Yana I, Seiki M. Clusterin, an abundant serum factor, is a possible negative regulator of MT6MMP/MMP-25 produced by neutrophils. J Biol Chem 2003;278:36350–7. Reyes-Moreno C, Boilard M, Sullivan R, Sirard MA. Characterization and identification of epididymal factors that protect ejaculated bovine sperm during in vitro storage. Biol Reprod 2002;66:159–66.
2747
29. Ibrahim NM, Gilbert GR, Loseth KL, Crabo BG. Correlation between clusterin-positive spermatozoa determined by flow cytometry in bull semen and fertility. J Androl 2000;21:887–94. 30. O’Bryan MK, Murphy BF, Liu DY, Clarke GN, Baker HW. The use of anticlusterin monoclonal antibodies for the combined assessment of human sperm morphology and acrosome integrity. Hum Reprod 1994;9:1490–6. 31. Jenne DE, Lowin B, Peitsch MC, B€ottcher 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;266:11030–6. 32. Clark AM, Griswold MD. Expression of clusterin/ sulfated glycoprotein-2 under conditions of heat stress in rat Sertoli cells and a mouse Sertoli cell line. J Androl 1997;18:257–63.
2748
Thacker et al.
33. Caputo E, Manco G, Mandrich L, Guardiola J. A novel aspartyl proteinase from apocrine epithelia and breast tumors. J Biol Chem 2000;275: 7935–41. 34. Autiero 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;197:268–71. 35. Schaller J, Akiyama K, Kimura H, Hess D, Affolter M, Rickli EE. Primary structure of a new actin-binding protein from human seminal plasma. Eur J Biochem 1991;196:743–50. 36. Caputo E, Carratore V, Ciullo M, Tiberio C, Mani JC, 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;265:664–70.
Correspondence
37. Yoshida K, Yamasaki T, Yoshiike M, Takano S, Sato I, Iwamoto T. Quantification of seminal plasma motility inhibitor/semenogelin inhuman seminal plasma. J Androl 2003;24:878–84. 38. Bergamo P, Balestrieri M, Cammarota G, Guardiola J, Abrescia P. CD4-mediated anchoring of the seminal antigen gp17 onto the spermatozoon surface. Hum Immunol 1997;58:30–41. 39. Loir M, Lanneau M. Structural function of the basic nuclear proteins in ram spermatids. J Ultrastruct Res 1984;86:262–72. 40. Hammadeh ME, Hamad MF, Montenarh M, Fischer-Hammadeh C. Protamine contents and P1/ P2 ratio in human spermatozoa from smokers and non-smokers. Hum Reprod 2010;25:2708–20.
Vol. 95, No. 8, June 30, 2011