BACKGROUND: Poor semen quality is one of the main causes of infertility. We have generated a set of monoclonal antibodies to human sperm and used them to investigate sperm quality. Some of these antibodies found differences in the expression of proteins between normal sperm and pathological sperm displaying severe defects. One of them was the Hs-14 antibody. The aim of this paper was to determine the target protein of the Hs-14 monoclonal antibody and to investigate the expression of the Hs-14-reacting protein on the sperm of asthenozoospermic men with sperm motility defect and of healthy normozoospermic men. METHODS: Indirect immunofluorescence, one-dimensional and two-dimensional polyacrylamide gel electrophoresis, immunoblotting and mass spectrometry. RESULTS: The Hs-14 antibody binds fibronectin, β-tubulin and valosin-containing protein - new name for this protein is transitional endoplasmic reticulum ATPase (TERA). Since the Hs-14 reaction with TERA remained the strongest at the highest antibody dilution, and Hs-14 consistently labelled the same spot or band as the monospecific anti-TERA antibody on immunoblots, we assume that TERA is an Hs-14-specific protein. Binding of fibronectin and β-tubulin might represent nonspecific cross-reactivity or Hs-14 reaction with similar epitopes of these proteins. A significant difference (P < 0.001) in immunofluorescence staining with Hs-14 was found between the normozoospermic and asthenozoospermic men. CONCLUSION: The Hs-14 antibody enables discrimination between sterile or subfertile asthenozoospermic and fertile normozoospermic men. Decreased levels of TERA in men can be used as a biomarker of reduced fertility.

INTRODUCTION: La pauvre qualité de la semence est l’une des causes d’infertilité. Nous avons généré une série d’anticorps monoclonaux contre le sperme humain et nous l’avons utilisée pour examiner la qualité du sperme. Certains de ces anticorps ont montré des différences d’ expression des protéines entre le sperme normal et le sperme pathologique qui a des défauts sévères. L’un d’eux a été l’anticorps Hs-14. Le but de cet article était de déterminer la protéine cible de l’anticorps monoclonal Hs-14 et d’établir l’expression de la protéine réagissant avec Hs-14 sur le sperme des hommes asthénozoospermiques qui ont des défauts de la mobilité du sperme et sur celui des hommes normozoospermiques. MÉTHODES: Immunofluorescence indirecte, electrophorèse sur gel polyacrylamide à une ou deux dimensions, immunoblotting et spectrométrie de masse. RÉSULTATS: L’anticorps Hs-14 s’attache à la fibronectine, à la β-tubuline et à la protéine TERA (ATPase transitoire de réticulum endoplasmique). Etant donné que la réaction du Hs-14 avec TERA a été la plus forte à la dilution la plus grande de l’anticorps, et que Hs-14 marquait systématiquement la même tache ou bande que l’anticorps mono-spécifique anti-TERA sur les immunoblots, nous supposons que TERA est une protéine spécifique pour Hs-14. L’attachement à la fibronectine et à la β-tubuline pourrait représenter une réaction croisée non spécifique ou la réaction du Hs-14 avec des épitopes similaires de ces protéines. Une différence significative (P < 0.001) en immunofluorescence avec Hs-14 a été révélée entre hommes normozoospermiques et asthénozoospermiques. CONCLUSIONS: L’anticorps Hs-14 permet de différencier les hommes stériles ou subfertiles asthénozoospermiques des hommes fertiles normozoospermiques. Les niveaux de la TERA chez les hommes pourraient être utilisés comme un marqueur biologique d’une fertilité réduite.

Institute of Microbiology AS CR v v i Videnska 1083 142 20 Prague 4 Czech Republic

Laboratory of Reproductive Biology Institute of Biotechnology AS CR the Czech Academy of Sciences v v i Videnska 1083 142 20 Prague 4 Czech Republic

Zobrazit více v PubMed

WHO. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. 5th ed. Cambridge: Cambridge University Press; 2010.

Reddy KV, Meherji PK, Shahani SK. Integrin cell adhesion molecules on human spermatozoa. Indian J Exp Biol. 1998;36(5):456–63. PubMed

Gadkar S, Shah CA, Sachdeva G, Samant U, Puri CP. Progesterone receptor as anindicator of sperm function. Biol Reprod. 2002;67(4):1327–36. doi: 10.1095/biolreprod67.4.1327. PubMed DOI

Nasr-Esfahani MH, Salehi M, Razavi S, Mardani M, Bahramian H, Steger K, Oreizi F. Effect of protamine-2 deficiency on ICSI outcome. Reprod Biol Online. 2004;9(6):652–8. doi: 10.1016/S1472-6483(10)61776-2. PubMed DOI

Tepla O, Peknicova J, Koci K, Mika J, Mrazek M, Elzeinova F. Evaluation of reproductive potencial after intracytoplasmic sperm injection of varied human semen tested by antiacrosomal antibodies. Fertil Steril. 2006;86(1):113–20. doi: 10.1016/j.fertnstert.2005.12.019. PubMed DOI

Anahí Franchi N, Avedano C, Molina RI, Tissera AD, Maldonado CA, Oehninger S, Coronel CE. Beta-Microseminoprotein in human spermatozoa and its potential role in male fertility. Reproduction. 2008;136(2):157–66. doi: 10.1530/REP-08-0032. PubMed DOI

Sutovsky P, Lovercamp K. Molecular markers of sperm quality. Soc Reprod Fertil Suppl. 2010;67:247–56. PubMed

Peknicova J, Chladek D, Hozak P. Monoclonal antibodies against sperm intra- acrosomal antigens as markers for male infertility diagnostics and estimation of spermatogenesis. Am J Reprod Immunol. 2005;53:42–9. doi: 10.1111/j.1600-0897.2004.00245.x. PubMed DOI

Peknicova J, Kyselova V, Buckiova D, Boubelik M. Effect of an endocrine disruptor on mammalian fertility. Application of monoclonal antibodies against sperm proteins as markers for testing sperm damage. AJRI. 2002;47:311–8. PubMed

Kyselova V, Peknicova J, Boubelik M, Buckiova D. Body and organ weight, sperm acrosomal status and reproduction after genistein and diethylstibestrol treatment of CD1 mice in multigenerational study. Theriogenology. 2004;61:1307–25. doi: 10.1016/j.theriogenology.2003.07.017. PubMed DOI

Pixton KL, Deeks ED, Flesch FM, Moseley FLC, Bjorndahl L, Ashton R, Barratt CLR, Brevis IA. Sperm proteome mapping of a patient who experienced failed fertilization at IVF reveals altered expression of at least 20 proteins compared with fertile donors: case report. Hum Reprod. 2004;19(6):1438–47. doi: 10.1093/humrep/deh224. PubMed DOI

Shimizu Y, Kodama H, Fukuda J, Tanaka T. Evidence of proacrosine molecule abnormality as a possible cause of low acrosin activity and unexplained failure of fertilization in vitro. J Androl. 1997;18(3):281–8. PubMed

Capkova J, Elzeinova F, Novak P. Increased expression of secretory actin-binding protein on human spermatozoa is associated with poor semen quality. Hum Reprod. 2007;22(5):1396–404. doi: 10.1093/humrep/del511. PubMed DOI

Oliva R, Martínez-Heredia J, Estanyol JM. Proteomics in the study of the sperm cell composition, differentiation and function. Syst Biol Reprod Med. 2008;54(1):23–36. doi: 10.1080/19396360701879595. PubMed DOI

duPlessis SS, Kashou AH, Benjamin DJ, Yadav SP, Agarval A. Proteomics: a subcellular look at spermatozoa. Reprod Biol Endocrinol. 2011;9:36. doi: 10.1186/1477-7827-9-36. PubMed DOI PMC

Baker MA, Nixon B, Naumovski N, Aitken RJ. Proteomic insights into the maturation and capacitation of mammalian spermatozoa. Syst Biol Reprod Med. 2012;58(4):211–7. doi: 10.3109/19396368.2011.639844. PubMed DOI

Liao TT, Xiang Z, Zhu WB, Fan LQ. Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electophoresis and mass spectrometry. Asian J Androl. 2009;11(6):638–93. doi: 10.1038/aja.2009.45. PubMed DOI PMC

Thacker S, Yadav SP, Sharma RK, Kashou A, Williard B, Zhang D, Agarval A. Evaluation of sperm proteins in infertile men: a proteomic approach. Fertil Steril. 2011;95(8):2745–8. doi: 10.1016/j.fertnstert.2011.03.112. PubMed DOI

Peknicova J, Capkova J, Cechova D, Sulcova B. Preparation and characterization of a monoclonal antibody against boar acrosin. Folia Biol (Praha) 1986;32:282–5. PubMed

Koubek P, Elzeinova F, Sulc M, Linhart O, Peknicova J. Monoclonal antibody FsC-47 against carp sperm creatine kinase. Hybridoma. 2006;25(3):154–7. doi: 10.1089/hyb.2006.25.154. PubMed DOI

Peknicova J, Moos J. Monoclonal antibodies against sperm intra-acrosomal antigens labelling undamaged acrosomes of spermatozoa in immunofluorescence test. Andrologia. 1990;22(5):427–35. doi: 10.1111/j.1439-0272.1990.tb02022.x. PubMed DOI

Moos J, Peknicova J. Monoclonal antibodies against progesterone. Am J Reprod Med. 1988;16:88.

Dráber P, Dráberová E, Linhartová I, Viklický V. Differences in the exposure of C-and N- terminal tubulin domains in cytoplasmic microtubules detected with domain-specific monoclonal antibodies. J Cell Sci. 1989;92:519–28. PubMed

Grimm E, Breitling F, Little M. Location of the epitope for the alpha-tubulin monoclonal antibody TU-01. Biochim Biophys Acta. 1987;914:83–8. doi: 10.1016/0167-4838(87)90164-6. PubMed DOI

Shelanski ML, Gaskin F, Cantor CR. Microtule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973;70:765–8. doi: 10.1073/pnas.70.3.765. PubMed DOI PMC

Dráber P, Dráberová E, Viklický V. Immunostaining of human spermatozoa with tubulin domain-specific monoclonal antibodies. Recognition of a unique beta- tubulin epitope in the sperm head. Histochemistry. 1991;95:319–24. doi: 10.1007/BF00315749. PubMed DOI

Laemmli UK. Cleavage of structural proteins during assembly of bacteriofage T4. Nature. 1970;227:680–5. doi: 10.1038/227680a0. PubMed DOI

Towbin H, Staehelin T, Gordon G. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979;76:4350–4. doi: 10.1073/pnas.76.9.4350. PubMed DOI PMC

Glander HJ, Schaller J, Rohwedder A, Henkel R. Adhesion molecules and matrix proteins on human spermatozoa. Andrologia. 1998;30(4-5):289–96. doi: 10.1111/j.1439-0272.1998.tb01173.x. PubMed DOI

Wennemuth G, Schiemann PJ, Krause W, Gressner AM, Aumuller G. Influence of fibronectin on the motility of human spermatozoa. Int J Androl. 1997;20(1):10–6. doi: 10.1046/j.1365-2605.1997.00005.x. PubMed DOI

Diaz ES, Kong M, Morales P. Effect of fibronectin on proteasome activity, acrosome reaction, tyrosine phosphorylation and intracellular calcium concentrations of human sperm. Hum Reprod. 2007;22(5):1420–30. doi: 10.1093/humrep/dem023. PubMed DOI

Thys M, Nauwynck H, Maes D, Hoogewijs M, Vercauteren D, Rijsselaere T, Favoreel H, Van Soom A. Expression and putative function of fibronectin and its receptor (integrin alpha(5)beta(1) in male and female gametes during bovine fertilization in vitro. Reproduction. 2009;138(3):471–82. doi: 10.1530/REP-09-0094. PubMed DOI

Wennemuth G, Meinhardt A, Mallidis C, Albrecht M, Krause W, Renneberg H, Aumuller G. Assessment of fibronectin as a potencial new clinical tool in andrology. Andrologia. 2001;33(1):43. doi: 10.1046/j.1439-0272.2001.00370.x. PubMed DOI

Attia AM, Hassan A, Zalata A, Hagag M, Yousef KE, Mostala T. Seminal fibronectin In fertile and infertile males. Andrologia. 2011;43(6):387–91. doi: 10.1111/j.1439-0272.2010.01056.x. PubMed DOI

Eddy EM, O’Brian DA. The spermatozoon. In: Knobil E, Neil JD, editors. The physiology of reproduction. New York: Raven; 1994.

Virtanen I, Badley RA, Paasivuo R, Lehto VP. Distinct cytoskeletal domains revealed in sperm cells. J Cell Biol. 1984;99:1083–91. doi: 10.1083/jcb.99.3.1083. PubMed DOI PMC

Peknicova J, Pexiderova M, Kubatova A, Koubek P, Tepla O, Sulimenko T, Draber P. Expression of beta-tubulin epitope in human with pathological spermiogram. Fertil Steril. 2007;88(2):1120–8. doi: 10.1016/j.fertnstert.2006.12.070. PubMed DOI

Dai RM, Li CC. Valosin-containing protein is a multi-ubiquitin chain-targeting factor required in ubiquitin-proteasome degradation. Nat Cell Biol. 2001;3:740–4. doi: 10.1038/35087056. PubMed DOI

Geussova G, Kalab P, Peknicova J. Valosine containing protein is a substrate of cAMP-activated boar sperm tyrosine kinase. Mol Reprod Dev. 2002;63:366–75. doi: 10.1002/mrd.10156. PubMed DOI

Ficarro S, Chertihin O, Westbrook A, White F, Jayes F, Kalab P, Marto JA, Shabanowitz J, Herr JC, Hunt DF, Visconti PE. Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation. J Biol Chem. 2003;278(13):11579–89. doi: 10.1074/jbc.M202325200. PubMed DOI

Vigodner M, Shrivastava V, Gutstein LE, Schneider J, Nieves E, Goldstein M, Feliciano M, Callaway M. Localization and identification of sumoylated proteins in human sperm: excessive sumoylation is a marker of defective spermatozoa. Hum Reprod. 2013;28(1):210–23. doi: 10.1093/humrep/des317. PubMed DOI PMC

The Transgenerational Transmission of the Paternal Type 2 Diabetes-Induced Subfertility Phenotype