Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

Characterization of a human spermatozoa surface antigens recognized by HS56 monoclonal antibody

Authors
Seyyed Ahmad Reza Mahmoudi 1
Roya Ghods 1
Amir Hassan Zarnani 1
Mahnaz Heidari 2
Ali Ahmad Bayat 1
Ebrahim Torkabadi 2
Mohammad Mehdi Akhondi 1
Kazem Parivar 3
Mahmood Jeddi-Tehrani 1
Mohammad Reza Sadeghi 1
1 Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran
2 Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
3 Department of Biology, Faculty of Sciences, Islamic Azad University, Science & Research Campus, Tehran, Iran
Abstract
Introduction: Surface antigens of ovum and sperm have key role in the fertilization process. In this regard, study of these molecules and their biochemical, biophysical and physiological characteristics could be helpful in understanding the mechanism of fertilization. Moreover, many cases of infertility with unknown ethiology have been revealed to have defects in these molecules. This will further emphasize on the importance of molecules involved in fertilization. The aim of this study was to characterize the sperm sur-face antigen recognized by the monoclonal antibody HS56. Materials and Methods: Clone HS56 with known specificity to human sperm antigen were injected perit-oneally into the balb/c mice and the produced antibody was purified over protein- G affinity chromato-graphy column. The isotype of the monoclonal antibody (mAb) was determined by Enzyme-Linked Immu-nosorbent Assay (ELISA). Surface antigens extracted from human sperm by lithinm 3,5 diiodosalicylate (LIS) method were run on SDS-PAGE and the molecular weight of antigen recognized by HS56 mAb was determined by western blotting. For localization of this antigen, indirect immunofluorescence staining was performed. Finally involvement of this antigen in acrosome reaction was tested by inhibition assay. Wil-coxon test was used for statistical analysis. Results: By LIS extraction method many sperm antigens were isolated and seen on SDS-PAGE using ELISA, It was found that LIS extraction was a useful method for isolation of antigen recognized by HS56 mAb. The subclass of the mAb HS56 was shown to be IgG1. The molecular weight of the antigen was determined to be about 56±2 KDa and the molecule included disulfide bond (s) in its structure. The antigen of interest was localized on acrosome and midpeace region of human sperm. Finally our results showed that this antigen had no effect on acrosome reaction (P=0.11). Conclusion: Although the HS56 antigen does not play an important role in acrosome reaction, its further characterization with regard to sperm-oocyte binding may reveal a role in fertilization process.
Keywords
Acrosome reaction
Fertility
Monoclonal antibody
Sperm
Sperm surface antigens
Pinon R.Biology of human reproduction. Uni-versity Science Book. 2002;pp:147-149.
Boyse E.A., Old L.J. Some aspects of normal and abnormal cell surface genetics. Ann. Rev. Genet.1969;(3)269-290.
Myles D.G., Primakaff P., Bellve A.R. Surface domains of the Guina Pig sperm defined with monoclonal antibodies. Cell.1981;23(2)432-439.
Naz R.K. Application of sperm antigens in immunocontraception. Front Biosci.1996;1:87-95.
Boue F., Sullivan R. Cases of human infertility are associated with the absence of P34H an epidy- dimal sperm antigen. Biol Reprod.1996;(54)1018 -1024.
Boue F., Berabe B., De Lamirande. E., Gagnon C., Sullivan R. Human sperm-zona pellucida inte-raction is inhibited by an antiserum against a ham-ster sperm protein. Biol Reprod.1994;(51)577-587.
Holland M.K., Nixon B. The specificity of epi-dydimal secretory proteins. J. Reprod. Fertil. (Supplement).1998;(53)197-201.
Kirchhoff C. Gene expression in the epidy-dimis. Intern Rev Cyto.1999;(188)133-202.
Bohring C., Krause E., Haberman B., Kvaus W. Isolation and Identification of sperm membrane antigens recognized by antisperm anti-bodies and their possible role in immunological infertility disease. Mol Hum Reprod.2001;7(2) 113-118.
Johnson MH., Everitt B.J. Essential Reprodu-ction. 5th Edition, Section 4: Testicular function in the adult.2000;pp:53-68.
Alexander N.J., Bearwood D. An immunosor-ption assay for antibodies to spermatozoa: Comp-arison with agglutination and immobilization tests. Fertil Steril.1984;41(2)270-276.
Shapiro A.L., Vinuela E., Maizel J.B. Molec-ular weight estimation of polypeptide chains by electrophoresis in SDS-Polyacrilamide gels. Bioc-hem Biophys Res Commun.1967;(28) 815-820.
Risopatron J., Pena P., Miska W., Sanchez R. Evaluation of the acrosome reaction in human spermatozoa: Comparison of cytochemical and fluorescence techniques. Andrologia.2001;(33): 63-67.
Towbin H., Staehelin T., Gordon J. Electro-phoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheet. Proceduve and some applications. Proc Nat Acad Sci. USA.1979;76(9): 4350-4354.
Rajeev S.K., Reddy K.V. Sperm membrane protein profiles of fertile and infertile men: identi- fication and characterization of fertility-associated sperm antigen. Hum Reprod.2004;19(2)234-242.
Yoshiki T., Herr J.C., Lee C.G. Purification and characterization of a sperm antigen recognize-ed by HSA-5 monoclonal antibody. J Reprod Immun.1995;(29)209-222.
Isahakia M., Alexander N.J. Interspecies Crossreactivity of monoclonal antibodies directed against human sperm antigens. Bio Reprod.1984; (30):1015-1026.
Journal of Reproduction & Infertility
Volume 5, Issue 4
Autumn 2004
Pages 290-298