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. 1998 Jan 15;329(Pt 2):231–241. doi: 10.1042/bj3290231

Studies on glutathione S-transferases important for sperm function: evidence of catalytic activity-independent functions.

B Gopalakrishnan 1, S Aravinda 1, C H Pawshe 1, S M Totey 1, S Nagpal 1, D M Salunke 1, C Shaha 1
PMCID: PMC1219036  PMID: 9425104

Abstract

Our earlier studies reported the identification of a rat testicular protein of 24 kDa with significant similarity at the N-terminus with Mu class glutathione S-transferases (GSTs). Treatment of goat sperm with antisera against this protein identified immunoreactive sites on the spermatozoa and inhibited in vitro fertilization of goat oocytes by the antibody-treated sperm. The above observations indicated the presence of GST-like molecule(s) important for fertility related events on goat spermatozoa. In this study, we report the purification of goat sperm GSTs (GSP1) which were purified by glutathione affinity chromatography and were enzymically active towards 1-chloro-2,4,-dinitrobenzene, a general GST substrate, and ethacrynic acid, a substrate for Pi class GSTs. GSP1 resolved into three major components on reverse-phase HPLC: peaks 1 and 2 with molecular masses of 26.5 kDa and peak 3 with a molecular mass of 25.5 kDa, as determined by SDS/PAGE. Multiple attempts to obtain N-terminal sequences of the first two peaks failed, indicating N-terminal block; however, they reacted to specific anti-Mu-GST antisera on Western blots and ELISA, and not to anti-Pi-GST antisera, which provides evidence for the presence of Mu-GST-reactive sites on peaks 1 and 2. The third component showed 80% N-terminal similarity with human and rat GSTP1-1 over an overlap of 15 amino acids, and reacted to anti-Pi-specific antisera in ELISA. Sperm labelled with antibodies against a 10-mer and an 11-mer peptide, designed from the N-terminal sequences of Mu and Pi class GSTs respectively, showed the presence of both Mu- and Pi-GST on goat sperm surface at distinct cellular domains. Selective inhibition of Pi class GST by the Pi-specific antisera, either at 0 h or at 3 h after initiation of sperm capacitation, leads to a reduction in fertilization rates. In contrast, the inhibition of Mu class GST by specific antisera at 0 h does not inhibit fertilization, although such treatment at 3 h after the initiation of capacitation reduces fertilization rates. The results indicate that both Pi- and Mu-GSTs are involved in fertilization, but the Mu-GST sites essential for fertilization are exposed only after 3 h of capacitation. The enzymic activity of GSP1 or live spermatozoa is not inhibited by the two antisera. The inability of the antibodies to cause such inhibition indicates that the reduction in fertilization rates and acrosome reaction caused by the antibodies is through a mechanism which does not interfere with the catalytic activity of the molecule. Therefore we established the presence of Pi and Mu class GST on goat sperm, their localization and their possible function in fertility-related events.

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Selected References

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  1. Aravinda S., Gopalakrishnan B., Dey C. S., Totey S. M., Pawshe C. H., Salunke D., Kaur K., Shaha C. A testicular protein important for fertility has glutathione S-transferase activity and is localized extracellularly in the seminiferous tubules. J Biol Chem. 1995 Jun 30;270(26):15675–15685. doi: 10.1074/jbc.270.26.15675. [DOI] [PubMed] [Google Scholar]
  2. Avrameas S. Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry. 1969 Jan;6(1):43–52. doi: 10.1016/0019-2791(69)90177-3. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Brown R. E., Jarvis K. L., Hyland K. J. Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal Biochem. 1989 Jul;180(1):136–139. doi: 10.1016/0003-2697(89)90101-2. [DOI] [PubMed] [Google Scholar]
  5. Bryan H. D., Akruk S. R. A naphthol yellow S and erythrosin B staining procedure for use in studies of the acrosome reaction of rabbit spermatozoa. Stain Technol. 1977 Jan;52(1):47–51. doi: 10.3109/10520297709116742. [DOI] [PubMed] [Google Scholar]
  6. Campbell E., Takahashi Y., Abramovitz M., Peretz M., Listowsky I. A distinct human testis and brain mu-class glutathione S-transferase. Molecular cloning and characterization of a form present even in individuals lacking hepatic type mu isoenzymes. J Biol Chem. 1990 Jun 5;265(16):9188–9193. [PubMed] [Google Scholar]
  7. Cowan A. E., Primakoff P., Myles D. G. Sperm exocytosis increases the amount of PH-20 antigen on the surface of guinea pig sperm. J Cell Biol. 1986 Oct;103(4):1289–1297. doi: 10.1083/jcb.103.4.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DeJong J. L., Morgenstern R., Jörnvall H., DePierre J. W., Tu C. P. Gene expression of rat and human microsomal glutathione S-transferases. J Biol Chem. 1988 Jun 15;263(17):8430–8436. [PubMed] [Google Scholar]
  9. Evangelista M., Chersi A., Citro G. Specific recognition of human pi glutathione transferase by an antipeptide antibody. Biochim Biophys Acta. 1991 May 24;1074(1):214–216. doi: 10.1016/0304-4165(91)90064-n. [DOI] [PubMed] [Google Scholar]
  10. Fulcher K. D., Welch J. E., Klapper D. G., O'Brien D. A., Eddy E. M. Identification of a unique mu-class glutathione S-transferase in mouse spermatogenic cells. Mol Reprod Dev. 1995 Dec;42(4):415–424. doi: 10.1002/mrd.1080420407. [DOI] [PubMed] [Google Scholar]
  11. Fusi F. M., Bronson R. A. Sperm surface fibronectin. Expression following capacitation. J Androl. 1992 Jan-Feb;13(1):28–35. [PubMed] [Google Scholar]
  12. Gaunt S. J. A 28K-dalton cell surface autoantigen of spermatogenesis: characterization using a monoclonal antibody. Dev Biol. 1982 Jan;89(1):92–100. doi: 10.1016/0012-1606(82)90297-4. [DOI] [PubMed] [Google Scholar]
  13. Hayes J. D., Mantle T. J. Inhibition of hepatic and extrahepatic glutathione S-transferases by primary and secondary bile acids. Biochem J. 1986 Jan 15;233(2):407–415. doi: 10.1042/bj2330407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hayes J. D., Pulford D. J. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol. 1995;30(6):445–600. doi: 10.3109/10409239509083491. [DOI] [PubMed] [Google Scholar]
  15. Hussey A. J., Hayes J. D. Human Mu-class glutathione S-transferases present in liver, skeletal muscle and testicular tissue. Biochim Biophys Acta. 1993 Nov 10;1203(1):131–141. doi: 10.1016/0167-4838(93)90047-u. [DOI] [PubMed] [Google Scholar]
  16. Klys H. S., Whillis D., Howard G., Harrison D. J. Glutathione S-transferase expression in the human testis and testicular germ cell neoplasia. Br J Cancer. 1992 Sep;66(3):589–593. doi: 10.1038/bjc.1992.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Listowsky I., Abramovitz M., Homma H., Niitsu Y. Intracellular binding and transport of hormones and xenobiotics by glutathione-S-transferases. Drug Metab Rev. 1988;19(3-4):305–318. doi: 10.3109/03602538808994138. [DOI] [PubMed] [Google Scholar]
  19. Mannervik B., Danielson U. H. Glutathione transferases--structure and catalytic activity. CRC Crit Rev Biochem. 1988;23(3):283–337. doi: 10.3109/10409238809088226. [DOI] [PubMed] [Google Scholar]
  20. Manoharan T. H., Gulick A. M., Puchalski R. B., Servais A. L., Fahl W. E. Structural studies on human glutathione S-transferase pi. Substitution mutations to determine amino acids necessary for binding glutathione. J Biol Chem. 1992 Sep 15;267(26):18940–18945. [PubMed] [Google Scholar]
  21. Marquant-Le Guienne B., De Almeida M. Role of guinea-pig sperm autoantigens in capacitation and the acrosome reaction. J Reprod Fertil. 1986 Jul;77(2):337–345. doi: 10.1530/jrf.0.0770337. [DOI] [PubMed] [Google Scholar]
  22. Mukhtar H., Lee I. P., Bend J. R. Glutathione S-transferase activities in rat and mouse sperm and human semen. Biochem Biophys Res Commun. 1978 Aug 14;83(3):1093–1098. doi: 10.1016/0006-291x(78)91507-3. [DOI] [PubMed] [Google Scholar]
  23. Myles D. G., Primakoff P. Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization. J Cell Biol. 1984 Nov;99(5):1634–1641. doi: 10.1083/jcb.99.5.1634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Oliphant G., Reynolds A. B., Thomas T. S. Sperm surface components involved in the control of the acrosome reaction. Am J Anat. 1985 Nov;174(3):269–283. doi: 10.1002/aja.1001740308. [DOI] [PubMed] [Google Scholar]
  25. Olson G. E., Orgebin-Crist M. C. Sperm surface changes during epididymal maturation. Ann N Y Acad Sci. 1982;383:372–392. doi: 10.1111/j.1749-6632.1982.tb23179.x. [DOI] [PubMed] [Google Scholar]
  26. Pawshe C. H., Totey S. M., Jain S. K. A comparison of three methods of recovery of goat oocytes for in vitro maturation and feritilization. Theriogenology. 1994;42(1):117–125. doi: 10.1016/0093-691x(94)90668-9. [DOI] [PubMed] [Google Scholar]
  27. Phelps B. M., Myles D. G. The guinea pig sperm plasma membrane protein, PH-20, reaches the surface via two transport pathways and becomes localized to a domain after an initial uniform distribution. Dev Biol. 1987 Sep;123(1):63–72. doi: 10.1016/0012-1606(87)90428-3. [DOI] [PubMed] [Google Scholar]
  28. Primakoff P., Hyatt H., Myles D. G. A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida. J Cell Biol. 1985 Dec;101(6):2239–2244. doi: 10.1083/jcb.101.6.2239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ricci G., Lo Bello M., Caccuri A. M., Galiazzo F., Federici G. Detection of glutathione transferase activity on polyacrylamide gels. Anal Biochem. 1984 Dec;143(2):226–230. doi: 10.1016/0003-2697(84)90657-2. [DOI] [PubMed] [Google Scholar]
  30. Rochwerger L., Cuasnicu P. S. Redistribution of a rat sperm epididymal glycoprotein after in vitro and in vivo capacitation. Mol Reprod Dev. 1992 Jan;31(1):34–41. doi: 10.1002/mrd.1080310107. [DOI] [PubMed] [Google Scholar]
  31. Shaha C. Antibody-induced changes on rabbit sperm surface inhibit gamete interaction. Mol Reprod Dev. 1994 Aug;38(4):393–403. doi: 10.1002/mrd.1080380407. [DOI] [PubMed] [Google Scholar]
  32. Shaha C., Seshadri T., Suri A., Talwar G. P. Characterisation of 24-kD proteins from rat testes using polyclonal sera reactive to human sperm antigens. Mol Reprod Dev. 1991 Jul;29(3):302–311. doi: 10.1002/mrd.1080290314. [DOI] [PubMed] [Google Scholar]
  33. Shaha C., Sheshadri T., Talwar G. P., Suri A. Characterization of a monoclonal antibody against a 24KD antigen from rat testis important for fertility regulation. Hybridoma. 1989 Dec;8(6):647–660. doi: 10.1089/hyb.1989.8.647. [DOI] [PubMed] [Google Scholar]
  34. Shaha C., Suri A., Talwar G. P. Identification of sperm antigens that regulate fertility. Int J Androl. 1988 Dec;11(6):479–491. doi: 10.1111/j.1365-2605.1988.tb01022.x. [DOI] [PubMed] [Google Scholar]
  35. Shaha C., Suri A., Talwar G. P. Induction of infertility in female rats after active immunization with 24 kD antigens from rat testes. Int J Androl. 1990 Feb;13(1):17–25. doi: 10.1111/j.1365-2605.1990.tb00956.x. [DOI] [PubMed] [Google Scholar]
  36. Shaha C., Taneja M., Seshadri T. Monoclonal antibody against a human sperm protein recognizes multiple epitopes on rabbit and human sperm and blocks sperm function. Hybridoma. 1993 Dec;12(6):709–718. doi: 10.1089/hyb.1993.12.709. [DOI] [PubMed] [Google Scholar]
  37. Shur B. D. Glycosyltransferases as cell adhesion molecules. Curr Opin Cell Biol. 1993 Oct;5(5):854–863. doi: 10.1016/0955-0674(93)90035-o. [DOI] [PubMed] [Google Scholar]
  38. Switzer R. C., 3rd, Merril C. R., Shifrin S. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem. 1979 Sep 15;98(1):231–237. doi: 10.1016/0003-2697(79)90732-2. [DOI] [PubMed] [Google Scholar]
  39. Totey S. M., Pawshe C. H., Singh G. P. In vitro maturation and fertilization of buffalo oocytes (Bubalus bubalis ): Effects of media, hormones and sera. Theriogenology. 1993 May;39(5):1153–1171. doi: 10.1016/0093-691x(93)90014-v. [DOI] [PubMed] [Google Scholar]
  40. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Warholm M., Guthenberg C., von Bahr C., Mannervik B. Glutathione transferases from human liver. Methods Enzymol. 1985;113:499–504. doi: 10.1016/s0076-6879(85)13065-x. [DOI] [PubMed] [Google Scholar]
  42. Willingham M. C. Fluorescence labeling of surface antigens of attached or suspended tissue-culture cells. Methods Mol Biol. 1994;34:123–130. doi: 10.1385/0-89603285-x:123. [DOI] [PubMed] [Google Scholar]

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