Abstract
The sperm of the mussel Mytilus had hydrolytic activities against substrates for aminopeptidase. Acrosome reaction (AR) was suppressed in the presence of aminopeptidase substrate, Phe‐4‐methylcoumaryl‐7‐amide (MCA), and an aminopeptidase inhibitor, bestatin. Treatment of sperm with phosphatidylinositol‐specific phospholipase C (PI‐PLC) released aminopeptidase activity from sperm and suppressed AR. These results suggest that the enzyme is located on the sperm surface via glycosylphosphatidylinositol (GPI)‐anchor and is involved in the AR. Immunoblot analysis showed that tyrosine residues of 40, 59, 68, and 72 kDa proteins were phosphorylated during induction of the AR. The 40 kDa protein was also recognized by anti‐c‐Src antibody by immunoblotting. The tyrosine phosphorylation of these proteins was inhibited when sperm were inseminated in the presence of Phe‐MCA, and by PI‐PLC treatment. Treatment of sperm with tyrosine kinase activator, 9,10‐dimethyl‐1,2‐benzanthracene, induced AR, and its inhibitor, genistein, suppressed AR. These results suggest that tyrosine phosphorylation of 40, 59, 68, and 72 kDa proteins, induced by the interaction of GPI‐anchored aminopeptidase with oocyte surface, triggers AR in Mytilus sperm. Mol. Reprod. Dev. 67: 465–471, 2004. © 2004 Wiley‐Liss, Inc.
Keywords: fertilization, GPI‐anchored protein, tyrosine kinase
REFERENCES
- Archuleta MM, Schieven GL, Ledbetter JA, Deanin GG, Burchiel SW. 1993. 7,12‐Dimethylbenz[a]anthracene activates protein‐tyrosine kinases Fyn and Lck in the HPB‐ALL human T‐cell line and increases tyrosine phosphorylation of phospholipase C‐γ1, formation of inositol 1,4,5‐trisphosphate, and mobilization of intracellular calcium. Proc Natl Acad Sci USA 90: 6105–6109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Arienti G, Carlini E, Verdacchi R, Cosmi EV, Palmerini CA. 1997. Transfer of aminopeptidase activity from prostasomes to sperm. Biochim Biophys Acta 1336: 269–274. [DOI] [PubMed] [Google Scholar]
- Baldi E, Luconi M, Bonaccorsi L, Forti G. 2002. Signal transduction pathways in human spermatozoa. J Reprod Immunol 53: 121–131. [DOI] [PubMed] [Google Scholar]
- Breitbart H. 2002. Role and regulation of intracellular calcium in acrosomal exocytosis. J Reprod Immunol 53: 151–159. [DOI] [PubMed] [Google Scholar]
- Brown DA, London E. 1998. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 14: 111–136. [DOI] [PubMed] [Google Scholar]
- Cherr GN, Yudin AI, Overstreet JW. 2001. The dual functions of GPI‐anchored PH‐20: Hyaluronidase and intracellular signaling. Matrix Biol 20: 515–525. [DOI] [PubMed] [Google Scholar]
- Delmas B, Gelfi J, L'Haridon R, Vogel LK, Sjöström H, Norén O, Laude H. 1992. Aminopeptidase N is a major receptor for the enteropathogenic coronavirus TGEV. Nature 357: 417–420. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Godknecht A, Honegger TG. 1991. Isolation, characterization, and localization of a sperm‐bound N‐acetylglucosaminidase that is indispensable for fertilization in the ascidian, Phallusia mammillata . Dev Biol 143: 398–407. [DOI] [PubMed] [Google Scholar]
- Godknecht AJ, Honegger TG. 1995. Specific inhibition of sperm β‐N‐acetylglucosaminidase by the synthetic inhibitor N‐acetylglucosaminono‐1,5‐lactone O‐(phenylcarbomoyl)oxime inhibits fertilization in the ascidian, Phallucia mammillata . Dev Growth Differ 37: 183–189. [DOI] [PubMed] [Google Scholar]
- Harder T, Simons K. 1999. Clusters of glycolipid and glycosylphosphatidylinositol‐anchored proteins in lymphoid cells: Accumulation of actin regulated by local tyrosine phosphorylation. Eur J Immunol 29: 556–562. [DOI] [PubMed] [Google Scholar]
- Harder T, Scheiffele P, Verkade P, Simons K. 1998. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J Cell Biol 141: 929–942. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoshi M. 1984. Roles of sperm glycosidases and proteases in the ascidian fertilization In: Engels W, Clark WH, Jr., Fischer A, Olive PJW, Went DF, editors. Advances in invertebrate repdoduction. Amsterdam: Elsevier; pp 431–462. [Google Scholar]
- Hoshi M. 1986. Sperm glycosidase as a plausible mediator of sperm binding to the vitelline envelope in ascidians In: Hedrick JL, editor. The molecular and cellular biology of fertilization. New York: Plenum Press; pp 251–260. [DOI] [PubMed] [Google Scholar]
- Hua G, Tsukamoto K, Rasilo M‐L, Ikezawa H. 1998. Molecular cloning of a GPI‐anchored aminopeptidase N from Bombyx mori midgut: A putative receptor for Bacillus thuringiensis GryIA toxin. Gene 214: 177–185. [DOI] [PubMed] [Google Scholar]
- Kasahara K, Sanai Y. 2000. Functional roles of glycosphingolipids in signal transduction via lipid rafts. Glycoconj J 17: 153–162. [DOI] [PubMed] [Google Scholar]
- Knight PJK, Knowless BH, Ellar DJ. 1995. Molecular cloning of an insect aminopeptidase N that serves as a receptor for Bacillus thuringiensis CryIA(c) toxin. J Biol Chem 270: 17765–17770. [DOI] [PubMed] [Google Scholar]
- Kolb AF, Hegyi A, Maile J, Heister A, Hagemann M, Siddell SG. 1998. Molecular analysis of the coronavirus‐receptor function of aminopeptidase N. Adv Exp Med Biol 440: 61–67. [DOI] [PubMed] [Google Scholar]
- Longo FJ. 1983. Meiotic maturation and fertilization In: Wilbur KM, editor. The mollusca. Vol. 3. New York: Academic Press; pp 49–89. [Google Scholar]
- Low MG, Finean JB. 1978. Specific release of plasma membrane enzymes by a phosphatidylinositol‐specific phospholipase C. Biochim Biophys Acta 508: 565–570. [DOI] [PubMed] [Google Scholar]
- Matsumoto M, Hirata J, Hirohashi N, Hoshi M. 2002. Sperm–egg binding mediated by sperm α‐l‐fucosidase in the ascidian, Halocynthia roretzi . Zool Sci 19: 43–48. [DOI] [PubMed] [Google Scholar]
- Ohta K, Sato C, Matsuda T, Toriyama M, Vacquier VD, Lennarz WJ, Kitajima K. 2000. Co‐localization of receptor and transducer proteins in the glycosphingolipid‐enriched, low density, detergent‐insoluble membrane fraction of sea urchin sperm. Glycoconj J 17: 205–214. [DOI] [PubMed] [Google Scholar]
- Schaller J, Glander HJ. 2000. Flow cytometric analysis of enzymes in live spermatozoa before and after cryostorage. Andrologia 32: 357–364. [DOI] [PubMed] [Google Scholar]
- Simons K, Toomre D. 2000. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1: 31–41. [DOI] [PubMed] [Google Scholar]
- Soderberg C, Giugni TD, Zaia JA, Larsson S, Wahlberg JM, Moller E. 1993. CD13 (human aminopeptidase N) mediates human cytomegalovirus infection. J Virol 67: 6576–6585. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tamaki H, Osanai K. 1985. Re‐initiation of meiosis in Mytilus oocytes with acrosome reaction product of sperm. Bull Mar Biol Stn Asamushi, Tohoku Univ 18: 11–23. [Google Scholar]
- Togo T, Morisawa M. 1997. Aminopeptidase‐like protease released from oocytes affects oocyte surfaces and suppresses the acrosome reaction in establishment of polyspermy block in oocytes of the mussel Mytilus edulis . Dev Biol 182: 219–227. [DOI] [PubMed] [Google Scholar]
- Togo T, Osanai K, Morisawa M. 1995. Existence of three mechanisms for blocking polyspermy in oocytes of the mussel Mytilus edulis . Biol Bull 189: 330–339. [DOI] [PubMed] [Google Scholar]
- Urner F, Sakkas D. 2003. Protein phosphorylation in mammalian spermatozoa. Reproduction 125: 17–26. [DOI] [PubMed] [Google Scholar]
- Wassarman PM, Jovine L, Litscher ES. 2001. A profile of fertilization in mammals. Nat Cell Biol 3: E59–E64. [DOI] [PubMed] [Google Scholar]
- Yaoi K, Nakanishi K, Kadotani T, Imamura M, Koizumi N, Iwahana H, Sato R. 1999. cDNA cloning and expression of Bacillus thuringiensis CryIAa toxin binding 120 kDa aminopeptidase N from Bombyx mori . Biochim Biophys Acta 1444: 131–137. [DOI] [PubMed] [Google Scholar]
- Yasuhara T, Yokosawa H, Hoshi M, Ishii S. 1983. Sea urchin sperm aminopeptidase: Comparative studies of sperm‐associated and ‐solubilized enzymes. Biochem Int 7: 593–598. [PubMed] [Google Scholar]
- Yasuhara T, Yokosawa H, Ishii S. 1990. Purification and characterization of an aminopeptidase from sperm of the sea urchin, Strongylocentrotus intermedius. Ca2+‐dependent substrate specificity as a novel feature of the enzyme. J Biochem 107: 273–279. [DOI] [PubMed] [Google Scholar]
- Yasuhara T, Yokosawa H, Hoshi M, Ishii S. 1991. Involvement of a sperm aminopeptidase in fertilization of the sea urchin. Experientia 47: 100–103. [Google Scholar]
- Yeager CL, Ashmun RA, Williams RK, Cardellichio CB, Shapiro LH, Look AT, Holmes KV. 1992. Human aminopeptidase N is a receptor for human coronavirus 229E. Nature 357: 420–422. [DOI] [PMC free article] [PubMed] [Google Scholar]
