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Induction of the Acrosome Reaction in Black Tiger Shrimp (Penaeus monodon) Requires Sperm Trypsin-Like Enzyme Activity¹

Department of Anatomy⁴ and Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp),⁵ Faculty of Science, Mahidol University, Bangkok 10400, Thailand Chronic Disease,⁶ Ottawa Health Research Institute, Ottawa, Ontario K1Y 4E9, Canada Departments of Biochemistry/Microbiology/Immunology,⁷ Medicine,⁸ and Obstetrics and Gynecology,⁹ University of Ottawa, Ottawa, Ontario K1H 8M5, Canada

ABSTRACT

Trypsin-like enzymes in egg water (EW), a natural acrosome reaction (AR) inducer, are known for their importance in shrimp AR. In this report, we describe a unique phenomenon of the AR of black tiger shrimp (Penaeus monodon) sperm. It was completed within 45–60 sec and comprised only the acrosomal exocytosis and depolymerization of the sperm head anterior spike. We used peptidyl fluorogenic substrates to show the presence of trypsin-like enzymes in P. monodon EW and sperm, but minimal activities of chymotrypsin-like enzymes. In sperm, these trypsin-like enzymes existed both on the sperm surface and in the acrosome. The acrosomal enzyme was revealed as a 45-kDa band by fluorogenic substrate in-gel zymography. Although EW possessed high trypsin-like enzyme activities, they were not essential for the AR induction; EW pretreated with an irreversible trypsin inhibitor, or heat-inactivated EW (HI-EW), to abolish the trypsin-like activities could still induce the AR. The HI-EW-induced AR was inhibited by the presence of a membrane impermeant soybean trypsin inhibitor (SBTI) in the sperm suspension, indicating the significance of sperm-borne trypsin-like enzymes (on the surface and/or in the acrosome) in this AR process. However, pretreatment of sperm with SBTI followed by its removal from the suspension still allowed the AR to occur within 5 min of sperm exposure to HI-EW. Since trypsin-like activity of the SBTI-pretreated sperm surface at 5 min after SBTI removal was at the minimal level, our results suggest the importance of the acrosomal trypsin-like enzyme in the AR process.

acrosome reaction, egg water, fertilization, gamete biology, P. monodon, sperm, trypsin

³These authors contributed equally to this work.

¹Supported by funding from the National Science and Technology Development Agency of Thailand to B.W., the Thailand Research Fund to W.W. and P.S., Thailand Research Fund-Royal Golden Jubilee Ph.D. Program grant PHD/0275/2545 to R.V., and from the Commission on Higher Education to H.K. and S.I.

Correspondence: ²Wattana Weerachatyanukul, Department of Anatomy, Faculty of Science, Mahidol University, Rama 6 Rd., Payathai, Bangkok 10400, Thailand. FAX: 66 2 354 7168; e-mail: scwwy{at}mahidol.ac.th