Gamete Biology

Induction of the Acrosome Reaction in Black Tiger Shrimp, Penaeus monodon, Requires Sperm Trypsin-Like Enzyme Activity

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 described 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. Using peptidyl fluorogenic substrates, we showed 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 preheated (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 suggested the importance of the acrosomal trypsin-like enzyme in the AR process.

Key words: Gamete Biology • Acrosome reaction • Fertilization • Sperm