Using homologous recombination, we have previously produced male mice carrying a disruptive mutation (Acr ؊/؊ ) in the acrosin gene. Although Acr ؊/؊ mouse sperm lacking the acrosin protease activity still penetrated the zona pellucida and fertilized the egg, the mutant sperm exhibited a delay in penetration of the zona pellucida solely at the early stages after insemination. To further elucidate the role of acrosin in fertilization, we have examined the involvement of acrosin in the acrosome reaction of sperm using the Acr ؊/؊ mutant mice. When the ability of sperm to adhere (attach) and bind to the zona pellucida of cumulus-free eggs was assessed in vitro, no significant difference was observed among Acr ؉/؉ , Acr ؉/؊ , and Acr ؊/؊ mouse sperm. Immunocytochemical analysis demonstrated that the release of several acrosomal proteins from the acrosome of Acr ؊/؊ mouse sperm was significantly delayed during the calcium ionophore-and solubilized zona pellucidainduced acrosome reaction, despite normal membrane vesiculation. These data indicate that the delayed sperm penetration of the zona pellucida in the Acr ؊/؊ mouse results from the altered rate of protein dispersal from the acrosome and provide the first evidence that the major role of acrosin is to accelerate the dispersal of acrosomal components during acrosome reaction.The acrosome reaction of sperm, a fusion (vesiculation) event between the overlying plasma and outer acrosomal membranes, occurs following the binding of sperm to the zona pellucida (ZP), 1 an extracellular glycoprotein matrix surrounding the egg. This exocytotic reaction is required for fertilization, because only acrosome-reacted sperm are capable of penetrating ZP and of fusing with the egg plasma membrane (for review see Ref. 1). The acrosomal components, including hydrolytic enzymes, are released by the acrosome reaction and then interact initially with ZP to facilitate the sperm penetration of the glycoprotein matrix.Acrosin, an endoprotease with a trypsin-like substrate specificity, is localized in the acrosomal matrix as an enzymatically inactive zymogen, proacrosin, that is then converted into the active form as a consequence of the acrosome reaction (2-4). The physiological role of acrosin in fertilization has long been believed to be the limited proteolysis of the ZP, thus enabling the sperm to penetrate the ZP. Using homologous recombination, we have successfully produced male mice carrying a disruptive mutation in the acrosin gene (Acr) and found that the mouse sperm lacking the acrosin protease activity (Acr Ϫ/Ϫ ) still penetrate ZP and normally fertilize the egg (5). These data provide evidence that acrosin is not essential for sperm penetration of the ZP. However, as compared with Acr ϩ/ϩ and Acr ϩ/Ϫ mice, Acr Ϫ/Ϫ mouse sperm showed a delay in sperm penetration of the ZP solely at the early stages after insemination (5). A recent report using separate lines of Acr Ϫ/Ϫ mice (6) has confirmed that sperm lacking acrosin exhibit the delayed fertilization. Thus, these results imply ...

It has been reported that a significant delay in protein dispersal from the acrosomal matrix is observed in wild-type sperm by adding p-aminobenzamidine, a trypsin/acrosin inhibitor, to the incubation medium. The pattern of this delayed release was similar to that of the acrosin-deficient mutant mouse sperm (Yamagata et al., J. Biol. Chem., 273, 10470–4, 1998). In the present study, no further delay in protein dispersal was found when the acrosin-deficient sperm were treated with p-aminobenzamidine, indicating that among the p-aminobenzamidine-sensitive protease(s) only acrosin may function to accelerate this process. Although the acrosin-deficient sperm penetrated the zona pellucida (Baba et al., J. Biol. Chem., 269, 31845–9, 1994), the addition of p-aminobenzamidine to the fertilisation medium caused a significant inhibition of fertilisation in vitro. This indicates that there is a p-aminobenzamidine-sensitive protease(s) other than acrosin participating in the zona penetration step. Indeed, we demonstrated that a non-acrosin protease with a size of 42 kDa was present in the supernatant of the acrosome-reacted sperm suspension. The enzyme was inhibited by p-amimobenzamidine, diisopropyl fluorophosphate and Nα-tosyl-L-lysine chloromethyl ketone, and was apparently activated by acrosin.