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Acrosin, the trypsin-like serine protease in the sperm acrosome, was long viewed as a key enzyme required for zona pellucida penetration to fertilize eggs. However, gene disruption experiments in mice surprisingly showed that acrosin-disrupted males were fertile. Thus, the acrosin was considered to be not an essential enzyme for fertilization in mice. However, the involvement of acrosin in fertilization has been suggested in various species such as rat, bull, and pig. Moreover, it has been reported that serine protease (including acrosin) activity in mice is significantly weaker compared to other species, including rats. We analyzed the role of acrosin by disrupting the rat acrosin gene. It was found that, unlike in mice, acrosin was almost the sole source of serine protease in rat spermatozoa. Nevertheless, the acrosin-disrupted males were not infertile. However, the litter size from acrosin-disrupted males was decreased compared to heterozygous mutant rats. Further investigation using an in vitro fertilization system revealed that the acrosin-disrupted spermatozoa possessed an equal ability to penetrate the zona pellucida with wild-type spermatozoa, but the cumulus cell dispersal was slower compared to wild-type and heterozygous spermatozoa. This delay was presumed to be the cause of the small litter size of acrosin-disrupted male rats., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mammalian fertilization is a complex process that involves gamete recognition, penetration, and fusion. Biochemical studies that identified the role of acrosome components during sperm-ova interaction especially the zona pellucida (ZP) provided major advances in sperm cell biology. Acrosin (a typical serine protease) functions during fertilization in several significant ways which include: a) activation of acrosome components, b) secondary binding with the ZP, and c) hydrolysis of the ZP. However, studies using knockout (KO) acrosin-deficient mice cast doubt on the traditional role of acrosin in fertilization. The KO acrosin-deficient mice exhibit normal fecundity except for delayed fertilization. Despite the doubt cast on the traditional role of acrosin by the KO acrosin-deficient mouse studies, acrosin still remains a major protease involved in multiple processes of fertilization. In this review, we assess the functional profile of acrosin and briefly summarize recent findings on proteases involved in fertilization. We propose a refined scheme for the functional role of acrosin in fertilization. We particularly emphasize the role of acrosin in acrosome exocytosis and activation of other acrosome components based on advanced technology like structural X-ray analysis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Mammalian fertilization requires sperm to penetrate the cumulus to reach the oocyte. Although sperm hyaluronidase has long been believed to participate in the penetration process, our previous works revealed that neither of two sperm hyaluronidases, SPAM1 and HYAL5, are essential for fertilization. In this study, we have produced double-knockout mice lacking SPAM1 and either one of two sperm serine proteases, ACR and PRSS21, and characterized the mutant sperm. The SPAM1/ACR- and SPAM1/PRSS21-deficient males were fertile, whereas epididymal sperm of the mutant mice exhibited a reduced capacity to fertilize the oocytes in vitro. Despite normal motility, the ability of sperm to traverse the cumulus matrix was more severely impaired by the loss of SPAM1 and ACR or SPAM1 and PRSS21 than by the loss of only SPAM1. Moreover, SPAM1/ACR- and SPAM1/PRSS21-deficient sperm accumulated on the surface (outer edge) of the cumulus more abundantly than SPAM1-deficient sperm. These results suggest that ACR or PRSS21 or both may function cooperatively with SPAM1 in sperm/cumulus penetration.

Although sperm serine protease and proteasome have long been believed to play an important role in the fertilization process, the molecular mechanism is still controversial. In this study, we have produced double-knockout mice lacking two sperm serine proteases, ACR and PRSS21, to uncover the functional role of the trypsinlike activity in fertilization. The double-knockout male mice were subfertile, likely owing to the incompleteness of fertilization in the oviductal ampulla. Despite male subfertility, the mutant epididymal sperm exhibited the inability to undergo acrosomal exocytosis on the zona pellucida (ZP) surface and to traverse the ZP, thus resulting in the failure of fertilization in vitro. The double-knockout epididymal sperm were also defective in penetration through the cumulus matrix to reach the ZP. When epididymal sperm were artificially injected into the uterus of wild-type mice, the 2-cell embryos, which had previously been fertilized by double-knockout sperm, were recovered at a low but significant level. The mutant epididymal sperm were also capable of fertilizing the oocytes in the presence of uterine fluids in vitro. These data demonstrate that the trypsinlike protease activity of ACR and PRSS21 is essential for the process of sperm penetration through the cumulus matrix and ZP in vitro, and suggest that the female reproductive tract partially compensates for the loss of the sperm function. We therefore conclude that the sperm trypsinlike activity is still important but not essential for fertilization in vivo in the mouse.

Sperm trypsin-like proteases are known to play important roles in fertilization, but their detailed functions are still unknown. We previously explored the binding partners of sperm trypsin-like proteases, HrProacrosin and HrSpermosin, in the ascidian Halocynthia roretzi, and we isolated several candidate proteins on the vitelline coat. We found that some of these proteins are identical to the C-terminal coding region (CT) and von Willebrand factor type D (vWF-D) domain of vitellogenin. We also found that CT on the vitelline coat disappears after fertilization. Vitellogenin is a large lipid transfer protein that is enzymatically processed during vitellogenesis. Although the processed domains including phosvitin and lipovitellin are known to function as yolk nutrient proteins, the roles of the CT and vWF-D domain remain elusive. Our results showed that the CT and vWF-D domain of vitellogenin are processed and attached to the vitelline coat, which in turn participate in fertilization as the binding partners of sperm proteases., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Over 200 genes have been shown to be associated with infertility in mouse models. However, knockout mice reveal unexpected functional redundancy of some germ cell expressed genes. Single null mutations in mouse genes encoding four male germ cell proteins, transition protein 2 (Tnp2), proacrosin (Acr), histone H1.1 (H1.1), histone H1t (H1t) and sperm mitochondria-associated cysteine-rich protein (Smcp) have been generated and analysed. Tnp2 is believed to participate in the removal of the nuclear histones and initial condensation of the spermatid nucleus. Proacrosin is an acrosomal protease synthesized as a proenzyme and activated into acrosin during the acrosome reaction. The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues as well as in germ cells during the S-phase of the cell cycle. The histone gene Hist1h1t is expressed exclusively in spermatocytes and may have a function in establishing an open chromatin structure for the replacement of histones by transition proteins and protamines. Sperm mitochondria-associated cysteine-rich protein (Smcp) is a major structural element of the mitochondria in the midpiece of the sperm tail. Male mutant mice lacking any of these proteins show no apparent defects in spermatogenesis or fertility. To examine the synergistic effects of these proteins in spermatogenesis and during fertilization four lines of double knockout mice Hist1h1a/Mcsp, Hist1h1t/Mcsp, Tnp2/Mcsp and Acr/Mcsp were established. It was found that even when knockout mice are heterozygous for one allele (-/+) and homozygous for the other allele (-/-), mice were subfertile. Homozygous double knockout mice of all four lines are nearly infertile. However, in the four homozygous double knockout mouse lines, different characteristic abnormalities are prominently manifested: In Hist1h1a-/-/Mcsp-/- the migration of spermatozoa is disturbed in female genital tract, in Hist1h1t-/-/Mcsp-/- spermatozoa show morphological head abnormalities, in Tnp2-/-/Mcsp-/- the motility of sperm is affected, and in Acr-/-/Mcsp-/- the sperm-oocyte interaction is impaired. These findings indicate strongly that male germ cell expressed genes have synergistic effects on male fertility., (Copyright 2003 S. Karger AG, Basel)

The mouse zona pellucida glycoprotein, mZP2, is thought to be the secondary receptor on eggs for retention of acrosome-reacted sperm during fertilization. Here, we present evidence that one of its complementary binding proteins on sperm is proacrosin/acrosin. mZP2 binds to proacrosin null sperm considerably less effectively than to wild-type sperm. Binding is mediated by a strong ionic interaction between polysulphate groups on mZP2 and basic residues on an internal proacrosin peptide. The stereochemistry of both sulphate groups and basic amino acids determines the specificity of binding. Structurally relevant sulphated polymers and suramin, a polysulphonated anticancer drug, compete with mZP2 for complementary binding sites on proacrosin/acrosin in solid-phase binding assays. The same competitors also displace attached sperm from the zona pellucida of eggs in an in vitro fertilization system. This combination of genetic, biochemical and functional data supports the hypothesis that mZP2-proacrosin interactions are important for retention of acrosome-reacted sperm on the egg surface during fertilization. Safe mimetics of suramin have potential as non-steroidal antifertility agents.