Mouse Sperm Lacking Cell Surface Hyaluronidase PH-20 Can Pass through the Layer of Cumulus Cells and Fertilize the Egg

Abstract

The function of glycosylphosphatidylinositol-anchored sperm hyaluronidase PH-20 in fertilization has long been believed to enable acrosome-intact sperm to pass through the layer of cumulus cells and reach the egg zona pellucida. In this study, we have produced mice carrying a null mutation in the PH-20 gene using homologous recombination. Despite the absence of sperm PH-20, the mutant male mice were still fertile. In vitro fertilization assays showed that mouse sperm lacking PH-20 possess a reduced ability to disperse cumulus cells from the cumulus mass, resulting in delayed fertilization solely at the early stages after insemination. Moreover, SDS-PAGE of sperm extracts and subsequent Western blot analysis revealed the presence of other hyaluronidase(s), except PH-20, presumably within the acrosome of mouse sperm. These data provide evidence that PH-20 is not essential for fertilization, at least in the mouse, suggesting that the other hyaluronidase(s) may play an important role in sperm penetration through the cumulus cell layer and/or the egg zona pellucida, possibly in cooperation with PH-20, although the importance of sperm motility cannot be neglected.

Possible Function of the ADAM1a/ADAM2 Fertilin Complex in the Appearance of ADAM3 on the Sperm Surface

“…The eggs and sperm were incubated for 1-6 h at 37°C under 5% CO 2 in air. The status of the cumulus mass was microscopically assessed at time intervals according to the criterion described previously (27).…”

“…Anti-ADAM3 antiserum was produced in female New Zealand White rabbits by intradermal injection of recombinant protein containing the cysteine-rich and epidermal growth factor-like domains of ADAM3 between residues 487 and 670. The anti-ADAM3 antibody was affinity-purified as described (21,27). Mouse monoclonal antibodies against ADAM2 (9D2.2) and ADAM3 (7C1.2) were purchased from Chemicon.…”

“…Antibodies-Affinity-purified rabbit polyclonal antibodies against ADAM1a, ADAM1b, sp32, and PH-20 were prepared as described previously (21,27,28). Anti-ADAM3 antiserum was produced in female New Zealand White rabbits by intradermal injection of recombinant protein containing the cysteine-rich and epidermal growth factor-like domains of ADAM3 between residues 487 and 670.…”

Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass

“…A possible involvement of PH-20 in the binding of acrosomereacted (AR) sperm to ZP, known as secondary sperm͞ZP binding, has been also suggested (18). However, male mice lacking PH-20 (Ph20 Ϫ/Ϫ ) are still fertile, although epididymal sperm of Ph20 Ϫ/Ϫ mice show a delayed dispersal of cumulus cells from the cumulus mass in vitro (19). In addition, no significant difference in sperm binding to the ZP is found between wild-type and Ph20 Ϫ/Ϫ mice.…”

Testicular descent, sperm maturation and capacitation. Lessons from our most distant relatives, the monotremes

“…A number have been identified in eutherian mammals, including SPAM1 (formerly PH20; Chen et al 2006), SED1 (Ensslin and Shur 2003), α-d-mannosidase (Jin et al 1999), CRISP1 (Eberspaecher et al 1995), CD52 (Kirchhoff et al 1993), ASF (Thomas et al 1984), HE1 (Kirchhoff et al 1996) and p26H (Coutu et al 1996). However, whilst knockout mice for SPAM1 (Baba et al 2002) and SED1 (Ensslin and Shur 2003) have reduced fertility compared with wild-type, none of the knockouts is infertile. Whilst there have been no studies of epididymal proteins that interact with monotreme spermatozoa, there is evidence that epididymal proteins are involved in the formation of sperm bundles (see below).…”