Functional Roles of Mouse Sperm Hyaluronidases, HYAL5 and SPAM1, in Fertilization1

Abstract

Although sperm entry into the oocyte-cumulus complex and subsequent sperm penetration through the cumulus matrix to reach the oocyte zona pellucida are essential for mammalian fertilization, the molecular mechanism remains controversial. Previously, we have shown that mouse sperm lacking SPAM1 are capable of penetrating the cumulus matrix despite a delayed dispersal of cumulus cells. We also have identified another sperm hyaluronidase, HYAL5, as a candidate enzyme involved in sperm penetration through the cumulus. In the present study, we produced HYAL5-deficient mice to uncover the functional roles of HYAL5 and SPAM1 in fertilization. The HYAL5-deficient mice were fully fertile and yielded normal litter sizes. In vitro fertilization assays demonstrated that HYAL5-deficient epididymal sperm is functionally normal. We thus conclude that HYAL5 may be dispensable for fertilization. Comparative analysis among wild-type, HYAL5-deficient, and SPAM1-deficient epididymal sperm revealed that only SPAM1 is probably involved in sperm penetration through the cumulus matrix. Notably, the loss of SPAM1 resulted in a remarkably increased accumulation of sperm on the surface or outer edge of the cumulus. These data suggest that SPAM1 may function in sperm entry into the cumulus and sperm penetration through the cumulus matrix.

Membrane transfer from oocyte to sperm occurs in two CD9-independent ways that do not supply the fertilising ability of Cd9-deleted oocytes

“…However, the goal of the exchange between oocytes and sperm is not to supply spermatozoa in CD9 tetraspanin. In fact, as described previously (Kimura et al 2009, Ito et al 2010 and confirmed here, the CD9 tetraspanin is also expressed on mouse sperm at a high level and is localised on the equatorial segment after acrosome reaction. This result shows that CD9 is mostly detected at the surface of the fertilising sperm at…”

“…Contrary to what has been initially described (Chen et al 1999, Li et al 2004, spermatozoa do also express CD9 tetraspanin, as recently reported (Kimura et al 2009, Ito et al 2010 and as confirmed here. Indeed, we observed the presence of CD9 tetraspanin on cauda epididymal mature sperm.…”

Acrosome reaction in the cumulus oophorus revisited: involvement of a novel sperm-released factor NYD-SP8

“…It is the hydrolytic enzymes released following the AR that ensure sperms to disperse the cumulus mass and reach the zona layer (Talbot, 1985;Yanagimachi, 1988). Unfortunately, this hypothesis is challenged by the observation that the expression of hydrolytic enzymes thought to facilitate sperm penetration of the cumulus, PH-20 and Hyal5, were found on the surface of sperm membrane but not within the acrosome (Lin et al, 1994;Kim et al, 2005;Kimura et al, 2009), which means sperm do not necessarily undergo AR to release hydrolytic enzymes when passing through the cumulus layer. Recent results obtained from PH-20 null mice have revealed that mouse sperm lacking PH-20 are still fertile with only delayed penetration through the cumulus (Baba et al, 2002), while sperm lacking both PH-20 and Hyal5 have not yet been studied.…”

Hyaluronan-Degrading Activity of Mouse Sperm Hyaluronidase Is Not Required for Fertilization?

“…Mouse epididymal sperm contain at least two hyaluronidases, SPAM1 and HYAL5, glycosylphosphatidylinositol (GPI)-anchored on the plasma and/or acrosomal membranes [8][9][10][11]. We previously demonstrated that SPAM1 is localized only on the plasma membrane of sperm and that acrosome-reacted sperm still retain approximately half of SPAM1 [9].…”

“…On the basis of the subcellular distribution, both HYAL5 and SPAM1 on the plasma membrane may be involved in sperm penetration through the cumulus matrix, and acrosomal HYAL5 released by the acrosome reaction may play an important role in the local hyaluronan hydrolysis near or on the ZP surface to enable the proximal region of the sperm tail to move freely [10]. Inconsistent with these possibilities, male mice lacking either one of these two hyaluronidases (Spam1 -/-or Hyal5 -/-) have been shown to be fully fertile [9,11], providing evidence that neither SPAM1 nor HYAL5 is essential for fertilization in the mouse. However, in vitro fertilization (IVF) assays have revealed that Spam1 -/-sperm are distinguished from wild-type and Hyal5 -/-sperm by significant delays in entering the OCC, dispersing cumulus cells from the OCC and traversing the cumulus matrix to reach the ZP [11].…”