Hyaluronidase activity of macaque sperm assessed by an in vitro cumulus penetration assay

Abstract

A model system consisting of cynomolgus macaque sperm and ovulated hamster ova-cumulus complexes (OCCs) was utilized to study the role of the sperm protein PH-20 in cumulus penetration. The hyaluronidase activity of solubilized macaque sperm PH-20 was evaluated using an ELISA-like microplate assay prior to and following the addition of the hyaluronidase inhibitors heparin (0-100 microg/ml) and apigenin (250 microM), as well as the Ig fraction of a polyclonal antibody raised against purified recombinant macaque PH-20 (R10; 10-400 microg/ml). Sperm motility following exposure to enzyme inhibitors was evaluated using computer-aided sperm motility analysis. Macaque sperm were labeled with the permeant fluorescent nuclear dye, Hoechst 33342, and were coincubated with ovulated hamster OCCs for 30 min at 37 degrees C. The addition of heparin, apigenin, or R10 antibody to solubilized sperm extracts resulted in a linear dose-dependent decrease in hyaluronidase activity (P < .01). In the heterologous cumulus penetration assay, fluorescently labeled macaque sperm that were pretreated with heparin (1-100 microg/ml), apigenin (250 microM), or R10 antibody (Ig fraction, 10-400 microg/ml) demonstrated a dose-dependent decrease in the ability to penetrate hamster OCCs (P < 0.01), in the absence of effects on sperm motility. In the homologous assay, experiments using macaque OCCs and fluorescently labeled macaque sperm confirmed that the same concentrations of heparin and R10 antibody similarly suppressed spermatozoal cumulus penetration (P < .01). These results suggest that macaque sperm PH-20-derived hyaluronidase participates in cumulus penetration in this species, and that this model system is useful for further studies into primate gamete interaction.

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

“…In some experiments, we have examined the effects of apigenin on the motility and spontaneous acrosome reaction of sperm (data not shown). Apigenin did not affect these two cellular events of mouse sperm, as was the case for monkey sperm [13,14]. These results raise the following question: besides sperm motility, what is required for sperm penetration through the cumulus matrix?…”

“…3 and 4). Even in monkey sperm, apigenin incompletely inhibits sperm penetration through the cumulus; the sperm ability to traverse the cumulus is reduced but is not abrogated by apigenin [13,14]. In some experiments, we have examined the effects of apigenin on the motility and spontaneous acrosome reaction of sperm (data not shown).…”

“…As expected, the hyaluro-nan-degrading activities of HYAL5 and SPAM1 were not detectable when the gels were incubated in the presence of 0.5 and 1 mM apigenin. Thus, consistent with previous works [12][13][14], apigenin efficiently inhibited the hyaluronan-degrading activity of sperm hyaluronidase.…”

“…Partially inconsistent with the findings in monkey sperm [13,14], mouse sperm are capable of normally penetrating the cumulus matrix in the presence of apigenin (Figs. 3 and 4).…”

Influence of Canine Cumulus Oophorus on Homologous Sperm Motility.

“…Sperm penetration into cumuli in many species [24] is made possible by the release of hyaluronidase, an enzyme that hydrolyzes hyaluronic acid, from the sperm head, and the authors have reported that canine sperm also release hyaluronidase [18]. It has been discovered that hamster acrosome-reacted sperm can not penetrate cumuli [5,7] and that the acrosome reaction is induced by homologous zona pellucida (ZP) [27], but there have been no reports on detailed investigation of the relationship between canine sperm and homologous cumuli during fertilization.…”