Hyaluronic acid and the cumulus extracellular matrix induce increases in intracellular calcium in macaque sperm via the plasma membrane protein PH-20

Abstract

The hyaluronic acid (HA)-rich extracellular matrix (ECM) of the cumulus oophorus is known to facilitate fertilisation. It has been suggested that HA may enhance fertilisation in a number of species, and in macaque sperm, HA has been shown to increase the number of acrosome reactions that follow sperm binding to the zona pellucida. In this study, we investigated the effects of HA on intracellular Ca²⁺ in capacitated cynomolgus macaque sperm. Fluorometry studies using the intracellular Ca²⁺ indicator Fluo-3 showed that addition of 100 μg/ml of HA induced a rapid increase in intracellular Ca²⁺. This Ca²⁺ increase (approximately 2–3 times above basal levels) was inhibited by preincubation of sperm with Fab fragments of anti-recombinant PH-20 IgG. The frequency of acrosome reactions in sperm exposed to HA was not above control levels. A synthetic gel was prepared with similar viscosity to the cumulus and with HA trapped in its matrix. Video imaging of individual sperm was used to demonstrate that capacitated sperm swimming into the HA gel had increased intracellular Ca²⁺ levels. Preincubation of sperm with Fab fragments of anti-PH-20 IgG inhibited the increased intracellular Ca²⁺ levels induced by the HA gel. Sperm in control gel (no HA) did not show increased intracellular Ca²⁺, while sperm in gel containing anti-PH-20 IgG showed increased Ca²⁺ (positive control). Sperm loaded with Fluo-3 were allowed to interact with cynomologus macaque cumulus masses, and sperm within the cumulus ECM clearly showed increased intracellular Ca²⁺ that was inhibited when sperm were preincubated in anti-PH-20 Fab. Fluorescein isothiocyanate (FITC)-HA was found to bind to sperm over the acrosomal region, corresponding to PH-20 localisation, and this binding could be inhibited by preincubation of sperm with anti-PH-20 fragments. The results of this study show that HA increases intracellular Ca²⁺ in macaque sperm through interaction with plasma membrane PH-20. We propose that HA binding to plasma membrane PH-20 induces an aggregation of receptors that in turn results in intracellular signalling. As a result, sperm have higher basal CA²⁺ levels and are more responsive to induction of the acrosome reaction after binding to the zona pellucida.