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Effects of calpain and Rho-kinase inhibitors on the acrosome reaction and motility of fowl spermatozoa in vitro

Laboratory of Animal Reproduction, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan and ¹ Department of Molecular and Life Sciences, University of Abertay Dundee, Dundee DD1 1HG, UK

Correspondence should be addressed to Koji Ashizawa; Email: ashizawa{at}cc.miyazaki-u.ac.jp

At the avian body temperature of 40 °C, intact fowl spermatozoa require Ca²⁺ for the initiation of motility and a combination of both Ca²⁺ and homogenized inner perivitelline layer (IPVL) together to induce the acrosome reaction. Within the range of 1–100 µmol/l, neither PD 150606 (a Ca²⁺-dependent calpain inhibitor) nor Y-27632 (an inhibitor of Ca²⁺-dependent Rho-kinase) were able to inhibit the acrosome reaction induced by the presence of Ca²⁺ and IPVL. However, PD 150606, although not Y-27632, was able to inhibit sperm motility initiated by Ca²⁺, as well as motility initiated by calyculin A – a specific inhibitor of protein phosphatases, which also initiates sperm motility at 40 °C. The addition of PD 150606 did not reduce the ATP concentrations of intact spermatozoa, nor the motility of demembranated spermatozoa. Immunoblot analysis of sperm extract using a polyclonal antibody against calpain 12 revealed a cross-reacting protein of approximately 80 kDa. These results suggest that Rho-kinase is not involved in the regulation of the acrosome reaction or of motility in fowl spermatozoa. In contrast, calpain appears to be involved in the regulation of flagellar movement, but not izn that of the acrosome reaction. Furthermore, it seems that endogenous calpain is present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components.

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