Regulatory mechanisms of fowl sperm mobility: Possible role of endogenous myosin light chain kinase-like protein

Abstract

The motility of both intact and demembranated fowl spermatozoa was vigorous at 30°C, but decreased markedly following the addition of 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9), a specific inhibitor of myosin light chain kinase (MLCK). Furthermore, the presence of a MLCK substrate peptide also inhibited the motility of demembranated spermatozoa at 30°C. In contrast, the addition of N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-8) or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004), specific inhibitors of cAMP-dependent protein kinase, did not appreciably affect the motility of either intact or demembranated spermatozoa. Cyclic AMP-dependent protein kinase substrate peptides were also ineffective for the inhibition of motility of demembranated spermatozoa at 30°C. Immunoblotting of sperm extract, using an antibody to MLCK, revealed two major crossreacting proteins of 130 kDa and 61-64 kDa, which corresponded to the molecular mass of MLCK. In addition, immunogold particles, which reacted with the anti-MLCK antibody, were observed around or on the axoneme at the ultrastructural level. These results suggest that the phosphorylation of axonemal protein(s) by MLCK, or a MLCK-like protein, rather than by cAMP-dependent protein kinase, may be involved in the maintenance of fowl sperm. motility at 30°C.