Regulatory mechanisms of fowl sperm motility: possible role of endogenous myosin light chain kinase-like protein.

The motility of both intact and demembranated fowl spermatozoa was vigorous at 30 degrees 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 degrees C. In contrast, the addition of N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-8) or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide dihydrochloride (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 degrees 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 degrees C.

Presence of protein phosphatase type 1 and its involvement in temperature-dependent flagellar movement of fowl spermatozoa.

Even in the presence of ATP, the motility of demembranated fowl spermatozoa was negligible at the avian body temperature of 40 degrees C. Motility could be restored by the addition of calyculin A, okadaic acid, specific inhibitors of phosphatase type 1 (PP1) and PP-2A, and inhibitor 1 or inhibitor 2, which are specific inhibitors of protein phosphatase type 1 (PP1). Demembranated spermatozoa, stimulated by calyculin A or okadaic acid, lost their motility following the addition of 1 mM CaCl2, but this was restored gradually by the stepwise addition of EGTA. Immunoblotting of sperm extract using an antibody to PP1 revealed a major cross-reacting protein of 36-37 kDa, which corresponded to the molecular weight of the known catalytic subunit of PP1. These results suggest that PP1 present in the fowl sperm axoneme may be involved in the inhibition of fowl sperm motility at 40 degrees C via Ca(2+)-dependent regulatory systems.