| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Uniquely, fowl spermatozoa display the phenomenon of reversible, temperature-dependent motility inhibition : in simple salt solutions in vitro, they are immotile at the avian body temperature of 40-41ﾟC,but regain motility on reduction of the temperature to 30ﾟC.However, the intracellular molecular cascades involved in the regulation of motility remain to be determined.
Intracellular free Ca^<2+> seems to be essential for the maintenance of motility, since the motility of intact spermatozoa at 40ﾟC can be restored by the addition of Ca^<2+>. Furthermore, the motility of intact spermatozoa loaded with an intracellular Ca^<2+> chelator, BAPTA/AM is negligible at 30ﾟC,but can be instantly restored by the subsequent addition of Ca^<2+>. However, even in the presence of Ca^<2+>, the addition of calmodulin antagonists such as W-7 and trifluoperazine inhibited sperm motility at 30ﾟC and 40ﾟC
The activation of protein phosphatase type 1 (PP1), present in the fowl sperm axoneme, may be involved i
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n the inhibition of fowl sperm motility at 40ﾟC,since, in addition to calyculin A and okadaic acid, specific inhibitors of PP1, inhibitors 1 and 2, also stimulated the motility of demembranated spermatozoa at 40ﾟC.Furthermore, the involvement of myosin light chain kinase (MLCK), or a MLCK-like protein, has been proposed to be a candidate of Ca^<2+>/calmodulin dependent protein kinase, since the motility of both intact and demembranated fowl spermatozoa at 30ﾟC decreased markedly following the addition of ML-9, a specific inhibitor of MLCK,but not following the addition of H-8, a specific inhibitor or cAMP-dependent protein kinase (PKA). A marked difference in phosphorylation was observed in a 30 kDa protein. This protein was strongly phosphorylated after the addition of Kemptide, a PKA substrate peptide as well as control samples but only slightly phosphorylated in the presence of a myosin light chain kinase (MLCK) substrate peptide.
These results suggest that phosphorylation by Ca^<2+>/calmodulin-dependent protein kinases, rather tha PKA,is likely to be a regulatory step in the maintenance of fowl sperm motility, and that phosphorylation of a 30 kDa protein by MLCK or a MLCK-like proteinis may be involved in the regulation of flagellar movement at 30ﾟC. Less
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