| Research Abstract |
In the presence of ATP, the motility of demembranated fowl spermatozoa was vigorous at 30ﾟC, both before and after rapid freezing at -70ﾟC or -196ﾟC without cryoprotectants. The rate of ATP consumption of the demembranated spermatozoa treated by freezing and thawing was, at approximately 200 nmol ATP hydrolysis/10^9 spermatozoa/mm, also similar to that of the untreated spermatozoa. At 400 C, neither frozen-thawed spermatozoa nor control spermatozoa were motile, but motility could be restored by the decreasing the temperature to 30ﾟC or by the addition of inhibitors of protein phosphatases.
The motility of demembranated fowl spermatozoa decreased markedly following the addition of recombinant protein phosphatase type I (PP-1) supplemented with Mn^2^+. Phosphorylation of demembranated fowl sperm proteins during incubation with [gamma-^3^2P]ATP at 30ﾟC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A marked difference in phosphorylation status was obs
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erved in approximately 116, 86, 79, 50 and 29-kDa proteins. These proteins were dephosphorylated in the presence of PP-1 and Mn^2^+ compared with those in control samples.
Intact fowl spermatozoa maintained vigorous movement at 30ﾟC with or without Ca^2^+. In contrast, the motility was reversibly inhibited at 40ﾟC without Ca^2^+, but could be immediately restored by the addition of Ca^2^+. However, the addition of verapamil, a specific Ca^2^+ channel blocker, before the addition of Ca^2^+, could not obtain fully motile spermatozoa at 40ﾟC.Under these conditions, the intracellular free Ca^2^+ concentration was lower than that of the control (no addition of verapamil), as measured by a fluorescent Ca^2^+ indicator, fura-2. The addition of Sr^2^+, which appears to stimulate the release of Ca^2^+ from the mitochondria, was also effective for the stimulation of motility at 40ﾟC, and induced a concomitant increase in the intracellular free Ca^2^+ concentrations.
These observations suggest that the substance(s) involved in the temperature-dependent immobilization of fowl spermatozoa are tightly associated with the axoneme, and that PP-I-mediated dephosphorylation of some of these proteins of the axoneme and/or accessory cytoskeletal components of fowl spermatozoa may be involved in the inhibition of motility. Furthermore, it is suggested that intracellular free Ca^2^+ play a pivotal role in regulating fowl sperm motility. Less
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