Temperature-dependent flagellar motility of demembranated, cytosol-free fowl spermatozoa.

A rapid and gentle procedure for preparing demembranated, cytosol-free sperm models was applied to fowl spermatozoa. Intact spermatozoa were introduced to a Triton X-100-containing extraction medium layered on top of a discontinuous Percoll gradient in a 1.5 ml microfuge tube. After brief exposure to the extraction medium, spermatozoa were separated from the plasma membrane and detergent-soluble components by centrifugation through a 55% Percoll layer, finally collecting on top of a 90% Percoll cushion from where they were recovered. Optimum conditions consisted of a Triton X-100 concentration in the extraction medium of 0.15%, duration of demembranation time of 1.5 min and ATP concentration in the reactivation medium of 0.5 mM. Demembranated sperm models obtained by this procedure could be reactivated, and the motility at 30 degrees C was more than 60%, but negligible at 40 degrees C. These values were similar to those obtained from the conventional method, in which centrifugation is not carried out, and which results in some of the cytosolic components being transferred to the reactivation medium along with the spermatozoa. Inhibition of motility was observed following the addition of EGTA or myosin light chain kinase (MLCK) substrate peptide at 30 degrees C, whilst the presence of protein phosphatase inhibitors, such as calyculin A or okadaic acid, permitted the restoration of motility at 40 degrees C. These results demonstrate that the axoneme and/or accessory cytoskeletal components are directly involved in the temperature-dependent regulatory system of fowl sperm motility in the absence of plasma membrane and/or soluble components of cytoplasm.