Intracellular Signal Transduction Pathways in the Regulation of Fowl Sperm Motility: Evidence for the Involvement of Phosphatidylinositol 3-Kinase (PI3-K) Cascade

The possible role of PI3-K in the reversible temperature-dependent immobilization of fowl sperm motility was investigated by using PI3-K inhibitor (LY294002) and its inactive analogue (LY303511). The existence of the PI3-K in fowl spermatozoa was also confirmed by Western blotting analysis. Fowl sperm motility in TES/NaCl buffer remained negligible at the avian body temperature of 40 degrees C but was maintained vigorously when the temperature was decreased to 30 degrees C. At 30 degrees C, no stimulation or inhibition of motility was observed after the addition of 2 mM CaCl2 and 10 mu M LY294002 or LY303511: around 70-80% of spermatozoa remained motile. In contrast, at 40 degrees C, the motility of spermatozoa was activated immediately after the addition of Ca2+, but the subsequent addition of LY294002 inhibited the motility again. The addition of LY303511 did not appreciably affect the Ca2+-supplemented sperm motility, which was maintained for at least 15 min. The ATP concentrations of spermatozoa after the addition of LY294002 + Ca2+ or LY303511 + Ca2+ were almost the same values compared with those of Ca2+ alone at 40 degrees C, suggesting that the addition of LY294002 was not simply affecting membrane damage or inhibiting energy production in the spermatozoa, but may be acting on some part of the motility-regulating cascade. Immunoblotting of sperm extract using an antibody to PI3-K revealed a major cross-reacting protein of 85 kDa, which corresponds to the molecular weight of the subunit of PI3-K. These results suggest that PI3-K may be positively involved in the calcium-regulated maintenance of flagellar movement of fowl spermatozoa at 40 degrees C.