Identification and analysis of temperature-sensitive proteins : the, temperature dependent immobilization of fowl spermatozoa at body temperature as a model

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The motility of spermatozoa is controlled by phosphorylation-dephosphorylation of specific proteins existing in flagella. Protein phosphorylation is mediated by protein kinases and dephosphorylation is mediated by protein phosphatases. Although it has been proposed that the reversible temperature-dependent immobilization of fowl spermatozoa is related to the activation of protein phosphatase, it has not been clarified in detail yet. The motility of spermatozoa in TES/NaCl buffer with or without IPVL at 40 ℃ was almost negligible, due to the temperature-dependent immobilization of fowl spermatozoa. However, sperm motility was restored at 40 ℃ following the addition of calyculin A, but not by fenvalerate or deltamethrin. The AR was stimulated by IPVL extracts at 40 ℃, but only in the presence of Ca^<2+>, calyculin A, fenvalerate or deltamethrin, the latter three phosphatase inhibitors stimulating the AR in a dose-dependent manner in the range of 10-1000 nM. At 30 ℃, the effects of PP2A activators (C2 - Ceramide and C6 - Ceramide) on the motility of spermatozoa, were not clear. At 40 ℃, both intact and demembranated sperm motility were almost negligible, regardless of the presence of Ceramides. Furthermore, stimulation of motility by Ca^<2+> was gradually inhibited following the addition of Ceramides. The same tendency was observed even when calyculin A was used instead of Ca^<2+>.These results suggest that Ca^<2+> may act via activation of protein phosphorylation pathways and that the Ca^<2+> -activated intracellular molecular mechanisms for the regulation of AR are different from those for restoration of sperm motility ; i.e., protein dephosphorylation by PP2B, in addition to PP1 and/or PP2A, in the former and PP1 and/or PP2A alone in the latter case.