Yasuhiro Tsuzuki

PRESENCE OF PROTEIN PHOSPHATASE TYPE 1 AND ITS INVOLVEMENT IN TEMPERATURE-DEPENDENT FLAGELLAR MOVEMENT OF FOWL SPERMATOZOA

Even in the presence of ATP, the motility of demembranated fowl spermatozoa was negligible at the avian body temperature of 40°C. Motility could be restored by the addition of calyculin A, okadaic acid, specific inhibitors of phosphatase type 1 (PP1) and PP‐2A, and inhibitor 1 or inhibitor 2, which are specific inhibitors of protein phosphatase type 1 (PP1). Demembranated spermatozoa, stimulated by calyculin A or okadaic acid, lost their motility following the addition of 1 mM CaCl2, but this was restored gradually by the stepwise addition of EGTA. Immunoblotting of sperm extract using an antibody to PP1 revealed a major cross‐reacting protein of 36–37 kDa, which corresponded to the molecular weight of the known catalytic subunit of PP1. These results suggest that PP1 present in the fowl sperm axoneme may be involved in the inhibition of fowl sperm motility at 40°C via Ca2+‐dependent regulatory systems.

Regulation of flagellar motility by temperature-dependent phosphorylation of a 43 kDa axonemal protein in fowl spermatozoa

Phosphorylation of fowl sperm proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after incubating the demembranated spermatozoa with [gamma-32P]ATP at 30 degrees C or 40 degrees C. A marked difference of phosphorylation between 30 degrees C and 40 degrees C was observed in a 43 kDa protein. This protein was slightly phosphorylated at 40 degrees C, but strongly phosphorylated at 30 degrees C in a cAMP-independent manner. The motility of demembranated spermatozoa was negligible at 40 degrees C, but vigorous movement was observed at 30 degrees C. These results showed that phosphorylation of a 43 kDa protein is likely to be a regulatory step in the maintenance of fowl sperm motility.

Effects of tyrosine kinase inhibitor on the motility and ATP concentrations of fowl spermatozoa

The possible role of tyrosine kinase in the regulation of fowl sperm motility was investigated by using a stable analogue of erbstatin, methyl 2,5‐dihydroxycinnamate (2,5‐MeC), a specific inhibitor of tyrosine kinase. This inhibited the motility of intact spermatozoa at 30°C in a dose‐dependent manner. In contrast, the motility of demembranated spermatozoa was not inhibited by the same concentrations of 2,5‐MeC. At 40°C, both intact and demembranated spermatozoa were almost immotile with or without 2,5‐MeC. Additionally, intact spermatozoa, stimulated by the addition of Ca2+ or calyculin A, a specific inhibitor of protein phosphatases, lost their motility with the subsequent addition of 2,5‐MeC at 40°C. However, unlike the motility, the ATP concentrations of spermatozoa were maintained in about 30–35 nmol ATP/109 cells during these incubation periods. The activity of tyrosine kinase of spermatozoa at 30°C, estimated by measuring the phosphorylation of a synthetic peptide substrate, RR‐SRC, was 0.17 pmol/min per milligram of protein. This activity was lower than that of fowl testes or chick brain but higher than that of chick liver. These results suggest that tyrosine kinase activity, which is not retained in the axoneme and/or accessory cytoskeletal components, may be involved in the maintenance of flagellar movement of fowl spermatozoa at 30°C. Mol. Reprod. Dev. 49:196–202, 1998.

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.

Inhibition of flagellar motility of demembranated fowl spermatozoa by protease substrates.

We investigated the effects of various protease substrates on the motility of demembranated fowl spermatozoa. In the presence of ATP, the motility of demembranated spermatozoa was vigorous at 30 degrees C, but decreased markedly following the addition of protease substrates, such as N alpha-carbobenzoxy-L-lys-thiobenzyl ester (BLT), N-benzoyl-phe-val-arg p-nitroanilide or N alpha-benzoyl-D,L-arg p-nitroanilide (BAPNA) in a dose-dependent manner, within the range 0-1 mM. The subsequent addition of 100 ng/ml trypsin released the inhibitory effect of protease substrates within 10 s. Phosphorylation or dephosphorylation of several proteins of demembranated spermatozoa was observed following the addition of protease substrates, however, no consistent patterns of protein phosphorylation or dephosphorylation were associated with the inhibition of motility. These results suggest that endogenous protease activity is instrumental in the maintenance of fowl sperm motility and that the site of action of this protease is in the axoneme and/or accessory cytoskeletal components. This enzyme may not act directly on the phosphorylation of sperm proteins involved in the regulation of motility.

Regulatory system of temperature-dependent flagellar motility is retained after freezing and thawing of demembranated fowl spermatozoa

Abstract In the presence of adenosine 5′-triphosphate (ATP), the motility of demembranated fowl spermatozoa was vigorous at 30 °C, both before and after rapid freezing at −70 °C or −196 °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/min, also similar to that of the untreated spermatozoa. At 40 °C, neither frozen-thawed spermatozoa nor control spermatozoa were motile, but motility could be restored by decreasing the temperature to 30 °C or by the addition of inhibitors of protein phosphatases. These observations suggest that the substance(s) involved in the temperature-dependent immobilization of fowl spermatozoa are closely associated with the axoneme, and that physical damage to the demembranated spermatozoa, such as freezing and thawing, is not enough to remove the substance(s).