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The possible role of protein-carboxyl methylation in the regulation of flagellar movement of fowl spermatozoa
Animal Reproduction Science 58 Ž2000. 137–146 www.elsevier.comrlocateranireprosci
The possible role of protein-carboxyl methylation in the regulation of flagellar movement of fowl spermatozoa K. Ashizawa) , M. Miyamoto, Y. Tsuzuki Laboratory of Animal Reproduction, Faculty of Agriculture, Miyazaki UniÕersity, Miyazaki 889-2192, Japan Received 5 March 1999; received in revised form 5 October 1999; accepted 19 October 1999
Abstract Both intact and demembranated fowl spermatozoa were incubated at 308C and 408C with adenosine, 3-deazaadenosine and homocysteine thiolactone. This combination of products is known to block intracellular protein-carboxyl methylation reaction. The motility of intact spermatozoa incubated at 308C was vigorous but decreased markedly after the addition of 100 mM adenosineq 100 mM 3-deazaadenosineq 100 mM homocysteine thiolactone. During this incubation period, the intracellular ATP concentrations of spermatozoa were maintained at approximately 40 nmol ATPr10 9 cells, in spite of the inhibition of motility. The motility of demembranated spermatozoa at 308C was not inhibited by the same concentrations of blocker. At 408C, the motility of intact spermatozoa without any effectors was almost negligible. The addition of blocker did not appreciably affect the motility of spermatozoa, which remained almost negligible. In contrast, motility became vigorous even at 408C when intact spermatozoa were suspended in fluid to which had been added 1 mM CaCl 2 or 100 nM calyculin A, a specific inhibitor of protein phosphatase-type 1 and -type 2. Stimulation of motility by Ca2q or calyculin A was inhibited by the presence of a blocker. Contrary to that of intact spermatozoa, the motility of demembranated spermatozoa stimulated by protein phosphatase inhibitor at 408C was not inhibited by the presence of a blocker. These results suggest that protein-carboxyl methylation may be involved in the regulation of fowl sperm motility. Furthermore, it appears that the methylating enzyme may be present in the cytoplasmic matrix andror the plasma membrane but not retained in the axoneme andror accessory cytoskeletal components. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Spermatozoa; Protein-carboxyl methylation; Motility; Temperature
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Corresponding author. Fax: q81-985-58-2884. E-mail address: [email protected] ŽK. Ashizawa..
0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 9 . 0 0 0 8 5 - 8
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1. Introduction Unlike mammalian spermatozoa, fowl spermatozoa become immotile at the avian body temperature of 40–418C. Motility is restored by decreasing the temperature or by the addition of Ca2q at 408C ŽMunro, 1938; Ashizawa and Nishiyama, 1978; Takeda, 1982; Wishart and Ashizawa, 1987; Ashizawa et al., 1989a; Thomson and Wishart, 1989, 1991.. Ca2q is the major stimulatory factor in body fluids such as seminal plasma, or the fluid of the female ovarian pocket taken at the time of ovulation, in which spermatozoa can maintain their motility even at 408C ŽAshizawa and Wishart, 1987, 1992.. The axoneme andror accessory cytoskeletal components themselves appear to be directly involved in the temperature-dependent regulatory system, since the motility of demembranated spermatozoa is, as with intact spermatozoa, negligible at 408C and restored at 308C ŽAshizawa et al., 1989a,b, 1996, 1998b.. However, the intracellular molecular mechanisms involved in immobilization and restoration of motility are unknown. Reversible covalent modifications play a central role in regulating protein activities. The most common chemistry involves phosphorylation and dephosphorylation at serine, threonine and tyrosine residues. Phosphorylation at these side chains has been shown to be essential to the control of a wide range of cellular processes, including metabolism, motility, growth and gene expression. Another type of modification involves methylation and demethylation at carboxyl groups ŽFloer and Stock, 1994.. Substantial evidence implicates a role for protein phosphorylation by cAMP and cAMP-dependent protein kinase in the initiation and activation of sperm flagellar motility in a variety of species Žfor reviews, see Tash, 1989; Tash and Means, 1983; Tash and Bracho, 1994; Brokaw, 1987; Lindemann and Kanous, 1989; Morisawa, 1994.. However, unlike that of other species, the motility of demembranated fowl spermatozoa was not restored by the addition of cAMP at 408C ŽAshizawa et al., 1989b, 1992b., and the presence of cAMP-dependent protein kinase substrate peptides did not appreciably affect the motility at 308C ŽAshizawa et al., 1995a,b.. In contrast, it has been proposed that the phosphorylation of axonemal andror accessory cytoskeletal proteinŽs. by myosin light chain kinase ŽMLCK. or an MLCK-like protein may be involved in some part of the regulatory cascade of fowl sperm motility ŽAshizawa et al., 1995a,b.. Tyrosine kinase activity, which is not retained in the axoneme andror accessory cytoskeletal components, may also be involved in the maintenance of flagellar movement of fowl spermatozoa at 308C ŽAshizawa et al., 1998a.. With regard to protein dephosphorylation, it has been suggested that inhibition of mammalian sperm motility by Ca2q may be due to the activation of protein phosphatase-type 2B ŽPP-2B., a Ca2qrcalmodulin-dependent protein phosphatase ŽTash et al., 1988.. More recent work has demonstrated that the activation of protein phosphatase-type 1 ŽPP-1., one of serinerthreonine phosphatases, may be dominant in the involvement for the temperature-dependent regulation of fowl sperm motility ŽAshizawa et al., 1994b, 1997.. This was because in addition to calyculin A and okadaic acid Žspecific inhibitors of PP-1 and PP-2A., the PP-1-specific inhibitors 1 and 2 ŽCohen, 1989. also stimulated the motility of demembranated spermatozoa at 408C ŽAshizawa et
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al., 1994b., and the motility decreased markedly following the addition of recombinant PP-1 supplemented with Mn2q at 308C ŽAshizawa et al., 1997.. Whereas the protein phosphorylation and dephosphorylation system has been extensively studied, the protein-carboxyl methylation and demethylation system has received much less attention, in spite of the presence of a methylating enzyme, protein-carboxyl methylase, and its substrate, methyl acceptor proteinŽs., in rat, rabbit and human spermatozoa ŽBouchard et al., 1980; Sastry and Janson, 1983.. Therefore, it has been suggested that the presence of all components of the protein-carboxyl methylation system in spermatozoa and the localization of protein-carboxyl methylase and some of its substrates in the sperm tail may be consistent with their involvement in sperm motility ŽBouchard et al., 1980.. Indeed, a highly significant relationship Ž r s 0.78; P - 0.001. was observed between the levels of sperm protein-carboxyl methylase activity and motility in human spermatozoa of infertile patients ŽGagnon et al., 1986.. The addition of protein-carboxyl methylation blockers caused a time-dependent progressive inhibition of intact sperm motility in rabbit and human ŽGagnon et al., 1983; Sastry and Janson, 1983.. In addition, such blockers inhibited the acrosome reactions of hamster spermatozoa ŽMeizel, 1981.. Thus, Castaneda et al. Ž1983. suggested that the conditions that lead to capacitation in vitro might be associated with a marked change in endogenous protein-carboxyl methylation. The role of protein-carboxyl methylation in the temperature-dependent immobilization and restoration of fowl spermatozoa has yet to be determined. In the following experiment, therefore, attempts were made to investigate the effects of protein-carboxyl methylation blocker on the motility and ATP concentrations of fowl spermatozoa.
2. Materials and methods 2.1. Animals and preparation of spermatozoa Commercial White Leghorn roosters ŽBabcock strain, Akagi Poultry Breeding Farm, Miyazaki, Japan. were used throughout the study. All birds were housed in individual cages and fed ad libitum on a commercial breeder diet. They were exposed to a photoperiod of 14 h light:10 h dark. Semen was collected by the method of Bogdonoff and Shaffner Ž1954.. Samples of semen pooled from four to six males were diluted approximately 10-fold in 150 mM NaCl with 20 mM TES Ž N-Triswhydroxymethylx-methyl-2-aminoethanesulfonic acid. at pH 7.4 and centrifuged at 700 = g for 15 min at room temperature Ž20–258C.. The washed spermatozoa were reconstituted in the same buffers to give a final concentration of approximately 1 = 10 9 cellsrml. Samples of 3–4 ml were poured into 30 ml Erlenmeyer flasks with a screw cap. 2.2. Chemicals Adenosine, adenosine 5X-triphosphate ŽATP., 3-deazaadenosine, desiccated firefly tails, dithiothreitol ŽDTT., DL-homocysteine thiolactone, p-nitrophenyl phosphate, potas-
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sium glutamate, TES and Triton X-100 were purchased from Sigma ŽSt. Louis, MO.. Calyculin A and okadaic acid were obtained from Wako, ŽOsaka, Japan.. Other chemicals were of reagent grade from Nacalai Tesque ŽKyoto, Japan.. 2.3. Analysis of intact and demembranated sperm motility Intact spermatozoa diluted at 100-fold Žapproximately 1 = 10 7 cellsrml. with TESrNaCl buffer described above were incubated aerobically in a shaking water bath at 308C and 408C for 1 h with or without protein-carboxyl methylation blocker, i.e., 100 mM adenosineq 100 mM 3-deazaadenosineq 100 mM homocysteine thiolactone. After the incubation, motility of spermatozoa was measured at 308C or 408C. The effects of the addition of CaCl 2 or calyculin A, an inhibitor of protein phosphatase-type 1 and -type 2, in the presence or absence of the blocker were also examined at 408C. Demembranation and reactivation of spermatozoa were performed according to the method described previously ŽAshizawa et al., 1989b.. The extraction medium used consisted of 0.1% Žvrv. Triton X-100, 200 mM sucrose, 25 mM potassium glutamate, 1 mM MgSO4 , 1 mM DTT and 20 mM Tris–HCl buffer ŽpH 7.9.. The reactivation medium consisted of 0.5 mM ATP, 200 mM sucrose, 25 mM potassium glutamate, 1.5 mM MgSO4 , 1 mM DTT and 20 mM Tris–HCl buffer ŽpH 7.9.. To examine the effects of protein-carboxyl methylation blocker, 100 mM adenosineq 100 mM 3-deazaadenosine q 100 mM homocysteine thiolactone were added to the reactivation medium at 308C or 408C and incubated for 15 min. In addition, at 408C, the following were added to the blocker-treated spermatozoa: calyculin A, okadaic acid Žspecific inhibitors of protein phosphatase-type 1 and -type 2., p-nitrophenyl phosphate Ža non-specific phosphatase substrate. and CaCl 2 q EGTA. The suspension of intact or demembranated spermatozoa was placed into a microscope slide chamber ŽSekisui Chemical, UR-157 type, Tokyo, Japan. on a thermostatically-controlled warm plate, and the motility of spermatozoa was recorded by videomicroscopy Žmagnification on the 12-in. black and white monitor was approximately= 600.
Table 1 The motility of intact or demembranated fowl spermatozoa and ATP concentrations of intact spermatozoa incubated with protein-carboxyl methylation blocker at 308C Each value represents the mean Ž"s.e.m.. of five samples of spermatozoa. Within columns, values with different superscripts differ significantly Ž P - 0.01. from each other. Substances Žconc..
Motility Ž%.
ATP concentrations Žnmolr10 9 spermatozoa.
Intact spermatozoa None Žcontrol. Adenosine Ž100 mM.q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM.
68.5"5.2 a 14.7"2.1b
39.3"3.9 a 40.6"2.2 a
Demembranated spermatozoa None Žcontrol. Adenosine Ž100 mM.q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM.
63.9"4.5a 61.8"5.6 a
– –
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at 308C or 408C ŽKatz and Overstreet, 1981.. Measurements were made on a total of 200–300 spermatozoa, distributed uniformly among three or more fields, to determine the percentage of vigorously motile spermatozoa. 2.4. Analysis of intact sperm ATP concentrations ATP content in spermatozoa was assayed by firefly bioluminescence in a boiled extract ŽWishart, 1982.. Numbers of spermatozoa were estimated by the method of Wishart and Ross Ž1985., using a double-beam spectrophotometer ŽShimadzu, Model UV-150-02, Kyoto, Japan.. The concentration of ATP was expressed in terms of nmol ATPr10 9 spermatozoa. 2.5. Statistical analysis Percentage of motility was transformed using arc sine transformation. The results were analyzed by Duncan’s multiple-range tests ŽDuncan, 1955.. 3. Results 3.1. Effects of protein-carboxyl methylation blocker on the motility of intact and demembranated fowl spermatozoa The motility of intact spermatozoa incubated at 308C was vigorous but decreased markedly after the addition of 100 mM adenosineq 100 mM 3-deazaadenosineq 100 mM homocysteine thiolactone ŽTable 1.. However, the motility of demembranated spermatozoa at 308C was not inhibited by the same concentrations of blocker ŽTable 1.. At 408C, the motility of intact spermatozoa without any effectors was almost negligible ŽTable 2.. The addition of adenosineq 3-deazaadenosineq homocysteine thiolactone did not appreciably affect the motility of intact spermatozoa, which remained almost
Table 2 The motility and ATP concentrations of intact fowl spermatozoa incubated with protein-carboxyl methylation blocker at 408C Each value represents the mean Ž"s.e.m.. of five samples of spermatozoa. Within columns, values with different superscripts differ significantly Ž P - 0.01. from each other. Substances Žconc.. None Žcontrol. Adenosine Ž100 mM.q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM. CaCl 2 Ž1 mM. Calyculin A Ž100 nM. CaCl 2 Ž1 mM.qadenosine Ž100 mM. q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM. Calyculin A Ž100 nM.qadenosine Ž100 mM. q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM.
Motility Ž%.
ATP concentrations Žnmolr10 9 spermatozoa.
4.7"1.9 a 5.1"2.3 a
44.8"4.6 a 42.9"3.9 a
58.2"3.5 b 52.4"5.3 b 8.7"2.2 a
33.6"3.5a 35.5"3.7 a 43.2"4.9 a
7.1"1.6 a
42.5"3.2 a
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negligible ŽTable 2.. In contrast, motility became vigorous even at 408C when intact spermatozoa were suspended in fluid to which had been added 1 mM CaCl 2 or 100 nM calyculin A. However, stimulation of motility by Ca2q or calyculin A was inhibited by the presence of adenosineq 3-deazaadenosineq homocysteine thiolactone ŽTable 2.. The motility of demembranated spermatozoa under control conditions was inhibited at 408C as well as the case for intact spermatozoa. The addition of adenosineq 3deazaadenosineq homocysteine thiolactone did not appreciably affect the motility of demembranated spermatozoa without regard to the presence or absence of Ca2q and EGTA ŽTable 3.. However, only the presence of calyculin A and okadaic acid, specific inhibitors of protein phosphatase-type 1 and -type 2, or p-nitrophenyl phosphate, a non-specific phosphatase substrate, permitted reactivation of demembranated spermatozoa at 408C ŽTable 3.. In addition, contrary to the intact spermatozoa, this stimulation of motility by protein phosphatase inhibitors or substrate was not inhibited by the presence of adenosineq 3-deazaadenosineq homocysteine thiolactone ŽTable 3.. 3.2. Effects of protein-carboxyl methylation blocker on the ATP concentrations of intact fowl spermatozoa The ATP concentrations of intact spermatozoa were similar in control solution or following exposure to adenosineq 3-deazaadenosineq homocysteine thiolactone at 308C
Table 3 The motility of demembranated fowl spermatozoa incubated with protein-carboxyl methylation blocker at 408C Each value represents the mean Ž"s.e.m.. of five samples of spermatozoa. Within columns, values with different superscripts differ significantly Ž P - 0.01. from each other. Substances Žconc.. None Žcontrol. Adenosine Ž100 mM.q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM. CaCl 2 Ž1 mM. EGTA Ž1 mM. CaCl 2 Ž1 mM.qEGTA Ž1 mM. Calyculin A Ž100 nM. Okadaic acid Ž1000 nM. p-Nitrophenyl phosphate Ž10 mM. CaCl 2 Ž1 mM.qEGTA Ž1 mM.qadenosine Ž100 mM. q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM. Calyculin A Ž100 nM.qadenosine Ž100 mM. q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM. Okadaic acid Ž1000 nM.qadenosine Ž100 mM. q3-deazaadenosine Ž100 mM.qhomocysteine thiolactone Ž100 mM. p-Nitrophenyl phosphate Ž10 mM. qadenosine Ž100 mM.q3-deazaadenosine Ž100 mM.qhomocysteine thiolactone Ž100 mM.
MotilityŽ%. 1.9"0.7 a 1.5"0.9 a 1.3"0.5a 0.8"0.8 a 1.6"0.7 a 61.5"5.2 b 59.8"4.7 b 63.4"6.1b 2.1"0.8 a
60.7"6.3 b
51.9"5.6 b 59.2"5.3 b
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ŽTable 1. and 408C ŽTable 2.. When the addition of CaCl 2 or calyculin A alone, the ATP concentrations of spermatozoa appeared to decrease slightly ŽTable 2., presumably due to the restoration of motility at 408C. In contrast to the effect on motility, the presence of adenosineq 3-deazaadenosineq homocysteine thiolactone with CaCl 2 or calyculin A did not induce the decrease of ATP concentrations compared with those of the addition of CaCl 2 or calyculin A alone at 408C ŽTable 2..
4. Discussion The protein-carboxyl methylation and determination system is composed of three macromolecular components: a methylating enzyme named protein-carboxyl methylase, a demethylating enzyme named protein methylesterase, and substrates named methyl acceptor proteins. The combination of adenosine, 3-deazaadenosine and homocysteine thiolactone has been shown to block intracellular protein-carboxyl methylation reaction ŽCantoni et al., 1979.. The addition of adenosineq 3-deazaadenosineq homocysteine thiolactone caused a time-dependent progressive inhibition of rabbit sperm protein-carboxyl methylation activity. This inhibition was associated with a concomitant decrease in intact sperm motility, although the intracellular ATP concentrations have not been measured ŽGagnon, 1990.. Similar results were obtained in human spermatozoa ŽSastry and Janson, 1983.. In the study reported here, the motility of intact fowl spermatozoa incubated at 308C was also decreased markedly after the addition of 100 mM adenosineq 100 mM 3deazaadenosineq 100 mM homocysteine thiolactone. At 408C, stimulation of intact sperm motility by Ca2q or calyculin A was inhibited by the presence of blocker. During these incubation periods, the intracellular ATP concentrations of spermatozoa were maintained at approximately 40–43 nmol ATPr10 9 cells, in spite of the inhibition of motility. Therefore, it appears that the blocker is not simply inhibiting energy production in these spermatozoa, but may be acting on some part of the regulatory cascade which thus appears to involve protein-carboxyl methylation reaction. Unlike that of intact spermatozoa, the motility of demembranated fowl spermatozoa at 308C was not inhibited by the same concentrations of blocker. At 408C, the motility of demembranated spermatozoa stimulated by protein phosphatase inhibitors was not inhibited by the presence of blocker. Hence, the methylating enzyme may be present in the cytoplasmic matrix andror the plasma membrane but not retained in the axoneme andror accessory cytoskeletal components. However, the location of the substrateŽs. for this enzyme still remains unclear. It may be in the cytoplasmic matrix andror plasma membrane or in the axoneme andror accessory cytoskeletal components. It has, however, been shown that protein-carboxyl methylase and methyl acceptor proteins appeared to be predominantly present in the Triton X-100 soluble fraction of rabbit sperm tail, whereas protein methylesterase was not solubilized by nonionic detergent ŽBouchard et al., 1980.. Earlier studies have clearly shown that the stimulatory effects of temperature or protein phosphatase-type 1 inhibitors on fowl sperm motility act on demembranated spermatozoa — i.e., on the axoneme andror accessory cytoskeletal components
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ŽAshizawa et al., 1989a,b, 1994b.. It seems therefore that in intact fowl spermatozoa, stimulation of motility by these effectorsrconditions depends on the ‘‘permissive’’ effect of a proteinŽs. ‘‘upstream’’ of the axoneme andror accessory cytoskeletal components, which is active on the protein-carboxyl methylation. The intracellular free Ca2q seems to be essential for the maintenance of intact fowl sperm motility ŽAshizawa et al., 1992a.. Moreover, Ashizawa et al. Ž1994a. suggested that calmodulin, which occurs in the cytoplasmic matrix andror the plasma membrane but not in the axoneme andror accessory cytoskeletal components, is a prominent candidate for the signal transducer in Ca2q-stimulated motility. This was because the motility of intact spermatozoa loaded with an intracellular Ca2q chelator, 1, 2-bis Ž2-aminophenoxy. ethaneN, N, N X , N X-tetraacetic acid, tetraacetoxymethyl ester ŽBAPTA-AM., was negligible at 308C, but could be immediately restored by the subsequent addition of excess Ca2q to the medium, and even in the presence of Ca2q, the subsequent addition of calmodulin antagonists such as N-Ž6-aminohexyl.-5-chloro-1-naphthalenesulfonamide hydrochloride ŽW-7. and trifluoperazine inhibited the motility of intact but not demembranated spermatozoa ŽAshizawa et al., 1994a.. Calmodulin has been shown to be one of the substrates of protein-carboxyl methylase ŽGagnon et al., 1981; Johnson et al., 1985; Brunauer and Clarke, 1986; Vincent and Siegel, 1987.. Therefore, it is possible that the addition of adenosineq 3-deazaadenosineq homocysteine thiolactone might inhibit the methylation of cytosolic calmodulin and hence inhibit the motility of intact fowl spermatozoa, although the precise mechanisms of such a process remain to be determined.
Acknowledgements The authors thank Dr. G.J. Wishart, University of Abertay Dundee, Scotland, UK, for kindly reviewing the manuscript. This study was supported by a grant from the Ministry of Education, Science and Culture, Japan ŽNo. 11460130..
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Meizel, S., 1981. Inhibition of the hamster sperm acrosome reaction by transmethylation inhibitors. J. Exp. Zool. 217, 443–446. Morisawa, M., 1994. Cell signaling mechanisms for sperm motility. Zool. Sci. 11, 647–662. Munro, S.S., 1938. Fowl sperm immobilization by a temperature-media interaction and its biological significance. Q.J. Exp. Physiol. 27, 281–287. Sastry, B.V., Janson, V.E., 1983. Depression of human sperm motility by inhibition of enzymatic methylation. Biochem. Pharmacol. 32, 1423–1432. Takeda, A., 1982. Studies on reversible inactivation of cock spermatozoa by temperature. I. Effects of several factors on reversible inactivation. Jpn. Poult. Sci. 19, 26–36. Tash, J.S., 1989. Protein phosphorylation: the second messenger signal transducer of flagellar motility. Cell Motil. Cytoskeleton 14, 332–339. Tash, J.S., Bracho, G.E., 1994. Regulation of sperm motility: emerging evidence for a major role for protein phosphatases. J. Androl. 15, 505–509. Tash, J.S., Krinks, M., Patel, J., Means, R.L., Klee, C.B., Means, A.R., 1988. Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm. J. Cell Biol. 106, 1625–1633. X X Tash, J.S., Means, A.R., 1983. Cyclic adenosine 3 , 5 monophosphate, calcium and protein phosphorylation in flagellar motility. Biol. Reprod. 28, 75–104. Thomson, M.F., Wishart, G.J., 1989. Elucidation of the mechanism responsible for the temperature-dependent reversible inactivation of the motility of fowl spermatozoa. Br. Poult. Sci. 30, 687–692. Thomson, M.F., Wishart, G.J., 1991. Temperature-mediated regulation of calcium flux and motility in fowl spermatozoa. J. Reprod. Fertil. 93, 385–391. Vincent, P.L., Siegel, F.L., 1987. Carboxylmethylation of calmodulin in cultured pituitary cells. J. Neurochem. 49, 1613–1622. Wishart, G.J., 1982. Maintenance of ATP concentrations in and of fertilizing ability of fowl and turkey spermatozoa in vitro. J. Reprod. Fertil. 66, 457–462. Wishart, G.J., Ashizawa, K., 1987. Regulation of motility of fowl spermatozoa by calcium and cAMP. J. Reprod. Fertil. 80, 607–611. Wishart, G.J., Ross, F.H., 1985. Characterization of a spectrophotometric technique for the estimation of fowl and turkey sperm motility. Gamete Res. 11, 169–178.
The possible role of protein-carboxyl methylation in the regulation of flagellar movement of fowl spermatozoa K. Ashizawa) , M. Miyamoto, Y. Tsuzuki Laboratory of Animal Reproduction, Faculty of Agriculture, Miyazaki UniÕersity, Miyazaki 889-2192, Japan Received 5 March 1999; received in revised form 5 October 1999; accepted 19 October 1999
Abstract Both intact and demembranated fowl spermatozoa were incubated at 308C and 408C with adenosine, 3-deazaadenosine and homocysteine thiolactone. This combination of products is known to block intracellular protein-carboxyl methylation reaction. The motility of intact spermatozoa incubated at 308C was vigorous but decreased markedly after the addition of 100 mM adenosineq 100 mM 3-deazaadenosineq 100 mM homocysteine thiolactone. During this incubation period, the intracellular ATP concentrations of spermatozoa were maintained at approximately 40 nmol ATPr10 9 cells, in spite of the inhibition of motility. The motility of demembranated spermatozoa at 308C was not inhibited by the same concentrations of blocker. At 408C, the motility of intact spermatozoa without any effectors was almost negligible. The addition of blocker did not appreciably affect the motility of spermatozoa, which remained almost negligible. In contrast, motility became vigorous even at 408C when intact spermatozoa were suspended in fluid to which had been added 1 mM CaCl 2 or 100 nM calyculin A, a specific inhibitor of protein phosphatase-type 1 and -type 2. Stimulation of motility by Ca2q or calyculin A was inhibited by the presence of a blocker. Contrary to that of intact spermatozoa, the motility of demembranated spermatozoa stimulated by protein phosphatase inhibitor at 408C was not inhibited by the presence of a blocker. These results suggest that protein-carboxyl methylation may be involved in the regulation of fowl sperm motility. Furthermore, it appears that the methylating enzyme may be present in the cytoplasmic matrix andror the plasma membrane but not retained in the axoneme andror accessory cytoskeletal components. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Spermatozoa; Protein-carboxyl methylation; Motility; Temperature
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Corresponding author. Fax: q81-985-58-2884. E-mail address: [email protected] ŽK. Ashizawa..
0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 9 . 0 0 0 8 5 - 8
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1. Introduction Unlike mammalian spermatozoa, fowl spermatozoa become immotile at the avian body temperature of 40–418C. Motility is restored by decreasing the temperature or by the addition of Ca2q at 408C ŽMunro, 1938; Ashizawa and Nishiyama, 1978; Takeda, 1982; Wishart and Ashizawa, 1987; Ashizawa et al., 1989a; Thomson and Wishart, 1989, 1991.. Ca2q is the major stimulatory factor in body fluids such as seminal plasma, or the fluid of the female ovarian pocket taken at the time of ovulation, in which spermatozoa can maintain their motility even at 408C ŽAshizawa and Wishart, 1987, 1992.. The axoneme andror accessory cytoskeletal components themselves appear to be directly involved in the temperature-dependent regulatory system, since the motility of demembranated spermatozoa is, as with intact spermatozoa, negligible at 408C and restored at 308C ŽAshizawa et al., 1989a,b, 1996, 1998b.. However, the intracellular molecular mechanisms involved in immobilization and restoration of motility are unknown. Reversible covalent modifications play a central role in regulating protein activities. The most common chemistry involves phosphorylation and dephosphorylation at serine, threonine and tyrosine residues. Phosphorylation at these side chains has been shown to be essential to the control of a wide range of cellular processes, including metabolism, motility, growth and gene expression. Another type of modification involves methylation and demethylation at carboxyl groups ŽFloer and Stock, 1994.. Substantial evidence implicates a role for protein phosphorylation by cAMP and cAMP-dependent protein kinase in the initiation and activation of sperm flagellar motility in a variety of species Žfor reviews, see Tash, 1989; Tash and Means, 1983; Tash and Bracho, 1994; Brokaw, 1987; Lindemann and Kanous, 1989; Morisawa, 1994.. However, unlike that of other species, the motility of demembranated fowl spermatozoa was not restored by the addition of cAMP at 408C ŽAshizawa et al., 1989b, 1992b., and the presence of cAMP-dependent protein kinase substrate peptides did not appreciably affect the motility at 308C ŽAshizawa et al., 1995a,b.. In contrast, it has been proposed that the phosphorylation of axonemal andror accessory cytoskeletal proteinŽs. by myosin light chain kinase ŽMLCK. or an MLCK-like protein may be involved in some part of the regulatory cascade of fowl sperm motility ŽAshizawa et al., 1995a,b.. Tyrosine kinase activity, which is not retained in the axoneme andror accessory cytoskeletal components, may also be involved in the maintenance of flagellar movement of fowl spermatozoa at 308C ŽAshizawa et al., 1998a.. With regard to protein dephosphorylation, it has been suggested that inhibition of mammalian sperm motility by Ca2q may be due to the activation of protein phosphatase-type 2B ŽPP-2B., a Ca2qrcalmodulin-dependent protein phosphatase ŽTash et al., 1988.. More recent work has demonstrated that the activation of protein phosphatase-type 1 ŽPP-1., one of serinerthreonine phosphatases, may be dominant in the involvement for the temperature-dependent regulation of fowl sperm motility ŽAshizawa et al., 1994b, 1997.. This was because in addition to calyculin A and okadaic acid Žspecific inhibitors of PP-1 and PP-2A., the PP-1-specific inhibitors 1 and 2 ŽCohen, 1989. also stimulated the motility of demembranated spermatozoa at 408C ŽAshizawa et
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al., 1994b., and the motility decreased markedly following the addition of recombinant PP-1 supplemented with Mn2q at 308C ŽAshizawa et al., 1997.. Whereas the protein phosphorylation and dephosphorylation system has been extensively studied, the protein-carboxyl methylation and demethylation system has received much less attention, in spite of the presence of a methylating enzyme, protein-carboxyl methylase, and its substrate, methyl acceptor proteinŽs., in rat, rabbit and human spermatozoa ŽBouchard et al., 1980; Sastry and Janson, 1983.. Therefore, it has been suggested that the presence of all components of the protein-carboxyl methylation system in spermatozoa and the localization of protein-carboxyl methylase and some of its substrates in the sperm tail may be consistent with their involvement in sperm motility ŽBouchard et al., 1980.. Indeed, a highly significant relationship Ž r s 0.78; P - 0.001. was observed between the levels of sperm protein-carboxyl methylase activity and motility in human spermatozoa of infertile patients ŽGagnon et al., 1986.. The addition of protein-carboxyl methylation blockers caused a time-dependent progressive inhibition of intact sperm motility in rabbit and human ŽGagnon et al., 1983; Sastry and Janson, 1983.. In addition, such blockers inhibited the acrosome reactions of hamster spermatozoa ŽMeizel, 1981.. Thus, Castaneda et al. Ž1983. suggested that the conditions that lead to capacitation in vitro might be associated with a marked change in endogenous protein-carboxyl methylation. The role of protein-carboxyl methylation in the temperature-dependent immobilization and restoration of fowl spermatozoa has yet to be determined. In the following experiment, therefore, attempts were made to investigate the effects of protein-carboxyl methylation blocker on the motility and ATP concentrations of fowl spermatozoa.
2. Materials and methods 2.1. Animals and preparation of spermatozoa Commercial White Leghorn roosters ŽBabcock strain, Akagi Poultry Breeding Farm, Miyazaki, Japan. were used throughout the study. All birds were housed in individual cages and fed ad libitum on a commercial breeder diet. They were exposed to a photoperiod of 14 h light:10 h dark. Semen was collected by the method of Bogdonoff and Shaffner Ž1954.. Samples of semen pooled from four to six males were diluted approximately 10-fold in 150 mM NaCl with 20 mM TES Ž N-Triswhydroxymethylx-methyl-2-aminoethanesulfonic acid. at pH 7.4 and centrifuged at 700 = g for 15 min at room temperature Ž20–258C.. The washed spermatozoa were reconstituted in the same buffers to give a final concentration of approximately 1 = 10 9 cellsrml. Samples of 3–4 ml were poured into 30 ml Erlenmeyer flasks with a screw cap. 2.2. Chemicals Adenosine, adenosine 5X-triphosphate ŽATP., 3-deazaadenosine, desiccated firefly tails, dithiothreitol ŽDTT., DL-homocysteine thiolactone, p-nitrophenyl phosphate, potas-
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sium glutamate, TES and Triton X-100 were purchased from Sigma ŽSt. Louis, MO.. Calyculin A and okadaic acid were obtained from Wako, ŽOsaka, Japan.. Other chemicals were of reagent grade from Nacalai Tesque ŽKyoto, Japan.. 2.3. Analysis of intact and demembranated sperm motility Intact spermatozoa diluted at 100-fold Žapproximately 1 = 10 7 cellsrml. with TESrNaCl buffer described above were incubated aerobically in a shaking water bath at 308C and 408C for 1 h with or without protein-carboxyl methylation blocker, i.e., 100 mM adenosineq 100 mM 3-deazaadenosineq 100 mM homocysteine thiolactone. After the incubation, motility of spermatozoa was measured at 308C or 408C. The effects of the addition of CaCl 2 or calyculin A, an inhibitor of protein phosphatase-type 1 and -type 2, in the presence or absence of the blocker were also examined at 408C. Demembranation and reactivation of spermatozoa were performed according to the method described previously ŽAshizawa et al., 1989b.. The extraction medium used consisted of 0.1% Žvrv. Triton X-100, 200 mM sucrose, 25 mM potassium glutamate, 1 mM MgSO4 , 1 mM DTT and 20 mM Tris–HCl buffer ŽpH 7.9.. The reactivation medium consisted of 0.5 mM ATP, 200 mM sucrose, 25 mM potassium glutamate, 1.5 mM MgSO4 , 1 mM DTT and 20 mM Tris–HCl buffer ŽpH 7.9.. To examine the effects of protein-carboxyl methylation blocker, 100 mM adenosineq 100 mM 3-deazaadenosine q 100 mM homocysteine thiolactone were added to the reactivation medium at 308C or 408C and incubated for 15 min. In addition, at 408C, the following were added to the blocker-treated spermatozoa: calyculin A, okadaic acid Žspecific inhibitors of protein phosphatase-type 1 and -type 2., p-nitrophenyl phosphate Ža non-specific phosphatase substrate. and CaCl 2 q EGTA. The suspension of intact or demembranated spermatozoa was placed into a microscope slide chamber ŽSekisui Chemical, UR-157 type, Tokyo, Japan. on a thermostatically-controlled warm plate, and the motility of spermatozoa was recorded by videomicroscopy Žmagnification on the 12-in. black and white monitor was approximately= 600.
Table 1 The motility of intact or demembranated fowl spermatozoa and ATP concentrations of intact spermatozoa incubated with protein-carboxyl methylation blocker at 308C Each value represents the mean Ž"s.e.m.. of five samples of spermatozoa. Within columns, values with different superscripts differ significantly Ž P - 0.01. from each other. Substances Žconc..
Motility Ž%.
ATP concentrations Žnmolr10 9 spermatozoa.
Intact spermatozoa None Žcontrol. Adenosine Ž100 mM.q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM.
68.5"5.2 a 14.7"2.1b
39.3"3.9 a 40.6"2.2 a
Demembranated spermatozoa None Žcontrol. Adenosine Ž100 mM.q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM.
63.9"4.5a 61.8"5.6 a
– –
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at 308C or 408C ŽKatz and Overstreet, 1981.. Measurements were made on a total of 200–300 spermatozoa, distributed uniformly among three or more fields, to determine the percentage of vigorously motile spermatozoa. 2.4. Analysis of intact sperm ATP concentrations ATP content in spermatozoa was assayed by firefly bioluminescence in a boiled extract ŽWishart, 1982.. Numbers of spermatozoa were estimated by the method of Wishart and Ross Ž1985., using a double-beam spectrophotometer ŽShimadzu, Model UV-150-02, Kyoto, Japan.. The concentration of ATP was expressed in terms of nmol ATPr10 9 spermatozoa. 2.5. Statistical analysis Percentage of motility was transformed using arc sine transformation. The results were analyzed by Duncan’s multiple-range tests ŽDuncan, 1955.. 3. Results 3.1. Effects of protein-carboxyl methylation blocker on the motility of intact and demembranated fowl spermatozoa The motility of intact spermatozoa incubated at 308C was vigorous but decreased markedly after the addition of 100 mM adenosineq 100 mM 3-deazaadenosineq 100 mM homocysteine thiolactone ŽTable 1.. However, the motility of demembranated spermatozoa at 308C was not inhibited by the same concentrations of blocker ŽTable 1.. At 408C, the motility of intact spermatozoa without any effectors was almost negligible ŽTable 2.. The addition of adenosineq 3-deazaadenosineq homocysteine thiolactone did not appreciably affect the motility of intact spermatozoa, which remained almost
Table 2 The motility and ATP concentrations of intact fowl spermatozoa incubated with protein-carboxyl methylation blocker at 408C Each value represents the mean Ž"s.e.m.. of five samples of spermatozoa. Within columns, values with different superscripts differ significantly Ž P - 0.01. from each other. Substances Žconc.. None Žcontrol. Adenosine Ž100 mM.q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM. CaCl 2 Ž1 mM. Calyculin A Ž100 nM. CaCl 2 Ž1 mM.qadenosine Ž100 mM. q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM. Calyculin A Ž100 nM.qadenosine Ž100 mM. q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM.
Motility Ž%.
ATP concentrations Žnmolr10 9 spermatozoa.
4.7"1.9 a 5.1"2.3 a
44.8"4.6 a 42.9"3.9 a
58.2"3.5 b 52.4"5.3 b 8.7"2.2 a
33.6"3.5a 35.5"3.7 a 43.2"4.9 a
7.1"1.6 a
42.5"3.2 a
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negligible ŽTable 2.. In contrast, motility became vigorous even at 408C when intact spermatozoa were suspended in fluid to which had been added 1 mM CaCl 2 or 100 nM calyculin A. However, stimulation of motility by Ca2q or calyculin A was inhibited by the presence of adenosineq 3-deazaadenosineq homocysteine thiolactone ŽTable 2.. The motility of demembranated spermatozoa under control conditions was inhibited at 408C as well as the case for intact spermatozoa. The addition of adenosineq 3deazaadenosineq homocysteine thiolactone did not appreciably affect the motility of demembranated spermatozoa without regard to the presence or absence of Ca2q and EGTA ŽTable 3.. However, only the presence of calyculin A and okadaic acid, specific inhibitors of protein phosphatase-type 1 and -type 2, or p-nitrophenyl phosphate, a non-specific phosphatase substrate, permitted reactivation of demembranated spermatozoa at 408C ŽTable 3.. In addition, contrary to the intact spermatozoa, this stimulation of motility by protein phosphatase inhibitors or substrate was not inhibited by the presence of adenosineq 3-deazaadenosineq homocysteine thiolactone ŽTable 3.. 3.2. Effects of protein-carboxyl methylation blocker on the ATP concentrations of intact fowl spermatozoa The ATP concentrations of intact spermatozoa were similar in control solution or following exposure to adenosineq 3-deazaadenosineq homocysteine thiolactone at 308C
Table 3 The motility of demembranated fowl spermatozoa incubated with protein-carboxyl methylation blocker at 408C Each value represents the mean Ž"s.e.m.. of five samples of spermatozoa. Within columns, values with different superscripts differ significantly Ž P - 0.01. from each other. Substances Žconc.. None Žcontrol. Adenosine Ž100 mM.q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM. CaCl 2 Ž1 mM. EGTA Ž1 mM. CaCl 2 Ž1 mM.qEGTA Ž1 mM. Calyculin A Ž100 nM. Okadaic acid Ž1000 nM. p-Nitrophenyl phosphate Ž10 mM. CaCl 2 Ž1 mM.qEGTA Ž1 mM.qadenosine Ž100 mM. q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM. Calyculin A Ž100 nM.qadenosine Ž100 mM. q3-deazaadenosine Ž100 mM. qhomocysteine thiolactone Ž100 mM. Okadaic acid Ž1000 nM.qadenosine Ž100 mM. q3-deazaadenosine Ž100 mM.qhomocysteine thiolactone Ž100 mM. p-Nitrophenyl phosphate Ž10 mM. qadenosine Ž100 mM.q3-deazaadenosine Ž100 mM.qhomocysteine thiolactone Ž100 mM.
MotilityŽ%. 1.9"0.7 a 1.5"0.9 a 1.3"0.5a 0.8"0.8 a 1.6"0.7 a 61.5"5.2 b 59.8"4.7 b 63.4"6.1b 2.1"0.8 a
60.7"6.3 b
51.9"5.6 b 59.2"5.3 b
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ŽTable 1. and 408C ŽTable 2.. When the addition of CaCl 2 or calyculin A alone, the ATP concentrations of spermatozoa appeared to decrease slightly ŽTable 2., presumably due to the restoration of motility at 408C. In contrast to the effect on motility, the presence of adenosineq 3-deazaadenosineq homocysteine thiolactone with CaCl 2 or calyculin A did not induce the decrease of ATP concentrations compared with those of the addition of CaCl 2 or calyculin A alone at 408C ŽTable 2..
4. Discussion The protein-carboxyl methylation and determination system is composed of three macromolecular components: a methylating enzyme named protein-carboxyl methylase, a demethylating enzyme named protein methylesterase, and substrates named methyl acceptor proteins. The combination of adenosine, 3-deazaadenosine and homocysteine thiolactone has been shown to block intracellular protein-carboxyl methylation reaction ŽCantoni et al., 1979.. The addition of adenosineq 3-deazaadenosineq homocysteine thiolactone caused a time-dependent progressive inhibition of rabbit sperm protein-carboxyl methylation activity. This inhibition was associated with a concomitant decrease in intact sperm motility, although the intracellular ATP concentrations have not been measured ŽGagnon, 1990.. Similar results were obtained in human spermatozoa ŽSastry and Janson, 1983.. In the study reported here, the motility of intact fowl spermatozoa incubated at 308C was also decreased markedly after the addition of 100 mM adenosineq 100 mM 3deazaadenosineq 100 mM homocysteine thiolactone. At 408C, stimulation of intact sperm motility by Ca2q or calyculin A was inhibited by the presence of blocker. During these incubation periods, the intracellular ATP concentrations of spermatozoa were maintained at approximately 40–43 nmol ATPr10 9 cells, in spite of the inhibition of motility. Therefore, it appears that the blocker is not simply inhibiting energy production in these spermatozoa, but may be acting on some part of the regulatory cascade which thus appears to involve protein-carboxyl methylation reaction. Unlike that of intact spermatozoa, the motility of demembranated fowl spermatozoa at 308C was not inhibited by the same concentrations of blocker. At 408C, the motility of demembranated spermatozoa stimulated by protein phosphatase inhibitors was not inhibited by the presence of blocker. Hence, the methylating enzyme may be present in the cytoplasmic matrix andror the plasma membrane but not retained in the axoneme andror accessory cytoskeletal components. However, the location of the substrateŽs. for this enzyme still remains unclear. It may be in the cytoplasmic matrix andror plasma membrane or in the axoneme andror accessory cytoskeletal components. It has, however, been shown that protein-carboxyl methylase and methyl acceptor proteins appeared to be predominantly present in the Triton X-100 soluble fraction of rabbit sperm tail, whereas protein methylesterase was not solubilized by nonionic detergent ŽBouchard et al., 1980.. Earlier studies have clearly shown that the stimulatory effects of temperature or protein phosphatase-type 1 inhibitors on fowl sperm motility act on demembranated spermatozoa — i.e., on the axoneme andror accessory cytoskeletal components
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ŽAshizawa et al., 1989a,b, 1994b.. It seems therefore that in intact fowl spermatozoa, stimulation of motility by these effectorsrconditions depends on the ‘‘permissive’’ effect of a proteinŽs. ‘‘upstream’’ of the axoneme andror accessory cytoskeletal components, which is active on the protein-carboxyl methylation. The intracellular free Ca2q seems to be essential for the maintenance of intact fowl sperm motility ŽAshizawa et al., 1992a.. Moreover, Ashizawa et al. Ž1994a. suggested that calmodulin, which occurs in the cytoplasmic matrix andror the plasma membrane but not in the axoneme andror accessory cytoskeletal components, is a prominent candidate for the signal transducer in Ca2q-stimulated motility. This was because the motility of intact spermatozoa loaded with an intracellular Ca2q chelator, 1, 2-bis Ž2-aminophenoxy. ethaneN, N, N X , N X-tetraacetic acid, tetraacetoxymethyl ester ŽBAPTA-AM., was negligible at 308C, but could be immediately restored by the subsequent addition of excess Ca2q to the medium, and even in the presence of Ca2q, the subsequent addition of calmodulin antagonists such as N-Ž6-aminohexyl.-5-chloro-1-naphthalenesulfonamide hydrochloride ŽW-7. and trifluoperazine inhibited the motility of intact but not demembranated spermatozoa ŽAshizawa et al., 1994a.. Calmodulin has been shown to be one of the substrates of protein-carboxyl methylase ŽGagnon et al., 1981; Johnson et al., 1985; Brunauer and Clarke, 1986; Vincent and Siegel, 1987.. Therefore, it is possible that the addition of adenosineq 3-deazaadenosineq homocysteine thiolactone might inhibit the methylation of cytosolic calmodulin and hence inhibit the motility of intact fowl spermatozoa, although the precise mechanisms of such a process remain to be determined.
Acknowledgements The authors thank Dr. G.J. Wishart, University of Abertay Dundee, Scotland, UK, for kindly reviewing the manuscript. This study was supported by a grant from the Ministry of Education, Science and Culture, Japan ŽNo. 11460130..
References Ashizawa, K., Hashiguchi, A., Tsuzuki, Y., 1992a. Intracellular free Ca2q concentration in fowl spermatozoa and its relationship with motility and respiration in spermatozoa. J. Reprod. Fertil. 96, 395–405. Ashizawa, K., Hashimoto, K., Tsuzuki, Y., 1997. Regulation of fowl sperm flagellar motility by protein phosphatase type 1 and its relationship with dephosphorylation of axonemal andror accessory cytoskeletal proteins. Biochem. Biophys. Res. Commun. 235, 108–112. Ashizawa, K., Higashio, M., Tsuzuki, Y., 1998a. Effects of tyrosine kinase inhibitor on the motility and ATP concentrations of fowl spermatozoa. Mol. Reprod. Dev. 49, 196–202. Ashizawa, K., Katayama, S., Tsuzuki, Y., 1992b. Regulation of flagellar motility by temperature-dependent phosphorylation of a 43 kDa axonemal protein in fowl spermatozoa. Biochem. Biophys. Res. Commun. 185, 740–745. Ashizawa, K., Maeda, S., Okauchi, K., 1989a. The mechanisms of reversible immobilization of fowl spermatozoa at body temperature. J. Reprod. Fertil. 86, 271–276. Ashizawa, K., Nishiyama, H., 1978. Effects of temperature on the vigour of motility, oxygen consumption and duration of motility of fowl spermatozoa under aerobic conditions. Jpn. Poult. Sci. 15, 264–266.
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