First published online November 17, 2005
Journal of Experimental Biology 208, 4549-4556 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01921
K+-independent initiation of motility in chum salmon sperm treated with an organic alcohol, glycerol
M. Morita1,*,
M. Fujinoki2 and
M. Okuno1,
1 Department of Biology, Graduate School of Arts and Sciences, University of
Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan
2 Department of Physiology, Dokkyo University School of Medicine, Mibu,
Tochigi 321-0293, Japan
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Fig. 1. Effect of ions on glycerol-treated sperm. Sperm were incubated in a
glycerol solution for 10 s exactly. Then, 1 vol of this suspension was diluted
in 40 vol of an activation solution (KCl 100 mmol l–1, KCl 50
mmol l–1+NaCl 50 mmol l–1, KCl 10 mmol
l–1+NaCl 90 mmol l–1 or NaCl 100 mmol
l–1). Gray bar, percentage motility of intact sperm; black
bars, glycerol-treated sperm. Intact sperm showed motility only with 100 mmol
l–1 NaCl. Values are means ±
S.D.(N=5).
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Fig. 2. Effect of organic alcohol on sperm motility. (A) Chemical formulae of three
organic alcohols. (B) Effects of ethylene glycol and erythritol on sperm
motility. Sperm were first incubated with one of these alcohol solutions (1.3
mol l–1) and then diluted into 100 mmol l–1
KCl solution. Values are means ± S.D.
(N=5).
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Fig. 3. Effect of extracellular Ca2+ concentration on glycerol-treated
sperm. Glycerol-treated sperm were suspended in 100 mmol l–1
NaCl solutions containing various concentration of Ca2+. Values are
means ± S.D. (N=5).
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Fig. 4. Relationship between swimming velocity and beat frequency of
glycerol-treated and intact sperm. Photographs were taken from 5 s after
dilution to the end of sperm motility. Velocity and beat frequency of intact
and glycerol-treated sperm were calculated from photographs. (A) Relationship
between swimming velocity and beat frequency of glycerol-treated (open
circles) and intact sperm (filled squares). The regressions are
y=7.0x–11.1 for intact sperm
(r2=0.8, P<0.0001, N=49) and
y=2.2x+22.6 for glycerol-treated sperm
(r2=0.6, P<0.0001, N=35). (B,C)
Trajectories taken at 0.5 s exposures for intact (B) and glycerol-treated(C)
sperm. Schema of the trajectory are represented below each photograph. Bars,
100 µm (N=3).
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Fig. 5. Effect of inhibitors on glycerol-treated sperm. Sperm were incubated for 30
min in ASP containing (A) CCCP, an uncoupler of the mitochondrial electron
transport chain for ATP synthesis; (B) SQ22536, an adenylyl cyclase inhibitor;
(C) H-89, a PKA inhibitor. Gray bars, percentage motility of intact sperm;
black bars, glycerol-treated sperm. Motility was determined from video
recordings taken 5 s after motility initiation. Values are means ±
S.D. (N=3).
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Fig. 6. Cyclic AMP synthesis in glycerol-treated and intact sperm. Sperm cAMP
levels were measured 15 s after suspending intact and glycerol-treated sperm
into various solutions. Glycerol-treated sperm were diluted in 100 mmol
l–1 KCl, 100 mmol l–1 NaCl and 300 mmol
l–1 KCl solutions. Intact sperm were diluted in 100 mmol
l–1 NaCl and KCl solutions. Gray bars, percentage motility of
intact sperm; black bars, glycerol-treated sperm. Values are means ±
S.D. (N=3).
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Fig. 7. Detection of phosphorylation in (A) demembranated and (B) glycerol-treated
sperm treated with various solutions. 32P-labelled proteins were
separated by a 12.5% separating gel. Demembranated sperm were reactivated in
the presence of cAMP. Glycerol-treated sperm showed motility in the 100 mmol
l–1 NaCl and KCl solutions. Bars on the left side show
molecular mass standards.
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© The Company of Biologists Ltd 2005
