First published online November 17, 2005
Journal of Experimental Biology 208, 4411-4418 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01906
Increase in intracellular pH induces phosphorylation of axonemal proteins for activation of flagellar motility in starfish sperm
Ayako Nakajima1,
Masaya Morita2,
Akihiro Takemura2,
Shinji Kamimura1 and
Makoto Okuno1,*
1 Department of Life Sciences, Graduate School of Arts and Sciences,
University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan
2 Sesoko Station, Tropical Biosphere Research Center, University of the
Ryukyus, Sesoko, Motobu, Okinawa 905-0227, Japan
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Fig. 1. Effect of histidine (His) on the flagellar motility activation of
Asterina pectinifera sperm. Sperm were suspended in (A) ASW
containing 0–10 mmol l–1 histidine or (B) Na-free ASW
containing 0–40 mmol l–1 histidine, and the percentage
of motile sperm was calculated. Values are means ±
S.D.; N=3.
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Fig. 2. Effect of increased [pH]i on the activation of A.
pectinifera sperm flagellar motility. Sperm were suspended in CC
solutions containing 0–20 mmol l–1 NH4Cl,
which is known to increase [pH]i. The percentage of motile sperm
was calculated. Values are means ± S.D.;
N=7.
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Fig. 3. Measurement of [pH]i using a fluorescent pH indicator, carboxy
SNARF-1. (A) The effect of histidine and NH4Cl on sperm
[pH]i. Carboxy SNARF-1 loaded sperm were suspended in Na-free ASW
containing 0 or 10 mmol l–1 histidine (white bars), ASW
containing 0 or 10 mmol l–1 histidine (gray bars), or choline
chloride (CC) solution containing 0 or 20 mmol l–1
NH4Cl (black bars). Values are means ±
S.D.; N=2. Sperm motility (percentage of motile
sperm) in each solution is given under the graph; – and + indicate
generally immotile and >60% motile, respectively. (B) Fluorescence
micrographs of carboxy SNARF-1 loaded sperm suspended in CC solution
containing 0 (left) or 20 mmol l–1 (right) NH4Cl.
Micrographs represent the fluorescent intensity at 640 nm with 470–550
nm excitation. Bars, 10 µm.
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Fig. 4. Effect of pH on the reactivation of demembranated A. pectinifera
sperm. Sperm were suspended in CC solution (CC treatment, black bars), CC
solution containing 20 mmol l–1 NH4Cl
(NH4Cl treatment, gray bars), or ASW containing 10 mmol
l–1 histidine (histidine treatment, white bars), and then
demembranated. The demembranated sperm were diluted with the reactivation
solution of various pH (ranging 7.0–8.0), and the percentage of motile
sperm calculated. Values are means ± S.D.;
N=5 (CC treatment and NH4Cl treatment); N=3
(histidine treatment).
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Fig. 5. Incorporation of 32P during motility activation of demembranated
flagella (axonemes). Sperm were suspended in CC solution (CC treatment) or CC
solution containing 20 mmol l–1 NH4Cl
(NH4Cl treatment) before the preparation of axonemes. The axonemes
were diluted with the reactivation solutions (pH range 7.0–8.0)
containing [ -32P]ATP, and axonemal proteins labeled with
32P were detected. (A) 32P-labeled proteins in the
CC-treated sperm axonemes. (B) 32P-labeled proteins in the
NH4Cl-treated sperm axoneme. An equal amount of protein (15 µg)
was loaded on each lane in A and B. Numbers on the left of the gel show the
positions of molecular mass markers (kDa). (C) Relative intensity of the 25
kDa band. The band intensities in the CC-treated sperm axoneme (white bars)
and in the NH4Cl-treated sperm axoneme (black bars) were compared.
Band intensity was quantitated by densitometry and the maximum intensity of
the band was taken as 1. Relative intensities of the 32 kDa band (D) and the
45 kDa band (E) are also shown.
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© The Company of Biologists Ltd 2005
