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First published online December 10, 2003
Journal of Experimental Biology 207, 337-345 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00748

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Acclimation of sperm motility apparatus in seawater-acclimated euryhaline tilapia Oreochromis mossambicus

Masaya Morita¹, Akihiro Takemura² and Makoto Okuno^1,*

¹ Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
² Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, 3422 Sesoko, Motobu, Okinawa 905-0227, Japan

View larger version (24K): [in a new window]   Fig. 1. Effects of osmolality and extracellular Ca2+ on motility in sperm of seawater-acclimated tilapia Oreochromis mossambicus. Sperm were suspended in different concentrations of electrolytes (NaCl and KCl) and nonelectrolyte (mannitol) containing 10 mmol l-1 Hepes-NaOH (pH 8.0). The percentage of motile sperm was measured from video recordings. (A) Motility in NaCl, KCl and mannitol solutions. Filled squares, NaCl; filled triangles, KCl; filled circles, mannitol. (B) The effect of Ca2+ on motility. Filled squares, NaCl alone; open triangle, NaCl solutions containing 2 mmol l-1 CaCl2; filled diamond, NaCl solutions containing 10 mmol l-1 CaCl2; open diamond, NaCl solutions containing 5 mmol l-1 EGTA. Filled arrowheads indicate the osmotic pressure of seminal plasma, i.e. isotonic osmolality. Open arrows and filled arrows indicate the osmotic pressure of seawater. Values are means ± S.D.; N=150 sperm from 5 fish for each point.

View larger version (18K): [in a new window]   Fig. 2. Effect of Ca2+ concentration on sperm motility in hypertonic conditions. (A) FWT and (B) SWT sperm. Squares, hypertonic solution containing 200 mmol l-1 NaCl; diamonds, 250 mmol l-1 NaCl (500 mOsm kg-1); triangles, 350 mmol l-1 NaCl (700 mOsm kg-1); circles, 500 mOsm kg-1 (1000 mOsm kg-1). Values are means ± S.D.; N=150 sperm from 5 fish for each for each point.

View larger version (28K): [in a new window]   Fig. 3. Swimming velocity, beat frequency and wave form of FWT and SWT sperm. (A) Swimming velocity, (B) beat frequency and (C) waveform of FWT and SWT sperm in hypotonic (50 mmol l-1 NaCl) and hypertonic (300 mmol l-1 NaCl) conditions with or without Ca2+. Values are means ± S.D. (N=4). ND, not determined. *The velocity of FWT and SWT sperm in the hypotonic condition+Ca2+ (A) were significantly different, and of SWT sperm in hypotonic+Ca2+ and hypertonic solutions (P<0.01).

View larger version (16K): [in a new window]   Fig. 4. Effect of (A) cAMP (10 µmol l-1), cGMP (10 µmol l-1), Ca2+ (10-4 mol l-1 free Ca2+) and reactivation solution (B) various concentrations of Ca2+ on reactivation of sperm motility in demembranated sperm. The demembranated sperm were suspended in reactivation solution containing 220 µmol l-1 Mg-ATP2-, 175 mmol l-1 potassium acetate, 1 mmol l-1 free Mg2+, 1 mmol l-1 DTT, 0.5 mmol l-1 EGTA, 0.5 mmol l-1 EDTA, 20 mmol l-1 Hepes-NaOH (pH 8.0). Values are means ± S.D. (N=5 in A; N=7 in B). *Significantly different (P<0.01).

View larger version (20K): [in a new window]   Fig. 5. Effect of osmolality and Ca2+ on motility in (A) FWT and (B) SWT demembranated sperm. The demembranated sperm were suspended in reactivation solutions containing 220 µmol l-1 Mg-ATP2-, 75-650 mmol l-1 potassium acetate, 1 mmol l-1 free Mg2+, 1 mmol l-1 DTT, 0.5 mmol l-1 EGTA, 0.5 mmol l-1 EDTA, 20 mmol l-1 Hepes-NaOH (pH 8.0). Open triangles, 10-4 mol l-1 Ca2+; open squares, 10-7 mol l-1 Ca2+; filled circles, 10-8 mol l-1 Ca2+. Values are means ± S.D. (N=7).

View larger version (45K): [in a new window]   Fig. 6. Changes in [Ca2+]i in SWT sperm subjected to hypotonic or hypertonic conditions, indicated by fluo-3 AM. Sperm were incubated with 500 µmol l-1 fluo-3 AM in artificial seminal plasma (ASP) for 2 h. Approximately 90% of sperm moved after dilution into Ca2+-containing hypotonic or hypertonic conditions: (A) 50 mmol l-1 NaCl + 5 mmol l-1 CaCl2 or (D) 300 mmol l-1 NaCl + 10 mmol l-1 CaCl2. Motility was suppressed in Ca2+-free hypotonic or hypertonic solutions: (C) 50 mmol l-1 NaCl + 5 mmol l-1 EGTA or (B) 300 mmol l-1 NaCl. Arrows, head and sleeve regions; arrowheads, flagella. (E) In hypertonic conditions the sleeve structure of SWT was expanded but that of FWT was shrunken. Bars, 10 µm. For details of microscopy, see Materials and methods.

View larger version (42K): [in a new window]   Fig. 7. Protein phosphorylation and dephosphorylation at serine (A) and threonine (B) residues in motility-feasible conditions. Sperm were diluted into either 50 mmol l-1 NaCl + 5 mmol l-1 CaCl2 (lane b) or 300 mmol l-1 NaCl + 10 mmol l-1 CaCl2 (lane c). Protein of dry sperm (lane a) was eluted after removal of seminal plasma by centrifugation. Sperm were motile in both the hypotonic solutions (50 mmol l-1 NaCl) and the hypertonic solution (300 mmol l-1 NaCl). Sperm were then collected and subjected to western blotting with (A) anti-phosphoserine antibody or (B) phosphothreonine antibody. Numbers on the left indicate molecular mass (kDa) obtained from molecular markers. Motility is shown below the lanes.