QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Morita, M. Articles by Okuno, M. Search for Related Content PubMed PubMed Citation Articles by Morita, M. Articles by Okuno, M.

Requirement of Ca²⁺ on activation of sperm motility in euryhaline tilapia Oreochromis mossambicus

M. Morita¹, A. Takemura² and M. 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 Ryukyu, 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 tilapia Oreochromis mossambicus. Sperm were suspended in solutions of 10 mmoll-1 Hepes-NaOH, pH 8.0, containing different concentrations of electrolytes and a nonelectrolyte (mannitol) to give the required osmolality. The percentage of motile sperm was measured from video recordings. (A) Motility in the absence of CaCl2. Electrolytes were NaCl (filled squares) and KCl (filled triangles). Filled circles, nonelectrolyte (mannitol). (B) The effect of Ca2+ on motility. Filled squares, NaCl alone; filled triangles, NaCl + 2 mmoll-1 CaCl2; filled circles, NaCl + 10 mmoll-1 CaCl2; open squares, NaCl + 5 mmoll-1 EGTA. Arrows indicate the osmotic pressure of seminal plasma, i.e. isotonic osmolality. Values are means ± S.D.; N=150 sperm from 5 fish for each point.

View larger version (35K): [in a new window]   Fig. 2. Change of [Ca2+]i in hypotonic or hypertonic condition indicated by fluo-3 AM. Sperm were incubated with 500 µmoll-1 fluo-3 AM in ASP (artificial seminal plasma) for 2 h. (A,B) Approximately 90% of sperm showed movement after dilution in hypotonic conditions: NaCl 50 mmoll-1 + CaCl2 5 mmoll-1 (A) or NaCl 50 mmoll-1 + EGTA 5 mmoll-1 (B). Confocal micrographs were taken approximately 30 min after the onset of activation, when almost all sperm had stopped moving. (C,D) In hypertonic conditions (300 mmoll-1 NaCl), sperm did not move even in the presence of Ca2+ (D) and [Ca2+]i was not increased, as indicated by the absence of fluorescence. Upper panels, phase contrast micrographs; lower photos, fluorescence micrographs. (E) Diagram of sleeve structure expanded in hypotonic conditions (as in A and B) but was shrunk in hypertonic conditions (as in C and D). Bars, 20 µm.

View larger version (18K): [in a new window]   Fig. 3. Effect of (A) cAMP, cGMP and Ca2+, and (B) various concentrations of Ca2+ on demembranated sperm motility. Demembranated sperm were suspended in reactivation solutions containing 220 µmoll-1 Mg-ATP2+, 175 mmoll-1 potassium acetate, 1 mmoll-1 free Mg2+, 1 mmoll-1 DTT, 0.5 mmoll-1 EGTA, 0.5 mmoll-1 EDTA, 20 mmoll-1 Hepes-NaOH (pH 8.0). (A) Solutions contained 10 µmoll-1 cAMP, 10 µmoll-1 cGMP, 10-4 moll-1 free Ca2+ or reactivation solution only (ATP). (B) Effect of [Ca2+] on reactivation of sperm motility. Values are means ± S.D.; N=5 (A); N=7 (B).

View larger version (62K): [in a new window]   Fig. 4. Motility-dependent dephosphorylation at serine residues in hypotonic conditions. Sperm were diluted in either 300 mmoll-1 NaCl solution (lane A), 50 mmoll-1 NaCl + 5 mmoll-1 CaCl2 (lane B), or 50 mmoll-1 NaCl + 5 mmoll-1 EGTA (lane C). Sperm were motile in the hypotonic solutions (containing 50 mmoll-1 NaCl; B,C) and immotile in the hypertonic solution (300 mmoll-1 NaCl; A). Sperm were collected and subjected to western blotting with anti-phosphoserine antibody. Numbers on the left indicate molecular mass markers. Motility is shown below the lanes.

View larger version (61K): [in a new window]   Fig. 5. Motility-dependent phosphorylation or dephosphorylation at threonine residues in hypotonic conditions. Sperm were diluted either in 300 mmoll-1 NaCl solution (lane A) or 50 mmoll-1 NaCl + 5 mmoll-1 CaCl2 (lane B) or 50 mmoll-1 NaCl + 5 mmoll-1 EGTA (lane C). Sperm were motile in the hypotonic solutions (containing 50 mmoll-1 NaCl; B,C) and immotile in hypertonic solution (300 mmoll-1 NaCl; A). Sperm were collected and subjected to western blotting with anti-phosphothreonine antibody. Numbers on the left indicate molecular mass markers. Motility is shown below the lanes.