Increase of cGMP, cADP-ribose and inositol 1,4,5-trisphosphate preceding Ca2+ transients in fertilization of sea urchin eggs
Ritsu Kuroda1,
Kenji Kontani2,
Yasunari Kanda2,*,
Toshiaki Katada2,
Takashi Nakano1,
Yu-ichi Satoh3,
Norio Suzuki3 and
Hideyo Kuroda1,
1 Department of Environmental Biology and Chemistry, Faculty of Science, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan
2 Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 3-7-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
3 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Kita 10-jyo, Nishi 8-chome, Sapporo 060-0810, Japan
* Present address: Department of Pharmacology, National Defense Medical College, Namiki, Tokorozawa 359-8513, Japan
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Fig. 1. Simultaneous measurements of the changes in cGMP and IP3 contents of A. crassispina (A) and H. pulcherrimus (B) eggs, compared with [Ca2+]i, after insemination. cGMP (red) and IP3 (blue) values at each point after insemination were obtained on the same aliquot of egg suspension, and [Ca2+]i (broken line) is an average over five independent experiments for A. crassispina or nine for H. pulcherrimus. Arrows indicate the onset of the fertilization potential. Scales for cGMP are on the left axis in each panel, and scales for InsP3 and [Ca2+]i are on the right axis.
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Fig. 2. Averaged time courses of the changes in cGMP and IP3 contents of sea urchin eggs, compared with [Ca2+]i, after insemination. (A-C) A. crassispina and (D-F) H. pulcherrimus. cGMP (A,D) and IP3 (B,E) values are averages from three independent experiments. Error bars represent s.d. Arrows indicate the onset of fertilization potential.
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Fig. 3. Increases in cGMP (A,D) and IP3 (B,E) contents during the latent periods of Ca2+ transients (C,F) in A. crassispina (A-C) and H. pulcherrimus eggs (D-F). Data for the initial 50 seconds of Fig. 2 were normalized to the cGMP content, IP3 content or [Ca2+]i of the unfertilized eggs. Arrows indicate the onset of fertilization potential.
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Fig. 4. Effect of artificial [Ca2+]i elevation on IP3 production. A Ca2+-ionophore A23187 was applied to unfertilized H. pulcherrimus eggs in stdASW (A) or 0CaASW (B) to a final concentration of 10 µg/ml. Data for cGMP (white circles) and IP3 (black circles) are averages of two experiments. Bars indicate the highest and lowest values. [Ca2+]i time courses (broken line) are typical ones. [Ca2+]i in stdASW (A) rose far higher than 3 µM (impossible to measure with indo-1), and 7-8 minutes later began to descend very slowly. More than 90% eggs were observed to form the fertilization envelope. Note that IP3 levels rose along with the [Ca2+]i increase, but cGMP levels did not change significantly.
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Fig. 5. Comparison of the amino acid sequence deduced from the nucleotide sequence of a cDNA fragment from unfertilized A. crassispina eggs with the deduced sequence of soluble guanylate cyclase of rat kidney. The deduced amino acid sequences are indicated by single-letter code. Sequenced A. crassispina cDNA fragment was 338 base pairs in length. The amino acid sequence of soluble guanylate cyclase (EC 4.6.1.2) of rat kidney is taken from Yuen et al. (Yuen et al., 1990), and its catalytic domain is from residue 360 to 584 in the full length of 682 amino acid residues. The identical amino acids are indicated by asterisks below the sequences (55% match). Note the highly conservative homology in their deduced amino acid sequences.
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Fig. 6. Simultaneous measurements of cADPR change with the cGMP and IP3 changes in eggs after insemination. Two representative data from three experiments on A. crassispina (A,B) and two from four experiments on H. pulcherrimus (C,D) are shown. cGMP (red), cADPR (green) and IP3 (blue) at each sampling point were measured on the same aliquot of egg suspension. Scales for cGMP and cADPR are on the left axis in each panel and scales for InsP3 are on the right axis.
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Fig. 7. Effect of preincubation of eggs in LY83583 on the Ca2+ transients at fertilization. A. crassispina eggs were incubated in stdASW containing the indicated concentration of LY83583 for 30 minutes, and then an equal volume of sperm suspension in stdASW was added. An arrow indicates the onset of fertilization potential. Each trace is a typical one.
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Fig. 8. Comparison of time courses of changes in cGMP (A,D), cADPR (B,E) and IP3 (C,F) contents of LY83583 preincubated eggs with those of unincubated control eggs after insemination. A. crassispina eggs for LY83583-preincubation (black symbols) and control (white symbols) (A-C) were prepared from the same batch of sea urchins and H. pulcherrimus eggs for both experiments (D-F) were obtained from the same batch of eggs, and each set of eggs was processed in parallel. Data on H. pulcherrimus control eggs are the same with Fig. 6B. Note the inhibition of cADPR increase for the first minute in LY83583-preincubated eggs of both species. The reason for the cADPR spikes at 70 and 150 seconds in the presence of LY83583 is unknown.
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Fig. 9. Averaged time courses of the changes in cGMP (A), IP3 (B) contents and [Ca2+]i (C) in LY83583 preincubated A. crassispina eggs. cGMP and IP3 contents are averages of four independent experiments and [Ca2+]i data are averages of five and nine experiments for control and 200 µM LY83583, respectively. Error bars represent s.d. An arrow in C indicates the onset of fertilization potential. Asterisks indicate the points showing significant difference between LY83583 preincubated eggs (black symbols) and unincubated control eggs (white symbols) (P<0.05, t-test). Note almost no increase in cGMP and the diminution both of IP3 increase and Ca2+ transients.
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© The Company of Biologists Ltd 2001
