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First published online 27 November 2002
doi: 10.1242/jcs.00221

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External Ca²⁺ is predominantly used for cytoplasmic and nuclear Ca²⁺ increases in fertilized oocytes of the marine bivalve Mactra chinensis

¹ Department of Biology, Miyagi University of Education, Aoba-ku, Sendai, Miyagi 980-0845, Japan
² Misaki Marine Biological Station, the University of Tokyo, Miura, Kanagawa 238-0225, Japan

View larger version (19K): [in a new window]   Fig. 5. Temporal patterns of Ca2+ changes induced by 5-HT and Ins(1,4,5)P3. The oocyte injected with 10 kDa (A, into the cytoplasm) or 70 kDa CGD (B, into the nucleus) was exposed to 5-HT (final concentration: 100 nM) in FSW. In C, the oocyte injected with 10 kDa CGD plus 600 µM caged Ins(1,4,5)P3 was irradiated with UV light six times (arrows) in CaFSW. The F/F0 values were calculated in the whole oocyte (A,C) or in the nuclear region alone (B). An inset in A depicts the expanded Ca2+ pattern of the first transient just after the addition of 5-HT. GVBD did not occur in A or B. In contrast, GVBD occurred in C just after the recording period.

View larger version (30K): [in a new window]   Fig. 1. Temporal patterns of Ca2+ changes in the cytoplasm and nucleus in normally fertilized oocytes. To monitor the Ca2+ changes, 10 kDa CGD (A) or 70 kDa CGD (B) was injected into the cytoplasm, or 70 kDa CGD was injected into the nucleus (C). The relative fluorescence levels of CGD (F/F0 values) were calculated in the cytoplasmic and nuclear regions separately. The steep increases in F/F0 (arrowheads in B and C) indicate the leakage of 70 kDa CGD through the nuclear envelope. A pair of right panels shows fluorescence images before (top) and after (bottom) each fluorescence measurement. All of the oocytes underwent GVBD on schedule (at 10 minutes) and then developed to early trochophores.

View larger version (20K): [in a new window]   Fig. 4. Effect of Ca2+-free seawater (CaFSW) on sperm-induced Ca2+ changes. The oocyte where 10 kDa (A) or 70 kDa CGD (B) was injected into the cytoplasm was inseminated in FSW, which was replaced by CaFSW at 4 minutes after fertilization. The F/F0 values were calculated in the cytoplasmic and nuclear regions separately. The steep increase in F/F0 (arrowhead in B) indicates the leakage of 70 kDa CGD through the nuclear envelope. The two oocytes underwent GVBD on schedule.

View larger version (71K): [in a new window]   Fig. 3. Ca2+ dynamics during the rising phase of the initial Ca2+ transient at fertilization. The data in A, B and C were obtained from the oocytes for Fig. 1A, 1C and 2B, respectively. Sequential fluorescence images were acquired every 0.4 seconds, normalized by dividing them by the resting image just before each Ca2+ increase in a pixel-to-pixel manner, and expressed as pseudocolor images. The zero time in each montage was defined as the time of the first visible Ca2+ increase at fertilization.

View larger version (81K): [in a new window]   Fig. 6. Ca2+ dynamics during the rising phase of 5-HT-induced Ca2+ transients. The data in A and B were obtained from the first Ca2+ transient (initiated at 0) and the fifth transient (initiated at 1278 seconds), respectively, in the same oocyte for Fig. 5A. In C, a rising pattern of the third transient (initiated at 537 seconds) in the oocyte for Fig. 5B was investigated. Sequential fluorescence images were acquired every 0.4 seconds, normalized by dividing them by the resting image just before each Ca2+ increase in a pixel-to-pixel manner, and expressed as pseudocolor images. The zero time in each montage was defined as the initiation time of each Ca2+ transient, which is independent of the time of 5-HT addition.

View larger version (21K): [in a new window]   Fig. 2. Effects of low Ca2+ seawater (LCaSW), D-600, and heparin on sperm-induced Ca2+ changes. The oocyte where 10 kDa CGD was injected into the cytoplasm was preincubated for 6-8 minutes in LCaSW (A), in FSW containing 100 µM D-600 (B), or in LCaSW containing 100 µM D-600 (C), and then inseminated. In D, the oocyte injected with 10 kDa CGD plus 20 mg/ml heparin was inseminated in FSW. The F/F0 values were calculated in the cytoplasmic and nuclear regions separately (A,D) or in the whole oocyte (B,C). GVBD was prominently delayed (occurred at 25 minutes after fertilization) in A, or prevented in B and C. In contrast, GVBD took place on schedule in D.

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