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First published online August 18, 2003
doi: 10.1242/10.1242/dev.00649

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Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH

Department of Biology, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan

View larger version (125K): [in a new window]   Fig. 1. Extrusion of polar bodies after spawning. Photographs were taken at the indicated times (minutes) after the injection of 1-MA into the body cavity of the recipient animal. (Top panels) Immediately after spawning (35 minutes after the injection of 1-MA into the body cavity), the oocyte was inseminated. The first and second polar bodies (arrowheads: 1stPB and 2ndPB) were formed after the elevation of the fertilization envelope, indicating that they were extruded after fertilization or spawning. (Middle and bottom panels) Oocytes spawned into SW at 95 or 155 minutes after the injection of 1-MA were inseminated. Similarly, polar bodies were extruded after spawning. Scale bar: 50 µm.

View larger version (59K): [in a new window]   Fig. 2. MI arrest in the ovaries of stimulated animals. (Left column) Oocytes spawned into SW at 45, 105 and 165 minutes after the injection of 1-MA into the body cavity of the animal were immediately collected and treated with extraction buffer, followed by fixation and staining with DAPI and an anti-tubulin antibody to visualize chromosomes (blue) and tubulin (green). (Right column) Oocytes incubated in normal SW for 30 minutes after spawning were similarly treated and stained at 75, 135, and 195 minutes. Representative figures are shown. (Top row) Prometaphase at 45 minutes and anaphase at 75 minutes. (Middle row) Metaphase at 105 minutes and anaphase at 135 minutes. (Bottom row) Metaphase at 165 minutes and anaphase at 195 minutes. Scale bar: 2 µm.

View larger version (26K): [in a new window]   Fig. 3. An increase of pHi during oocyte maturation induced by Na+/H+ antiporters coupling to 1-MA signal transduction. (A) Representative traces of pHi before and after 1-MA treatments. Fluorescence ratios of BCECF-dextran were measured every 30 seconds using a single oocyte before and after 1-MA treatments. Symbols represent oocytes treated with 1 µM 1-MA in normal SW (open circles, n=40), or in zero-Na+ artificial SW (0 NaSW) (closed diamonds, n=25) containing 480 mM choline chloride, 55 mM MgCl2, 10 mM CaCl2, 5 mM KCl, 2.5 mM KHCO3, pH 8.0 adjusted with KOH, or in normal SW with 0.6 mM EIPA (open diamonds, n=22). Open triangles represent oocytes in normal seawater without 1-MA treatment (n=11). (B) Representative traces of pHi after Gß (0.6 µM) microinjection. Gß purified from bovine brain was stored in 0.6% Na+ cholate, 100 mM NaCl, 20 mM Tris-HCl, pH 8.0, and microinjected as previously described (Chiba et al., 1993). Fluorescence ratios of a single oocyte before and after Gß microinjection were measured. Symbols represent oocytes microinjected with Gß in normal SW (open circles, n=22) or in zero-Na+ artificial SW (closed diamonds, n=6), or microinjected in normal SW with the buffer used for Gß (open triangles, n=8). (C) The fluorescence ratios of a single oocyte in artificial SW with (open diamonds, n=14) or without (open circles, n=40) 0.1 mM LY294002 were measured before and after 1-MA treatment.

View larger version (64K): [in a new window]   Fig. 4. Inhibition of polar body formation in zero-Na+ artificial SW after 1-MA addition. GVBD occurred 20 minutes after 1-MA addition both in normal SW and zero-Na+ artificial SW. In normal SW, the first and second polar body (arrowheads) were extruded 70-90 minutes and 100-120 minutes after 1-MA addition, respectively. In zero-Na+ artificial SW, polar bodies were not extruded.

View larger version (18K): [in a new window]   Fig. 5. Blockage of pHi increase of oocytes in the ovary of stimulated animals. (A) Ovaries were isolated from the stimulated animals 15 minutes before measurement of fluorescence ratios. They were immersed in zero-Na+ artificial SW and dissociated oocytes were obtained. Oocytes were continuously treated with zero-Na+ artificial SW for 15 minutes, during which time they were microinjected with BCECF. The zero-Na+ artificial SW was replaced by normal SW 60 minutes or 180 minutes after injection of 1-MA into the body cavity of the animal, followed by measurement of fluorescence ratios. (B) Ovaries were isolated from non-stimulated animals and immersed in zero-Na+ artificial SW. Then, oocytes in the ovaries were isolated and microinjected with BCECF. The zero-Na+ artificial SW was replaced by normal SW at 10 minutes, and again replaced by zero-Na+ artificial SW at 60 minutes. A small rise of pHi occurred when the zero-Na+ artificial SW was replaced by normal SW. (C) The pHi increase of oocytes from non-stimulated animals was induced by 1-MA in normal SW, which was replaced by zero-Na+ artificial SW without 1-MA at 65 minutes, and again replaced by normal SW at 93 minutes. High pHi induced by normal SW containing 1-MA was not affected by zero-Na+ artificial SW. (D) Ovaries were isolated from the stimulated animals and immersed in normal SW. Immediately, oocytes in the ovaries were isolated and microinjected with BCECF, and the pHi of oocytes was measured. pHi increased with time after removal of the ovary from the animal.

View larger version (61K): [in a new window]   Fig. 6. Effects of pH and the MAPK pathway on the destruction of cyclin B in a cell-free preparation. (A) The cell-free preparation at pH 7.0 or 7.3 with or without U0126 (0.1 mM) was incubated for 0, 20, 40, 60 and 85 minutes. After incubation, sample buffer for SDS-PAGE was added to stop the reaction. Then, the samples were analyzed by 10% SDS-PAGE, followed by immunoblotting with an anti-cyclin B antibody. (B) The MAPK activity of the cell-free preparation at pH 7.0 or 7.3 with or without U0126 (0.1 mM). Activation of MAPK was inhibited by U0126. MAPK was activated at pH 7.0 and 7.3 (arrow), and inactivated at pH 7.0 with U0126 (arrowhead). Each sample was analyzed by 12.5% SDS-PAGE, followed by immunoblotting with an anti-MAPK (ERK1) antibody.