Effect of Temperature Decline on the Cytoskeletal Organization of the Porcine Oocyte

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  • ブタ卵母細胞の細胞骨格の形成に及ぼす冷却の影響

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Abstract

The purpose of this study was to evaluate effect of cooling on the cytoskeletal organization affecting the distribution and structure of the meiotic spindle in the porcine metaphase II (MII) oocyte. Fluorescence staining was done for visualization of microtubules, microfilaments and chromosomes. In vitro-matured oocytes were either maintained at 37°C (for controls) or cooled abruptly to 5 or 18°C. Microtubules were preferentially labeled at the meiotic spindle and microfilaments were mainly detected at the cortex of the MII oocyte. The majority of controls (87%, n=97) had a barrel-shaped spindle, whereas the remaining 13% showed a broad, box-shaped spindle. After exposure to 18°C for 30 min, 94% of oocytes (n=48) possessed either a box-shaped spindle (63%) with a shortened interpolar distance (7.4 ± 0.4 μm vs. 9.8 ± 0.2 μm for controls P<0.05), or no spindle (31%). Exposure of oocytes to 5°C for 30 min caused complete disassembly of the spindle in 81% of the oocytes (n=103). Such spindle-disassembled oocytes had increased intensity of microtubule signaling in the cytoplasm and irregular staining of cortical microfilaments. Cortical microfilament staining decreased in intensity in 64% (n=33) and 89% (n=74) of the oocytes cooled to 18 and 5°C, respectively. When oocytes were rewarmed at 37°C after exposure to 5°C for 30 min, the spindles were not restored to normal in any oocyte (n=138) regardless of the rewarming duration (0, 1, 10, 20 or 60 min), although microtubules and microfilaments were reassembled. However, more oocytes contained dispersed chromosomes with no spindle and collapsed microfilament architecture, as rewarming time increased beyond 20 min. These results suggest that in porcine MII oocytes both microtubular spindle and cortical microfilaments are irreversibly affected by a 5°C cold shock, and that the polar microtubule-organizing centers malfunction even after rewarming.<br>

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