Saint-Dizier Marie, Reynaud Karine, Chastant-Maillard Sylvie
UMR 1198 INRA/ENVA Biologie du Développement et Reproduction, Ecole Nationale Vétérinaire d'Alfort, 7 Avenue du Général de Gaulle, 94704 Maisons-Alfort Cedex, France.
Mol Reprod Dev. 2004 Jun;68(2):205-12. doi: 10.1002/mrd.20062.
In contrast to the majority of mammals, canine oocytes are ovulated at immature germinal vesicle (GV) stage and complete meiotic maturation to metaphase II during 48-72 hr within the oviducts. This study aims to characterize meiotic maturation process in bitch oocytes, with both morphological and biochemical approaches. The follow-up of chromatin and microtubules during maturation was described, and MPF and MAP kinase activities were quantified at different stages of maturation. Since bitch oocyte cytoplasm is darkly pigmented, the first step was to setup an appropriate staining method for DNA. We thus compared the efficiency of two visualization techniques and demonstrated that propidium iodide coupled to confocal microscopy was a better method than Hoechst/fluorescence microscopy for nuclear stage observation (determination rates: 98.6 vs. 69.5%, respectively; P < 0.01, n = 1622 oocytes). Microtubule organization, evaluated by tubulin immunodetection, revealed subcortical and perinuclear alpha-tubulin and asters in GV oocytes and a clear network of microtubules in GVBD oocytes. In MI and MII oocytes, a symmetrical, barrel-shaped, and radially located spindle was observed. MPF and MAP kinase activities were assayed concomitantly using histone H1 and MBP as substrates. Kinase activities were detected at low levels in oocytes at GV and GVBD stages and were significantly higher at MI and MII stages. In conclusion, despite the particular pattern of meiotic resumption in canine oocytes (ovulated at GV stage), cytoskeleton/chromatin organization and kinase activities follow a similar pattern to those observed in other mammalian species.
与大多数哺乳动物不同，犬类卵母细胞在未成熟的生发泡（GV）阶段排卵，并在输卵管内48 - 72小时内完成减数分裂成熟至中期II。本研究旨在通过形态学和生化方法对母犬卵母细胞的减数分裂成熟过程进行表征。描述了成熟过程中染色质和微管的后续变化，并在成熟的不同阶段对MPF和MAP激酶活性进行了定量。由于母犬卵母细胞的细胞质色素沉着较深，第一步是建立一种合适的DNA染色方法。因此，我们比较了两种可视化技术的效率，并证明碘化丙啶与共聚焦显微镜结合是一种比Hoechst/荧光显微镜更好的核阶段观察方法（确定率分别为98.6%和69.5%；P < 0.01，n = 1622个卵母细胞）。通过微管蛋白免疫检测评估的微管组织显示，GV卵母细胞中有皮质下和核周α - 微管蛋白以及星状体，GVBD卵母细胞中有清晰的微管网络。在MI和MII卵母细胞中，观察到一个对称的、桶状的、径向定位的纺锤体。使用组蛋白H1和MBP作为底物同时测定MPF和MAP激酶活性。在GV和GVBD阶段的卵母细胞中检测到低水平的激酶活性，而在MI和MII阶段显著更高。总之，尽管犬类卵母细胞减数分裂恢复的模式特殊（在GV阶段排卵），但其细胞骨架/染色质组织和激酶活性遵循与其他哺乳动物物种相似的模式。
