Laboratory of Animal Reproduction, Faculty of Veterinary Sciences, University of Chile, Santiago, Chile.
Theriogenology. 2011 Jan 15;75(2):346-53. doi: 10.1016/j.theriogenology.2010.09.005. Epub 2010 Nov 12.
The objective was to evaluate mitochondrial distribution, and its relationship to meiotic development, in canine oocytes during in vitro maturation (IVM) at 48, 72, and 96 h, compared to those that were non-matured or in vivo matured (ovulated). The distribution of active mitochondria during canine oocyte maturation (both in vitro and in vivo) was assessed with fluorescence and confocal microscopy using MitoTracker Red (MT-Red), whereas chromatin configuration was concurrently evaluated with fluorescence microscopy and DAPI staining. During IVM, oocytes exhibited changes in mitochondrial organization, ranging from a fine uniform distribution (pattern A), to increasing clustering spread throughout the cytoplasm (pattern B), and to a more perinuclear and cortical distribution (pattern C). Pattern A was mainly observed in germinal vesicle (GV) oocytes (96.4%), primarily in the non-matured group (P < 0.05). Pattern B was seen in all ovulated oocytes which were fully in second metaphase (MII), whereas in IVM oocytes, ∼64% were pattern B, irrespective of duration of culture or stage of nuclear development (P > 0.05). Pattern C was detected in a minor percentage (P < 0.05) of oocytes (mainly those in first metaphase, MI) cultured for 72 or 96 h. In vitro matured oocytes had a minor percentage of pattern B (P < 0.05) and smaller mitochondrial clusters in IVM oocytes than ovulated oocytes, reaching only 4, 11, and 17% of MII at 48, 72, and 96 h, respectively. Thus, although IVM canine oocytes rearranged mitochondria, which could be related to nuclear maturation, they did not consistently proceed to MII, perhaps due to incomplete IVM, confirming that oocytes matured in vitro were less likely to be competent than those matured in vivo.
目的是评估犬卵母细胞在体外成熟（IVM）48、72 和 96 小时期间的线粒体分布及其与减数分裂发育的关系，并与未成熟或体内成熟（排卵）的卵母细胞进行比较。使用 MitoTracker Red（MT-Red）通过荧光和共聚焦显微镜评估犬卵母细胞成熟（体外和体内）过程中活性线粒体的分布，同时使用荧光显微镜和 DAPI 染色评估染色质构型。在 IVM 过程中，卵母细胞的线粒体组织发生变化，从均匀分布（A 型）到细胞质中逐渐分散的聚集（B 型），再到核周和皮质分布（C 型）。A 型主要在生发泡期（GV）卵母细胞中观察到（96.4%），主要存在于未成熟组中（P < 0.05）。B 型见于所有完全处于第二次减数分裂中期（MII）的排卵卵母细胞，而在 IVM 卵母细胞中，约 64%为 B 型，无论培养时间或核发育阶段如何（P > 0.05）。C 型仅在培养 72 或 96 小时的少数卵母细胞（主要是那些处于第一次减数分裂中期，MI）中检测到（P < 0.05）。体外成熟的卵母细胞中 B 型的比例较小（P < 0.05），并且 IVM 卵母细胞中的线粒体簇较小，分别在 48、72 和 96 小时时达到 MII 的 4%、11%和 17%。因此，尽管 IVM 犬卵母细胞重新排列了线粒体，这可能与核成熟有关，但它们并没有一致地进入 MII 期，这可能是由于 IVM 不完全，证实体外成熟的卵母细胞不如体内成熟的卵母细胞有能力。
