Lee H S, Yin X J, Jin Y X, Kim N H, Cho S G, Bae I H, Kong I K
Department of Animal Science & Technology, Sunchon National University, 315 Maegok-dong, Suncheon, JeonNam Province 540-742, South Korea.
Anim Reprod Sci. 2008 Jan 30;103(3-4):336-47. doi: 10.1016/j.anireprosci.2006.12.016. Epub 2006 Dec 17.
This study investigated the effect of deriving oocytes from different stages of the estrous cycle on oocyte diameter, germinal vesicle (GV) chromatin configuration, and in vitro meiotic competence in canine oocytes. Cumulus oocyte complexes (COCs) were recovered from both ovaries during anestrous, follicular, and luteal phases and in vivo ovulated oocytes. The diameter of canine oocyte was compared with or without the zona pellucida (ZP) before in vitro maturation (IVM). Also, GV chromatin configuration was evaluated before (0 h) or 72 h after IVM by fixation with 3.7% formaldehyde supplemented with 10 microg/ml Hoechst 33342 for 30 min. COCs were matured in TCM199 supplemented with 10% fetal bovine serum (FBS), 0.6 mM cysteine, 0.2 mM pyruvic acid, 50 microg/ml gentamycin sulfate, and 20 microg/ml 17beta-estradiol (E(2)) at 39 degrees C and 5% CO(2) in air for 72 h. The diameter of in vivo ovulated oocytes with the ZP (167.5+/-12.7 microm) or without ZP (133.9+/-5.3 microm) was significantly greater (p<0.05) than those of anestrous, follicular, and luteal oocytes (with ZP, 151.2+/-7.4, 153.1+/-8.8 and 152.8+/-5.4 microm, respectively; without ZP, 115.3+/-7.6, 122.1+/-4.9 and 114.3+/-6.6 microm, respectively). At 0 h, the GV-II configuration was more prevalent in oocytes from anestrual ovaries than from follicular or luteal ovaries or in vivo ovulated oocytes (63.6% versus 14.8%, 33.0%, and 0.0%; p<0.05), whereas the proportion of oocytes with the GV-V configuration was higher in follicular phase and ovulated oocytes than in oocytes from anestrus and luteal phase (57.4% and 100% versus 2.0% and 22.7%; p<0.05). However, oocytes in luteal phase exhibited diverse GV configurations (10.3%, 33.0%, 16.5%, 13.4%, and 22.7% in GV-I, GV-II, GV-III, GV-IV, and GV-V, respectively). After 72 h post-IVM, a greater percentage of in vivo ovulated oocytes progressed to MII than those oocytes collected during anestrous, follicular, and luteal phases (50.0% versus 5.5%, 11.5%, and 9.1%; p<0.05). In conclusion, the oocyte diameter, GV chromatin configuration, and meiotic maturation of canine COCs are related to the oocyte source. These results indicated that the oocyte source could be critical to nuclear progression to MII stage in canines.
本研究调查了从发情周期不同阶段获取卵母细胞对犬类卵母细胞直径、生发泡（GV）染色质构型及体外减数分裂能力的影响。在乏情期、卵泡期和黄体期以及体内排卵的卵母细胞中，从双侧卵巢回收卵丘卵母细胞复合体（COCs）。在体外成熟（IVM）前，比较有或无透明带（ZP）的犬类卵母细胞直径。此外，通过用补充有10μg/ml Hoechst 33342的3.7%甲醛固定30分钟，在IVM前（0小时）或IVM后72小时评估GV染色质构型。COCs在补充有10%胎牛血清（FBS）、0.6mM半胱氨酸、0.2mM丙酮酸、50μg/ml硫酸庆大霉素和20μg/ml 17β-雌二醇（E₂）的TCM199中，于39℃、5% CO₂的空气中培养72小时。有ZP的体内排卵卵母细胞直径（167.5±12.7μm）或无ZP的体内排卵卵母细胞直径（133.9±5.3μm）显著大于乏情期、卵泡期和黄体期的卵母细胞（有ZP时分别为151.2±7.4、153.1±8.8和152.8±5.4μm；无ZP时分别为115.3±7.6、122.1±4.9和114.3±6.6μm；p<0.05）。在0小时时，GV-II构型在乏情期卵巢的卵母细胞中比卵泡期或黄体期卵巢的卵母细胞或体内排卵卵母细胞中更普遍（63.6%对14.8%、33.0%和0.0%；p<0.05），而具有GV-V构型的卵母细胞比例在卵泡期和排卵卵母细胞中高于乏情期和黄体期卵母细胞（57.4%和100%对2.0%和22.7%；p<0.05）。然而，黄体期的卵母细胞表现出多种GV构型（GV-I、GV-II、GV-III、GV-IV和GV-V分别为10.3%、33.0%、16.5%、13.4%和22.7%）。IVM后72小时，体内排卵卵母细胞进入MII期的百分比高于乏情期、卵泡期和黄体期收集的卵母细胞（50.0%对5.5%、11.5%和9.1%；p<0.05）。总之，犬类COCs的卵母细胞直径、GV染色质构型及减数分裂成熟与卵母细胞来源有关。这些结果表明，卵母细胞来源可能对犬类细胞核进展到MII期至关重要。
