De los Reyes Monica, de Lange Johanna, Miranda Pedro, Palominos Jaime, Barros Claudio
Laboratory of Animal Reproduction, Faculty of Veterinary Sciences. University of Chile, Casilla 2 Correo 15 Santiago, Chile.
Theriogenology. 2005 Jul 1;64(1):1-11. doi: 10.1016/j.theriogenology.2004.11.008. Epub 2004 Dec 19.
The IVM of canine oocytes is characterized by low rates of metaphase II. The objective of this study was to evaluate the effects of hCG on meiotic development of canine oocytes for culture periods up to 96 h. Oocytes were collected after ovariohysterectomy. Only oocytes >110 microm in diameter, with a homogeneous dark cytoplasm and three or more layers of compact cumulus cells were used. For IVM, the COCs were cultured in TCM-199+10% fetal calf serum, without (medium A control) or supplement with 10 IU/mL of hCG (medium B), or with a combination of both media (treatment B/A). The COCs were randomly allocated into three groups. The first and second groups were cultured in either medium A or B, respectively for 24, 48, 72, and 96 h. Oocytes of the third group (treatment B/A) were incubated in medium with hCG (medium B) the first 48 h and then transferred to medium without hCG (medium A) for an additional 24 or 48 h. The proportion of COCs with cumulus cell expansion was also evaluated before fixation. Oocytes were stained with propidium iodide prior to nuclear assessment (with epifluorescence microscopy). COCs with cumulus expansion were evident after 48 h of culture. The proportion of COCs with cumulus expansion was higher (P<0.05) for media containing hCG (B or B/A) than for meda lacking hCG (A); this difference was maintained for 72 and 96 h in culture. In media A, B and B/A, 23.3, 31.7 and 29.5%, respectively, of oocytes were at metaphase II after 72 h, with 20.7, 33.1 and 43.4% at this stage after 96 h. The advancement of meiosis was directly proportional to the time of incubation; the highest percentage (P<0.05) of oocytes at metaphase II was observed after 96 h of culture when 10 IU/mL hCG was present for only the first 48 h of culture.
犬卵母细胞的体外成熟（IVM）特点是处于减数分裂中期II的比率较低。本研究的目的是评估人绒毛膜促性腺激素（hCG）对犬卵母细胞长达96小时培养期减数分裂发育的影响。在卵巢子宫切除术后收集卵母细胞。仅使用直径大于110微米、细胞质均匀深色且有三层或更多层紧密卵丘细胞的卵母细胞。对于IVM，将卵丘卵母细胞复合体（COCs）培养于含10%胎牛血清的TCM - 199中，一种是不添加（培养基A对照）或添加10 IU/mL hCG（培养基B），或两种培养基联合使用（处理B/A）。将COCs随机分为三组。第一组和第二组分别在培养基A或B中培养24、48、72和96小时。第三组（处理B/A）的卵母细胞在最初48小时培养于含hCG的培养基（培养基B）中，然后转移至不含hCG的培养基（培养基A）中再培养24或48小时。在固定前也评估了具有卵丘细胞扩展的COCs的比例。在进行核评估（用落射荧光显微镜）之前，用碘化丙啶对卵母细胞进行染色。培养48小时后，具有卵丘扩展的COCs明显可见。含hCG的培养基（B或B/A）中具有卵丘扩展的COCs比例高于不含hCG的培养基（A）（P<0.05）；这种差异在培养72和96小时时依然存在。在培养基A、B和B/A中，72小时后分别有23.3%、31.7%和29.5%的卵母细胞处于减数分裂中期II，96小时后此阶段的比例分别为20.7%、33.1%和43.4%。减数分裂的进展与培养时间成正比；当在培养的前48小时仅存在10 IU/mL hCG时，培养96小时后观察到处于减数分裂中期II的卵母细胞比例最高（P<0.05）。
