Kitiyanant Yindee, Saikhun Jumnian, Pavasuthipaisit Kanok
Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.
Theriogenology. 2003 Apr 15;59(8):1775-86. doi: 10.1016/s0093-691x(02)01235-9.
Oocyte maturation and somatic cell nuclear transfer (NT) studies conducted in the domestic cat can provide valuable insights that are relevant to the conservation of endangered species of felids. The present investigation focuses on the in vitro maturation (IVM) of domestic cat oocytes stimulated by insulin-like growth factor-I (IGF-I) and their possible use as recipient cytoplasts for somatic cell NT. In Experiment I, the effects of IGF-I on cat oocyte IVM were monitored. Cumulus-oocyte complexes (COCs) were recovered in TALP-HEPES medium following ovarian follicular aspiration and were classified under a stereomicroscope into four grades using criteria based on cumulus cell investment and the uniformity of ooplasm. The COCs were either cultured in Dulbecco's modified Eagle medium (DMEM) alone as a control group or supplemented with 100 ng/ml IGF-I. After culturing for 32-34 h, oocytes were denuded and maturation rate was evaluated by observing the extrusion of the first polar body and staining with aceto-orcein. The percentages of maturation of Grades 1 and 2 oocytes were significantly increased (P<0.05) in IGF-I supplemented medium compared with medium alone (85.8 versus 65.5 and 70.3 versus 51.8, respectively) whereas the maturation rates of Grades 3 and 4 oocytes were not different. The IVM of Grade 1 oocytes was significantly higher (P<0.05) than for all other grades in both control and experimental groups. In Experiment II, the in vitro development of cat NT embryos using cumulus cells, fetal or adult fibroblasts as donor nuclei was investigated. The IVM oocytes in medium containing IGF-I were enucleated and fused with cumulus cells, fetal or adult fibroblasts between passages 2 and 4 of culture. Reconstructed embryos were cultured and monitored every 24h for progression of development through Day 9. There was no significant difference in the percentage of fusion of NT embryos using different donor nuclei whereas the cleavage rates of NT embryos reconstructed with fetal fibroblasts and cumulus cells were significantly higher (P<0.05) than those reconstructed with adult fibroblasts (72.5 and 70.7% versus 54.8%, respectively). Development of NT embryos reconstructed with adult fibroblast to the morula stage was significantly lower (P<0.05) compared with cumulus cell or fetal fibroblast donor cells (25.8% versus 37.9 or 47.5%, respectively). However, no difference was observed in development to the blastocyst stage. These results demonstrated that IGF-I promoted the IVM of domestic cat oocytes. The enucleated IVM oocytes could be used as recipient cytoplasm for fetal and adult somatic cell nuclei resulting in the production of cloned cat embryos.
在家猫身上进行的卵母细胞成熟和体细胞核移植（NT）研究可以提供与猫科濒危物种保护相关的宝贵见解。本研究聚焦于胰岛素样生长因子-I（IGF-I）刺激下家猫卵母细胞的体外成熟（IVM）及其作为体细胞核移植受体细胞质的可能性。在实验I中，监测了IGF-I对猫卵母细胞IVM的影响。通过卵巢卵泡抽吸在TALP-HEPES培养基中回收卵丘-卵母细胞复合体（COCs），并在体视显微镜下根据卵丘细胞包裹情况和卵质均匀性标准将其分为四个等级。COCs要么单独培养于杜氏改良 Eagle 培养基（DMEM）中作为对照组，要么添加100 ng/ml IGF-I。培养32 - 34小时后，去除卵母细胞周围的卵丘细胞，通过观察第一极体的排出并用醋酸洋红染色来评估成熟率。与单独培养基相比，添加IGF-I的培养基中1级和2级卵母细胞的成熟率显著提高（P<0.05）（分别为85.8%对65.5%和70.3%对51.8%），而3级和4级卵母细胞的成熟率没有差异。在对照组和实验组中，1级卵母细胞的IVM均显著高于所有其他等级（P<0.05）。在实验II中，研究了使用卵丘细胞、胎儿或成年成纤维细胞作为供体细胞核的猫NT胚胎的体外发育情况。将含有IGF-I的培养基中IVM的卵母细胞去核，并与培养第2至4代的卵丘细胞、胎儿或成年成纤维细胞融合。对重构胚胎进行培养，并每24小时监测一次发育进程直至第9天。使用不同供体细胞核的NT胚胎融合率没有显著差异，而用胎儿成纤维细胞和卵丘细胞重构的NT胚胎的分裂率显著高于用成年成纤维细胞重构的胚胎（分别为72.5%和70.7%对54.8%）。与卵丘细胞或胎儿成纤维细胞供体细胞相比，用成年成纤维细胞重构的NT胚胎发育至桑椹胚阶段的比例显著较低（P<0.05）（分别为25.8%对37.9%或47.5%）。然而，在发育至囊胚阶段方面未观察到差异。这些结果表明，IGF-I促进了家猫卵母细胞的IVM。去核的IVM卵母细胞可作为胎儿和成年体细胞核的受体细胞质，从而产生克隆猫胚胎。
