Gall Laurence, Dedieu Thierry, Chesne Patrick, Ruffini Sylvie, Sevellec Claude, Peynot Nathalie, Renard Jean Paul, Heyman Yvan
Unité de Biologie de la Fécondation, Station de Physiologie Animale, INRA, 78350 Jouy-en-Josas, France.
Unité de Biologie du Développement, INRA, 78350 Jouy-en-Josas, France.
Dev Growth Differ. 1996 Oct;38(5):517-525. doi: 10.1046/j.1440-169X.1996.t01-4-00008.x.
When in vitro-matured oocytes were enucleated, aged and kept at 10°C before reconstitution, the in vitro development of nuclear transfer embryos to the blastocyst stage did not differ from that obtained with in vitro fertilization. This suggests that these recipient cytoplasts constitute a suitable environment for the development of the nuclear transplant. The aim of the present study was to investigate, at the biochemical level, the result of the preparation of recipient oocytes, including enucleation, ageing and cooling. For this purpose the phosphorylation profiles of four groups of in vitro-matured bovine oocytes (aged oocytes, aged-cooled oocytes, enucleated-aged oocytes and enucleated-aged-cooled oocytes (recipient cytoplasts)) were analyzed. These recipient cytoplasts exhibited a phosphorylation profile similar to that of activated oocytes. Maturation promoting factor (MPF) activity, which was high in young metaphase II oocytes, in aged oocytes, in enucleated-aged oocytes and in aged-cooled oocytes, dropped to the basal level in enucleated-aged-cooled oocytes (recipient cytoplasts), while mitogen-activated protein kinase (MAPK) activity remained elevated. The combination of enucleation, ageing and cooling following oocyte in vitro maturation resulted in an interphase-like stage cytoplasm having a phosphorylation profile and low MPF activity similar to activated oocytes, but exhibiting high MAPK activity.
当体外成熟的卵母细胞去核、老化并在重构前于10°C保存时，核移植胚胎体外发育至囊胚阶段的情况与体外受精获得的结果并无差异。这表明这些受体细胞质构成了核移植发育的适宜环境。本研究的目的是在生化水平上研究受体卵母细胞制备（包括去核、老化和冷却）的结果。为此，分析了四组体外成熟的牛卵母细胞（老化卵母细胞、老化冷却卵母细胞、去核老化卵母细胞和去核老化冷却卵母细胞（受体细胞质））的磷酸化谱。这些受体细胞质表现出与活化卵母细胞相似的磷酸化谱。成熟促进因子（MPF）活性在年轻的中期II卵母细胞、老化卵母细胞、去核老化卵母细胞和老化冷却卵母细胞中较高，而在去核老化冷却卵母细胞（受体细胞质）中降至基础水平，而丝裂原活化蛋白激酶（MAPK）活性仍保持升高。卵母细胞体外成熟后进行去核、老化和冷却，导致细胞质呈现类似间期的阶段，其磷酸化谱和低MPF活性与活化卵母细胞相似，但MAPK活性较高。
