62 SPINDLE MORPHOGENESIS AND THE MORPHOLOGY OF CHROMOSOMES IN MOUSE NUCLEAR TRANSFER: AN ABNORMAL START IN CLONING OF MICE

Abstract

Cloning of mammals by somatic nuclear transfer (NT) was achieved more than 6 years ago. Nonetheless, the success rate is very low. Although spindles and nuclear mitotic apparatus protein (NuMA) are thought to play important roles in the cell cycle and maybe reprogramming after NT, the spindle and NuMA morphogenesis after NT is not well understood. The aim of this study was to examine the spindle, NuMA morphogenesis and chromosome morphology in mouse NT. Maturated mouse oocytes were collected from mature B6D2F1 females 16 h after hCG injection. The donor nuclei used in this study were cumulus cells. A donor nucleus was injected into an oocyte by means of a piezo pulse system. For double labeling, DNA was stained with PI (red) or Hoechst 33342 (blue); tubulin, NuMA, and phosphorylated histone H3 were stained with FITC or alexa 488 (green). All samples were visualized with a BioRad Radiance 2100 confocal scanning laser microscope. In this study, the spindle, NuMA morphogenesis, chromosome morphology, and histone H3 phosphorylation were identified using more than 3000 maturated mouse oocytes in five experimental studies to examine (1) the morphological changes of spindle and NuMA in NT oocytes at 10, 30, 60, 90, 120, 180, and 360 min after injection of somatic cell nuclei into intact or enucleated maturated oocytes; (2) the localization and morphology of spindles and NuMA of NT embryo followed by activation at 1, 2, and 6 h after somatic nuclear injection; (3) the effects of the timing of activation and donor cell membrane on the spindle morphogenesis; (4) the effects of intact nuclear membrane or its breakdown by micropipette on spindle morphogenesis; and (5) the correlation between donor cell chromosome condensation and histone H3 phosphorylation after injection into enucleated maturated oocytes. As control, the NuMA morphogenesis during the early pronuclear stages of NT was compared with those of ICSI oocytes. We consistently observed that abnormal spindle morphogenesis occurred during the early stages of mouse NT. Most of the NT oocytes began with monopolar spindle and monopolar NuMA (90–95%), and this phenomenon occurred 10 min after NT. Only 5–10% of NT oocytes started with bipolar spindles. However, monopolar spindles were transformed into bipolar spindles during 30–60 min after NT by bipolarization of centrosomal NuMA. After activation, NuMA was localized in the perispindle region, and finally concentrated inside the pronuclei of embryo. This spindle morphogenesis was independent of the presence or absence of metaphase II [not like in rhesus monkeys, whereas meiotic spindle removal depletes the ooplasm of NuMA and HSET, both vital for mitotic spindle pole formation (Lanza R et al., 2003 Science, 300, 297)]. The breakdown of the donor nuclear membrane by micropipette resulted in the increase of bipolar spindle formation in NT. The initiation of chromosome condensation and histone H3 phosphorylation were observed 30 min after NT and the maximum of histone H3 phosphorylation was occurred 60 to 90 min after NT. Future studies are required to elucidate the mechanism of monopolar spindle formation, chromosomal abnormalities in NT, and the relation of the monopolar phenomenon to the success rate in mammalian cloning.