NuMA distribution and microtubule configuration in rabbit oocytes and cloned embryos

Abstract

The assembly of microtubules and the distribution of NuMA were analyzed in rabbit oocytes and early cloned embryos. a-Tubulin was localized around the periphery of the germinal vesicle (GV). After germinal vesicle breakdown (GVBD), multi-arrayed microtubules were found tightly associated with the condensed chromosomes and assembled into spindles. After the enucleated oocyte was fused with a fibroblast, microtubules were observed around the introduced nucleus in most reconstructed embryos and formed a transient spindle 2-4 h post-fusion (hpf). A mass of microtubules surrounded the swollen pseudo-pronucleus 5 hpf and a normal spindle was formed 13 hpf in cloned embryos. NuMA was detected in the nucleus in germinal vesicle-stage oocytes, and it was concentrated at the spindle poles in both meiotic and mitotic metaphase. In both donor cell nucleus and enucleated oocyte cytoplasm, NuMA was not detected, while NuMA reappeared in pseudo-pronucleus as reconstructed embryo development proceeded. However, no evident NuMA staining was observed in the poles of transient spindle and first mitotic spindle in nuclear transfer eggs. These results indicate that NuMA localization and its spindle pole tethering function are different during rabbit oocyte meiosis and cloned embryo mitosis.

Meiotic spindle assembly and chromosome segregation in oocytes

“…Remarkably, the function of NuMa in tethering microtubule minus ends is conserved in acentriolar spindles. Indeed, the microtubule-associated protein NuMa accumulates at the poles in rabbit, human, and mouse oocytes (Yan et al, 2006;Alvarez Sedó et al, 2011;Kolano et al, 2012). In mouse oocytes, NuMa is required for the formation of barrel-shaped spindle poles as well as microtubule minus-end cohesion (Fig.…”

Effect of Donor Cell Type on Nuclear Remodelling in Rabbit Somatic Cell Nuclear Transfer Embryos

“…1992); however, when somatic cells were introduced by NT, the majority of embryos had abnormal PCC configurations (Chesne et al. 2002; Yan et al. 2006).…”

“…In rabbit, after fusing a somatic nucleus with a mature oocyte, the cloned construct undergoes maturation-promoting factor activation, nuclear membrane breakdown, chromosome condensation and the formation of a metaphase spindle-like structure. When early-stage embryo blastomeres at G0 ⁄ G1 phase were used as donor cells, metaphase chromosomes and spindle were intact in most cases of PCC (Collas et al 1992); however, when somatic cells were introduced by NT, the majority of embryos had abnormal PCC configurations (Chesne et al 2002;Yan et al 2006). Similar to the previous results, our data showed that most of the somatic cell nuclei would undergo PCC 1 h post-fusion with nonactivated enucleated oocytes and chromosomes in most cases misaligned with the spindle or individual chromosomes located near the spindle poles regardless of which donor cell we used.…”

“…2002; Wakayama et al. 2003; Van Thuan 2006; Yan et al. 2006), such as misalignment of a metaphase plate with the spindle and location of an individual chromosome near the spindle poles (Wakayama et al.…”

“…After donor nuclei are introduced into recipient ooplasm, metaphase-like structures have been observed in mouse (Wakayama et al 2003;Van Thuan 2006) and rabbit NT-embryos (Chesne et al 2002;Yin et al 2002). Most cloned embryos observed to date exhibit a range of chromosomal abnormalities (Yin et al 2002;Wakayama et al 2003;Van Thuan 2006;Yan et al 2006), such as misalignment of a metaphase plate with the spindle and location of an individual chromosome near the spindle poles (Wakayama et al 2003). These deficiencies in somatic nuclear reprogramming after NT might emanate, at least in part, from failure to remodel the somatic nucleus morphologically into a functional embryonic nucleus (Moreira et al 2003).…”