Shinozawa Tadahiro, Mizutani Eiji, Tomioka Ikuo, Kawahara Manabu, Sasada Hiroshi, Matsumoto Hiromichi, Sato Eimei
Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
Mol Reprod Dev. 2004 Jul;68(3):313-8. doi: 10.1002/mrd.20083.
In the present study, we examined the developmental ability of enucleated zygotes, MII oocytes, and parthenogenetically activated oocytes at pronuclear stages (parthenogenetic PNs) as recipient cytoplasm for rat embryonic cell nuclear transfer. Enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts, whereas the development of embryos reconstituted with MII oocytes and parthenogenetic PNs was arrested at the two-cell stage. Previous observations in rat two-cell embryos suggested that the distribution of microtubules is involved in two-cell arrest. Therefore, we also examined the distribution of microtubules using immunofluorescence. At the two-cell stage after nuclear transfer into enucleated zygotes, microtubules were distributed homogeneously in the cytoplasm during interphase, and normal mitotic spindles were observed in cleaving embryos from the two- to four-cell stage. In contrast, embryos reconstituted with MII oocytes and parthenogenetic PNs showed aberrant microtubule organization. In enucleated zygotes, fibrous microtubules were distributed homogeneously in the cytoplasm. In contrast, dense microtubules were localized at the subcortical area in the cytoplasm and strong immunofluorescence intensity was observed at the plasma membrane, while very weak intensity was detected in the central part of enucleated MII oocytes. In enucleated parthenogenetic PNs, high-density and fibrous microtubules were distributed in the subcortical and central areas, respectively. Pre-enucleated parthenogenetic PNs also showed lower intensity of microtubule immunofluorescence in the central cytoplasm than zygotes. In conclusion, the results of the present study showed that zygote cytoplasm is better as recipient than MII oocyte and parthenogenetic PNs for rat two-cell embryonic cell nuclear transfer to develop beyond four-cell stage. Furthermore, microtubule organization is involved in the development of reconstituted embryos to overcome the two-cell arrest.
在本研究中，我们检测了去核受精卵、MII期卵母细胞和孤雌激活卵母细胞在原核期（孤雌原核）作为大鼠胚胎细胞核移植受体细胞质时的发育能力。以去核受精卵作为接受二细胞期细胞核的受体细胞质可使胚胎发育至囊胚期，而用MII期卵母细胞和孤雌原核重构的胚胎发育则停滞在二细胞期。先前对大鼠二细胞胚胎的观察表明，微管的分布与二细胞期停滞有关。因此，我们还利用免疫荧光检测了微管的分布情况。将细胞核移植到去核受精卵后，在二细胞期，间期细胞质中的微管分布均匀，并且在二细胞至四细胞期的分裂胚胎中观察到正常的有丝分裂纺锤体。相比之下，用MII期卵母细胞和孤雌原核重构的胚胎显示出微管组织异常。在去核受精卵中，纤维状微管均匀分布于细胞质中。相比之下，致密微管位于去核MII期卵母细胞细胞质的皮质下区域，在质膜处观察到较强的免疫荧光强度，而在去核MII期卵母细胞的中央部分检测到的强度非常弱。在去核孤雌原核中，高密度和纤维状微管分别分布在皮质下区域和中央区域。预去核的孤雌原核在中央细胞质中的微管免疫荧光强度也低于受精卵。总之，本研究结果表明，对于大鼠二细胞胚胎细胞核移植以发育至四细胞期以上，受精卵细胞质作为受体比MII期卵母细胞和孤雌原核更好。此外，微管组织参与了重构胚胎的发育以克服二细胞期停滞。
