Płusa Berenika, Grabarek Joanna B, Karasiewicz Jolanta, Modliński Jacek A
Department of Experimental Embryology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, Wólka Kosowska, Poland.
Mol Reprod Dev. 2005 Apr;70(4):429-37. doi: 10.1002/mrd.20216.
The aim of this study was to investigate the fate of an additional female genome introduced to a dividing zygote. Maternal chromatin in the form of karyoplasts containing a metaphase II spindle were fused to zygotes blocked in anaphase or telophase of the first cleavage. Permanent preparations made 20-40 min after fusion at anaphase revealed that the donor maternal chromosomes had entered anaphase or telophase in 16 out of 18 cases. A further two groups of embryos that were fused at either anaphase or anaphase/telophase were cultured to the first division. Division occurred 50 min after fusion in both groups of embryos (86 and 85.1%, respectively), of which most divided to two cells (80 and 71.6% of total) and the remainder divided to three cells. About two thirds of two-cell embryos contained an extra nucleus in one blastomere. Nuclei containing donor maternal chromosomes reached a similar size to recipient nuclei in 68% of embryos derived from anaphase-blocked zygotes, in contrast to 31.1% of embryos derived from anaphase/telophase-blocked embryos. Replication of DNA in donor nuclei closely followed the timing and intensity of that in control embryos. When fixed 24 hr after fusion, one third of embryos were still at the two-cell stage, with one or both blastomeres showing a single metaphase plate of the second cleavage. In the remaining embryos, three or four cells were present, some containing two nuclei. Blastocysts developed in 50% of fused embryos and three young were born after transfer of cleaving hybrid embryos to recipients. Chromosome preparations from bone marrow of the young contained 3-4 tetraploid metaphase plates per several hundred plates counted compared with none in control embryos. In conclusion, additional maternal chromosomes can be introduced at the late-dividing zygote and join the embryonic cell cycles during subsequent divisions. This method may provide a useful approach for studying changes specific to the maternal genome during early cell cycles of the mammalian embryo.
本研究的目的是调查引入正在分裂的受精卵中的额外雌性基因组的命运。将含有中期II纺锤体的核质体形式的母本染色质与在第一次卵裂后期或末期阻滞的受精卵融合。在后期融合后20 - 40分钟制作的永久制片显示,在18例中有16例供体母本染色体进入了后期或末期。另外两组分别在后期或后期/末期融合的胚胎被培养至第一次分裂。两组胚胎在融合后50分钟均发生分裂(分别为86%和85.1%),其中大多数分裂为两个细胞(分别占总数的80%和71.6%),其余分裂为三个细胞。约三分之二的二细胞胚胎在一个卵裂球中含有一个额外的核。在源自后期阻滞受精卵的胚胎中,68%含有供体母本染色体的核达到与受体核相似的大小,相比之下,源自后期/末期阻滞胚胎的胚胎中这一比例为31.1%。供体核中DNA的复制紧密跟随对照胚胎中的时间和强度。融合后24小时固定时,三分之一的胚胎仍处于二细胞阶段,一个或两个卵裂球显示出第二次卵裂的单个中期板。在其余胚胎中,有三个或四个细胞,一些细胞含有两个核。50%的融合胚胎发育成囊胚,将分裂的杂交胚胎移植到受体后有三只幼崽出生。所产幼崽骨髓的染色体制片显示,每数百个计数的制片中有3 - 4个四倍体中期板,而对照胚胎中没有。总之,额外的母本染色体可在分裂后期的受精卵中引入,并在随后的分裂过程中加入胚胎细胞周期。该方法可能为研究哺乳动物胚胎早期细胞周期中母本基因组特有的变化提供一种有用的途径。