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Methods: MII, GV and IVM-MII were vitrified. After thawed, they underwent in vitro maturation, in vitro fertilization, embryo cultivation or fixed for immunofluorescence. The rates of survival, maturation, fertilization, blastulation and cytoskeleton with normal configuration were calculated.

玻璃化冷冻生殖泡期卵、成熟中期卵及体外成熟卵,解冻复苏后,分别作体外成熟、体外受精、胚胎培养或固定作免疫荧光标记,统计复苏率、成熟率、受精率、囊胚率、细胞骨架正常率。

Methods: mⅱ,gv and ivm-mⅱ were vitrified. after thawed, they underwent in vitro maturation, in vitro fertilization,embryo cultivation or fixed for immunofluorescence. the rates of survival, maturation, fertilization, blastulation and cytoskeleton with normal configuration were calculated.

玻璃化冷冻生殖泡期卵、成熟中期卵及体外成熟卵ivm-mⅱ),解冻复苏后,分别作体外成熟、体外受精、胚胎培养或固定作免疫荧光标记,统计复苏率、成熟率、受精率、囊胚率、细胞骨架正常率。

Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastocysts followed by fusion of cytoplasm and nucleus and cleavage in vitro. During the fusion of cytoplasm, the DNA methylation levels of the fused embryos are as high as these of two-cell diploid embryos in vivo Then the embryos are rapidly demethylated when the nucleus begin to fuse, resulting in the lowest DNA methylation levels when the nucleus are fused completely. After that, the DNA methylation levels of the fused embryos are gradually increased until the morula stage. However, whereas an asymmetric distribution of DNA methylation is established in vivo-derived blastocysts with a higher methylation level in the inner cell mass than that in the trophectoderm, we can not detect the asymmetric distribution in most in vitro-derived tetraploid blastocysts.

结果表明:利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程,在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样,呈现高度甲基化状态;在细胞核开始融合的时候,甲基化水平急速下降,在细胞核完全融合的时候甲基化水平达到最低点;随着胚胎继续分裂,胚胎甲基化水平逐渐增加,在桑葚胚期甲基化水平最高;但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别,这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。

Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastocysts followed by fusion of cyto-plasm and nucleus and cleavage in vitro. During the fusion of cytoplasm, the DNA methylation levels of the fused embryos are as high as these of two-cell diploid embryos in vivo Then the embryos are rapidly demethylated when the nucleus begin to fuse, resulting in the lowest DNA methylation levels when the nucleus are fused completely. After that, the DNA methy-lation levels of the fused embryos are gradually increased until the morula stage. However, whereas an asymmetric distribu-tion of DNA methylation is established in vivo-derived blastocysts with a higher methylation level in the inner cell mass than that in the trophectoderm, we can not detect the asymmetric distribution in most in vitro-derived tetraploid blastocysts.

结果表明:利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程,在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样,呈现高度甲基化状态;在细胞核开始融合的时候,甲基化水平急速下降,在细胞核完全融合的时候甲基化水平达到最低点;随着胚胎继续分裂,胚胎甲基化水平逐渐增加,在桑葚胚期甲基化水平最高;但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别,这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。

Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastcysts fellowed by fusion of cytoplasm and nucleus and cleavage in vitro.After fusion of cytoplasm, the DNA methylation levels of the fused embryos was very high as well as two-cell diploid embryos in vivo.Then the embryos was rapiddly demethylated when the nucleus begin to fuse, resulting the lowest DNA methylation levels when the nucleus fused completely.After that, the DNA methylation levels of fused embryos were gradually increased until the blastocysts stage.However, whereas an asymmetric distribution of DNA methylation was established in an vivo-derived blastocysts with a higher methylation level in the inner cell mass than in the trophectoderm, in most vitro-derived tetraploid blastocysts, we can not detect the asymmetric distribution.

结果表明:利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程,在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样,呈现高度甲基化状态;在细胞核开始融合的时候,甲基化水平急速下降,在细胞核完全融合的时候甲基化水平达到最低点;随着胚胎继续分裂,胚胎甲基化水平逐渐增加,在囊胚期甲基化水平最高;但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别,这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。

Observing the mature embryo sac of the mutant: It was found thatthe embyo sac appeared in typical polygonum type and more than 90% of the embryo sac hadintegral inner structure. They had egg apparatus, two polar nucleus and three antipodal cells , and their position in the embryonic sac was normal .

幼穗分化、成熟胚囊观察发现,90%以上的胚囊具有完整的内部结构,有卵器、两个极核及多个反足细胞,且位置正常,胚囊呈现典型的蓼型结构;同时还观察到雌、雄器官的发育是同步的。

Polycystic ovary syndrome is the commonest endocrinopathy in women of reproductive age.Its features are chronic anovulation or several ovulate,muticystic augmentation of ovaries,high male hormone.Its clinical manifestation has heterogeneity.

多囊卵巢综合征是育龄妇女最常见的内分泌疾病,PCOS患者以长期不排卵或稀发排卵、卵巢多囊性增大、高雄激素血症为基本特征,临床表现异质性。

When electric fusion method was used for nuclear transfer, the fusion rate (46. 0%), cleavage rate (53. 9%) and blastocyte development rate (10.9%) of adult ear fibroblasts were significantly lower than that of fetal fibroblasts (64. 5%, 70.1%, 21. 6% respectively), fetal skin cells (71. 5%, 70.8%, 22. 1% respectively) and ovary granulosa cells (88. 2%, 79. 1%, 25. 5% respectively). There was no significant difference among other donor cells in the cleavage and blastocyst development rate of resconstituted embryos.

当用电融合法进行核移植时,成体耳部成纤维细胞的融合率(46.0%),卵裂率(53.9%)和囊胚发育率(10.9%)均显著低于胎儿成纤维细胞(64.5%,70.1%和21.6%),胎儿皮肤细胞(71.5%,70.8%和22.1%),以及卵巢颗粒细胞(88.2%,79.1%和25.5%);另外三种细胞间的卵裂率,囊胚发育率无显著差异,但卵巢颗粒细胞的融合率显著高于胎儿成纤维细胞和胎儿皮肤细胞(88.2%vs 64.4%,71.5%,P<0.05)。

Analysis of mitochondrial DNA composition of the panda-rabit cloned embryos found that mitochondria from both panda somatic cells and rabbit ooplasm co-existed in early blastocyst, but mitochondria from rabbit ooplasm decreased and those from panda donor cells dominated in early fetuses after implantation.

结果显示,应用卵裂球电融合方法可以制作一系列的多倍体胚胎;小鼠囊胚的形成与胚胎中的细胞数目无直接关系,卵裂球的电融合和染色体数目的增加不会改变胚胎的发育进程;而胚胎细胞中的核/质比可能是控制胚胎囊胚化的一个重要因素。

The effects of different temperatures (37, 38, 39) on parthenogenetic embryo cleavage and blastocyst rate and the effects of different time (2, 3, 4 h) on parthenogenetic embryo cleavage and blastocyst rate were studied.

方法]研究不同温度(37、38、39℃)对孤雌胚胎卵裂率、囊胚率的影响;不同时间(2、3、4h)对孤雌胚胎卵裂率、囊胚率的影响。

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