O'Neill G T, Rolfe L R, Kaufman M H
Institute of Animal Physiology and Genetics Research, Roslin, Scotland.
Mol Reprod Dev. 1991 Nov;30(3):214-9. doi: 10.1002/mrd.1080300308.
The brief exposure of recently ovulated mouse oocytes to M16 embryo culture medium supplemented with strontium chloride (M16 Sr2+) for 2-10 min was observed to induce a high incidence of parthenogenesis. A lower incidence of activation and a significant rate of oocyte degeneration was observed when oocytes were incubated in M16 Sr2+ medium for 20-60 min. The majority of the oocytes exposed to this agent for 2-10 min developed as single-pronuclear haploid parthenogenones. The incidence of this parthenogenetic class was reduced as the duration of exposure to M16 Sr2+ was increased from 2 to 30 min. Under these conditions a greater proportion of the activated oocytes developed as two-pronuclear diploid parthenogenones, due to failure of second polar body extrusion. The activation frequency and the proportionate incidence of the pathways of parthenogenetic development observed following the exposure of ovulated oocytes to calcium-free M16 medium differed significantly from that induced by exposure to M16 Sr2+. Cytogenetic analysis of the single-pronuclear haploid class of Sr(2+)-induced parthenogenones at metaphase of the first-cleavage mitosis has shown that this agent did not induce a significant increase in the incidence of chromosome segregation errors during the completion of the second meiotic division. Analysis of the developmental potential of the two-pronuclear class of diploid Sr(2+)-induced parthenogenones during the preimplantation stages of embryogenesis revealed that their cell number and rate of cell division were less than those of fertilised embryos retained either in vivo or in vitro. The novel methods of activating oocytes indicated in this study present new opportunities to improve the efficiency of embryo cloning techniques with the ruminant species.
将刚排卵的小鼠卵母细胞短暂暴露于添加了氯化锶的M16胚胎培养基(M16 Sr2+)中2 - 10分钟,发现可诱导较高的孤雌生殖发生率。当卵母细胞在M16 Sr2+培养基中孵育20 - 60分钟时,激活发生率较低且卵母细胞退化率显著升高。大多数暴露于该试剂2 - 10分钟的卵母细胞发育为单原核单倍体孤雌生殖体。随着暴露于M16 Sr2+的时间从2分钟增加到30分钟,这种孤雌生殖类型的发生率降低。在这些条件下,由于第二极体排出失败,更大比例的激活卵母细胞发育为双原核二倍体孤雌生殖体。排卵后的卵母细胞暴露于无钙M16培养基后观察到的激活频率和孤雌生殖发育途径的相应发生率与暴露于M16 Sr2+诱导的情况有显著差异。对Sr(2+)诱导的单原核单倍体孤雌生殖体在第一次卵裂有丝分裂中期的细胞遗传学分析表明,该试剂在第二次减数分裂完成过程中未诱导染色体分离错误发生率显著增加。对Sr(2+)诱导的双原核二倍体孤雌生殖体在胚胎发育植入前阶段的发育潜能分析表明,它们的细胞数量和细胞分裂速率低于体内或体外保留的受精胚胎。本研究中指出的激活卵母细胞的新方法为提高反刍动物胚胎克隆技术的效率提供了新机会。