Vitrification of Bovine Blastocysts Produced In Vitro Inflicts Selective Damage to the Inner Cell Mass
Vitrification of Bovine Blastocysts Produced In Vitro Inflicts Selective Damage to the Inner Cell Mass
复制标题
DOI：
10.1111/j.1439-0531.2007.01026.x
复制
发表时间：
2009-04-01
影响因子：
1.7
通讯作者：
Diez, C.
中科院分区：
文献类型：
--
作者：
Gomez, E.;Munoz, M.;Diez, C.
与来自活动物的胚胎相反，体外产生的胚胎在冷冻保存后经历增加的损伤和降低的存活率，特别是当用血清产生时。在含有血清的培养基中，视黄酸增加内细胞团和滋养外胚层中的细胞数量，而不改变它们在牛囊胚中的相对比例。在这项工作中，在无血清的培养基中，我们分析了维甲酸对囊胚发育和玻璃化冷冻存活的贡献，并发现与滋养外胚层相比，内团中的细胞减少。用视黄酸（0.7和1.4 μ m）处理第6天体外产生的桑椹胚24小时，随后在无添加剂的情况下再培养24小时。第8天的囊胚生产和孵化囊胚中的细胞计数不受视黄酸的影响。然而，第7天扩大，玻璃化冷冻胚胎与维甲酸1.4 μ m的存活率低于对照组时，培养后升温。玻璃化处理大大减少了内团中的细胞数量（p < 0.0001），而滋养外胚层中的细胞保持不变。应采用囊胚细胞分类计数分析代替非特异性的总细胞测定，以准确了解实质性变化。我们发现内细胞团的强烈减少可以解释为什么在体外存活到冷冻保存有时几乎不能提供移植到受体后胚胎活力的信息。
In contrast to the embryos derived from live animals, the embryos produced in vitro undergo increased damage and reduced survival after cryopreservation, particularly when produced with serum. In medium containing serum, retinoic acid increases cell numbers in the inner cell mass and the trophectoderm without altering their relative proportions in the bovine blastocyst. In this work, in medium without serum, we analyzed the contribution of retinoic acid to the development of blastocyst and survival to vitrification, and found a strong cell reduction in the inner mass when compared to the trophectoderm. Day-6 in vitro-produced morulae were treated for 24 h with retinoic acid (0.7 and 1.4 mu m) and subsequently cultured without additives for a further 24 h period. Day-8 blastocyst production and cell counts in hatched blastocysts were unaffected by retinoic acid. However, Day-7 expanded, vitrified embryos produced with retinoic acid 1.4 mu m survived at lower rates than controls when cultured after warming. Vitrification greatly reduced cell numbers in the inner mass (p < 0.0001), while cells in the trophectoderm remained unaltered. Differential cell counts analysis in blastocysts should be taken up to replace unspecific determination of total cells to appreciate substantial modifications in their exact terms. The strong reduction we found in the inner cell mass could explain why in vitro survival to cryopreservation is sometimes scarcely informative on the viability of the embryo after transfer to recipients.