当前位置: X-MOL 学术Biochem. Biophys. Res. Commun. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
The use of a two-step removal protocol and optimized culture conditions improve development and quality of zona free mouse embryos
Biochemical and Biophysical Research Communications ( IF 3.1 ) Pub Date : 2021-09-07 , DOI: 10.1016/j.bbrc.2021.09.013
Weihong Fan 1 , Misato Homma 1 , Renliang Xu 2 , Hiroki Kunii 1 , Hanako Bai 3 , Manabu Kawahara 3 , Toshikazu Kawaguchi 2 , Masashi Takahashi 3
Affiliation  

The zona pellucida (ZP) plays an important role in both the fertilization and embryonic development. For the successful handling of early stage blastomeres for differentiation analysis, the production of identical twins or quadruplets, nuclear transfer or gene introduction requires the removal of the ZP (ZPR). Although single use of either acidic Tyrode's solution or pronase are commonly used for ZPR, long-term exposure to these agents can result in the inhibition of development with the collapse of the three-dimensional blastomere structure. Here, we demonstrate the benefits of using a two-step combined ZPR method, which relies upon a customized well-of-well (cWOW) system with smaller well size, on developmental competence and the quality of the zona free (ZF) mouse embryos.

We first isolated 2-cell embryos using acid Tyrode's solution and then cultured these embryos using either commercially available or cWOW, which had a smaller microwell size. The rate of blastocyst was significantly increased by use of cWOW when compared to other culture systems. Then we evaluated the use of a two-step ZPR protocol, relying on acid Tyrode's solution and proteinase K, and subsequent culture in the cWOW system. Although acid Tyrode's solution treatment alone reduced ZPR time, blastomere morphology became wrinkled, significant decrease in blastocyst rate associated with increased number of apoptotic cells and increased expression of apoptosis-related genes were observed. Using proteinase K alone increased ZPR time and significantly decreased the blastocyst rate, but did not induce an increase in apoptotic cell number or apoptosis-related gene expression. In contrast, two-step method significantly reduced ZPR time and improved blastocyst rate by increasing the total number of cells in these wells an reducing the number of apoptotic cells in these experiments. These results suggest that the two-step ZPR protocol is beneficial for reducing the toxic effects of zona removal on ZF embryo development and quality when combined with a suitable culture system.



中文翻译:

使用两步去除方案和优化的培养条件改善无透明带小鼠胚胎的发育和质量

透明带 (ZP) 在受精和胚胎发育中都起着重要作用。为了成功处理早期卵裂球以进行分化分析、生产同卵双胞胎或四胞胎、核转移或基因导入,需要去除 ZP (ZPR)。尽管 ZPR 通常使用酸性 Tyrode's 溶液或链霉蛋白酶,但长期接触这些药物会导致发育抑制,导致 3D 卵裂球结构崩溃。在这里,我们展示了使用两步组合 ZPR 方法的好处,该方法依赖于具有较小孔尺寸的定制孔 (cWOW) 系统、发育能力和无带 (ZF) 小鼠胚胎的质量.

我们首先使用酸性 Tyrode 溶液分离 2 细胞胚胎,然后使用市售或 cWOW 培养这些胚胎,cWOW 具有较小的微孔尺寸。与其他培养系统相比,使用 cWOW 显着提高了囊胚率。然后我们评估了两步 ZPR 协议的使用,依赖于酸性 Tyrode 溶液和蛋白酶 K,以及随后在 cWOW 系统中的培养。尽管单独用酸性 Tyrode 溶液处理减少了 ZPR 时间,但卵裂球形态变得皱巴巴的,观察到与凋亡细胞数量增加和凋亡相关基因表达增加相关的囊胚率显着降低。单独使用蛋白酶 K 增加了 ZPR 时间并显着降低了囊胚率,但没有诱导凋亡细胞数量或凋亡相关基因表达的增加。相比之下,两步法通过增加这些孔中的细胞总数并减少这些实验中的凋亡细胞数量,显着减少了 ZPR 时间并提高了囊胚率。这些结果表明,当与合适的培养系统相结合时,两步 ZPR 协议有利于减少透明带去除对 ZF 胚胎发育和质量的毒性影响。

更新日期:2021-09-09
down
wechat
bug