The use of a two-step removal protocol and optimized culture conditions improve development and quality of zona free mouse embryos
The use of a two-step removal protocol and optimized culture conditions improve development and quality of zona free mouse embryos
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使用两步去除方案和优化的培养条件可改善无透明带小鼠胚胎的发育和质量
DOI：
10.1016/j.bbrc.2021.09.013
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发表时间：
2021
影响因子：
3.1
通讯作者：
Takahashi Masashi
中科院分区：
文献类型：
--
作者：
Fan Weihong;Homma Misato;Xu Renliang;Kunii Hiroki;Bai Hanako;Kawahara Manabu;Kawaguchi Toshikazu;Takahashi Masashi
透明细胞在受精和胚胎发育过程中起着重要作用。为了成功处理早期卵裂球进行分化分析，产生同卵双胞胎或四胞胎，核移植或基因导入需要去除ZP（ZPR）。虽然单一使用酸性台氏溶液或链霉蛋白酶通常用于ZPR，但长期暴露于这些试剂可导致发育抑制，三维卵裂球结构塌陷。在这里，我们证明了使用两步组合ZPR方法的好处，它依赖于一个定制的孔中孔（cWOW）系统，具有较小的孔尺寸，对发育能力和无菌（ZF）小鼠胚胎的质量。我们首先使用酸性台氏溶液分离2-细胞胚胎，然后使用商业上可获得的或cWOW，其中有一个较小的微孔尺寸培养这些胚胎。与其他培养系统相比，使用cWOW可显著提高囊胚率。然后，我们评估了两步ZPR方案的使用，该方案依赖于酸台氏溶液和蛋白酶K，以及随后在cWOW系统中的培养。虽然单独的酸性台氏溶液处理减少了ZPR时间，但观察到卵裂球形态变得褶皱，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著降低。单独使用蛋白酶K增加了ZPR时间，并显着降低囊胚率，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威尔斯孔中的细胞总数和减少这些实验中的凋亡细胞的数量，显著减少了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组合时，两步ZPR方案有利于降低去除胚胎对ZF胚胎发育和质量的毒性作用。
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.