Na/K-ATPase β1 subunit expression is required for blastocyst formation and normal assembly of trophectoderm tight junction-associated proteins
Na/K-ATPase β1 subunit expression is required for blastocyst formation and normal assembly of trophectoderm tight junction-associated proteins
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DOI：
10.1074/jbc.m700696200
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发表时间：
2007-04-20
影响因子：
4.8
通讯作者：
Watson, Andrew J.
中科院分区：
文献类型：
--
作者：
Madan, Pavneesh;Rose, Keeley;Watson, Andrew J.
Na/K-ATPase在介导胚泡形成中起重要作用。尽管在小鼠着床前发育过程中存在多种Na/K-ATPaseα和β亚型的表达，但仅有α1和β1亚型定位于滋养外胚层的基侧膜区。本研究的目的是利用小干扰RNA(SiRNA)寡核苷酸对小鼠1细胞受精卵进行显微注射，选择性地下调Na/KATPaseβ1亚单位的表达。实验包括未注射对照和两组显微注射Stealth(TM)Na/K-ATPaseβ1亚单位寡核苷酸或非特异性Stealth(TM)siRNA作为对照。发育到2-、4-、8-和16-细胞期和桑拿期在三组之间没有差异。然而，显微注射Na/K-ATPaseβ1亚单位siRNA寡核苷酸的胚胎中只有2.3%发育到囊胚期，而对照组和未注射对照组的这一比例分别为73%和91%。用逆转录-聚合酶链式反应和整体免疫荧光方法验证了Na/K-ATPaseβ1亚单位表达下调，证明在siRNA显微注射的胚胎中没有检测到Na/K-ATPaseβ1亚单位mRNAs和蛋白质。β1亚单位siRNA显微注射胚胎与对照组的聚合嵌合体实验表明，对囊胚形成的阻断是可逆的。比较3组间Na/K-ATPaseβ1、紧密连接相关蛋白occludin和ZO-1的分布。蛋白质分布在对照组之间没有差异，但在Na/K-ATPaseβ1亚单位siRNA显微注射胚胎中，这三种多肽都显示出异常的分布。我们的结果表明，Na/K-ATPase的β1亚基是胚泡形成所必需的，在着床前发育过程中，该亚基也是维持紧密连接相关多肽的正常分布和定位所必需的。
Na/K-ATPase plays an important role in mediating blastocyst formation. Despite the expression of multiple Na/K-ATPase alpha and beta isoforms during mouse preimplantation development, only the alpha 1 and beta 1 isoforms have been localized to the basolateral membrane regions of the trophectoderm. The aim of the present study was to selectively down-regulate the Na/KATPase beta 1 subunit employing microinjection of mouse 1 cell zygotes with small interfering RNA (siRNA) oligos. Experiments comprised of non- injected controls and two groups microinjected with either Stealth (TM) Na/K- ATPase beta 1 subunit oligos or nonspecific Stealth (TM) siRNA as control. Development to the 2-, 4-, 8-, and 16-cell and morula stages did not vary between the three groups. However, only 2.3% of the embryos microinjected with Na/K-ATPase beta 1 subunit siRNA oligos developed to the blastocyst stage as compared with 73% for control-injected and 91% for non-injected controls. Na/K-ATPase beta 1 subunit down-regulation was validated by employing reverse transcription-PCR and whole-mount immunofluorescence methods to demonstrate that Na/K-ATPase beta 1 subunit mRNAs and protein were not detectable in beta 1 subunit siRNA-microinjected embryos. Aggregation chimera experiments between beta 1 subunit siRNA-microinjected embryos and controls demonstrated that blockade of blastocyst formation was reversible. The distribution of Na/K-ATPase beta 1 and tight junction-associated proteins occludin and ZO-1 were compared among the three treatment groups. No differences in protein distribution were observed between control groups; however, all three polypeptides displayed an aberrant distribution in Na/K-ATPase beta 1 subunit siRNA-microinjected embryos. Our results demonstrate that the beta 1 subunit of the Na/K-ATPase is required for blastocyst formation and that this subunit is also required to maintain a normal Na/K-ATPase distribution and localization of tight junction-associated polypeptides during preimplantation development.