Laboratory of Animal Breeding, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Reproduction. 2012 Mar;143(3):377-87. doi: 10.1530/REP-11-0364. Epub 2011 Dec 7.
In the course of experiments to identify and characterize the factors that function in bovine conceptuses during peri-attachment periods, various transcripts related to the epithelial-mesenchymal transition (EMT) were found. In this study, RNA was extracted from different sets of days 17, 20, and 22 (day 0=day of estrous) bovine conceptuses and subjected to real-time PCR analysis as well as Western blotting, from which abundances of N-cadherin (CDH2), vimentin, matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) (MMP2), and matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) (MMP9) mRNAs were determined on day 22, concurrent with (CDH1) mRNA and protein downregulation. Transcription factors in EMT processes were then analyzed and changes in snail homolog 2 (Drosophila) (SNAI), zinc finger E-box binding homeobox 1 (ZEB1), zinc finger E-box binding homeobox 2 (ZEB2), twist homolog 1 (Drosophila) (TWIST1), twist homolog 2 (Drosophila) (TWIST2), and Kruppel-like factor 8 (KLF8) transcripts were found in day 22 conceptuses, while confirming SNAI2 expression by Western blotting. Immunohistochemical analysis revealed that the day 22 trophectoderm expressed the mesenchymal markers N-cadherin and vimentin as well as the epithelial marker cytokeratin. In attempts to identify the molecular mechanisms by which the trophectoderm expressed EMT-related genes, growth factor receptors associated with EMT were analyzed. Upregulation of the growth factor receptor transcripts, fibroblast growth factor receptor 1 (FGFR1), platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), platelet-derived growth factor receptor, beta polypeptide (PDGFRB), and transforming growth factor, beta receptor II (70/80 kDa) (TGFBR2) mRNAs, was found on day 22. The analysis was extended to determine the integrin (ITG) transcripts and found high levels of integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) (ITGA4), integrin, alpha 8 (ITGA8), integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) (ITGB3), and integrin, beta 5 (ITGB5) mRNAs on day 22. These observations indicate that after the conceptus-endometrium attachment, EMT-related transcripts as well as the epithelial marker cytokeratin were present in the bovine trophectoderm and suggest that the implantation process for noninvasive trophoblasts requires not only extracellular matrix expression but also partial EMT.
在鉴定和描述附着前期牛胚泡中起作用的因子的实验过程中，发现了与上皮-间充质转化（EMT）相关的各种转录本。在这项研究中，从不同天数的第 17、20 和 22 天（0 天=发情日）的牛胚泡中提取 RNA，并进行实时 PCR 分析和 Western blot 分析，从第 22 天确定了 N-钙粘蛋白（CDH2）、波形蛋白、基质金属蛋白酶 2（明胶酶 A，72 kDa 明胶酶，72 kDa 型 IV 胶原酶）（MMP2）和基质金属蛋白酶 9（明胶酶 B，92 kDa 明胶酶，92 kDa 型 IV 胶原酶）（MMP9）mRNA 的丰度，同时伴随（CDH1）mRNA 和蛋白下调。然后分析 EMT 过程中的转录因子，并发现第 22 天胚泡中 snail 同源物 2（果蝇）（SNAI）、锌指 E 框结合同源框 1（ZEB1）、锌指 E 框结合同源框 2（ZEB2）、 twist 同源物 1（果蝇）（TWIST1）、 twist 同源物 2（果蝇）（TWIST2）和 Kruppel 样因子 8（KLF8）转录本的变化，同时通过 Western blot 证实 SNAI2 的表达。免疫组织化学分析显示，第 22 天的滋养层表达了间充质标志物 N-钙粘蛋白和波形蛋白以及上皮标志物细胞角蛋白。为了确定滋养层表达 EMT 相关基因的分子机制，分析了与 EMT 相关的生长因子受体。发现第 22 天的生长因子受体转录物，成纤维细胞生长因子受体 1（FGFR1）、血小板衍生生长因子受体，α 多肽（PDGFRA）、血小板衍生生长因子受体，β 多肽（PDGFRB）和转化生长因子，β 受体 II（70/80 kDa）（TGFBR2）mRNA 上调。分析还扩展到确定整合素（ITG）转录本，并发现整合素，α 4（抗原 CD49D，VLA-4 受体的α 4 亚基）（ITGA4）、整合素，α 8（ITGA8）、整合素，β 3（血小板糖蛋白 IIIa，抗原 CD61）（ITGB3）和整合素，β 5（ITGB5）mRNA 水平在第 22 天很高。这些观察结果表明，在胚泡-子宫内膜附着后，牛滋养层中存在与 EMT 相关的转录本以及上皮标志物细胞角蛋白，这表明非侵袭性滋养层的植入过程不仅需要细胞外基质的表达，还需要部分 EMT。
