Goossens Karen, Tesfaye Dawit, Rings Franca, Schellander Karl, Hölker Michael, Van Poucke Mario, Van Zeveren Alex, Lemahieu Isabel, Van Soom Ann, Peelman Luc J
Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium.
Reprod Fertil Dev. 2010;22(2):395-404. doi: 10.1071/RD09080.
The expression of the cytoskeleton protein Keratin 18 (KRT18) starts at the onset of bovine blastocyst formation. KRT18 is solely expressed in the trophectoderm and can therefore be used as a marker for trophectodermal differentiation. In the present study, the expression of KRT18 was suppressed by RNA interference to probe its functional importance in bovine blastocyst formation. Microinjection of KRT18 double-stranded RNA into the cytoplasm of zygotes resulted in reduced KRT18 mRNA (76% reduction) and protein expression at the blastocyst stage and a lower developmental competence (41% reduction in the percentage of blastocyst formation) compared with non-injected and phosphate-buffered saline (PBS)-injected controls. KRT18 downregulation was associated with reduced mRNA expression of KRT8, the binding partner of KRT18, but had no effect on the expression of KRT19, CDH1 and DSP, other genes involved in intermediate filament and cytoskeleton formation. The results of the present study demonstrated that KRT18 knockdown in preimplantation embryos results in reduced blastocyst formation, but no further morphological aberrations were observed with regard to the biological function of KRT18. These observations could be due to the function of KRT18 being replaced by that of another gene, the surviving blastocysts expressing the minimum level of KRT18 required for normal blastocyst development or the possibility that further aberrations may occur later in development.
细胞骨架蛋白角蛋白18（KRT18）的表达始于牛囊胚形成之时。KRT18仅在滋养外胚层中表达，因此可作为滋养外胚层分化的标志物。在本研究中，通过RNA干扰抑制KRT18的表达，以探究其在牛囊胚形成中的功能重要性。与未注射和注射磷酸盐缓冲盐水（PBS）的对照组相比，向受精卵细胞质中显微注射KRT18双链RNA导致囊胚阶段KRT18 mRNA表达降低（降低76%）和蛋白质表达降低，发育能力也较低（囊胚形成率降低41%）。KRT18的下调与KRT18的结合伴侣KRT8的mRNA表达降低有关，但对KRT19、CDH1和DSP（参与中间丝和细胞骨架形成的其他基因）的表达没有影响。本研究结果表明，着床前胚胎中KRT18的敲低导致囊胚形成减少，但未观察到KRT18生物学功能方面的进一步形态学异常。这些观察结果可能是由于KRT18的功能被另一个基因取代，存活的囊胚表达正常囊胚发育所需的最低水平的KRT18，或者是由于在发育后期可能会出现进一步异常的可能性。
