Research On Gene Differential Expression In Sister Blastomeres In2-Cell Mouse Embryo

It is well known that the first embryonic cell lineage differentiation, or, the emergence oftrophectoderm and inner cell mass, is a key events in preimplantation development ofmammalian embryo. Trophectoderm cells form extraembryonic tissues, inner cell massdeveloped to be embryos and one part of extraembryonic tissues. Lineage tracing experimentsdemonstrated that the differentiation can be traced back to2-cell stage. The2-cell stageblastomere that is first to divide will preferentially contribute its progeny to the embryonicpart of the blastocyst. Progeny from the later dividing2-cell stage blastomere that contributespredominantly to the abembryonic part of the blastocyst. The asynchronous cleavage and thespindle rotation will arise during the transition of2-cell to4-cell stage. That is the reason whymany researchers focus on the study of gene differential expression in sisters blastomeresduring2-cell stage. However, the progress of the research is so slowly because of thedifficulties in sampling and the lower amout of samples. Until now, no gene was identified tobe related to the direction and order of blastomeres cleavage division. Our studies focused onthe asymmetrical distribution of genes in sister blastomeres during the2-cell late stage. Wefound some genes that related to the direction and order of blastomeres cleavage division. Ourstudies shed light on the mechanism of the process of early embryos development.1. Our work display the change of genes expression during the2-cell late stage by genechip, and found that the differential expression genes, such as Wnt; Notch and MAPK, arerelated to the signal path.12of interested genes were selected to test chip result bysemi-quantitative RT-PCR method. Beta-actin was used as internal control for the certificationof RNA integrity. The results showed that except for Beta-actin, Axin1, Cdc25c, Cdkn2d,Mta2, Pin4and Yy1, specific band of other five genes cannot be detected by RT-PCR. Theresults showed that the results obtained from semi-quantitative PCR are basically inconsistence with the results of gene chip. At the same time, it is also proved thatsemi-quantitative PCR methods are reliable and can be used as a subsequent examination ofgene expression.2. The results of one cell RT-PCR showed that the mRNA expression of Axin1, Cdc25c,Cdkn2d and Mta2in blastomere2is higher than blastomere1while the mRNA expression ofYy1and Pin4in blastomere2is lower than blastomere149h post-hCG. These results demonstrated that the asymmetrical distribution of mRNA indeed exists between the sisterblastomeres.3. Axin1、Cdc25c and Cdkn2d were chosen to better observe the distribution of the genesrelated to the direction and order of blastomere cleavage division by real time PCR andRT-PCR. The results showed that Axin1、Cdc25c and Cdkn2d mRNA were all highlyexpressed in the same blastomere. This demonstrated that the asymmetry of embryos wereexist in the2-cell, and the asymmetry may be related to the direction and order of blastomerecleavage division and the establishment of embryonic-abembryonic axis (E-Ab axis).4. Three plasmids including pET28a-Axin1, pET28a-Cdc25c and pET28a-Cdkn2d werecorrectly constructed in this experiment and respective proteins were expressed by IPTGinduction in Ecoli. BL21. Rabbit antibodies of anti Cdc25c/Cdkn2d and duck antibodies ofanti Axin1were prepared by immunizing with purified fusion protein. Western blot analysisshowed that the antibody purified by ammonium sulfate law have good specificity. Whereafter,subcellular localization and expression level of the endogenous proteins Axin1, Cdc25c andCdkn2d in sister blastomeres were detected by the double staining immunofluorescence byAxin1and Cdc25c or Axin1and Cdkn2d, meanwhile DAPI counterstain.Immunofluorescence staining assay indicated that Axin1displayed punctate distribution in thecytoplasma of later2-cell stage embryos, and the expression of Axin1was more or lessasymmetry among the512-cell late embryos. Cdc25c was found in the cell nuclei in122-celllate stage embryos, and the expression is also asymmetry between the two blastomeres.Cdkn2d was found in cell cytomere but not in the locations touched between the twoblastomeres near the cytomembrane in392-cell late stage embryos, and the expression wasalso asymmetry between the two blastomeres. The asymmetrical distribution of these proteinsis consistent with corresponding mRNA. These results suggested that the three proteins maybe having direct or indirect interaction in some unknown way, and this affect the developmentof early embryos.5. Observation of the shape of4-cell embryos indicated that all of the4-cell embryos ofnormal oestrus mouse are regular tetrahedral embryos. However, there are two kinds of4-cellembryos in superovulation mouse. Most of them were regular tetrahedral embryos and few ofthem were non-regular tetrahedral embryos. The in vitro culture showed that different kind of4-cell embryos can develop into blastocyst. The expression of Beta-actin、Axin1、Cdc25c、Cdkn2d、Mta2、Pin4and Yy1mRNA were higher in regular tetrahedral embryos than in thenon-regular tetrahedral embryos compared by semi-quantitative RT-PCR analysis. Thesuperovulation could affect the genes expression in blastomere during2-cell stage, and theninfluenced the orientation and the order of blastomere division from the2-to the4-cell stage. But there affect only in the level of expression without in the future development.