Vrije Universiteit Brussel

The pre-implantation period is a crucial phase in the reproduction of the Human Being, during which the embryo prepares itself to implant in the uterus. During this five day period, the fertilized egg undergoes a series of divisions, which lead to the formation of a blastocyst. A blastocyst consists of two types of cell: 1) the totipotent or undifferentiated germ cells which form the embryo itself and which can under lab conditions develop into human embryonic stem cells and 2) the differentiated trofectoderm cells which are necessary for the implantation in the uterus, and later for the formation of the placenta. A large percentage of human embryos do not implant in the uterus. One of the many causes for this can be genetic. The pre-implantation embryo has two sources of genetic information: 1) the maternal genetica and 2) the embryonic genetica. The maternal genetica consists of the genes from the unfertilized egg cell and these determine the first two days of the embryos' development. The embryonic genetica consists of the genetic material of both the egg cell and the sperm (gametes). The embryonic genetics become active on the third day of pre-implantation development. Genes are strands of DNA which cells use as the basis for production of proteins which in turn determine the nature of the cell. The transition from protein to cell is called Expression.

Research into the expression of genes in human pre-implantatation embryos is restricted by ethical and practical issues. As a result of this, animal models are all too often substituted for studies on human genes. In the research centre for reproductive genetics at the VUB, patients can give their specific permission for gametes and embryos unsuitable for clinical use, to be used for this scientific research. Our research centre has a stem cell lab, where various human stem cells have been cultivated (Mateizel et al., 2005). In this manner, we have at our disposal, human gametes, embryos and stem cells, in which we can study the expression of genes. In this way, we have been able to study the expression of the fertility related gene DAZL, which is necessary for the development of gametes in humans and animals. Our results indicated that DAZL does not only undergo expression in in gametes and stem cells, as was previously known, but also during every stage of the pre-implantation development (Caufman et al., 2005a). During the third day of the pre-implantation development, large differences in expression were determined, both within an embryo and in various embryos compared to one another. These differences reflect clearly, the transition from maternal to embryonic genetic influence. In blastocysts, it appeared that the expression of DAZL was directly related to the quality of the embryo. In well formed blastocysts, DAZL underwent expression in the germ cells and the trofectoderm. Because the trofectoderm can never form gametes, DAZL may have another function aside from the development of gametes.

Another gene in which we studied expression, is OCT-4. OCT-4 is regarded to be a indicator for undifferentiated cells and therefore a stem cell indicator. This property is shown particularly in 1) studies in mice in which this was proved and 2) the fact that the expression of OCT-4 is necessary to keep human stem cells in an undifferentiated state. Our study showed that OCT-4 is formed later than was previously thought, namely on day four of the pre-implantation development and that the expression of OCT-4 in humans is not restricted to undifferentiated cells, because OCT-4 also expresses in the trofectoderm and in in-vitro differentiated human embryonic stem cells (Cauffman et al., 2005b). Therefore, the observation in mice that OCT-4 is extrapolated as a specific indicator for the undifferentiated status in innaccurate. An additional difference between mouse and human OCT-4 is that in humans, two forms of the gene exist (OCT-3A en OCT-3B) and in the mouse, only one. Recent research has identified differences in the expressions of OCT-3A en OCT-3B (Cauffman et al., in preparation).

Apart from OCT-4 there are many more indicators which are regularly used to characterize human embryonic cells, such as NANOG, SOX-2, REX-1, ... . The majority of these stem cell indicators have been extrapolated from mouse models to human models. Nevertheless, recent studies have identified significant differences between mice and humans at the level of gene expression. Thorough research of these indicators in humans is indicated. When one of these indicators in proven to be exclusively produced in undifferentiated cells, it will be able to be used to examine whether all cells in the early pre-implantation embryo are undifferentiated or totipotent.

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