Although the mouse embryo can be grown successfully from the 2-cell to the blastocyst stage in a defined medium containing BSA, that development is not completely normal. Only certain F1 hybrid embryos can grow from the 1-cell to the blastocyst stage in vitro, most strains exhibiting a 2-cell block to further development. To determine the mechanisms which govern critical developmental events, embryos must be able to be studied and manipulated in vitro. It is therefore imperative that we improve the current level of in vitro culture such that embryos undergo completely parallel development in vivo and in vitro. It is first necessary to establish criteria for normal in vivo embryonic development and compare in vitro grown embryos to these standards. Among the criteria for assessing normal development will be gross morphological appearance, cleavage rates and cell numbers, ability of blastocysts to outgrow in vitro, size of the inner cell mass, normality of biochemical and cellular differentiation and finally ability of the blastocyst to give rise to a normal fetus upon transfer to pseudopregnant females. Once the criteria for normal development have been established, a variety of media will be tested on CF-1 and F1 hybrid embyros to determine the medium which best supports normal embyronic development. The composition of that medium will then be modified by appropriate additions or substitutions (serum, energy sources, growth factors, EDTA). In addition, the environment of the cultures will be modified with respect to gaseous composition, culture dishes and agitation to see if development can be enhanced by these alterations. Co-culture of embryos with other embryos or certain established cell lines will be undertaken. Embryo or cell line conditioned medium will also be tested for its effect on in vitro embryonic development. The in vivo environment of the embryos, the oviductal fluid, will be analyzed by HPLC and atomic absorption. This analysis will be performed on intact oviducts and oviductal segments to see if there are changes in the embryonic environment as the embryos move down the oviduct into the uterus. Peak fractions can be collected by HPLC and used in in vitro reconstitution experiments using defined media.
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