Mouse Zygote Development in Culture Medium without Protein in the Presence of Ethylenediaminetetraacetic Acid1

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Abstract
Development of zygotes from a hybrid-inbred (B6D2F1) and two random-bred (CD1 and CF1) strains of mice were compared after culture in several modifications of a simple, chemically defined medium based on Earle''s Balanced Salts Solution. When cultured without the addition of protein or the chelating agent, ethylenediamine tetraacetic acid (EDTA), none of the zygotes reached the blastocyst stage. The addition of EDTA or protein significantly improved embryo development to blastocysts (p < 0.05). The degree of improvement was dependent upon the strain of the female (85% or 91% for B6D2F1, 56% or 45% for CD1, and 19% or 28% for CF1, respectively). The addition of protein to the media in the presence of EDTA did not further improve embryo development. In all supportive conditions, zygotes from B6D2F1 females developed to blastocysts better than those from CD1 or CF1 females; embryos of the latter strain exhibited the lowest rates of development in vitro. Glycine and alanine (20.mu.M) partially substituted for EDTA; the decreased hybrid-inbred embryo development to blastocysts (20% and 26%, respectively) obtained in the presence of the amino acids suggested, however, that the stimulatory effect of EDTA on embryo developments was other as a source of fixed nitrogen. The rates of development observed with an alternate chelating agent, citric acid (.ltoreq. 20% vs. 83% blastocysts, p < 0.01), although better than the unsupplemented medium, were significantly less effective than EDTA-supplemented medium (83% blastocysts, p < 0.01). The results of this study suggest that the protective effect of proteins in culture medium may be more important than their nutritive role. The protein-free, chemically defined culture medium used in this report provides as basic milieu to study the embryonic requirements of those strains that do not achieve maximal development in the culture conditions routinely used.