Development potential of mouse embryos conceived in vitro and cultured in ethylenediaminetetraacetic acid with or without amino acids or serum.

Mouse ova were inseminated in vitro in modified Earle's balanced salts solution (EBSS) supplemented with 10 or 100 microM EDTA and 4 mg/ml BSA. After 4 h exposure to sperm, the ova were transferred to five different culture conditions based on albumin-free EBSS supplemented with 10 microM EDTA minus or plus amino acids, or with 100 microM EDTA minus or plus amino acids, or with human cord serum. After 44 h of culture, four-cell embryos from each culture group were transferred in cohorts of five into the left oviduct of pseudopregnant recipients (13-16 per culture condition). Two-cell embryos developed in vivo were similarly transferred to a separate group of recipients to serve as controls. The pregnancy rates following transfer of embryos cultured in 10 microM EDTA minus or plus amino acids or in 100 microM EDTA plus amino acids (38%, 43%, and 50%, respectively) were not significantly different from those of the in vivo control group (43%). The pregnancy rates following transfer of embryos cultured in 100 microM EDTA plus amino acids (21%) or plus cord serum (8%) were significantly lower (p less than 0.01) than those of the other groups. The overall yield of fetuses from total embryos transferred was significantly higher (p less than 0.01) for the groups developed in 100 microM EDTA plus amino acids (29%) and in vivo (26%) compared with embryos developed in 10 or 100 microM EDTA with no amino acids, 10 microM EDTA plus amino acids, or 100 microM EDTA plus cord serum (15%, 15%, 9%, and 3%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse zygote development in culture medium without protein in the presence of ethylenediaminetetraacetic acid.

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, ethylenediaminetetraacetic acid (EDTA), none of the zygotes reached the blastocyst stage. The addition of EDTA or protein significantly improved embryo development to blastocysts (p less than 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 microM) 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 development was other than as a source of fixed nitrogen. The rates of development observed with an alternate chelating agent, citric acid (less than or equal to 20% vs. 83% blastocysts, p less than 0.01), although better than the unsupplemented medium, were significantly less effective than EDTA-supplemented medium (83% blastocysts, p less than 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of 'delayed implanting' mouse embryos in vitro.

Blastocysts were recovered from mice with experimentally produced delay of implantation. The dormant embryos were incubated in vitro for up to 24 h in medium containing [3H]uridine and supplemented with mouse serum or bovine serum albumin. Outgrowth of the trophoblast cells occurred in the presence of serum but not with bovine serum albumin. In contrast, the rate of incorporation of [3H]uridine into RNA by the embryos increased steadily throughout the period of incubation and was not influenced by the presence of serum. The change in incorporation of [3H]uridine was due to an increase in the overall rate of RNA synthesis and serum therefore has not effect on this aspect of embryo activation. The observation that a stimulatory serum factor is necessary for outgrowth of dormant embryos in vitro, but is not required for increased metabolic activity, indicates that these two aspects of embryo activation are regulated differently in vivo. With the assumption that trophoblast outgrowth and the changes in metabolic activity in vitro are analogous to the events that occur when embryonic diapause is terminated in vivo, it is suggested that the process of embryo activation after delayed implantation proceeds in a stepwise fashion with each phase being controlled in different ways.