Amino Acids and Ammonium Regulate Mouse Embryo Development in Culture1

The regulation of 1-cell mouse embryo development in culture by amino acids was investigated. When the 20 amino acids in Eagle's medium were present, blastocyst formation at 72 h (9%; p < 0.01), and blastocyst cell number (66; p < 0.05) and hatching (45%; p < 0.05) after 96 h of culture were significantly increased, compared to control embryos grown in the absence of amino acids (0%, 60, and 23%, respectively). The beneficial effect of Eagle's amino acids was attributed primarily to the non-essential group. In the presence of non-essential amino acids, blastocyst formation (54%; p < 0.001) and cell number after 72 h of culture (33; p < 0.05), and blastocyst cell number (69; p < 0.01) and hatching (68%; p < 0.01) after 96 h of culture were all significantly greater than for embryos cultured with all amino acids (9%, 26, 66, and 45%, respectively). In the absence of glutamine, essential amino acids significantly reduced blastocyst cell number after 96 h (53; p < 0.05). Continual culture in the presence of amino acids reduced the cleavage rate after around 72 h of in vitro culture; this decrease was not observed in the absence of amino acids. Transfer of embryos to fresh medium after 48–72 h of culture resulted in increases in the percentage of blastocysts formed and in blastocyst cell numbers. These data are consistent with the build-up of an inhibitory compound in the medium, possibly ammonium, an end-product of amino acid metabolism. Ammonium titrated in the medium caused a significant reduction in the percentage of embryos forming morulae (0.62 mM; p < 0.05) and blastocysts (0.15 mM; p < 0.05). Blastocyst cell numbers were significantly reduced by 75 μM (p < 0.05) ammonium. Using an ultramicrofluorometric technique, the production of ammonium by individual embryos was determined. In the absence of amino acids, ammonium was produced at low levels (approximately 0.1 pmol/embryo/h) by all stages of development except the zygote. In the presence of amino acids, ammonium production significantly increased at the 2-cell stage (0.5 pmol/embryo/h) and blastocyst (1.0 pmol/embryo/h). Analysis of culture media revealed that significant quantities of ammonium were generated by the spontaneous breakdown of amino acids at 37°C, which increased linearly with time, generating around 0.3 mM ammonium after 96 h. A potentially inhibitory level of ammonium in culture medium was detectable after 24 h of culture. The data demonstrate that mouse embryonic development is both stimulated and inhibited by specific amino acids and that the breakdown of amino acids in the medium can compromise embryonic development in vitro.