Alleviation of the '2-cell block' and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters

Abstract

The role of amino acids, ethylenediaminetetraacetic acid (EDTA), transferrin, oxygen, glucose, glutamine, taurine and ammonium in CF1 mouse zygote development in culture was examined. Non-essential amino acids and glutamine were shown to alleviate the 2-cell block in culture, and acted in synergy with EDTA to facilitate development to the blastocyst stage. In the presence of amino acids and EDTA, transferrin conferred no beneficial effect. Development of zygotes was significantly impaired if amino acids were removed from the collection medium, even when they were subsequently cultured in the presence of amino acids. Zygote development to the blastocyst stage was significantly improved when modular incubator chambers were used compared to using a conventional incubator, and when an oxygen concentration of 7% was used as opposed to 20%. Addition of taurine to medium containing non-essential amino acids had no effect on embryo development, whereas the removal of glutamine and/or glucose from the culture medium significantly reduced blastocyst cell number. Removal of glucose from the culture medium also resulted in a significant decrease in implantations. Ammonium, generated from the breakdown of amino acids, significantly reduced blastocyst development. EDTA was found to confer its beneficial effects during the first 48 h of culture, and indeed was inhibitory during the second 48 h, resulting in loss of subsequent viability. In summary, the data demonstrate that development of CF1 zygotes to the blastocyst stage is readily achievable. In the presence of non-essential amino acids and glutamine the removal of glucose is detrimental to CF1 mouse embryo development in culture and reduces subsequent viability. Optimal development and maintenance of viability requires more than one culture medium to support the preimplantation period.