Removal of embryo-toxic ammonium from the culture medium by in situ enzymatic conversion to glutamate.
An enzymatic method for removing embryo-toxic ammonium from culture medium has been developed. Ammonium, produced by both embryo metabolism and spontaneous breakdown of amino acids at 37 degrees C, is transaminated by glutamate dehydrogenase to nontoxic glutamate. Initially, the individual components of the transamination reaction were titrated against mouse embryo development in vitro to determine embryo-safe levels. ADP, an allosteric activator of glutamate dehydrogenase, was found to inhibit embryo development and was therefore omitted from the final formulation (alpha-ketoglutarate, 0.44 mM; glutamate dehydrogenase, 0.375 U; NADH, 0.12 mM). It was found that 0.30 mM ammonium could be removed from the culture medium in situ in 3 h. In situ removal of ammonium significantly increases both blastocyst cell number, implantation, fetal development, and fetal weight after transfer. Removal of ammonium by the conventional method of renewing the culture medium also increased blastocyst cell number but did not affect postimplantation development. In conclusion, it is possible to alleviate the toxic effects of ammonium in vitro on pre- and postimplantation mouse embryo development by its transamination in situ, thereby facilitating the continual exposure to embryo-derived factor(s) which stimulates both pre- and postimplantation development.
Lane
M
M
Laboratories of Human and Animal Reproductive Biology, Monash University, Monash Medical Centre, Clayton, Victoria, Australia.
Gardner
D K
DK
D000818:Animals; D001755:Blastocyst / Q000187:drug effects / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000378:metabolism; D005314:Embryonic and Fetal Development / Q000187:drug effects*; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D000644:Quaternary Ammonium Compounds / Q000378:metabolism* / Q000633:toxicity
D017077:Culture Media, Conditioned; D000644:Quaternary Ammonium Compounds; D018698:Glutamic Acid; D005969:Glutamate Dehydrogenase