title: Intracellular pH of the mouse preimplantation embryo: amino acids act as buffers of intracellular pH.
abstract: The inclusion of specific amino acids in conventional culture media has been shown to enhance mammalian embryo development in vitro. Amino acids have been shown to confer their benefits to the preimplantation embryo in a number of different ways. However, their ability to buffer intracellular pH (pHi) has not been investigated. Thus, the aim of this study was to determine if amino acids regulate pHi in the mouse preimplantation embryo. pHi was determined using carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. Incubation with 5,5-dimethyl-2,4-oxazol-idinedione (DMO), a non-metabolizable weak acid, resulted in a significant intracellular acidification in the zygote, 2-, 4- and 8-16-cell embryo. However, in the presence of groups of amino acids, the degree of acidification due to DMO was markedly reduced in the mouse embryo up to the 4-cell stage. Specifically, non-essential amino acids and glutamine had the greatest capacity to buffer pHi in the early embryo. The ability of amino acids to buffer pHi was not apparent from the 8-16-cell stage onwards. In contrast to the precompacted embryo, the morula did not undergo a significant decrease in pHi until exposed to DMO concentrations > or = 10 mM in the absence of amino acids. This may be due to the generation of a permeability seal during compaction, thus enabling the morula to regulate its own pHi. This regulatory ability could either be reversed by causing the morula to decompact, or created by inducing premature compaction in the 8-16-cell embryo. Data presented in this study indicate that amino acids act as buffers of pHi in the early embryo and play a key role in regulating cell physiology. Further evidence for this was provided by the result that only those embryos cultured in 30 mM DMO in the presence of non-essential amino acids and 1 mM glutamine did not block at the 2-cell stage, but grew on to develop into expanded blastocysts.
authors.affiliation: Physiology Department, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: L J
authors.initials: LJ
authors.lastname: Edwards
authors.forename: D A
authors.initials: DA
authors.lastname: Williams
authors.forename: D K
authors.initials: DK
authors.lastname: Gardner
mesh_terms: D000596:Amino Acids; D000818:Animals; D001578:Benzopyrans; D001755:Blastocyst; D002021:Buffers; D004624:Embryo Transfer; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D018613:Microscopy, Confocal; D009284:Naphthols; D012235:Rhodamines
chemical_list: D000596:Amino Acids; D001578:Benzopyrans; D002021:Buffers; D005456:Fluorescent Dyes; D009284:Naphthols; D012235:Rhodamines; C091666:seminaphthorhodaminefluoride