Dipeptide glutamine functions as an organic osmolyte in mouse preimplantation embryos but does not support blastocyst hatching at the same rate as individual component amino acids

Abstract

Dipeptide glutamine is included in human embryo culture media to reduce ammonia production. However, dipeptides have not been examined to determine if they can fulfill known physiologic roles of individual, non-dipeptide amino acids. Our objective was to examine whether dipeptide alanyl-glutamine (ala-glu) can act as an organic osmolyte in mammalian embryos as efficiently as its component individual amino acids. Prospective experimental study. Thawed 1-cell mouse embryos were cultured in groups of 10 in 500ul in 1 of 4 media treatments for 96h in ∼6% CO2, 5% O2, 89% N2. Positive controls (Pos Con) consisted of amino acid-free Human Tubal Fluid (HTF; 280mOsm). Treatment media included HTF with elevated NaCl to increase osmolality (320mOsm) and either 1mM dipeptide ala-glu or 1mM alanine + 1mM glutamine (ala+glu). Negative controls (Neg Con) consisted of 320mOsm with no amino acids. Blastocyst development was assessed and sizes determined using Cronus software. Data were collected over 5 replicates and analyzed using ANOVA and Tukey analysis. Pos Con resulted in 79.0% ± 5.9 blastocyst development while Neg Con media resulted in significantly lower rates of blastocyst formation (20.1% ±7.3), p<0.05. Both ala-glu and ala+glu resulted in similar rates of blastocysts (47.9%±4.5 and 52.2%7.3, respectively), which were significantly lower than Pos Con, but higher than Neg Con, p<0.05. While there were no differences in rates of expanded blastocysts between treatments, Pos Con and ala+glu had similar rates of hatching blastocysts, significantly greater than Neg Con, p<0.05. Ala-glu yielded similar rates of hatching to ala+glu and Neg Con, but were significantly lower than Pos Con. Because osmolality impacts cell volume and blastocyst expansion is a marker of quality, we measured resulting blastocyst size as a more precise indicator of size and further indicator of osmolyte function. There were no significant differences in blastocyst perimeter between treatments (Pos Con: 421um±16.7, ala-glu: 380.2um±13.2, ala+glu: 385.5±14.9, Neg Con: 348.94±18.0). The dipeptide ala-glu can function as an organic osmolyte and rescue embryo development under high osmolality conditions. This is the first report of dipeptide glutamine acting as an osmolyte. However, as indicated by hatching rates, the dipeptide may not be as efficient as its component amino acids. The mechanism by which embryos transport and utilize dipeptide amino acids may lend insight into these findings, yet remain to be identified. These data may have implications in future formulation of human embryo culture media.Tabled 1Dipeptide glutamine acts as an osmolyte in mouse embryos.Treatment% Total Blast% Expanded Blast% Hatching BlastBlast PerimeterPos Con (280mOsm)79.0±5.9a25.8±8.526.0±3.6a421.1±16.7Dipeptide Ala-Glu (320mOsm)47.9±4.5b19.5±6.56.8±1.9bc380.2±132Ala+Glu (320mOsm)52.2±7.3b15.4±3.920.5±7.4ab385.5±14.9Neg Con (320mOsm)20.1±7.3c6.1±4.02.0±2.0c348.9±18.0 Open table in a new tab