Mouse blastocysts produce glutamine from glutamate in the presence of ammonium, but consume glutamine under oxidative stress (#264)
During embryo culture, ammonium is generated by amino acid metabolism and from spontaneous deamination of amino acids at 37°C. Ammonium has been shown to be embryo toxic, however relatively few studies have investigated the mechanism(s) by which the early embryo can dispose of ammonium. In order to study ammonium metabolism at the blastocyst stage we have (i) examined the metabolic effect of inhibiting glutamine synthetase (glutamine being a possible sink for ammonium) and (ii) investigated the effects of ammonium in the presence of atmospheric oxygen, another known source of oxidative stress to the embryo.
Zygotes from F1 hydrid mice (C57BL x CBA) were cultured in groups of 10 in 20 μL drops of culture medium G1 at 37°C for 48 h before transfer to medium G2 for a further 24 h. Embryos which developed to the early blastocyst stage after 72 h were cultured in groups of 3 in 2 μL drops of G2 for a further 24 h, and their amino acid utilisation determined by LC-MS.
Amino acid utilisation by blastocysts was determined after culture with 0, 150 and 300 μM ammonium chloride (in either 5% or 20% oxygen), and with or without 500 μM methionine sulfoximine (MSO), an inhibitor of glutamine synthetase.
In the presence of MSO, ammonium production was significantly increased (P<0.001) and glutamate was no longer consumed. Although glutamine synthetase inhibition with MSO had no effect on amino acid overall turnover (the sum of appearance and disappearance), it significantly decreased glutamine formation (P<0.01). Ammonium and oxygen independently altered overall amino acid turnover (P<0.05). Together, 5% oxygen and ammonium promoted glutamine production, whereas in the presence of 20% oxygen and ammonium, glutamine was consumed (P<0.01).
This study has examined ammonium production and potential mechanisms of disposal by mouse blastocysts. The data indicates that the mouse blastocyst uses glutamate to trap ammonium as glutamine and this pathway is compromised in the presence of exogenous ammonium and atmospheric oxygen.