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Glutamine Transport by Mouse Inner Cell Masses M

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Glutamine Transport by Mouse Inner Cell Masses M Glutamine transport by mouse inner cell masses M. B. Jamshidi and P. L. Kaye Department of Physiology and Pharmacology, The University of Queensland, Brisbane, QLD 4072, Australia Mouse blastocysts take up glutamine by specific transport systems. Glutamine is an important precursor for macromolecular synthesis and a potential alternative fuel to glucose. This study compared glutamine uptake in blastocysts and isolated inner cell masses and characterized the major participating systems in the latter. Inner cell masses take up glutamine by facilitated transport systems. The identity of these was investigated using substrate competition and kinetic studies. Na +-dependent uptake of 13 \g=m\molglutamine l \m=-\1 was inhibited by 60% by 1 mmol tryptophan l\m=-\1, 25% by 1 mmol 2-amino-2\x=req-\ norbornanecarboxylic acid l\m=-\1and 50% by 1 mmol lysine l\m=-\1. Furthermore, 1 mmol 2-methyl(amino)isobutyric acid (MeAIB) l \m=-\1 inhibited uptake by 29%. Kinetic analysis of MeAIB-resistant uptake revealed a predominant Na+-dependent facilitated uptake system with Km and Vmax values of 434 \m=+-\72 \g=m\moll\m=-\1 and 237 \m=+-\38 fmol per inner cell mass per 10 min, respectively. The inhibition of Na +-dependent uptake by tryptophan, lysine and the analogue 2-amino-2-norbornanecarboxylic acid suggests that most uptake of glutamine by inner cell masses occurs via the same system that predominates in whole blastocysts, Bo, +. The period of assay was so brief that significant participation of the inner cell mass in whole blastocyst uptake was precluded showing that system Bo,+ is expressed by both the trophectoderm and inner cell mass components of the blastocyst. However, MeAIB inhibited uptake by inner cell masses but not by blastocysts. This MeAIB-sensitive 1 uptake had a Km value of 4.3 \m=+-\1.7 mmol l \m=-\ and a Vmax value of 451 \m=+-\119 fmol per inner cell mass per 10 min. These characteristics suggest the first embryonic appearance of system A, which is a common Na+ -dependent transporter in many somatic cells. Significant inhibition of Na+-independent glutamine uptake by tryptophan suggests participation of the ubiquitous system L, while inhibition by lysine suggests that bo,+ may also be expressed in inner cell masses. Thus glutamine uptake by inner cell masses occurs via several transport systems expressed in whole blastocysts, including Na+-dependent system Bo, +, and probably Na+-independent systems L and bo,+, but is supplemented by the first expression of Na+-dependent system A in these cells from which the fetus later develops. Introduction Mouse embryos express facilitated transport systems capable of carrying this amino acid across the plasma mem¬ Glutamine is a precursor for protein and pyrimidine biosyn¬ brane from the two-cell stage, and uptake increases signifi¬ thesis in addition to being implicated in nitrogen metabolism. cantly as differentiation of the blastocyst occurs (Gardner et al, In many cells, conversion of glutamine to a-ketoglutarate can 1989; Chatot et al, 1990; Lewis and Kaye, 1992). However, it provide a significant source of energy and lead to conversion is not known whether, in the blastocyst, this glutamine can be of the carbon backbone into other amino acids. Glutamine is provided to cells of the inner cell mass, since there is no necessary for the culture of many cells in vitro and is required knowledge of glutamine uptake activity in this tissue. at concentrations 5-10 times higher than other amino acids The aim of this study was to classify glutamine transport + (Eagle, 1955). systems in inner cell mass cells, using studies of Na require¬ During preimplantation development, glutamine may pro¬ ment, substrate inhibition profiles and kinetics of uptake by vide an alternative to glucose as an energy source for bovine immunosurgically isolated inner cell masses. blastocysts (Rieger and Guay, 1988) and it has been suggested that it is an source in mouse energy preimplantation embryos Materials and Methods at all stages (Chatot et al, 1990). In mice, glutamine is abundant in oviduct fluid (Gardner and Leese, 1990). Embryos * Correspondence and reprint requests. Quackenbush mice were randomly bred in our own animal Revised manuscript received 7 February 1995. house with lights on between 06:00 and 18:00 h. At 10 weeks Downloaded from Bioscientifica.com at 09/27/2021 02:09:25AM via free access or cell masses were collected, washed of age, mice were superovulated by an injection with 10 iu The blastocysts inner of 2 ml ice cold pregnant mares' serum gonadotrophin i.p. (Folligon: Intervet, thoroughly by transfer through four changes Lane Cove, NSW) at 10:00 h; followed 48 h later by 10 iu hCG M2 and placed in separate vials containing 0.2 ml H20 to (Chorulon: Intervet). At this time, the injected females were which was added 1 ml of Optiphase 'Hisafe 3' scintillation FSA placed with single males of the same strain and the presence of cocktail (Optiphase 'Hisafe' 3: LKB; Laboratory Supplies, was determined in a a vaginal plug 23 h later indicated mating. Blastocysts (96 h Loughborough) before the radioactivity 40% after hCG treatment) were collected from the mated mice and Packard 1900 CA liquid scintillation counter at efficiency, washed in M2 medium (Fulton and Whittingham, 1978; with an absolute background of 4—7 c.p.m. modified as described by Hobbs and Kaye, 1985). Kinetic were on cell masses isolated from studies performed inner [ blastocysts that had developed in vivo and blastocysts that had Efflux of HJglutamine developed from two-cell embryos (48 h after hCG treatment) Inner cell masses were preincubated for 10 min at 37°C 20 or 1 1 during 48-52 h culture in vitro in µ droplets of BMOC2 in M2 containing 100 µ mmol [3H]glutamine ~~ modified as described Pemble and Ci 1 collected and washed in M2, before medium (Brinster, 1965), by (0.5 ~ ), rapidly Kaye (1986), under paraffin oil in an atmosphere of H20- transfer to M2 lacking glutamine at 37°C. At various times saturated 5% C02, 5% 02 and 90% N2 at 37°C. Previous inner cell masses were collected, washed in ice-cold M2 and experiments confirmed that under these conditions at least 50% radioactivity was determined as above. of two-cell embryos (48 h after hCG treatment) developed to blastocysts by 96 h after hCG treatment. Competition by other amino acids Blastocysts or inner cell masses were incubated for 10 min masses 1 Isolation of inner cell one of in M2 containing 9-13 µ [3H]glutamine 1 ~ and 1 1 : 2-amino- The zonae pellucidae were removed by brief exposure to the following at mmol ~ bicyclic analogue, Tyrode's solution (pH 2.5; modified as described by Hogan 2-norbornanecarboxylic acid (BCH); lysine (Lys); trypto¬ et al, 1986) before immediate transfer through three 2 ml phan (Trp); or 2-methyl(amino)isobutyric (MeAIB), and above. washes of M2. The zonae-free blastocysts were then placed in [3H]glutamine uptake was determined as rabbit anti-mouse spleen cell antiserum diluted 1:10 (v:v) in M2 The was in a New Zealand for 13 min. antiserum produced Standardization White rabbit, bled 10 days after three injections i.V., 6 days apart, of Quackenbush mouse spleen cells in 0.5 ml 0.9% (w/v) The specific radioactivity of each drop was determined from sterile saline (Solter and Knowles, 1975; Harvey and Kaye, samples of the medium collected at the end of the uptake 1990). The blastocysts were then washed with fresh M2 and period and the known non-radiolabelled glutamine concen¬ placed in guinea-pig serum diluted 1:10 in M2 for 13 min (ICN tration, allowing for [3H]glutamine contribution. A sample of Biomedicals, Costa Mesa), as a source of complement. Inner cell the final wash was used to determine the experimental back¬ same as the masses were freed of lysed trophectoderm cells by passage ground radioactivity, which was found to be the through a fine hand drawn pipette. About 75% of blastocysts absolute background. yielded suitable inner cell masses. Statistical analyses medium Na+-free M2 In cases where the data were not normally distributed, they The statistical M2 medium, free of Na+ (M2—Na+ ), was prepared by were normalized by logarithmic transformation. equimolar replacement of NaCl with choline chloride and significance of differences between means was assessed by NaHC03 with KHC03. The hepes used to buffer this medium multiple-range test (Fisher's protected LSD) after analysis of variance of normalized data. Kinetic for saturable was adjusted to pH 7.4 with KOH, and sodium Iactate was parameters omitted. Previous experiments had shown that omission of glutamine uptake were determined by fitting data to the Iactate did not affect blastocyst development over 4 h in vitro, equation or in and amino acid transport rates blastocysts (Hobbs Kaye, = + + K0[glyn] ({ Vmax[gln]} {Km [gin]} " ') 1986). The medium was added to the freeze-dried radiolabelled using non-linear regression, where is the initial rate or uptake substrate, mixed and then transferred to the culture dish and in fmol per inner cell mass per 10 min, K0 is the rate constant preincubated under paraffin oil at 37°C, for at least 1 h. for the non-saturable transport, and Km (µ 1_I) and Vmax (fmol per inner cell mass per 10 min) values are the kinetic parameters describing the saturable transport component Glutamine uptake (Hobbs and Kaye, 1990). Glutamine uptake was determined by transferring blasto¬ cysts or inner cell masses to 20 µ drops of M2 containing Results 9-13 µ [3H]glutamine 1_1 (33-55 Ci mmol-1, Amersham Bucks) under in a Petri dish that Initial confirmed that over 95% of 13 µ International, liquid paraffin experiments' inner cell masses 10 min had been equilibrated at 37°C.
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