Effect of insulin on glucose uptake by murine embryos in low glucose medium

THERIOGENOLOGY EFFECT OF IMSULIN ON GLUCOSE UPTAKE BY MURINE EHBRYOS IN LOU GLUCOSE NEDIUN J.E. Butler and J.E. Williams Animal Science Department University of Idaho Moscow, ID 83843 USA Previous studies have indicated that insulin does not increase glucose uptake by preimplantation embryos. However, a criticism of these studies has been that the relatively high glucose concentrations of most culture media may mask a physiological effect of insulin. The objective of this study therefore was to determine the effect of insulin on glucose uptake by embryos in low glucose medium. Blastocysts were obtained from nonsuperovulated, random bred females. Culture medium consisted of M-16 medium modified to contain 0.1 mM glucose and 4 mg/ml bovine serum albumin (BSA) with (trt) or without (control) 1.0 ug/ml bovine insulin. For each of 10 replicates, six embryos were obtained from each of two females. If more than six blastocysts were obtained from a female, the embryos used were randomly selected. To eliminate possible donor female effects, an equal number of embryos from each female were assigned to treatment and control groups. Individual embryos were placed in 20 nl droplets of modified M-16 on siliconized glass slides under saline-equilibrated paraffin oil. Two droplets without embryos were included in each replicate to serve as assay blanks. Embryos were incubated for 3 h at 37°C in a humidified atmosphere of 5% CO,. A 5.8 nl sample was then removed from each droplet and added to a 60 nl reaction droplet containing 3.7 mM MgSO '7H,O, 0.6 mM nicotinamide adenine dinucleotide phosphate (NADP), 0.5 mM a%enosine 5'-triphosphate (ATP) 0.5 mM dithiothreitol (DTT), 12 U/ml hexokinase and 6 U/ml glucose-6-phbsphate dehydrogenase, in 50 mM EPPS buffer (N-(2-hydroxyethyl)-piperazine-Nf-3-propanesulfonicacid), pH 8.0. The reaction was allowed to proceed to completion (15 min) at 37'C. Glucose concentration was determined by the stoichiometric production of NADPH during the conversion of glucose to 6-phosphogluconate. NADPH was determined fluorometrically using a Nikon Diaphot microscope equipped for quantitative fluorescence. A standard curve was included in each replicate. Mean values (+SEM) for glucose uptake were: TREATMENT

NUMBER OF EMBRYOS

GLUCOSE UPTAKE (oMOLES/EMBRY0/3H)

CONTROL

60

1.55 + 0.05

INSULIN

60

1.62 + 0.05

Although insulin-treated embryos exhibited slightly higher uptake values, these differences were not significant (P=O.37). The lack of treatment effect was not due to inadequate exposure time to insulin since similar results were obtained when embryos were maintained in insulincontaining medium for 12 h prior to measuring uptake. Likewise, insulin contamination of the BSA was not a factor since replacement of the BSA with polyvinylpyrrolidone (PVP) had no effect. It is concluded from these experiments that insulin does not significantly increase glucose uptake by preimplantation mouse blastocysts in vitro. This work was supported by USPHS grant HD24556

JANUARY1990

VOL.33NO.l

203