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OOCYTE AND EMBRYO DEVELOPMENTGLUCOSE TRANSPORT IN MURINE BLASTOCYSTS IS STIMULATED BYGRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF)Sjoblom, C. l , Wikland, M. l and Robertson, S.A?lFertilitetscentrum, Carlandersplaten 1 40229, Goteborg, Sweden, and 2Department ofObstetrics &Gynaecology and Reproductive Medicine Unit, The University ofAdelaide, South Australia 5005.INTRODUCTION _ 6Glucose is a major energy substrate in compacted .s~5pre-implantation embryos, and growth factorsfI)ftIincluding insulin and IGF appear to promote m4anabolic activity, survival and proliferation in C) 3blastomeres through increasing the activity of ~facilitative glucose transporters (1). The cytokine ~ 2GM-CSF has been shown to stimulate the growth ~ '0 1and development ofboth mouse and human pre-Eimplantation embryos through binding to the 0.- Q. 0chain ofthe GM-CSF receptor (2,3). In other cell 0,08 0,4 2,0 10 Con (ng/ml)lineages, including Xenopus oocytes and melanoma Fig. I Effect ofdose ofGM-CSF on 3-H* OMG uptakecells, ligation ofthis receptor can stimulate glucose in murine blastocysts (mean±SD pmoles 3-H* OMG /uptake through a kinase-independent pathway (4). blastocyst) *p< 0.005The aim ofthis study was to investigate the effect of~l\GM-CSF on glucose transport in mouse pre- . ~.rJ' ~implantation embryos. .', C,~ '{"('" (30) (21) (8) (8) (10) (10) (17)MA TERIALS AND METHODS i..,.r/~. g 5Blastocysts (n=369 in total) recovered from 'isuperovulated 4 week old Balb/c x C57BV6 female j5 4mice at 97 h post hCG after mating with CBA x ~ 3C57BV6 males were allocated into HTF media with 0addition ofrecombinant murine (rm)GM-CSF (0.08, ~ 20.4, 2.0, 10 ng/ml) or various other cytokines ~including IGF, macrophage-CSF (M-CSF),interleukin (IL)-2, IL-IO and IL-3 (concentrationsECo 0shown in Fig. 2). After 1 hour incubationblastocysts were transferred for 4 min into mediawith non-metabolisable analogue 3-H* ortho-methylglucose (3-H* OMG, 25 mM), then washed inglucose-free media and assessed by betascintillation counting.RESULTS3-H* OMG uptake into blastocysts was significantlyincreased by incubation in rmGM-CSF (Fig 1). Theresponse to GM-CSF was dose-dependent with amaximum effect (50% increase) at 2 ng/ml. Theextent ofthis effect was comparable to that ofIGFused at a dose previously found to have optimaleffects (1). None ofthe other cytokines tested hadan effect on glucose uptake (Fig 2).REFERENCES1. Pantaleon M et at. (1996) Mot.Reprod.Dev. 44:71-762. Sjoblom C, et al. (1999) Hum. Reprod. 14:3069-3076.4. Robertson S et al., (1998) Proc. ASRB Abstract 62.3. Ding DX et at (1994) PNAS USA. 91:2537-41.GM·CSF2nglml(36)(46)(66)IGF-1 M-CSF IL-213 Ps:IIml 200IUlml 200IU/ml(47)IL·10200IU/ml(51)IL·3200IUlmlConFig 2 Effect ofcytoldnes on 3-H*OMG uptake in murineblastocysts (mean±SD pmoles 3-H* OMG / blastocyst)*p< 0.005CONCLUSIONThese data show that GM-CSF can promote glucoseuptake in murine blastocysts, presumably throughbinding to trophectoderm cells via the a-chain ofthe GM-CSF receptor. Improved glucoseavailability might thus provide a mechanismwhereby GM-CSF can improve blastomere survivalin human and murine pre-implantation embryos (2).Conversely, blastocyst culture in cytokine deficientmedia might be associated with blastomereapoptosis due to suboptimal glucose availability.Further experiments are required to delineate theeffect of GM-CSF on specific glucose transportermolecules in blastocysts.Materials and methodsThe NH4+ concentration of media (mOdified~synthetic oviduct fluid (SOF) supplemented witheither 20% HS or amino acids) was determinedover 5 days using a clinical bichromatic ratetechnique (Dimension, Dade Behring, USA))Assays were kindly conducted by staff at th~Dept. of Clinical Chemistry, QEH, Woodvill