Single Embryo Measurement of Basal-and Insulin-Stimulated Glucose Uptake

  • Maggie M-Y. Chi
  • Kelle H. Moley
Part of the Methods in Molecular Biology™ book series (MIMM, volume 83)

Abstract

Working with single-mouse embryos at the blastocyst stage is difficult from a biochemical standpoint because of the paucity of tissue. Each blastocyst contains only 25 ng of total protein (1) and has a volume of approx 160 pL (2,3). In order to measure glucose uptake, many groups have pooled embryos to increase the signal of radioactive 2-deoxyglucose transported into the blastocyst. The advantage of this technique is that single-embryo uptake can be measured using not radioactivity but enzymatic cycling reactions that are based on the amplification of a fluorescent signal. This signal is a pyridine nucleotide.

Keywords

MgCl2 NADPH NADH Haas NADP 

References

  1. 1.
    Turner, K., Rogers, A. W., and Lenton, E. A. (1994) Effect of culture in vitro and organ culture on the dry mass of preimplantation mouse embryos. Reprod. Fertil. Dev. 6, 229–234.PubMedCrossRefGoogle Scholar
  2. 2.
    Lewis, H. and Wright, E. D. (1925) Carnegie Contrib. Embryol. 25, 113–143.Google Scholar
  3. 3.
    Barbehenn, E. K., Wales, R. G., and Lowry, O. H. (1978) Measurement of metabolites in single preimplantation embryos; a new means to study metabolic control in early embryos. J. Embryol. Exp. Morphol. 43, 29–46.PubMedGoogle Scholar
  4. 4.
    Negelein, E. H. and Hass, E. (1935) Biochem. Z. 282, 206–220.Google Scholar
  5. 5.
    Greengard P. (1956) Nature 178, 632–643.PubMedCrossRefGoogle Scholar
  6. 6.
    Passonneau, J. V and Lowry, O. H. (1993) Enzymatic Analysis, Humana, Totowa, NJ.Google Scholar
  7. 7.
    Chi, M. M.-Y., Manchester, J. K., Carter, J. G.; Pusateri, M. E., McDougal, D. B., and Lowry, O. H. (1993) A refinement of the Akabayashi-Saito-Kato modification of the enzymatic methods for 2-deoxyglucose and 2-deoxyglucose 6-phosphate. Anal. Biochem. 209, 335–338.PubMedCrossRefGoogle Scholar
  8. 8.
    Chi, M. M., Manchester, J. K., Basuray, R, Mahendra, S., Strickler, R C, McDougal, D. B., Jr., et al. (1993) An unusual active hexose transport system in human and mouse preimplantation embryos. Proc. Natl. Acad. Sci. USA 90, 10,023–10,025.PubMedCrossRefGoogle Scholar
  9. 9.
    Chi, M. M., Schlein, A. L., and Moley, K. H. (2000) High insulin-like growth factor 1 (IGF-1) and insulin concentrations trigger apoptosis in the mouse blastocyst via down-regulation of the IGF-1 receptor. Endocrinology 141, 4784–4792.PubMedCrossRefGoogle Scholar
  10. 10.
    Chi, M. M., Pingsterhaus, J., Carayannopoulos, M., and Moley, K. H. (2000) Decreased glucose transporter expression triggers BAX-dependent apoptosis in the murine blastocyst. J. Biol. Chem. 275, 40,252–40,257.PubMedCrossRefGoogle Scholar
  11. 11.
    Moley, K. H., Chi, M., and Mueckler, M. (1998) Maternal hyperglycemia alters glucose transport and utilization in mouse preimplantation embryos. Am. J. Physiol. 275, E38–E47.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2003

Authors and Affiliations

  • Maggie M-Y. Chi
    • 1
  • Kelle H. Moley
    • 1
  1. 1.Department of Obstetrics and Gynecology, Department of Cell Biology and PhysiologyWashington University School of MedicineSt Louis

Personalised recommendations