Introduction

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 blasto-cyst. 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.

This technique was first described in 1935 by Negelein and Haas for determining glucose-6-phosphate dehydrogenase activity based on the increase in absorption in the near-ultraviolet (UV) as NADPH was produced (4). Greengard was the first to describe fluorometric pyridine nucleotide methods for measuring metabolites (5). Since that time, an extensive list of enzymes and metabolites has been measured with the aid of NAD and NADP (6). With the use of auxiliary enzymes, almost every substance of biological significance can be measured via a pyridine nucleotide system. Pyridine nucleotides have unusual properties that make them useful for analytic purposes (6). First, they act as natural oxidizing and reducing agents in many specific enzyme systems. Using the correct enzyme as a catalyst, selective oxidation or reduction of a substrate can be achieved in the presence of several other compounds. It is a rare substrate that cannot be linked by auxillary reactions to one using NAD or NADP in this way. Second, the reduced form, NADH or NADPH, not only absorbs near-UV light but also is flu orescent, whereas the oxidized form is not. Moreover, this fluorescence can be measured accurately at concentrations as low as 10 M and this redox state or pyridine nucleotide can be measured with great sensitivity. Third, the reduced forms can be destroyed in acid, without affecting the oxidized forms, and the oxidized forms, can be destroyed entirely by alkali without affecting the reduced form. This means that at the end of a reaction the excess pyridine nucleotide of the reagent mix can be destroyed and only the generated product can be measured. Finally, two methods exist for measuring the pyridine nucleotides. Both the oxidized and reduced forms can be converted to highly fluorescent forms in strong alkali, allowing accurate measurements down to 10~8 M. In contrast, much greater sensitivity by orders of magnitude can be attained by enzymatic cycling in which the pyridine nucleotide acts as the catalytic intermediate for a two-enzyme system. This is the technique used to measure the femtamolar and picomolar of quantities of metabolites in the individual blastocyst.

For measuring glucose uptake, 2-deoxyglucose (2-DG) is used as a tracer and the concentration of 2-DG transported into the blastocyst is measured. As described in detail in this chapter, 2-DG is linked to the production of NADPH by two reactions, and two further reactions are used to eliminate endogenous glucose and glucose-6-phosphate from the reaction measuring DG. These reactions generate NADPH, which is then cycled enzymatically. Finally, a byproduct of the cycling reaction is measured fluorometrically. These techniques have been used to measure glucose uptake in a wide variety of tissues, including preimplantation embryos (7-11).

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