Notes

1. This strain responds well to superovulation with 30-40 embryos per animal under the most optimal conditions. Mice should be ordered at 3 wk of age but allowed to adjust to their surroundings for 4-5 d prior to superovulation.

2. The heat plus alkali completely inactivates the G6PDH before the last step to avoid an upward drift in fluorescence.

3. All cycling reagent components except the imidazole HCl and the enzymes can be stored at 20-fold concentrated stock solution in 50 mM imidazole HCl (pH 7.0) for many weeks at -80°C. The most unstable component is a-ketoglutarate. The 100 mM imidazole HCl (pH 7.0) and the enzymes are added just before use, and the reagent is then kept on ice to minimize the reagent blank from any trace of NADP+ that may be present.

4. Calculations are based on internal standards and are therefore independent of variation in enzyme activities, temperature, or incubation times. Exact proportionality between readings and NADPH concentrations are kept far below the Michaelis constants for the enzymes.

5. Measurements are expressed as millimoles per kilogram wet weight, with the value of 160 ng or 160 pL per embryo used in the calculation. These values are the means reported by Lewis and Wright (2) and Barbehenn et al. (3) for mouse preimplanta-tion embryos. . To express the data as picomole per embryo, the values can be multiplied by 0.16.

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