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Nonmitochondrial oxygen utilization by rabbit blastocysts and surface production of superoxide radicals

A minimum value for nonmitochondrial oxygen utilization in rabbit blastocysts at day 6 post coitum was determined by measuring oxygen consumption in the presence of cyanide. A microcathode oxygen electrode was used to monitor oxygen concentration continuously during blastocyst incubation in a newly devised culture medium, and the uninhibited blastocyst was found to consume 2.79 ± 0.09 µl O₂ h^–1 cm^–2. This rate was reduced by 51% in the presence of 1 mmol KCN l^–1. The addition of nitroblue tetrazolium to the cyanide-containing medium reduced net oxygen consumption by an additional 23% as the nitroblue tetrazolium was reduced to formazan. The ability of rabbit blastocysts to reduce nitroblue tetrazolium in the presence of cyanide was investigated using a spectrophotometric assay. Fractionation of blastocyst cells revealed that the enzymatic activity chiefly responsible for formazan production partitioned with the membrane/particulate fraction and could be solubilized by the detergent NP40. The enzyme was NAD(P)H-dependent, did not require divalent cations for activity, and appeared to contain no haeme moiety. The rate of formazan production in the spectrophotometric assay was markedly reduced by the presence of superoxide dismutase. The oxygen electrode and spectrophotometer data indicate that there is a superoxide-generating NAD(P)H oxidase on the blastocyst surface. Calculations based on the average surface area of rabbit blastocysts at day 6 show that these embryos can produce at least 8 nmoles of superoxide per embryo h^–1. Potential deciduogenic effects of blastocyst-derived superoxide and its dismutated product, hydrogen peroxide, are discussed.