NONMITOCHONDRIAL OXYGEN UTILIZATION BY RABBIT BLASTOCYSTS AND SURFACEPRODUCTION OF SUPEROXIDE RADICALS

A minimum value for nonmitochondrial oxygen utilization in rabbit blas tocysts at day 6 past coitum was determined by measuring oxygen consum ption in the presence of cyanide. A microcathode oxygen electrode was used to monitor oxygen concentration continuously during blastocyst in cubation in a newly devised culture medium, and the uninhibited blasto cyst was found to consume 2.79 +/- 0.09 mu l O-2 h(-1) cm(-2). This ra te was reduced by 51% in the presence of 1 mmol KCN l(-1). The additio n of nitroblue tetrazolium to the cyanide-containing medium reduced ne t 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 usin g a spectrophotometric assay. Fractionation of blastocyst cells reveal ed that the enzymatic activity chiefly responsible for formazan produc tion partitioned with the membrane/particulate fraction and could be s olubilized by the detergent NP40. The enzyme was NAD(P)H-dependent, di d not require divalent cations for activity, and appeared to contain n o haeme moiety. The rate of formazan production in the spectrophotomet ric 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 a t day 6 show that these embryos can produce at least 8 nmoles of super oxide per embryo h(-1) Potential deciduogenic effects of blastocyst-de rived superoxide and its dismutated product, hydrogen peroxide, are di scussed.