Effect of estradiol injection on the cytoplasmic fractions of the myometrium in the ovariectomized rat.

Abstract

Unless otherwise stated all materials and methods were as described previously (14, 15). Experiments consisted in studying with respect to time the effects of a single dose of estradiol on the myofilament and nucleus fraction (N), the mitochondrial fraction (M), the microsomal fraction (P), and the soluble fraction (S) of the ovariectomized rat myometrium. The separation between these fractions, which was effected by differential centrifugation, is not clear cut, there being unavoidable overlapping. Thus fraction N contains some unbroken cells, and although fraction M is essentially devoid of myofilaments it is contaminated with collagen fibres and microsomal material. Fraction P is also contaminated with mitochondrial material as evidenced by its relatively high succinic dehydrogenase activity in the ovariectomized state. Estradiol monobenzoate (supplied in 1 mg. ampoules by the British Drug Houses, Ltd.) was diluted with arachis oil immediately before use. Each rat was injected subcutaneously with 10 #g. of estradiol contained in 0.5 ml. of oil. Depending on the material available, batches of 6 to 12 uteri were pooled for each experiment. All uteri were slit open with fine scissors, and the endometrium was removed by scraping with a scalpel. Succinic dehydrogenase was assayed colourimetritally with KaFe(CN)6 as electron acceptor and was determined only in fractions M and P, the activity in fi-actions N and S being negligible. ATPasO activity was determined by measuring the amount of inorganic phosphate liberated after 6 minutes of incubation with 5 mM ATP and 5 mM MgSO4. In the extraction of phospholipids the waterethanol mixture was omitted for fear of dissolving low molecular weight RNA. Lipids were therefore separated with two 95 per cent ethanol extractions in the