Great Britain Biogenesis of Oestrogens from [ 14 C ] Testosterone in the Mitochondrial Fraction of the Human Placenta

oxygen are essential for the reactions (Ryan, 1959). Among the numerous subsequent studies on the mechanism of oestrogen biosynthesis in vitro in the human placenta (Longchampt, Gual, Ehrenstein & Dorfman, 1960; Morato, Hayano, Dorfman & Axelrod, 1961; Gual, Morato, Hayano, Gut & Dorfman, 1962; Morato, Raab, Brodie, Hayano & Dorfman, 1962; Wilcox & Engel, 1965; Akhtar & Skinner, 1968; Townsley & Brodie, 1968) none has described oestrogen formation in the mitochondrial fraction. This communication reports the conversion of [4-l40]testosterone into radioactive oestrone and oestradiol-17fl in high yield by carefully prepared mitochondrial fractions from human placenta. Full-term placentae were dissected free from foetal membranes and cut into small pieces, which were washed repeatedly with 0.9% NaCl. This tissue, after disruption (Waring Blendor) in 0-25Msucrose in 0-01 M-tris-HCl buffer, pH 7-7 (3ml./g. of tissue), was homogenized either in a PotterElvehjem-type homogenizer or in a Sorvall OmniMixer. After filtration through gauze the homogenate was centrifuged at 700g for 10min. and the supernatant was then centrifuged at 6000g for 10min. to yield a crude mitochondrial fraction. The 6000g supernatant was centrifuged at 10OOOg for 45min., the pellet discarded and the microsomal fraction sedimented by centrifugation at 6000Og for 75min. The mitochondrial fraction was washed twice in 10vol. of 0-20M-mannitol and 0-07m-sucrose in 0-01M-tris-HCl buffer, pH7-7, and sedimented by centrifugation at 100OOg for 10min.; the 'fluffy layer' of light mitochondria was discarded each time. This washing procedure was repeated three times with centrifugation at 100OOg for 15min. to yield the mitochondrial fraction that was used in the studies on oestrogen formation. Allmann, Bachmann, Orme-Johnson, Tan & Green (1968) used similar techniques to obtain mitochondrial fractions from rat and bovine liver tissue. The microsomal fraction was washed twice in 20vol. of 1-15% (w/v) KCI and sedimented by centrifugation at 60000g for 50min. each time. All the operations described above were done at -4O The washed mitochondrial fractionwas suspended in 0-05M-potassium phosphate buffer, pH7-4, at a concentration equivalent to 9mg. of mitochondrial protein/ml. Similarly the microsomal fraction was suspended at a concentration equivalent to 37mg. of microsomal protein/ml. Measurement of mitochondrial and microsomal protein gave values of 1-8mg. and 3-7mg. respectively/g. wet wt. of placental tissue. Protein was estimated by the method of Lowry, Rosebrough, Farr & Randall (1951) as modified by Eggstein & Kreutz (1955), with bovine serum albumin as standard. Incubations were carried out in a Dubnoff metabolic shaking incubator at 370 in an atmosphere of air. Each reaction flask contained [4-14C]testosterone (200,ug., 0-81,uc) dissolved in ethanol (0-5ml.), NADP+ (20,tmoles), glucose 6-phosphate dehydrogenase (10units) and glucose 6-phosphate disodium salt (100,umoles), each dissolved in 0-05M-potassium phosphate buffer, pH7-4, to giveafinalvolume of 7ml. NADPHwasgenerated by incubation for 30min. Mitochondria (68mg. of protein) or microsomes (73mg. of protein) were then added with buffer to give a final volume of 15-Oml. The mixture, containing 725 umoles of potassium phosphate, was incubated for a further 60min. The mitochondrial incubations were carried out in duplicate. At the end of the incubation period the reaction