Metabolism of free and esterified cholesterol by Leydig-cell tumour mitochondria

1. Experiments were designed to localize intracellularly the enzymes and sterol substrates required for steroidogenesis in Leydig-cell tumours. Subcellular fractions were prepared by differential centrifugation of tumour homogenates. Both free and esterified cholesterol were associated primarily with the fractions sedimenting at 1400g(av.) and the lipid layer floating on the surface of the isolation tubes; they were not found in the mitochondria, where the conversion of cholesterol into pregnenolone occurred. 2. Hydrolysis of esterified cholesterol was required before it could be oxidized to pregnenolone. 3. An enzyme capable of hydrolysing cholesterol esters was located external to the mitochondria. 4. Mitochondria were subfractionated by allowing them to swell in 0.02m-phosphate buffer (pH7.2) and separating the inner and outer membranes by sedimentation in sucrose gradients. The outer membrane, identified by its content of monoamine oxidase, contained most of the cholesterol associated with the mitochondria. The inner membrane, identified by its content of succinate dehydrogenase, contained the cholesterol side-chain-cleaving enzyme and very little cholesterol. 5. Accumulation of sterols by the mitochondria was studied by incubating this fraction with labelled free and esterified cholesterol suspended in lipid-free bovine serum albumin. Two phases of cholesterol accumulation were observed. The first phase, requiring 10–15min, was independent of the incubation temperature, and was inhibited by the presence of bovine serum albumin in the incubation medium. The second phase of accumulation was independent of the serum albumin content of the medium but was inhibited by low incubation temperature. 6. Esterified cholesterol was not accumulated by the mitochondria after the initial rapid binding phase. 7. The findings suggest that cholesterol was not rapidly accumulated by the mitochondrial fraction in vitro and that mechanisms may be required to facilitate cholesterol transport into mitochondria in intact tumour cells during the periods in which steroidogenesis is stimulated maximally

Influence of luteinizing hormone and adenosine 3′:5′-cyclic monophosphate on the metabolism of free and esterified cholesterol in mouse Leydig-cell tumours

1. Male C57B1/6J mice bearing Leydig-cell tumours known to synthesize steroids in response to luteinizing hormone (LH) were given intravenous injections of [1,2-(3)H]cholesterol (50–100μCi per animal). Single-cell suspensions were prepared from the tumours 5–9 days after the injection of [(3)H]cholesterol and were incubated at 37°C in foetal calf serum supplemented with 50mm-Tris–HCl, pH7.4. At various times after the start of incubation cells were collected by filtration of portions of the suspension and their sterols analysed. Within 10min after LH (5μg/ml) or 3′:5′-cyclic AMP (20mm) was added to the cell suspensions an increased conversion of ester cholesterol into free cholesterol could be demonstrated. 2. To observe this rapid effect of LH it was necessary to incubate the cells for 60min before addition of hormone. 3. The specific radioactivity of testosterone produced was approximately equal to that of the intracellular cholesterol regardless of the presence or absence of LH. 4. The amount of free cholesterol produced in response to LH was far greater than that needed for steroid synthesis. 5. Free cholesterol, but not esterified cholesterol, was released into the incubation medium linearly with time and this release was unaffected by LH. LH may stimulate steroidogenesis in part by increasing the concentration of free cholesterol within the cell

Ovulating-inducing activity of FSH in the rat

Ovine LH or FSH administered 44 hr after a priming dose of PMS induced ovulation inhypophysectomized immature rats. A well-characterized and highly specific LH antiserum (A/S) showing no cross reactivity with NIH FSH, TSH, GH or pr.olactin or with serum and tissue proteinextracts, was used to test the ability of NIH-FSH or LH-free FSH to cause ovulation. Theminimal effective dose (MEID) of LH A/S blocking ovulation induced by 10 ug LH was 3.0 ul per rat whereas that for blocking ovulation induced by FSH (100 ug) was 1.0 ul. While the MEID of the LH A/S for inhibition of ovarian weight gain was about 3 times greater than forinhibition of ovulation induced by LH, that for suppression of ovarian weight gain and inhibition of ovulation induced by FSH were the same. Incubation of FSH with variable amounts of LH A/Sfollowed by removal of the LH A/S with anti-rabbit gamma globulin resulted in preparations having no LH contamination as indicated by the double immunodiffusion test. The ovulation inducing capacity of this LH free FSH was negligible