The compartmentation of non-esterified and esterified cholesterol in the superovulated rat ovary.
1. The specific radioactivities of non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined in slices of superovulated rat ovary after incubation with [1-(14)C]acetate in vitro for various times. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal, and (during the fourth hour of incubation) exceeded those of the non-esterified cholesterol and the esterified cholesterol by factors of 2.8 and 7.6 respectively. 2. After separation of homogenates of superovulated rat ovary slices previously incubated with [(14)C]acetate into subcellular fractions by differential centrifugation, the specific radioactivities of non-esterified cholesterol in the cytosol, mitochondria, lipid-containing storage granules and microsomal fraction were 1220, 1510, 1420 and 4020d.p.m./mumol respectively; the corresponding values for the specific radioactivity of the esterified cholesterol were 600, 700, 730 and 760d.p.m./mumol. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal in all fractions; the corresponding mean specific radioactivity of progesterone+20alpha-hydroxypregn-4-en-3-one was 6150d.p.m./mumol. 3. By using glutamate dehydrogenase and cytochrome (a+a(3)) as mitochondrial markers, the presence of cholesterol side-chain cleavage enzyme was demonstrated in microsomal fraction free of mitochondrial contamination. 4. The specific radioactivities of ovarian non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined up to 8h after the intravenous injection of [4-(14)C]cholesterol into superovulated rats. At all times the specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal to the specific radioactivity of non-esterified cholesterol and exceeded, by up to 3.3-fold, that of the esterified cholesterol. 5. It is concluded that non-esterified cholesterol formed from [(14)C]acetate in the endoplasmic reticulum equilibrates slowly with non-esterified cholesterol in other subcellular fractions, and is preferentially converted into steroids. Such a mechanism presupposes the operation of a microsomal cholesterol side-chain cleavage enzyme using non-esterified cholesterol as its substrate. Unrelated evidence is presented in support of the existence of such an enzyme. The results are discussed in the light of other biochemical and electron-microscopic findings relating to the compartmentation of cholesterol in steroidogenic tissues.
D000085:Acetates / Q000378:metabolism; D000818:Animals; D001940:Breast / Q000473:pathology; D002458:Cell Fractionation; D002498:Centrifugation; D002784:Cholesterol / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003594:Cytoplasmic Granules / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004952:Esters / Q000378:metabolism; D005260:Female; D066298:In Vitro Techniques; D008861:Microsomes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D010053:Ovary / Q000378:metabolism*; D011278:Pregnanes / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D051381:Rats; D013261:Sterols / Q000378:metabolism
D000085:Acetates; D004952:Esters; D011278:Pregnanes; D013261:Sterols; D011374:Progesterone; D002784:Cholesterol