Patricia M. Stevenson

Properties of bovine luteal cytochrome P-450scc incorporated into artificial phospholipid vesicles

Bovine luteal cytochrome P-450scc was purified and incorporated into artificial phosphatidylcholine vesicles. The vesicle reconstituted cytochrome used membrane bound cholesterol as substrate and cholesterol binding varied with the phosphatidylcholine fatty acyl composition and was stimulated by the presence of cardiolipin in the vesicle. 22R -hydroxycholesterol and 20 alpha, 22R - dihydroxycholesterol bound to the cytochrome up to 300 times more tightly than cholesterol and decreased the affinity of the cytochrome for CO by 100-200-fold. The properties of the cytochrome closely paralleled those reported for cytochrome P-450scc purified from the bovine adrenal gland.

Properties of ferredoxin reductase and ferredoxin from the bovine corpus luteum

Ferredoxin reductase and ferredoxin were purified from the bovine corpus luteum and their properties compared to the corresponding adrenal proteins. The luteal and adrenal proteins had similar absorbance spectra and molecular weights. Evidence was obtained from spectrophotometric titrations for formation of 1:1 complexes between luteal ferredoxin reductase and ferredoxin and between ferredoxin and cytochrome P-450scc. Adrenal ferredoxin reductase and ferredoxin were equally as effective as luteal ferredoxin reductase and ferredoxin in supporting cholesterol side-chain cleavage by luteal cytochrome P-450scc.

Ferredoxin and cytochrome P-450scc concentrations in granulosa cells of porcine ovaries during follicular cell growth and luteinization

The concentrations of cytochrome P-450scc and ferredoxin, two of the three proteins which comprise the mitochondrial steroidogenic electron transport chain, were measured in granulosa and luteal cells from porcine ovaries by an immunoblot procedure. During the follicular phase of the ovarian cycle the concentration of cytochrome P-450scc increased 5-fold and ferredoxin increased 3-fold. When the large follicles developed into corpora lutea the cytochrome P-450scc concentration increased a further 7-fold while ferredoxin increased only 3-fold. These changes were coincident with an overall 4-fold increase in the concentration of ferredoxin reductase during follicular cell development and luteinization. Analysis of the data revealed that the concentration of ferredoxin, which shuttles electrons from ferredoxin reductase to cytochrome P-450scc, was always adequate to saturate both the reductase and cytochrome P-450scc. This came about from a co-ordinate increase in the concentration of cytochrome P-450scc and the concentration of ferredoxin minus ferredoxin reductase.

The differential development of mitochondrial cytochrome P-450 and the respiratory cytochromes in rat ovary

The cytochrome concentrations in ovarian mitochondria were determined in immature and superovulated rats. The respiratory cytochromes (b, c + c1 and a + a3) were found to increase approximately 3-fold during the transition from immature follicle to active steroid-producing corpus luteum. At the same time, mitochondrial cytochrome P-450 exhibited a 10-fold increase in concentration, reflecting the ability of the gland to synthesize steroids. This different rate of synthesis of mitochondrial cytochrome P-450 and respiratory cytochromes may be regarded as a biochemical differentiation step necessary for the granulosa cells to develop steroidogenic competence.

Methanolysis of cholesteryl esters: Conditions for quantitative preparation of methyl esters

Abstract We have investigated the conditions required to obtain a quantitative yield of methyl esters from cholesteryl esters by alkaline methanolysis. Methanolysis of the cholesteryl ester for 60 min at room temperature with 1 m methoxide reagent ensured complete reaction. Some ester hydrolysis always accompanied methanolysis and necessitated acid-catalyzed methylation of the resultant fatty acids after completion of the alcoholysis. Analysis of the composition of methyl ester product and remaining cholesteryl ester substrate before methanolysis had gone to completion showed selective hydrolysis of some fatty acid cholesteryl esters and illustrates the importance of obtaining a quantitative yield of methyl esters following methanolysis.

Changes in catalase activity and concentration during ovarian development and differentiation

The ovaries of immature rats were used to prepare a peroxisome-enriched fraction by differential centrifugation. Following gonadotropin stimulation, which caused large numbers of follicles to develop into corpora lutea, the specific activity of catalase in the peroxisome-enriched fraction increased 5-fold, while catalase recovered in the post-30,000 x g supernatant did not increase in activity. The increase in catalase specific activity in the peroxisome enriched fraction was shown to be due to an increased concentration of the enzyme as determined by Western blotting. Catalase in pig granulosa cells also increased in specific activity as the follicles aged and luteinized. This increase appeared to parallel increases in the concentration of cytochrome P-450scc. We conclude there is a differential regulation of the peroxisomal and cytosolic pools of rat ovarian catalase.

The composition and distribution of lipid granules in the rat ovary

Lipid granules in ovaries of immature rats were confined to the interstitial tissue, and comprised 70% cholesteryl esters and 20% triacylglycerols, the balance being phospholipid and free cholesterol. Following treatment with gonadotropin the interstitial granules disappeared as cholesteryl esters were hydrolysed, but reformed in the follicle as it developed, first in the theca, then in the outer granulosa and finally in the inner cells. The cholesteryl ester: triacylglycerol ratio fell during follicular growth, but in the corpus luteum the ratio in the widely distributed granules was 1:1. Esterified fatty acids in both cholesteryl esters and triacylglycerols became longer and more unsaturated as development progressed. The same progression of granules across the follicles was evident in ovaries of normal adult rats. We concluded that lipid granules in interstitial tissue supplied the substrates for synthesis of new cells in adjacent developing follicles, and those in corpora lutea were a prerequisite for steroidogenic competence.

Free and esterified cholesterol concentration and cholesteryl ester composition in the ovaries of maturing and superovulated immature rats

The cholesterol and cholesteryl ester concentration and cholesteryl ester composition were determined in the ovaries of immature rats, sexually mature rats and superovulated immature rats. The immature rat ovary accumulated cholesteryl esters, and long-chain polyunsaturated fatty acids were preferentially incorporated into these esters. The cholesteryl esters decreased in concentration and changed in composition with the onset of the first estrous cycle. Superovulation of immature rats, by injection of 50 I.U. pregnant mare serum gonadotropin, caused a decrease in the cholesteryl ester concentration of the ovary within 24 h and specific depletion of some esters, particularly those of 20 : 1 and 22 : 6 acids. Human choriogonadotropin, administered 54 h later, induced synchronous luteinization of the ovaries and was followed by increases in the concentration of free and esterified cholesterol and preferential accumulation of the esters of 20 : 4, 22 : 4, 4, 22 : 5 and 22 : 6 acids. Acute stimulation of luteinized ovaries by a second injection of the rats with 25 I.U. of human choriogonadotropin resulted in preferential hydrolysis of the esters of 18 : 1, 20 : 4, 22 : 4 and 22 : 5 acids.

Creatine kinase, steroidogenesis and the developing ovarian follicle.

Creatine kinase activity was present throughout follicular development and luteinization in rat ovary. The activity of creatine kinase in ovary was about 1/4 that of brain and 1/15th that of heart. The ovary contained the same creatine kinase isoenzyme as brain, and the type did not change during follicular development. Creatine kinase was found to be concentrated in the steroidogenic thecal and luteal cells of the ovary. The activity of creatine kinase was stimulated 23% by choriogonadotropin in luteal tissue.

Changes in coenzyme A and carnitine concentrations in superovulated rats

Changes in the concentrations of total coenzyme A, acetyl CoA, free carnitine and acetylcarnitine were measured in ovaries from immature rats before and after superovulation with 50 I.U. pregnant mares serum gonadotropin. In addition, the concentrations of total CoA and total acid-soluble carnitine were measured in liver, adrenal glands and skeletal muscle from the same rats. Ovarian concentrations of total CoA, free carnitine and acetylcarnitine increased 3-fold on gonadotropin stimulation, whereas there was no marked change in total CoA and acid-soluble carnitine concentrations in the other organs. In ovary, the ratio of free CoA to acetyl CoA was about 2:1 during the growth period of follicular development and during active steroidogenesis in the luteal phase, but less than 1 when replication stopped and ovulation occurred. These results show that during periods of high energy demand the ovary has a good capacity to accommodate fatty acid oxidation, and supports the evidence that fatty acids are the major source of reducing equivalents for steroidogenesis at these times.