Abstract
Substrate turnover rates by cytochrome P-450scc were measured in mitochondria isolated from corpora lutea and granulosa cells of follicles. Hydroxycholesterol substrates were added to the mitochondria to test the degree of saturation of the cytochrome with endogenous cholesterol during pregnenolone synthesis. 25-Hydroxycholesterol proved unsuitable for this since it was converted into pregnenolone with a maximum velocity of only 25% of that for cholesterol. 20 alpha-Hydroxycholesterol was found to be suitable providing correction was made for the one less hydroxylation required to convert this substrate into pregnenolone, compared to cholesterol. Mitochondria isolated from large follicles and corpora lutea displayed biphasic time courses for pregnenolone synthesis from endogenous cholesterol with a rapid phase lasting for 2-4 min and a slow phase which was linear for at least 30 min. Only a single rapid phase was observed for these mitochondria in the presence of 20 alpha-hydroxycholesterol. From the degree of stimulation of the substrate turnover rate by this steroid, it was concluded that the endogenous cholesterol concentration was saturating during the fast phase for large follicles but subsaturating in luteal mitochondria. Time courses for pregnenolone synthesis by mitochondria isolated from granulosa cells of small and medium follicles were linear for 30 min and gave a substrate turnover rate of 16-18 mol of steroid/min/mol of cytochrome P-450scc, similar to the turnover rates under saturating substrate conditions determined for large follicles and corpora lutea. The substrate turnover rate for cytochrome P-450scc in medium follicles was not increased by the addition of 20 alpha-hydroxycholesterol, indicating that the cholesterol concentration in the steroidogenic pool of these mitochondria was saturating and remained so over the 30-min duration of the incubation. It is therefore unlikely that gonadotropin stimulation of granulosa cells of small to medium follicles could acutely regulate pregnenolone synthesis by increasing the rate of transfer of cholesterol into a steroidogenic pool. This study shows that as the cytochrome P-450scc concentration in porcine ovarian mitochondria increases during follicular growth and luteinization there is a decrease in the fractional saturation of the cytochrome with cholesterol.
Highlights
From the Raine Center for the Study of Perinatal andDevelopmental Biology, Department of Biochemistry, the University of Western Australia, Nedlands 6009, Perth, Australia
Follicular growth is accompanied by increases in both this steroid, it wcaosncluded that theendogenous cho- cholesterol side chain cleavage activity and the concentration lesterol concentration was saturating during the fast of adrenodoxin reductase, adrenodoxin, and cytochrome P
The method for measuring cytochromeoxidase activity hasalso been described before turnover rate was increased to3.8 mol of steroid/min/mol of Pregnenolone Synthesis from Endogenous Cholesterol and 25-HydroxycholesterolbyLutealMitochondria-Pregnenolone synthesis from endogenous cholesterolwas biphasic with an initial rapid phasewhich lasted for [2,3] min followed by a slow phase which was linear for 30 min (Fig. 1).The rapid phase gave a substrate turnover rate for cytochrome P-450, of 7.8 mol of steroid/min/mol of cytochrome P-450, while for the slow phase the turnoverwas only one-seventh of this
Summary
Pregnenolone Synthesis from Endogenous Cholesterol and 25-HydroxycholesterolbyLutealMitochondria-Pregnenolone synthesis from endogenous cholesterolwas biphasic with an initial rapid phasewhich lasted for [2,3] min followed by a slow phase which was linear for 30 min (Fig. 1).The rapid phase gave a substrate turnover rate for cytochrome P-450,,, of 7.8 mol of steroid/min/mol of cytochrome P-450,, while for the slow phase the turnoverwas only one-seventh of this. Pregnenolone Synthesis from Endogenous Cholesterol and 20a-Hydroxycholesterolby Mitochondria from Granulosa Cells of Large Follicles-The time course for pregnenolone synthesis by these mitochondria was biphasic with a rapid phaselasting [3,4] min giving a substrate turnover rate by the cytochrome of 16.5 mol of steroid/min/mol of cytochrome P-450,,,(Fig. 7). This is similar tothe turnover rate from endogenous cholesterol by mitochondria from medium follicles The initial rate of pregnenolone synthesis by mitochondria from large follicles was stimulated by 40% by 20a-hydroxycholesterol (Fig. 7)
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