Estrogen Increases Precursor for Pregnenolone Synthesis with Temperature-Sensitive Occupancy of P-450scc in Mitochondria of Rabbit Corpus Luteum*

Abstract

To examine the mechanism of estrogen's direct stimulation of steroidogenesis in the rabbit corpus luteum, we tested the hypothesis that the effect of estrogen on progestin production occurs at the site of processing of the precursor for pregnenolone (i.e. cholesterol) in the mitochondrion. For this purpose, we manipulated a model of estrogen stimulation by 1) removing sc estradiol-filled polydimethylsiloxane capsules from superovulated rabbits on day 9 of pseudopregnancy or 2) leaving the capsules in place to preserve a chronic estrogen stimulus. In the estrogen-deprived rabbits, the serum progesterone level fell precipitously in vivo within 24 h, but in rabbits with chronic estrogen stimulation, serum progesterone levels remained high. Our results show that the loss in progestin production caused by estrogen deprivation could not be attributed to loss of the mitochondrial cytochrome P-450 side-chain cleavage enzyme (P-450scc), a common rate-limiting step in progestin synthesis in many steroidogenic tissues. In addition, we confirmed that there was no loss in the catalytic activity of this enzyme. Treatment with aminoglutethimide in vivo followed by electron paramagnetic resonance spectroscopic analysis of mitochondria (prepared in aminoglutethimide-free buffers) showed that incubation of isolated mitochondria at 37 C and pH 6.2 caused an increased high spin state (g = 8.2 signal) and a concomitant decreased low spin state. This shift from low to high spin states, which is indicative of cholesterol-P-450scc complex formation, occurred in the luteal mitochondria from both estrogen-deprived and estrogen-stimulated rabbits. In further studies to localize estrogen's regulatory point, we determined that the initial (first minute) rate of production of pregnenolone (per mg protein or per U P-450scc) from endogenous precursor proceeded equally fast in mitochondria from estrogen-deprived and those from estrogen-stimulated rabbits. However, the rapid pregnenolone production in the estrogen-deprived group lasted for a shorter time and, after 30 min, yielded less pregnenolone per mg protein or per U P-450scc than did mitochondria from estrogen-stimulated rabbits. Addition of 25-hydroxycholesterol did not increase the initial rate of pregnenolone formation, indicating that precursor availability is not limiting during the initial period. In aggregate, these observations suggest that the effect of estrogen on progestin production in the rabbit corpus luteum is not regulation of the movement of cholesterol to the catalytic site on the inner mitochondrial membrane, even though this is a step in the regulation of protein hormone-stimulated steroidogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of the Steroidogenic Acute Regulatory Protein in the Corpus Luteum of the Rabbit: Dependence upon the Luteotropic Hormone, Estradiol-17β1 1

“…Thus, only by further synthesis and processing of additional precursor can cholesterol continue to be transferred to the inner mitochondrial membrane. In rabbits, estradiol increases the supply of cholesterol in the mitochondria of the corpus luteum [10]. Further, estradiol alters the physical structure of the mitochondria: luteal mitochondria of rabbits acutely deprived of estrogen have less surface area along the inner mitochondrial membrane [32].…”

“…Initially, it was postulated that key enzymes in the steroidogenic pathway might be estrogen-responsive. However, enzymes such as 3-hydroxy-3-methylglutaryl coenzyme A reductase [9], cytochrome P-450 side-chain cleavage complex (P450,cc) [10,11], and acyl-CoA:cholesterol acyltransferase and cholesterol ester hydrolase [12] do not appear to be tightly regulated by estradiol. Holt [1] proposed that estradiol might act to stimulate the delivery of cholesterol to its site of cleavage, the P450Scc complex, which is located on the matrix side of the inner mitochondrial membrane.…”

Control and Trafficking of Cholesterol and Other Lipids within the Ovary

“…A clear effect of estradiol, which acts as a gonadotropin by promoting progesterone synthesis in the rabbit luteal cell, is to increase the amounts of precursor cholesterol reserves available within the luteal mitochondria. 22 Lambeth 23 has suggested that passage of cholesterol from the outer to the inner mitochondrial membrane is facilitated by the increased proximity of the two membranes as a consequence of steroidogenic stimuli (ACTH in the case of adrenal cortex cells). Several investigators have provided evidence that SCP 2 facilitates cholesterol movement across the critical gap between the mitochondrial membranes.…”

“…It is the cellular lipid-binding proteins that have either the tertiary structure, or that bind additional elements, that can target the ligand-protein complexes to specific sites within organelles of the cell and result in sequestration of cholesterol and other lipids into selected compartments (e.g., the inner mitochondrial membrane). 22,23 The cellular content of these proteins, which establishes the capacity of a cell to utilize the various types of lipids (e.g., cholesterol for production of steroid hormones), is regulated by events that control transcription and translation of cellular lipid-binding proteins. 28 A novel prospect for cellular lipid-trafficking proteins is the role that they may play in regulating enzyme expression in sterol metabolizing pathways.…”

“…[46][47][48] In the rabbit corpus luteum, estradiol acts directly to enhance progesterone production via estrogen receptor-mediated effects on cholesterol storage in lipid droplets and mitochondria. 22,48 The levels of enzyme activity in the pathway of cholesterol intake, storage, and conversion to pregnenolone are relatively insensitive to estradiol. 22,49 Therefore, mechanisms of cholesterol translocation within the cell and in the subcellular organelles have been proposed as being estrogensensitive and responsible for the relatively abrupt loss in rabbit luteal progestin output that can be caused in a reversible manner by deprivation and reimposition of an estrogen stimulus.…”