Estrogen-Mediated Mitochondrial Cholesterol Transport and Metabolism to Pregnenolone in the Rabbit Luteinized Ovary1

We investigated the mechanisms of luteotropic actions of estradiol on steroidogenesis. To this end, we examined, in vitro, the metabolism of cholesterol from endogenous or exogenous sources for pregnenolone production in rabbit luteinized ovarian cell mitochondria isolated from pseudopregnant animals in various states of stimulation by estradiol. We found that estradiol-mediated regulation of mitochondrial cholesterol metabolism for pregnenolone production differs from the mechanisms of regulation reported for steroidogenic protein/polypeptide hormones in the following respects: 1) in the estradiol-sensitive, luteinized-ovary, rabbit model, temporary blockage of cytochrome P-450 cholesterol side-chain cleavage enzyme by aminoglutethimide treatment in vivo has no effect on mitochondrial pregnenolone production in vitro after the aminoglutethimide is removed, indicating no additional capacity for upstream cholesterol storage; 2) preincubating mitochondria at 37 degrees C fails to increase subsequent pregnenolone synthesis in response to the addition of isocitrate; and 3) exogenously added cholesterol does not readily enter the steroidogenic pool of cholesterol unless the endogenous cholesterol pool is first depleted. These new observations indicate that estradiol increases the usable steroidogenic cholesterol pool in rabbit ovarian mitochondria. Also, 1) they are consistent with a putative requirement for the participation of one or more estrogen-sensitive protein factors to enhance cholesterol trafficking to the inner mitochondrial membrane, and 2) they complement the observation of estrogen-dependent expression of steroidogenic acute regulatory protein in rabbit luteal tissue.