MECHANISMS OF REGULATION OF OVARIAN STEROL-METABOLISM BY INSULIN-LIKEGROWTH-FACTOR TYPE-II - IN-VITRO STUDIES WITH SWINE GRANULOSA-CELLS

The present studies were designed to investigate the nature of the act ions of insulin-like growth factor-II (IGF-II) on granulosa cell stero idogenesis and assess the potential facilitative interactions between IGF-II and other major regulators of ovarian sterol metabolism, viz. e strogen, FSH, and low density lipoprotein (LDL). In serum-free first p assage monolayer cultures of swine granulosa cells, human recombinant IGF-II stimulated progesterone production with a half-maximally effect ive concentration of 4.6 +/- 1.2 ng/ml (0.61 +/- 0.16 nm) between 0-48 h of culture and 27 +/- 5.7 ng/ml (3.6 +/- 0.76 nm) between 48-96 h. Maximal progesterone accumulation increased 12-fold over that in untre ated cultures (48-96 h). Over the latter interval, IGF-I stimulated pr ogesterone production approximately 10-fold, with a significantly lowe r ED. of 6.1 +/- 0.70 ng/ml (0.78 +/- 0.09 nm; P < 0.01 vs. IGF-II eff ect). IGF-II (100 ng/ml) enhanced progesterone biosynthesis approximat ely 2-fold in the presence of 25-hydroxycholesterol, suggesting that I GF-II increases the effective activity of the mitochondrial cholestero l side-chain cleavage enzyme. IGF-II (100 ng/ml) augmented human LDL-p romoted progesterone production approximately 18-fold between 0-48 h o f culture and approximately 6-fold between 48-96 h. In addition, IGF-I I showed time-dependent stimulatory effects on the rates of [I-125]iod o-LDL internalization, and the amounts of cell-associated and degraded lipoprotein. IGF-II increased by approximately 10-fold the number of specific high affinity LDL receptors on granulosa cells, with no appar ent change in their binding affinity, as assessed in equilibrium compe tition studies. Coadministration of IGF-II and FSH (100 ng/ml) or estr adiol (E2; 1 mug/ml) for 2 days increased progesterone production syne rgistically. Cotreatment with FSH or E2 for 4 days decreased the ED50 of IGF-II's stimulation of progesterone accumulation by 61% and 50%, r espectively (P < 0.01). Synergistic interactions also existed between IGF-II and 8-bromo-cAMP, which indicates that IGF-II can act in part a t cellular loci distal to cAMP generation. Northern blot analysis of t otal RNA isolated from granulosa cells treated with IGF-II (100 ng/ml) , FSH (100 ng/ml), or IGF-II plus FSH for 2 days revealed 5-, 7-, or 8 -fold increases, respectively, in the amount of cytochrome P450 choles terol side-chain cleavage enzyme mRNA. The same treatments produced 6- fold increases in the level of LDL receptor mRNA, as determined by sol ution hybridization/RNase protection assays. In summary, immature swin e granulosa cells are highly responsive to the actions of IGF-II in vi tro. Nanomolar concentrations of IGF-II can enhance progesterone biosy nthesis by 12-fold. IGF-II's mechanisms of action include facilitation of sterol delivery via increased LDL binding and metabolism and conco mitantly higher steady state cellular concentrations of LDL receptor m RNA. Moreover, IGF-II augments sterol utilization in progesterone bios ynthesis by increasing levels of cholesterol side-chain cleavage enzym e mRNA. Given the evidence for intrafollicular production of IGF-II in human and porcine ovaries, paracrine and autocrine effects of IGF-II may be physiologically significant in the developing Graafian follicle .