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

The present studies were designed to investigate the nature of the actions of insulin-like growth factor-II (IGF-II) on granulosa cell steroidogenesis and assess the potential facilitative interactions between IGF-II and other major regulators of ovarian sterol metabolism, viz. estrogen, FSH, and low density lipoprotein (LDL). In serum-free first passage monolayer cultures of swine granulosa cells, human recombinant IGF-II stimulated progesterone production with a half-maximally effective 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 untreated cultures (48-96 h). Over the latter interval, IGF-I stimulated progesterone production approximately 10-fold, with a significantly lower ED. of 6.1 +/- 0.70 ng/ml (0.78 +/- 0.09 nm; P < 0.01 vs. IGF-II effect). IGF-II (100 ng/ml) enhanced progesterone biosynthesis approximately 2-fold in the presence of 25-hydroxycholesterol, suggesting that IGF-II increases the effective activity of the mitochondrial cholesterol side-chain cleavage enzyme. IGF-II (100 ng/ml) augmented human LDL-promoted progesterone production approximately 18-fold between 0-48 h of culture and approximately 6-fold between 48-96 h. In addition, IGF-II showed time-dependent stimulatory effects on the rates of [I-125]iodo-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 apparent change in their binding affinity, as assessed in equilibrium competition studies. Coadministration of IGF-II and FSH (100 ng/ml) or estradiol (E2; 1 mug/ml) for 2 days increased progesterone production synergistically. Cotreatment with FSH or E2 for 4 days decreased the ED50 of IGF-II's stimulation of progesterone accumulation by 61% and 50%, respectively (P < 0.01). Synergistic interactions also existed between IGF-II and 8-bromo-cAMP, which indicates that IGF-II can act in part at cellular loci distal to cAMP generation. Northern blot analysis of total 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 cholesterol side-chain cleavage enzyme mRNA. The same treatments produced 6-fold increases in the level of LDL receptor mRNA, as determined by solution hybridization/RNase protection assays. In summary, immature swine granulosa cells are highly responsive to the actions of IGF-II in vitro. Nanomolar concentrations of IGF-II can enhance progesterone biosynthesis by 12-fold. IGF-II's mechanisms of action include facilitation of sterol delivery via increased LDL binding and metabolism and concomitantly higher steady state cellular concentrations of LDL receptor mRNA. Moreover, IGF-II augments sterol utilization in progesterone biosynthesis by increasing levels of cholesterol side-chain cleavage enzyme 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.