Insulin-like growth factor I and the control of steroidogenesis in the rabbit corpus luteum.

Abstract

The objective of this research is to investigate the hypothesis that insulin-like growth factor I (IGF-I) regulates steroidogenesis in the rabbit corpus luteum. Using a primary culture of rabbit luteal cells (obtained three days after ovulation) the effects of IGF-I on progesterone accumulation in culture medium and on the amount and synthesis of the enzyme cholesterol side-chain cleavage cytochrome P-450 (P-450scc), the rate-limiting enzyme for progesterone synthesis were investigated. The effects of IGF-I in luteal cells were compared to those of luteinizing hormone (LH), which is known to stimulate progesterone production in luteal cells of all species. IGF-I (human recombinant) increased progesterone accumulation by luteal cells in a dose-dependent manner over a four-day period of culture. The time-course for the stimulatory effect of IGF-I was different from that of LH; in response to IGF-I, an increase in progesterone was detectable at 36 hours, whereas LH stimulated progesterone within 2 hours. In the presence of 25-hydroxycholesterol, a soluble substrate for progesterone synthesis, luteal cells that had been exposed to IGF-I accumulated more progesterone than control cells or cells that were cultured with LH suggesting that IGF-I affects one or more enzymes involved in the synthesis of progesterone. To determine if IGF-I affects P-450scc synthesis, cells were cultured with ($\sp{35}$S) methionine, the enzyme was immunoprecipitated, and subjected to gel electrophoresis followed by autoradiography. In cells cultured with IGF-I, the incorporation of ($\sp{35}$S) methionine into P-450scc was 3-fold higher after both 24 and 72 hours in culture than that in control cells. Using immunoblot analysis, the amount of P-450scc in cells cultured in medium 199 decreased by 50% and 80% after 24 and 72 hours of culture, compared to initial values (cells freshly removed from fetal bovine serum). In contrast, the amount of P-450scc in cells cultured with IGF-I decreased by only 20% after 24 hours of culture, and remained at this value after 72 hours. These data indicate a potential role for IGF-I in the regulation of cholesterol side-chain cleavage cytochrome P-450 synthesis, and of steroidogenesis in rabbit luteal cells.