INVESTIGATION OF THE MECHANISM OF IGF-INDEPENDENT ACTIONS OF IGFBP-4
RJ Wright1, CM Perks2, JMP Holly2 & HD Mason1
IGFs act as amplifiers of gonadotrophin (Gn) action in the ovary, but their activity is modulated by IGFBPs. In addition, we have shown IGF-independent inhibitory effects of IGFBP-4 on ovarian steroidogenesis. The aim of this study was to investigate the possible mechanism of action of IGFBP-4 in ovarian cells.
Granulosa cells (GC) and theca were harvested from intact follicles (2.5-14mm) dissected from 8 pairs of ovaries collected with ethics committee approval. GC were also collected from IVF-flush. Cells were plated at 50,000 per well in 200microlitres of Medium 199. Theca was incubated as 2-3mg explants in 1ml M199. Cells from pooled small or large separate follicles were incubated with 5ng/ml Gn or 1mM 8-bromo-cAMP +/- 5ng/ml IGFBP-4 for a range of time points (3-48h). Following treatment with IGFBP-4, the cells were either restimulated with Gn or cell viability was assessed by trypan blue exclusion or MTT assay. Steroid and lactate accumulation in the medium was then determined.
IGFBP-4 inhibited Gn-mediated steroidogenesis in GC within 3h of culture. However, no inhibition of 8-bromo-cAMP-stimulated steroid production was observed. IGFBP-4 also inhibited the effects of Gn on lactate accumulation in GC and theca after 48h. Cells exposed to IGFBP-4 could be restimulated by Gn and showed no difference in cell viability from controls.
In conclusion, IGFBP-4 has effects on more than one metabolic pathway in the ovary. The fact that IGFBP-4 effects are seen within 3h would suggest that IGFBP-4 is inhibiting steroidogenesis by a mechanism not involving synthesis of new proteins. It appears that IGFBP-4 is blocking steroidogenesis at a point upstream of cAMP in the Gn-mediated steroid pathway.
This project is supported by the Wellcome Trust.