The IGF/IGFBPs are important in follicle growth and selection, with IGFBP-4 being one of the major players in this system. We have shown potent, IGF-independent, inhibitory effects of IGFBP-4 on steroidogenesis, explaining its' preferential proteolysis in healthy follicles. Two isoforms of IGFBP-4 exist, non-glycosylated and glycosylated, at approximately 24 and 28kDa, with different susceptibility to proteolysis. We therefore investigated the production of IGFBP-4 isoforms by ovarian cells.
160 follicles of 3-25mm were dissected from 13 pairs of ovaries collected with ethics committee approval. Granulosa cells (GC) were plated at 50,000 per well in 200microlitres Medium 199 with 10-7M testosterone while theca (T) was incubated as 2-3mg explants in 1ml M199. GC and T were incubated from pooled small or separate large follicles with medium changes every 24-48h. Conditioned medium (CM) from various time points was immunoblotted for IGFBP-4.
Intact IGFBP-4 was found in all CM. The 28kDa glycosylated isoform predominated in GCCM throughout. However, 3 separate isoforms of IGFBP-4 were seen in TCM, including a more heavily glycosylated form at approximately 30kDa, not previously described. The levels of this isoform increased from day 3. Non-glycosylated IGFBP-4 was seen at all time points, predominating from day 3 of culture. The glycosylated 28kDa form, together with fragmented IGFBP-4, appeared in the first days of culture, disappearing completely by day 7.
In summary, a 28kDa glycosylated form predominated in GCCM, whereas a 24kDa non-glycosylated form prevailed in TCM. The profile of IGFBP-4 isoforms in TCM varied over time with an apparently new form appearing between 3-8 days of culture.
In conclusion, it appears that post-translational modification of IGFBP-4 is different in GC and T and over time of culture. These forms vary in their susceptibility to proteolysis and this may be important for the compartmentalisation of IGFBP-4 actions in the ovary.
This project is supported by the Wellcome Trust.
03 - 04 Dec 2001
Society for Endocrinology