Modulation of the insulin-like growth factor-binding proteins by follicle size in the human ovary

The IGFs are believed to play an important role in the regulation of steroidogenesis and follicular maturation in the human ovary. The activities of the IGFs are regulated by a family of binding proteins (IGFBPs) which are subject to a number of potential post-translational modifications. The aim of this study was to investigate both the production and modification of the IGFBPs in follicular fluid and in medium conditioned by granulosa cells and theca from individual follicles at different stages of maturation. In follicular fluid from healthy, dominant follicles there was an increase in the amount of IGFBP-2, -3 and -4 present as lower molecular weight forms (23 kDa, 29 kDa and 16.5 kDa respectively) in comparison to that seen in atretic follicles from the same ovary. Furthermore for IGFBP-4, this fragmentation was confirmed to be attributable to the presence of a specific protease which could be inhibited not only by the addition of metal ion chelators or serine protease inhibitors, but also by the addition of other recombinant unsaturated IGFBPs, particularly IGFBP-3. IGF-I did not modulate the activity of the IGFBP-4 protease in solution but was able to prevent the inhibition seen with IGFBP-3. Analysis of granulosa cell conditioned medium from the same series of healthy and atretic follicles revealed that IGFBP-2 and -4 were the predominant IGFBPs with no fragments seen on immunoblotting. In contrast, IGFBP-3 in conditioned medium from theca of atretic follicles was always found as an intact doublet, but was found partially fragmented (30 and 32 kDa) in medium conditioned by theca from healthy, dominant follicles with the proportion of IGFBP-3 in this lower molecular weight or fragmented doublet increasing with follicular maturation. A similar situation was also found for IGFBP-4 with the progressive increase in the amount of the 15 and 16.5 kDa fragments. IGFBP-2 was always found to be intact. Finally, IGFBP production from stroma explants was also examined. This revealed a wide variation in IGFBP pattern between different ovaries, although there was a remarkable degree of consistency between different stroma explant cultures from the same ovary. Immunoblotting for IGFBP-3 revealed that, where present, it existed as both an intact and a lower molecular weight doublet and that IGFBP-2 was again always found to be intact. In conclusion we have demonstrated alterations in the proteolytic modification of the IGFBPs which differ in the various follicular compartments and are closely linked to the stage of follicular development.