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 s teroidogenesis and follicular maturation in the human ovary. The activ ities of the IGFs are regulated by a family of binding proteins (IGFBP s) which are subject to a number of potential post-translational modif ications. The aim of this study was to investigate both the production and modification of the IGFBPs in follicular fluid and in medium cond itioned by granulosa cells and theca from individual follicles at diff erent stages of maturation. In follicular fluid from healthy, dominant follicles there was an increase in the amount of IGFBP-2, -3 and -4 p resent as lower molecular weight forms (23 kDa, 29 kDa and 16.5 kDa re spectively) in comparison to that seen in atretic follicles from the s ame ovary. Furthermore for IGFBP-4, this fragmentation was confirmed t o be attributable to the presence of a specific protease which could b e inhibited not only by the addition of metal ion chelators or serine protease inhibitors, but also by the addition of other recombinant uns aturated IGFBPs, particularly IGFBP-3. IGF-I did not modulate the acti vity of the IGFBP-4 protease in solution but was able to prevent the i nhibition seen with IGFBP-3. Analysis of granulosa cell conditioned me dium from the same series of healthy and atretic follicles revealed th at IGFBP-2 and -4 were the predominant IGFBPs with no fragments seen o n immunoblotting. In contrast, IGFBP-3 in conditioned medium from thec a of atretic follicles was always found as an intact doubler, but was found partially fragmented (30 and 32 kDa) in medium conditioned by th eca from healthy, dominant follicles with the proportion of IGFBP-3 in this lower molecular weight or fragmented doublet increasing with fol licular maturation. A similar situation was also found for IGFBP-4 wit h the progressive increase in the amount of the 15 and 16.5 kDa fragme nts. 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 rema rkable 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 dou bler and that IGFBP-2 was again always found to be intact. In conclusi on we have demonstrated alterations in the proteolytic modification of the IGFBPs which differ in the various follicular compartments and ar e closely linked to the stage of follicular development.