ESTROGEN-DOMINANT BUT NOT ANDROGEN-DOMINANT HUMAN OVARIAN FOLLICULAR-FLUID CONTAINS AN INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-4 PROTEASE

Estrogen-dominant follicular fluid (FFe) and granulosa-luteal cell con ditioned media, in contrast to androgen-dominant FF (FFa), contain bar ely detectable levels of insulin-like growth factor binding protein-4 (IGFBP-4) by ligand binding techniques. The current study was designed to evaluate the possibility of an IGFBP-4 protease in FFe, which may alter the affinity of IGFBP-4 for insulin-libe growth factors (IGFs), rendering IGFBP-4 undetectable by ligand binding techniques. FFe and F Fa were obtained from regularly menstruating women, and FFe was also o btained during in vitro fertilization procedures. Mixing experiments w ere performed by using human recombinant IGFBP-4 or IGFBP-4 in nonpreg nancy serum (NPS) as substrate and FF as the source of the putative pr otease. Incubation of NPS at 37 C for 5 h in the presence of FFa resul ted in the reduction of IGFBP-4 to barely detectable levels when analy zed by Western Ligand blotting, with no change occurring in the levels of the other binding proteins present in NPS. In contrast, incubation of FFa with NPS under similar conditions had no effect on the levels of IGFBP-4. The disappearance of IGFBP-4 when NPS was mixed with FFe e xhibited optimal pH-dependence at pH 7-9. Inhibition of the putative p rotease by aprotinin, ethylenediaminetetraacetic acid, and 1,10-phenan throline supports its identification as a metalloserine protease. West ern immunoblot analysis detected the presence of a proteolytic fragmen t of approximately 17-18 kDa after incubation of recombinant IGFBP-4 i n the presence of FFe but not in the presence of FFa. Similar incubati on of other recombinant human IGFBPs did not reveal their degradation, further suggesting that the protease in FFe is specific for IGFBP-4. These data demonstrate the presence of an IGFBP-4-specific metalloseri ne protease in FFe but not in FFa, and they suggest that proteolytic c leavage may be responsible for effectively decreasing levels of inhibi tory IGFBP-4 and thus increasing bioavailability of IGF peptides withi n estrogen-dominant follicles. The importance of this mechanism may li e in providing the dominant follicle with more available IGFs to syner gize with gonadotropins in stimulating estradiol production and in inh ibiting this synergy in androgen-dominant and atretic follicles.