Estrogen- but not androgen-dominant human ovarian follicular fluid contains an insulin-like growth factor binding protein-4 protease.

Abstract

Estrogen-dominant follicular fluid (FFe) and granulosa-luteal cell conditioned media, in contrast to androgen-dominant FF (FFa), contain barely 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-like growth factors (IGFs), rendering IGFBP-4 undetectable by ligand binding techniques. FFe and FFa were obtained from regularly menstruating women, and FFe was also obtained during in vitro fertilization procedures. Mixing experiments were performed by using human recombinant IGFBP-4 or IGFBP-4 in nonpregnancy serum (NPS) as substrate and FF as the source of the putative protease. Incubation of NPS at 37C for 5 h in the presence of FFe resulted in the reduction of IGFBP-4 to barely detectable levels when analyzed 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 exhibited optimal pH-dependence at pH 7-9. Inhibition of the putative protease by aprotinin, ethylenediaminetetraacetic acid, and 1,10-phenanthroline supports its identification as a metalloserine protease. Western immunoblot analysis detected the presence of a proteolytic fragment of approximately 17-18 kDa after incubation of recombinant IGFBP-4 in the presence of FFe but not in the presence of FFa. Similar incubation 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 metalloserine protease in FFe but not in FFa, and they suggest that proteolytic cleavage may be responsible for effectively decreasing levels of inhibitory IGFBP-4 and thus increasing bioavailability of IGF peptides within estrogen-dominant follicles. The importance of this mechanism may lie in providing the dominant follicle with more available IGFs to synergize with gonadotropins in stimulating estradiol production and in inhibiting this synergy in androgen-dominant and atretic follicles.

Manipulation of Human Ovarian Function: Physiological Concepts and Clinical Consequences*

“…IGFBP-3 was shown to exhibit structural similarity with the FSH-binding inhibitor (240), and the IG-FBP profile in follicle fluid has been described to vary during follicle development, independent from changes in serum (241). Moreover, proteases capable of specifically decreasing the level of IGFBP-4 could be demonstrated in estrogendominant follicle fluid only (242), suggesting that more bioavailable IGF-II is available to synergize with gonadotropins in the dominant follicle. It should be noted, however, that growth of follicles could be induced by exogenous FSH in a patient with Laron-type dwarphism (low endogenous IGF-I secretion due to a familial GH receptor defect) (243), suggesting that IGF-I is not required for normal ovarian function.…”

Proteolytic degradation of IGF-binding protein (IGFBP)-2 in equine ovarian follicles: involvement of pregnancy-associated plasma protein-A (PAPP-A) and association with dominant but not subordinated follicles

“…The decrease in IGFBP-4 levels is essentially due to an increase in intrafollicular proteolytic activity in ovine, bovine, porcine, equine and human species (Chandrasekher et al 1995, Besnard et al 1996b, Spicer et al 2001. In preovulatory follicles from these species, the IGFBP-4 protease has been identified as pregnancy-associated plasma protein-A (PAPP-A) (Mazerbourg et al 2001).…”

Clinical Implications of Insulin-Like Growth Factors and Their Binding Proteins in Reproduction.

“…Proteases for IGFBPs, which lower the affinity of IGFBPs for the IGFs, are postulated to increase bioavailable IGF peptide for synergistic action with gonadotropins, promoting steroidogenesis and follicle development [76][77][78]. IGFBP-specific proteases have been identified in various systems [reviewed in Ref.…”

“…IGFBP-specific proteases have been identified in various systems [reviewed in Ref. 7], and in the human ovary an IGFBP-4 protease is present in estrogen-dominant follicular fluids but not in androgen-dominant follicular fluids [76,79]. The proteolytic activity of human follicular fluids correlates negatively with the ratio of androstenedione/E2 [79].…”