CHARACTERIZATION AND HORMONAL-REGULATION OF GRANULOSA CELL-DERIVED INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-4

OBJECTIVE: Because of the potential importance of insulin-like growth factor binding protein-4 (IGFBP-4) to ovarian physiology and the obvio us limitations imposed by in vivo-exclusive experimental paradigms, we set out to delineate the characteristics and hormonal regulation of g ranulosa cell-derived IGFBP-4 under in vitro circumstances. METHODS: G ranulosa cells obtained by follicular puncture of the ovaries from die thylstilbestrol-primed intact immature rats were subjected to culture for up to 72 hours. Insulin-like growth factor binding protein-4 mRNA extracted from culture was subjected to Northern blot hybridization. D ata normalization was assured by reprobing with the hamster Chinese ha mster ovary B (CHOB) cDNA, and the IGFBP-4/CHOB ratio was calculated. Conditioned culture media were subjected to Western ligand blot before and after immunoprecipitation with a rat IGFBP-4 directed polyclonal antiserum (alpha-B104). RESULTS: Immunoprecipitation studies revealed granulosa cell-derived IGFBP-4 to be composed of a major 24-kDa specie s as well as a relatively minor 27-kDa moiety. Given cultures of untre ated granulosa cells from immature estrogen-treated rats, transcripts corresponding to IGFBP-4 displayed an initial temporary decline culmin ating in a 6-hour nadir (a decrease of 67%; P < .05) followed by relat ively prompt recovery (within 24 hours) to levels comparable to those noted at the outset of the culture (time 0). However, additional (albe it statistically insignificant) increments were noted at the 48-hour ( but not 72-hour) time point. Treatment of granulosa cells with increas ing concentration of FSH resulted in decrements of up to 30% (P < .05) in the steady-state levels of IGFBP-4 transcripts. A modest, biphasic , time-dependent response was noted for IGFBP-4 transcripts after trea tment with high-dose FSH (100 ng/mL), an effect characterized by 24- a nd 48-hour increments (51% [P < .05] and 26% [P = .052] over untreated controls, respectively) and a 72-hour decrement (25%; P = .16). The c oncurrent provision of the C-19 aromatase substrate androstenedione (1 0(-7) mol/L) to the culture medium from 72 hours enhanced the inhibito ry effect of FSH (100 ng/mL) for a maximal decrement in IGFBP-4 transc ripts of 49% (P < .05). Treatment with insulin-like growth factor (IGF )-I produced limited inhibition (up to 26%) of the steady-state levels of IGFBP-4 transcripts (P < .05). CONCLUSION: Findings indicate the e xistence of heterogenously-sized IGFBP-4 species, of which the 27-kDa (as distinct from the 24-kDa) IGFBP-4 moiety constitutes a relatively minor component. The steady-state levels of granulosa cell-derived IGF BP-4 transcripts display relatively limited regulation in response to treatment with either FSH or IGF-I.