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Characterization and Hormonal Regulation of Granulosa Cell-Derived Insulin-Like Growth Factor Binding Protein-4

Lechoslaw T. Putowski, MD, PhD

Paul J. Fielder, PhD

Ron G. Rosenfeld, MD

Richard M. Rohan, PhD

Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Maryland School of Medicine, Baltimore, Maryland; Division of Pediatric Endocrinology, Stanford University Medical Center, Stanford, California

Eli Y. Adashi, MD

Division of Reproductive Endocrinology, Departments of Obstetrics/Gynecology and Physiology, University of Maryl and School, 405 W. Redwood Street, 3rd Floor, Baltimore, MD 21201; Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Maryland School of Medicine, Baltimore, Maryland; Division of Pediatric Endocrinology, Stanford University Medical Center, Stanford, California

Objective: Because of the potential importance of insulin-like growth factor binding protein-4 (IGFBP-4) to ovarian physiology and the obvious limitations imposed by in vivo-exclusive experimental paradigms, we set out to delineate the characteristics and hormonal regulation of granulosa cell-derived IGFBP-4 under in vitro circumstances.

Methods: Granulosa cells obtained by follicular puncture of the ovaries from diethylstilbestrol-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. Data normalization was assured by reprobing with the hamster Chinese hamster 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 (B104).

Results: Immunoprecipitation studies revealed granulosa cell-derived IGFBP-4 to be composed of a major 24-kDa species as well as a relatively minor 27-kDa moiety. Given cultures of untreated granulosa cells from immature estrogen-treated rats, transcripts corresponding to IGFBP-4 displayed an initial temporary decline culminating in a 6-hour nadir (a decrease of 67%; P < .05) followed by relatively prompt recovery (within 24 hours) to levels comparable to those noted at the outset of the culture (time 0). However, additional (albeit statistically insignificant) increments were noted at the 48-hour (but not 72-hour) time point. Treatment of granulosa cells with increasing 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 treatment with high-dose FSH (100 ng/mL), an effect characterized by 24-and 48-hour increments (51% [P < .05] and 26% [P = .052] over untreated controls, respectively) and a 72-hour decrement (25%; P = .16). The concurrent provision of the C₁₉ aromatase substrate androstenedione (10^-7 mol/L) to the culture medium from 72 hours enhanced the inhibitor effect of FSH (100 ng/mL) for a maximal decrement in IGFBP-4 transcripts 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 existence of heterogenously-sized IGFBP-4 species, of which the 27-kDa (as distinct from the 27-kDa) IGFBP-4 moiety constitutes a relatively minor component. The steady-state levels of granulosa cell-derived IGFBP-4 transcripts display relatively limited regulation in response to treatment with either FSH or IGF-I.

Key Words: Insulin-like growth factors • carrier proteins • gonadotropins

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