Characterization and hormonal regulation of granulosa cell-derived insulin-like growth factor binding protein-4

Doo Seok Choi, Lechoslaw T. Putowski, Paul J. Fielder, Ron G. Rosenfeld, Richard M. Rohan, Eli Y. Adashi

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

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) dna, 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 (α-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 outsets 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 a 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 culture medium from 72 hours enhanced the inhibitory effect of FSH (100 ng/mL) for a maximal decrement in IGFBP-4 transcript 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: Finding indicate the existence 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 IGFBP-4 transcripts display relatively limited regulation in response to treatment with either FSH or IGF-L.

Original languageEnglish (US)
Pages (from-to)145-151
Number of pages7
JournalJournal of the Society for Gynecologic Investigation
Volume3
Issue number3
DOIs
StatePublished - May 1 1996

Keywords

  • Insulin-like growth factors
  • carrier proteins
  • gonadotropins

ASJC Scopus subject areas

  • Obstetrics and Gynecology

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