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 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 (alpha-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 untrated 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 inhibitory 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).


CONCLUSIONS
Findings indicate the existence of heterogeneously-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-I.
Choi
D
D
Department of Obstetrics and Gynecology, University of Maryland School of Medicine, Baltimore 21201, USA.
Putowski
L T
LT
Fielder
P J
PJ
Rosenfeld
R G
RG
Rohan
R M
RM
Adashi
E Y
EY
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D017931:DNA Primers; D015342:DNA Probes; D004054:Diethylstilbestrol / Q000494:pharmacology*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000096:biosynthesis* / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis; D012741:Sexual Maturation; D012756:Sheep; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D017931:DNA Primers; D015342:DNA Probes; D018974:Insulin-Like Growth Factor Binding Protein 4; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004054:Diethylstilbestrol; D005640:Follicle Stimulating Hormone