The modulation of granulosa cell function by follicular fluid constituents is incompletely characterized. Therefore, we examined follicular fluid pooled from small follicles (FF8) of sexually immature pigs for regulatory properties utilizing hormonally responsive porcine granulosa cells under serum-free conditions in vitro. FF8 contains heat-labile ornithine decarboxylase (ODC)-stimulating factors equipotent with FSH, LH, or prostaglandin E2 (PGE2). Significant activity is preserved after extensive dialysis or 99.5% adsorption of steroids by charcoal. ODC stimulation by submaximal concentrations of FFS is additive to that of saturating doses of LH, FSH, PGE2, or epidermal growth factor. Maximal concentrations of FF8 exhibit synergism with saturating levels of FSH, LH, or PGE2 in ODC stimulation. Preliminary molecular weight sizing of the FF8-stimulating activity by membrane ultrafiltration revealed two active fractions with discrete, saturable, concentration-dependent responses: a high molecular weight substance (>300, 000 daltons), and a low molecular weight moiety (<10, 000 daltons) stable after lyophilization. No activity was detectable in the 10, 000 to 300, 000-dalton fraction after 4-fold concentration. The mechanism of FF8 action was studied. The relationship of cAMP to FF8-stimulated ODC activity is complex. The phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine (0.25 HIM), significantly potentiates stimulation of ODC by FF8. However, the effects of maximally stimulating concentrations of 8-bromo bromocAMP (0.5 mM) are additive to those of FF8. Further analysis of the active fractions of FF8 indicated that enhancement of maximal 8-bromo-cAMP stimulation occurred with the <10, 000-dalton moiety but not with the >300, 000-dalton fraction. Similarly, 3-isobutyl-l-methylxanthine enhancement occurred with the high rather than the low molecular weight moiety. Thus, the action of the <10, 000 (but not the >300, 000)-dalton fraction appears independent of cAMP. Further investigation of the mechanism of ODC stimulation by FF8 demonstrated that cycloheximide (10 μg/ml) reduced ODC augmentation by FF8 to undetectable assay levels; actinomycin D (1 μg/ml) and a-amanitin (1 μg/ml) diminished ODC activity to below unstimulated control levels. To assess possible posttranslational enzyme stabilization, the t1/2 of enzyme activity decay after 95% protein synthesis inhibition with cycloheximide was studied. ODC activity decreased with a t1/2 of 44.7 min after follicular fluid stimulation and 41.7 min after supramaximal FSH. In summary, FF8 contains heat-labile nonsteroidal factors capable of stimulating ODC activity in isolated granulosa cells under serum-free conditions in vitro. Indirect evidence suggests that these factors differ from other well characterized stimulators of ODC in this cell system and that the mechanism of action of one moiety may be independent of cAMP.
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