Divergent Effects of 1-Oleoyl-2-Acetylglycerol and Tumor-Promoting Phorbol Ester on Rat Granulosa Cell Steroidogenesis in Vitro

Abstract

The present study was undertaken to compare the effects of diacylglycerol (synthetic 1-oleoyl-2-acetylglycerol; DG) and TPA (12-O-tetradecanoylphorbol 13-acetate) on FSH- and dibutyryl cyclic AMP ((Bu)₂cAMP)-stimulated granulosa cell pregnenolone (P₅), progesterone (P), and 20 α-hydroxypregn-4-en-3-one (20 α-OH-P) secretion. Granulosa cells from immature rats pretreated with pregnant mare’s serum gonadotropin (PMSG) were incubated for up to 24 h with DG (0−80 μg/ml) or TPA (0−80 μg/ml) in the presence or absence of FSH (150 ng/ml) or (Bu)₂cAMP (1.5 mM). DG, when continually present in the culture medium (MEM), significantly stimulated basal P₅ (in the presence of 25 μM cyanoketone to block further metabolism), P, and 20 α-OH-P secretion during 6 h and 24 h of incubation. Pretreatment with TPA for 1 h caused a substantial increase in the subsequent progestin (P + 20 α-OH-P) secretion. However, the phorbol ester had little or no effect on steroid secretion during 6 h of incubation, significantly inhibited the secretion of P₅ and P, but stimulated 20 α-OH-P production in 24 h.

DG and TPA exerted divergent effects on FSH- and (Bu)₂cAMP-stimulated progestin secretion. Accumulation of P₅ throughout the culture periods (1−24 h) was markedly increased by DG (20 μg/ml) but significantly inhibited in the presence of TPA (40 ng/ml). DG (5−80 μg/ml) significantly enhanced FSH-stimulated progestin secretion during 6 h and 24 h of incubation and increased (Bu),cAMP-induced steroid secretion during a 24-h culture period. In contrast, TPA (5−80 ng/ml) dose-dependently inhibited FSH- and (Bu)₂cAMP-stimulated progestin secretion during both 6 h and 24 h of incubation. The inhibitory action of TPA was evident also after 1 h of pretreatment with this phorbol ester, although the stimulatory effect of DG was observed only during continual presence of the phospholipid. The current studies demonstrate that, although TPA mimics certain effects of DG on basal steroid production, marked differences exist between the actions of the two kinase C activators on gonadotropin- and (Bu)₂,cAMP-stimulated granulosa cell steroidogenesis in vitro. These findings indicate that caution must be exercised when TPA is used as a pharmacologic probe for protein kinase C activation.