EVIDENCE THAT MEHP INHIBITS RAT GRANULOSA-CELL FUNCTION BY A PROTEIN-KINASE C-INDEPENDENT MECHANISM

We have recently shown that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the reproductive toxicant di-(ethylhexyl) phthala te (DEHP), inhibited FSH- but not forskolin-, isoproterenol-, or chole ra toxin-stimulated granulosa cell cAMP accumulation in vitro. In addi tion, MEHP also inhibited FSH-stimulated progesterone production, a cA MP-dependent process. Similar to MEHP, the protein kinase C (PKC) acti vator, 12-0-tetradecanoyl-phorbol 13-acetate (TPA) has been shown to i nhibit rat granulosa cell cAMP accumulation in a FSH-specific manner, and decrease FSH-stimulated progesterone production. Due to the simila rity with respect to inhibition of cAMP accumulation, we conducted stu dies to determine if the inhibitory actions of MEHP on granulosa cell function are mediated via activation of PKC. Treatment of granulosa ce lls for 48 h with 100-mu-M MEHP produced no effect on forskolin- or is oproterenol-stimulated progesterone production, indicating that MEHP d oes not have a post-cyclic AMP site of action with respect to progeste rone inhibition. Unlike the FSH-specific effect seen with MEHP, treatm ent with 10 nM TPA inhibited FSH-, forskolin-, and isoproterenol-stimu lated progesterone production. In addition, maximally inhibitory conce ntrations of TPA and MEHP caused significantly greater inhibition of F SH-stimulated cAMP accumulation than either compound alone. Finally, a ddition of the progesterone precursor, pregnenolone, reversed the FSH- stimulated progesterone production inhibition by MEHP, but not that by TPA. Taken together, these data indicate that the inhibitory effects of MEHP on granulosa cell function are independent of phorbol ester-se nsitive PKC activation.