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) phthalate (DEHP), inhibited FSH- but not forskolin-, isoproterenol-, or cholera toxin-stimulated granulosa cell cAMP accumulation in vitro. In addition, MEHP also inhibited FSH-stimulated progesterone production, a cAMP-dependent process. Similar to MEHP, the protein kinase C (PKC) activator, 12-0-tetradecanoyl-phorbol 13-acetate (TPA) has been shown to inhibit rat granulosa cell cAMP accumulation in a FSH-specific manner, and decrease FSH-stimulated progesterone production. Due to the similarity with respect to inhibition of cAMP accumulation, we conducted studies to determine if the inhibitory actions of MEHP on granulosa cell function are mediated via activation of PKC. Treatment of granulosa cells for 48 h with 100-mu-M MEHP produced no effect on forskolin- or isoproterenol-stimulated progesterone production, indicating that MEHP does not have a post-cyclic AMP site of action with respect to progesterone inhibition. Unlike the FSH-specific effect seen with MEHP, treatment with 10 nM TPA inhibited FSH-, forskolin-, and isoproterenol-stimulated progesterone production. In addition, maximally inhibitory concentrations of TPA and MEHP caused significantly greater inhibition of FSH-stimulated cAMP accumulation than either compound alone. Finally, addition 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-sensitive PKC activation.