TYROSINE KINASE INHIBITOR AG18 ARRESTS FOLLICLE-STIMULATING HORMONE-INDUCED GRANULOSA-CELL DIFFERENTIATION - USE OF REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION ASSAY FOR MULTIPLE MESSENGER RIBONUCLEIC-ACIDS

A sensitive assay of multiple mRNAs by reverse transcriptase-polymeras e chain reaction was adopted to study the hormonally regulated express ion of steroidogenic enzymes in primary rat granulosa cells in culture . As little as 15-60 ng total RNA prepared from cultured cells were re verse transcribed in the presence of pd(T)(5), and polymerase chain re action was conducted in the presence of specific oligonucleotide pairs designed to identify cDNAs of steroidogenic enzymes. In combination w ith Northern blot analysis of cholesterol side-chain cleavage cytochro me P450 (P450scc) message, it is shown that a novel protein kinase inh ibitor, tyrphostin AG18, arrests the FSH-induced accumulation of P450s cc mRNA. This inhibition is dose dependent (IC50, 15 mu M) and reversi ble. The addition of 80 mu M AG18 to cells containing high levels of P 450scc mRNA caused a rapid decline of the cytochrome message (t(1/5), 5 h), similar to the effect of 30 mu g/ml alpha-amanitin. However, con comitant addition of the two drugs did not accelerate the mRNA degrada tion process, suggesting that AG18 does not affect message stabilizati on. Tyrphostin AG18 did not affect mRNA species that are net FSH induc ible, such as the ribosomal protein L19, or the constitutively express ed low levels of steroid 5 alpha-reductase mRNA. Moreover, even the ex tremely high levels of P450scc mRNA in granulosa-lutein cells, being c AMP independent and terminally differentiated a few hours after LH sur ge, were not affected by the addition of AG18 in culture. In contrast, two additional key and FSH-inducible steroidogenic enzymes, ie. aroma tase cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase-I, were i nhibited by AG18 at their mRNA levels. These results suggest that an a s yet undetermined tyrosine kinase pathway is involved in the cAMP-dep endent signal transduction pathway of FSH action, so that the presence of AG18 does not allow FSH induction of gene expression to occur.