FUNCTIONAL OVARIAN AND PLACENTAL ISOFORMS OF PORCINE AROMATASE

Functional isoforms of porcine aromatase cytochrome P-450 were cloned from placenta, and ovarian theca interna and granulosa tissues, and fu ll length cDNAs were expressed in vitro. Porcine theca and granulosa e xpressed an identical form of P-450arom. This ovarian cDNA encoded for a protein of 501 amino acids, two amino acids shorter at the N-termin al end than placental P-450arom isoform (503 residues). Overall, the t wo isoforms exhibited 93% nucleotide and 87% amino acid identity with each other, and both were highly homologous, at the nucleotide and ami no acid levels, to human and bovine P-450arom, also 503 amino acid pro teins. Analysis of the predicted amino acid sequence further suggested that the regions of the cDNAs, corresponding to presumed exons III, V and IX, assuming conservation of intron-exon boundaries with the huma n P-450arom gene, were conserved in the porcine placental and ovarian enzymes, while sequence variance occurred in all other putative exons. In vitro expression indicated that the cDNA encoding porcine placenta l P-450arom was almost 10-fold more active in the synthesis of estrone from androstenedione than was the-ovarian isoform which synthesized m ore 19OH-androstenedione than estrone. Western analysis of transfected Cos1 cells suggested that the differences in activity were not due to levels of expression of the cDNAs since similar levels of immunodetec table protein were observed in cells transfected with each construct. Both isoforms were sensitive to inhibition of activity by the specific aromatase inhibitors, 4OH-androstenedione and CGS16949A. In addition, activity of the enzyme encoded by the ovarian P-450arom cDNA was supp ressed by etomidate, an inhibitor of cytochrome P-450 11 beta-hydroxyl ase, but the placental P-450arom isoform was not. These functional dif ferences were consistent with observations made in similar experiments involving P-450arom activity in freshly homogenized tissues. These da ta provide evidence of the existence of distinct, intraspecies isoform s of P-450arom, the first described in any species and suggest that pi gs possess a unique mechanism for regulating androgen metabolism.