Functional aromatase expression in porcine adrenal gland and testis

Abstract

The expression of aromatase cytochrome P450 (P450(arom)) in the adrenal glands, testes, and placentas of fetal and newborn pigs was investigated. Western immunoblot analysis detected a single 48-50-kDa protein band in these tissues as well as in other porcine tissues known to express P450(arom) including Day 12 tubular conceptuses, theca interna, and granulosa. Slight differences in migration suggested that the P450(arom) protein expressed in the testis was larger than that in the adrenal gland, which was, in turn, larger than that in placenta, theca, and granulosa. Consistent with P450(arom) expression in these tissues, a cDNA encoding porcine P450(arom) hybridized to a 2.3-kb transcript in Northern analyses of porcine blastocysts, placentas, and fetal and newborn adrenal glands and testes, as well as in theca and granulosa tissues from preovulatory follicles. No differences in transcript size were detectable among tissues. The identity of P450(arom) transcripts was confirmed by sequence analysis of partial cDNA clones amplified from porcine fetal adrenal glands, testes, and placentas according to the RACE procedure. The sequences of the adrenal and testis clones were identical but differed from the placental sequence, which represented the first 85 amino acids of porcine P450(arom). Specifically, the adrenal and testis clones expressed transcripts that resembled the ovarian isoform of porcine P450(arom), rather than the porcine placental isoform, predicting a two-amino acid deletion and 12 predicted amino acid substitutions. P450(arom) activity was examined to further define expression in these tissues. Activity in adrenal, testis, and placental homogenates was inhibited by 4-hydroxyandrostenedione (4OH-A4) whereas inhibition by etomidate was demonstrated in the adrenal and testis homogenates but not in the placental homogenates. The sensitivity of activity in the newborn porcine adrenal glands and testes to inhibition by etomidate was similar to that of ovarian P450(arom) activity. The level of P450(arom) activity was highest in the placenta and lowest in the adrenal gland, and no effect of fetal sex was noted in either tissue. Immunocytochemical studies localized the expression of P450(arom) in the adrenal gland of newborns to cells at the corticomedullary junction and, with greater intensity, to cells around the developing medullary lobules. The same cells expressed cytochrome P450 17α- hydroxylase, the expression of which also extended throughout the zona fasciculata. The interstitial cells were the site of P450(arom) expression in the testis, but no expression could be detected in any cells within the spermatic tubules. These data demonstrate that fetal and newborn porcine adrenal glands and testes express an active P450(arom) that resembles the isoform expressed in the ovary. The localization of adrenal expression suggests a possible role in medullary maturation and function in the fetal and newborn pig.