Catalytic differences between porcine blastocyst and placental aromatase isozymes

Abstract

Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO cells using the mammalian cell transfection method. Using an`in-cell' assay (a 3 H-water release method), catalytic parameters of the porcine placental aromatase were found to be very similar to those of the human enzyme; however, the activity of the blastocyst isozyme was found to be one-thirtieth that of the placental isozyme. Product isolation assay (using testosterone as the substrate) revealed that the major steroid products were 17b-estradiol and 19-nortestosterone. The product ratio of estradiol/19-nortestosterone was found to be 94 : 6 for the porcine placental form, 6 : 94 for the porcine blastocyst form, and 92 : 8 for the human wild-type aromatase. Therefore, the porcine blastocyst aromatase isozyme catalyzes mainly androgen 19-desmethylation rather than aromatization. In addition, inhibition profile analyses on the placental and blastocyst isozymes were performed using three steroidal inhibitors [4-hydroxyandro-stenedione (4-OHA), 7a-(4 H -amino)phenylthio-1,4-androstandiene-3,17-dione (7a-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)]. While the two isozymes of porcine aromatase share 93% amino-acid sequence identity, our results indicate that the two porcine aromatase isozymes have distinct responses to various aromatase inhibitors.

Estrogen Synthesis in Fetal Sheep Brain: Effect of Maternal Treatment with an Aromatase Inhibitor1

“…The pig (Sus scrofa) expresses three distinct isozymes of the aromatase protein that are specific for the gonads, placenta, and embryo [26]. Although aromatase isozymes in pigs share Ͼ90% amino acid sequence identity, they exhibit distinct catalytic properties and responses to different aromatase inhibitors [27]. To our knowledge, the possibility that sheep express isozymes of aromatase has never been explored.…”

The presence of 19-norandrostenedione and its sulphate form in yolk-sac fluid of the early equine conceptus

“…Sulphoconjugation may serve to regulate the biological availability of 19-norA for whatever role the 'free' steroid might play. It has been reported that 19-norA inhibited the conversion of androgen to estrogen by aromatase, perhaps by competing for binding to the active site of the enzyme [22]. The well known anabolic action of norandrogens also has to be considered.…”

“…No difference seems to exist for the localization of aromatase protein and transcript for the early equine embryo when compared to the porcine embryo, despite the transient nature of the enzyme activity in the latter [28]. Special interest lies in the report that the major product of the blastocyst form in the pig was the 19-norandrogen when transfected CHO cells were incubated with [4-14 C]-testosterone [22]. Our finding of high concentrations of 19-norA in y-s fluid of the mare gives support to these in vitro studies.…”

“…We now report that large amounts of 19-norA, and even greater quantities of the steroid in the sulphate fraction, were found in extracts of y-s fluid of the early equine conceptus. These findings are considered to be reflective of the equine equivalent of the blastocyst form of the porcine aromatase which catalyzes mainly androgen 19-demethylation rather than aromatization [22].…”

“…Interest was heightened when a developmental switch in expression from blastocyst to endometrial/placental-type P450arom gene was demonstrated in the pig and horse [20]; it was reported later that the isoforms of porcine aromatase are encoded by three distinct genes [21]. Catalytic differences between porcine blastocyst and placental aromatase were revealed in a collaborative effort by three groups of investigators [22]. Attention is drawn particularly to the finding that the product ratio of estradiol/19-nortestosterone, from testosterone as substrate, was 94:6 for the porcine placental form and, conversely, 6:94 for the porcine blastocyst form.…”