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A J Conley, W E Rainey, and J I Mason

ABSTRACT

This study examined fetal steroidogenic enzyme expression and function during pregnancy in the pig. Northern and Western analyses were performed to detect the cytochrome P450 enzyme 17α-hydroxylase/17–20 lyase (P450c17) and that for cholesterol side-chain cleavage (P450scc), as well as 3β-hydroxysteroid dehydrogenase (3β-HSD) expression in several porcine fetal tissues. The data demonstrate higher steroidogenic enzyme expression in the fetal adrenal glands and testes than in the placenta at all stages of development examined. Although steroidogenic enzyme expression was maintained throughout gestation in both the fetal adrenals and the testes, adrenal P450c17 expression was higher in the early and late stages when compared with the intermediate stages of fetal development. The stimulation of fetal adrenal steroidogenic enzyme expression in the later stage fetuses was accompanied by increased expression of P450c17 in both the fetal testes and placenta. The expression of 3β-HSD by porcine fetal testes was low compared with that of the fetal adrenal gland at all stages of development. Adrenal explants and cultured cells secreted cortisol and androstenedione but much lower amounts of corticosterone, dehydroepiandrosterone and aldosterone. Secretion of cortisol and androstenedione by adrenal explants was maintained by ACTH for 5 days of culture but declined in controls. In cultured porcine fetal adrenal cells, ACTH and angiotensin II stimulated the secretion of multiple steroids. Porcine fetal testis explants and cultured cells secreted testosterone, dehydroepiandrosterone and androstenedione, but were only moderately responsive to trophic stimulation by LH. In general, the data suggest that the fetal adrenal glands and the fetal testes have the potential to contribute significantly to the production of steroids during pregnancy in pigs.

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M M Pasquarette, P M Stewart, M L Ricketts, K Imaishi, and J I Mason

ABSTRACT

The type 2 isoform of 11 β-hydroxysteroid dehydrogenase (11β-HSD2), which catalyzes the conversion of cortisol to hormonally inactive cortisone in man, is principally expressed in the placenta and mineralocorticoid target tissues, kidney and colon. To date, few studies have addressed the regulation of this novel 11β-HSD2 isoform. We have characterized the nature and regulation of the 11β-HSD activity expressed in a human cytotrophoblastic cell line, the JEG-3 choriocarcinoma cell. The 11β-HSD activity in JEG-3 cell homogenates required NAD+ as cofactor with NADP ineffective and demonstrated a high affinity for cortisol (apparent K m 31 nm). Incubation of JEG-3 cells with forskolin and dibutyryl cyclic AMP increased 11β-HSD2 activity several-fold in a time-dependent manner, while treatment with phorbol ester had little, if any, effect on 11β-HSD2 activity. Northern blot analysis of RNA isolated from JEG-3 cells after these treatments demonstrated a marked increase in a 1·9 kb 11β-HSD2 mRNA species in cells treated with forskolin for 24 h. We conclude that 11β-HSD2 is regulated by activation of the protein kinase A pathway, but not the protein kinase C pathway in human choriocarcinoma cells, and that this regulation occurs at a pretranslational level. JEG-3 cells provide an excellent model for further studies on the regulation of 11β-HSD2 gene expression in human trophoblast tissue.