ABSTRACT

The interconversion of oestrone and oestradiol, androstenedione and testosterone, and dehydroepi-androsterone and 5-androstene-3β,17β-diol in mammalian tissues is catalysed by 17β-hydroxysteroid oxidoreductase (17β-HSOR). To identify tissue sites of 17β-HSOR activity in the human fetus, microsomal fractions from 15 different fetal tissues obtained from first and second trimester pregnancies were used for evaluation of enzymatic activity by use of [17α-³H] oestradiol as the substrate and NADP⁺ as the co-factor. With these reagents, the enzyme-catalysed reaction led to the production of both non-radiolabelled oestrone and NADP³H in equimolar amounts; the radioactivity associated with NADP³H was used to quantify 17β-HSOR activity. Activity of 17β-HSOR was present in microsomes of all the tissues evaluated. The specific activity of the enzyme was highest in liver and placental microsomes. The interconversion of oestradiol and oestrone in microsomal fractions of nine different fetal tissues was studied by the use of substrates labelled with tritium at stable nuclear positions ([6,7-³H]oestradiol and [6,7-³H]oestrone). The products, [³H]oestrone and [³H]oestradiol, were quantified by the use of established techniques; other metabolites formed in these incubations were not identified. The reductive pathway of metabolism (oestrone to oestradiol) appeared to be favoured in microsomal fractions prepared from placenta, fetal zone of the adrenal gland and, possibly, lung. The oxidative pathway (oestradiol to oestrone) appeared to be favoured in microsomes prepared from liver, intestine, stomach, kidney, brain and heart. 17β-HSOR activity in fetal liver also was assessed by the use of fresh and frozen-thawed tissue, homogenate, subcellular fractions, and, also, in primary hepatocytes maintained in culture; the specific activity of the enzyme was highest in the microsomal fraction of liver tissue and 17β-HSOR activity in liver microsomes was linear with time of incubation up to 1 h. In hepatocytes, the enzymatic activity was linear with time of incubation up to 2 h and with cell number up to 2·⁵ × 105 cells/ml; the apparent Michaelis constant of hepatocyte 17β-HSOR for oestradiol was 11 μmol/l. The specific activity of 17β-HSOR did not change after pretreatment of hepatocytes for 24 h with insulin, glucagon or dexamethasone.

Journal of Endocrinology (1989) 123, 509–518

ABSTRACT

Aromatase cytochrome P-450 (P-450_AROM) is the enzyme in the steroidogenic pathway controlling the formation of oestrogens from 19 carbon steroid precursors. Aromatase is present in various tissues of the human fetus. The liver is second only to the placenta in the level of P-450_AROM activity in the fetus. In this study we examined the effects of type 1 transforming growth factor-β (TGFβ) on P-450_AROM expression in human fetal (HF) hepatocytes. The HF hepatocytes were dispersed into single cells which were placed into monolayer cell culture until confluent. Cells were then rinsed and treated in serum-free media with dibutyryl cyclic AMP (Bu₂cAMP) for 72 h. Treatment with Bu₂cAMP (2 mmol/l) caused a fivefold increase in aromatase activity in hepatocytes. The increase in aromatase activity apparently represented an increase in P-450_AROM enzyme as determined by immunoblotting using an antibody directed against human placental aromatase. TGFβ blocked basal, as well as Bu₂cAMP increases, in aromatase activity by over 50%. The effect of TGFβ was dose-dependent with maximal inhibition observed using 2–5 ng TGFβ/ml. Immunodetectable P-450_AROM decreased in parallel with activity following TGFβ treatment. The mechanism of TGFβ action was not through increasing phosphodiesterase (PDE) breakdown of cAMP since inhibition of PDE had no effect on TGFβ action. Finally we examined the level of P-450_AROM mRNA using competitive polymerase chain reaction amplification. Bu₂cAMP increased mRNA levels of P-450_AROM by 2·5-fold, while TGFβ inhibited this induction by 35%. The results of this investigation demonstrated that TGFβ is a potent regulator of P-450_AROM expression in HF hepatocytes.

Journal of Endocrinology (1992) 133, 311–320

Abstract

A tumour of the left adrenal gland was identified in a woman who presented with virilization and secondary amenorrhea. Preoperatively, the plasma levels of dehydroepiandrosterone sulphate, dehydroepiandrosterone, androstenedione, testosterone, 5α-dihydrotestosterone and 5-androstene-3β,17β-diol were elevated two- to fourfold whereas those of urinary 17-ketosteroids were elevated more than tenfold. The production rate of dehydroepiandrosterone sulphate was more than 16 times that in normal women whereas those of dehydroepiandrosterone, testosterone and androstenedione were approximately twofold greater; plasma testosterone was derived almost entirely from the peripheral conversion of androstenedione. Blood was obtained by catheterization of the ovarian veins, left adrenal gland vein and inferior vena cava (at two different sites) and plasma steroid levels were determined: testosterone and cortisol levels were elevated in all blood samples whereas those of androstenedione, dehydroepiandrosterone sulphate and 11-desoxycortisol were approximately six- to eightfold, 1·5-fold and nine- to 22-fold higher in the effluent of the left adrenal gland/tumour compared with the levels in the other compartments. Blood was collected hourly for 24 h to determine steroid levels under basal conditions and, also, after ACTH treatment. Plasma cortisol levels increased markedly upon ACTH administration and fell to very low levels 11 h later, but those of androstenedione, testosterone, dehydroepiandrosterone, 5-androstene-3β,17β-diol and dehydroepiandrosterone sulphate were not affected by ACTH treatment. A histological diagnosis of cortical adenoma of the extirpated tumour was made. Tissue explants and adenoma cells were maintained in culture to characterize the steroid-metabolizing properties of the tumour. The secretion of dehydroepiandrosterone sulphate by tissue explants was high initially, but declined to almost undetectable levels after 5 days in culture. In the presence of ACTH, dehydroepiandrosterone sulphate secretion remained elevated throughout the entire study up to 5 days. Basal secretion of dehydroepiandrosterone sulphate, androstenedione, 11-desoxycortisol, cortisol, testosterone and 11β-hydroxyandrostenedione by adenoma cells was either very low or undetectable. In the presence of ACTH, dibutyryl cyclic AMP or cholera toxin the secretion of dehydroepiandrosterone sulphate, androstenedione and 11-desoxycortisol increased markedly with time in culture up to 3 days, whereas the other steroids were undetected in the medium. A homogenate of adenoma tissue metabolized testosterone to androstenedione, but the conversion of androstenedione to testosterone was minimal. The findings of this study served to establish that virilization in this woman was due, at least in part, to excess testosterone – and testosterone-derived 5α-dihydrotestosterone – produced at extra-adrenal tissue sites almost exclusively through metabolism of tumour-secreted androstenedione. The excess production of steroid prohormones in this woman was due to autonomous tumour steroidogenesis. The remarkable feature was the degree of virilization resulting from a modest increase in biologically potent androgens.

Journal of Endocrinology (1994) 140, 297–307

Abstract

Activin and inhibin are structurally related dimeric glycoproteins belonging to the transforming growth factor-β superfamily of proteins which are synthesized and secreted by the granulosa cells of the ovary. Although initially characterized by their ability to influence FSH secretion from pituitary cells, paracrine regulatory roles of these factors on neighboring ovarian theca interna have been suggested. While inhibin has been shown to increase and activin to decrease the production of androgens, the mechanisms of action are not well defined, partly due to difficulties in obtaining adequate numbers of thecal cells from individual patients or animal models.

Using a unique human ovarian thecal-like tumor (HOTT) cell culture model system we investigated the biochemical and molecular mechanisms controlling C19 steroidogenesis and the effects of activin and inhibin on the activity and expression of key ovarian thecal steroidogenic enzymes, cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD) and 17α-hydroxylase/17,20 lyase cytochrome P450 (P450c17). Steroid production, level of steroidogenic enzyme mRNA expression, and enzyme activity following treatment with forskolin, inhibin-A and activin-A were examined.

Basal steroid production, enzyme activities, and steroidogenic enzyme mRNA levels were not markedly different following treatment with activin (25 ng/ml) or inhibin (25 ng/ml) alone. Forskolin (10 μm) markedly increased production of both androstenedione (fivefold) and progesterone (threefold) as well as the activity of 3βHSD (sevenfold), and P450c17 (sevenfold) over basal. Forskolin stimulated the expression of mRNA for P450scc (fourfold), 3βHSD (threefold), and P450c17 (eightfold) over basal. Androstenedione accumulation was decreased by 60% in the forskolin plus activin group compared with forskolin alone, while progesterone production was maintained. This was attributed to a reduction of P450c17 mRNA (45% of forskolin alone) and activity (45% of forskolin alone). In contrast, co-treatment with forskolin and inhibin increased androstenedione production by 40% while decreasing progesterone by 40% compared with forskolin alone. Concomitantly, this was associated with a higher P450c17 mRNA expression (1·5-fold) and activity (two-fold) but with minimal effects on the mRNA for 3βHSD and P450scc. HOTT cell responses to activin (0·05–50 ng/ml) and inhibin (0·05–50 ng/ml) in the presence of forskolin demonstrated dose-dependent effects on the steroid accumulation, enzymatic activity and mRNA expression of P450c17. Additionally, the differences seen on mRNA expression of steroidogenic enzymes in response to these factors were time-dependent.

In summary, forskolin stimulated C19 steroid production from HOTT cells by increasing the expression of all steroidogenic enzymes examined. Inhibin and activin exerted differential effects on the expression of these enzymes which resulted in alterations in the steroid profile toward production of C19 steroids in the case of inhibin and away from C19 steroids in the case of activin. The influence of these important intraovarian factors on the expression of P450c17, a pivotal enzyme in thecal cell production of C19 steroids, could impact greatly on the follicular milieu of a normal developing follicle as well as in pathophysiological disorders such as polycystic ovarian syndrome.

Journal of Endocrinology (1996) 148, 213–221

SUMMARY

Studies of the electrolyte concentrations of uterine fluid samples from spayed rats, after injection of ovarian hormones, showed a much higher potassium concentration after treatment with oestrogen (42·3 m-equiv./1.) than with progesterone (20·8 m-equiv./1.). There was an even more pronounced change in the sodium: potassium ratio, which fell from 7·3 to 1 with progesterone to 2·5 to 1 with oestrogen. These findings are supported by recalculation of the results of Heap & Lamming (1962).

It is suggested that these changes in the Na⁺ and K⁺ concentrations of uterine fluid produce changes in the membrane potential of the endometrium which could account for delayed implantation of the negatively charged blastocyst under progesterone dominance and implantation under oestrogen dominance.

ABSTRACT

Opioid peptides have been shown to modulate various parameters of both the humoral and cellular arms of the immune system. The modulatory capacity of the peptides can often be substantially reduced in the presence of naloxone, an opioid receptor antagonist, indicating a classical ligand-receptor interaction. In order to characterize these interactions further, we investigated the characteristics of opioid receptors on a macrophage cell line, P388d₁. A δ-class opioid receptor was found with an M _r of 58 000. We also identified opioid receptors on MOLT-4 (T-cell) and IM-9 (B cell) cell lines as well as thymocytes and T celland B cell-enriched populations. Using the central (brain) κ-selective agonist, U-69,593, it was also determined that P388d₁ cells possess κ-like opioid receptors. Scatchard analysis of the binding of [³H]U-69,593 revealed a single population of sites with a dissociation constant of 17 ± 3 (s.e.m.) nmol/l and a total number of binding sites of 53·8 ± 1·0 (s.e.m.) fmol/10⁶ cells. Moreover, the racemic κ-selective agonist U-50,488H was able to displace 50% of [³H]U-69,593 binding at 8·0 nmol/l, whereas other opioid ligands such as [Met]-enkephalinamide (δ-selective) and [d-Ala²,N - Me - Phe⁴,Gly⁵ - ol] - enkephalin (μ - selective) were ineffective displacers of [³H]U-69,593 except at high concentrations.

Journal of Endocrinology (1989) 122, 161–168