mRNA Expression Of Steroidogenic Enzymes Research Articles

In vitro release of aldosterone and cortisol in human adrenal adenomas correlates to mRNA expression of steroidogenic enzymes for genes CYP11B2 and CYP17.

Adenomas of the adrenal cortex cause different disorders depending on the main steroid synthesized and released. The aim of this research is to increase our understanding of the pathophysiology of steroidogenesis in adrenocortical disorders by comparing the release of steroids from adrenocortical adenomas in vitro with the messenger RNA (mRNA) expression of steroid synthesizing enzymes. Fourteen patients with adrenal tumors were included in the present study; nine were diagnosed with primary aldosteronism and three with Cushing's syndrome. Two patients had an adrenal tumor discovered on computed tomography (CT) during workup for an unrelated disease. Serum cortisol, plasma aldosterone, and urinary catecholamines were normal. Tissue was taken for in vitro steroid release, and aldosterone and cortisol in the medium after a 1-hour incubation were determined. Oligonucleotide probes with sequences complementary to mRNAs encoding for the steroid synthesizing enzymes 11 beta-hydroxylase (CYP11B1), 18-hydroxylase (CYP11B2), 17 alpha-hydroxylase (CYP17), and 21-hydroxylase (CYP21) were synthesized (Genset, Paris, France) and in situ hybridization was performed. Moderate expression of CYP11B2 and low expression of CYP11B1 were seen in the zona glomerulosa. The zona fasciculata of the control adrenals expressed a high signal of CYP11B1, whereas the expression of CYP11B2 was very low. There was considerable variation in aldosterone release from the aldosteronomas, whereas the tumors from the Cushing patients showed no detectable release of aldosterone. In contrast, tumors from patients with primary aldosteronism, Cushing's syndrome, and no hyperfunction all had the ability to synthesize and release cortisol in vitro. The highest cortisol release was found in tumors from patients with Cushing's syndrome, but also the nonhyperfunctioning tumors and some of the aldosteronomas released significant amounts of cortisol. The two patients with highest release of aldosterone in vitro showed the highest expression of CYP11B2 and the lowest expression of CYP11B1 and CYP17. The remaining aldosteronomas had low expression of CYP11B2, similar to the two other groups. Expression of CYP11B1 was high as expected in the Cushing adenomas, but also the two nonhyperfunctioning tumors and some of the aldosteronomas showed a moderate expression. Adenomas from Cushing's syndrome, nonhyperfunctioning adenomas, and some of the aldosterone-producing adenomas had moderate to high expression of CYP17. This paper presents new means for functional characterization of adrenocortical tumors. Diagnosis of an aldosteronoma is often difficult, and with the advent of these methods it is possible to determine the functional capacity of a tumor, once it is removed. This is of special interest if the patient remains hypertensive postoperatively, and it is not clear whether the patient indeed had a functioning tumor.

Feed restriction and insulin treatment affect subsequent luteal function in the immediate postovulatory period in pigs: progesterone production in vitro and messenger ribonucleic acid expression for key steroidogenic enzymes.

Progesterone production and release in vitro, and mRNA expression for key steroidogenic enzymes, were studied in luteal tissue recovered in the immediate postovulatory period from cyclic gilts allocated to one of three treatments: moderate feed restriction during the first (RH) or second week of the estrous cycle, with (HR+I) or without (HR) concomitant injections of long-acting insulin. Time of feed restriction affected neither progesterone production or release, nor mRNA expression for several key steroidogenic enzymes. However, luteal tissue from RH but not from HR gilts responded to LH stimulation by increasing progesterone production and release (P: < 0.05). Insulin treatment increased progesterone production and release, restored luteal tissue responsiveness to LH, up-regulated steroidogenic enzyme mRNA expression, and down-regulated the tissue inhibitor of metalloproteinase-I mRNA expression in HR+I compared with HR gilts (P: < 0.05). In vitro progesterone production and gene expression were affected by time of tissue collection after ovulation in RH and HR gilts but not in HR+I gilts, and were correlated with temporal changes in oviductal and peripheral plasma progesterone concentrations. Inherent differences in luteal function therefore appear to mediate latent effects of nutrition and insulin treatment on circulating progesterone concentrations in the critical postovulatory period in gilts.

The effect of hypoxia from birth on the regulation of aldosterone in the 7-day-old rat: plasma hormones, steroidogenesis in vitro, and steroidogenic enzyme messenger ribonucleic acid.

Adaptation to hypoxia in the neonate requires an appropriate adrenocortical response. The purpose of this study was to examine the adaptation of the aldosterone pathway in rat pups exposed to hypoxia in vivo from birth to 7 days of age. Neonatal rats (with their lactating dams) were exposed to normoxia (21% O2) or hypoxia (12% O2) continuously for 7 days from birth. Trunk blood was collected, and entire adrenal glands were processed from 7-day-old rats to study the activity of the steroidogenic pathway in dispersed cells and isolated mitochondria, for measurement of expression of the steroidogenic enzyme messenger RNAs (mRNAs) by RT-competitive PCR and in situ hybridization histochemistry, for measurement of zona glomerulosa width by immunohistofluorescent staining for P450c11AS protein, and for measurement of mitochondrial number and distribution by transmission electron microscopy. Exposure to hypoxia for 7 days from birth resulted in a marked increase in plasma ACTH, corticosterone, and aldosterone with no change in PRA. Aldosteronogenesis and P450c11AS activity were both augmented in dispersed cells; this effect was lost in isolated mitochondria (from entire adrenal glands) using a permeable substrate for P450c11AS. There was no significant effect of hypoxia on expression of the steroidogenic enzyme mRNAs measured by RT-competitive PCR or in situ hybridization histochemistry. Finally, hypoxia had no effect on mitochondrial number or stereology as assessed by transmission electron microscopy or on zona glomerulosa width as assessed by staining for P450c11AS protein. We conclude that, as opposed to that in adults, hypoxia in the neonate results in an augmentation of aldosteronogenesis. This effect is not accounted for by a change in steroidogenic enzyme mRNA expression, zona glomerulosa width (i.e. hyperplasia), or mitochondrial number or distribution. This functional augmentation of aldosteronogenesis may be due to a change in mitochondrial permeability to steroid substrates and/or the effect of cytosolic factors that control mitochondrial steroidogenesis.

Gene expression of steroidogenic enzymes in chicken embryonic gonads

This paper reviews the molecular aspects of sex differentiation in birds and describes the sex-reversal effects of aromatase inhibitor on mRNA expression of steroidogenic enzymes. Although it is unknown whether the sex-determining gene resides in the W chromosome or whether a gene-dosage mechanism is involved in sex determination, it is clear that anti-Müllerian hormone (AMH) and androgen play a key role for testicular development, whereas estrogen is important for ovarian formation. Our in situ hybridization results demonstrated that mRNA expression of P450aromatase first takes place on day 6.5 of incubation exclusively in the female gonad. Treatment with aromatase inhibitor in ovo suppressed mRNA expression in genetic females. This suggests that ovarian development is promoted by aromatase expression via estrogen production. On the other hand, previous reports demonstrated that formation of the testis is accompanied by Sox9 and AMH mRNA expression, which start on days 6 and 8, respectively. This suggests that Sox9 plays an important role for the male determining pathway which leads to AMH expression and formation of the testis. J. Exp. Zool. 281:450–456, 1998. © 1998 Wiley-Liss, Inc.

Gene expression of steroidogenic enzymes in chicken embryonic gonads

This paper reviews the molecular aspects of sex differentiation in birds and describes the sex-reversal effects of aromatase inhibitor on mRNA expression of steroidogenic enzymes. Although it is unknown whether the sex-determining gene resides in the W chromosome or whether a gene-dosage mechanism is involved in sex determination, it is clear that anti-Müllerian hormone (AMH) and androgen play a key role for testicular development, whereas estrogen is important for ovarian formation. Our in situ hybridization results demonstrated that mRNA expression of P450aromatase first takes place on day 6.5 of incubation exclusively in the female gonad. Treatment with aromatase inhibitor in ovo suppressed mRNA expression in genetic females. This suggests that ovarian development is promoted by aromatase expression via estrogen production. On the other hand, previous reports demonstrated that formation of the testis is accompanied by Sox9 and AMH mRNA expression, which start on days 6 and 8, respectively. This suggests that Sox9 plays an important role for the male determining pathway which leads to AMH expression and formation of the testis.

Developmental and hormonal regulation of rat theca-cell differentiation factor secretion in ovarian follicles.

We have recently presented data demonstrating that preantral follicles secrete a peptide (or family of peptides) that stimulates ovarian theca-interstitial cell (TIC) androgen production by an LH-independent mechanism. The purpose of the study reported here was to study the gonadotropin and developmental regulation of this thecal differentiating factor(s) (TDF) and to determine whether follicle-conditioned medium (FCM) containing TDF bioactivity could stimulate LH receptor and steroidogenic enzyme mRNA expression in TIC. Preantral follicles devoid of theca were obtained by limited enzymatic dispersal of 26-day-old rat ovaries. Follicles were cultured (5 follicles/well) in 96-well plates containing serum-free medium to generate FCM containing bioactive TDF. To bioassay for TDF activity, isolated TIC were cultured (2 days) with 50% FCM; then androsterone production was measured by RIA. Recombinant FSH (rFSH, 0.3-100 mlU/ml) increased TDF bioactivity in a dose-dependent fashion, stimulating maximum androsterone production (20 ng/ml) at 30 mlU/ml. To determine the time course of the production of TDF bioactivity, FCM was collected from follicle cultures treated with and without rFSH at 1, 6, 12, 18, 24, 30, 36, 42, and 48 h. FCM from follicles cultured without rFSH caused a progressive increase in androsterone production to a peak (8 ng/ml) at 18 h followed by a decline to baseline by 48 h. A similar time course was observed for the first 18 h with the rFSH-treated FCM, but androsterone production continued to increase to a level twice that of the untreated FCM (18 ng/ml) at 36 h of culture. In the presence of 100 mlU/ml of rFSH, TDF bioactivity was produced by preantral follicles with > or = 2 layers of granulosa cells but not by small antral follicles, preovulatory follicles, or corpora lutea, demonstrating that production of TDF bioactivity is developmentally regulated. To determine whether FCM could stimulate mRNA expression in TIC, LH receptor, cholesterol side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and 17 alpha-hydroxylase (P450(17) alpha) mRNAs were measured by reverse transcription polymerase chain reaction assays. FCM stimulated LH-receptor, P450scc, 3 beta-HSD, and P450(17) alpha mRNAs above controls. Our data demonstrate that the production of TDF bioactivity is increased by FSH during a specific stage in follicular development when the theca interna is rapidly differentiating, but its production stops when the follicle develops an antrum. Treatment of TIC with FCM stimulates the expression of the mRNAs coding for LH receptors and the steroidogenic enzymes P450scc, 3 beta-HSD, and P450(17) alpha, mimicking the events that occur during normal thecal differentiation. Thus, it seems likely that TDF is involved in the regulation of initial thecal differentiation in preantral follicles.

Inhibin and activin differentially regulate androgen production and 17α-hydroxylase expression in human ovarian thecal-like tumor cells

Activin and inhibin are structurally related dimeric glycoproteins belonging to the transforming growth factor-beta 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), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 17 alpha-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 microM) markedly increased production of both androstenedione (fivefold) and progesterone (threefold) as well as the activity of 3 beta HSD (sevenfold), and P450c17 (sevenfold) over basal. Forskolin stimulated the expression of mRNA for P450scc (fourfold), 3 beta HSD (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 (twofold) but with minimal effects on the mRNA for 3 beta HSD 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.