3 beta-hydroxysteroid dehydrogenase isomerase (3beta-HSD) activity in the rat sciatic nerve: kinetic analysis and regulation by steroids.

We have shown that progesterone (PROG) has a stimulatory effect on myelin formation after sciatic nerve injury. PROG is synthesized from pregnenolone (PREG) by the enzyme 3 beta-hydroxysteroid dehydrogenase isomerase (3beta-HSD). At the occasion of the 15th International Symposium of the Journal of the Steroid Biochemistry and Molecular Biology, we presented some of our recent results demonstrating, expression and activity of the enzyme 3beta-HSD in the rat sciatic nerve. We determined the kinetic properties of 3beta-HSD and its regulation by PROG and estradiol. The expression of 3beta-HSD protein was assessed by Western-blot analysis, and the 3beta-HSD activity was evaluated by incubating homogenates with [3H]-PREG as substrate and NAD(+) as cofactor. Levels of steroids formed were calculated either by extrapolation of the relationship between the tritiated peaks obtained by thin layer chromatography (TLC) and the initial amount of PREG, or by gas chromatography-mass spectrometry (GC-MS) determination. A rapid increase in PROG formation was found between 0 and 50min of incubation and no significant change was observed between 1 and 4h. The calculated K(m) value was close to the values obtained for the 3beta-HSD types I and IV isoforms. Trilostane caused a potent inhibition of the rate of conversion of PREG to PROG. When we tested the effects of progesterone and estradiol on 3beta-HSD activity, a significant inhibition was obtained.

Characterization and regulation of the 3beta-hydroxysteroid dehydrogenase isomerase enzyme in the rat sciatic nerve.

In the peripheral nervous system, progesterone (PROG) has a stimulatory effect on myelination. It could be derived from local synthesis, as Schwann cells in culture express the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and convert pregnenolone (PREG) to PROG. Although 3beta-HSD mRNA can be detected by RT-PCR in peripheral nerves, the activity of the enzyme has so far not been demonstrated and characterized in nerve tissue. In this study, we show that homogenates prepared from rat sciatic nerves contain a functional 3beta-HSD enzyme and we have analysed its kinetic properties and its regulation by steroids. The activity of 3beta-HSD in homogenates was evaluated using 3H-labelled PREG as a substrate and NAD+ as a cofactor, the levels of steroids formed were calculated either by extrapolating the relationship between tritiated peaks obtained by TLC to the initial amount of PREG, or by gas chromatography/mass spectrometry determination. A rapid increase in PROG formation was found between 0 and 50 min of incubation and no further significant changes were observed between 1 and 4 h. The calculated Km value (1.06 +/- 0.19 microm) was close to the values described for the 3beta-HSD type-I and type-IV isoforms. Trilostane, a competitive inhibitor of the 3beta-HSD caused a potent inhibition of the rate of conversion of PREG to PROG (IC50 = 4.06 +/- 2.58 microm). When the effects of different steroids were tested, both oestradiol and PROG significantly inhibited the conversion of PREG to PROG.

3 Beta-hydroxysteroid dehydrogenase expression in rat spinal cord.

In adult male rats, 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3beta-HSD) expressing cells were identified in the spinal cord from the cervical to the sacral segments. An in situ hybridization study, using an oligonucleotide common to the four known isoforms of rat 3beta-HSD, revealed its mRNA in gray matter. Measurements of optical densities in autoradiograms showed the following regional distribution: dorsal horn (layers I-III) > central canal (layer X) > or = ventral horn (layers VIII-IX) > ventral funiculus = lateral funiculus. At the cellular level, the number of grains was higher on the large motoneurons than on small neurons of the dorsal horn, but the grain density per cell was similar. Further evidence for the expression of 3beta-HSD in the spinal cord was obtained by western blot analysis, which revealed an immunoreactive protein of approximately 45 kDa in the dorsal and ventral parts of the spinal cord. Castration and adrenalectomy did not influence the expression of 3beta-HSD mRNA and protein. Gas chromatography/mass spectrometry measurements showed higher levels of pregnenolone and progesterone in the spinal cord than in the plasma. After castration and adrenalectomy, their levels remained elevated in the spinal cord, suggesting that these neurosteroids may be synthesized locally. The wide distribution of 3beta-HSD, and the high levels of pregnenolone and progesterone in the spinal cord even after castration and adrenalectomy, strongly suggest a potential endogenous production of progesterone and an important signalling function of this steroid in the spinal cord.

Progesterone stimulates Krox-20 gene expression in Schwann cells.

The gene of the zinc finger transcription factor Krox-20 (Egr-2) is expressed in Schwann cells and plays an important role in myelination of peripheral nerves. We have shown that progesterone promotes myelination in the regenerating sciatic nerve and in cocultures of Schwann cells and sensory neurones. To determine whether progesterone regulates Krox-20 expression, we measured its effects on Krox-20 mRNA levels in the MSC80 mouse Schwann cell line by semi-quantitative RT-PCR. Although low levels of Krox-20 mRNA are detectable in MSC80 cells cultured in defined medium, treatment with 10(-6) M progesterone induces a rapid (15 min) and transient increase in the levels of Krox-20 mRNA. Lower doses of progesterone (10(-9), 10(-8) and 10(-7) M) are also effective in increasing Krox-20 mRNA. Other steroids including testosterone, dexamethasone, and estradiol are ineffective when added to the culture medium at 10(-6) M for 1 h. The induction of Krox-20 mRNA was also observed with the selective progesterone agonist Organon 2058 and was abolished by treating the MSC80 Schwann cells with the progesterone antagonist RU486, indicating that progesterone induces Krox-20 mRNA expression by binding to its intracellular receptor. The induction of Krox-20 by progesterone was also demonstrated in primary cultures of Schwann cells isolated from neonatal rat sciatic nerves, at the mRNA level by RT-PCR and at the protein level by immunohistochemistry. As Krox-20 is a necessary step for the initiation of myelin formation in peripheral nerves, its stimulation by progesterone suggests an important signalling function for this steroid in myelination.

Neurosteroids: expression of functional 3beta-hydroxysteroid dehydrogenase by rat sensory neurons and Schwann cells.

Steroids which are synthesized within the nervous system, such as progesterone, have been termed 'neurosteroids'. Levels of progesterone are much larger in peripheral nerves of rats and mice than in plasma, and persist after removal of the steroidogenic endocrine glands. Schwann cells are a source of progesterone: when isolated from embryonic dorsal root ganglia, they can synthesize progesterone from pregnenolone, the obligate precursor of all steroids. Locally produced progesterone has been shown to play an important role in myelination of peripheral nerve. We show here that sensory neurons from embryonic dorsal root ganglia also express 3beta-hydroxysteroid dehydrogenase and can convert [3H]pregnenolone to [3H]progesterone. Moreover, when cultured under different conditions and incubated for 24 h in the presence of 100 nM [3H]pregnenolone, they produce 5-10 times more [3H]progesterone than Schwann cells. The conversion of pregnenolone to progesterone by neurons is further increased by a diffusible factor produced by Schwann cells. Sensory neurons can also metabolize progesterone to 5alpha-dihydroprogesterone, but unlike Schwann cells, they do not produce 3alpha,5alpha-tetrahydroprogesterone, a potent positive allosteric modulator of gamma-aminobutyric acid type A receptors. We also show that cells isolated from the adult nervous system still have the capacity to convert [3H]pregnenolone to progesterone and its 5alpha-reduced metabolites: neurons and Schwann cells purified from dorsal root ganglia of 6 week old male rats show a similar pattern of pregnenolone metabolism to cells isolated from 18 day old embryos. These findings further support the important role of progesterone in the development and regeneration of the peripheral nervous system.

A key enzyme in the biosynthesis of neurosteroids, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD), is expressed in rat brain.

In rat brain, the presence of pregnenolone and progesterone, not attributable to peripheral glandular sources, has been demonstrated and thus the two compounds can be classified as neurosteroids. In vitro experiments have shown the conversion of pregnenolone, a 3 beta-hydroxy-delta 5-ene steroid, into progesterone, a delta 4-oxo steroid, thus demonstrating a 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) enzymatic activity. The conversion of 3 beta-hydroxy-delta 5-derivatives into the corresponding delta 4-oxo steroids by 3 beta-HSD is an essential step in the biosynthesis of all steroid hormones in endocrine glands. To date, four isoforms of 3 beta-HSD have been characterized in the rat. We report here the selective expression of a 3 beta-HSD isoform in rat brain. An in situ hybridization study, using an oligonucleotide common to the 4 known isoforms, demonstrated 3 beta-HSD mRNA in neurons of the olfactory bulb, striatum, cortex, thalamus, hypothalamus, septum, habenula, hippocampus and cerebellum. The cerebellum showed the highest level of 3 beta-HSD mRNA corresponding to a transcript of 1.8 kb. Nucleotide sequencing of PCR-amplified cDNA fragments from cerebellar mRNA indicated the expression of an isoform of 3 beta-HSD cDNA very closely related to the isoform I expressed in the adrenals and gonads. Further evidence for the expression of 3 beta-HSD gene in the brain was demonstrated utilizing anti-peptide 3 beta-HSD antibodies which revealed an immunoreactive protein of approximately 45 kDa in the cerebellum. Our results demonstrate for the first time the expression of the enzyme 3 beta-HSD in the brain, at both the mRNA and protein levels. Since several neuroactive neurosteroids are substrates or products of the 3 beta-HSD enzymatic activity, our findings offer new possibilities to study the regulatory mechanisms governing their biosynthesis in the brain.

Androgen biosynthesis in the stomach: expression of cytochrome P450 17 alpha-hydroxylase/17,20-lyase messenger ribonucleic acid and protein, and metabolism of pregnenolone and progesterone by parietal cells of the rat gastric mucosa.

Dehydroepiandrosterone (DHEA) and its conjugates persist in the rat brain, for up to 1 month after ablation of both adrenals and gonads. Since DHEA synthesis in brain from pregnenolone (PREG) was excluded, we have considered other tissular sources including the digestive tract. In situ hybridization with specific oligonucleotide probes showed that the parietal cells of the gastric mucosa, contrary to other cell types, strongly expressed P450(17) alpha messenger RNA. Expression of the enzyme in the parietal cells was confirmed by immunocytochemistry with specific antibodies. An intense reaction was observed in the stomach of adult males and of cyclic or pregnant females. Access to food did not influence the intensity of immunostaining. It appeared at postnatal days 16-21, then the number of positive cells increased rapidly and leveled off at adult age. Parietal cells were released by pronase digestion of everted stomachs from adult male and female rats and were purified by density gradient centrifugation on Nycodenz. 5 x 10(4) to 1.6 x 10(6) cells were incubated with either 1 microM 14C-PREG or 14C-progesterone (14C-PROG) at 37 C under 95% O2-5% CO2, for 10-180 min. PREG was converted to 17-OH PREG and to androstenediol, whereas PROG was converted to 17-OH PROG and to testosterone. Only minute amounts of either DHEA or androstenedione, respectively, were detected at any incubation time, indicating their fast conversion to the corresponding 17 beta-hydroxysteroids. 3H-25-OH cholesterol was not metabolized to 3H-PREG, and 14C-PREG was not converted to 14C-PROG, in accordance with negative immunocytochemical results with antibodies to cytochrome P450scc and 3 beta-hydroxysteroid dehydrogenase delta 5-->4-isomerase (3 beta-HSD). In conclusion, the parietal cells, which are known as the source of gastric acid secretion, can synthesize testosterone from PROG and androstenediol from PREG. The physiological relevance of such conversions remains to be established.

Suppressed expression of the cytochrome P45017 alpha protein in the testicular feminized (Tfm) mouse testes.

The testes of testicular feminized (Tfm) mice synthesize and secrete abnormally low amounts of testosterone, as a consequence of selectively decreased cytochrome P450(17 alpha) activity. To investigate the mechanism of this deficiency, three steroidogenic enzymes were immunolabeled in the testes of normal and Tfm adult (2.5-6 month old) mice. Cholesterol side-chain cleavage cytochrome P450 (P450scc) and delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) were detected in the Leydig cells of both normal and Tfm mice whereas, in contrast to normal mice, only a small proportion of Leydig cells were immunostained for cytochrome P450-17 alpha-hydroxylase, C17-->20 lyase (P450(17 alpha)) in the testes of Tfm mice. The numbers of cells differed from male to male and interestingly were markedly higher in the right testis. Explants of testes from Tfm mice were kept in organ culture at 32 degrees C for 45 h, with or without dibutyryl cyclic AMP (100 or 500 mumol/l). All Leydig cells remained positive for P450scc and 3 beta-HSD, and P450(17 alpha) became detectable in the majority of Leydig cells in both left and right testes, showing that the lack of expression of P450(17 alpha) protein in Tfm mouse testes in vivo is not structural but is a regulatory phenomenon.

Astrocytes and neurosteroids: metabolism of pregnenolone and dehydroepiandrosterone. Regulation by cell density.

The rat central nervous system (CNS) has previously been shown to synthesize pregnenolone (PREG) and convert it to progesterone (PROG) and 7 alpha-hydroxy-PREG (7 alpha-OH PREG). Astrocytes, which participate to the regulation of the CNS function, might be involved in the metabolism of neurosteroids. Purified type 1 astrocytes were obtained from fetal rat forebrain with the use of selective culture conditions and were identified by immunostaining with specific antibodies (GFAP+, A2B5-). They were plated at low, intermediate, or high densities (2.5-5 x 10(5), 1-2 x 10(6), or 4-8 x 10(6) cells/dish, respectively) and maintained for 21 d. They were then incubated with 14C-PREG and 14C-DHEA for 24 h and the steroids extracted from cells and media were analyzed. Most radioactive derivatives were released into incubation media. Two metabolic pathways were mainly observed. PREG and DHEA were oxidized to PROG and androstenedione (ADIONE), respectively, [3 beta-hydroxysteroid-dehydrogenase, delta 5-->4 3-ketosteroid-isomerase (3 beta-HSD) activity], and converted to 7 alpha-OH PREG and 7 alpha-OH DHEA, respectively (7 alpha-hydroxylase activity). After low density plating, the formation of PROG and ADIONE was approximately 10% of incubated radioactivity, tenfold larger than that of 7 alpha-hydroxylated metabolites. In contrast, after high density plating, low levels of PROG and ADIONE were formed, whereas the conversion to either 7 alpha-OH PREG or 7 alpha-OH DHEA was > or = 50%. The results expressed per cell indicated that the 3 beta-HSD activity was almost completely inhibited at high cell density, in contrast to the 7 alpha-hydroxylation which was maintained or increased. The pattern of steroid metabolism was related to cell density at the time of measurement and not to an early commitment of cells: when primary cultures were plated at high density (8 x 10(6) cells/dish), then subcultured after several dilutions (3-, 9-, or 27-fold), the 3 beta-HSD activity was recovered only at low density. Furthermore, when 5 x 10(5) cells were centrifuged and the resulting clusters were plated, 3 beta-HSD activity was decreased, whereas steroid 7 alpha-hydroxylation was enhanced. This implies that cell density per se, but neither cell number nor a diffusible factor(s) is involved in the regulation of steroid metabolism. We conclude that astrocytes in culture metabolize PREG and DHEA, and that the metabolic conversions and, therefore, the related enzymatic activities depend on cell-to-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunoreactive cytochrome P-450(17 alpha) in rat and guinea-pig gonads, adrenal glands and brain.

The cytochrome P-450(17 alpha)-hydroxylase, 17----20 lyase (P-450(17 alpha)) is the key enzyme responsible for the biosynthesis of androgens in steroidogenic organs. Its cellular localization has been examined with an immunohistochemical technique. In immature rat ovary, P-450(17 alpha) was first detected in sparse interstitial cells on postnatal Day 8. The number of immunoreactive interstitial cells increased thereafter and the intensity of P-450(17 alpha) staining in these cells was highest at 3 weeks of age. The intensity of staining then started to decline and was very faint at Day 35. From 6 weeks on, the distribution of immunoreactive P-450(17 alpha) was of the adult type: it was detected exclusively in the thecal cells of the large antral, preovulatory, follicles. P-450(17 alpha) was not detectable during pregnancy except on the day of parturition, when thecal cells were transiently immunoreactive. The staining had vanished 24 h after delivery. Human chorionic gonadotrophin (hCG), injected into immature females on Days 24 to 26, induced P-450(17 alpha) prematurely in thecal cells. When injected on Days 12 to 14 of pregnancy, hCG also induced P-450(17 alpha) in the thecal cells surrounding the largest follicles, whereas the interstitial and luteal cells were not immunostained. The antiprogestin RU486, injected on Day 16 of pregnancy, reinstated P-450(17 alpha) (and P-450scc) immunoreactivity in the thecal cells. Oestradiol selectively suppressed P-450(17 alpha) expression in the thecal cells of RU486-treated females. In immature guinea-pig ovary, P-450(17 alpha) was immunostained in thecal cells, not in interstitial cells, although the interstitial cells expressed the delta 5-3 beta-hydroxysteroid dehydrogenase. P-450(17 alpha) was also immunolocalized in the Leydig cells of rat and guinea-pig testes, and in the guinea-pig adrenal cortex (zonae fasciculata and reticularis), but not in the rat adrenal cortex. P-450(17 alpha) was not detectable in the brain of either rat or guinea-pig.

Cell-specific variations and hormonal regulation of immunoreactive cytochrome P-450scc in the rat ovary.

Specific rabbit antibodies to the bovine cholesterol side-chain cleavage cytochrome P-450 (P-450scc) were used to cross-react with the enzyme in the rat ovary. The luteal cells of cyclic, pregnant, and pseudopregnant rats were immunostained. P-450scc was also expressed in the interstitial cells of prepubertal and cyclic adult rats, and in the thecal cells lining the preovulatory follicles. In cyclic females, RU 486 and oestradiol increased the intensity of P-450scc immunostaining. The granulosa cells of ovarian follicles whatever their stage of development, including preovulatory follicles, were not labelled, except after ovulation. The intensity of immunostaining of thecal and interstitial cells decreased during early pregnancy or pseudopregnancy, and disappeared after Day 9, whereas these cells were intensely labelled 24 h after parturition. The immunostaining of thecal and interstitial cells was again detected in 18-day pregnant rats, treated with the antiprogesterone RU 486. It is therefore concluded that both oestradiol and progesterone are involved in P-450scc regulation.

The cholesterol side-chain cleavage complex in human brain white matter.

Abstract Specific antibodies obtained in rabbits after injection of bovine cholesterol side-chain cleavage enzymes cytochrome P-450scc, adrenodoxin and adrenodoxin-reductase were used for immunohistochemical studies in human brain. The three enzymes were co-localized in the white matter of the cerebellum. This observation strongly suggests the existence of steroidogenic activity in human oligodendrocytes, as previously reported in the rat, and suggests that the concept of 'neurosteroids' can be applied to Delta5-3ss-hydroxysteroid metabolites of cholesterol that accumulate in human brain.

Neurosteroids: cytochrome P-450scc in rat brain.

The steroid hormones corticosterone and testosterone are supplied to the central nervous system by endocrine glands, the adrenals and gonads. In contrast, the 3 beta-hydroxy-delta 5-derivatives of cholesterol, pregnenolone and dehydroepiandrosterone, accumulate in the rat brain through mechanisms independent of peripheral sources. Immunohistochemical studies have been performed with specific antibodies to bovine adrenal cytochrome P-450scc, which is involved in cholesterol side-chain cleavage and pregnenolone formation. The enzyme was localized in the white matter throughout the brain. Scarce clusters of cell bodies were also stained in the entorhinal and cingulate cortex and in the olfactory bulb. These observations strongly support the existence of "neurosteroids," which have been posited on the basis of biochemical, physiological, and behavioral studies.

Alkaline phosphatase activity in the membrane of Xenopus laevis oocytes: effects of steroids, insulin, and inhibitors during meiosis reinitiation.

The mechanism of steroid hormone-induced reinitiation of meiosis in Xenopus laevis oocytes in vitro involves interaction of the hormone with an ooplasma membrane receptor and early changes of enzymatic activities (adenylate cyclase, p48 protein kinase). In full-grown (stage 6) oocytes, we have observed cytochemically, at the ultrastructural level, alkaline phosphatase activity in the ooplasma membrane of microvilli, its decrease by 2 hr of progesterone action, and its complete disappearance at the time of germinal vesicle breakdown (GVBD). Insulin (30 micrograms/ml) also provoked a decrease of phosphatase activity, although it did not promote GVBD under these circumstances. When oocytes were exposed simultaneously to progesterone (1 microM) and insulin (30 micrograms/ml), the enzymatic activity disappeared earlier than with any one of them, correlating with the faster occurrence of GVBD. Inhibitors of alkaline phosphatase activity and competitive substrates potentiated progesterone action on GVBD. Insulin and beta-glycerophosphate potentiating activities were additive. These results suggest that the ooplasma membrane alkaline phosphatase may be implicated in the course of reinitiation of meiosis in X. laevis oocytes.

Testosterone-induced meiotic maturation of Xenopus laevis oocytes: evidence for an early effect in the synergistic action of insulin.

Meiosis reinitiation in oocytes (stage 5-6 of Dumont) isolated free of follicle cells by collagenase treatment from ovarian pieces of Xenopus laevis, was studied in observing the germinal vesicle breakdown (GVBD) provoked by progesterone and testosterone (0.1 nM-1 microM), alone or in association with insulin (30 micrograms/ml). Testosterone, was more active than progesterone to elicit GVBD in vitro, raising the question of the relative roles of both steroids in the physiological maturation process in vivo. The potentiating effect of insulin, already observed on progesterone action, was also demonstrated upon testosterone effect; the results suggested that it occurs during the early phase of hormone-induced meiosis reinitiation.

[Potentiation of the effect of steroid hormones (progesterone and testosterone) inducing the reinitiation of meiosis in Xenopus oocytes in vitro, by inhibitors of phosphatase activity]. / Potentialisation de l'effet des hormones stéroïdes (progestérone et testostérone) inductrices de la reprise de la méiose de l'ovocyte de Xénope in vitro, par des inhibiteurs de l'activité phosphatasique.

The induction of meiosis reinitiation by steroid hormones (progesterone and testosterone) in Xenopus laevis oocytes was studied in vitro in presence of inhibitors of phosphatase activity such as beta-glycerophosphate, considered as a competitive inhibitor, and the two ions, Zn++ and MoO--4. Kinetics of the germinal vesicle breakdown indicating the reinitiation of meiosis, have shown that while these phosphatase inhibitors were not active by themselves under the present experimental conditions, they enhanced the process elicited by progesterone or testosterone.