Progesterone as a neurosteroid: synthesis and actions in rat glial cells.

The central nervous system (CNS) and the peripheral nervous system (PNS) are targets for steroid hormones where they regulate important neuronal functions. Some steroid hormones are synthesized within the nervous system, either de novo from cholesterol, or by the metabolism of precursors originating from the circulation, and they were termed 'neurosteroids'. The sex steroid progesterone can also be considered as a neurosteroid since its synthesis was demonstrated in rat glial cell cultures of the CNS (oligodendrocytes and astrocytes) and of the PNS (Schwann cells). Both types of glial cells express steroid hormone receptors, ER, GR and PR. As in target tissue, e.g. the uterus, PR is estrogen-inducible in brain glial cell cultures. In the PNS, similar PR-induction could not be seen in pure Schwann cells derived from sciatic nerves. However, a significant PR-induction by estradiol was demonstrated in Schwann cells cocultured with dorsal root ganglia (DRG), and we will present evidence that neuronal signal(s) are required for this estrogen-mediated PR-induction. Progesterone has multiple effects on glial cells, it influences growth, differentiation and increases the expression of myelin-specific proteins in oligodendrocytes, and potentiates the formation of new myelin sheaths by Schwann cells in vivo. Progesterone and progesterone analogues also promotes myelination of DRG-Neurites in tissue culture, strongly suggesting a role for this neurosteroid in myelinating processes in the CNS and in the PNS.

Intracellular distribution of a cytoplasmic progesterone receptor mutant and of immunophilins cyclophilin 40 and FKBP59: effects of cyclosporin A, of various metabolic inhibitors and of several culture conditions.

The effect of cyclosporin A (CsA) on the intracellular distribution of a mutated NLS minus rabbit progesterone receptor (PRm) and the receptor-associated immunophilins, cyclophilin 40 (Cyp40) and FKBP59, was tested in Lc13 cells by indirect immunofluorescent staining. PRm, which is cytoplasmic in absence of progesterone, is shifted to the nucleus by the hormone as well as by CsA, but not by FK506 or Rapamycin [I. Jung-Testas, M.-C. Lebeau, E.E. Baulieu. C.R. Acad. Sci. Paris 318 (1995) 873-878]. However the time course of nuclear import due to CsA and its sensitivity to N-ethyl maleimide (NEM) and to a calmodulin inhibitor (W7) was different from those observed for the hormonal effect. Cyp40 in Lc13 cells is localized mainly in the nucleoli. CsA treatment increased nucleolar staining, while NEM and W7 caused it to decrease; after actinomycin D (1 microM) nucleolar staining of Cyp40 disappeared. FKBP59 is mainly cytoplasmic and concentrated in the perinuclear region, never in the nucleoli. CsA, actino D and W7 treatment did not influence FKBP59 localization. In serum-deprived medium FKBP59 was cytoplasmic, but when the culture medium was enriched (20% serum, insulin and EGF) FKBP59 became perinuclear and hsp 86 was partly shifted to the nucleus, but PRm remained cytoplasmic. CsA has an effect on PRm distribution, while it does not influence Cyp40 and FKBP59 localization. In presence of actino D the labelling of Cyp40 disappears from the nucleoli, while the distribution of PRm and FKBP59 is unaffected. Growth factors influence FKBP59 but not PRm or Cyp40. These results suggest that these proteins shuttle independently and that their association is transient.

Steroid hormone receptors and steroid action in rat glial cells of the central and peripheral nervous system.

The nervous system is a target for sex steroid hormones which have profound actions on the growth, maturation, differentiation and functioning of brain cells. We found that some steroids, termed "neurosteroids", are synthesized within the brain by glial cells. The term "neurosteroids" designates their site of synthesis--the nervous system, either de novo from cholesterol or from steroid hormone precursors. The biological effects of steroid hormones are mediated by specific high-affinity intracellular receptors, which, after hormone binding, function as activated transcription factors. The presence of such receptors was shown in primary cultures of oligodendrocytes and astrocytes, derived from forebrains (CNS), and in Schwann cells, derived from sciatic nerves (PNS), of newborn rats. In glial cells of the CNS, progesterone-, glucocorticoid-, estrogen and androgen-receptors (PR, GR, ER, AR) were demonstrated and of these receptors, only PR was estrogen-inducible. In glial cells of the PNS, the presence of PR and ER was shown, but the PR in Schwann cell cultures was not inducible by estrogen treatment. Different effects of steroids on glial cell growth and differentiation during primary culture were observed. In particular, a striking increase of myelin-specific proteins such as myelin basic protein (MBP) and cyclic nucleotide phosphodiesterase (CNPase) was observed when oligodendrocytes, the myelinating glial cells of the CNS, were cultured in the presence of progesterone, as determined by indirect immunofluorescence staining and immunoblotting. Insulin also increases MBP and CNP-ase in oligodendrocytes and the combined treatment (insulin + progesterone) promotes a strong synergistic stimulation (14-fold increase) of myelin protein expression. Estradiol also increases MBP- and CNPase expression in oligodendrocytes, although to a lesser extent than progesterone. In the search for optimal stimulation of myelin-protein expression, several progesterone analogues were tested and the results are discussed.

Neuronal signals are required for estrogen-mediated induction of progesterone receptor in cultured rat Schwann cells.

Rat glial cells from the central nervous system (CNS) and the peripheral nervous system (PNS) express steroid hormone receptors. Whereas progestin receptors (PR) in cultured glial cells of the CNS are estrogen-inducible, similar increase of PR in cultured Schwann cells, the glial cells of the PNS, prepared from newborn rat sciatic nerves, could not be demonstrated. In the present work we have used fetal dorsal root ganglion cultures to study the effect of estrogen and its antagonist ICI 164,384 on the expression of PR in rat Schwann cells. The PR levels were measured by hormone binding in whole cell assays or in cell cytosol, 18 h after excision of the ganglion from the cultures. Treatment of DRG-Schwann cell cultures with estradiol (E2) increased PR levels from about 60 to 160 fmol per mg cytosol protein, in untreated and estrogen-treated cells, respectively. This increase was dose-dependent; maximal induction was obtained at 50 nM E2-concentration. Treatment of the cultures with the antagonist ICI 164,384 completely inhibited the estrogen-induction of PR, whereas ICI alone did not influence receptor levels in Schwann cells. The estrogen-induction of PR was dependent on the presence of dorsal root ganglion during the period of estrogen treatment. Excision of the neuronal mass from the cultures caused a rapid decrease and disappearance of estrogen-inducible progestin receptors, whereas the concentration of non-inducible PR-binding sites remained unchanged. Estradiol had no influence on DRG-Schwann cell proliferation, only replated secondary Schwann cells showed a slightly higher level of proliferation in presence of 100 nM E2 and 5 microM forskolin. Receptors for estrogen (ER) were also demonstrated in DRG-Schwann cells by ligand binding experiments. Specific ER-binding was 36 +/- 8 fmol bound estradiol per mg cytosol protein. Finally, both PR and ER were visualized in Schwann cells by indirect immunofluorescence staining using specific anti-receptor antibodies. These findings suggest that the expression of estrogen-inducible progestin receptors in cultured glial cells of the PNS is mediated via intracellular estrogen receptors and that it requires the presence of neuronal signal(s).

Demonstration of progesterone receptors in rat Schwann cells.

We have recently shown that progesterone promotes myelin formation in peripheral nerves of rodents. In this study, we demonstrate the presence of progesterone receptors (PR) in primary cultures of rat Schwann cells, the glial cells of the PNS, prepared from sciatic nerves of 4-5 days old rats. After 3 weeks of culture, the presence of PR was measured by whole cell assay after incubating living cells for 1 h at 37 degrees C with [3H]-Organon 2058 as a ligand, and about 5000 specific binding sites per cell were found. In contrast to the PR of rat glial cells from the central nervous system (CNS), which is induced by estrogens, treatment of Schwann cells with estradiol did not increase the PR-binding, even after exposure of cells to high doses of estrogen under various culture conditions. Progesterone receptors were also visualized in Schwann cells by indirect immunofluorescence staining with a monoclonal anti-PR antibody. Again, treatment of the cells with estradiol did not increase the immunofluorescence staining of the PR. Specific PR binding was also measured in sciatic nerves of adult female rats. Cytosol was prepared and labeled with [3H]-Organon 2058 for 15 h at 2 degrees C. After treatment with dextran-coated charcoal, specific ligand binding was about 30 fmol/mg cytosolic protein. When castrated adult female rats were treated with estradiol (20 micrograms EB/day for 3 days), no PR-induction was observed in the cytosol of sciatic nerves. In contrast, PR-binding sites in cytosols prepared from pituitary gland and uteri of the same animals were significantly increased by the estrogen.

Progesterone as a neurosteroid: actions within the nervous system.

1. Some progesterone is synthesized within both the central and the peripheral nervous systems, where it regulates neurotransmission and important glial functions, such as the formation of myelin. Progesterone can thus be designated a "neurosteroid." 2. Steroids act not only on the brain, but also on peripheral nerves, which offer many advantages to study the biological significance of locally produced neurosteroids: their remarkable plasticity and regenerative capacity and their relatively simple structure. 3. By using the regenerating mouse sciatic nerve as a model, we have shown that progesterone synthesized by rat Schwann cells promotes the formation of new myelin sheaths. Progesterone also increases the number of myelinated axons when added at a low concentration to cocultures of Schwann cells and sensory neurons. 4. These findings show a function on myelination for locally produced progesterone and suggest a new pharmacological approach of myelin repair.

Cyclosporin A promotes nuclear transfer of a cytoplasmic progesterone receptor mutant.

The effects of cyclosporin A (CsA), FK506 and rapamycin (Rapa) on the intracellular localization of a mutated rabbit progesterone receptor (PR) which lacks the main constitutive nuclear localization signal (NLS) (delta 638-642) and is cytoplasmic in the absence of progesterone (Prog), were assayed by indirect immunofluorescence in Lcl3 cells, a mouse L-cell line stably expressing this mutant. CsA alone, at 5-10 microM concentrations, induced almost complete nuclear transfer of the PR-mutant within 18 h. In contrast, FK506 and Rapa at the same concentrations had no effect. This nuclear transfer induced by CsA was concentration and time dependent and was independent of protein synthesis. It was not a potentiation of hormone action since it took place in the absence of hormone, including in serum-free culture conditions. The implications of this specific effect of CsA are discussed.

Progesterone synthesis and myelin formation by Schwann cells.

Progesterone is shown here to be produced from pregnenolone by Schwann cells in peripheral nerves. After cryolesion of the sciatic nerve in male mice, axons regenerate and become myelinated. Blocking either the local synthesis or the receptor-mediated action of progesterone impaired remyelination. Administration of progesterone or its precursor, pregnenolone, to the lesion site increased the extent of myelin sheath formation. Myelination of axons was also increased when progesterone was added to cultures of rat dorsal root ganglia. These observations indicate a role for locally produced progesterone in myelination, demonstrate that progesterone is not simply a sex steroid, and suggest a new therapeutic approach to promote myelin repair.

Actions of steroid hormones- and growth factors on glial cells of the central and peripheral nervous system.

Primary cultures of oligodendrocytes and astrocytes and purified cultures of Schwann cells were prepared respectively from forebrain and sciatic nerves of newborn rats. The effects of steroid hormones and growth factors on glial cell growth and on the production of myelin-specific proteins and lipids were investigated. Progesterone (P, 100 nM) decreased the proliferation of glial cells of the central nervous system. This inhibitory effect of P was abolished by the simultaneous administration of the antagonist RU486, thus suggesting a receptor-mediated action of the hormone. The expression of myelin-specific proteins, including the myelin basic protein (MBP) and the 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase), and of a myelin-specific lipid, galactocerebroside (Gal C), was also measured during cell differentiation under different hormonal conditions. The expression of MBP in oligodendrocytes was increased by P, and this effect was not blocked by RU486. The combined application of P and insulin promoted a synergistic stimulation of MBP expression. Insulin, by itself, also increased the number of MBP-positive oligodendrocytes in culture. The effects of P and insulin appeared to be selective as dexamethasone, dehydroepiandrosterone, pregnanolone and epidermal growth factor (EGF) had no effect. Only estradiol (E2, 500 nM) increased the number of MBP-immunoreactive cells, but in contrast to P, only a small synergism between E2 and insulin on MBP expression was observed. The expression of CNPase, another myelin-specific protein, was also increased by P and, here again, a synergy between P and insulin could be observed. In contrast, the expression of Gal C, a myelin-specific lipid, was not modified by P or other steroid hormones. Moreover, the increase in Gal C-positive cells observed in response to insulin alone was not further potentiated by P. Glial cells of the peripheral nervous system, namely Schwann cells, are also sensitive to steroid hormones. Schwann cells contain estrogen receptors, and E2 stimulates their proliferation in the presence of forskolin or dibutyryl cyclic AMP (dbcAMP). The mitogenic effect of E2 was abolished by the pure antiestrogen ICI-164,384. Insulin, at micromolar concentration, also stimulated Schwann cell growth when forskolin or dbcAMP were present in the culture medium. The mitogenic effect of insulin was mediated by insulin-like growth factor I (IGF-I) receptors. Indeed, at a physiological nanomolar concentration, IGF-I but not insulin or IGF-II, increased the proliferation of Schwann cells in synergy with forskolin. In addition, Schwann cells express receptors for IGF-I.

[Inhibition of the anti-glucocorticosteroid effect of RU486 on the growth of mouse fibroblasts in culture by the immunosuppressor kf506]]. / Inhibition par l'immunosuppresseur FK506 de l'effet antiglucocorticostéroïde du RU486 sur la croissance de fibroblastes de souris en culture.

The growth of mouse L-929 fibroblasts in culture is inhibited by dexamethasone, a synthetic glucocorticosteroid, and this effect is itself suppressed by the antiglucocorticosteroid RU486 (mifepristone). Neither RU486 nor the immunosuppressant FK506 alone influence L-929 growth, and FK506 does not modify dexamethasone action. However, FK506 suppresses the antiglucocorticosteroid activity of RU486. The implication of the "anti-antagonist" activity of FK506 in its immunosuppressant properties has still to be explained. The role of the recently cloned, FK506-binding, p59 immunophilin, which binds to the heat shock protein hsp90 which itself interacts with the glucocorticosteroid receptor, is discussed.

Insulin-like growth factor I: a mitogen for rat Schwann cells in the presence of elevated levels of cyclic AMP.

To develop effective procedures for improving the regeneration of peripheral nerves and for preventing the formation of neurofibromas, it is necessary to identify the different mitogens that stimulate the proliferation of Schwann cells. Insulin-like growth factor I (IGF-I), which is a potent autocrine growth factor in many tissues, is synthesized by proliferating Schwann cells. However, the role of IGF-I in stimulating their division is still uncertain. Here we show that nanomolar concentrations of IGF-I stimulate the growth of Schwann cells in primary culture. IGF-I alone was uneffective, but in the presence of forskolin (5 microM) or dibutyryl cyclic AMP (dbcAMP, 10 microM), it became a potent mitogen. Neither IGF-II nor epidermal growth factor (EGF) were effective even in the presence of forskolin. Insulin also stimulated Schwann cell proliferation in the presence of forskolin, but only at micromolar concentration. Receptors for IGF-I were visualized on the Schwann cell surface by indirect immunofluorescence staining using anti-human IGF-I receptor antibodies. Their presence was also assessed by binding assays using [125I]-IGF-I as a ligand. Scatchard analysis showed a single class of high-affinity receptors (Kd = 1.5 nM). Competitions studies with unlabeled IGF-I or insulin indicated a half-maximal displacement of [125I]-IGF-I by IGF-I at about 5 nM, while insulin was about 500-fold less effective. The number of binding sites for IGF-I was increased by exposing cells for 3 days to forskolin (-forskolin: about 5,100; + forskolin: about 12,200 binding sites/cell).(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of rat Schwann cell proliferation by estradiol: synergism between the estrogen and cAMP.

Schwann cells dissociated from sciatic nerves of 4- to 5-day-old rats were established in long-term primary cultures. After treatment with anti-Thy-1.1 and complement, pure Schwann cell cultures were obtained as confirmed by indirect immunofluorescence staining of the S-100 antigen. The effect of estradiol on Schwann cell proliferation was tested. The estrogen alone had no effect on cell multiplication, but in the presence of forskolin (5 microM) or dibutyryl cyclic AMP (dbcAMP, 1 microM), estradiol (100 nM) became a potent mitogen. This effect was specific since when the anti-estrogen ICI-164,384 (200 nM) was added to the cultures, it completely inhibited the mitogenic effect of estradiol. In the absence of the estrogen, ICI-164,384 did not inhibit dbcAMP-induced cell growth. The effect of progesterone was also tested in the presence of forskolin, and no influence on cell multiplication was seen. Specific and saturable binding sites for estradiol were measured in Schwann cells by whole cell assay after labeling cells at 37 degrees C with [3H]estradiol and about 3000 specific binding sites per cell were found. Treatment of the cultures with forskolin (5 microM) for 48 or 72 h, but not for shorter times, resulted in a two-fold increase in estradiol binding. Estrogen receptors were visualized inside Schwann cells with a monoclonal anti-ER antibody by indirect immunofluorescence staining. Double-label immunofluorescence of ER and S-100 showed that ER immunostaining was restricted to spindle-shaped Schwann cells expressing the S-100 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurosteroids in rat sciatic nerves and Schwann cells.

Pregnenolone (PREG) and corticosterone were measured in sciatic nerves and plasma of adult male rats by radioimmunoassays. Concentrations of PREG were higher in nerves than in plasma, whereas those of corticosterone were lower. In contrast to corticosterone, PREG was not reduced by castration and adrenalectomy, strongly suggesting a local synthesis of this steroid independent of glandular sources. In fact, Schwann cells in culture produced PREG from 25-hydroxy-cholesterol. They also converted PREG and dehydroepiandrosterone to the respective 7 alpha-hydroxylated metabolites and to 5-androstene-3 beta,17 beta-diol. The metabolism of steroids was increased when Schwann cells were grown in the presence of insulin and forskolin. These findings may have important implications for understanding the regeneration of peripheral nerves.

Low density lipoprotein-receptors in primary cultures of rat glial cells.

Newborn rat glial cells in primary culture contain an active cholesterol side chain cleavage cytochrome P450. Cholesterol can be supplied either by biosynthesis or derive from low density lipoproteins (LDL), which bind apolipoprotein Band E (apoB,E) (LDL)-receptors and undergo receptor-mediated endocytosis. Using antibodies to purified human plasma LDL and antibodies to bovine adreno-cortical LDL-receptor, the presence of LDL-receptors was demonstrated on rat glial cells after 3-4 weeks of primary culture, by ligand blotting, immunoblotting, and indirect immunofluorescence staining. The latter approach indicated that oligodendrocytes express higher levels of LDL-receptors than astrocytes present in the same culture. The immunofluorescence staining was observed not only at the cell surface, but also within the cytoplasm, suggesting that the LDL-receptor complexes had been internalized. Western blotting of LDL-receptors extracted from glial cells indicated a band of approximately 130 kDa, the size expected for intact receptors. Their functionality was shown by the conversion of [3H]cholesterol linoleate, incorporated into reconstituted LDL and added to the cell cultures, to [3H]pregnenolone and/or its 20 alpha-hydroxy-metabolite. This is the first characterization of functional LDL-receptors on isolated, well characterized, normal brain cells.

Demonstration of steroid hormone receptors and steroid action in primary cultures of rat glial cells.

Primary cultures of rat glial cells were established from newborn rat forebrains. A mixed population of oligodendrocytes and astrocytes was obtained, as confirmed by indirect immunofluorescence staining with specific markers for each cell type. Receptors were measured 3 weeks after primary culture in glial cells cultured in the presence or not of 50 nM estradiol and we have identified progesterone, glucocorticoid, estrogen, and androgen receptors (PR, GR, ER and AR), but only PR was inducible by the estrogen treatment. This estrogen-induction of PR was more dramatic in glial cells derived from female offsprings than from males, as measured by binding studies and by immunohistochemical techniques with the KC 146 anti-PR monoclonal antibody. The antiestrogen tamoxifen inhibited the estrogen induction, but had no effect by itself on PR concentration. Specific binding sites for PR, GR, ER and AR were measured by whole cell assays after labeling cells with, respectively, [3H]R5020, [3H]dexamethasone, [3H]OH-tamoxifen or [3H]R1881. PR and GR were also analyzed by ultracentrifugation and after exposure of cells to agonists, both receptors were recovered from cytosol as a 9S form, and from the nuclear high-salt, tungstate ions-containing fraction as a 4-6S form. In contrast, when the antiprogestin- and antiglucocorticosteroid RU486 was used as a ligand, a non-activated 8.5S receptor complex was found for both receptors in this nuclear fraction. The 8.5S complex of the GR was further analyzed in the presence of specific antibodies and, in addition to GR, the presence of the heat shock protein hsp90 and of a 59 kDa protein was found. During primary culture, the effects of progesterone (P) and estradiol (E2) were tested on glial cell multiplication, morphology and differentiation. Cell growth was inhibited by P and stimulated by E2. Both hormones induced dramatic morphologic changes in oligodendrocytes and astrocytes and increased synthesis of the myelin basic protein in oligodendrocytes and of the glial fibrillary acidic protein in astrocytes.

Estrogen-inducible progesterone receptor in primary cultures of rat glial cells.

The presence of an estrogen-inducible progesterone receptor was demonstrated in primary cultures of newborn rat glial cells by biochemical and immunohistochemical techniques. The progesterone receptor (PR) was measured 3-4 weeks after primary culture in estradiol-containing or control medium. Cells were labeled with the synthetic progestin [3H]R5020 followed by ultracentrifugation analysis of the cellular extracts. A "9 S" PR was observed in the cytosol and a "4-5 S" PR was found in the nuclear high salt, tungstate ions containing extract of estradiol-treated cells. When the antiprogestin [3H]RU486 was used instead of [3H]R5020 as a ligand, a 9 S PR was also found in the cytosol, but a nonactivated "8.5 S" receptor complex was identified in the high salt nuclear fraction in presence of tungstate ions. The levels of PR, as measured by whole cell assay, were significantly increased when glial cells were cultured in the presence of 50 nM estradiol, as compared to nonestradiol-treated controls. The estrogen induction of PR was suppressed by the antiestrogen tamoxifen, but tamoxifen by itself had no effect on PR concentration. When the glucocorticosteroid receptor and PR were measured in parallel after estradiol treatment of the same primary culture, only the levels of PR were increased. The PR was visualized inside glial cells by immunohistochemical studies with a monoclonal antibody specific for the B-form of PR (KC 146), which was recognized by fluorescein-linked or biotinylated secondary antibodies. Strong staining was observed in estradiol-treated cultures, when compared to a weaker staining in control cultures. This is the first demonstration of PR in rat glial cells, and we present evidence of its induction by estradiol in primary cultures.

Neurosteroids: biosynthesis, metabolism and function of pregnenolone and dehydroepiandrosterone in the brain.

Pregnenolone (P) and dehydroepiandrosterone (D) accumulate in the brain as unconjugated steroids and their sulfate (S) and fatty acid (L) esters. The microsomal acyl-transferase activity is highest in immature (1-3 weeks old) male rats. The immunocytochemical and biochemical evidence for P biosynthesis by differentiated oligodendrocytes is reviewed. The importance of P synthesis for its brain accumulation is assessed by the intracysternal injection of the inhibitor aminoglutethimide. Primary glial cell cultures convert P to 20-OH-P, PL, progesterone, 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnane-20-one (Polone). Astroglial cell cultures also produce these metabolites, whereas neurons from 17-day mouse embryos only form 20-OH-P. P and D are converted to the corresponding 7 alpha-hydroxylated metabolites by a very active P-450 enzyme from rat brain microsomes. Several functions of neurosteroids are documented. P decreases in olfactory bulb of intact male rats exposed to the scent of estrous females. D inhibits the aggressive behavior of castrated male mice towards lactating female intruders. The D analog 3 beta-methyl-androst-5-en-17-one, which cannot be metabolized into sex steroids and is not demonstrably androgenic or estrogenic is at least as efficient as D. Both compounds elicit a marked decrease of PS in rat brain. The Cl- conductance of gamma-aminobutyric (GABAA) receptor is stimulated by GABA agonists, an effect which is enhanced by Polone and antagonized by PS. Thus, P metabolites in brain as well as steroids of extraencephalic sources may be involved physiologically in GABAA receptor function. The neurosteroids accumulated in brain may be precursors of sex steroid hormones and progesterone receptors have been localized in glial cells. P and D do not bind to any known intracellular receptor. A heat stable P binding protein has been found in brain cytosol with distinct ligand specificity. A binding component specific for steroids sulfates, including Polone S, DS and PS, in the order of decreasing affinity is localized in adult rat brain synaptosomal membranes. Its relationship to the GABAA receptor is under current investigation.

The nonactivated progesterone receptor is a nuclear heterooligomer.

The discovery of the nuclear localization of estradiol and progesterone receptors in the absence of the steroid hormone has led to reconsideration of the model of cytoplasmic to nuclear translocation of these receptors upon exposure to hormone. Unoccupied nonactivated receptors are thought to be weakly bound to nuclei of target cells from which they are leaking during tissue fractionation and thus found in the cytosol fraction of homogenates in a nontransformed heterooligomeric "8-9 S" form, which includes hsp90. However, no direct biochemical evidence has yet been obtained for the presence of such heterooligomers in the target cell nucleus, possibly because it dissociates in high ionic strength medium used for extraction of the nuclear receptor. We took advantage of the combined stabilizing effects of tungstate ions and antiprogestin RU486 to extract a nuclear non-DNA binding nontransformed 8.5 S-RU486-progesterone receptor complex from estradiol-treated immature rabbit uterine explants incubated with the antagonist. As demonstrated by immunological criteria and by irreversible cross-linking with dimethylpimelimidate, the complex contained, in addition to the hormone binding unit, hsp90, and p59, another nonhormone binding protein. Control experiments carried out with the progestin R5020 yielded the expected nuclear transformed DNA binding 4.5 S-R5020-progesterone receptor complex. These results offer evidence for two distinct forms of steroid receptor in target cell nuclei. Besides the classical "4 S" agonist-receptor complex, tightly bound to the DNA-chromatin structure and in all probability able to trigger the hormonal response, we have observed in the RU486-bound state a non-DNA binding nontransformed 8.5 S form, presumably already present in the nucleus in the absence of hormone and representing the native nonactive form of the receptor.

Neurosteroids: biosynthesis of pregnenolone and progesterone in primary cultures of rat glial cells.

Cells dissociated from newborn rat forebrains were established in long term primary cultures. The cultures were made up almost exclusively of oligodendrocytes and astrocytes, as confirmed by indirect immunofluorescence staining with monoclonal antibodies to galactocerebroside and glial fibrillary acidic protein, respectively. After 3 weeks of culture, the oligodendrocytes were also highly immunoreactive to monospecific polyclonal antibodies against cytochrome P450scc, an enzyme involved in the conversion of cholesterol to pregnenolone (P). Biosynthesis of [3H]cholesterol, [3H]P, and [3H]Pregn-5-ene-3 beta, 20 alpha-diol was demonstrated in these primary cultures by incubating cells with [3H]mevalonolactone in the presence of mevinolin and trilostane. The activity of the 2',3'-cyclic nucleotide 3'-phosphodiesterase enzyme, a documented indicator of oligodendrocyte differentiation, increased rapidly after day 10 of culture, together with the onset of steroid biosynthetic activity. Both reached a maximum at 3 weeks of culture and remained stable up to 6.5 weeks. In the absence of trilostane, [3H]P was converted by glial cell cultures to [3H]progesterone, [3H]5 alpha-pregnane-3,20-dione, and [3H]3 alpha-hydroxy-5 alpha-pregnan-20-one. The demonstration of P, pregn-5-ene-3 beta,20 alpha-diol, and progesterone synthesis by normal rat glial cells, once oligodendrocytes have undergone their differentiation process, brings additional support to the concept of "neurosteroids."