I. Introduction

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Steroid hormones, which are synthesized in the adrenal gland, gonads and placenta, exert a large array of biological effects on the nervous system. In particular, steroid hormones play an important role in the development, growth, maturation, and differentiation of the central nervous system (CNS)² and peripheral nervous system (PNS) (for review, McEwen, 1994). Depending on their chemical nature and concentration, steroids can induce either protective or deleterious effects on nerve cells (Uno et al., 1989; Yu, 1989; Jones, 1993; Sapolsky, 1996; Green et al., 1997; Seckl, 1997; Kimonides et al., 1998). These actions had long been exclusively ascribed to steroids produced by endocrine glands, which can easily cross the blood-brain barrier to act on the CNS. However, a series of studies conducted by Baulieu and coworkers have shown that the rat brain is capable of synthesizing various steroid hormones such as pregnenolone (⁵P) and dehydroepiandrosterone (DHEA) from cholesterol (Baulieu, 1981). These authors first demonstrated the existence of high amounts of ⁵P and DHEA in the brain of castrated and adrenalectomized rats (Corpéchot et al., 1981, 1983). Thereafter, they found that the cerebral concentrations of ⁵P and DHEA are not affected by administration of adrenocorticotropic hormone or suppression of circulating glucocorticoids by dexamethasone (Robel and Baulieu, 1985). They also showed that the levels of ⁵P and DHEA in the brain undergo circadian variations which are not synchronized with those of circulating adrenal steroids (Robel et al., 1986). Finally, the immunohistochemical localization of cytochrome P-450 side chain cleavage (scc) in rat oligodendrocytes and the observation that the enzyme was biologically active (i.e., capable of converting cholesterol into ⁵P) unambiguously demonstrated that steroids can be synthesized within the CNS (Le Goascogne et al., 1987). The term neurosteroids has been coined to designate steroids that are newly synthesized from cholesterol or another early precursor in the nervous system, and are thus still present in substantial amounts after removal of peripheral steroidogenic glands (Robel and Baulieu, 1994). Neurosteroids occur in the nervous system as unconjugated steroids, sulfated esters of steroids, or fatty acid esters of steroids (Jo et al., 1989). These various forms of steroids are involved in the control of metabolic, behavioral, and psychical processes including cognition, stress, anxiety, and sleep (Majewska, 1992; Baulieu and Robel, 1996).

Besides their actions at the transcriptional level (McEwen, 1994), neuroactive steroids may act on nerve cells via two types of membrane receptors. Steroids can exert allosteric modulation of receptors for neurotransmitters such as -aminobutyric acid (GABA)_A receptors (Majewska, 1992), nicotinic receptors (Valera et al., 1992), muscarinic receptors (Klangkalya and Chan, 1988), N-methyl-D-asparate (NMDA) receptors (Wu et al., 1991), and  receptors (Monnet et al., 1995). In addition, it has been proposed that neuroactive steroids may act on nerve cells via proper membrane receptors coupled to G proteins (Orchinik et al., 1992) or through specific membrane sites using calcium as an intracellular messenger (Ramirez and Zheng, 1996). The recent demonstration that progesterone and 5-dihydroprogesterone directly inhibit oxytocin receptor function suggests that neurosteroids may also interact with various membrane-bound neuropeptide receptors (Grazzini et al., 1998).

	II. Biochemical Pathways of Steroid Biosynthesis in Endocrine Glands

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All steroid hormones derive from cholesterol which is provided by blood as low-density and high-density lipoproteins. A small proportion of cholesterol can also be produced directly in steroidogenic cells from acetate. The biochemical reactions responsible for the synthesis of steroids are controlled by various families of enzymes including hydroxylases (desmolases), oxydo-reductases (dehydrogenases), sulfotransferases (sulfokinases), and sulfuryl transferases (Fig. 1). Molecular cloning of the enzymes responsible for biosynthesis of steroid hormones has revealed, for some of these enzymes, the existence of multiple isoforms which are differentially expressed in steroidogenic tissues (Miller, 1988; Labrie et al., 1994). It has also been shown that various peripheral organs, such as the digestive tract (Dalla-Valle et al., 1992; Le Goascogne et al., 1995), the liver (Martel et al., 1994), and the prostate (Bélanger et al., 1989), can express at a low level the genes encoding several steroidogenic enzymes, suggesting that the production of bioactive steroids is not restricted to steroidogenic endocrine glands. The mechanisms regulating the expression of steroidogenic enzymes have been studied in great detail in the adrenal gland and gonads (Miller, 1988; Güse-Behling et al., 1992; Penning, 1997).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Biosynthesis of steroid hormones in endocrine glands. P-450scc, P450c17, 3-HSD, 17-HSD, 11OHase, 11-hydroxylase; 18OHase, 18-hydroxylase; 18-HSOR, 18-hydroxysteroid oxidoreductase; 21OHase, 21-hydroxylase.

	III. Cytochrome P-450scc

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The scc of cholesterol leading to the formation of ⁵P is catalyzed by cholesterol-desmolase, an enzymatic complex composed of cytochrome P-450scc (P-450scc) which possesses a hydroxylasic activity, adrenodoxine or ferredoxine, and adrenodoxine reductase (Fig. 1). Using an antibody raised against bovine adrenal P-450scc, Le Goascogne et al. (1987) have shown the presence of immunoreactive elements in the white matter throughout the rat brain, an observation which is rightly considered as the fundamental discovery that paved the way for further research on neurosteroids. The fact that glial cells in primary culture are capable of converting cholesterol into ⁵P has subsequently demonstrated that the immunoreactive P-450scc localized in the rodent brain actually corresponds to an active form of the enzyme (Jung-Testas et al., 1989). In addition, the occurrence of the mRNAs encoding for P-450scc and adrenodoxine has been evidenced in the CNS of mammals by means of various approaches including reverse transcription-polymerase chain reaction (RT-PCR), ribonuclease protection assays, and in situ hybridization (Mellon and Deschepper, 1993; Compagnone et al., 1995a). The P-450scc gene is expressed at a particularly high concentration in the cerebral cortex and, to a lesser extent, in the amygdala, hippocampus, and midbrain. The distribution of P-450scc mRNA is similar in the brain of female and male rats (Mellon and Deschepper, 1993; Compagnone et al., 1995a). The P-450scc gene is also expressed in the nervous system of developing rodent embryos, specifically in the cell lineages derived from the neural crest and in sensory structures of the PNS (Compagnone et al., 1995a). Recently, Ukena et al. (1998) have shown the presence of P-450scc in Purkinje cells of neonatal and adult rats, indicating that the gene is not only expressed in glial cells but also in neurons. The detection of relatively high amounts of ⁵P in the frog hypothalamus (Mensah-Nyagan et al., 1994) and the localization of an active form of P-450scc in the quail brain (Tsutsui and Yamazaki, 1995; Usui et al., 1995) have shown that the gene encoding P-450scc is also actively expressed in the CNS of nonmammalian vertebrates.

Recent studies have revealed the existence of substantial differences between the transcriptional factors regulating the expression of P-450scc in the C6 glial cell line and in steroid-secreting cells of endocrine glands (Mellon and Deschepper, 1993; Papadopoulos, 1993). Specifically, the steroidogenic factor SF-1, which plays a crucial role in the control of the expression of all steroid hydroxylase genes including P-450scc (Clemens et al., 1994; Ikeda et al., 1994), and the basal transcriptional factor Sp1 are not expressed in C6 glioma cells (Zhang et al., 1995). In fact, SF-1 has been detected in discrete cerebral areas which do not contain P-450scc mRNA (Mellon and Deschepper, 1993; Ikeda et al., 1994), indicating that SF-1 is not involved in the regulation of P-450scc gene expression in neural tissues as it is in steroidogenic glands. In contrast, cAMP which controls steroid biosynthesis in peripheral tissues (Moore et al., 1990; Watanabe et al., 1994) appears to modulate neurosteroidogenesis in C6 glioma cells (Papadopoulos and Guarneri, 1994). These observations reveal that the mechanisms regulating P-450scc gene expression in endocrine cells and nerve cells exhibit both differences and similarities.

Studies performed in rat indicate that the biological activity of P-450scc may be controlled by peripheral-type benzodiazepine receptor (PBR) ligands in the CNS (Guarneri et al., 1992) as previously shown in classical steroidogenic tissues (Krueger and Papadopoulos, 1990; Papadopoulos et al., 1990). In fact, PBRs, which facilitate the translocation of cholesterol from the external surface of mitochondria to the internal membrane, cause indirect stimulation of P-450scc activity (Papadopoulos, 1993). The observation that benzodiazepines activate neurosteroid biosynthesis has suggested that endogenous ligands of PBR (endozepines) could be involved in the regulation of P-450scc activity (Papadopoulos et al., 1992; Korneyev et al., 1993). The major natural ligand of PBR, diazepam-binding inhibitor (DBI), is a 11-kDa polypeptide whose gene is highly expressed in steroid-secreting tissues (Rhéaume et al., 1990; Brown et al., 1992; Rouet-Smih et al., 1992; Toranzo et al., 1994) as well as in C6 glioma cells (Alho et al., 1994) and in astrocytes (Tong et al., 1991; Malagon et al., 1992, 1993; Slobodyansky et al., 1992; Lihrmann et al., 1994; Patte et al., 1995; Lamacz et al., 1996). Proteolytic cleavage of DBI generates several bioactive peptides including the triakontatetraneuropeptide (TTN) DBI[17-50], the octadecaneuropeptide (ODN) DBI[33-50], and the triakontaseptaneuropeptide DBI[39-75] (Ferrero et al., 1986; Slobodyansky et al., 1994; Tonon et al., 1994; Patte et al., 1999). A study performed on isolated mitochondria from C6 glioma cells has revealed that DBI and TTN both stimulate the formation of ⁵P (Papadopoulos et al., 1992). These results strongly suggest that endozepines play an important role in the regulation of P-450scc activity in the nervous system.

	IV. 3-Hydroxysteroid Dehydrogenase

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The enzymatic complex 3-hydroxysteroid dehydrogenase/⁵-⁴ isomerase (3-HSD), which catalyzes the conversion of ⁵-3-hydroxysteroids into ⁴-3-ketosteroids, plays a crucial role in the biosynthesis of all classes of steroid hormones (Fig. 1). Molecular cloning of the cDNAs encoding 3-HSD has revealed the existence in human of two isoforms of the enzyme: type I 3-HSD which is mainly expressed in the placenta (Luu-The et al., 1989) and type II 3-HSD which is predominantly expressed in the adrenal gland and gonads (Rhéaume et al., 1991). Four types of 3-HSD cDNAs (types I-IV) have been characterized in the rat (Zhao et al., 1990, 1991; Mason, 1993) and six types (types I-VI) in the mouse (Simard et al., 1996; Abbaszade et al., 1997). The rodent type III 3-HSD isoform possesses the structural features common to all 3-HSD but does not display the expected classical 3-HSD activity; in fact, this isoenzyme behaves as a 3-ketosteroid reductase using NADPH as a cofactor, i.e., it is responsible for the conversion of saturated 3-ketosteroids into 3-hydroxy metabolites (Labrie et al., 1992). The enzyme 3-HSD is also present in various tissues such as the skin (Dumont et al., 1992), mammary gland (Rhéaume et al., 1991), and prostate (Bartsch et al., 1990).

The first data suggesting the existence of 3-HSD in the CNS have been provided by Weidenfield et al. (1980) who showed that homogenates of rat amygdala and septum are capable of converting ⁵P into progesterone. The formation of androstenedione from DHEA, which is also catalyzed by 3-HSD (Fig. 1), confirmed the presence of the enzyme in the rat brain (Robel et al., 1986). The biological activity of 3-HSD has also been detected in primary cultures of rodent oligodendrocytes (Jung-Testas et al., 1989) and neurons (Bauer and Bauer, 1989). The first immunohistochemical localization of 3-HSD in the CNS has been performed in the European green frog Rana ridibunda by using an antiserum raised against type I human placental 3-HSD (Mensah-Nyagan et al., 1994). This antiserum had been previously applied for the immunocytochemical localization of 3-HSD in classical steroid-producing organs of mammals such as the adrenal, testis, ovary, and placenta (Dupont et al., 1990a-c). Although the antibodies were raised against type I human placental 3-HSD (Luu-The et al., 1989), they also recognize other 3-HSD isotypes, in particular, type II 3-HSD (Dupont et al., 1990a-c) which is predominantly expressed in the adrenal and gonads (Lachance et al., 1991). It thus appears that the immunoreactive material detected in the frog brain may correspond to different variants of the 3-HSD family. The occurrence of large amounts of ⁴-3-ketosteroids (progesterone and 17-hydroxyprogesterone) in the frog brain and the capability of frog hypothalamic explants to catalyze the conversion of tritiated pregnenolone ([³H]⁵P) into progesterone demonstrate that the 3-HSD-immunoreactive material detected in the CNS actually corresponds to an active form of the enzyme (Mensah-Nyagan et al., 1994). In situ hybridization studies have revealed that the mRNAs encoding for 3-HSD in the rat brain are localized in the olfactive bulb, nucleus accumbens, hippocampus, area of medulla bordering the fourth ventricle as well as in the thalamus, hypothalamus, and cerebellum (Dupont et al., 1994; Guennoun et al., 1995). Immunocytochemical data have shown that, in the frog brain, the 3-HSD gene is exclusively expressed in neurons (Fig. 2). Similarly, in the rat CNS, 3-HSD mRNAs were only detected in neuronal cell bodies (Dupont et al., 1994; Guennoun et al., 1995) (Fig. 3). It should be noted however that the presence of 3-HSD and its mRNAs has recently been found in rodent Schwann cells by immunocytochemistry and RT-PCR (Guennoun et al., 1997). In addition, 3-HSD activity has been demonstrated in primary cultures of rat astrocytes and oligodendrocytes (Jung-Testas et al., 1989; Kabbadj et al., 1993). These observations indicate that glial cells, which do not possess 3-HSD in situ, may acquire the ability of expressing the 3-HSD genes when they are maintained in culture. Alternatively, it is possible that other 3-HSD isoenzymes distinct from isotypes I and II are present in brain glial cells. To solve this question, it will be necessary to identify the mRNAs encoding for the different 3-HSD isoforms in cultured rat astrocytes and oligodendrocytes.

View larger version (56K): [in this window] [in a new window]   Fig. 2.   Confocal laser scanning microscope photomicrographs of 3-HSD-immunoreactive neurons in the frog diencephalon. A, frontal section through the anterior preoptic area (Poa) showing 3-HSD-positive cell bodies close to the third ventricle (III). Original magnification, 700×. B, high magnification of a 3-HSD-immunoreactive neuron in the ventral hypothalamic nucleus (VH). Original magnification, 1300×.

View larger version (116K): [in this window] [in a new window]   Fig. 3.   Autoradiographic localization of 3-HSD mRNAs by in situ hybridization on frontal sections of the rat brain at the level of the nucleus prepositus hypoglossi. A, dense accumulation of silver grains is observed in a subpopulation of neurons (arrows) located in the vicinity of the fourth ventricle (V). Ependymal cells (E) do not express the 3-HSD gene. Original magnification, 560×. B, pretreatment of the brain section with RNase abolished autoradiographic labeling. Original magnification, 560×. Reprinted from Dupont et al. (1994) with permission from Academic Press, Inc.

The mechanisms of regulation of 3-HSD gene transcription have been extensively studied in peripheral steroidogenic tissues (Labrie et al., 1994; Guérin et al., 1995; Mason et al., 1997). In contrast, until recently, nothing was known concerning the control of 3-HSD activity in the CNS. The observation that, in the frog, numerous hypothalamic neurons contain simultaneously 3-HSD- and PBR-like imunoreactivities (Do-Régo et al., 1998) suggested that the endogenous ligands of PBR may control 3-HSD activity. As a matter of fact, it was found that the endozepine TTN causes a dose-dependent stimulation of the conversion of ⁵P into 17-hydroxyprogesterone, indicating that TTN enhances 3-HSD activity (Do-Régo et al., 1998). The effect of TTN was mimicked by the PBR agonist 4'-chlorodiazepam and inhibited by the PBR antagonist 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinoline carboxamide (PK11195; Benavides et al., 1984; Zavala and Lenfant, 1987; Costa et al., 1994). In contrast, flumazenil, a central-type benzodiazepine receptor antagonist (Brodgen and Goa, 1991), did not affect TTN-evoked neurosteroid secretion (Do-Régo et al., 1998) (Fig. 4). Altogether, these data indicate that TTN stimulates the biological activity of 3-HSD in hypothalamic neurons through activation of PBR likely located at the plasma membrane level.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Effect of the triakontatetraneuropeptide TTN and other benzodiazepine receptor ligands on the conversion of [3H]5P into 4-3-ketoneurosteroids. Frog hypothalamic explants were incubated for 2 h with 24 µCi/ml of [3H]5P and the radioactive newly synthesized 4-3-ketosteroids were separated by HPLC analysis. The endozepine TTN and the PBR agonist Ro-5-4864 both induced a marked increase of neurosteroid biosynthesis. The PBR antagonist PK11195 (104 M) reduced the basal synthesis of neurosteroids and significantly attenuated the effect of TTN. The central-type benzodiazepine receptor antagonist flumazenil (105 M) also reduced the basal synthesis of neurosteroids but did not affect the stimulatory action of TTN on 4-3-ketoneurosteroid formation. P < .05; , P < .01; , P < .001; NS, not statistically different.

	V. Cytochrome P-450c17

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The enzymatic system 17-hydroxylase/17,20 lyase (cytochrome P-450c₁₇) is responsible for the transformation of C₂₁ steroids (⁵P, progesterone) into C₁₉ steroids (DHEA and androstenedione, respectively) (Fig. 1). It is now clearly demonstrated that, in classical steroid-producing glands, these reactions are catalyzed by a single microsomal enzyme coupled to a cytochrome reductase, cytochrome P-450c₁₇ (P-450c₁₇ or P-450₁₇), which possesses both 17-hydroxylase and 17,20 lyase activities. The lyase bioactivity of this enzymatic complex is modulated by phosphorylation and depends on the lipidic environment (Nakajin et al., 1981; Miller, 1988; Namiki et al., 1988).

The early observation that the rat brain contains high concentrations of DHEA (Corpéchot et al., 1981) suggested the existence of P-450c₁₇ activity in the CNS of mammals. However, biochemical and immunocytochemical studies aimed at demonstrating the presence of the enzyme in the brain have long remained unsuccessful (Baulieu and Robel, 1990; Le Goascogne et al., 1991; Mellon and Deschepper, 1993). In 1994, it was demonstrated that frog hypothalamic explants are capable of converting [³H]⁵P into [³H]17-hydroxyprogesterone (Mensah-Nyagan et al., 1994). This observation provided the first evidence for the presence of P-450c₁₇ in the CNS. Subsequently, P-450c₁₇ mRNAs have been detected by RT-PCR in the brain of rat embryos (Compagnone et al., 1995b). P-450c₁₇-like immunoreactivity has also been observed in various neuronal populations of the pontine nucleus, the locus ceruleus and the spinal cord in mouse embryos. In contrast, conflicting data have been reported in adults: according to Compagnone et al. (1995b), P-450c₁₇ gene is only expressed in the PNS of rat and mouse, whereas other studies have described the presence of P-450₁₇ mRNAs in various brain regions of adult rodents (Strömstedt and Waterman, 1995).

The regulation of P-450c₁₇ gene expression in peripheral steroidogenic tissues is controlled by androgens (Burgos-Trinidad et al., 1997), insulin-like growth factor type I (Naseeruddin and Hornsby, 1990), catecholamines (Ehrhart-Bornstein et al., 1991; Güse-Behling et al., 1992; Haidan et al., 1998), cAMP, and protein kinase C activators (McAllister and Hornsby, 1988; Cheng et al., 1992). In contrast, the mechanisms regulating the expression of the P-450c₁₇ gene in the brain of mammals have not yet been determined. In a recent study, it has been shown that, in the frog hypothalamus, TTN stimulates the conversion of [³H]⁵P into [³H]17-hydroxyprogesterone, indicating that endozepines can increase P-450c₁₇ activity in nerve cells (Do-Régo et al., 1998).

	VI. 17-Hydroxysteroid Dehydrogenase

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The enzyme 17-HSD plays a pivotal role in the biosynthesis and the inactivation of sex steroid hormones by catalyzing the interconversion of 17-ketosteroids (androstenedione, estrone) and 17-hydroxysteroids (testosterone, 17-estradiol) (Fig. 1). Molecular cloning of the 17-HSD cDNAs and biochemical characterization of the enzyme activity have revealed the existence of seven isoforms designated types I to VII (Andersson, 1995; Blomquist, 1995; Andersson and Moghrabi, 1997; Biswas and Russell, 1997; Nokelainen et al., 1998). Type I, III, and V isoenzymes catalyze almost exclusively reductive reactions, leading to the formation of active steroids such as testosterone and 17-estradiol. Conversely, the type II and IV isoenzymes, which preferentially catalyze the oxidative reaction, are responsible for the synthesis of androstenedione and estrone (Andersson, 1995). Type VI 17-HSD oxidizes essentially 5-androstane-3,17-diol to androsterone. This latter 17-HSD isoform shares 65% sequence identity with retinol dehydrogenase 1 which catalyzes the oxidation of retinol to retinal (Biswas and Russell, 1997). Type VII 17-HSD, which catalyzes the conversion of estrone to estradiol, has been initially described as a prolactin receptor-associated protein because of a high (89%) sequence homology (Duan et al., 1996, 1997; Nokelainen et al., 1998). Recently, it has been shown that the Ke 6 protein, which is intimately linked to the development of cysts in the kidney and liver (Aziz et al., 1996), efficiently catalyzes the reduction of estrone and also the oxidation of estradiol and testosterone in an NAD-dependent manner, indicating that the Ke 6 protein is a potential eighth member of the 17-HSD isozyme family (Fomitcheva et al., 1998). Five isoforms of 17-HSD have been cloned in humans and their cDNAs structurally characterized. Type I 17-HSD, which was isolated for the first time from a human placental library, has subsequently been identified in the ovary and mammary gland (Martel et al., 1992). The type II isoenzyme, which was isolated from prostate and placental cDNA libraries, is also present in the endometrium, liver, small intestine as well as in the kidney, pancreas, and colon (Casey et al., 1994). In contrast, the type III 17-HSD gene is exclusively expressed in the testis (Geissler et al., 1994). Molecular cloning of human type IV 17-HSD revealed that this isoenzyme is expressed in the liver, kidney and, to a lesser extent, in the endometrium and testis (Adamski et al., 1995). Recently, the cDNA encoding for the type V 17-HSD isoenzyme has been characterized in humans using a placental cDNA library (Labrie et al., 1997). Different isoforms of 17-HSD were also detected in various peripheral tissues of rodents (Normand et al., 1995) and pig (Adamski et al., 1992; Leenders et al., 1994a,b).

The existence of a 17-HSD activity in the mammalian brain has long been known (Reddy, 1979; Resko et al., 1979) but it is only recently that the cellular distribution of the enzyme in the CNS has been described (Pelletier et al., 1995). Immunocytochemical mapping of 17-HSD has also been determined in the frog brain using antibodies against human placental type I 17-HSD (Mensah-Nyagan et al., 1996a,b). In the CNS of both mammals and amphibians, type I 17-HSD is exclusively expressed in glial cells (Pelletier et al., 1995; Mensah-Nyagan et al., 1996a,b) (Fig. 5). In the rat brain, 17-HSD-like immunoreactivity is widely distributed in ependymocytes and astrocytes of the hippocampus, cerebral cortex, thalamus, and hypothalamus, whereas, in the frog brain, the immunoreactive material is only located in ependymocytes of the telencephalon. Whether these species differences reflect authentic variations in the anatomical distribution of 17-HSD in the CNS of mammals and amphibians or whether they can be ascribed to the presence, in the frog brain, of distinct 17-HSD isoforms which cannot be detected with the antibodies against type I 17-HSD remains unkown. In this respect, it should be noticed that the five isoforms of 17-HSD cloned in various vertebrate species do not exhibit the same cellular distribution or functional characteristics in peripheral steroidogenic tissues (Andersson, 1995; Andersson and Moghrabi, 1997; Puranen et al., 1997). In addition, in the CNS of mammals, 17-HSD is mainly involved in the inactivation of sex steroid hormones (Reddy, 1979; Resko et al., 1979; Martini et al., 1996), whereas, in the brain of amphibians, this enzyme is responsible for the synthesis of testosterone (Mensah-Nyagan et al., 1996a,b). In the frog Rana ridibunda, a series of observations have demonstrated that biosynthesis of testosterone actually occurs in the CNS: 1) high amounts of testosterone have been detected in the medial pallium and the hypothalamus, and the concentrations of testosterone are not affected by castration; 2) endogenous testosterone extracted from the telencephalon has been chemically characterized by combining HPLC analysis, gas chromatography and mass spectrometry; and finally 3) synthesis of [³H]testosterone and [³H]5-DHT from [³H]⁵P by frog telencephalon explants has been demonstrated in vitro (Mensah-Nyagan et al., 1996a,b). Formation of androgens and estrogens from a distant precursor such as [³H]⁵P or [³H]DHEA has also been shown in primary cultures of avian nerve cells (Vanson et al., 1996), indicating that 17-HSD-like activity responsible for the synthesis of sex steroids is present in the CNS of various groups of vertebrates. Taken together, these data suggest the expression of new classes of 17-HSD isoforms in the nervous system of nonmammalian vertebrates or the occurrence of a biological activity distinct from that of 17-HSD isoenzymes present in the mammalian brain. Molecular cloning of the various 17-HSD genes in representative submammalian species is clearly required to investigate their expression in the CNS.

View larger version (78K): [in this window] [in a new window]   Fig. 5.   Immunocytochemical localization of 17-HSD in the CNS of rat and frog. A, 17-HSD-positive glial cells in the rat cerebellar cortex (CC). Reprinted from Pelletier et al. (1995) with kind permission from Elsevier Science-NL (Sara Burgerhartstraat 25, 1055 KV Amsterdam, the Netherlands). Original magnification, 80×. B, 17-HSD-immunoreactive ependymal cells bordering the lateral ventricle (V) in the frog telencephalon. MP, medial pallium. Original magnification, 500×.

In peripheral tissues, the expression of 17-HSD is regulated at the transcriptional level by sex steroid hormones (Peltoketo et al., 1996), growth factors (Ghersevich et al., 1994; Jantus-Lewintre et al., 1994), retinoic acid (Piao et al., 1995), and cAMP (Tremblay and Baudouin, 1993). Whether these different factors are also involved in the control of 17-HSD gene expression in nerve cells has not yet been investigated.

	VII. 5-Reductase

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The enzyme 5-reductase (5-R) is a microsomal NADPH-dependent protein which acts specifically on steroids possessing a C₄-C₅ double bond and a ketone group at the C₃ position. This enzyme catalyzes the transfer of two hydrogens from NADPH causing the reduction of the C₄-C₅ double bond and the formation of 5-reduced metabolites. In particular, 5-R catalyzes the conversion of testosterone, the main circulating androgen, into dihydrotestosterone (5-DHT) and the transformation of progesterone into dihydroprogesterone (5-DHP) (Fig. 1). In humans, two distinct cDNAs encoding type I and type II 5-R have been cloned from a prostate library; these cDNAs exhibit an overall sequence identity of 60% (Andersson and Russell, 1990; Andersson et al., 1991). The 5-RI gene, located on chromosome 5, is mainly expressed in the skin (Luu-The et al., 1994), notably in the pubic skin and the scalp (Andersson and Russell, 1990; Jenkins et al., 1992). The 5-RII gene is predominantly expressed in the prostate and gonads (Thigpen et al., 1993; Luu-The et al., 1994; Mowszowicz et al., 1995). Deletion in the 5-RII gene causes male pseudohermaphroditism, indicating that 5-RII is involved in the determination of the sexual phenotype during embryogenesis (Andersson et al., 1991). In rat, 5-RI and 5-RII cDNAs have been cloned from a prostate library but the two genes are actually transcribed in distinct cell types: 5-RI mRNAs are localized in the basal epithelial cells whereas 5-RII mRNAs are found in stromal cells (Andersson and Russell, 1990; Berman and Russell, 1993).

In vitro studies have shown the existence of 5-R bioactivity in brain tissue and specially in primary cultures of nerve cells (Saitoh et al., 1982; Melcangi et al., 1993; Martini et al., 1996; Negri-Cesi et al., 1996a,b). Northern blot analysis has shown the occurrence of high concentrations of 5-RI mRNAs but relatively low amounts of 5-RII mRNAs in rat brain extracts (Normington and Russell, 1992; Lephart, 1993). The anatomical distribution of 5-R in the rat brain was first investigated using an antibody raised against human 5-RI (Luu-The et al., 1994). The presence of 5-R-like immunoreactivity has been found in astrocytes, ependymocytes, and tanycytes within various brain regions including the hypothalamus, thalamus, hippocampus, cerebral cortex, and circumventricular organs (Pelletier et al., 1994). At the ultrastructural level, the immunoreactive material appeared to be distributed throughout the cytoplasm of glial cells without any particular association with mitochondria (Pelletier et al., 1994). This observation is consistent with previous subcellular fractionation studies which had shown that 5-R activity was mostly associated with the microsomal fraction (Lephart, 1993). Using an antibody raised against rat 5-RI, Tsuruo et al. (1996) have recently reported that 5-R-like immunoreactivity is mainly contained in oligodendrocytes of the white matter and in ependymocytes bordering the cerebral ventricles. Collectively, these data indicate that, in the CNS of mammals, the 5-R gene is primarily expressed in glial cells. However, biochemical studies have shown that neurons from rat embryos in primary culture exhibit 5-R activity (Melcangi et al., 1994). These observations suggest that the 5-R genes may be transcribed in distinct cell types of the CNS according to development stages. Alternatively, the expression of the 5-R genes may be up-regulated in cultured neurons.

In all classes of vertebrates, conversion of gonadal testosterone into 5-DHT in the brain of male individuals is necessary for the induction of various behavioral effects. Since the occurrence of 17-HSD-like immunoreactivity has been demonstrated within glial cells in the frog brain (Mensah-Nyagan et al., 1996a,b), it would be interesting to investigate the cellular localization of 5-R in the CNS of amphibians to examine whether the same cells may simultaneously synthesize testosterone and convert it into 5-DHT. Concurrently, in the mammalian brain, the consecutive catalytic actions of 5-R and 3-hydroxysteroid dehydrogenase on progesterone leads to the formation of allopregnanolone (Baulieu et al., 1996), a potent modulator of GABA_A receptors, which controls various psychical processes (Schumacher et al., 1996; Patchev et al., 1996). Expression of the 5-R gene in nerve cells may thus have important implications in the control of neurophysiological functions in vertebrates.

Neuroanatomical studies have revealed that, in mammals, 5-R is present in various regions of the CNS where androgen (Arnold and Gorski, 1984; Clark et al., 1988; Roselli et al., 1996a,b) and estrogen receptors are located (Pelletier et al., 1988; Balthazart et al., 1989; Torran-Allerand et al., 1992; Yuan et al., 1995). However, it is generally accepted that, in the brain, there is no sexual dimorphism in the expression of 5-R; in addition, castration or sex steroid hormone administration does not affect 5-R activity (Wilson, 1975; Celotti et al., 1983; Lephart, 1993; Negri-Cesi et al., 1996b). A remarkable exception has been reported in monkey in which castration induces a selective increase of the biological activity of 5-R in the basolateral amygdala but not in other regions of the CNS (Roselli et al., 1987). These observations suggest that, in discrete brain areas, sex steroids may control 5-R expression and/or activity as described in the rat adrenal gland (Lephart et al., 1991). Concurrently, suppression of hypothalamic (nor-) adrenergic neurotransmission by pharmacological blockers and surgical deafferentation of the hypothalamus do not affect 5-R activity, indicating that the expression of the enzyme is not regulated by extrinsic neural inputs (Celotti et al., 1983). However, incubation of glial cells with 8Br-cAMP, but not phorbol esters, causes a significant increase of 5-DHT formation (Celotti et al., 1992; Negri-Cesi et al., 1996b). These data suggest that a protein kinase A is involved in the regulation of 5-R activity in nerve cells, although the neural factors responsible for the activation of this transduction pathway remain unknown.

	VIII. Aromatase

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The conversion of androgens into estrogens is catalyzed by aromatase (Fig. 1), an enzymatic complex which comprises two proteins, i.e., a specific form of cytochrome (cytochrome P-450aromatase) responsible for the binding of the C₁₉ steroid substrate and the formation of the phenolic A-ring characteristic of estrogens, and a flavoprotein (NADPH-cytochrome P-450reductase) which transfers reducing equivalents from NADPH to any microsomal form of cytochrome (for review, Nelson et al., 1993). Aromatase activity occurs in various tissues including the placenta (Fournet-Dulguerov et al., 1987), ovary (McNatty et al., 1976; Lephart et al., 1995), testis (Fritz et al., 1976; Valladares and Payne, 1979; Levallet and Carreau, 1997), and adipocytes (Simpson et al., 1989). The aromatase gene, which has been cloned in humans, is composed of 17 exons and is localized at the q21.1 level of chromosome 15 (Means et al., 1989; Harada et al., 1990; Toda et al., 1990). Molecular cloning of aromatase cDNAs in various vertebrate taxa has revealed the existence of a single enzyme in most species including trout (Tanaka et al., 1992), chicken (McPhaul et al., 1988), rat (Hickey et al., 1990), mouse (Terashima et al., 1991), bovine (Hinshelwood et al., 1993), and humans (Corbin et al., 1988; Harada, 1988). A remarkable exception has been reported in pig which possesses two distinct isoforms of aromatases (Corbin et al., 1995; Conley et al., 1997).

It has long been known that conversion of androstenedione into estrone occurs in the rat brain, indicating the presence of aromatase activity in the CNS (Naftolin et al., 1972, 1975; Roselli et al., 1985). Immunocytochemical studies have recently shown that aromatase is expressed in neurons and not in glial cells (Lephart, 1996). In the brain of birds, a good correlation has been observed between the localization of aromatase-like immunoreactivity and the distribution of aromatase activity. Particularly, in the Japanese quail, aromatase-positive neurons are located in the preoptic area where an intense enzymatic activity is also found (Balthazart et al., 1990a,b, 1991b, 1992). Conversely, in mammals, especially in rodents, mismatches have been reported between the localization of aromatase-positive neurons and the distribution of enzymatic activity in the CNS. For instance, high levels of aromatase activity are detected in the median preoptic area and the ventromedian nucleus of rat, two regions which are virtually devoid of aromatase-immunoreactive neurons (Shinoda et al., 1989a,b; Balthazart et al., 1991a; Sanghera et al., 1991). It should be noted however that, during ontogenesis, aromatase-positive neurons have been visualized in the preoptic area, the ventromedian nucleus and the arcuate nucleus at embryonic day 13 (E₁₃), E_16, and E₁₉, respectively. In these regions, the number of aromatase-positive neurons increases during gestation, peaks before birth, and decreases or vanishes during the two first postnatal weeks (Tsuruo et al., 1994). These data reveal the existence of spatio-temporal variations in the level of transcription of the aromatase gene during development.

The mechanisms controlling aromatase expression and bioactivity in the CNS have been investigated during ontogenesis and in the adult. Because of the high affinity of the enzyme for testosterone, various research groups have examined the effects of androgens on aromatase gene transcription during embryogenesis (Callard et al., 1980; Paden and Roselli, 1987; Lephart et al., 1992; Roselli and Resko, 1993). Their studies revealed that, in rodent embryos, neither testosterone nor 5-DHT had any influence on aromatase gene expression in cultured hypothalamic neurons (Abe-Dohmae et al., 1994; Negri-Cesi et al., 1996a). In contrast, aromatase activity in the CNS appears to be modulated by androgens, although controversial data have been reported in the literature: Lephart et al. (1992) have observed that androgens are capable of reducing aromatase activity in rat embryo hypothalamic explants, whereas Beyer et al. (1994b) have described a stimulatory effect of testosterone on estrogen formation in cultured mouse fetal diencephalic neurons. Depending on the species and/or the environmental milieu, androgens may thus exert opposite effects on aromatase activity in the developing brain. In adult individuals, androgens clearly play a crucial role in the regulation of aromatase gene transcription and aromatase activity in the CNS of amphibians (Moore et al., 1994), birds (Harada et al., 1992; Panzica et al., 1996), and mammals (Negri-Cesi et al., 1996a,b). In particular, it has been demonstrated that castration significantly reduces the amount of aromatase mRNAs and activity in the quail (Harada et al., 1992) and rat brain (Abdelgadir et al., 1994; Roselli et al., 1997). Reciprocally, administration of testosterone increases the level of aromatase mRNA and the number of aromatase-immunoreactive neurons (Harada et al., 1992; Abdelgadir et al., 1994), indicating the importance of testosterone in the regulation of aromatase expression in the CNS. Since estrogens stimulate the expression of androgen receptors and increase the duration of androgen receptor occupation in the rat brain (Roselli and Fasasi, 1992), it is conceivable that estrogens and androgens may exert a coordinate action in the control of aromatase gene expression in the CNS. Concurrently, an effect of dopamine on aromatase activity has been demonstrated in the quail preoptic area, indicating that neurotransmitters may regulate reproductive behavior by modulating estrogen formation in the brain (Baillien and Balthazart, 1997). Finally, the fact that a large population of aromatase-positive neurons are located in the preoptic-septal complex (Shinoda et al., 1989b; Balthazart et al., 1990a,b, 1991a,b; Sanghera et al., 1991; Jakab et al., 1993, 1994; Beyer et al., 1994a,c; Foidart et al., 1995), where a number of neuropeptides controlling sexual behavior are present (Wehrenberg et al., 1989; DeVries, 1990; Kalra et al., 1990; Kawata et al., 1991; Albers et al., 1992; Andersen et al., 1992; King and Millar, 1992; Sherwood et al., 1993; Winslow et al., 1993; Moore et al., 1994), suggests that some of these neuropeptides could be involved in the regulation of aromatase gene transcription and/or activity in the CNS.

	IX. Sulfotransferase and Sulfatase

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Sulfate conjugation of steroids is catalyzed by sulfotransferases or sulfokinases, a family of cytosolic enzymes which transfer the sulfate moiety from the universal donor molecule 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a hydroxyl group of the steroid substrates. In contrast, sulfatase is responsible for the hydrolysis of sulfated steroids leading to the formation of unconjugated steroids (Fig. 1). Hydroxysteroid sulfonates act as potent regulators of neuronal activity. In particular, pregnenolone sulfate (⁵PS) and dehydroepiandrosterone sulfate (DHEAS) modulate the functions of GABA_A receptors (Majewska, 1992), NMDA receptors (Wu et al., 1991; Weaver et al., 1997),  receptors (Monnet et al., 1995), and voltage-gated calcium channels (Ffrench-Müllen and Spence, 1991; Ffrench-Müllen et al., 1994). The fact that the inhibitory action of ⁵PS on calcium channel currents in pyramidal neurons is abolished after substitution of the sulfate moiety by an acetate (Ffrench-Müllen et al., 1994) demonstrates the importance of the sulfate group in the neurogenic activity of 3-hydroxysteroids.

Molecular cloning of sulfotransferase cDNAs has revealed the existence of multiple isoforms which have differential affinity for various steroid substrates and are expressed in a tissue-specific manner. The steroid sulfotransferase superfamily comprises four classes of enzymes: 1) hydroxysteroid sulfotransferases (HST) that act on primary and secondary alcohols of hydroxysteroids such as cholesterol, ⁵P and DHEA, 2) estrone sulfotransferases that transfer the sulfonate moiety on the 3-hydroxyl group of estrogens, 3) steroid sulfotransferases that have a broad specificity, and 4) cortisol sulfotransferases that act on the 21-hydroxyl group of glucocorticosteroids (for reviews, Webb, 1992; Strott, 1996).

The human sulfatase gene has been cloned and mapped to the Xp22.3 chromosome, proximal to the pseudoautosomal region (Ballabio and Shapiro, 1995). Recent molecular cloning studies have also characterized the complete gene of rat sulfatase (Li et al., 1996) and a mouse sulfatase cDNA (Salido et al., 1996). These results revealed that the overall genomic organization of rat and human sulfatases is very similar, except that the insertion site for intron 1 in the rat is 26 bp upstream from that in humans.

The existence of sulfotransferase-like activity has long been demonstrated in the primate brain (Knapstein et al., 1968). Similarly, early studies have shown the occurrence of sulfatase bioactivity in the CNS of vertebrates including human (Kishimoto and Sostek, 1972; Iwamori et al., 1976). Consistent with these findings, high amounts of ⁵P, DHEA, and their sulfated esters (⁵PS and DHEAS) have been detected in the brain of castrated and adrenalectomized rats, suggesting the presence of sulfotransferase and sulfatase activities in the CNS (Corpéchot et al., 1981, 1983). In vitro studies have confirmed the existence of sulfotransferase (Rajkowski et al., 1997) and sulfatase bioactivity (Park et al., 1997) in the mammalian brain. However, the anatomical localization of these enzymes in the brain of vertebrates has long remained unknown. Recently, the cellular distribution of sulfotransferase has been investigated in the CNS of the European green frog Rana ridibunda using an antiserum raised against rat liver HST (Beaujean et al., 1999). Two populations of HST-immunoreactive neurons have been detected in the anterior preoptic area and in the magnocellular preoptic nucleus of the hypothalamus. A dense bundle of HST-positive glial processes is also present in the ventral hemispheric zone. In addition, frog telencephalon and hypothalamus homogenates are capable of synthesizing ⁵PS and DHEAS (Fig. 6), as demonstrated by pulse-chase experiments using [³⁵S]PAP and [³H]⁵P or [³H]DHEA as precursors (Beaujean et al., 1999). Concurrently, the presence of sulfatase mRNAs has been visualized in the cortex, hindbrain, and thalamus of mouse fetuses during the last week of gestation (Compagnone et al., 1997). In the adult bovine brain, sulfatase activity is particularly abundant in the midbrain and hypothalamus (Park et al., 1997), suggesting that the sites of expression of the enzyme in the CNS may vary during development and/or may differ from one species to the other. These studies indicate that brain neurons and/or glial cells express both sulfotransferase and sulfatase activities which play an important role in the regulation of the functions of neuroactive steroids.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Biosynthesis of 5PS (A) and DHEAS (B) in the frog hypothalamus in vitro. Frog hypothalamic homogenates were incubated for 3 h with [3H]5P and [35S]PAPS (A) or with [3H]DHEA and [35S]PAPS (B). The steroids were extracted in a mixture of water and dichloromethane (v/v) and the aqueous phase, containing sulfated steroids, was analyzed by HPLC using a hexane/tetrahydrofuran (THF) gradient. The ordinates indicate the radioactivity (3H and 35S) measured in the HPLC fractions. The dashed linerepresents the gradient of secondary solvent (% THF). The arrows indicate the elution position of standard steroids. Reprinted from Beaujean et al. (1999) with permission from the Journal of Neurochemistry, Lippincott-Raven Publishers.

	X. 11-Hydroxysteroid Dehydrogenase

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The transformation of physiologically active glucocorticoids (cortisol, corticosterone) into inactive metabolites (cortisone, 11-dehydrocorticosterone) is catalyzed by 11-hydroxysteroid dehydrogenase (11-HSD), a microsomal NADP⁺-dependent enzyme. High levels of 11-HSD activity are present in the kidney and salivary gland (Edwards et al., 1988; Monder et al., 1989), as well as in the liver, lung, and testis (Phillips et al., 1989). Molecular cloning of the cDNAs encoding 11-HSD revealed the existence of two isoforms of the enzyme (type I 11-HSD or 11-HSDI and type II 11-HSD or 11-HSDII) in humans (Tannin et al., 1991; Albiston et al., 1994), sheep (Yang et al., 1992; Agarwal et al., 1994), rat (Agarwal et al., 1989; Zhou et al., 1995), and mouse (Rajan et al., 1995; Cole, 1995). Type I 11-HSD isozyme utilizes NADPH as a cofactor and is capable of functioning (in addition of the classical 11-HSD activity) as an 11-reductase by regenerating active glucocorticoids in cultured cells (Agarwal et al., 1989; Duperrex et al., 1993; Low et al., 1994). Type II 11-HSD is an exclusive glucocorticoid-inactivating enzyme whose bioactivity is NAD-dependent (Brown et al., 1993; Rusvai and Naray-Fejes-Toth, 1993).

The presence of 11-HSD activity has been demonstrated in various areas of the CNS including the cerebellum, hippocampus, neocortex, amygdala, and brainstem (Grosser and Axelrod, 1968; Miyabo et al., 1973; Moisan et al., 1990, 1992; Lakshmi et al., 1991). Northern blot analysis, using a rat liver 11-HSDI cDNA probe has revealed the presence of a single mRNA band in the rat brain (Moisan et al., 1990, 1992). The 11-HSDI gene is actively expressed in various neuronal populations of the cerebellum, hippocampus, cerebral cortex, and hypothalamus (Moisan et al., 1992). The location of 11-HSDI mRNA in these brain areas coincides exactly with the regional distribution of the enzymatic activity (Lakshmi et al., 1991). Northern blot experiments using human kidney and placental 11-HSDII cDNAs did not reveal the presence of type II 11