- Email: [email protected]
5α-Reductase Isozymes in the Central Nervous System
5a-reductase isozymes in the central nervous system Angelo Poletti, Anna Coscarella, Paola Negri-Cesi, Alessandra Colciago, Fabio Celotti, and Luciano Martini Istituto di Endocrinologia, Universita` di Milano, via Balzaretti 9, 20133 Milano, Italy The enzyme 5a-reductase (5a-R) activates several D4–3keto steroids to more potent derivatives which may also acquire new biological actions. Testosterone gives rise to the most potent natural androgen dihydrotestosterone (DHT), and progesterone to dihydroprogesterone (DHP), a precursor of the endogenous anxiolytic/anesthetic steroid tetrahydroprogesterone (THP). Two isoforms of 5a-R, with a limited degree of homology, different biochemical properties and distinct tissue distribution have been cloned: 5a-R type 1 and type 2. In androgendependent structures DHT is almost exclusively formed by 5a-R type 2; 5a-R type 1 is widely distributed in the body, with the highest levels in the liver, and may be involved in steroid catabolism. In the brain, the roles of the two isozymes are still largely unknown. This brief review will summarize recent experimental data from our laboratory which try to assign possible functional roles to the process of 5a-reduction, and to the two 5a-R isoforms in the CNS. (Steroids 63:246–251, 1998) © 1998 by Elsevier Science Inc. Keywords: testosterone; 5a-reductase; flutamide; neurons; rat brain
Introduction The transformation of testosterone into dihydrotestosterone (DHT) by the enzyme 5a-reductase (5a-R) plays an important physiological role in peripheral androgen-dependent structures; for instance, it controls the development and the function of the prostate, the seminal vesicle, etc. The intracellular formation of DHT precedes the activation of the androgen receptor (AR), which is essential for receptormediated transcription of androgen-responsive genes. In the prostate, this metabolic transformation is a mechanism for amplification of the androgenic signal, since DHT possesses an affinity for the AR four times higher than that of testosterone.1– 4 Moreover, the activated AR is also stabilized by DHT,3,4 and DHT exhibits a fivefold slower dissociation rate from the hormone binding domain of the receptor than its precursor testosterone. Consequently, DHT activates transcription of androgen-dependent genes at concentrations significantly lower than testosterone. The enzyme(s) responsible for its formation are thus important components of the intracellular system for responding to androgens. Two isoforms of the 5a-R (5a-R type 1 and 5a-R type 2) have been cloned (see Reference 5 for review). The human 5a-R type 1 gene comprises five exons and four introns, and produces a protein of 259 amino acids. The structure of the 5a-R type 2 gene is similar to that of the 5a-R type 1, but the resulting protein is of 254 amino acids. Protein sequences deduced from their cDNAs show modest homology Address reprint requests to Angelo Poletti, Istituto di Endocrinologia, via Balzaretti 9, 20133 Milano, Italy. Steroids 63:246 –251, 1998 © 1998 by Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
(about 47%), a predicted molecular weight of 28 –29 kDa, and a high number of hydrophobic amino-acid residues responsible for the intrinsic membrane localization of the enzymes. Both isozymes are able to 5a-reduce all D4 –3keto steroids, but with different kinetics and specificity. Androgens, progestogens and corticosteroids can all be 5a-reduced; their affinity for the type 1 isoform is in the micromolar range, much lower than that for the type 2 isoform, which is in the nanomolar range.6 On the other hand, the conversion yields (Vmax) are much higher for the type 1 isoform for all substrates. For both isozymes, progesterone is the preferred substrate, followed by testosterone and at a considerable distance corticosterone. The two isoforms exhibit different pH optima, with 5a-R type 1 active over a wide pH range (from 5 to 8) and 5a-R type 2 showing a narrow pH optimum around 5.5, with a very low activity at pH 7.5. The two isozymes also show differential sensitivity to synthetic inhibitors, like the selective blocker of the 5a-R type 2 finasteride,5 and the non-specific inhibitor suramine, a drug that acts on the NADPH binding sites of the 5a-R7; however, both inhibitors are most potent toward the type 2 isozyme.5,8 Only preliminary data are available on the efficacy of inhibitors specific for the type 1 isoform.9 Reflecting their different biochemical profiles, the two 5a-R isozymes might be expected to have different physiological roles, even thought they catalyse the same reaction. Studies on their tissue- and cell-specific expression, as well as on the transcriptional control of the two genes, may thus be of help to determine at least some specific functional roles of the two isozymes. In human, the type 1 gene is highly expressed in the liver and in non-genital skin, but 0039-128X/98/$19.00 PII S0039-128X(98)00018-X
5a-reductase isozymes in the CNS: Poletti et al. poorly expressed in androgen target tissues.6 In rats, this isoform is widely distributed throughout various tissues, with the highest levels in the liver. In contrast, in both man and rat, the type 2 isoform appears to be mainly concentrated in androgen-dependent structures such as the prostate (where it is present largely in the stromal component), the epididymis and the seminal vesicles; low levels, if any, have been detected in other tissues.5 Interestingly, a genetically defective type 2 isoform produces a syndrome of male pseudohermaphroditism (Imperato–McGinley syndrome), characterized by aplasia of the prostate and ambiguity of the external genitalia.10,11 The presence of the type 1 isozyme does not seem to be able to replace the inactive type 2 isoform. Several studies have been performed to determine the subcellular localization of the 5a-R, which appears to be associated with cellular membranes.12 A different subcellular localization of the two isozymes has been found by subcellular fractionation studies performed on yeast cells genetically transformed to specifically produce the two rat isozymes: the 5a-R type 1 appears to be associated with cell nuclei, whereas 5a-R type 2 is mainly found in microsomal fractions.12 Considerable 5a-R activity is also present in the central nervous system (CNS), but the physiological functions of the enzyme in the brain are still poorly understood. This review will summarize relevant results from our laboratory on possible functional roles for 5a-R in the CNS. The data will be presented in the following order: 1) 5a-R isozymes during ontogenesis and sexual differentiation of the brain and of the spinal cord; 2) involvement of 5a-R in stressevoked pathways; 3) 5a-R in LHRH-secreting neurons; 4) 5a-R in myelin; and 5) catabolic role of the enzyme.
Roles of the two 5a-Rs in the brain As previously mentioned, several studies indicate that an active 5a-R system is present in the brain. The majority of the data predate the discovery of the two isozymes, and were primarily obtained by measuring the activity of the 5a-R in various tissue samples at neutral pH, at which only the type 1 isozyme is fully functional. After the genes coding two isoforms were cloned, the kinetic constants for the activity of the enzyme in the hypothalamus,13 cerebral cortex, subcortical white matter and purified myelin membranes14 were shown to resemble those of the recombinant type 1 isoform.12,15 The presence of the type 2 enzyme could not however be excluded in the brain, in that its activity might have been masked by the higher Vmax of the type 1 isoform. It is also possible that the type 2 isozyme is expressed only in particular brain structure(s), and/or during some particular phase of life.
5a-R isozymes during ontogenesis and sexual differentiation of the brain Two isoforms in the brain. Gonadal steroids play an essential role in the process of brain differentiation, especially during embryonic development and in early postnatal life. In mammals, the undifferentiated CNS naturally evolves toward a female pattern, with sexual differentiation toward
the male pattern produced by androgens formed by the fetal testis. This process involves the organization of both endocrine and behavioral functions, including both the dimorphic regulation of gonadotropin, GH secretion, and the control of sexual and aggressive behavior. It is generally believed that the final effector of this process is estradiol, a steroid formed locally in the brain from testosterone by the neuronal enzyme aromatase (see References 16,17 for review); in this case, the differential process is mediated by the estrogen receptor (ER). However, DHT, formed in the CNS from testosterone by 5a-R, also exerts particular organizational effects on selected neuronal populations, and may be involved in the processes of sexual differentiation of specific brain regions. Testosterone might thus act as a differentiating agent via estrogens in some brain areas, nuclei or even single neurons, and via the formation of DHT in others. The fact that the formation of DHT occurs with high yields in some particular brain structures, such as the amygdala, the hypothalamus and the spinal cord (which are also particularly rich in AR),18 suggests that at least in these structures fetal and neonatal androgens may act via AR activated by DHT. At the cellular level, the direct role of androgens on sexual differentiation of the brain is supported by the observation that both testosterone and DHT modify the number of branching points in preoptic neurons in cultures,19,20 acting particularly as morphogenetic signals for the developing hypothalamic neurons containing aromatase, and thus influencing the plasticity and the synaptic connectivity of the hypothalamic aromatase system.21 At higher structural levels, DHT administered to male rats has been shown to decrease the volume of the sexually dimorphic nucleus of the accessory olfactory tract22 to the dimensions usually found in females; this dimorphism is counteracted by treatment of female with antiandrogens.22 Peri- and postnatally both DHT and estrogens are needed to induce a complete masculinization of the sexually dimorphic spinal nucleus of the bulbocavernosus in the rat lumbar spinal cord (References 23–26, see below). A complete characterization of the enzymatic systems involved in the activation of testosterone in the CNS is therefore required to elucidate of the process of brain sexual differentiation. With regard to the two isoforms of 5a-R, the type 1 isoform is expressed and active in several brain cell types cultured from fetal and perinatal rat CNS (mixed glia, type 1 astrocytes, oligodendrocytes, and neurons, which possess the highest levels of the enzyme).27,28 In situ hybridisation data have also shown the presence of 5a-R type 1 mRNA in selected regions of the brain.29 Maximal expression at very early stages of fetal development has been described in the proliferating zones close to the ventricular wall (gestational day, GD12); after birth, expression is highest in the pyramidal cell layer of the hippocampus, the subiculum, the cortical plate, the thalamus and the cerebellum. In older rats expression of the 5a-R type 1 mRNA and of the corresponding translated protein is detectable in white matter structures, such as optic chiasm and corpus callosum,14,29–31 with the enzyme strictly associated with myelin membranes.32 Neither glial nor neuronal cells in culture express the
Steroids, 1998, vol. 63, May/June
247
Steroids in prepubertal period 5a-R type 2 gene27; however, cultured cells may not completely represent the physiological situation, reflecting the absence in the artificial environment of paracrine, endocrine and/or neuronal regulation present in vivo. In fact, while 5a-R type 1 mRNA is always detectable in the whole brain (from GD14 to adult), expression of 5a-R type 2 mRNA is totally different. This isoform is undetectable at GD14, but increases after GD18 to peak at postnatal day (PN) 2, and then decreases gradually to low levels in adults.33 This pattern of expression appears to correlate with the levels of secretion of testosterone from the fetal testis, indicating that the 5a-R type 2 might be modulated by androgens. The fact that the transient expression of the 5a-R type 2 isoform overlaps the critical period of male development of the brain, when circulating androgens34 and AR in the CNS35 are higher, is consistent with this isoform of 5a-R being very important during a crucial period for the sexual differentiation of the brain. This hypothesis was strengthened by the observation that the AR antagonist flutamide abolishes the peak of the 5a-R type 2 expression normally present in the brain of male embryos at birth. Interestingly, the same antiandrogen was much less effective in females. Therefore, the mechanism of control of the type 2 isozyme appears different in male and in the female rat brain33; androgens appear to trigger 5a-R type 2 gene expression in males, whereas in females other control mechanisms must also be involved. Interestingly, 5a-R type 2 gene expression in cultured hypothalamic neurons is highly induced by testosterone and by the phorbol ester TPA; 5a-R type 1 expression remained unchanged during treatment.33 The induction of 5a-R type 2 by testosterone appears to be due to the activation of AR, because DHT is able to mimic this effect, while estradiol (which in neurons can derive from testosterone, but which acts via ER) was unable to induce 5a-R type 2 gene expression (Poletti et al., unpublished). Spinal cord. Several lines of evidence obtained in rodents,25,36,37 amphibians24 and man38 indicate that the medulla oblongata and spinal cord show elevated 5a-R activity. We have recently shown that the 5a-R activity (at neutral pH) is differentially distributed in the cervical, thoracic and lumbosacral tracts of the spinal cord in adult male and female rats.39 In both sexes activity increases progressively in the cranio-caudal direction. RT-PCR analysis performed on total RNA from spinal cord confirmed the presence of the type 1 isoform in amounts similar to those found in the whole brain; it also showed 5a-R type 2 mRNA to be highly expressed in spinal cord, in amounts much higher than those usually found in whole adult brain. It is possible that the presence of 5a-R type 1 in the spinal cord is linked to the high content of myelinated structures in this region (see below). The localization of the type 2 isozyme in spinal motorneurons has been recently demonstrated both in cultured hybrid motoneuron cell lines (NSC34, kindly provided by Dr. N.L. Cashman)40 and on spinal cord tissue slices by in situ hybridization (Poletti et al., unpublished results). Therefore, the selective expression of the type 2 isoform in motoneurons may serve to potentiate the effects that androgens exert on motoneuron growth, development and regeneration.23,41,42
248
Steroids, 1998, vol. 63, May/June
5a-R in stress-evoked pathways The activity of the enzyme 5a-R in the CNS, as in many peripheral androgen target structures, is frequently coupled to that of a second enzyme, 3a-hydroxy-steroid dehydrogenase (3a-HSD); the two enzymes may act as a unit because they work in parallel to regulate the intracellular concentrations of important steroid molecules. This system is highly versatile, in the sense that every D4 –3keto steroid may be first 5a-reduced and subsequently 3a-hydroxylated. Through this pathway, DHT derived from testosterone is converted to 5a-androstane-3a,17b-diol (3a-diol); DHP derived from progesterone is metabolized to allopregnanolone (THP); deoxycorticosterone (DOC) following transformation to dihydroDOC (DHDOC) gives rise to tetrahydroDOC (THDOC). THP and THDOC are two potent regulators of neuronal functions, since they possess sleepinducing and anesthetic/anxiolytic properties.43 The hypnotic effect of progesterone and deoxycorticosterone (DOC) in the rat was described over forty years ago; at the same time these effects were also observed for the two 5areduced 3a-hydroxylated steroids THP and THDOC.44,45 It has now been demonstrated that steroids possessing CNS depressant properties are unable to interact with the classic intracellular receptors for progesterone and for corticoids, and may act through membrane receptors; their action appears to be largely mediated by the GABAA receptor complex.46,47 On this background, it is thus possible that the 5a-R isozymes play physiological roles in situations in which elevated plasma levels of progesterone and/or of corticoids occur. For instance, during stress increased plasma levels of corticoids may provide substrate for the formation of tetrahydroderivatives which act on the GABAA receptor to produce a sedative (anxiolytic) effect.48 Similarly, at time of parturition when the 5a-R type 2 isoform is highly expressed in the fetal brain, plasma levels of progesterone are extremely high, and provide substrate for the production of anesthetic compounds.49 The 5a-R type 2 may also be responsible for the state of sedation which occasionally is seen during pregnancy.49 Consistent with this hypothesis, during late pregnancy female rats also express high levels of 5a-R type 2 mRNA in whole brain (Poletti A. et al., unpublished). These results may also provide a possible molecular explanation for the altered behavioral responses in women at the end of the menstrual cycle and postpartum (premenstrual syndrome, postpartum depression), when there is a significant decrease in 5areduced metabolites of progesterone.49 –51 The low levels of 5a-R type 2 expression in the brain of adult animals is in apparent contrast with these possibilities. Selective expression of this isoform in a few localized brain areas has however been recently observed in adult animals with 5a-R type 2 selectively expressed in the hypothalamus; it appears also to be expressed in the hippocampus after acute stress (Negri-Cesi et al., unpublished). The action of 5a-R type 2 might thus be crucial for local intracerebral formation of active anxiolytic/anesthetic steroids.
5a-R in LHRH-secreting neurons Hormonal steroids are potent modulators of the secretion of LHRH, the primary regulatory component of the hypo-
5a-reductase isozymes in the CNS: Poletti et al. thalamic-pituitary-gonadal axis. This control is exerted via different pathways: indirectly via neurotransmitters/neuropeptides, directly via steroid receptors located within the LHRH synthesizing neurons. LHRH synthesis and release are modulated in vivo by gonadal steroids, and several steroid hormone responsive elements have been identified in the promoter region of the LHRH gene, underlying the possibility of a direct effect of sex steroids on these specialized neurons. Receptors for gonadal steroid have not been detected so far in normal LHRH synthesizing cells.52,53 However the LHRH-secreting neuronal cell line GT1–154 possesses high affinity, low capacity binding sites both for estrogens and androgens.55 A doubling of androgen binding is observed after estradiol treatment, indicating that the binding of estradiol in GT1–1 cells is probably linked to a functional receptor. Expression of classic estrogen56 and androgen57 receptors has subsequently been confirmed by RT-PCR in some clones of the GT1 cells. The absence of ER in LHRH-secreting neurons is postulated on the basis of double labeling immunohistochemical studies52 utilizing antibodies against LHRH and ERa; the recent discovery of a transcriptionally active ERb (with low homology with the ERa in the N-terminal transactivation domain),58,59 which is highly expressed in the brain and particularly in the hypothalamus,60 may lead to a different interpretation in terms of the estrogen sensitivity of LHRH neurons. No detectable aromatase activity is present in GT1–1 cells,55 so that any effect of estradiol acting on such cells must reflect an endocrine or paracrine mechanism, through the transport of estradiol from the circulation or formed in surrounding neurons. On the other hand, GT1–1 cells actively 5a-reduce testosterone to DHT and 3a-diol; 5a-R activity (at neutral pH) in GT1–1 cells is however much less than in cultures of fetal neurons (whereas 3a-HSD activity is 2–3 times higher).55 This discrepancy has now been explained by the demonstration that in GT1–1 cells the 5a-R enzymatic reaction shows a narrow pH optimum around 5.557 (which represents the optimum for the 5a-R type 2) and the Km for testosterone is very similar to that observed for the recombinant type 2 isozyme expressed in yeast12; it is noteworthy that this isoform is usually restricted to classic androgen target tissues. The function of the androgen-responsive machinery in GT1–1 cells is still obscure. As previously noted androgens are necessary during fetal and/or early neonatal life to masculinize the hypothalamic centers which control male-type gonadotropin and GH secretion,61 as well as male and female sexual behavior,62 effects normally mediated by their aromatization to estrogens.63 GT1–1 cells, however, do not possess aromatase, but produce high levels of 5a-R type 2, responsible for intracellular DHT formation; this steroid has also been implicated as a modulator of neuronal differentiation. DHT, by controlling neurite outgrowth in the hypothalamus, might influence the formation of the sex specific neuronal network originating from or reaching LHRH-producing cells (see section 1).
5a-R in myelin As previously mentioned, in the rat brain the highest levels of 5a-R activity are found associated with white matter
structures (see Reference 31 for review), a location linked to the presence of myelin sheaths.14,28,32 During the late (postnatal) stages of ontogeny, 5a-R enzymatic activity appears to parallel the process of myelinization.31,32 The Km values determined in myelin for 5a-R activity (substrate progesterone: male 0.5 mM/female 0.6 mM; substrate testosterone: male 1.1 mM/female 1.5 mM) are all in the micromolar range,32 and are identical to those found for recombinant 5a-R type 1.5,12 A polyclonal antibody raised against a synthetic hydrophilic peptide deduced from the amino-acid sequence of type 1 5a-R specifically recognized the enzymatic protein in the myelin sheaths of the rat optic nerve.32 However, the role of the 5a-R type 1 in myelin membranes is still unclear. Progesterone, the preferred substrate of 5a-R, is able to induce myelin basic protein (MBP), a major component of myelin64,65; this action is probably a genomic effect, since P receptors have been detected in oligodendrocytes,66 and specific glucocorticoid/progesterone response elements have been identified on the MBP promoter,67 leading us to hypothesize a role for 5a-R during myelination.
Catabolic role of 5a-R 5a-reduction of the A ring is an irreversible process, and allows the subsequent 3a- or (3b)-reduction of the 3-oxo group by 3a-HSD or 3b-HSD; while 3a-hydroxylation may give origin to active steroids, 3b-hydroxylation leads to the formation of inactive compounds.31 In general, reduction at the 3 position decreases binding affinity for intracellular receptors. In concert with other enzymes (see below), the 5a-R/3a(3b)-HSD system may thus participate in the catabolism of high concentrations of potentially neurotoxic steroids (e.g., glucocorticoids, which may induce apoptotic processes in particular neuronal populations in the hippocampus; see later). 5a-R type 1, present at all stages of development in various brain cell types,27 efficiently metabolizes androgens, progesterone and the glucocorticoids only when they reach a high concentration inside the cells.6 Moreover, in the rat brain, 5a-R type 1 is highly concentrated in the myelin membranes of axons14,15,32 which help to protect neurons from toxic insults, regulating the types and the amounts of substances reaching the axons; 5a-R type 1 may thus work as a component of the myelin filter. This leads to the hypothesis that the type 1 isoform plays an essentially catabolic role, protecting neurons from excessive levels of steroid hormones. A protective role for 5a-R type 1 has recently described by Mahendroo and colleagues68 in that transgenic mice carrying an inactive form of 5a-R type 1 produce toxic levels of estradiol, derived from increased testosterone bioavailability for aromatization, since testosterone is not removed by 5a-reduction.68,69 This results in a significant decrease in the number of live fetuses; the toxic role of estradiol has been confirmed by the finding that antiestrogens administered to the mothers reversed the effect.68,69 The 5a-R type 1 may thus serve to protect the brain and other structures (e.g., blood vessels) from the damaging effects of excess of circulating estrogens,70 –72 or glucocorticoids.73–76
Steroids, 1998, vol. 63, May/June
249
Steroids in prepubertal period
Conclusions The two isoforms of 5a-R appear to play different physiological roles and to regulate distinct brain events. Type 1 5a-R is constitutively expressed in the brain, and in adulthood appears mainly localized in the myelin membranes. This localization combined with the biochemical properties of the isoform (high capability of conversion, low affinity for the substrates) suggest a catabolic (protective) rather than an activating role of this isozyme in the brain. Type 2 5a-R is transiently expressed in the perinatal period, and at least in males its expression is controlled by androgens. Testosterone similarly induces high levels of expression of 5a-R type 2 in cultured hypothalamic neurons. In adults the expression of 5a-R type 2 appears to be confined in the hypothalamus, and in the hippocampus after stress. Type 2 enzymatic activity is particularly elevated in LHRH-secreting neurons suggesting a possible involvement of 5a-reduced metabolites, derived through this pathway, on LHRH dynamics. Elevated levels of expression of 5a-R type 2 have also been found in the spinal cord, with highest expression in motoneurons. In conclusion, we propose that 5a-R type 2 might participate in the perinatal differentiation of the brain toward a male pattern. The peak of 5a-R type 2 expression in the perinatal period also suggests an involvement in the formation of anxiolytic/anesthetic steroids at the time of parturition.
10. 11.
12.
13. 14.
15.
16. 17.
18. 19.
Acknowledgments Grants funding TeleThon-Italy (A.96); CT96.03105.CT04 (Special Projects ACRO 95.00395.PF39; FATMA 95.00868.PF41; AGING 95.01020.PF40) by MURST and by AIRC.
20. 21. 22.
References 1.
2.
3.
4. 5. 6. 7. 8. 9.
250
Trapman J, Klaassen P, Kuiper GGJM, van der Korput JAGM, Faber PW, van Rooij HCJ, Van Kessel AG, Voorhorst MM, Mulder E, Brinkmann AO (1988). Cloning, structure and expression of a cDNA encoding the human androgen receptor. Biochem Biophys Res Commun 153:241–248. Lubahn DB, Joseph DR, Sullivan PM, Willard HF, French FS, Wilson EM (1988). Cloning of human androgen receptor complementary DNA and localization to the X chromosome. Science (Washington DC) 240:327–330. Kovacs WJ, Griffin JE, Weaver DD, Carlson BR, Wilson, JD (1984). A mutation that causes lability of the androgen receptor under conditions that normally promote transformation to the DNA binding state. J Clin Invest 73:1095–1104. Grino PB, Griffin JE, Wilson JD (1990). Testosterone at high concentration interacts with the human androgen receptor similarly to dihydrotestosterone. Endocrinology 126:1165–1172. Russell DW, Wilson JD (1994). Steroid 5a-reductase: two genes/ two enzymes. Ann Rev Biochem 63:25–61. Normington K, Russell DW (1992). Tissue distribution and kinetic characteristics of rat steroid 5a-reductase isozymes. Evidence for distinct physiological functions. J Biol Chem 267:19548–19554. Taylor MF, Bhattacharyya AK, Collins DC (1995). Inhibition of rat steroid 5a-reductase (isozyme 1) by suramin. Steroids 60:452–456. Poletti A, Rabuffetti M, Martini L (1996). Effect of suramin on the biologic activity of the two isoforms of the rat 5a-reductase Steroids 61:504–505. Schwartz JI, Tanaka WK, Wang DZ, Geissler LA, Hafkin B, Gertz BJ (1997). MK-386, an inhibitor of 5a-reductase type 1, reduces
Steroids, 1998, vol. 63, May/June
23. 24. 25. 26.
27. 28.
29. 30.
dihydrotestosterone concentrations in serum and sebum without affecting dihydrotestosterone concentrations in semen. J Clin Endocr Metab 82:1373–1377. Imperato-McGinley J, Guerrero L, Gautier T, Peterson RE (1974). Steroid 5a-reductase deficiency in man: an inherited form of male pseudohermaphroditism. Science (Washington DC) 186:1213–1215. Katz MD, Cai L, Zhu Y, Herrera C, DeFillo-Ricart M, Shackleton CHL, Imperato-McGinley J (1995). The biochemical and phenotypic characterization of females homozygous for 5a-reductase-2 deficiency. J Clin Endocrinol Metab 80:3160–3167. Poletti A, Celotti F, Motta M, Martini L (1996). Characterization and subcellular localization of rat 5a-reductases type 1 and type 2 expressed in yeast Saccharomyces cerevisiae. Biochem J 314:1047– 1052. Campell JS, Karavolas HJ (1989). The kinetic mechanism of the hypothalamic progesterone 5a-reductase. J Steroid Biochem 32: 283–289. Poletti A, Celotti F, Melcangi RC, Ballabio M, Martini L (1990). Kinetics properties of the 5a-reductase of testosterone in the purified myelin, in the sub-cortical white matter, and in the cerebral cortex of the male rat brain. J Steroid Biochem 35:97–101. Poletti A, Rabuffetti M, Celotti F (1996). The 5a-reductase in the rat brain. In: Gennazzani AR, Petraglia F, Purdy RH (eds), The Brain: Source and Target for Sex Steroid Hormones. London, Parthenon Publishing, UK, pp. 123–133. Lephart ED (1996). A review of brain aromatase cytochrome P450. Brain Res Rev 22:1–26 . Negri-Cesi P, Poletti A, Celotti F (1996). Metabolism of steroids in the brain: a new insight in the role of 5a-reductase and aromatase in the brain differentiation and functions. J Steroid Biochem Mol Biol 58:455–466. Martini L (1982). The 5a-reduction of testosterone in the neuroendocrine structures. Biochemical and physiological implications. Endocrine Rev 3:1–25. Goldstein LA, Sengelaub DR (1994). Differential effects of dihydrotestosterone and estrogens on the development of motoneuron morphology in a sexually dimorphic rat spinal cord. J Neurobiol 25:878–892. Takani K, Kawashima S (1993). Culture of rat brain preoptic area neurons: effects of sex steroids Int J Dev Neurosci 11:63–70. Beyer C, Hutchison JB (1997). Androgens stimulate the morphological maturation of embryonic hypothalamic aromataseimmunoreactive neurons in the mouse. Dev Brain Res 98:74–81. Valencia A, Collado P, Cales JM, Segovia S, Perez Laso C, Rodriguez Zafra M, Guillamon A (1992). Postnatal administration of dihydrotestosterone to the male rat abolishes sexual dimorphism in the accessory olfactory bulb: a volumetric study. Brain Res 68:132– 135. Matsumoto A, Micevych PE, Arnold P (1988). Androgen regulates synaptic input to motoneurones of the adult rat spinal cord. J Neurosci 8:4168–4176. Jurman ME, Erulkar SD, Krieger NR (1982). Testosterone 5areductase in spinal cord of Xenopus laevis. J Neurochem 38:657– 661. Hauser KF, McLusky NJ, Toran-Allerand CD (1987). Androgen action in fetal spinal cultures: metabolic and morphologic aspects. Brain Res 406:62–72. Goldstain LA, Sengelaub DR (1992). Timing and duration of dihydrotestosterone treatment affect the development of motoneuron number and morphology in a sexually dimorphic rat spinal nucleus. J Comp Neurol 326:147–157. Poletti A, Negri-Cesi P, Melcangi RC, Colciago A, Martini L, Celotti F (1997). Expression of androgen-activating enzymes in cultured cells of developing rat brain. J Neurochem 68:1298–1303. Melcangi RC, Celotti F, Castano P, Martini L (1993). Differential localization of the 5a-reductase and 3a-hydroxysteroiddehydrogenase in neuronal and glial cultures. Endocrinology 132: 1252–1259. Lauber ME, Lichtensteiger W (1996). Ontogeny of 5a-reductase (type 1) messenger ribonucleic acid expression in rat brain: early presence in germinal zones. Endocrinology 137:2718–2729. Celotti F, Melcangi, RC, Negri-Cesi, P, Poletti, A (1991). Testosterone metabolism in brain cells and membranes. J Steroid Biochem Mol Biol 40:673–678.
5a-reductase isozymes in the CNS: Poletti et al. 31. 32. 33.
34. 35. 36. 37. 38. 39. 40.
41. 42. 43. 44. 45. 46. 47. 48. 49. 50.
51. 52. 53. 54. 55.
Celotti F, Melcangi RC, Martini L (1992). The 5a-reductase in the brain: Molecular aspects and relation to brain function. Front Neuroendocrinol 13:163–215. Poletti A, Celotti F, Rumio C, Rabuffetti M, Martini L (1997). Identification of type 1 5a-reductase in myelin membranes of male and female rat brain. Mol Cell Endocrinol 129:181–190. Poletti A, Negri-Cesi P, Rabuffetti M, Colciago A, Celotti F, Martini L (1998). Transient expression of the type 2 5a-reductase isozyme in the brain of the late fetal and early post-natal life. Endocrinology, 139:2171–2178. Weisz J, Ward IL (1980). Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses and neonatal offspring. Endocrinology 106:306–316. Meaney MJ, Aitken DH, Jensen LK, McGinnis MY, McEwen BS (1985). Nuclear and cytosolic androgen receptor levels in the limbic brain of neonatal male and female rats. Dev Brain Res 23:179–185. MacLusky NJ, Clark CR, Shanabroug M, Naftolin F (1987). Metabolism of androgens in the spinal cord of the rat. Brain Res 422:83–91. MacLusky NJ, Naftolin F (1981). Sexual differentiation of the central nervous system. Science (Washington DC) 211:1294–1302. Thigpen AE, Silver RI, Guileyardo JM, Casey ML, McConnel JD, Russell DW (1993). Tissue distribution and ontogenicity of steroid 5a-reductase isozyme expression. J Clin Invest 92:903–910. Celotti F, Negri-Cesi P, Poletti A (1997). Steroid metabolism in the mammalian brain: 5a-reduction and aromatization. Brain Res Bull 44:365–375. Cashman NR, Durham HD, Blusztajn JK, Oda K, Tabira T, Shaw IT, Dahrouge S, Antel JP (1992). Neuroblastoma 3 spinal cord (NSC) hybrid cell lines resemble developing motor neurons. Dev Dynamics 194:209–221. Yu WA (1989). Administration of testosterone attenuates neuronal loss following axotomy in the brain-stem nuclei of female rats. J Neurosci 9:3908–3914. Jones KJ (1994). Androgenic enhancement of motor neuron regeneration. Ann NY Acad Sci 743:141–164. Mendelson WB, Martin JV, Perlis M, Wagner R, Majewska MD, Paul SM (1987). Sleep induction by an adrenal steroid in the rat. Psychopharmacology 93:226–229. Selye H (1941). Anesthetic effect of steroid hormones. Proc Soc Exp Biol Med 46:116–121. Selye H (1942). Correlation between the chemical structure and the pharmacological actions of the steroids. Endocrinology 30:437–453. Majewska MD, Harrison NL, Schwartz RD, Barker JL, Paul SM (1986). Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science (Washington DC) 232:1004–1007. Harrison NL, Majewska MD, Harrington JW, Barker JL (1987). Structure-activity relationships for steroid interaction with the GABAA receptor complex. J Pharmacol Exp Therap 241:346–353. Majewska MD (1992). Neurosteroids: Endogenous bimodal modulators of the GABAA receptor. Mechanism of action and physiological significance. Prog Neurobiol 38:379–395. 49. Paul SM, Purdy RH (1992). Neuroactive steroids. FASEB J 6:2311–2322. Rosciszewske D, Buntner B, Guz I, Zawisza L (1986). Ovarian hormones, anticonvulsant drugs, and seizure during the menstrual cycle in women with epilepsy. J Neurol Neurosurg Psychiatry 49:47–51. Majewska MD, Force-Rice F, Kalkay G (1989). Pregnancy-induced alteration of GABAA receptor sensitivity in maternal brain: An antecedent of post-partum “blues”? Brain Res 482:397–401. Shivers BD, Harlan RE, Morrel JI, Pfaff DW (1983). Absence of oestradiol concentration in cell nuclei of LHRH-immunoreactive neurons. Nature (London) 304:345–347. Huang X, Harlan RE (1993). Absence of androgen receptors in LHRH immunoreactive neurons. Brain Res 624:309–311. Mellon PL, Windle JJ, Goldsmith PC, Padula CA, Roberts JL, Weiner RI (1990). Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5:1–10. Poletti A, Melcangi RC, Negri-Cesi P, Maggi R, Martini L (1994).
56.
57.
58. 59. 60. 61.
62. 63. 64.
65. 66.
67. 68. 69. 70.
71. 72.
73. 74. 75. 76.
Steroid binding and metabolism in the LHRH-producing neuronal cell line GT1–1. Endocrinology 135:2623–2628. Lopez FJ, Merchenthaler I, Liposits Z, Negro-Vilar A. (1996). Steroid imprinting and modulation of sexual dimorphism in the luteinizing hormone-releasing hormone neuronal system. Cell Mol Neurobiol 16:129–141. Poletti A, Zanisi M, Maggi R, Martini L (1997). Are luteinizing hormone-releasing hormone secreting neurons androgen-target cells? 79th Annual Meeting of the Endocrine Society Meeting. Abstracts. Minneapolis, MN, USA. June 11–14. Mosselman S, Polman J, Dijkema R (1996). ERb: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA (1996). Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930. Shughrue PJ, Komm B, Merchenthaler I (1996). The distribution of estrogen receptor b mRNA in the rat hypothalamus. Steroids 61: 678–681. Ho KY, Evans WS, Blizzard RM, Veldhuis JD, Merriam GR, Samojlik E, Furlanetto R, Rogol AD, Kaiser DL, Thorner MO (1987). Effects of sex and age on the 24-hour profile of growth hormone secretion in man: importance of endogenous estradiol concentrations. J Clin Endocrinol Metab 64:51–58. Kawata M (1995). Roles of steroid hormones and their receptors in structural organization in the nervous system. Neurosci Res 24:1– 46. McEwen BS (1991). Steroid hormones are multifactorial messengers to the brain. Trends Endocrinol Metab 2:62–67. Jung-Testas I, Schumacher M, Robel P, Baulieu EE (1994). Actions of steroid hormones and growth factors on glial cells of the central and peripheral nervous system. J Steroid Biochem Mol Biol 48:145– 154. Verdi JM, Kampf K, Campagnoni AT (1989). Translational regulation of myelin protein synthesis by steroids. J Neurochem 52:321– 324. Koenig HL, Schumacher M, Ferzaz B, Do Thi AN, Ressouches A, Guennoun R, Jung-Testas I, Robel P, Akwa Y, Baulieu EE (1995). Progesterone synthesis and myelin formation by Schwann cells. Science (Washington DC) 268:1500–1503. Campagnoni AT, Verdi JM, NeilVerity A, Amur-Umarjee S (1990). Posttranscriptional events in the expression of myelin protein genes. Ann NY Acad Sci USA 605:270–279. Mahendroo MS, Cala Km, Landrum CP, Russell DW (1997). Fetal death in mice lacking 5a-reductase type 1 caused by estrogen excess. Mol Endocrinol 11:917–927. Mahendroo MS, Cala KM, Russell DW (1996). 5a-Reduced androgens play a key role in murine parturition. Mol Endocrinol 10:380– 392. Brawer JR, Naftolin F, Martin J, Sonnenschein C (1978). Effects of a single injection of estradiol valerate on the hypothalamic arcuate nucleus and on reproductive function in the female rat. Endocrinology 103:501–512. Brawer JR, Schipper H, Naftolin F (1980). Ovary-dependent degeneration in the hypothalamic arcuate nucleus. Endocrinology 107: 274–279. Naftolin F, Garcia-Segura LM, Keefe D, Leranth C, MacLusky NJ, Brawer JR (1990). Estrogen effects on the synaptology and neural membranes of the rat hypothalamic arcuate nucleus. Biol Reprod 42:21–28. Sapolsky RM (1996). Why stress is bad for your brain. Science (Washington DC) 273:749–750. Yehuda R (1997). Stress and glucocorticoid. Science (Washington DC) 275:1662–1663. Sapolsky RM, Pulsinelli WA (1985). Glucocorticoids potentiate ischemic injury to neurons: therapeutic implications. Science (Washington DC) 229:1397–1400. Sapolsky RM, Packan DR, Vale WW (1988). Glucocorticoid toxicity in the hippocampus: in vitro demonstration. Brain Res 453: 369–371.
Steroids, 1998, vol. 63, May/June
251
Introduction The transformation of testosterone into dihydrotestosterone (DHT) by the enzyme 5a-reductase (5a-R) plays an important physiological role in peripheral androgen-dependent structures; for instance, it controls the development and the function of the prostate, the seminal vesicle, etc. The intracellular formation of DHT precedes the activation of the androgen receptor (AR), which is essential for receptormediated transcription of androgen-responsive genes. In the prostate, this metabolic transformation is a mechanism for amplification of the androgenic signal, since DHT possesses an affinity for the AR four times higher than that of testosterone.1– 4 Moreover, the activated AR is also stabilized by DHT,3,4 and DHT exhibits a fivefold slower dissociation rate from the hormone binding domain of the receptor than its precursor testosterone. Consequently, DHT activates transcription of androgen-dependent genes at concentrations significantly lower than testosterone. The enzyme(s) responsible for its formation are thus important components of the intracellular system for responding to androgens. Two isoforms of the 5a-R (5a-R type 1 and 5a-R type 2) have been cloned (see Reference 5 for review). The human 5a-R type 1 gene comprises five exons and four introns, and produces a protein of 259 amino acids. The structure of the 5a-R type 2 gene is similar to that of the 5a-R type 1, but the resulting protein is of 254 amino acids. Protein sequences deduced from their cDNAs show modest homology Address reprint requests to Angelo Poletti, Istituto di Endocrinologia, via Balzaretti 9, 20133 Milano, Italy. Steroids 63:246 –251, 1998 © 1998 by Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
(about 47%), a predicted molecular weight of 28 –29 kDa, and a high number of hydrophobic amino-acid residues responsible for the intrinsic membrane localization of the enzymes. Both isozymes are able to 5a-reduce all D4 –3keto steroids, but with different kinetics and specificity. Androgens, progestogens and corticosteroids can all be 5a-reduced; their affinity for the type 1 isoform is in the micromolar range, much lower than that for the type 2 isoform, which is in the nanomolar range.6 On the other hand, the conversion yields (Vmax) are much higher for the type 1 isoform for all substrates. For both isozymes, progesterone is the preferred substrate, followed by testosterone and at a considerable distance corticosterone. The two isoforms exhibit different pH optima, with 5a-R type 1 active over a wide pH range (from 5 to 8) and 5a-R type 2 showing a narrow pH optimum around 5.5, with a very low activity at pH 7.5. The two isozymes also show differential sensitivity to synthetic inhibitors, like the selective blocker of the 5a-R type 2 finasteride,5 and the non-specific inhibitor suramine, a drug that acts on the NADPH binding sites of the 5a-R7; however, both inhibitors are most potent toward the type 2 isozyme.5,8 Only preliminary data are available on the efficacy of inhibitors specific for the type 1 isoform.9 Reflecting their different biochemical profiles, the two 5a-R isozymes might be expected to have different physiological roles, even thought they catalyse the same reaction. Studies on their tissue- and cell-specific expression, as well as on the transcriptional control of the two genes, may thus be of help to determine at least some specific functional roles of the two isozymes. In human, the type 1 gene is highly expressed in the liver and in non-genital skin, but 0039-128X/98/$19.00 PII S0039-128X(98)00018-X
5a-reductase isozymes in the CNS: Poletti et al. poorly expressed in androgen target tissues.6 In rats, this isoform is widely distributed throughout various tissues, with the highest levels in the liver. In contrast, in both man and rat, the type 2 isoform appears to be mainly concentrated in androgen-dependent structures such as the prostate (where it is present largely in the stromal component), the epididymis and the seminal vesicles; low levels, if any, have been detected in other tissues.5 Interestingly, a genetically defective type 2 isoform produces a syndrome of male pseudohermaphroditism (Imperato–McGinley syndrome), characterized by aplasia of the prostate and ambiguity of the external genitalia.10,11 The presence of the type 1 isozyme does not seem to be able to replace the inactive type 2 isoform. Several studies have been performed to determine the subcellular localization of the 5a-R, which appears to be associated with cellular membranes.12 A different subcellular localization of the two isozymes has been found by subcellular fractionation studies performed on yeast cells genetically transformed to specifically produce the two rat isozymes: the 5a-R type 1 appears to be associated with cell nuclei, whereas 5a-R type 2 is mainly found in microsomal fractions.12 Considerable 5a-R activity is also present in the central nervous system (CNS), but the physiological functions of the enzyme in the brain are still poorly understood. This review will summarize relevant results from our laboratory on possible functional roles for 5a-R in the CNS. The data will be presented in the following order: 1) 5a-R isozymes during ontogenesis and sexual differentiation of the brain and of the spinal cord; 2) involvement of 5a-R in stressevoked pathways; 3) 5a-R in LHRH-secreting neurons; 4) 5a-R in myelin; and 5) catabolic role of the enzyme.
Roles of the two 5a-Rs in the brain As previously mentioned, several studies indicate that an active 5a-R system is present in the brain. The majority of the data predate the discovery of the two isozymes, and were primarily obtained by measuring the activity of the 5a-R in various tissue samples at neutral pH, at which only the type 1 isozyme is fully functional. After the genes coding two isoforms were cloned, the kinetic constants for the activity of the enzyme in the hypothalamus,13 cerebral cortex, subcortical white matter and purified myelin membranes14 were shown to resemble those of the recombinant type 1 isoform.12,15 The presence of the type 2 enzyme could not however be excluded in the brain, in that its activity might have been masked by the higher Vmax of the type 1 isoform. It is also possible that the type 2 isozyme is expressed only in particular brain structure(s), and/or during some particular phase of life.
5a-R isozymes during ontogenesis and sexual differentiation of the brain Two isoforms in the brain. Gonadal steroids play an essential role in the process of brain differentiation, especially during embryonic development and in early postnatal life. In mammals, the undifferentiated CNS naturally evolves toward a female pattern, with sexual differentiation toward
the male pattern produced by androgens formed by the fetal testis. This process involves the organization of both endocrine and behavioral functions, including both the dimorphic regulation of gonadotropin, GH secretion, and the control of sexual and aggressive behavior. It is generally believed that the final effector of this process is estradiol, a steroid formed locally in the brain from testosterone by the neuronal enzyme aromatase (see References 16,17 for review); in this case, the differential process is mediated by the estrogen receptor (ER). However, DHT, formed in the CNS from testosterone by 5a-R, also exerts particular organizational effects on selected neuronal populations, and may be involved in the processes of sexual differentiation of specific brain regions. Testosterone might thus act as a differentiating agent via estrogens in some brain areas, nuclei or even single neurons, and via the formation of DHT in others. The fact that the formation of DHT occurs with high yields in some particular brain structures, such as the amygdala, the hypothalamus and the spinal cord (which are also particularly rich in AR),18 suggests that at least in these structures fetal and neonatal androgens may act via AR activated by DHT. At the cellular level, the direct role of androgens on sexual differentiation of the brain is supported by the observation that both testosterone and DHT modify the number of branching points in preoptic neurons in cultures,19,20 acting particularly as morphogenetic signals for the developing hypothalamic neurons containing aromatase, and thus influencing the plasticity and the synaptic connectivity of the hypothalamic aromatase system.21 At higher structural levels, DHT administered to male rats has been shown to decrease the volume of the sexually dimorphic nucleus of the accessory olfactory tract22 to the dimensions usually found in females; this dimorphism is counteracted by treatment of female with antiandrogens.22 Peri- and postnatally both DHT and estrogens are needed to induce a complete masculinization of the sexually dimorphic spinal nucleus of the bulbocavernosus in the rat lumbar spinal cord (References 23–26, see below). A complete characterization of the enzymatic systems involved in the activation of testosterone in the CNS is therefore required to elucidate of the process of brain sexual differentiation. With regard to the two isoforms of 5a-R, the type 1 isoform is expressed and active in several brain cell types cultured from fetal and perinatal rat CNS (mixed glia, type 1 astrocytes, oligodendrocytes, and neurons, which possess the highest levels of the enzyme).27,28 In situ hybridisation data have also shown the presence of 5a-R type 1 mRNA in selected regions of the brain.29 Maximal expression at very early stages of fetal development has been described in the proliferating zones close to the ventricular wall (gestational day, GD12); after birth, expression is highest in the pyramidal cell layer of the hippocampus, the subiculum, the cortical plate, the thalamus and the cerebellum. In older rats expression of the 5a-R type 1 mRNA and of the corresponding translated protein is detectable in white matter structures, such as optic chiasm and corpus callosum,14,29–31 with the enzyme strictly associated with myelin membranes.32 Neither glial nor neuronal cells in culture express the
Steroids, 1998, vol. 63, May/June
247
Steroids in prepubertal period 5a-R type 2 gene27; however, cultured cells may not completely represent the physiological situation, reflecting the absence in the artificial environment of paracrine, endocrine and/or neuronal regulation present in vivo. In fact, while 5a-R type 1 mRNA is always detectable in the whole brain (from GD14 to adult), expression of 5a-R type 2 mRNA is totally different. This isoform is undetectable at GD14, but increases after GD18 to peak at postnatal day (PN) 2, and then decreases gradually to low levels in adults.33 This pattern of expression appears to correlate with the levels of secretion of testosterone from the fetal testis, indicating that the 5a-R type 2 might be modulated by androgens. The fact that the transient expression of the 5a-R type 2 isoform overlaps the critical period of male development of the brain, when circulating androgens34 and AR in the CNS35 are higher, is consistent with this isoform of 5a-R being very important during a crucial period for the sexual differentiation of the brain. This hypothesis was strengthened by the observation that the AR antagonist flutamide abolishes the peak of the 5a-R type 2 expression normally present in the brain of male embryos at birth. Interestingly, the same antiandrogen was much less effective in females. Therefore, the mechanism of control of the type 2 isozyme appears different in male and in the female rat brain33; androgens appear to trigger 5a-R type 2 gene expression in males, whereas in females other control mechanisms must also be involved. Interestingly, 5a-R type 2 gene expression in cultured hypothalamic neurons is highly induced by testosterone and by the phorbol ester TPA; 5a-R type 1 expression remained unchanged during treatment.33 The induction of 5a-R type 2 by testosterone appears to be due to the activation of AR, because DHT is able to mimic this effect, while estradiol (which in neurons can derive from testosterone, but which acts via ER) was unable to induce 5a-R type 2 gene expression (Poletti et al., unpublished). Spinal cord. Several lines of evidence obtained in rodents,25,36,37 amphibians24 and man38 indicate that the medulla oblongata and spinal cord show elevated 5a-R activity. We have recently shown that the 5a-R activity (at neutral pH) is differentially distributed in the cervical, thoracic and lumbosacral tracts of the spinal cord in adult male and female rats.39 In both sexes activity increases progressively in the cranio-caudal direction. RT-PCR analysis performed on total RNA from spinal cord confirmed the presence of the type 1 isoform in amounts similar to those found in the whole brain; it also showed 5a-R type 2 mRNA to be highly expressed in spinal cord, in amounts much higher than those usually found in whole adult brain. It is possible that the presence of 5a-R type 1 in the spinal cord is linked to the high content of myelinated structures in this region (see below). The localization of the type 2 isozyme in spinal motorneurons has been recently demonstrated both in cultured hybrid motoneuron cell lines (NSC34, kindly provided by Dr. N.L. Cashman)40 and on spinal cord tissue slices by in situ hybridization (Poletti et al., unpublished results). Therefore, the selective expression of the type 2 isoform in motoneurons may serve to potentiate the effects that androgens exert on motoneuron growth, development and regeneration.23,41,42
248
Steroids, 1998, vol. 63, May/June
5a-R in stress-evoked pathways The activity of the enzyme 5a-R in the CNS, as in many peripheral androgen target structures, is frequently coupled to that of a second enzyme, 3a-hydroxy-steroid dehydrogenase (3a-HSD); the two enzymes may act as a unit because they work in parallel to regulate the intracellular concentrations of important steroid molecules. This system is highly versatile, in the sense that every D4 –3keto steroid may be first 5a-reduced and subsequently 3a-hydroxylated. Through this pathway, DHT derived from testosterone is converted to 5a-androstane-3a,17b-diol (3a-diol); DHP derived from progesterone is metabolized to allopregnanolone (THP); deoxycorticosterone (DOC) following transformation to dihydroDOC (DHDOC) gives rise to tetrahydroDOC (THDOC). THP and THDOC are two potent regulators of neuronal functions, since they possess sleepinducing and anesthetic/anxiolytic properties.43 The hypnotic effect of progesterone and deoxycorticosterone (DOC) in the rat was described over forty years ago; at the same time these effects were also observed for the two 5areduced 3a-hydroxylated steroids THP and THDOC.44,45 It has now been demonstrated that steroids possessing CNS depressant properties are unable to interact with the classic intracellular receptors for progesterone and for corticoids, and may act through membrane receptors; their action appears to be largely mediated by the GABAA receptor complex.46,47 On this background, it is thus possible that the 5a-R isozymes play physiological roles in situations in which elevated plasma levels of progesterone and/or of corticoids occur. For instance, during stress increased plasma levels of corticoids may provide substrate for the formation of tetrahydroderivatives which act on the GABAA receptor to produce a sedative (anxiolytic) effect.48 Similarly, at time of parturition when the 5a-R type 2 isoform is highly expressed in the fetal brain, plasma levels of progesterone are extremely high, and provide substrate for the production of anesthetic compounds.49 The 5a-R type 2 may also be responsible for the state of sedation which occasionally is seen during pregnancy.49 Consistent with this hypothesis, during late pregnancy female rats also express high levels of 5a-R type 2 mRNA in whole brain (Poletti A. et al., unpublished). These results may also provide a possible molecular explanation for the altered behavioral responses in women at the end of the menstrual cycle and postpartum (premenstrual syndrome, postpartum depression), when there is a significant decrease in 5areduced metabolites of progesterone.49 –51 The low levels of 5a-R type 2 expression in the brain of adult animals is in apparent contrast with these possibilities. Selective expression of this isoform in a few localized brain areas has however been recently observed in adult animals with 5a-R type 2 selectively expressed in the hypothalamus; it appears also to be expressed in the hippocampus after acute stress (Negri-Cesi et al., unpublished). The action of 5a-R type 2 might thus be crucial for local intracerebral formation of active anxiolytic/anesthetic steroids.
5a-R in LHRH-secreting neurons Hormonal steroids are potent modulators of the secretion of LHRH, the primary regulatory component of the hypo-
5a-reductase isozymes in the CNS: Poletti et al. thalamic-pituitary-gonadal axis. This control is exerted via different pathways: indirectly via neurotransmitters/neuropeptides, directly via steroid receptors located within the LHRH synthesizing neurons. LHRH synthesis and release are modulated in vivo by gonadal steroids, and several steroid hormone responsive elements have been identified in the promoter region of the LHRH gene, underlying the possibility of a direct effect of sex steroids on these specialized neurons. Receptors for gonadal steroid have not been detected so far in normal LHRH synthesizing cells.52,53 However the LHRH-secreting neuronal cell line GT1–154 possesses high affinity, low capacity binding sites both for estrogens and androgens.55 A doubling of androgen binding is observed after estradiol treatment, indicating that the binding of estradiol in GT1–1 cells is probably linked to a functional receptor. Expression of classic estrogen56 and androgen57 receptors has subsequently been confirmed by RT-PCR in some clones of the GT1 cells. The absence of ER in LHRH-secreting neurons is postulated on the basis of double labeling immunohistochemical studies52 utilizing antibodies against LHRH and ERa; the recent discovery of a transcriptionally active ERb (with low homology with the ERa in the N-terminal transactivation domain),58,59 which is highly expressed in the brain and particularly in the hypothalamus,60 may lead to a different interpretation in terms of the estrogen sensitivity of LHRH neurons. No detectable aromatase activity is present in GT1–1 cells,55 so that any effect of estradiol acting on such cells must reflect an endocrine or paracrine mechanism, through the transport of estradiol from the circulation or formed in surrounding neurons. On the other hand, GT1–1 cells actively 5a-reduce testosterone to DHT and 3a-diol; 5a-R activity (at neutral pH) in GT1–1 cells is however much less than in cultures of fetal neurons (whereas 3a-HSD activity is 2–3 times higher).55 This discrepancy has now been explained by the demonstration that in GT1–1 cells the 5a-R enzymatic reaction shows a narrow pH optimum around 5.557 (which represents the optimum for the 5a-R type 2) and the Km for testosterone is very similar to that observed for the recombinant type 2 isozyme expressed in yeast12; it is noteworthy that this isoform is usually restricted to classic androgen target tissues. The function of the androgen-responsive machinery in GT1–1 cells is still obscure. As previously noted androgens are necessary during fetal and/or early neonatal life to masculinize the hypothalamic centers which control male-type gonadotropin and GH secretion,61 as well as male and female sexual behavior,62 effects normally mediated by their aromatization to estrogens.63 GT1–1 cells, however, do not possess aromatase, but produce high levels of 5a-R type 2, responsible for intracellular DHT formation; this steroid has also been implicated as a modulator of neuronal differentiation. DHT, by controlling neurite outgrowth in the hypothalamus, might influence the formation of the sex specific neuronal network originating from or reaching LHRH-producing cells (see section 1).
5a-R in myelin As previously mentioned, in the rat brain the highest levels of 5a-R activity are found associated with white matter
structures (see Reference 31 for review), a location linked to the presence of myelin sheaths.14,28,32 During the late (postnatal) stages of ontogeny, 5a-R enzymatic activity appears to parallel the process of myelinization.31,32 The Km values determined in myelin for 5a-R activity (substrate progesterone: male 0.5 mM/female 0.6 mM; substrate testosterone: male 1.1 mM/female 1.5 mM) are all in the micromolar range,32 and are identical to those found for recombinant 5a-R type 1.5,12 A polyclonal antibody raised against a synthetic hydrophilic peptide deduced from the amino-acid sequence of type 1 5a-R specifically recognized the enzymatic protein in the myelin sheaths of the rat optic nerve.32 However, the role of the 5a-R type 1 in myelin membranes is still unclear. Progesterone, the preferred substrate of 5a-R, is able to induce myelin basic protein (MBP), a major component of myelin64,65; this action is probably a genomic effect, since P receptors have been detected in oligodendrocytes,66 and specific glucocorticoid/progesterone response elements have been identified on the MBP promoter,67 leading us to hypothesize a role for 5a-R during myelination.
Catabolic role of 5a-R 5a-reduction of the A ring is an irreversible process, and allows the subsequent 3a- or (3b)-reduction of the 3-oxo group by 3a-HSD or 3b-HSD; while 3a-hydroxylation may give origin to active steroids, 3b-hydroxylation leads to the formation of inactive compounds.31 In general, reduction at the 3 position decreases binding affinity for intracellular receptors. In concert with other enzymes (see below), the 5a-R/3a(3b)-HSD system may thus participate in the catabolism of high concentrations of potentially neurotoxic steroids (e.g., glucocorticoids, which may induce apoptotic processes in particular neuronal populations in the hippocampus; see later). 5a-R type 1, present at all stages of development in various brain cell types,27 efficiently metabolizes androgens, progesterone and the glucocorticoids only when they reach a high concentration inside the cells.6 Moreover, in the rat brain, 5a-R type 1 is highly concentrated in the myelin membranes of axons14,15,32 which help to protect neurons from toxic insults, regulating the types and the amounts of substances reaching the axons; 5a-R type 1 may thus work as a component of the myelin filter. This leads to the hypothesis that the type 1 isoform plays an essentially catabolic role, protecting neurons from excessive levels of steroid hormones. A protective role for 5a-R type 1 has recently described by Mahendroo and colleagues68 in that transgenic mice carrying an inactive form of 5a-R type 1 produce toxic levels of estradiol, derived from increased testosterone bioavailability for aromatization, since testosterone is not removed by 5a-reduction.68,69 This results in a significant decrease in the number of live fetuses; the toxic role of estradiol has been confirmed by the finding that antiestrogens administered to the mothers reversed the effect.68,69 The 5a-R type 1 may thus serve to protect the brain and other structures (e.g., blood vessels) from the damaging effects of excess of circulating estrogens,70 –72 or glucocorticoids.73–76
Steroids, 1998, vol. 63, May/June
249
Steroids in prepubertal period
Conclusions The two isoforms of 5a-R appear to play different physiological roles and to regulate distinct brain events. Type 1 5a-R is constitutively expressed in the brain, and in adulthood appears mainly localized in the myelin membranes. This localization combined with the biochemical properties of the isoform (high capability of conversion, low affinity for the substrates) suggest a catabolic (protective) rather than an activating role of this isozyme in the brain. Type 2 5a-R is transiently expressed in the perinatal period, and at least in males its expression is controlled by androgens. Testosterone similarly induces high levels of expression of 5a-R type 2 in cultured hypothalamic neurons. In adults the expression of 5a-R type 2 appears to be confined in the hypothalamus, and in the hippocampus after stress. Type 2 enzymatic activity is particularly elevated in LHRH-secreting neurons suggesting a possible involvement of 5a-reduced metabolites, derived through this pathway, on LHRH dynamics. Elevated levels of expression of 5a-R type 2 have also been found in the spinal cord, with highest expression in motoneurons. In conclusion, we propose that 5a-R type 2 might participate in the perinatal differentiation of the brain toward a male pattern. The peak of 5a-R type 2 expression in the perinatal period also suggests an involvement in the formation of anxiolytic/anesthetic steroids at the time of parturition.
10. 11.
12.
13. 14.
15.
16. 17.
18. 19.
Acknowledgments Grants funding TeleThon-Italy (A.96); CT96.03105.CT04 (Special Projects ACRO 95.00395.PF39; FATMA 95.00868.PF41; AGING 95.01020.PF40) by MURST and by AIRC.
20. 21. 22.
References 1.
2.
3.
4. 5. 6. 7. 8. 9.
250
Trapman J, Klaassen P, Kuiper GGJM, van der Korput JAGM, Faber PW, van Rooij HCJ, Van Kessel AG, Voorhorst MM, Mulder E, Brinkmann AO (1988). Cloning, structure and expression of a cDNA encoding the human androgen receptor. Biochem Biophys Res Commun 153:241–248. Lubahn DB, Joseph DR, Sullivan PM, Willard HF, French FS, Wilson EM (1988). Cloning of human androgen receptor complementary DNA and localization to the X chromosome. Science (Washington DC) 240:327–330. Kovacs WJ, Griffin JE, Weaver DD, Carlson BR, Wilson, JD (1984). A mutation that causes lability of the androgen receptor under conditions that normally promote transformation to the DNA binding state. J Clin Invest 73:1095–1104. Grino PB, Griffin JE, Wilson JD (1990). Testosterone at high concentration interacts with the human androgen receptor similarly to dihydrotestosterone. Endocrinology 126:1165–1172. Russell DW, Wilson JD (1994). Steroid 5a-reductase: two genes/ two enzymes. Ann Rev Biochem 63:25–61. Normington K, Russell DW (1992). Tissue distribution and kinetic characteristics of rat steroid 5a-reductase isozymes. Evidence for distinct physiological functions. J Biol Chem 267:19548–19554. Taylor MF, Bhattacharyya AK, Collins DC (1995). Inhibition of rat steroid 5a-reductase (isozyme 1) by suramin. Steroids 60:452–456. Poletti A, Rabuffetti M, Martini L (1996). Effect of suramin on the biologic activity of the two isoforms of the rat 5a-reductase Steroids 61:504–505. Schwartz JI, Tanaka WK, Wang DZ, Geissler LA, Hafkin B, Gertz BJ (1997). MK-386, an inhibitor of 5a-reductase type 1, reduces
Steroids, 1998, vol. 63, May/June
23. 24. 25. 26.
27. 28.
29. 30.
dihydrotestosterone concentrations in serum and sebum without affecting dihydrotestosterone concentrations in semen. J Clin Endocr Metab 82:1373–1377. Imperato-McGinley J, Guerrero L, Gautier T, Peterson RE (1974). Steroid 5a-reductase deficiency in man: an inherited form of male pseudohermaphroditism. Science (Washington DC) 186:1213–1215. Katz MD, Cai L, Zhu Y, Herrera C, DeFillo-Ricart M, Shackleton CHL, Imperato-McGinley J (1995). The biochemical and phenotypic characterization of females homozygous for 5a-reductase-2 deficiency. J Clin Endocrinol Metab 80:3160–3167. Poletti A, Celotti F, Motta M, Martini L (1996). Characterization and subcellular localization of rat 5a-reductases type 1 and type 2 expressed in yeast Saccharomyces cerevisiae. Biochem J 314:1047– 1052. Campell JS, Karavolas HJ (1989). The kinetic mechanism of the hypothalamic progesterone 5a-reductase. J Steroid Biochem 32: 283–289. Poletti A, Celotti F, Melcangi RC, Ballabio M, Martini L (1990). Kinetics properties of the 5a-reductase of testosterone in the purified myelin, in the sub-cortical white matter, and in the cerebral cortex of the male rat brain. J Steroid Biochem 35:97–101. Poletti A, Rabuffetti M, Celotti F (1996). The 5a-reductase in the rat brain. In: Gennazzani AR, Petraglia F, Purdy RH (eds), The Brain: Source and Target for Sex Steroid Hormones. London, Parthenon Publishing, UK, pp. 123–133. Lephart ED (1996). A review of brain aromatase cytochrome P450. Brain Res Rev 22:1–26 . Negri-Cesi P, Poletti A, Celotti F (1996). Metabolism of steroids in the brain: a new insight in the role of 5a-reductase and aromatase in the brain differentiation and functions. J Steroid Biochem Mol Biol 58:455–466. Martini L (1982). The 5a-reduction of testosterone in the neuroendocrine structures. Biochemical and physiological implications. Endocrine Rev 3:1–25. Goldstein LA, Sengelaub DR (1994). Differential effects of dihydrotestosterone and estrogens on the development of motoneuron morphology in a sexually dimorphic rat spinal cord. J Neurobiol 25:878–892. Takani K, Kawashima S (1993). Culture of rat brain preoptic area neurons: effects of sex steroids Int J Dev Neurosci 11:63–70. Beyer C, Hutchison JB (1997). Androgens stimulate the morphological maturation of embryonic hypothalamic aromataseimmunoreactive neurons in the mouse. Dev Brain Res 98:74–81. Valencia A, Collado P, Cales JM, Segovia S, Perez Laso C, Rodriguez Zafra M, Guillamon A (1992). Postnatal administration of dihydrotestosterone to the male rat abolishes sexual dimorphism in the accessory olfactory bulb: a volumetric study. Brain Res 68:132– 135. Matsumoto A, Micevych PE, Arnold P (1988). Androgen regulates synaptic input to motoneurones of the adult rat spinal cord. J Neurosci 8:4168–4176. Jurman ME, Erulkar SD, Krieger NR (1982). Testosterone 5areductase in spinal cord of Xenopus laevis. J Neurochem 38:657– 661. Hauser KF, McLusky NJ, Toran-Allerand CD (1987). Androgen action in fetal spinal cultures: metabolic and morphologic aspects. Brain Res 406:62–72. Goldstain LA, Sengelaub DR (1992). Timing and duration of dihydrotestosterone treatment affect the development of motoneuron number and morphology in a sexually dimorphic rat spinal nucleus. J Comp Neurol 326:147–157. Poletti A, Negri-Cesi P, Melcangi RC, Colciago A, Martini L, Celotti F (1997). Expression of androgen-activating enzymes in cultured cells of developing rat brain. J Neurochem 68:1298–1303. Melcangi RC, Celotti F, Castano P, Martini L (1993). Differential localization of the 5a-reductase and 3a-hydroxysteroiddehydrogenase in neuronal and glial cultures. Endocrinology 132: 1252–1259. Lauber ME, Lichtensteiger W (1996). Ontogeny of 5a-reductase (type 1) messenger ribonucleic acid expression in rat brain: early presence in germinal zones. Endocrinology 137:2718–2729. Celotti F, Melcangi, RC, Negri-Cesi, P, Poletti, A (1991). Testosterone metabolism in brain cells and membranes. J Steroid Biochem Mol Biol 40:673–678.
5a-reductase isozymes in the CNS: Poletti et al. 31. 32. 33.
34. 35. 36. 37. 38. 39. 40.
41. 42. 43. 44. 45. 46. 47. 48. 49. 50.
51. 52. 53. 54. 55.
Celotti F, Melcangi RC, Martini L (1992). The 5a-reductase in the brain: Molecular aspects and relation to brain function. Front Neuroendocrinol 13:163–215. Poletti A, Celotti F, Rumio C, Rabuffetti M, Martini L (1997). Identification of type 1 5a-reductase in myelin membranes of male and female rat brain. Mol Cell Endocrinol 129:181–190. Poletti A, Negri-Cesi P, Rabuffetti M, Colciago A, Celotti F, Martini L (1998). Transient expression of the type 2 5a-reductase isozyme in the brain of the late fetal and early post-natal life. Endocrinology, 139:2171–2178. Weisz J, Ward IL (1980). Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses and neonatal offspring. Endocrinology 106:306–316. Meaney MJ, Aitken DH, Jensen LK, McGinnis MY, McEwen BS (1985). Nuclear and cytosolic androgen receptor levels in the limbic brain of neonatal male and female rats. Dev Brain Res 23:179–185. MacLusky NJ, Clark CR, Shanabroug M, Naftolin F (1987). Metabolism of androgens in the spinal cord of the rat. Brain Res 422:83–91. MacLusky NJ, Naftolin F (1981). Sexual differentiation of the central nervous system. Science (Washington DC) 211:1294–1302. Thigpen AE, Silver RI, Guileyardo JM, Casey ML, McConnel JD, Russell DW (1993). Tissue distribution and ontogenicity of steroid 5a-reductase isozyme expression. J Clin Invest 92:903–910. Celotti F, Negri-Cesi P, Poletti A (1997). Steroid metabolism in the mammalian brain: 5a-reduction and aromatization. Brain Res Bull 44:365–375. Cashman NR, Durham HD, Blusztajn JK, Oda K, Tabira T, Shaw IT, Dahrouge S, Antel JP (1992). Neuroblastoma 3 spinal cord (NSC) hybrid cell lines resemble developing motor neurons. Dev Dynamics 194:209–221. Yu WA (1989). Administration of testosterone attenuates neuronal loss following axotomy in the brain-stem nuclei of female rats. J Neurosci 9:3908–3914. Jones KJ (1994). Androgenic enhancement of motor neuron regeneration. Ann NY Acad Sci 743:141–164. Mendelson WB, Martin JV, Perlis M, Wagner R, Majewska MD, Paul SM (1987). Sleep induction by an adrenal steroid in the rat. Psychopharmacology 93:226–229. Selye H (1941). Anesthetic effect of steroid hormones. Proc Soc Exp Biol Med 46:116–121. Selye H (1942). Correlation between the chemical structure and the pharmacological actions of the steroids. Endocrinology 30:437–453. Majewska MD, Harrison NL, Schwartz RD, Barker JL, Paul SM (1986). Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science (Washington DC) 232:1004–1007. Harrison NL, Majewska MD, Harrington JW, Barker JL (1987). Structure-activity relationships for steroid interaction with the GABAA receptor complex. J Pharmacol Exp Therap 241:346–353. Majewska MD (1992). Neurosteroids: Endogenous bimodal modulators of the GABAA receptor. Mechanism of action and physiological significance. Prog Neurobiol 38:379–395. 49. Paul SM, Purdy RH (1992). Neuroactive steroids. FASEB J 6:2311–2322. Rosciszewske D, Buntner B, Guz I, Zawisza L (1986). Ovarian hormones, anticonvulsant drugs, and seizure during the menstrual cycle in women with epilepsy. J Neurol Neurosurg Psychiatry 49:47–51. Majewska MD, Force-Rice F, Kalkay G (1989). Pregnancy-induced alteration of GABAA receptor sensitivity in maternal brain: An antecedent of post-partum “blues”? Brain Res 482:397–401. Shivers BD, Harlan RE, Morrel JI, Pfaff DW (1983). Absence of oestradiol concentration in cell nuclei of LHRH-immunoreactive neurons. Nature (London) 304:345–347. Huang X, Harlan RE (1993). Absence of androgen receptors in LHRH immunoreactive neurons. Brain Res 624:309–311. Mellon PL, Windle JJ, Goldsmith PC, Padula CA, Roberts JL, Weiner RI (1990). Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5:1–10. Poletti A, Melcangi RC, Negri-Cesi P, Maggi R, Martini L (1994).
56.
57.
58. 59. 60. 61.
62. 63. 64.
65. 66.
67. 68. 69. 70.
71. 72.
73. 74. 75. 76.
Steroid binding and metabolism in the LHRH-producing neuronal cell line GT1–1. Endocrinology 135:2623–2628. Lopez FJ, Merchenthaler I, Liposits Z, Negro-Vilar A. (1996). Steroid imprinting and modulation of sexual dimorphism in the luteinizing hormone-releasing hormone neuronal system. Cell Mol Neurobiol 16:129–141. Poletti A, Zanisi M, Maggi R, Martini L (1997). Are luteinizing hormone-releasing hormone secreting neurons androgen-target cells? 79th Annual Meeting of the Endocrine Society Meeting. Abstracts. Minneapolis, MN, USA. June 11–14. Mosselman S, Polman J, Dijkema R (1996). ERb: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA (1996). Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930. Shughrue PJ, Komm B, Merchenthaler I (1996). The distribution of estrogen receptor b mRNA in the rat hypothalamus. Steroids 61: 678–681. Ho KY, Evans WS, Blizzard RM, Veldhuis JD, Merriam GR, Samojlik E, Furlanetto R, Rogol AD, Kaiser DL, Thorner MO (1987). Effects of sex and age on the 24-hour profile of growth hormone secretion in man: importance of endogenous estradiol concentrations. J Clin Endocrinol Metab 64:51–58. Kawata M (1995). Roles of steroid hormones and their receptors in structural organization in the nervous system. Neurosci Res 24:1– 46. McEwen BS (1991). Steroid hormones are multifactorial messengers to the brain. Trends Endocrinol Metab 2:62–67. Jung-Testas I, Schumacher M, Robel P, Baulieu EE (1994). Actions of steroid hormones and growth factors on glial cells of the central and peripheral nervous system. J Steroid Biochem Mol Biol 48:145– 154. Verdi JM, Kampf K, Campagnoni AT (1989). Translational regulation of myelin protein synthesis by steroids. J Neurochem 52:321– 324. Koenig HL, Schumacher M, Ferzaz B, Do Thi AN, Ressouches A, Guennoun R, Jung-Testas I, Robel P, Akwa Y, Baulieu EE (1995). Progesterone synthesis and myelin formation by Schwann cells. Science (Washington DC) 268:1500–1503. Campagnoni AT, Verdi JM, NeilVerity A, Amur-Umarjee S (1990). Posttranscriptional events in the expression of myelin protein genes. Ann NY Acad Sci USA 605:270–279. Mahendroo MS, Cala Km, Landrum CP, Russell DW (1997). Fetal death in mice lacking 5a-reductase type 1 caused by estrogen excess. Mol Endocrinol 11:917–927. Mahendroo MS, Cala KM, Russell DW (1996). 5a-Reduced androgens play a key role in murine parturition. Mol Endocrinol 10:380– 392. Brawer JR, Naftolin F, Martin J, Sonnenschein C (1978). Effects of a single injection of estradiol valerate on the hypothalamic arcuate nucleus and on reproductive function in the female rat. Endocrinology 103:501–512. Brawer JR, Schipper H, Naftolin F (1980). Ovary-dependent degeneration in the hypothalamic arcuate nucleus. Endocrinology 107: 274–279. Naftolin F, Garcia-Segura LM, Keefe D, Leranth C, MacLusky NJ, Brawer JR (1990). Estrogen effects on the synaptology and neural membranes of the rat hypothalamic arcuate nucleus. Biol Reprod 42:21–28. Sapolsky RM (1996). Why stress is bad for your brain. Science (Washington DC) 273:749–750. Yehuda R (1997). Stress and glucocorticoid. Science (Washington DC) 275:1662–1663. Sapolsky RM, Pulsinelli WA (1985). Glucocorticoids potentiate ischemic injury to neurons: therapeutic implications. Science (Washington DC) 229:1397–1400. Sapolsky RM, Packan DR, Vale WW (1988). Glucocorticoid toxicity in the hippocampus: in vitro demonstration. Brain Res 453: 369–371.
Steroids, 1998, vol. 63, May/June
251