Transient expression of the 5alpha-reductase type 2 isozyme in the rat brain in late fetal and early postnatal life

The enzyme 5alpha-reductase plays a key role on several brain functions controlling the formation of anxiolytic/anesthetic steroids derived from progesterone and deoxycorticosterone, the conversion of testosterone to dihydrotestosterone, and the removal of excess of potentially neurotoxic steroids. Two 5alpha-reductase isoforms have been cloned: 5alpha-reductase type 1 is widely distributed in the body, and 5alpha-reductase type 2 is confined to androgen-dependent structures. In this study, the gene expression of the two 5alpha-reductase isozymes has been analyzed in fetal, postnatal, and adult rat brains by RT-PCR followed by Southern analysis. 5Alpha-reductase type 1 messenger RNA is always detectable in the rat brain [from gestational day 14 (GD14) to adulthood]. 5Alpha-reductase type 2 messenger RNA expression is undetectable on GD14, increases after GD18, peaks on postnatal day 2, then decreases gradually, becoming low in adulthood. This pattern of expression appears to be correlated with the rate of production of testosterone by the testis. The possible control by androgens of gene expression of the two isozymes has been studied in brain tissues of animals exposed in utero to the androgen antagonist flutamide; the sex of the animals was determined by genetic sex screening of the SRY gene located on the Y-chromosome. In the brain of male embryos, flutamide treatment inhibited the expression of 5alpha-reductase type 2; this effect was much less pronounced in females. Moreover, 5alpha-reductase type 2 gene expression in cultured hypothalamic neurons is highly induced by testosterone and by the phorbol ester 12-O-tetradecanoyl-phorbol-13 acetate. The transient, androgen-regulated, expression of 5alpha-reductase type 2 overlaps the critical period of development, which may be important for sexual differentiation of the brain and for the formation of anxiolytic/anesthetic steroids involved in the stress responses associated with parturition.     

Steroid 5alpha-reductase type 1 immunolocalized in the rat peripheral nervous system and paraganglia

Steroid 5alpha-reductase is an enzyme that converts a number of steroids with a C-4, 5 double bond and C-3 ketone to 5alpha-reduced metabolites. This enzyme has been suggested to play a role in brain development and myelination in the rat nervous system. In the present study, we examined the cellular and subcellular localization of the enzyme immunocytochemically in the rat peripheral nervous system and paraganglia using a polyclonal antibody against rat 5alpha-reductase type 1. Light and electron microscopical studies localized 5alpha-reductase in the Schwann cells of myelinated and unmyelinated nerve fibres, the satellite cells of the ganglia, the enteric glial cells and the supporting/sustentacular cells of the paraganglia. In the myelinated nerve fibres, immunoreactivity was observed in the outer loops, the nodes of Ranvier and the Schmidt-Lanterman incisures. Subcellularly, the immunoreactivity was localized in the cytoplasm of various glial cells. No immunoreactivity was observed in the myelin membrane, the axon or the neuronal perikaryon. These findings suggest that 5alpha-reductase is widely distributed in glial cells, and that, in addition to myelination, 5alpha-reduced steroids play a role in some glial functions in the peripheral nervous system.     

Hypothalamic aromatase cytochrome P450 and 5alpha-reductase enzyme activities in pregnant and female rats

The metabolism of steroid hormones in the medial basal hypothalamus (MBH) is known to play a critical role in neural development, the modulation of neuroendocrine function and regulating sexual behavior. While the important biological functions of the aromatase enzyme are well established, the importance of brain 5alpha-reductase has been revealed and elucidated only in the last few years. The distribution and regulation of brain aromatase and 5alpha-reductase enzyme activities have been investigated for the most part in male rats. Therefore, in the present study, MBH aromatase cytochrome P450 and 5alpha-reductase activities were characterized in pregnant and female rats during postnatal development under various hormonal conditions. MBH aromatase activity was determined in each tissue sample using the 'tritiated water release' assay, whereas, the 5alpha-reductase rates were determined by thin layer chromatography and scintillation counting of the isolated 5alpha-metabolites. Both activities were highest in infantile animals, then declined with increasing postnatal age; whereas, in aged non-cycling or ovariectomized/adrenalectomized (Ovx/Adx) rats high rates of androgen metabolism were seen in MBH tissue. No significant alterations in MBH aromatase were observed when the 5alpha-reductase pathway was blocked in pregnant animals during late gestation with a known 5alpha-reductase inhibitor (Proscar). However, plasma estradiol levels were significantly increased in the Proscar-treated animals. These results indicate that: 1) the decreasing MBH aromatase and 5alpha-reductase profile (in infantile to adult cycling animals) is developmentally regulated, 2) evidently, there is a divergent regulatory mechanism controlling MBH aromatase versus 5alpha-reductase in aged animals where the aromatase activity increased in aged non-cycling and Ovx/Adx rats while 5alpha-reductase rates remained at moderate levels and, 3) apparently, the 5alpha-reductase pathway is not involved in regulating MBH aromatase activity during late pregnancy.     

An analysis of automatic teller machine usage by older adults: a structured interview approach

Sex differences in the activity of aromatase cytochrome P450 (CYP19) in the rat brain have been reported during pre- and postnatal development. It is unclear, however, whether these differences are reflected by corresponding differences in specific mRNA levels. To address this question, we have examined aromatase mRNA levels in specific regions of male and female rat brains by means of in situ hybridization (ISH). At prenatal stages of development, i.e. at gestational day 18 (GD18) and GD20, aromatase mRNA was detected in several preoptic, hypothalamic and limbic brain regions. Semiquantitative analysis of aromatase mRNA did not reveal sex differences in any of these regions. In contrast, clear-cut sex differences were determined at postnatal day (PN) 2; male animals expressed significantly more aromatase mRNA in the bed nucleus of stria terminalis (BST) and the sexually dimorphic nucleus of the preoptic area (SDN). Smaller but still significant differences (females > males) were obtained in the medial preoptic area (MPO). At PN6, sex differences of aromatase mRNA signals (males > females) were still present in the BST, but were no longer detectable in the SDN and the MPO. At PN15 and in adult animals, aromatase mRNA levels were similar in BST and medical amygdaloid nucleus of male and female rats. Since aromatase mRNA expression decreases during postnatal development, no ISH signals could be detected anymore in MPO, SDN and ventromedial hypothalamic nucleus. Our results are consistent with the concept that differential regulation of aromatase mRNA expression might be important for the establishment of different neuronal circuitry in male and female animals.     

Cell-type-specific expression of rat steroid 5 alpha-reductase isozymes

The enzyme steroid 5 alpha-reductase (EC 1.3.99.5) is a component of an intercellular signaling pathway that determines cell fate in the primordium of the mammalian reproductive tract. During male phenotypic sexual differentiation, the dihydrotestosterone product of this enzyme binds to the androgen receptor and initiates development of the external genitalia and prostate. Genes encoding two isozymes of steroid 5 alpha-reductase with different biochemical properties and tissue distributions have recently been isolated. In the current study, we utilize in situ hybridization analysis to determine cell-type-specific expression patterns of the 5 alpha-reductase isozyme mRNAs in two androgen target tissues (regenerating ventral prostate and epididymis) and a peripheral tissue (liver). In regenerating ventral prostate, the type 1 mRNA is expressed in basal epithelial cells whereas expression of the type 2 mRNA is largely confined to stromal cells. These results were confirmed by immunohistochemical analysis and are consistent with distinct roles played by the isozymes in the prostate. In the epididymis, both 5 alpha-reductase isozyme mRNAs are expressed in epithelial cells. Only the type 1 mRNA is present in the liver. This mRNA is distributed in a striking spatial gradient extending from hepatocytes surrounding the portal triad (high expression) to those surrounding the central vein (low to absent expression). These findings demonstrate cell-type-specific expression of the steroid 5 alpha-reductase isozymes and underscore their distinct and overlapping functions in androgen physiology.     

Brain formation of oestrogen in the mouse: sex dimorphism in aromatase development

Steroid sex hormones have an organizational role in gender-specific brain development. Aromatase, converting testosterone (T) to oestradiol-17 beta (E2), is a key enzyme in the brain and the regulation of this enzyme is likely to determine availability of E2 effective for neural differentiation. In rodents, oestrogens are formed very actively during male perinatal brain development. This paper reviews work on the sexual differentiation of the brain aromatase system in vitro. Embryonic day 15 mouse hypothalamic culture aromatase activity (AA: mean Vmax = 0.9 pmol/h/mg protein) is several times greater than in the adult, whereas apparent Km is similar for both (approximately 30-40 nM). Using microdissected brain areas and cultured cells of the mouse, sex differences in hypothalamic AA during both early embryonic and later perinatal development can be demonstrated, with higher E2 formation in the male than in the female. The sex differences are brain region-specific, since no differences between male and female are detectable in cultured cortical cells. AA quantitation and immunoreactive staining with an aromatase polyclonal antibody both identify neuronal rather than astroglial localizations of the enzyme. Kainic acid eliminates the gender difference in hypothalamic oestrogen formation indicating, furthermore, that this sex dimorphism is neuronal. Gender-specific aromatase regulation is regional in the brain. Oestrogen formation is specifically induced in cultured hypothalamic neurones of either sex by T, since androgen has no effect on cortical cells. Androgen is clearly involved in the growth of hypothalamic neurones containing aromatase. It appears that differentiation of the brain involves maturation of a gender-specific network of oestrogen-forming neurones.     

Sex differences in the regulation of embryonic brain aromatase

Oestrogen formed from androgen by aromatization plays a critical role in the sexual differentiation of the male brain and behaviour. A question which has still to be answered is what regulates the gender-specific changes in aromatase activity forming oestrogen during sensitive periods of brain growth. Using a primary cell culture technique and sexed embryos, we have shown that in the fetal mouse brain, oestrogen formation in the male is neuronal rather than glial and aromatase activity is regionally localized, being higher in the hypothalamus than in the cortex. The aromatase activity measured from cells in culture has the same enzyme binding affinity (apparent Km approximately 40 nM) as intact brain samples. Neurones developing in the embryonic male brain (embryonic day (ED) 15) contain higher aromatase activity (Vmax, 895 fmol/h/mg protein) than the female (Vmax, 604). Although a sex difference exists at early stages of embryonic development (ED 13), the embryonic aromatase system is regulated by steroids later in fetal development. The developing aromatase-containing neuroblasts probably form processes which connect to other aromatase neurones. Immunoreactive staining with an aromatase polyclonal antibody identifies an increase in numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies following testosterone treatment. Testosterone treatment also causes both stimulation of neurite growth and branching as well as functional maturation of aromatase neurones. In particular, there is an increase in aromatase activity per neurone as well as a dramatic increase in the number of neurones expressing the enzyme. Both the functional and morphological changes depend on androgen receptor stimulation for several days in vitro. This conclusion is supported by colocalization studies which reveal a high number of fetal hypothalamic aromatase neurones co-expressing androgen receptor. We conclude that testosterone influences the growth of male hypothalamic neurones containing aromatase at a sensitive period of brain development. Endogenous steroid inhibitors of aromatase, probably formed within the neuroglia, also play a role in the control of oestrogen production. An endogenous 5alpha-reduced metabolite of testosterone, 5alpha-androstanedione, is almost as potent in inhibiting neuronal hypothalamic aromatase activity (Ki = 23 nM) as the synthetic non-steroidal inhibitors such as the imidazole, fadrozole, and the triazoles, arimidex and letrozole. It is clear that the oestrogen-forming capacity of the male hypothalamus has the special characteristics and plasticity of regulation which could affect brain differentiation at specific steroid-sensitive stages in ontogeny.     

Photodynamic therapy for rheumatoid arthritis?

Songbirds have emerged as extremely important animals for investigating sex steroid hormone effects on the central nervous system. The masculinizing effects of exogenous estrogen on the neural circuits controlling song in female zebra finches are well documented. There is evidence that estrogens are necessary for the full activation of singing behavior in several species. These kinds of studies have led us and others to investigate the mechanisms whereby estrogens are made available to the brains of songbirds during development and adulthood. In this article, I review results of some of these studies examining the estrogen synthetic enzyme aromatase and its expression and activity in brain and in other tissues of songbirds. I will discuss some results and thoughts we have about the interactions of aromatase with the two remaining androgen-metabolizing enzymes in the avian brain, 5alpha-reductase, the enzyme that converts T into the active androgen 5alpha-dihydrotestosterone (DHT); and 5beta-reductase, the enzyme that converts T into the inactive 5beta-DHT. Finally, I will consider some ideas raised by these studies concerning potential sources of the androgen substrate for brain aromatization as well as some possible new functions that aromatase might be playing in the songbird telencephalon.     

Neuroblastoma and Alzheimer's disease brain cells contain aromatase activity

Human brain steroidogenic mechanisms, particularly aromatase, have been investigated in healthy and diseased conditions. Aromatase activity was measured in differentiated and undifferentiated neuroblastoma cell lines from mouse (TMN) and human (5H SY5Y) and in human post mortem brain samples. Neuroblastomas show much higher aromatase activity than human brain samples. Homogenates of adult human male and female cortex and frontal and temporal areas of both Alzheimer's and control patients all show considerably lower activity. The temporal area has significantly higher aromatase activity than the frontal. Aromatisation activity in differentiated neuroblastoma cells of both species is lower than in undifferentiated cells. These results are consistent with an inverse relationship between brain estrogen formation and stage of neuronal differentiation and the hypothesis that aromatase may be involved in the early stages of neuronal growth. Significant but variable activities of other androgen-metabolising enzymes, such as 5 alpha-reductase, 3 alpha/beta-hydroxysteroid dehydrogenases, and 17 beta-hydroxysteroid dehydrogenase, which generate a spectrum of regulatory molecules, are also found.     

The 5alpha-reductase in the central nervous system: expression and modes of control

The present paper will summarize two important aspects of the interactions between steroids and the brain, which have recently been studied in the authors' laboratory. In particular the paper will consider data on: (1) the significance of the two isoforms of the 5alpha-R during brain ontogenesis and development, and (2) the cross-talk between glial and neuronal elements, particularly in relation to the metabolism of sex hormones. (1) The data obtained have shown that the 5alpha-R type 1 enzyme is constitutively expressed in the rat CNS at all stages of brain development. Moreover, the expression of the 5alpha-R type 1 is similar in males and in females, and does not appear to be controlled by androgens. The gene expression of the 5alpha-R type 2 is totally different. This isoform appears to be expressed in the rat brain almost exclusively in the late fetal/early post-natal life and is controlled by testosterone. (2) The present data show that two cell lines derived respectively from a rat glioma (C6 cell line) and from a human astrocytoma (1321N1 cell line) are able to convert testosterone and progesterone into their corresponding 5alpha-reduced metabolites dihydrotestosterone and dihydroprogesterone. The possibility that secretory products of normal and tumoral brain cells might be able to influence steroid metabolism occurring in the two glial cell lines previously mentioned has been considered.     

Expression of the brain creatine kinase gene in rat RT4 peripheral neurotumor cell lines and its modulation by cell confluence

Creatine kinases (CK) catalyze the reversible transfer of a high energy phosphate group between creatine phosphate and ADP to regenerate ATP in cell types where the requirements for ATP are extensive and/or sudden. Previously, we have shown in primary rat brain cell cultures that brain CK (CKB) mRNA levels are highest in astrocytes and oligodendrocytes and much lower in neuronal cells. However, little is known of the factors which regulate CKB expression in the central nervous system and peripheral nervous system. To begin to investigate these factors, we asked in this report (1) if this pattern of CKB expression was also characteristic of some established glial and neuronal cell lines derived from the PNS; (2) whether CKB expression could be rapidly modulated by culture conditions, and (3) if CKB is expressed in cells with characteristics of glial cell progenitors. In subconfluent cells, CKB mRNA and enzyme activity were found to be high in both the rat RT4 peripheral neurotumor stem cell RT4-AC36A and its glial cell derivative RT4-D6. Conversely, CKB mRNA and activity were 5- and 8-fold lower, respectively, in the neuronal derivative RT4-E5 and, more dramatically, CKB was undetectable in neuronal RT4-B8 cells. Maintaining RT4-D6 glial cells at confluence rapidly increased CKB enzyme activity by 7-fold, such that D6 cells contained about 25% of the CKB level in lysates prepared from either whole adult rat brain or primary cultures of rat brain astrocytes. The levels of CKB mRNA and immunoreactive protein were also correspondingly increased in confluent D6 cells. These confluence-mediated increases in CKB appeared to be due to cell-cell contact and not the depletion of serum growth factors or an increase in intracellular cAMP. This study indicates that CKB expression is highest in cells displaying glial properties and can be rapidly modulated by appropriate culture conditions. The results are discussed in relation to the factors which may regulate CKB expression in vivo.     

Biomechanical analysis of limited intercarpal fusion for the treatment of Kienb?ck''s disease: a three-dimensional theoretical study

Using mRNA differential display technique, we have found a differentially expressed band in rat brain, designated HAP2G1, which was the strongest one induced in response to peripheral administration of lipopolysaccharide (LPS). Sequence analysis showed that HAP2G1 cDNA is the rat homologue of the human alpha-chemokine IP-10. Using RT-PCR technique and in situ hybridization, we demonstrate that IP-10 mRNA was expressed only in brain tissue of rats treated with LPS and not in control brain tissue. Using semi-quantitative PCR, we found that both cultured astrocytes and microglia express IP-10 mRNA after treatment with LPS. LPS-induced IP-10 mRNA reached peak levels in rat brain and in cultured microglia at approximately 3 h after treatment with LPS. At 10 h, IP-10 mRNA was markedly decreased, and at 24 h it was low but still detectable by PCR or in situ hybridization. In contrast to unstimulated microglia, unstimulated astrocytes constitutively expressed IP-10 mRNA at a low level. Increased IP-10 expression could possibly be involved in the microglia response to inflammatory stimuli in vivo.     

Patient psychological and information needs when the diagnosis is diabetes

Both aromatase and 5 alpha-reductase activities were found by whole-cell assay in osteoblast-like cells, MG-63 and HOS. Aromatase activity was measured by the [3H] water release method, and the formation of 5 alpha-androstanedione from androstenedione was expressed as 5 alpha-reductase activity. When CGS16949A, an inhibitor of aromatase, was added to the incubation medium at a concentration of 2 x 10(-9) M, sufficient to completely inhibit placental aromatase activity, only 63% to 68% inhibitions were observed. When progesterone, a competitive inhibitor of 5 alpha-reductase, was added at a concentration of 10(-5) M, 28% to 40% inhibitions were recorded. Because the release of [3H] from [1 beta-3H] androstenedione into water by 5 alpha-reductase is reported, results from the present study suggest that the measurement of aromatase activity in osteoblasts by the [2H] water release method may overestimate aromatase activity owing to the inclusion of 5 alpha-reductase activity. The results also suggest that osteoblast cells may play an important role in bone metabolism by transforming androgens into estrogens and more biologically active androgen derivatives.     

Fetal death in mice lacking 5alpha-reductase type 1 caused by estrogen excess

Female mice deficient in steroid 5alpha-reductase type 1 have a decreased litter size. The average litter in homozygous deficient females is 2.7 pups vs. 8.0 pups in wild type controls. Oogenesis, fertilization, implantation, and placental morphology appear normal in the mutant animals. Fetal loss occurs between gestation days 10.75 and 11.0 commensurate with a midpregnancy surge in placental androgen production and an induction of 5alpha-reductase type 1 expression in the decidua of wild type mice. Plasma levels of androstenedione and testosterone are 2- to 3-fold higher on gestation day 9, and estradiol levels are chronically elevated by 2- to 3-fold throughout early and midgestation in the knockout mice. Administration of an estrogen receptor antagonist or inhibitors of aromatase reverse the high rate of fetal death in the mutant mice, and estradiol treatment of wild type pregnant mice causes fetal wastage. The results suggest that in the deficient mice, a failure to 5alpha-reduce androgens leads to their conversion to estrogens, which in turn causes fetal death in midgestation. These findings indicate that the 5alpha-reduction of androgens in female animals plays a crucial role in guarding against estrogen toxicity during pregnancy.