5α-reductase 1 mRNA levels are positively correlated with TRAMP mouse prostate most severe lesion scores.

BACKGROUND: The contribution of 5α-reductase 1 and 5α-reductase 2 to prostate cancer development and progression is not clearly understood. TRAMP mice are a common prostate cancer model, in which 5α-reductase 1 and 5α-reductase 2 expression levels, along with prostate lesions scores, have not been investigated at different time points to further understand prostate carcinogenesis. METHOD/PRINCIPAL FINDINGS: To this end, 8-, 12-, 16-, and 20-week-old male C57BL/6TRAMP x FVB mice prostate most severe and most common lesion scores, 5α-reductase 1 and 5α-reductase 2 in situ hybridization expression, and Ki-67, androgen receptor, and apoptosis immunohistochemistry levels were measured. Levels of these markers were quantified in prostate epithelium, hyperplasia, and tumors sections. Mice developed low- to high-grade prostatic intraepithelial neoplasia at 8 weeks as the most severe and most common lesions, and moderate- and high-grade prostatic intraepithelial neoplasia at 12 and 16 weeks as the most severe lesion in all lobes. Moderately differentiated adenocarcinoma was observed at 20 weeks in all lobes. Poorly differentiated carcinoma was not observed in any lobe until 12-weeks-old. 5α-reductase 1 and 5α-reductase 2 were not significantly decreased in tumors compared to prostate epithelium and hyperplasia in all groups, while proliferation, apoptosis, and androgen receptor were either notably or significantly decreased in tumors compared with prostate epithelium and hyperplasia in most or all groups. Prostate 5αR1 levels were positively correlated with adjusted prostate most severe lesion scores. CONCLUSION: Downregulation of androgen receptor and 5α-reductase 2, along with upregulation of 5α-reductase 1 in tumors may promote prostatic intraepithelial neoplasia and prostate cancer development in TRAMP mice.

Cellular localization of 5α-reductase in the rat cerebellum.

The enzyme 5α-reductase catalyzes the transformation of progesterone, testosterone, and deoxycorticosterone into 5α-reduced metabolites, which are recognized as neurosteroids in the brain with variable potential neuroactivity. Two isoforms of 5α-reductase were identified in rodents, and, of these, 5α-reductase type 1 (5α-R1) is abundantly expressed in the brain. To understand the multiple influences of neurosteroids in the central nervous system, we need to know their region-specific synthesis. The present study reports the detailed localization of 5α-R1 in the adult rat cerebellum. The occurrence of 5α-R1 was detected by reverse transcription-polymerase chain reaction. The enzyme activity was also detected by thin layer chromatography. Immunocytochemistry showed 5α-R1 immunoreactive cells in all cerebellar layers. Multiple immunolabeling revealed that 5α-R1 was mainly localized in glia, such as astrocytes and oligodendrocytes. The most intense immunoreactivity for 5α-R1 was found in Bergmann glia, and the processes of these glia were associated with dendrites of both Purkinje cells and interneurons in the molecular layer. The 5α-R1 in the cerebellum was expressed consistently throughout different ages and sexes, in both gonadectomized and hypophysectomized rats. Thus, 5α-R1 may contribute to the formation and maintenance of the cerebellar neurons through 5α-reduced metabolites, which are synthesized through a complex interaction between neurons and glia.

Expression of aromatase and two isozymes of 5α-reductase in the developing green anole forebrain.

Neural steroids, as well as the enzymes that produce these hormones, are important for sexual differentiation of the brain during development. Aromatase converts testosterone into oestradiol. 5α-reductase converts testosterone to 5α-dihydrotestosterone and occurs in two isozymes: type 1 (5αR1) and type 2 (5αR2). Each of these enzymes is present in the developing brain in many species, although no work has been carried out examining the expression of all three enzymes in non-avian reptiles with genetic sex determination. In the present study, we evaluated mRNA expression of neural aromatase, 5αR1 and 5αR2, on the day of hatching and at day 50 in one such lizard, the green anole. We describe the distribution of these enzymes throughout the brain and the quantification of mRNA expression in three regions that control adult sexual behaviours: the preoptic area (POA) and ventromedial amygdala (AMY), which are involved in male displays, as well as the ventromedial hypothalamus, which regulates female receptivity. Younger animals had a greater number (POA) and density (AMY) of 5αR1 mRNA expressing cells. We detected no effects of sex or age on aromatase or 5αR2. In comparison with data from adults, the present results support the idea that the green anole forebrain has not completely differentiated by 50 days after hatching and that 5αR1 may play a role in the early development of regions important for masculine function.

Aromatase and 5α-reductase type 2 mRNA in the green anole forebrain: an investigation of the effects of sex, season and testosterone manipulation.

Aromatase and 5α-reductase (5αR) catalyze the synthesis of testosterone (T) metabolites: estradiol and 5α-dihydrotestosterone, respectively. These enzymes are important in controlling sexual behaviors in male and female vertebrates. To investigate factors contributing to their regulation in reptiles, male and female green anole lizards were gonadectomized during the breeding and non-breeding seasons and treated with a T-filled or blank capsule. In situ hybridization was used to examine main effects of and interactions among sex, season, and T on expression of aromatase and one isozyme of 5αR (5αR2) in three brain regions that control reproductive behaviors: the preoptic area, ventromedial nucleus of the amygdala and ventromedial hypothalamus (VMH). Patterns of mRNA generally paralleled previous evaluations of intact animals. Although no main effects of T were detected, interactions were present in the VMH. Specifically, the density of 5αR2 expressing cells was greater in T-treated than control females in this region, regardless of season. Among breeding males, blank-treated males had a denser population of 5αR2 positive cells than T-treated males. Overall, T appears to have less of a role in the regulation of these enzymes than in other vertebrate groups, which is consistent with the primary role of T (rather than its metabolites) in regulation of reproductive behaviors in lizards. However, further investigation of protein and enzyme activity levels are needed before specific conclusions can be drawn.

Isolation rearing-induced reduction of brain 5α-reductase expression: relevance to dopaminergic impairments.

Isolation rearing (IR), a well-established rat model of early chronic psychosocial stress, engenders marked behavioral alterations related to changes of dopamine (DA) neurotransmission in cortical and subcortical brain regions. Stress-induced shifts in γ-aminobutyric acid (GABA)-ergic signaling have been implicated in the dysregulation of DA release. The neurosteroid 3α-hydroxy-5α-pregnan-20-one (allopregnanolone/AP), synthesized from progesterone by the action of the rate-limiting enzyme 5α-reductase (5AR), is a potent positive allosteric modulator of GABA(A) receptor function. Thus, alterations of 5AR activity/expression may impact upon DA neurotransmission. We studied the effects of IR on the 5AR expression/function and extracellular concentrations of DA and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the rat nucleus accumbens (NAcc) and medial prefrontal cortex (mPFC). Immediately after weaning, male rats were subjected to either IR or social rearing (SR) conditions for 5-8 weeks. Compared to SR, IR rats exhibited significantly lower protein expression of 5AR isoforms (1 and 2) in both brain regions and reduced brain, but not plasma, content of AP and allotetrahydrodeoxycorticosterone, the 5α-reduced metabolite of deoxycorticosterone. IR-exposed rats also exhibited higher levels of DA and DOPAC in the NAcc shell, but not in mPFC, when compared to SR rats. The 5AR inhibitor finasteride (FIN, 100 mg/kg, i.p.) enhanced DA and DOPAC content in the NAcc shell of SR, but not IR rats. FIN, however, elicited equivalent increases in DA and DOPAC levels in the mPFC of both groups. These results show that IR induces changes in expression/activity of brain 5AR which, in a brain-region specific manner, may partially underlie the alterations in DA signaling induced by this manipulation. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Chronic treatment with polychlorinated biphenyls (PCB) during pregnancy and lactation in the rat Part 2: Effects on reproductive parameters, on sex behavior, on memory retention and on hypothalamic expression of aromatase and 5alpha-reductases in the offspring.

The gender-specific expression pattern of aromatase and 5alpha-reductases (5alpha-R) during brain development provides neurons the right amount of estradiol and DHT to induce a dimorphic organization of the structure. Polychlorinated biphenyls (PCBs) are endocrine disruptive pollutants; exposure to PCBs through placental transfer and breast-feeding may adversely affect the organizational action of sex steroid, resulting in long-term alteration of reproductive neuroendocrinology. The study was aimed at: a) evaluating the hypothalamic expression of aromatase, 5alpha-R1 and 5alpha-R2 in fetuses (GD20), infant (PN12), weaning (PN21) and young adult (PN60) male and female rats exposed to PCBs during development; b) correlating these parameters with the time of testicular descent, puberty onset, estrous cyclicity and copulatory behavior; c) evaluating possible alterations of some non reproductive behaviors (locomotion, learning and memory, depression/anxiety behavior). A reconstituted mixture of four indicator congeners (PCB 126, 138, 153 and 180) was injected subcutaneously to dams at the dose of 10 mg/kg daily from GD15 to GD19 and then twice a week till weanling. The results indicated that developmental PCB exposure produced important changes in the dimorphic hypothalamic expression of both aromatase and the 5alpha-Rs, which were still evident in adult animals. We observed that female puberty onset occurs earlier than in control animals without cycle irregularity, while testicular descent in males was delayed. A slight but significant impairment of sexual behavior and an important alteration in memory retention were also noted specifically in males. We conclude that PCBs might affect the dimorphic neuroendocrine control of reproductive system and of other neurobiological processes.

Effects of testosterone on brain mRNA levels of steroid 5alpha-reductase isozymes in early postnatal life of rat.

The enzyme 5alpha-reductase (5alpha-R) (EC 1.3.99.5) exists as two isoforms, 5alpha-R type 1 (5alpha-R1) and 5alpha-R type 2 (5alpha-R2), and both are present in the brain. 5alpha-R1 has been proposed as a constitutive enzyme that essentially plays a catabolic and neuron protective role whereas 5alpha-R2 has been associated with sexually dimorphic functions of the male. In this work, we studied the effects of testosterone (T), the masculinizing hormone of the central nervous system (CNS), on mRNA levels of both 5alpha-R isoforms in the prefrontal cortex of male and female rats during the postnatal sexual differentiation of the CNS in the rat, using one-step quantitative RT-PCR coupled with laser-induced fluorescence capillary electrophoresis (LIF-CE). We found an increase in 5alpha-R2 mRNA levels in both male and female rats after T treatment, while 5alpha-R1 mRNA levels were decreased in the same experimental conditions. Our results clearly indicated that T regulates the expression of both 5alpha-R1 and 5alpha-R2 genes in an opposite manner and independently of the sex. This could point to a crucial role of T in the sexual dimorphism for both 5alpha-R isozymes in the neonatal brain. These results open up a new research line that could improve understanding of the role of 5alpha-R isozymes in the physiology of the CNS.

Five-alpha-reductase expression in benign and malignant urothelium: correlation with disease characteristics and outcome.

OBJECTIVES: To evaluate 5-alpha-reductase (5alphaR) expression in benign and malignant urothelium and to assess the relationship between 5alphaR expression and tumor stage, tumor grade, and clinical outcome in patients with urothelial carcinoma/transitional cell carcinoma. METHODS: We performed immunohistochemistry for 5alphaR on 53 urothelial specimens from 36 patients with transitional cell carcinoma treated at our institution between June 2002 and July 2003. For each tumor and the adjacent nontumor urothelium, a semiquantitative staining score was calculated. We used t tests and analysis of variance to compare the staining score across groups. Kaplan-Meier and logistic regression analyses were performed to assess the relationship between 5alphaR expression and clinical outcome. RESULTS: 5alphaR was expressed throughout the non-neoplastic urothelium. Nontumor urothelium had greater mean staining scores than did tumor specimens (160.1 versus 105.5, P <0.01). Low staining scores were associated with high grade (P <0.05), Stage pT3, pT4, and pTis (P <0.05), and disease progression (P <0.05). A staining score less than the median was a risk factor for progression (odds ratio 6.2, P <0.01) on univariate regression analysis. Patients with a staining score less than the median had a greater likelihood of disease progression (log-rank P <0.05) and cause-specific mortality (log-rank P <0.05). CONCLUSIONS: We demonstrated 5alphaR expression in human urothelium and found that expression is decreased in transitional cell carcinoma in relation to tumor grade and stage. Decreased 5alphaR expression was associated with disease progression and cause-specific mortality.

Increased A-ring reduction of glucocorticoids in obese Zucker rats: effects of insulin sensitization.

OBJECTIVE: Obesity is associated with altered glucocorticoid metabolism, which may impact on hypothalamic-pituitary-adrenal axis activity. Here we characterize hepatic 5alpha- and 5beta-reductase in obese rats and their responses to insulin sensitization. RESEARCH METHODS AND PROCEDURES: Hepatic A-ring reductase protein and mRNA were assessed in lean and obese Zucker rats after insulin sensitization with metformin or rosiglitazone (n = 7 to 8/group). RESULTS: Hepatic 5alpha-reductase 1 and 5beta-reductase mRNA and protein (p < 0.01) were increased in obese rats. Insulin sensitization ameliorated increased 5alpha-reductase 1 mRNA in obese rats (p < 0.01) and partially reversed increased 5beta-reductase activity. DISCUSSION: Hepatic clearance of glucocorticoids by 5alpha- and 5beta-reductase is increased in obese Zucker rats, and this increase in clearance is attenuated by insulin sensitization. This increased hepatic clearance may underpin compensatory activation of the hypothalamic-pituitary-adrenal axis in obesity.

Ontogeny of 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase in the frog brain.

The distribution of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 5alpha-reductase (5alpha-R) has been studied in the frog brain during development. Soon after hatching, 3beta-HSD- and 5alpha-R-immunoreactive (ir) cells appeared first in the olfactory bulb and in the rhombencephalon. Subsequently, 3beta-HSD-ir cells were seen in the hypothalamus and cerebellum, whereas 5alpha-R-ir cells were visualized in the pallium, preoptic nucleus, posterocentral nucleus, cerebellum, and pituitary gland. At stages XIII-XVIII, additional 3beta-HSD- and 5alpha-R-ir cells appeared in several regions of the telencephalon, diencephalon, and mesencephalon. At stages XIX-XXI, the number of 5alpha-R-ir cells increased in the preoptic nucleus. These observations indicate that biosynthesis of biologically active steroids occurs in the brain of tadpoles, suggesting that neurosteroids may play a role in brain development.

5alpha-reductase in human embryonic kidney cell line HEK293: evidence for type II enzyme expression and activity.

Human 5alpha-reductase catalyses the last step in androgen biosynthesis, namely the reduction of testosterone (T) to the more potent androgen dihydrotestosterone (DHT). The enzyme is therefore considered to be an important drug target for androgen related diseases such as benign prostatic hyperplasia and prostate cancer. The present study displays evidence that the human embryonic kidney cell line HEK293 which is frequently used in recombinant target protein expression contains an endogenous 5alpha-reductase type II activity. After an incubation of 24 h 1 x 10(6) HEK293 cells converted 23% of the substrate 4-androstene-3,17-dione (7.5 nM) to the product 5alpha-androstane-3,17-dione. Reverse transcription polymerase chain reaction was carried out to identify the mRNA of the isoform responsible for the 5alpha-reductase activity. Only with type II specific primers a fragment with the predicted size was amplified, while with type I specific primers no band could be observed. An antiserum against human 5alpha-reductase type II was raised by immunizing a rabbit with a hemocyanin-conjugated peptide corresponding to amino acid 29 to 44 of the type II enzyme. Western blot analysis of different fractions of a HEK293 homogenate performed with this antiserum detected a band at 45 kDa in the nuclear and microsomal fraction corresponding to 5alpha-reductase type II protein.

Progesterone enhances motor, anxiolytic, analgesic, and antidepressive behavior of wild-type mice, but not those deficient in type 1 5 alpha-reductase.

The importance of progesterone's (P(4)) metabolism by the 5 alpha-reductase type I enzyme was examined in homozygous and heterozygous 5 alpha-reductase type I knockout mice and their wild-type siblings. P(4) (1.0 mg) or vehicle was administered and effects on motor, anxiety, nociceptive, and depression behavior were observed. After testing, whole-brain progesterone and 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha,5 alpha-THP) levels were determined by radioimmunoassay. Motor behavior in the horizontal crossing and open field tasks of 5 alpha-reductase-deficient mice administered P(4) was similar to vehicle control mice and significantly reduced compared to wild-type mice administered P(4). In the open field, 5 alpha-reductase-deficient mice administered P(4) had a similar number of central entries as did vehicle control mice, both were lower than central entries of P(4)-administered wild-type mice. However, in the plus maze, P(4) to 5 alpha-reductase-deficient or wild-type mice significantly increased open arm activity compared to vehicle-administered control mice. P(4) to wild-type, but not 5 alpha-reductase-deficient mice, significantly increased latencies to lick front and back paws in response to radiant heat stimuli compared to vehicle administration to control mice. In the forced swim test, 5 alpha-reductase-deficient mice administered P(4) were similar to vehicle control mice and the latency to immobility was significantly decreased, and the duration of immobility was significantly increased, compared to wild-type mice administered P(4). Thus, these data suggest metabolism by the 5 alpha-reductase type I enzyme may mitigate P(4)'s effects on some tasks of motor, anxiety, nociception, and depression behavior.