Methylated CpG dinucleotides in the 5-α reductase 2 gene may explain finasteride resistance in benign prostatic enlargement patients.

The inhibition of 5-α reductase type 2 (SRD5A2) by finasteride is commonly used for the management of urinary obstruction resulting from benign prostatic enlargement (BPE). Certain BPE patients showing no SRD5A2 protein expression are resistant to finasteride therapy. Our previous work showed that methylated cytosine-phosphate-guanine (CpG) islands in the SRD5A2 gene might account for the absence or reduction of SRD5A2 protein expression. Here, we found that the expression of the SRD5A2 protein was variable and that weak expression of the SRD5A2 protein (scored 0-100) occurred in 10.0% (4/40) of benign adult prostates. We showed that the expression of SRD5A2 was negatively correlated with DNA methyltransferase 1 (DNMT1) expression. In vitro SRD5A2-negative BPH-1 cells were resistant to finasteride treatment, and SRD5A2 was re-expressed in BPH-1 cells when SRD5A2 was demethylated by 5-Aza-2'-deoxycytidine (5-Aza-CdR) or N-phthalyl-L-tryptophan (RG108). Furthermore, we determined the exact methylation ratios of CpG dinucleotides in a CpG island of SRD5A2 through MassArray quantitative methylation analysis. Ten methylated CpG dinucleotides, including four CpG dinucleotides in the promoter and six CpG dinucleotides in the first exon, were found in a CpG island located from -400 bp to +600 bp in SRD5A2, which might lead to the silencing of SRD5A2 and the absence or reduction of SRD5A2 protein expression. Finasteride cannot exert a therapeutic effect on patients lacking SRD5A2, which may partially account for the resistance to finasteride observed in certain BPE patients.

Early upregulation of AR and steroidogenesis enzyme expression after 3 months of androgen-deprivation therapy.

BACKGROUND: Androgen deprivation therapy (ADT) is a standard treatment for advanced prostate cancer (PCa). However, PCa recurrence and progression rates during ADT are high. Until now, there has been no evidence regarding when progression begins. This study evaluated the gene expression of intraprostatic androgen receptor (AR) and steroidogenic enzymes in the early stages of ADT. METHODS: Prostate tissue samples were taken from PCa patients with urinary retention who received ADT (ADT-PCa; n = 10) and were further subgrouped into ADT ≤12 months (n = 4) and ADT > 12 months (n = 6). The ADT-PCa tissues were then compared with BPH (n = 12) and primary (no treatment) PCa tissues (n = 16). mRNA for gene expression analysis of AR and steroidogenic enzymes was extracted from formalin-fixed paraffin embedded (FFPE) tissues and analyzed by real-time PCR. Protein expression was evaluated by immunohistochemistry with specific antibodies. RESULTS: AR gene expression was higher in the ADT-PCa group than in the BPH or primary PCa group. Both the ADT ≤12 and > 12 months subgroups had significantly higher relative gene expression levels of AR (p < 0.01 and 0.03, respectively) than the primary PCa group. In the ADT-PCa group, AR protein expression showed an increasing trend in the ADT ≤12 months subgroup and was significantly elevated in the ADT > 12 months subgroup compared with the PCa group (100%; p < 0.01). Half (50%) of the patients in the ADT ≤12 months subgroup were found to have upregulation of AR, and one showed upregulation beginning at 3 months of ADT. A trend toward elevated relative gene expression of SRD5A3 was also apparent in the ADT groups. CONCLUSION: AR and steroidogenic enzymes are upregulated in ADT-PCa patients as early as 3 months, without PSA elevation. Steroidogenic enzymes, particularly SRD5A3, were also upregulated before PSA rose.

Paraquat exposure delays stem/progenitor Leydig cell regeneration in the adult rat testis.

Paraquat, a widely used nonselective herbicide, is a serious hazard to human health. However, the effects of paraquat on the male reproductive system remain unclear. In this study, adult male Sprague Dawley rats were intraperitoneally injected ethane dimethane sulfonate (EDS, 75 mg/kg) to initiate a regeneration of Leydig cells. EDS-treated rats were orally exposed to paraquat (0.5, 2, 8 mg/kg/day) from post-EDS day 17 to day 28 and effects of paraquat on Leydig and Sertoli cell functions on post-EDS day 35 and day 56 were investigated. Paraquat significantly decreased serum testosterone levels at 2 and 8 mg/kg. Paraquat lowered Leydig cell Hsd17b3, Srd5a1, and Hsd11b1 mRNA levels but increased Hsd3b1 on post-EDS day 35. Paraquat lowered Cyp11a1, Cyp17a1, and Hsd11b1 but increased Srd5a1 on post-EDS day 56. However, paraquat did not alter Leydig cell number and PCNA labeling index. Epididymal staining showed that few sperms were observed in paraquat-treated rats. Primary culture of adult Leydig cells showed that paraquat diminished testosterone output and induced reactive oxygen species generation at 1 and 10 µM and apoptosis rate at 10 µM. In conclusion, a short-term exposure to paraquat delays Leydig cell regeneration from stem/progenitor Leydig cells, causing low production of testosterone and an arrest of spermatogenesis.

5α-Reductase isozymes and aromatase mRNA levels in plucked hair from young women with female pattern hair loss.

Female pattern hair loss (FPHL) is an important hair disorder, especially when young women are affected. However, pharmacological treatments are not successful in all women. Androgens, especially dihydrotestosterone (DHT), may play a role in FPHL, but many women with this disorder have normal serum androgen levels. It therefore appears that hair follicle levels of DHT depend on in situ testosterone (T) metabolism. Because T can be converted to DHT or estradiol (E2) by 5α-reductase (5α-R) and aromatase, respectively, these enzymes would determine DHT and E2 concentrations and their ratio. We propose and apply a low-invasive, sensitive and precise method for the absolute quantification of mRNA levels of aromatase and 5α-R isozymes (type 1, type 2 and type 3) in plucked hair from young women with FPHL. Normoandrogenic women with FPHL and controls were studied. Plucked hair samples were obtained by trichogram from vertex scalp and mRNA levels quantified by real-time RT-PCR. We revealed for the first time the presence of 5α-R3 mRNA in human hair. Interestingly, one, two, or even three 5α-R isozymes were increased in some women with FPHL but not in others, which may explain the lack of response to 5α-R inhibitors in some FPHL cases. Aromatase mRNA levels were significantly lower in women with FPHL than in controls. It may therefore produce a reduction in oestrogen levels and an increase in the androgen/oestrogen ratio in hair. The proposed low-invasive technique offers a molecular aetiologic diagnosis of FPHL for the selection of more appropriate pharmacological treatments with early predicted effectiveness.

Characterization of 5α-reductase activity and isoenzymes in human abdominal adipose tissues.

INTRODUCTION: The substrate for the generation of 5α-dihydrotestosterone (DHT) is either androstenedione (4-dione) which is first converted to androstanedione and then to DHT through 17-oxoreductase activity, or testosterone, which is directly converted to DHT. Three 5α-reductase isoenzymes have been characterized and designated as types 1, 2 and 3 (SRD5A1, 2 and 3). OBJECTIVE: To define the predominant source of local DHT production in human adipose tissues, identify 5α-reductase isoenzymes and test their impact on preadipocyte differentiation. METHODS: Cultures of omental (OM) and subcutaneous (SC) preadipocytes were treated for 0, 6 or 24h with 30nM (14)C-4-dione or (14)C-testosterone, with and without 500nM 5α-reductase inhibitors 17-N,N-diethylcarbamoyl-4-methyl-4-aza-5-androstan-3-one (4-MA) or finasteride. Protein level and mRNA abundance of 5α-reductase isoenzymes/transcripts were examined in whole SC and OM adipose tissue. HEK-293 cells stably transfected with 5α-reductase type 1, 2 or 3 were used to test 5α-reductase inhibitors. We also assessed the impact of 5α-reductase inhibitors on preadipocyte differentiation. RESULTS: Over 24h, DHT formation from 4-dione increased gradually (p<0.05) and was significantly higher compared to that generated from testosterone (p<0.001). DHT formation from both 4-dione and testosterone was blocked by both 5α-reductase inhibitors. In whole adipose tissue from both fat compartments, SRD5A3 was the most highly expressed isoenzyme followed by SRD5A1 (p<0.001). SRD5A2 was not expressed. In HEK-293 cells, 4-MA and finasteride inhibited activity of 5α-reductases types 2 and 3 but not type 1. In preadipocyte cultures where differentiation was inhibited by 4-dione (p<0.05, n=7) or testosterone (p<0.05, n=5), the inhibitors 4-MA and finasteride abolished these effects. CONCLUSION: Although 4-dione is the main source of DHT in human preadipocytes, production of this steroid by 5α-reductase isoenzymes mediates the inhibitory effect of both 4-dione and testosterone on preadipocyte differentiation.

GEMMs shine a light on resistance to androgen deprivation therapy for prostate cancer.

Androgen deprivation therapy (ADT) for advanced prostate cancer inexorably leads to resistance, and clinically useful biomarkers are lacking. The value of genetically engineered mice for coclinical studies is clearly demonstrated in a recent publication that reveals XAF1, XIAP, and SRD5A1 as novel predictive biomarkers and therapeutic targets for ADT resistance.

Expanding the use of fluorogenic enzyme reporter substrates to imaging metabolic flux changes: the activity measurement of 5α-steroid reductase in intact mammalian cells.

The study of dynamic properties of metabolic and signaling networks is hindered by the lack of methods for imaging metabolic fluxes in individual intact cells. We describe a novel optical approach for measuring the changes of metabolic fluxes in cells, based on a two-substrate competition between a physiological substrate and a fluorogenic reporter substrate. We have constructed a model cell system for a two-step metabolic pathway involved in the metabolism of testosterone. Potent androgen testosterone is converted by steroid 5α-reductase to DHT (5α-dihydrotestosterone), which is subsequently metabolized to 3α-diol (3α,17ß-androstanediol) by the reductase AKR1C2 (aldo-ketoreductase 1C2), for which we have previously developed the fluorogenic reporter substrate Coumberone. Despite the medicinal importance of 5α-reductase, there are presently no probes or methods for the continuous activity readout of this enzyme in cells. We show that the activity of 5α-R1 (5α-reductase type 1) can be measured in COS-1 cells via the changes of DHT flux. Our system enables a measurement of 5α-reductase activity in cells, via either fluorimetry or fluorescence microscopy, with a wide dynamic range of activities, and provides a continuous optical assay for evaluation of small molecule inhibitors for this important enzyme. Furthermore, this paper demonstrates a novel optical approach to measuring metabolic flux changes in living cells and expands the utility of fluorogenic enzyme reporter substrates: optical reporters can measure not only the activity of the target enzyme but also the activity of other enzymes upstream in the pathway, for which there are no probes available.

Determination of rat 5alpha-reductase type 1 isozyme activity and its inhibition by novel steroidal oxazolines.

The 5alpha-reductase type 1 isozyme is a key enzyme in the metabolism of the androgen steroid hormones and inhibitors of this enzyme represent a new pharmacological treatment for several androgen dependent diseases. We developed a radiosubstrate in vitro incubation method for the determination of 5alpha-reductase type 1 activity using rat liver microsomes as an enzyme source. With this method we have studied the inhibiting activity of novel (5' S)-17beta-(4,5-dihydrooxazol-5-yl)androst-5-en-3-one compounds containing various derivatized phenyl substituents coupled to the exo -heterocyclic moiety. Tests revealed moderate inhibitory actions compared to finasteride, nevertheless, results provide interesting structure-activity relationship data.

New therapeutic approaches for epilepsies, focusing on reorganization of the GABAA receptor subunits by neurosteroids.

Neurosteroids such as allopregnanolone (THP) act as positive allosteric modulators of gamma-aminobutyric acid (GABA)A receptors and have exerted anticonvulsant properties. However, their role in the regulation of epileptogenesis is unclear. It has been shown that circulating levels of THP fluctuate during development and seizure episodes. Furthermore, both chronic administration of THP and its withdrawal transiently increase expression of the alpha4 subunit of the GABAA receptor in the brain. The steroidogenic enzymes, 5-alpha-reductase (5aR) and 3-alpha-hydroxysteroid dehydrogenase (3aHSD) have been identified as well, indicating that various cell types are involved in the biosynthesis of neuroactive steroids in the brain. The purpose of the present study is to examine how GABAA receptor-modulating neurosteroids contribute to the epileptogenesis by using the epileptic mutant EL mouse. Male EL mice and control animals, DDY mice, were used. EL mice show secondary generalized seizures, which initiate primarily at the parietal cortex and generalize through the hippocampus. In the interictal period during development, changes of THP, 5aR, 3aSDH, and GABAA receptor alpha4, gamma2, and delta subunits were investigated by western blotting in the hippocampus. In EL mice, levels of the neurosteroid THP and the steroidogenic enzymes 5aR and 3aSDH significantly increased at 3 weeks of age, and rapidly decreased thereafter (5-10 weeks). The sharp withdrawal was observed before mice experienced frequent seizures. In contrast, GABAA alpha4, gamma2, and delta expressions were upregulated (3-8 weeks). In the brain of EL mice, positive neurosteroids such as THP were withdrawn before mice experienced repetitive seizures, which may likely be a trigger for ictogenesis and epileptogenesis. Furthermore, reorganization of the GABAA receptor subunits may lead to a hypersensitivity of the receptor to neurosteroids. Therefore, GABAA receptor-regulating neurosteroids may be a promising target for the development of novel antiepileptic agents.

Clinical characterization and analysis of the SRD5A2 gene in six Korean patients with 5alpha-reductase type 2 deficiency.

AIMS: The aim of this study was to perform a 5alpha-reductase type 2 gene (SRD5A2) analysis in 6 Korean patients with external genitalia ranging from predominantly female to male in whom 5alpha-reductase type 2 deficiency was suspected. PATIENTS: Six patients from five unrelated families participated, and all of their parents were non-consanguineous. Three patients presented with ambiguous genitalia at birth, and 2 were referred owing to delayed puberty. The other patient was presented incidentally during an operation for inguinal hernia. Basal and post-human chorionic gonadotropin-stimulated serum testosterone and dihydrotestosterone levels were determined, but neither the levels nor ratio yielded enough information for differential diagnosis. Confirmative diagnosis was achieved by SRD5A2 gene analysis. RESULTS: Four different pathologic mutations were identified. All have already been reported, and are located in exon 1 (p.Q6X), exon 4 (p.G203S and c.655delT), and exon 5 (p.R246Q). p.R246Q was the most frequently identified mutation in our study, and c.655delT has been detected only in Korean patients to date. CONCLUSION: The molecular analysis is the most reliable method for a correct diagnosis of 5alpha-reductase type 2 deficiency. Identification of mutations also enables genetic counseling for families at risk.

Levels of 5alpha-reductase type 1 and type 2 are increased in localized high grade compared to low grade prostate cancer.

PURPOSE: In the prostate testosterone is converted to dihydrotestosterone by 5alpha-reductase type 1 and/or 2. Although 5alpha-reductase type 2 is predominant in normal prostates, type 1 is increased in cancer vs benign tissue. It is unclear whether 5alpha-reductase type 1/2 levels correlate with cancer grade. We compared the relative expression of 5alpha-reductase type 1 and 2 in localized high and low grade prostate cancer. MATERIALS AND METHODS: Immunostaining for 5alpha-reductase type 1/2 was evaluated in 64 prostate tissues from untreated men with localized prostate cancer. The percent of tumor area with moderate-high intensity staining was estimated for each Gleason pattern in the tissues. Adjacent benign tissue was evaluated in 26 prostate cancer specimens. RESULTS: Moderate-high staining for 5alpha-reductase type 1 increased from 18.8% +/- 2.9% (mean +/- SEM) in 34 Gleason pattern 3 cancers to 31.0% +/- 4.1% in 30 Gleason pattern 4/5 cancers (p = 0.016). Staining for 5alpha-reductase type 2 increased from 22.9% +/- 3.0% in 34 Gleason pattern 3 cancers to 39.2% +/- 4.1% in 30 Gleason pattern 4/5 cancers (p = 0.002). Compared to benign prostatic hyperplasia tissues staining for 5alpha-reductase type 1 was greater than 3-fold higher and staining for 5alpha-reductase type 2 was significantly lower in benign tissue adjacent to cancer (p = 0.006 and 0.0236, respectively). CONCLUSIONS: Levels of 5alpha-reductase type 1 and 2 are increased in localized high vs low grade prostate cancer. Levels of 5alpha-reductase type 1 are higher in benign tissue adjacent to cancer than in benign prostatic hyperplasia. These results raise the possibility that increased 5alpha-reductase type 1 in localized high grade cancers may contribute to the decreased effectiveness of the 5alpha-reductase type 2 selective inhibitor finasteride against high grade prostate cancer in the Prostate Cancer Prevention Trial.

5Alpha-reductase type 1 and aromatase in breast carcinoma as regulators of in situ androgen production.

Previous in vitro studies demonstrated that bioactive androgen 5alpha-dihydrotestosterone (DHT) exerted antiproliferative effects through an interaction with androgen receptor (AR) in breast carcinoma cells. However, AR status has not been examined in association with DHT concentration in breast carcinoma tissues, and significance of androgenic actions remains unclear in breast carcinomas. Therefore, in our study, we first examined intratumoral DHT concentrations in 38 breast carcinoma tissues using liquid chromatography/electrospray tandem mass spectrometry. Intratumoral DHT concentration was positively associated with 5alpha-reductase type 1 (5alphaRed1), and negatively correlated with aromatase. We then examined clinical significance of AR and 5alphaRed1 status in 115 breast carcinoma tissues by immunohistochemistry. Breast carcinomas positive for both AR and 5alphaRed1 were inversely associated with tumor size or Ki-67. These patients showed significant associations with a decreased risk of recurrence and improved prognosis for overall survival, and the AR / 5alphaRed1 status was demonstrated an independent prognostic factor. Moreover, we examined possible regulation of DHT production by aromatase in in vitro studies. DHT synthesis from androstenedione in MCF-7 cells was significantly inhibited by coculture with aromatase-positive stromal cells, which was significantly reversed by addition of aromatase inhibitor exemestane. These results suggest that intratumoral DHT concentration is mainly determined by 5alphaRed1 and aromatase in breast carcinoma tissues, and antiproliferative effect of DHT may primarily occur in the cases positive for both AR and 5alphaRed1. Aromatase inhibitors may be more effective in these patients, possibly due to increasing local DHT concentration with estrogen deprivation.

Diagnosis of hyperandrogenism: biochemical criteria.

Biochemical derangements in ovarian, adrenal, and peripheral androgen production and metabolism play an important role in underlying causes of hyperandrogenism. Specific diagnostic serum markers such as testosterone (total) and dehydroepiandrosterone sulfate (DHEAS), respectively, may be helpful in the diagnosis of ovarian and adrenal hyperandrogenism, respectively. Validated immunoassays or mass spectrometry assays should be used to quantify testosterone, DHEAS and other principal androgens. Free testosterone measurements, determined by equilibrium dialysis or the calculated method, are advocated for routine evaluation of more subtle forms of hyperandrogenism. The skin, with its pilosebaceous units (PSUs), is an important site of active androgen production. A key regulator in PSUs is 5alpha-reductase, which transforms testosterone or androstenedione to dihydrotestosterone (DHT). DHT in blood is not effective in indicating the presence of hyperandrogenism. However, distal metabolites of DHT have been shown to be good markers of clinical manifestations of hirsutism, acne and alopecia. Assays for these peripheral markers need improvement for routine clinical testing.

Content of 5-alpha-reductase (type 1 and type 2) mRNA in dermal papillae from the lower abdominal region in women with hirsutism.

BACKGROUND: Androgens influence the growth of terminal hair. The dermal papilla contains androgen receptors and the enzymes 5-alpha-reductase types 1 and 2. Both of these enzymes convert testosterone to the more active androgen, 5-alpha-dihydrotestosterone. The male distribution pattern of terminal hair in females is termed hirsutism. It is most common among women with hyperandrogenism; however, it may also affect patients with normal androgen levels (idiopathic hirsutism). OBJECTIVES: The aim of this study was to assess the expression of 5-alpha-reductase types 1 and 2 mRNA in dermal papillae from the lower abdominal skin in women with hirsutism. METHODS: The study included 42 subjects, 24 with a diagnosis of polycystic ovary syndrome (PCOS) and 18 with idiopathic hirsutism (IH). In all patients, free serum testosterone was measured. RESULTS: The mean +/- SD concentration of free serum testosterone was 7.2 +/-5.3 pmol/L in the total group of patients, 10.8 +/- 4.0 pmol/L in patients with PCOS, and 2.5 +/- 1.7 pmol/L in patients with IH. Quantitative analysis was then performed for the mRNA of 5-alpha-reductase types 1 and 2, both of which were found within the dermal papillae from the lower abdominal skin region. The number of mRNA copies/microg of total RNA for 5-alpha-reductase type 1 was statistically significantly higher than that for type 2 in both groups of examined patients. We also demonstrated a positive correlation between the number of mRNA copies/microg of total RNA for 5-alpha-reductase types 1 and 2 and the concentration of free serum testosterone in women with PCOS and IH. Considering all patients together, we found a positive correlation between the number of mRNA copies/microg of total RNA for 5-alpha-reductase type 2 and the concentration of free serum testosterone. There was also a tendency towards a positive correlation between the number of mRNA copies/microg of total RNA for 5-alpha-reductase type 1 and the concentration of free serum testosterone. CONCLUSION: The results of our study suggest that testosterone increases expression of 5-alpha-reductase types 1 and 2 in dermal papillae from the lower abdominal region in patients with hirsutism.

Determination method for steroid 5alpha-reductase activity using liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry.

A determination method for steroid 5alpha-reductase activity using liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) in the positive-ion mode has been developed. The rat prostatic enzyme source was used and the enzymatically formed 5alpha-dihydrotestosterone and 5a-androstane-3alpha,17beta-diol were determined by LC/APCI-MS using absolute calibration curve method. The sum of the formed products was used as a measurement of the enzyme activity. This method was applied to kinetic study of this enzyme and inhibitory experiments using Finasteride as a model inhibitor.

Differential alterations in 5alpha-reductase type 1 and type 2 levels during development and progression of prostate cancer.

BACKGROUND: In the prostate, conversion of testosterone to dihydrotestosterone (DHT), by the enzymes 5alpha-reductase types 1 and 2 (5alphaR1, 5alphaR2) is required for normal growth and probably also for development of prostate cancer (PCa). Finasteride, a 5alphaR2 inhibitor, was shown to reduce the prevalence of PCa in the Prostate Cancer Prevention Trial. However, inhibition of both 5alphaR isoenzymes causes a greater decrease in serum DHT. The aim of this study was to assess differential expression of these enzymes at various stages of PCa development. METHODS: Immunostaining for 5alphaR1 and 5alphaR2, using specific, well-validated antibodies, was evaluated in 26 benign prostatic hyperplasia (BPH) (16 for 5alphaR2), 53 primary PCa (21 for 5alphaR2), 18 prostatic intraepithelial neoplasia (PIN), 12 primary PCa treated with neoadjuvant androgen ablation, 15 locally recurrent PCa specimens, and 18 PCa metastases. RESULTS: The mean area of moderate plus high intensity staining for 5alphaR1 increased from 4.8 +/- 2.8% of total epithelial area in BPH, to 18.9 +/- 5.7% in PIN, 17.0 +/- 3.2% in primary cancer, 38.0 +/- 7.3% in recurrent cancer, and 55.8 +/- 8.5% in PCa metastases. The mean staining area for 5alphaR2 decreased from 58.8 +/- 7.2% in BPH, to 21.1 +/- 5.5% in PIN and 34.8 +/- 6.7% in primary PCa. Staining for 5alphaR2 was increased in recurrent cancer and PCa metastases compared to primary PCa, at 58.7 +/- 5.2% and 69.2 +/- 8.7%, respectively. CONCLUSIONS: 5alphaR1 immunostaining is increased and 5alphaR2 immunostaining is decreased during development of PCa. In addition, there is increased expression of both 5alphaR isozymes in recurrent and metastatic cancers, suggesting that both isozymes may be important in the development and progression of PCa.

Quantitation of mRNA levels of steroid 5 alpha-reductase isoenzymes in the rat brain by "one-step" RT-PCR and capillary electrophoresis.

The enzyme 5alpha-reductase (5alpha-R) is present in many mammalian tissues, including the brain. The physiological importance of 5alpha-R in the brain derives from its capability to convert testosterone (T) to a more potent androgen, dihydrotestosterone (DHT), and to convert progesterone to its 5alpha-reduced derivative, precursors of allopregnanolone, potent allosteric modulator of the gamma-aminobutyric acid receptor (GABA(A)-R). 5alpha-R occurs as two isoforms, 5alpha-R type 1 (5alpha-R1) and 5alpha-R type 2 (5alpha-R2). We present an accurate, rapid, and modestly labor-intensive method to precisely quantitate 5alpha-R mRNA species in the cerebral cortex of the rat. This approach combines the high specificity of "one-step" reverse transcription-polymerase chain reaction (RT-PCR) with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). Both cDNA synthesis and PCR amplification are performed with the same enzyme and site-specific primers, improving the efficiency of cDNA synthesis. The specific target mRNA and a mimic DNA fragment, used as a competitive internal standard, were co-amplified in a single reaction in which the same primers are used. The method presented in this paper enables a more efficient quantitative determination of 5alpha-R mRNA isozymes, and may lead to a better understanding of the role of 5alpha-R isozymes in the physiology of the central nervous system.

Quantitation of mRNA levels of steroid 5alpha-reductase isozymes in the rat brain by "one-step" RT-PCR and capillary electrophoresis.

The enzyme 5alpha-reductase (5alpha-R) is present in many mammalian tissues, including the brain. The physiological importance of 5alpha-R in the brain derives from its capability to convert testosterone (T) to a more potent androgen, dihydrotestosterone (DHT), and to convert progesterone to its 5alpha-reduced derivative, precursors of allopregnanolone, potent allosteric modulator of the gamma-aminobutyric acid receptor (GABA(A)-R). 5alpha-R occurs as two isoforms, 5alpha-R type 1 (5alpha-R1) and 5alpha-R type 2 (5alpha-R2). We present an accurate, rapid, and modestly labor-intensive method to precisely quantitate 5alpha-R mRNA species in the cerebral cortex of the rat. This approach combines the high specificity of "one-step" reverse transcription-polymerase chain reaction (RT-PCR) with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). Both cDNA synthesis and PCR amplification are performed with the same enzyme and site-specific primers, improving the efficiency of cDNA synthesis. The specific target mRNA and a mimic DNA fragment, used as a competitive internal standard, were co-amplified in a single reaction in which the same primers are used. The method presented in this paper enables a more efficient quantitative determination of 5alpha-R mRNA isozymes, and may lead to a better understanding of the role of 5alpha-R isozymes in the physiology of the central nervous system.

Expression of estrogen receptors and enzymes involved in sex steroid metabolism in the rat tibia during sexual maturation.

Estrogens are essential for bone mass accrual but their role before sexual maturation has remained elusive. Using in situ hybridization and immunohistochemistry, we investigated the expression of both estrogen receptor (ER) alpha and beta mRNA and protein as well as several mRNAs coding for enzymes involved in sex steroid metabolism (aromatase, type I and II 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), steroid sulfatase (STS) and type I 5 alpha-reductase) on sections of tibial metaphyses before (1- and 4-week-old), during (7-week-old) and after (16-week-old) sexual maturation in female and male rats. ER alpha and ER beta mRNA and protein were detected in metaphyseal bone in lining cells, osteoblasts, osteoclasts and some osteocytes with no apparent differences in expression during development or between the sexes. In contrast, aromatase, type I and II 17 beta-HSD and type I 5 alpha-reductase mRNAs were first detected in osteoblasts, osteoclasts and occasionally in osteocytes from sexual maturation (7-week-old rat) and onwards. Only STS was present before sexual maturation. To study the significance of ER alpha and beta expression in bone before sexual maturation when circulating sex steroid levels are low, 26-day-old female and male rats underwent gonadectomy or 17 beta-estradiol (E(2)) supplementation (0.5 mg/21 days) during 3 weeks. Following gonadectomy, trabecular bone volume (TBV) was lower in males (P=0.03) and there was a trend towards reduction in females (P=0.057). E(2) supplementation increased tibial TBV compared with controls in both genders as assessed by Masson-Goldner staining. These data suggest that the presence of ERs in bone cells before sex maturation might be of significance for bone mass accrual. Furthermore, based on the mRNA expression of the crucial enzymes aromatase and type I 17 beta-HSD, we suggest that bone cells in the tibial metaphysis acquire the intrinsic capacity to metabolize sex steroids from sexual maturation onwards. This process may contribute to the beneficial effects of estrogen on bone mass accrual, possibly by intracrinology.

Quantitation of mRNA levels of steroid 5alpha-reductase isozymes: A method that combines one-step reverse transcription-polymerase chain reaction and separation by capillary electrophoresis.

We developed an accurate, rapid, and modestly labor-intensive method to precisely quantitate mRNA species by one-step reverse transcription-polymerase chain reaction (RT-PCR). This approach combines the high specificity of quantitative competitive PCR with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). Both cDNA synthesis and PCR amplification are performed with the same enzyme and site-specific primers, improving the efficiency of cDNA synthesis. The specific target mRNA and a mimic DNA fragment, used as a competitive internal standard, were coamplified in a single reaction in which the same primers are used. The 5' forward primers were end-labeled with 6-carboxy-fluorescein (6-FAM). The ratio of fluorescence intensity between amplified products of the target cDNA and the competitive DNA was determined quantitatively after separation by CE and fluorescence analysis. Using this method, we have been able to precisely quantify the mean amount of steroid 5alpha-reductase (5alpha-R) isozyme mRNA levels in ventral prostate of the rat, detecting 10-fold difference for 5alpha-R1 and 50-fold difference for 5alpha-R2, respectively, in comparison with our previously reported two-step method. Because the competitive RT-PCR presented in this paper enables a more efficient quantitative determination of mRNAs, low-level gene expression could be quantified.