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Identification of type 1 5α-reductase in myelin membranes of male and female rat brain
Molecular and Cellular Endocrinology 129 (1997) 181 – 190
Identification of type 1 5a-reductase in myelin membranes of male and female rat brain Angelo Poletti a,*, Fabio Celotti a, Cristiano Rumio b, Monica Rabuffetti a, Luciano Martini a a
Istituto di Endocrinologia, Uni6ersita` degli Studi di Milano, 6ia Balzaretti 9, 20133 Milano, Italy b Istituto di Anatomia Umana, 6ia Mangiagalli 31, 20133 Milano, Italy Received 3 January 1997; accepted 24 February 1997
Abstract The formation of the 5a-reduced metabolites of testosterone (T) and of progesterone (P) is a very active process in the brain, since the enzyme 5a-reductase (5a-R) is present in almost any central nervous system (CNS) structure. A particularly elevated 5a-R activity has been shown in myelin sheaths. Two isoforms of the enzyme have been cloned, with different localisation as well as different biochemical properties. The present study was performed to determine whether both isoforms of the 5a-R, or only one of them, are/is responsible for the enzymatic activity observed in myelin. Kinetic analyses have been performed on purified myelin membranes prepared from the male or female rat brain, using both T and P as substrates. The 5a-R present appears to possess a pH optimum at basic values. The Vmax values obtained in the Lineweaver – Burk analysis were comparable in male and female preparations independently on whether T or P were used as the substrates, suggesting that a single enzymatic form is present in all samples examined; the Km obtained using [14C]T (Km: male 1.14 mM; female 1.46 mM) or [14C]P (Km: male 0.5 mM; female 0.64 mM) as substrates, were in good agreement with those obtained for the recombinant type 1 isoform. These data suggest that the type 1 isoform is the most relevant 5a-R present in myelin. To confirm this, a new polyclonal antibody was raised against the type 1 5a-R enzymatic protein, and used in immunohistochemical studies. The experiments were performed on the optic nerve, a myelinated structure very rich in 5a-R activity and the results clearly indicated the presence of a specific type 1 enzyme immunoreactivity in the myelin sheaths of axons. © 1997 Elsevier Science Ireland Ltd. Keywords: 5a-reductase; Testosterone; Progesterone; Neurosteroids; Myelin
1. Introduction In the brain of developing and adult animals, hormonal steroids are involved in the regulation of several nervous and endocrine functions. Many of these actions are mediated through classical intracellular steroid receptors, which activate the transcription of specific genes. Some hormonal steroids, and in particular androgens, need to be locally transformed into ‘active metabolites’, in order to be able to exert their functions; the conversion into estradiol and/or dihydrotestosterone (DHT) mediates several of the effects of testosterone (T) [1]; the resulting steroids bind, re* Corresponding author. Tel.: + 39 2 29406576; fax: +39 2 29404927; e-mail: [email protected]
spectively, to the estrogenic or to the androgenic receptor [2]. On the other hand, sex steroids and their metabolites may also act by the modulation of some neurotransmitter systems, and may interact with membrane-linked binding sites which, however, have not been fully-characterised so far [3–6]. In this context, the best known example is provided by the binding of some 5a-reduced, 3a-hydroxylated progesterone and corticosteroid metabolites to the g-aminobutyric acid (GABA)A receptor [6]. For instance, the marked anaesthetic activity of 3a-hydroxy, 5a-pregnan-20-one (allopregnanolone or tetrahydroprogesterone, THP) is due to its interaction with the GABA-gated chloride ion channel of the GABAA receptor [7–9]; this compound is formed in the brain from progesterone [10,11].
0303-7207/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 0 3 - 7 2 0 7 ( 9 7 ) 0 4 0 5 6 - 2
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The 5a-reductase (5a-R), the enzymatic complex which transforms T into DHT, as well as progesterone (P) and the corticoids into their respective 5a-reduced metabolites is widely diffused in the brain. The highest levels of activity of the enzyme have been found to be associated with several white matter structures [12]. Moreover, it has been shown that the formation of DHT from T is very high in purified myelin membranes obtained from the central nervous system (CNS) [13] indicating that the elevated enzymatic activity found in the white matter is principally due to the presence of myelin sheaths. The 5a-R is a membrane-associated enzyme, which requires NADPH as a cofactor; two isoforms of the 5a-R have been identified and cloned both in human and in the rat. In this last species, the homology between the amino acid sequences of the two 5a-R isoforms, deduced from their respective cDNAs, is only 44% [15]. This fact is reflected in important differences in the functional and biochemical properties of the two isozymes, such as the pH optima for their reactions (alkaline for the type 1 isoform, with a broad range from pH 5–8; acidic for the type 2 isoform, with a typical narrow pH optimum at value of 5.0); the affinity for the various substrates (T, P, androstenedione, corticosterone, etc.), which is generally higher in the case of the type 2 isoform; and, finally, in the total capacity of conversion, which is greater for the type 1 than for the type 2 isozyme [15]. The two isozymes also differ in the chromosomal localisation of the respective coding genes, in their tissue and cellular distribution [16], and possibly in their subcellular compartmentalisation [17]. The presence of specific mRNAs coding for the two isoforms has been observed in the rat brain [16], but in different amounts during animal life. The expression of the type 1 isoform appears to be constant throughout life, while the type 2 isoform is maximally expressed in the late fetal life and in the early post-natal life (up to 4 weeks of life) (Poletti et al., unpublished results). Because of these differences, it is expected that the two 5a-R subtypes, even if they catalyse the same reactions, might exert different physiological functions. So far, no data are available indicating the distribution of the two 5a-R isoforms in the various CNS structures, and in particular in myelin membranes. The aim of the present work was: (1) to analyse whether the 5a-R, previously found in myelin sheaths using T as the substrate, is also able (like the enzyme found in all other structures analysed so far) to produce the 5aderivatives of P, (2) to determine possible sex differences in the activity of the enzyme, (3) to evaluate, by means of the analysis of kinetic parameters (determination of the constant of Michaelis – Menten, pH optima, etc.) whether both isoform(s) or only one form are/is present in the myelin and (4) to establish whether a polyclonal antibody raised against one of the two 5a-R
isoforms might recognise the corresponding isozyme in the myelin by immunohistochemical techniques. 2. Materials and methods
2.1. Animals Three-months old male or female Sprague–Dawley rats (Charles River, Italy) were used throughout these experiments. The animals were maintained in animal quarters with controlled temperature and humidity. The light schedule was 14 h light and 10 dark (lights on at 6.30 a.m.). Animals were fed a standard pellet diet and water was provided ad libitum.
2.2. Myelin purification The purification has been performed with the method of Keenan [18], with slight modifications. In brief, after decapitation, the brain was rapidly removed and homogenised at 5% (w/v) in 0.32 M sucrose at 0–4°C; the suspension (3.5 ml/each tube) was centrifuged at 75 000× g (28 000 rpm) for 10 min in a Beckman SW 55 Ti rotor. The pellet formed was resuspended in 0.85 M sucrose (4 ml/each tube) and centrifuged again in the same conditions. The floating crude myelin layer was collected, diluted with water to 4 ml/each tube and centrifuged at 42 000× g (21 000 rpm) for 5 min. The resulting pellet was resuspended in water and centrifuged as before twice. The final pellet was resuspended in 0.85 M sucrose and centrifuged at 75 000×g (28 000 rpm) for 10 min; the floating purified myelin layer was collected, washed twice with distilled water and recovered by a spin at 1 000 × g for 10 min. The purity of myelin was evaluated by electron microscopy as previously described [12].
2.3. Determination of 5a-reductase acti6ity 2.3.1. Incubation To determine the pH optimum of the enzymatic isoform present in the purified myelin membranes, the incubations were performed in phosphate buffer saline (PBS) solution (250 ml) at various pH (ranging from 3.5 and 8 to assess pH optimum and at pH 7.5 to determine kinetic parameters) in the presence of 1 mM NADPH and [14C]T (specific activity 56.9 mCi/ mmol, Amersham, UK). The incubation was carried out at 37°C in a Dubnoff metabolic shaker under a stream of O2 –CO2 98:2. After determination of the pH optimum, the kinetic studies were performed at pH 7.5 in the presence of 1 mM NADPH. The following incubation conditions were tested for [14C]P metabolism, since the working condition for [14C]T were previously obtained [13].
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The apparent Michaelis – Menten constant (Km) and the Vmax were calculated for the purified myelin utilising the following condition of incubation. Time of incubation: 1 h at 37°C. Substrate ([14C]T or [14C]P): from 0.5 to 3 mM; each experiment was performed utilising five different concentrations of the substrate and triplicate determinations. The amounts of protein incubated in these studies were about 0.1 mg for the purified myelin both from male and female rats. Vials without tissue provided the blanks for each concentration of substrate and protein content was evaluated, according to the method of Bradford [19].
2.3.2. Detection of metabolites At the end of the incubation the reaction was stopped by freezing the samples to −20°C. Tritium labelled DHT or DHP (about 5000 dpm each) were added to each sample in order to evaluate the recoveries. The metabolites formed were extracted twice with diethylether, non-radioactive DHT or DHP (40 mg/100 ml) were added to each sample in order to aid visualisation of the steroid on the thin layer chromatography (TLC) plates. Extracted samples were separated on a thin layer silica gel plate (Merck 60 F254, DC). The eluting mixture was dichloromethanediethylether (11:1 v/v) to determine [14C]DHT formation and benzenemethanol (19:1 v/v) followed by cyclohexane-N-butylacetate (1:2 v/v) to determine [14C]DHP formation. The spots of the 5a-reduced metabolites were identified with iodine vapours, scraped off and the radioactivity counted in a Packard 300C liquid scintillation spectrometer. Quench corrected dpm of the isotope were obtained by a calibration standard curve. The identification of the metabolite was performed by recrystallization to constant 3H/14C ratio as previously described [12]. It has been previously shown that the purified myelin does not possess 3a-hydroxysteroid dehydrogenase activity [13], which is generally associated to the soluble cellular cytosolic fraction, thus, in the incubation conditions described, usually the formation of 5a-androstane-3a, 17b-diol (3a-diol) or 5a-pregnane-3a, 17b-diol (THP) is not detectable (data not shown). Moreover, preliminary experiments were also performed using samples of purified myelin membranes incubated with either [14C]T or [14C]P, but without the addition of tritium labeled metabolites. The products of these incubations were then assayed by TLC analysis followed by autoradiography to determine the various metabolites formed. The results have shown that using these substrates 5a-DHT or 5a-DHP were respectively the principal metabolites (85 – 90% of total metabolites formed; data not shown). Because of this, it was felt appropriate to use only the 5a-DHT or 5a-DHP data in order to perform the kinetic analysis here to be described.
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2.3.3. Statistical analysis The experimental data concerning the different incubation conditions (tissue, time) were analysed by means of the linear regression analysis, the data obtained in the kinetic study utilising the Lineweaver–Burk plot with the computer programs Enzyme [14]. 2.4. Antibody preparation. The anti-5a-R1 polyclonal antibody was prepared in New Zealand white rabbits (Charles River, Italy) as follows: a peptide reproducing the sequence of the rat type 1 5a-R, from aa 176 to 189, with the following sequence: CTGYKIPRGGLFEYV, was synthesised; this sequence is not conserved in rat type 2 5a-R, and does not match with other published protein sequences. The synthetic peptide was coupled to bovine serum albumin (BSA) using Bis(sulfosuccimidyl)suberate (Pierce, Rockford, IL) as a cross-linking agent; the complex was purified on Sephadex G-25 columns (Pharmacia Biotech, Sweden). The coupled peptide was used as an antigen, and injected subdermally in rabbit at the doses of 100, 200 or 400 mg with Freund’s complete adjuvant (Sigma); a second immunisation step (using the same doses of peptide) was performed after 3 weeks using Freund’s incomplete adjuvant (Sigma). This was followed, after 3 additional weeks, by a treatment with the uncoupled peptide (at doses of 25, 50 or 100 mg) in Freund’s incomplete adjuvant (Sigma). At the end of the immunisation procedure the rabbits were injected intravenously with 200 ml of a sterile saline solution (0.9% NaCl) containing 1 mg/ml of the peptide. After 3 days, samples of blood were collected to determine the presence of positive antisera. The antisera were screened in immunodot assays against the synthetic peptide using a chemiluminescent detection system (ECL, Amersham, UK). To eliminate cross-reactivity with other non-specific rabbit antibodies, the anti-5a-R1 antibody was purified as follows: The total rabbit IgG were initially isolated from the sera by affinity chromatography, using protein A-sepharose columns (CL-4B, Pharmacia Biotech, Sweden). The purity was checked on a sample run on 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) [20] followed by Silver staining. The anti-5a-R1 antibody was then affinity purified on a HiTrap column (Pharmacia Biotech, Sweden) previously cross-linked to the synthetic peptide utilised for the immunisation procedure. The specificity and concentration of the purified antibody was tested again in immunodot blots, using the synthetic peptide of the type 1 5a-R cross-linked to the BSA. The specificity of the antibody was also tested on yeast cells expressing the recombinant type 1 5a-R [17] and in sections of prostatic tissue as described in the next paragraphs.
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2.5. Immunopurification of the recombinant type 1 5a-R expressed in yeast Saccharomyces cere6isiae Yeast cells (protease-deficient strain Saccharomyces cere6isiae BJ3505; genotype: MATa, pep4::HIS3 PR61D1.6R his3 lys2-208 trp1-D101 ura3-52 gal2) were transformed either with plasmid YEpR1 or plasmid YEpR2 using the lithium acetate protocol [17]. Transformants were selected by tryptophan prototrophy. BJ3505[YEpR1] produces the type 1 and BJ3505[YEpR2] produces the type 2 5a-R enzyme. The transformed cells producing either type 1 or type 2 5a-R were grown overnight at 30°C in CAAU medium (2% glucose, 0.1% casamino acids, 0.67% yeast nitrogen base, 0.001% adenine, 0.002% uracil); 2 × 105 cells/ml were inoculated in fresh CAAU containing 100 mM of CuSO4 (to induce CUP1 promoter) and grown until an A600nm of 1.0 (early mid-log phase). Spheroplasts were prepared using lyticase as previously described [17]. Cells were lysed by osmotic shock (30 min at 4°C) in low salt buffer (PBS; 10 mM potassium phosphate buffer, 150 mM KCl, 1 mM EDTA; pH 7.5) and sonicated 5 s at the lowest power; total protein were determined using the Bradford assay [19]. Fractions of the yeast cell lysates containing either type 1 or type 2 5a-R (500 mg of total protein), were dissolved in 1 ml of 0.1% Triton X-100 in PBS (1 ml), incubated for 2 h at room temperature, and then incubated overnight at 4°C in the presence of 30 ml of the immunoaffinity purified primary rabbit antibody against type 1 rat 5a-R; control samples, performed without antibody, were also included in the study. At the end of the incubation, the complex type 1 5a-R/antibody was precipitated with Protein A-sepharose (Pharmacia Biotech, Sweden), washed five times with immunopurification buffer (IPB: 0.5 M NaCl, 0.05 M Tris, 0.02% NaN3, 0.2% Triton X-100, pH 7.5) and three times with distilled water; the recovered proteins were resolved on 12% SDS-PAGE. The gel was stained using Silver staining.
2.6. Immunohistochemistry The yeast cells transformed or not with the plasmid bearing the 5a- R cDNA were grown as previously described [17]. The rat optic nerves and prostate were embedded in O.C.T. immediately after the animal sacrifice. They were then frozen at −20°C for 30 – 60 min, and sectioned with a freezing microtome. Each 15 mm section was collected on a slide and treated as follows: the sections were fixed with 4% formaldehyde in 0.15 M PBS, pH 7.2 for 10 min at room temperature, washed in PBS (3× 5 min) and post-fixed in methanol (5 min at −20°C) and acetone (3 min at − 20°C). They were then rinsed in PBS (3×5 min) and incubated with
normal goat serum 10% at 25°C for 10 min to block non-specific staining associated with the secondary antibody. Goat serum was removed by washing with PBS (3× 5 min). The sections were incubated with the immunoaffinity purified primary rabbit antibody against type 1 rat 5a-R (0.25 mg/ml), for 24 h in a humid atmosphere at 4°C. After rinsing in PBS (3 ×5 min) the samples were incubated for 1 h in a humid atmosphere at 37°C with rhodamine-conjugated goat anti-rabbit antibodies (Jackson Immunoresearch West Grove, PA, USA) diluted 1:400 in PBS, rinsed in PBS (3×5 min) and mounted in an anti-fading mounting medium (glycerol: PBS 90:10, containing 2.5 g DABCO (diazabicyclo(2,2,2)octane, Sigma). Controls were obtained by eliminating the first incubation, by maintaining the addition of the secondary goat anti-rabbit antibody.
2.7. Confocal microscopy. A Zeiss LSM 410 Invert microscope has been used in this study. The instrument was equipped by 543 nm He–Ne laser and a TRITC set of filters (FT 560 beam-splitter and a low-pass LP 590 emission filter). The images were obtained at 2 s scan speed with 16 averages, with a Plan-Neofluar 40/1.3 Oil lens. The distortions of the confocal images [21] were not significant because we used only single optic sections or very limited depth of scanning.
3. Results
3.1. Kinetic studies Preliminary experiments were performed on purified myelin membranes obtained from the rat CNS, to verify whether these preparations were able to convert P into DHP; the optimal conditions required for kinetic studies of the reaction were also established. The study of the variations of the 5a-R activity in purified myelin membranes as a function of pH was performed using T as the substrate. The data are shown in Fig. 1, which also illustrates (in the inset) the pH profiles of the 5a-R activities of lysates of yeast cells expressing the recombinant type 1 or type 2 5a-R. The capability of the purified myelin to 5a-reduce the substrate appears to increase when the pH reaches basic values. No peaks of activity were present at an acidic pH, around 5.0–5.5, indicating that the pH optimum for the 5a-R present in the myelin is in the alkaline range. This, as previously mentioned, is a characteristic of the type 1 5a-R, both in its native form or when expressed in mammalian or yeast cells [15–17]. There were no significant differences between male and female rat myelin preparations at any pH examined.
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Fig. 1. Variation of the myelin 5a-R activity as a function of the pH of incubation. The myelin preparations, obtained both from male and female rats, have been incubated in PBS buffer at different pH, in presence of 3 mM of [14C]T; the formation of [14C]DHT was measured as described in Section 2 and is expressed as mean9 S.E obtained from four different determinations. The profile of activity as a function of the pH obtained for yeast cell lysates producing the recombinant type 1 or type 2 5a-R is also reported.
To further verify whether the type 1 isozyme is the form of the 5a- R which predominates in purified myelin membranes, the kinetics of the enzymatic reaction was determined in the same myelin preparations, obtained respectively both from the male and the female rat brain. Fig. 2 shows the Lineweaver–Burk plots of the reciprocal of the initial velocity of the formation of [14C]DHP and [14C]DHT as a function of the reciprocal of the initial substrate (P and T) concentrations. The results of the experiments are summarized in Table 1, which shows the data of a single analysis, however, this study was repeated three times with very similar results. The apparent Michaelis – Menten constants (Km) determined for the 5a-R of P are similar in the myelin preparation obtained from the two sexes (male 0.5 mM; female 0.6 mM); similarly, no sex differences in these parameters were observed when T was used as substrate (male 1.1 mM; female 1.5 mM). In the case of T, the affinity was lower than that observed for
P, which appears to be the preferred substrate (see ratio Vmax/Km). It is also clear from the Km values, all in the micromolar range, that the affinities of the two substrates (P and T) for the enzyme present in myelin membranes are almost identical to those detected for the recombinant type 1 isoform of the 5a-R expressed either in mammalian cells or in a yeast cell system [15,17]. The values obtained for the maximal velocity of reaction (Vmax), an index of the total amount of active enzyme present in the preparation, were in the same range in the four groups considered (i.e., P males and females, T males and females).
3.2. Antibody characterization and immunofluorescence studies To confirm that the type 1 5a-R is present in myelin membranes, a polyclonal antibody was raised against this isoform, using a synthetic peptide reproducing a
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Fig. 2. Lineweaver –Burk plots of the 5a-R activity present in purified myelin membranes obtained both from male and female rat brains. The kinetics of the reaction were determined using either [14C]T or [14C]P as substrates. Each curve was obtained with five different increasing concentrations in triplicate. Non-limiting concentration of cofactor (1 mM of NADPH) were also added in the reaction. Details of the experiments are described in Section 2.
potentially antigenic and hydrophilic amino acidic sequence of the enzyme. Fig. 3 shows the immunodot blot obtained using 1 ml of different fractions (1 ml) of the purified antibody. Only the spots containing the synthetic peptide were specifically labelled by the antibody. The fraction 3, which appeared to have the highest concentration of the desired antibody, was chosen. The specificity of the immunopurified polyclonal antibody was further tested in immunopurification experiments using yeast cells expressing either recombinant type 1 or type 2 isoform [17]. The results are shown in Fig. 4. It appears that the antibody specifically recognizes only a single protein with a molecular weight of about 26 Kd in the samples of yeast cell lysates expressing type 1 5a-R (lane 3), but not in those expressing the type 2 5a-R (lane 2), indicating that it does not cross react with the second isoform of the rat 5a-R; the specificity of the antibody is also confirmed by the absence of immunoprecipitation of other yeast proteins. The specificity of the antibody for immunofluorescence experiments was also tested using yeast cells expressing type 1 5a-R and section of the rat anterior prostate. Fig. 5 shows the yeast S. cere6isiae transTable 1 Characterization of rat 5a-reductase present in purified myelin membranes [14C]progesterone
Vmax (ng/h/mg of protein) Km (mM) Vmax/Km R2
formed with a plasmid expressing the rat type 1 5a-R, observed with a phase contrast microscope. Fig. 6 shows the immunofluorescence of the same field after labelling with the antitype 1 5a-R antibody. It clearly appears that all yeast cells expressing the type 1 isoform are recognized by the antibody which is concentrated in the nuclear compartment of the cells; on the contrary yeast cells transformed with the recombinant type 2 isoform did not show any reaction (not shown). In the sample of rat anterior prostate (Fig. 7) a strong immunoreactivity was observed in the nuclei (or in the perinuclear region) of the epithelial cells of the alveoli. The nuclei of the alveolar cells show a characteristic annular fluorescence, in good agreement with the data of the literature [22]. This indicates that in the prostate the type 1 isoform is an epithelial enzyme. The nuclei of few cells, interpreted as fibroblasts of stromal connective, appears to be also fluorescent while the autofluorescence of collagen bundles is enhanced by confocal microscopy. The study on the myelin was performed, for convenience, on the rat optic nerve, a structure easy to obtain and very rich in myelin and in 5a-R activity. When observed with the confocal laser scanning microscope, the sections of the rat optic nerve (Figs. 8 and 9) show a strong fluorescence of the nerve fibres. The examination of single optical sections allowed the localisation of the fluorescence at the level of the myelin sheaths, while the axons were negative.
[14C]testosterone
Male
Female
Male
Female
38.5
45.5
28.6
35.7
0.5 76.9 0.94
0.6 71.0 0.99
1.1 25.1 0.96
1.5 24.5 0.94
Myelin membranes and kinetic analysis were performed as described in Section 2.
4. Discussion The data obtained in the present study using biochemical and immunohistochemical approaches demonstrated that the formation of the 5a-reduced derivatives of T and P occurring in myelin sheaths obtained from the rat brain is mainly due to the presence of the type 1 isoform of the rat 5a-R. The enzymatic activity found
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Fig. 3. Immunodot blot obtained by testing the affinity-purified rabbit polyclonal antibody anti-type 1 5a-R with the synthetic 5a-R type 1 peptide linked to BSA. The BSA peptide fraction represents the fraction utilized to immunize rabbits; the BSA fraction represents only the BSA not cross-linked to the synthetic peptide, the different fractions are those derived after elusion from the column of immunoaffinity purification. See Section 2 for details.
in purified myelin membranes presents the biochemical characteristics described in the literature for the recombinant type 1 rat 5a-R [15 – 17]. First of all, the pH optimum of the reaction, which allows to discriminate between the two known 5a-Rs isoforms, is in the basic range. Secondly, the affinity for the substrates correlates well with the kinetic parameters of the type 1 isoform, since the apparent Michaelis – Menten constants are all found in the micromolar range, moreover, the similarity of the Vmax values obtained in the kinetic studies when P and T are used as substrates indicates that only one form of the enzyme is probably responsible for both reactions [15,17]; if both are present, one would be largely predominant. No evidence of a sex
Fig. 4. Silver staining of a 12% SDS-PAGE loaded with samples of lysates of yeast cells expressing the two isoforms of the 5a-R after immunoprecipitation with the immunopurified polyclonal antibody raised against the type 1 5a-R. Lane 1, lysates of yeast cells expressing the type 1 5a-R without addition of the antibody; lane 2, lysates of yeast cells expressing the type 2 5a-R and lane 3 lysates of yeast cells expressing the type 1 5a-R after immunoprecipitation with the antibody; lane 4 low range molecular weight markers (14.4, 21.5, 31.0 and 45.0 kD). Ab indicates the band which corresponds to the antibody utilised for immunoprecipitation; type 1 5a-R indicates the specific product of the immunoprecipitation, a protein of about 26 kD.
difference in the amounts of 5a-R present in the purified myelin was obtained. In every experiment, the data were identical, independently on whether male or female rat brains were used for the purification of the myelin. The existence of the type 1 5a-R in the white matter, and in particular in the myelin, has been corroborated by immunohistochemical studies. A specific polyclonal antibody raised against a synthetic hy-
Fig. 5. Immunochemical detection of rat type 1 5a-R. Yeast cells expressing the recombinant rat type 1 5a-R observed with phase constrast microscope.
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Fig. 6. Immunochemical detection of rat type 1 5a-R. Yeast cells expressing the recombinant rat type 1 5a-R after immunocytochemical reaction with the polyclonal antibody.
drophilic peptide corresponding to a potentially antigenic site of the amino acid sequence of the type 1 enzyme 5a-R specifically recognised the enzymatic protein in the myelin sheaths of the rat optic nerve. Unfortunately, it was not possible to analyse whether also type 2 5a-R is present, even in low amounts, because a suitable antiserum against the type 2 isoform is not presently available. The significance of the presence of 5a-R in myelin membranes is still unclear. Oligodendrocytes, the cells responsible for myelin formation in the CNS, are known to contain 5a-R activity [12]. However, this enzyme is not generally bound to the external membranes of the cells, but is associated to the perinuclear region [17,22–24], as also shown by the data obtained in prostatic tissue in the present study. During myelinogenesis, myelin membranes are derived from the external plasma membrane of the oligodendrocytes. However, it is now accepted that myelin membranes are surrounded by, and infiltrated with, cytoplasmic channels (outer/lateral loops and longitudinal incisures) which may serve to translocate polyribosome complexes bearing specific mRNAs from the cell body of the oligodendrocytes to the myelin sheaths [25]. Because of this, some proteins (e.g., the myelin basic protein, MBP) may be synthesised in free polyribosomes localised in the myelinating extensions of the oligodendrocytes before being inserted in the myelin [26,27]. A
Fig. 7. Rat anterior prostate section, corresponding to a single optic section observed at CSLM (panel C). The epithelial cell nuclei are strongly fluorescent and present the characteristic ring aspect. He–Ne laser (543 nm with TRITC filters), lens 40/1.30 Ocl-Bar = 14 mm= 25 mm (× 560).
mechanism similar to that of the formation of MBP could explain the association of type 1 5a-R with the myelin membranes. The presence of the type 1, and the probable absence of the type 2 isoforms in myelin is
Fig. 8. Almost transversal section of a single rat optic nerve corresponding to a single optic section observed at CSLM. The labelling of myelin membranes appears evident. He – Ne laser (543 nm with TRITC filters), lens 63/1.10 Ocl-Bar =14 mm = 25 mm ( × 570).
A. Poletti et al. / Molecular and Cellular Endocrinology 129 (1997) 181–190
Fig. 9. Longitudinal section of rat optic nerve corresponding to a single optic section observed at CSLM. The labelling of myelin membranes appears evident. He–Ne laser (543 nm with TRITC filters), lens 63/1.10 Ocl-Bar= 14 mm= 25mm (× 570).
also corroborated by recent studies obtained using the reverse transcriptase-polymerase chain reaction (RTPCR) technique, that showed the presence of specific mRNA coding for type 1, but not for type 2 isozyme, in cultured rat oligodendrocytes [28], the cells responsible for myelin production. The role of the type 1 5a-R in myelin membranes in still unclear. It has been shown that P, the preferential substrate of the 5a-R, is able to induce MBP [29,30]. Probably, this action is directly mediated by a genomic effect, since P receptors have been detected in the oligodendrocytes [31], and specific glucocorticoid/ progesterone responsive elements have been identified on the MBP promoter [25]. The presence of high levels of type 1 5a-R in myelin membranes may lead then to the hypothesis that the conversion of 3keto-D4 steroids into their corresponding 5a-reduced metabolites may play a role in the control of the synthesis of proteins which are important components of myelin membranes. Also other 5a-reducible steroids (such as the glucocorticoids corticosterone and cortisol) may be involved in the transcriptional and post-transcriptional regulation of myelin genes [30,32 – 34]. Interestingly, the 5a-R is the first enzyme involved in the formation of the tetrahydro-derivatives of P and of the glucocorticoids, compounds which are further reduced in position 3 by the soluble cytoplasmic enzyme 3a-hydroxysteroid dehydrogenase. These steroids are highly active as anaesthetic and anxiolytic drugs [35], and these actions are mediated by the GABAA-gated chloride ion channel of the GABAAreceptor [7]. There-
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fore, the high levels of type 1 5a-R present in myelin membranes could serve as a part of the ‘neurosteroid synthesising structures’ of the CNS. Furthermore, a different role of the rat type 1 5a-R have been postulated some years ago [36,37] because of a certain degree of homology of the enzyme with the SC2 glycoprotein which is strongly expressed by many types of neurons, and is involved in synaptic function. The significance of this homology is still unclear, but the possibility that type 1 5a-R enzyme is a bifunctional protein should be taken into account. Even the distribution of the type 1 5a-R mRNA (analyzed by ‘in situ’ hybridization) during pre- and peri-natal stages [38] agrees with a possible non-enzymatic role in the development and differentiation of the rat CNS. It is also interesting to note that, in humans showing the Imperato–McGinley syndrome [39,40], a disease in which the type 2 enzyme is genetically absent, brain development or myelination are not affected. No specific genetic defects of the type 1 isozyme have been identified so far. This could mean either that the deficiency of the type 1 5a-R is clinically silent, or that the defect is not compatible with life. Finally, the presence in myelin of the 5a-R type 1 is relevant in view of the increasing use of 5a-R inhibitors in the therapy of prostatic diseases, like benign prostatic hyperplasia (BPH). Since the actual role of the type 1 enzyme in myelin is not yet known, it would be safer to utilise for the treatment of this and other diseases drugs which show a weak inhibitory effect on the type 1 enzyme, until studies on the action of selective type 1 5a-R and type 2 5a-R inhibitors on myelin will be available.
Acknowledgements We thank Dr Nancy L. Weigel for the helpful advice on antibody preparation and the choice of the synthetic peptide sequence of 5a-reductase, and for the revision of the manuscript. We are indebted to Professor Chillemi for the peptide synthesis. This work was supported by CNR funding Special Projects ACRO 94.01162.PF39: FATMA 95.00868.PF41: AGING 95.01020.PF40: BTBs 93.01103.PF70 and Bilateral project 94.02374. CT04.
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Identification of type 1 5a-reductase in myelin membranes of male and female rat brain Angelo Poletti a,*, Fabio Celotti a, Cristiano Rumio b, Monica Rabuffetti a, Luciano Martini a a
Istituto di Endocrinologia, Uni6ersita` degli Studi di Milano, 6ia Balzaretti 9, 20133 Milano, Italy b Istituto di Anatomia Umana, 6ia Mangiagalli 31, 20133 Milano, Italy Received 3 January 1997; accepted 24 February 1997
Abstract The formation of the 5a-reduced metabolites of testosterone (T) and of progesterone (P) is a very active process in the brain, since the enzyme 5a-reductase (5a-R) is present in almost any central nervous system (CNS) structure. A particularly elevated 5a-R activity has been shown in myelin sheaths. Two isoforms of the enzyme have been cloned, with different localisation as well as different biochemical properties. The present study was performed to determine whether both isoforms of the 5a-R, or only one of them, are/is responsible for the enzymatic activity observed in myelin. Kinetic analyses have been performed on purified myelin membranes prepared from the male or female rat brain, using both T and P as substrates. The 5a-R present appears to possess a pH optimum at basic values. The Vmax values obtained in the Lineweaver – Burk analysis were comparable in male and female preparations independently on whether T or P were used as the substrates, suggesting that a single enzymatic form is present in all samples examined; the Km obtained using [14C]T (Km: male 1.14 mM; female 1.46 mM) or [14C]P (Km: male 0.5 mM; female 0.64 mM) as substrates, were in good agreement with those obtained for the recombinant type 1 isoform. These data suggest that the type 1 isoform is the most relevant 5a-R present in myelin. To confirm this, a new polyclonal antibody was raised against the type 1 5a-R enzymatic protein, and used in immunohistochemical studies. The experiments were performed on the optic nerve, a myelinated structure very rich in 5a-R activity and the results clearly indicated the presence of a specific type 1 enzyme immunoreactivity in the myelin sheaths of axons. © 1997 Elsevier Science Ireland Ltd. Keywords: 5a-reductase; Testosterone; Progesterone; Neurosteroids; Myelin
1. Introduction In the brain of developing and adult animals, hormonal steroids are involved in the regulation of several nervous and endocrine functions. Many of these actions are mediated through classical intracellular steroid receptors, which activate the transcription of specific genes. Some hormonal steroids, and in particular androgens, need to be locally transformed into ‘active metabolites’, in order to be able to exert their functions; the conversion into estradiol and/or dihydrotestosterone (DHT) mediates several of the effects of testosterone (T) [1]; the resulting steroids bind, re* Corresponding author. Tel.: + 39 2 29406576; fax: +39 2 29404927; e-mail: [email protected]
spectively, to the estrogenic or to the androgenic receptor [2]. On the other hand, sex steroids and their metabolites may also act by the modulation of some neurotransmitter systems, and may interact with membrane-linked binding sites which, however, have not been fully-characterised so far [3–6]. In this context, the best known example is provided by the binding of some 5a-reduced, 3a-hydroxylated progesterone and corticosteroid metabolites to the g-aminobutyric acid (GABA)A receptor [6]. For instance, the marked anaesthetic activity of 3a-hydroxy, 5a-pregnan-20-one (allopregnanolone or tetrahydroprogesterone, THP) is due to its interaction with the GABA-gated chloride ion channel of the GABAA receptor [7–9]; this compound is formed in the brain from progesterone [10,11].
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The 5a-reductase (5a-R), the enzymatic complex which transforms T into DHT, as well as progesterone (P) and the corticoids into their respective 5a-reduced metabolites is widely diffused in the brain. The highest levels of activity of the enzyme have been found to be associated with several white matter structures [12]. Moreover, it has been shown that the formation of DHT from T is very high in purified myelin membranes obtained from the central nervous system (CNS) [13] indicating that the elevated enzymatic activity found in the white matter is principally due to the presence of myelin sheaths. The 5a-R is a membrane-associated enzyme, which requires NADPH as a cofactor; two isoforms of the 5a-R have been identified and cloned both in human and in the rat. In this last species, the homology between the amino acid sequences of the two 5a-R isoforms, deduced from their respective cDNAs, is only 44% [15]. This fact is reflected in important differences in the functional and biochemical properties of the two isozymes, such as the pH optima for their reactions (alkaline for the type 1 isoform, with a broad range from pH 5–8; acidic for the type 2 isoform, with a typical narrow pH optimum at value of 5.0); the affinity for the various substrates (T, P, androstenedione, corticosterone, etc.), which is generally higher in the case of the type 2 isoform; and, finally, in the total capacity of conversion, which is greater for the type 1 than for the type 2 isozyme [15]. The two isozymes also differ in the chromosomal localisation of the respective coding genes, in their tissue and cellular distribution [16], and possibly in their subcellular compartmentalisation [17]. The presence of specific mRNAs coding for the two isoforms has been observed in the rat brain [16], but in different amounts during animal life. The expression of the type 1 isoform appears to be constant throughout life, while the type 2 isoform is maximally expressed in the late fetal life and in the early post-natal life (up to 4 weeks of life) (Poletti et al., unpublished results). Because of these differences, it is expected that the two 5a-R subtypes, even if they catalyse the same reactions, might exert different physiological functions. So far, no data are available indicating the distribution of the two 5a-R isoforms in the various CNS structures, and in particular in myelin membranes. The aim of the present work was: (1) to analyse whether the 5a-R, previously found in myelin sheaths using T as the substrate, is also able (like the enzyme found in all other structures analysed so far) to produce the 5aderivatives of P, (2) to determine possible sex differences in the activity of the enzyme, (3) to evaluate, by means of the analysis of kinetic parameters (determination of the constant of Michaelis – Menten, pH optima, etc.) whether both isoform(s) or only one form are/is present in the myelin and (4) to establish whether a polyclonal antibody raised against one of the two 5a-R
isoforms might recognise the corresponding isozyme in the myelin by immunohistochemical techniques. 2. Materials and methods
2.1. Animals Three-months old male or female Sprague–Dawley rats (Charles River, Italy) were used throughout these experiments. The animals were maintained in animal quarters with controlled temperature and humidity. The light schedule was 14 h light and 10 dark (lights on at 6.30 a.m.). Animals were fed a standard pellet diet and water was provided ad libitum.
2.2. Myelin purification The purification has been performed with the method of Keenan [18], with slight modifications. In brief, after decapitation, the brain was rapidly removed and homogenised at 5% (w/v) in 0.32 M sucrose at 0–4°C; the suspension (3.5 ml/each tube) was centrifuged at 75 000× g (28 000 rpm) for 10 min in a Beckman SW 55 Ti rotor. The pellet formed was resuspended in 0.85 M sucrose (4 ml/each tube) and centrifuged again in the same conditions. The floating crude myelin layer was collected, diluted with water to 4 ml/each tube and centrifuged at 42 000× g (21 000 rpm) for 5 min. The resulting pellet was resuspended in water and centrifuged as before twice. The final pellet was resuspended in 0.85 M sucrose and centrifuged at 75 000×g (28 000 rpm) for 10 min; the floating purified myelin layer was collected, washed twice with distilled water and recovered by a spin at 1 000 × g for 10 min. The purity of myelin was evaluated by electron microscopy as previously described [12].
2.3. Determination of 5a-reductase acti6ity 2.3.1. Incubation To determine the pH optimum of the enzymatic isoform present in the purified myelin membranes, the incubations were performed in phosphate buffer saline (PBS) solution (250 ml) at various pH (ranging from 3.5 and 8 to assess pH optimum and at pH 7.5 to determine kinetic parameters) in the presence of 1 mM NADPH and [14C]T (specific activity 56.9 mCi/ mmol, Amersham, UK). The incubation was carried out at 37°C in a Dubnoff metabolic shaker under a stream of O2 –CO2 98:2. After determination of the pH optimum, the kinetic studies were performed at pH 7.5 in the presence of 1 mM NADPH. The following incubation conditions were tested for [14C]P metabolism, since the working condition for [14C]T were previously obtained [13].
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The apparent Michaelis – Menten constant (Km) and the Vmax were calculated for the purified myelin utilising the following condition of incubation. Time of incubation: 1 h at 37°C. Substrate ([14C]T or [14C]P): from 0.5 to 3 mM; each experiment was performed utilising five different concentrations of the substrate and triplicate determinations. The amounts of protein incubated in these studies were about 0.1 mg for the purified myelin both from male and female rats. Vials without tissue provided the blanks for each concentration of substrate and protein content was evaluated, according to the method of Bradford [19].
2.3.2. Detection of metabolites At the end of the incubation the reaction was stopped by freezing the samples to −20°C. Tritium labelled DHT or DHP (about 5000 dpm each) were added to each sample in order to evaluate the recoveries. The metabolites formed were extracted twice with diethylether, non-radioactive DHT or DHP (40 mg/100 ml) were added to each sample in order to aid visualisation of the steroid on the thin layer chromatography (TLC) plates. Extracted samples were separated on a thin layer silica gel plate (Merck 60 F254, DC). The eluting mixture was dichloromethanediethylether (11:1 v/v) to determine [14C]DHT formation and benzenemethanol (19:1 v/v) followed by cyclohexane-N-butylacetate (1:2 v/v) to determine [14C]DHP formation. The spots of the 5a-reduced metabolites were identified with iodine vapours, scraped off and the radioactivity counted in a Packard 300C liquid scintillation spectrometer. Quench corrected dpm of the isotope were obtained by a calibration standard curve. The identification of the metabolite was performed by recrystallization to constant 3H/14C ratio as previously described [12]. It has been previously shown that the purified myelin does not possess 3a-hydroxysteroid dehydrogenase activity [13], which is generally associated to the soluble cellular cytosolic fraction, thus, in the incubation conditions described, usually the formation of 5a-androstane-3a, 17b-diol (3a-diol) or 5a-pregnane-3a, 17b-diol (THP) is not detectable (data not shown). Moreover, preliminary experiments were also performed using samples of purified myelin membranes incubated with either [14C]T or [14C]P, but without the addition of tritium labeled metabolites. The products of these incubations were then assayed by TLC analysis followed by autoradiography to determine the various metabolites formed. The results have shown that using these substrates 5a-DHT or 5a-DHP were respectively the principal metabolites (85 – 90% of total metabolites formed; data not shown). Because of this, it was felt appropriate to use only the 5a-DHT or 5a-DHP data in order to perform the kinetic analysis here to be described.
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2.3.3. Statistical analysis The experimental data concerning the different incubation conditions (tissue, time) were analysed by means of the linear regression analysis, the data obtained in the kinetic study utilising the Lineweaver–Burk plot with the computer programs Enzyme [14]. 2.4. Antibody preparation. The anti-5a-R1 polyclonal antibody was prepared in New Zealand white rabbits (Charles River, Italy) as follows: a peptide reproducing the sequence of the rat type 1 5a-R, from aa 176 to 189, with the following sequence: CTGYKIPRGGLFEYV, was synthesised; this sequence is not conserved in rat type 2 5a-R, and does not match with other published protein sequences. The synthetic peptide was coupled to bovine serum albumin (BSA) using Bis(sulfosuccimidyl)suberate (Pierce, Rockford, IL) as a cross-linking agent; the complex was purified on Sephadex G-25 columns (Pharmacia Biotech, Sweden). The coupled peptide was used as an antigen, and injected subdermally in rabbit at the doses of 100, 200 or 400 mg with Freund’s complete adjuvant (Sigma); a second immunisation step (using the same doses of peptide) was performed after 3 weeks using Freund’s incomplete adjuvant (Sigma). This was followed, after 3 additional weeks, by a treatment with the uncoupled peptide (at doses of 25, 50 or 100 mg) in Freund’s incomplete adjuvant (Sigma). At the end of the immunisation procedure the rabbits were injected intravenously with 200 ml of a sterile saline solution (0.9% NaCl) containing 1 mg/ml of the peptide. After 3 days, samples of blood were collected to determine the presence of positive antisera. The antisera were screened in immunodot assays against the synthetic peptide using a chemiluminescent detection system (ECL, Amersham, UK). To eliminate cross-reactivity with other non-specific rabbit antibodies, the anti-5a-R1 antibody was purified as follows: The total rabbit IgG were initially isolated from the sera by affinity chromatography, using protein A-sepharose columns (CL-4B, Pharmacia Biotech, Sweden). The purity was checked on a sample run on 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) [20] followed by Silver staining. The anti-5a-R1 antibody was then affinity purified on a HiTrap column (Pharmacia Biotech, Sweden) previously cross-linked to the synthetic peptide utilised for the immunisation procedure. The specificity and concentration of the purified antibody was tested again in immunodot blots, using the synthetic peptide of the type 1 5a-R cross-linked to the BSA. The specificity of the antibody was also tested on yeast cells expressing the recombinant type 1 5a-R [17] and in sections of prostatic tissue as described in the next paragraphs.
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2.5. Immunopurification of the recombinant type 1 5a-R expressed in yeast Saccharomyces cere6isiae Yeast cells (protease-deficient strain Saccharomyces cere6isiae BJ3505; genotype: MATa, pep4::HIS3 PR61D1.6R his3 lys2-208 trp1-D101 ura3-52 gal2) were transformed either with plasmid YEpR1 or plasmid YEpR2 using the lithium acetate protocol [17]. Transformants were selected by tryptophan prototrophy. BJ3505[YEpR1] produces the type 1 and BJ3505[YEpR2] produces the type 2 5a-R enzyme. The transformed cells producing either type 1 or type 2 5a-R were grown overnight at 30°C in CAAU medium (2% glucose, 0.1% casamino acids, 0.67% yeast nitrogen base, 0.001% adenine, 0.002% uracil); 2 × 105 cells/ml were inoculated in fresh CAAU containing 100 mM of CuSO4 (to induce CUP1 promoter) and grown until an A600nm of 1.0 (early mid-log phase). Spheroplasts were prepared using lyticase as previously described [17]. Cells were lysed by osmotic shock (30 min at 4°C) in low salt buffer (PBS; 10 mM potassium phosphate buffer, 150 mM KCl, 1 mM EDTA; pH 7.5) and sonicated 5 s at the lowest power; total protein were determined using the Bradford assay [19]. Fractions of the yeast cell lysates containing either type 1 or type 2 5a-R (500 mg of total protein), were dissolved in 1 ml of 0.1% Triton X-100 in PBS (1 ml), incubated for 2 h at room temperature, and then incubated overnight at 4°C in the presence of 30 ml of the immunoaffinity purified primary rabbit antibody against type 1 rat 5a-R; control samples, performed without antibody, were also included in the study. At the end of the incubation, the complex type 1 5a-R/antibody was precipitated with Protein A-sepharose (Pharmacia Biotech, Sweden), washed five times with immunopurification buffer (IPB: 0.5 M NaCl, 0.05 M Tris, 0.02% NaN3, 0.2% Triton X-100, pH 7.5) and three times with distilled water; the recovered proteins were resolved on 12% SDS-PAGE. The gel was stained using Silver staining.
2.6. Immunohistochemistry The yeast cells transformed or not with the plasmid bearing the 5a- R cDNA were grown as previously described [17]. The rat optic nerves and prostate were embedded in O.C.T. immediately after the animal sacrifice. They were then frozen at −20°C for 30 – 60 min, and sectioned with a freezing microtome. Each 15 mm section was collected on a slide and treated as follows: the sections were fixed with 4% formaldehyde in 0.15 M PBS, pH 7.2 for 10 min at room temperature, washed in PBS (3× 5 min) and post-fixed in methanol (5 min at −20°C) and acetone (3 min at − 20°C). They were then rinsed in PBS (3×5 min) and incubated with
normal goat serum 10% at 25°C for 10 min to block non-specific staining associated with the secondary antibody. Goat serum was removed by washing with PBS (3× 5 min). The sections were incubated with the immunoaffinity purified primary rabbit antibody against type 1 rat 5a-R (0.25 mg/ml), for 24 h in a humid atmosphere at 4°C. After rinsing in PBS (3 ×5 min) the samples were incubated for 1 h in a humid atmosphere at 37°C with rhodamine-conjugated goat anti-rabbit antibodies (Jackson Immunoresearch West Grove, PA, USA) diluted 1:400 in PBS, rinsed in PBS (3×5 min) and mounted in an anti-fading mounting medium (glycerol: PBS 90:10, containing 2.5 g DABCO (diazabicyclo(2,2,2)octane, Sigma). Controls were obtained by eliminating the first incubation, by maintaining the addition of the secondary goat anti-rabbit antibody.
2.7. Confocal microscopy. A Zeiss LSM 410 Invert microscope has been used in this study. The instrument was equipped by 543 nm He–Ne laser and a TRITC set of filters (FT 560 beam-splitter and a low-pass LP 590 emission filter). The images were obtained at 2 s scan speed with 16 averages, with a Plan-Neofluar 40/1.3 Oil lens. The distortions of the confocal images [21] were not significant because we used only single optic sections or very limited depth of scanning.
3. Results
3.1. Kinetic studies Preliminary experiments were performed on purified myelin membranes obtained from the rat CNS, to verify whether these preparations were able to convert P into DHP; the optimal conditions required for kinetic studies of the reaction were also established. The study of the variations of the 5a-R activity in purified myelin membranes as a function of pH was performed using T as the substrate. The data are shown in Fig. 1, which also illustrates (in the inset) the pH profiles of the 5a-R activities of lysates of yeast cells expressing the recombinant type 1 or type 2 5a-R. The capability of the purified myelin to 5a-reduce the substrate appears to increase when the pH reaches basic values. No peaks of activity were present at an acidic pH, around 5.0–5.5, indicating that the pH optimum for the 5a-R present in the myelin is in the alkaline range. This, as previously mentioned, is a characteristic of the type 1 5a-R, both in its native form or when expressed in mammalian or yeast cells [15–17]. There were no significant differences between male and female rat myelin preparations at any pH examined.
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Fig. 1. Variation of the myelin 5a-R activity as a function of the pH of incubation. The myelin preparations, obtained both from male and female rats, have been incubated in PBS buffer at different pH, in presence of 3 mM of [14C]T; the formation of [14C]DHT was measured as described in Section 2 and is expressed as mean9 S.E obtained from four different determinations. The profile of activity as a function of the pH obtained for yeast cell lysates producing the recombinant type 1 or type 2 5a-R is also reported.
To further verify whether the type 1 isozyme is the form of the 5a- R which predominates in purified myelin membranes, the kinetics of the enzymatic reaction was determined in the same myelin preparations, obtained respectively both from the male and the female rat brain. Fig. 2 shows the Lineweaver–Burk plots of the reciprocal of the initial velocity of the formation of [14C]DHP and [14C]DHT as a function of the reciprocal of the initial substrate (P and T) concentrations. The results of the experiments are summarized in Table 1, which shows the data of a single analysis, however, this study was repeated three times with very similar results. The apparent Michaelis – Menten constants (Km) determined for the 5a-R of P are similar in the myelin preparation obtained from the two sexes (male 0.5 mM; female 0.6 mM); similarly, no sex differences in these parameters were observed when T was used as substrate (male 1.1 mM; female 1.5 mM). In the case of T, the affinity was lower than that observed for
P, which appears to be the preferred substrate (see ratio Vmax/Km). It is also clear from the Km values, all in the micromolar range, that the affinities of the two substrates (P and T) for the enzyme present in myelin membranes are almost identical to those detected for the recombinant type 1 isoform of the 5a-R expressed either in mammalian cells or in a yeast cell system [15,17]. The values obtained for the maximal velocity of reaction (Vmax), an index of the total amount of active enzyme present in the preparation, were in the same range in the four groups considered (i.e., P males and females, T males and females).
3.2. Antibody characterization and immunofluorescence studies To confirm that the type 1 5a-R is present in myelin membranes, a polyclonal antibody was raised against this isoform, using a synthetic peptide reproducing a
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Fig. 2. Lineweaver –Burk plots of the 5a-R activity present in purified myelin membranes obtained both from male and female rat brains. The kinetics of the reaction were determined using either [14C]T or [14C]P as substrates. Each curve was obtained with five different increasing concentrations in triplicate. Non-limiting concentration of cofactor (1 mM of NADPH) were also added in the reaction. Details of the experiments are described in Section 2.
potentially antigenic and hydrophilic amino acidic sequence of the enzyme. Fig. 3 shows the immunodot blot obtained using 1 ml of different fractions (1 ml) of the purified antibody. Only the spots containing the synthetic peptide were specifically labelled by the antibody. The fraction 3, which appeared to have the highest concentration of the desired antibody, was chosen. The specificity of the immunopurified polyclonal antibody was further tested in immunopurification experiments using yeast cells expressing either recombinant type 1 or type 2 isoform [17]. The results are shown in Fig. 4. It appears that the antibody specifically recognizes only a single protein with a molecular weight of about 26 Kd in the samples of yeast cell lysates expressing type 1 5a-R (lane 3), but not in those expressing the type 2 5a-R (lane 2), indicating that it does not cross react with the second isoform of the rat 5a-R; the specificity of the antibody is also confirmed by the absence of immunoprecipitation of other yeast proteins. The specificity of the antibody for immunofluorescence experiments was also tested using yeast cells expressing type 1 5a-R and section of the rat anterior prostate. Fig. 5 shows the yeast S. cere6isiae transTable 1 Characterization of rat 5a-reductase present in purified myelin membranes [14C]progesterone
Vmax (ng/h/mg of protein) Km (mM) Vmax/Km R2
formed with a plasmid expressing the rat type 1 5a-R, observed with a phase contrast microscope. Fig. 6 shows the immunofluorescence of the same field after labelling with the antitype 1 5a-R antibody. It clearly appears that all yeast cells expressing the type 1 isoform are recognized by the antibody which is concentrated in the nuclear compartment of the cells; on the contrary yeast cells transformed with the recombinant type 2 isoform did not show any reaction (not shown). In the sample of rat anterior prostate (Fig. 7) a strong immunoreactivity was observed in the nuclei (or in the perinuclear region) of the epithelial cells of the alveoli. The nuclei of the alveolar cells show a characteristic annular fluorescence, in good agreement with the data of the literature [22]. This indicates that in the prostate the type 1 isoform is an epithelial enzyme. The nuclei of few cells, interpreted as fibroblasts of stromal connective, appears to be also fluorescent while the autofluorescence of collagen bundles is enhanced by confocal microscopy. The study on the myelin was performed, for convenience, on the rat optic nerve, a structure easy to obtain and very rich in myelin and in 5a-R activity. When observed with the confocal laser scanning microscope, the sections of the rat optic nerve (Figs. 8 and 9) show a strong fluorescence of the nerve fibres. The examination of single optical sections allowed the localisation of the fluorescence at the level of the myelin sheaths, while the axons were negative.
[14C]testosterone
Male
Female
Male
Female
38.5
45.5
28.6
35.7
0.5 76.9 0.94
0.6 71.0 0.99
1.1 25.1 0.96
1.5 24.5 0.94
Myelin membranes and kinetic analysis were performed as described in Section 2.
4. Discussion The data obtained in the present study using biochemical and immunohistochemical approaches demonstrated that the formation of the 5a-reduced derivatives of T and P occurring in myelin sheaths obtained from the rat brain is mainly due to the presence of the type 1 isoform of the rat 5a-R. The enzymatic activity found
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Fig. 3. Immunodot blot obtained by testing the affinity-purified rabbit polyclonal antibody anti-type 1 5a-R with the synthetic 5a-R type 1 peptide linked to BSA. The BSA peptide fraction represents the fraction utilized to immunize rabbits; the BSA fraction represents only the BSA not cross-linked to the synthetic peptide, the different fractions are those derived after elusion from the column of immunoaffinity purification. See Section 2 for details.
in purified myelin membranes presents the biochemical characteristics described in the literature for the recombinant type 1 rat 5a-R [15 – 17]. First of all, the pH optimum of the reaction, which allows to discriminate between the two known 5a-Rs isoforms, is in the basic range. Secondly, the affinity for the substrates correlates well with the kinetic parameters of the type 1 isoform, since the apparent Michaelis – Menten constants are all found in the micromolar range, moreover, the similarity of the Vmax values obtained in the kinetic studies when P and T are used as substrates indicates that only one form of the enzyme is probably responsible for both reactions [15,17]; if both are present, one would be largely predominant. No evidence of a sex
Fig. 4. Silver staining of a 12% SDS-PAGE loaded with samples of lysates of yeast cells expressing the two isoforms of the 5a-R after immunoprecipitation with the immunopurified polyclonal antibody raised against the type 1 5a-R. Lane 1, lysates of yeast cells expressing the type 1 5a-R without addition of the antibody; lane 2, lysates of yeast cells expressing the type 2 5a-R and lane 3 lysates of yeast cells expressing the type 1 5a-R after immunoprecipitation with the antibody; lane 4 low range molecular weight markers (14.4, 21.5, 31.0 and 45.0 kD). Ab indicates the band which corresponds to the antibody utilised for immunoprecipitation; type 1 5a-R indicates the specific product of the immunoprecipitation, a protein of about 26 kD.
difference in the amounts of 5a-R present in the purified myelin was obtained. In every experiment, the data were identical, independently on whether male or female rat brains were used for the purification of the myelin. The existence of the type 1 5a-R in the white matter, and in particular in the myelin, has been corroborated by immunohistochemical studies. A specific polyclonal antibody raised against a synthetic hy-
Fig. 5. Immunochemical detection of rat type 1 5a-R. Yeast cells expressing the recombinant rat type 1 5a-R observed with phase constrast microscope.
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Fig. 6. Immunochemical detection of rat type 1 5a-R. Yeast cells expressing the recombinant rat type 1 5a-R after immunocytochemical reaction with the polyclonal antibody.
drophilic peptide corresponding to a potentially antigenic site of the amino acid sequence of the type 1 enzyme 5a-R specifically recognised the enzymatic protein in the myelin sheaths of the rat optic nerve. Unfortunately, it was not possible to analyse whether also type 2 5a-R is present, even in low amounts, because a suitable antiserum against the type 2 isoform is not presently available. The significance of the presence of 5a-R in myelin membranes is still unclear. Oligodendrocytes, the cells responsible for myelin formation in the CNS, are known to contain 5a-R activity [12]. However, this enzyme is not generally bound to the external membranes of the cells, but is associated to the perinuclear region [17,22–24], as also shown by the data obtained in prostatic tissue in the present study. During myelinogenesis, myelin membranes are derived from the external plasma membrane of the oligodendrocytes. However, it is now accepted that myelin membranes are surrounded by, and infiltrated with, cytoplasmic channels (outer/lateral loops and longitudinal incisures) which may serve to translocate polyribosome complexes bearing specific mRNAs from the cell body of the oligodendrocytes to the myelin sheaths [25]. Because of this, some proteins (e.g., the myelin basic protein, MBP) may be synthesised in free polyribosomes localised in the myelinating extensions of the oligodendrocytes before being inserted in the myelin [26,27]. A
Fig. 7. Rat anterior prostate section, corresponding to a single optic section observed at CSLM (panel C). The epithelial cell nuclei are strongly fluorescent and present the characteristic ring aspect. He–Ne laser (543 nm with TRITC filters), lens 40/1.30 Ocl-Bar = 14 mm= 25 mm (× 560).
mechanism similar to that of the formation of MBP could explain the association of type 1 5a-R with the myelin membranes. The presence of the type 1, and the probable absence of the type 2 isoforms in myelin is
Fig. 8. Almost transversal section of a single rat optic nerve corresponding to a single optic section observed at CSLM. The labelling of myelin membranes appears evident. He – Ne laser (543 nm with TRITC filters), lens 63/1.10 Ocl-Bar =14 mm = 25 mm ( × 570).
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Fig. 9. Longitudinal section of rat optic nerve corresponding to a single optic section observed at CSLM. The labelling of myelin membranes appears evident. He–Ne laser (543 nm with TRITC filters), lens 63/1.10 Ocl-Bar= 14 mm= 25mm (× 570).
also corroborated by recent studies obtained using the reverse transcriptase-polymerase chain reaction (RTPCR) technique, that showed the presence of specific mRNA coding for type 1, but not for type 2 isozyme, in cultured rat oligodendrocytes [28], the cells responsible for myelin production. The role of the type 1 5a-R in myelin membranes in still unclear. It has been shown that P, the preferential substrate of the 5a-R, is able to induce MBP [29,30]. Probably, this action is directly mediated by a genomic effect, since P receptors have been detected in the oligodendrocytes [31], and specific glucocorticoid/ progesterone responsive elements have been identified on the MBP promoter [25]. The presence of high levels of type 1 5a-R in myelin membranes may lead then to the hypothesis that the conversion of 3keto-D4 steroids into their corresponding 5a-reduced metabolites may play a role in the control of the synthesis of proteins which are important components of myelin membranes. Also other 5a-reducible steroids (such as the glucocorticoids corticosterone and cortisol) may be involved in the transcriptional and post-transcriptional regulation of myelin genes [30,32 – 34]. Interestingly, the 5a-R is the first enzyme involved in the formation of the tetrahydro-derivatives of P and of the glucocorticoids, compounds which are further reduced in position 3 by the soluble cytoplasmic enzyme 3a-hydroxysteroid dehydrogenase. These steroids are highly active as anaesthetic and anxiolytic drugs [35], and these actions are mediated by the GABAA-gated chloride ion channel of the GABAAreceptor [7]. There-
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fore, the high levels of type 1 5a-R present in myelin membranes could serve as a part of the ‘neurosteroid synthesising structures’ of the CNS. Furthermore, a different role of the rat type 1 5a-R have been postulated some years ago [36,37] because of a certain degree of homology of the enzyme with the SC2 glycoprotein which is strongly expressed by many types of neurons, and is involved in synaptic function. The significance of this homology is still unclear, but the possibility that type 1 5a-R enzyme is a bifunctional protein should be taken into account. Even the distribution of the type 1 5a-R mRNA (analyzed by ‘in situ’ hybridization) during pre- and peri-natal stages [38] agrees with a possible non-enzymatic role in the development and differentiation of the rat CNS. It is also interesting to note that, in humans showing the Imperato–McGinley syndrome [39,40], a disease in which the type 2 enzyme is genetically absent, brain development or myelination are not affected. No specific genetic defects of the type 1 isozyme have been identified so far. This could mean either that the deficiency of the type 1 5a-R is clinically silent, or that the defect is not compatible with life. Finally, the presence in myelin of the 5a-R type 1 is relevant in view of the increasing use of 5a-R inhibitors in the therapy of prostatic diseases, like benign prostatic hyperplasia (BPH). Since the actual role of the type 1 enzyme in myelin is not yet known, it would be safer to utilise for the treatment of this and other diseases drugs which show a weak inhibitory effect on the type 1 enzyme, until studies on the action of selective type 1 5a-R and type 2 5a-R inhibitors on myelin will be available.
Acknowledgements We thank Dr Nancy L. Weigel for the helpful advice on antibody preparation and the choice of the synthetic peptide sequence of 5a-reductase, and for the revision of the manuscript. We are indebted to Professor Chillemi for the peptide synthesis. This work was supported by CNR funding Special Projects ACRO 94.01162.PF39: FATMA 95.00868.PF41: AGING 95.01020.PF40: BTBs 93.01103.PF70 and Bilateral project 94.02374. CT04.
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