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Δ5-Δ4-isomerase in different regions of the avian brain
Brain Research 818 Ž1999. 536–542
Short communication
Expression and activity of 3b-hydroxysteroid dehydrogenaserD5-D4-isomerase in different regions of the avian brain Kazuyoshi Ukena a , Yoko Honda a , Yuto Inai a , Chie Kohchi b , Robert W. Lea c , Kazuyoshi Tsutsui a, ) a
Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima, UniÕersity, Higashi-Hiroshima 739-8521, Japan b Radioisotope Center, Hiroshima UniÕersity, Higashi-Hiroshima 739-8526, Japan c Department of Applied Biology, UniÕersity of Central Lancashire, Preston, PR1 2HE, Lancashire, UK Accepted 24 November 1998
Abstract Recently, we have demonstrated, using biochemical and immunochemical methods, that the quail brain possesses the cholesterol side-chain cleavage enzyme Žcytochrome P450scc. and produces pregnenolone and its sulfate ester. To clarify progesterone biosynthesis in the avian brain, therefore, we examined the expression of messenger RNA ŽmRNA. encoding for the enzyme 3b-hydroxysteroid dehydrogenaserD5-D4-isomerase Ž3b-HSD. and its enzymatic activity using the quail. RT-PCR analysis together with Southern hybridization indicated the expression of 3b-HSD mRNA in the brain of sexually mature birds but with no clear-cut sex difference. Employing biochemical techniques combined with HPLC analysis, the conversion of pregnenolone to progesterone was found in brain slices of mature males. Progesterone biosynthesis was increased in a time dependent manner and completely abolished by trilostane, a specific inhibitor of 3b-HSD. The enzymatic activity of 3b-HSD was greatest in the cerebrum and lowest in the mesencephalon. A specific RIA indicated that progesterone concentrations in the different brain regions closely followed the level of 3b-HSD activity. High levels of progesterone concentration were observed in the diencephalon and cerebrum with lowest values in the mesencephalon. Progesterone levels in the brain regions were significantly higher than those in the plasma. These results suggest that the avian brain possesses not only cytochrome P450scc but also 3b-HSD and produces progesterone. It is also indicated that progesterone biosynthesis in the avian brain may be region-dependent. q 1999 Elsevier Science B.V. All rights reserved. Keywords: 3b-Hydroxysteroid dehydrogenaserD5-D4-isomerase; Gene expression; Enzymatic activity; Progesterone; Quail brain; Reverse transcriptionpolymerase chain reaction
De novo steroidogenesis from cholesterol in the mammalian brain is now well established. For example, pregnenolone and dehydroepiandrosterone, as unconjugated steroids, and their fatty acid or sulfate esters accumulate within the brain in several mammalian species w7,8,12,18,31–33x. The brain content of these steroids is almost constant even after the removal of peripheral steroids. Certain structures in the mammalian brain have the capacity to metabolize cholesterol to pregnenolone w1–3,11,13,28,30x. The cytochrome P450 side-chain cleavage enzyme Ž P450scc. cleaves cholesterol to form pregnenolone, which is a 3b-hydroxy-D5-steroid and a main precursor of steroid hormones secreted by the steroido-
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genic glandular cells Žfor a review, see Ref. w25x.. Recent studies have further indicated that both P450scc protein and its messenger RNA ŽmRNA. are expressed in the rat brain w2,3,6,13,19,23,28,39 x. Considered with these extensive mammalian studies, we have recently demonstrated that the quail brain also possesses cytochrome P450scc and produces pregnenolone and its sulfate ester, using both biochemical and immunochemical techniques w37,38x. In addition, our immunohistochemical studies with this same avian species have shown that P450scc immunoreactive cells are distributed in several telencephalic, diencephalic and mesencephalic regions, such as the hyperstriatum accessorium ŽHA., the ventral portions of the archistriatum ŽA. and the corticoid area ŽC., the preoptic area ŽPOA., the anterior hypothalamus ŽAHy., the dorsolateral thalamus ŽDL. and the cerebellar cortex w38,40x. 3b-Hydroxysteroid dehydrogenaserD5 -D4 -isomerase Ž3b-HSD. is also a key enzyme for steroidogenesis in
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 2 9 6 - 7
K. Ukena et al.r Brain Research 818 (1999) 536–542
peripheral steroidogenic glands. The enzyme 3b-HSD catalyzes the oxidation and isomerization of 3b-hydroxy-D5steroids, such as pregnenolone and dehydroepiandrosterone. Therefore, the demonstration of the presence of 3b-HSD is essential in order to understand the concept of steroidogenesis in the brain. 3b-HSD mRNA expression andror activity of 3b-HSD have also been observed in the mammalian brain w9,10,13,34,42x. In non-mammalian vertebrates, 3b-HSD activity has recently been found in the brain of both avian w41x and amphibian species w24x. However, the expression of 3b-HSD mRNA and differences in its enzymatic reaction in several brain regions have not been investigated in the non-mammalian brain. In the present study, therefore, we have demonstrated the expression of mRNA encoding for the key steroidogenic enzyme 3b-HSD in the quail brain. 3b-HSD expression was confirmed by biochemical studies combined with HPLC analysis and the measurement of intrabrain progesterone. The second aim of this study was to determine differences in the enzymatic activity of 3b-HSD in different brain regions. Japanese quail Ž Coturnix japonica. was used in these studies. The birds were housed in a temperature-controlled room Ž25 " 28C. under a daily photoperiod of 16-h light and 8-h dark Žlong day; lights on at 0700 h., and given quail food and tap water ad libitum. Sexually mature male and female quails, 3 months of age, were used to examine 3b-HSD mRNA expression. Mature males were used on studies investigating the enzymatic reaction catalyzed by 3b-HSD and for the RIA of progesterone. Birds were isolated in individual cages approved in accordance with the Guide for the Care and Use of Laboratory Animals prepared by Hiroshima University, Japan. To determine the expression of mRNA encoding for 3b-HSD in the brain, RT- PCR analysis was performed using the male and female quails Ž n s 5 in each sex. according to a method described previously w14,39x. All birds were killed between 1000 and 1200 h. Total RNA of the brain was isolated by the guanidium thiocyanate–phenol–chloroform extraction method. Total RNA contains ribosomal RNA and mRNA. Fifteen micrograms of total RNA were reverse transcribed using Oligo dT primer and RT in a 30-ml reaction volume for 2 h at 378C. The reaction mixture contained 15 mg of total RNA, 50 mM Tris–HCl ŽpH 8.3., 75 mM KCl, 3 mM MgCl 2 , 10 mM dithiothreitol, 1 mM deoxynucleoside triphosphate ŽdNTP. mix, 1.5 mg of Oligo dT12 – 18 ŽPharmacia, Uppsala, Sweden., 15 U of ribonuclease inhibitor ŽWako, Osaka, Japan., and 400 U of moloney murine leukemia virus transcriptase ŽPromega, Osaka, Japan.. The reaction was stopped by incubating at 658C for 10 min, and the cDNA ethanol precipitated and redissolved in 15 ml of distilled water. For PCR, an aliquot of the cDNA solution corresponding to 2 mg of initial total RNA was used as a template in a 25-ml reaction mixture. The PCR mixture contained cDNA, 20 mM Tris–HCl ŽpH 8.0., 100 mM KCl, 0.1% Triton X-100,
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1.5 mM MgCl 2 , 0.2 mM dNTP mix, 25 pmol of each primer, and 1 U of Gene Taq polymerase ŽWako, Osaka, Japan.. After denaturation at 958C for 3 min, the mixture was subjected to 30 thermal cycling in a programmed temperature control system ŽPC700; ASTEC, Fukuoka, Japan. as follows: denaturation at 938C for 1 min, primer annealing at 568C for 1 min, and extension at 728C for 1 min. After the thermal cycling, the mixture was additionally incubated at 728C for 10 min. An 8-ml aliquot of each sample was electrophoresed through a 1.5% agarose gel. To confirm the identity of the amplified fragment, the gels were subjected to Southern analysis with a digoxigenin-labeled oligonucleotide probe, corresponding to the internal sequence of the target gene. Digoxigenin DNA labeling and detection were performed according to the manufacturer recommendations ŽBoehringer, Vienna, Austria.. Oligonucleotides used as PCR primer and probe for mRNA detection, were based on cDNA sequences of chicken 3b-HSD w27x and chicken b-actin, and were as follows: 3b-HSD sense primer 5X-TCAACGTGACAGGTACTCAG-3X Žnucleotide number 388–407 in Ref. w27x., 3b-HSD antisense primer 5X-CTCTTTGCCTCTTCCCATGT-3X Žnucleotide number 1125–1144 in Ref. w27x., 3b-HSD probe 5X-TACATCTCAGATGACACTCC-3X Žnucleotide number 855–874 in Ref. w27x., b-actin sense primer 5X-GAGACCTTCAACACCCCAG-3X Žnucleotide number 441–459, gb L08165., and b-actin antisense primer 5X-GACAGAGTACTTGCGCTCAG-3X Žnucleotide number 1066–1085, gb L08165.. The 3b-HSD sense and antisense primers give a 757 bp amplified fragment of chicken 3b-HSD gene. The b-actin primers give a 638 bp amplified fragment. Each RT-PCR analysis was repeated five times using independently extracted RNA samples from different animals. 3b-HSD activity of the quail brain was determined by measuring the conversion of w7 y3 Hx pregnenolone Žspecific activity, 19.9 Cirmmol, New England Nuclear, Boston, MA. to 3 H-progesterone using brain slices of different regions of the mature male. Biochemical analysis in the present study was performed according to a method cited previously w24x. The different brain regions, i.e., cerebrum, diencephalon, mesencephalon and cerebellum, were cut into slices using a razor and preincubated at 408C for 15 min in 1 ml of physiological saline Ž124 mM NaCl, 5 mM KCl, 1.24 mM KH 2 PO4 , 1.3 mM MgSO4 , 2.4 mM CaCl 2 , 26 mM NaHCO 3 , and 10 mM glucose.. Following this, the slices were incubated at 408C for 0, 30 or 60 min in 0.5 ml of physiological saline containing 50 pmol Ž1000,000 cpm. of w7-3 Hxpregnenolone and 4% propylene glycol. The incubation medium was constantly gassed with 95% O 2 and 5% CO 2 . At the end of the incubation period, 2-ml ethyl acetate was added and the slices homogenized with a glass–glass homogenizer. The homogenates were stirred for 15 min and centrifuged at 3000 = g for 5 min. The organic phase was removed and the extraction step repeated twice. The combined organic extracts were dried
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down, re-dissolved in 40% acetonitrile ŽACN., and filtrated through a membrane filter Ž0.45 mm: Ultrafree-MC, Millipore, Bedford, MA.. To detect labeled steroids formed from w7-3 Hxpregnenolone, filtrates were subjected to HPLC analysis using a reversed-phase column, LiCrospher 100 RP-18 Ž4.0 = 250 mm, Kanto Chemical, Tokyo, Japan.. The column was eluted with a 30-min linear gradient of 40–70% ACN at a flow rate of 0.7 mlrmin, followed by an isocratic elution of 70% ACN. The eluate was fractionated every 0.5 min from 28 min to 33 min and counted in a liquid scintillation counter. Reference standards of tritiated pregnenolone and progesterone were chromatographed under similar conditions to the tissue extracts and detected using a liquid scintillation counter. To confirm the involvement of 3b-HSD activity in the formation of the radioactive peak of progesterone, brain slices were incubated with saline containing trilostane ŽMochida, Tokyo, Japan., a specific inhibitor of 3b-HSD, and subjected to HPLC analysis in a similar manner as described earlier. Trilostane Ž2 = 10y2 M; 10 ml. dissolved in acetone, was added to 0.5 ml of the incubation medium and incubated with brain slices for 60 min. These biochemical analyses were repeated independently at least five times with tissue from different animals. To measure progesterone levels in the quail brain, 6 mature males were killed between 1000 and 1200 h and trunk blood collected into heparinized tubes. The blood was centrifuged at 1800 = g for 20 min at 48C and the plasma stored at y808C until assayed for progesterone. Immediately after blood collection, each different brain region, i.e., cerebrum, diencephalon, mesencephalon and cerebellum, was dissected out and weighed and then frozen in liquid nitrogen and stored as a sample at y808C. Extraction of progesterone was performed according to a method described previously w8,37–39x. Each brain sample was homogenized in 5 ml ice-cold phosphate-buffered saline ŽPBS: 10 mM phosphate buffer and 140 mM NaCl, pH 7.3. with a Teflon-glass homogenizer. Plasma Ž200 ml. was diluted with 5 ml cold PBS. Brain and plasma samples were then subjected to steroid extraction. To estimate steroid recovery during extraction, 2000 cpm of w1, 2, 6, 7-3 Hx progesterone Žspecific activity, 115 Cirmmol, New England Nuclear. was added to each sample together with 5-ml ethyl acetate and the tubes stirred for 30 min followed by centrifugation at 3000 = g for 5 min. The organic phase was subsequently removed and the extraction step repeated further twice. The combined organic extracts, which contained progesterone, were dried down and dissolved in 1-ml PBS containing 0.1% gelatin. The aqueous solution was divided into two aliquots: one aliquot for the estimation of recovery, the other for the measurement of progesterone. To measure progesterone concentration, aliquots of organic extracts were assayed in a progesterone RIA w5,8,15,37–39x using an antiserum to progesterone ŽScantibodies Laboratories, Santee, CA. and w1, 2, 6, 7-3 Hx
progesterone. The antiserum used in this assay cross-reacted with deoxycorticosterone at 3.3%, 17a-hydroxyprogesterone at 0.6%, and pregnenolone less than 0.1% and no chromatographic purification was performed. Separation of bound and free steroid was performed by centrifugation after reaction with the IgG SORB ŽThe Enzyme Center, Malden, MA.. The least detectable amount was 0.1 ngrml, and intraassay variation was estimated as less than 7%. The precision index Ž l. of a linear portion of the competition curve computed according to a method described previously w36,37x was 0.037. Results for 3b-HSD activity and progesterone concentration in different brain regions were expressed as the mean " S.E.M. and were analyzed for significance by using Duncan’s multiple range test or the Kruskal–Wallis test followed by Mann–Whitney’s U-test after verification of equality or inequality, respectively, of variances among the groups compared w4x. The expression of mRNA encoding for 3b-HSD in the brains of both male and female quails was examined by RT-PCR analysis. The initial RNA amount used in the RT-PCR was adjusted spectrophotometrically and RT-PCR for b-actin was performed as a control experiment. Gel electrophoresis of the RT-PCR product for the 3b-HSD gene identified a single band of 757 bp corresponding to 3b-HSD mRNA size in both male and female brains ŽFig. 1a.. Serial Southern hybridization confirmed that this band was 3b-HSD mRNA specific ŽFig. 1b.. No clear-cut sex difference in the 3b-HSD mRNA expression was detected in the brain ŽFig. 1.. Although the density of the band corresponding to 3b-HSD mRNA in the brain was less than that in the testis ŽFig. 1., suggesting that 3b-HSD is expressed in the brain of sexually mature quails in both sexes. To demonstrate the enzymatic activity of 3b-HSD in the brain, diencephalic slices of the male quail were incubated with tritiated pregnenolone as a precursor, and radioactive metabolites analyzed by reversed-phase HPLC. As shown in Fig. 2, the radioactive peak was detected approximately 2 min before the elution of the precursor steroid, pregnenolone. Tritiated progesterone used as a reference standard exhibited the same retention time under similar chromatographic conditions. The radioactive peak corresponding to progesterone increased in a time dependent manner from 0 min- to 60 min-incubation ŽFig. 2a, b, c.. In addition, 10y4 M trilostane, a specific inhibitor of 3b-HSD, completely abolished the production of this peak in 60 min-incubation samples ŽFig. 2c, d.. This evidence suggests that the observed radioactive metabolite is progesterone produced by the enzymatic activity of 3b-HSD in the diencephalon. To compare the ability of progesterone biosynthesis among different brain regions, each brain tissue was incubated with tritiated pregnenolone for 60 min. Progesterone formation was calculated from the radioactivity corresponding to progesterone and was expressed as pmolrgrh.
K. Ukena et al.r Brain Research 818 (1999) 536–542
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regions Ž P - 0.01, vs. diencephalon, mesencephalon, cerebellum; Fig. 4.. The data presented in this paper demonstrates the expression of mRNA encoding for 3b-HSD, a key steroidogenic enzyme, in the brain of the Japanese quail in both sexes. To the best of our knowledge, this is the first report showing the expression of 3b-HSD mRNA in the avian brain. Employing biochemical techniques combined with HPLC analysis in the present study, we have observed the conversion of pregnenolone to progesterone in brain slices, suggesting enzymatic activity of 3b-HSD in the quail brain. Considered with this, 3b-HSD activity has previously been reported in primary cultures of the telencephalon in another avian species, i.e., the zebra finch w41x.
Fig. 1. RT-PCR analysis of 3b-HSD mRNA in the brain of the male and female quails. The testis was also used as a positive control. Upper panel Ža. shows a result of the gel electrophoresis of RT-PCR products for chicken 3b-HSD, and middle panel Žb. shows an identification of the band by Southern hybridization using digoxigenin-labeled oligonucleotide probe for chicken 3b-HSD. cDNA corresponding to 2 mg total RNA extracted from the brain or testis was used for a PCR reaction. The lane labeled ‘No cDNA’ was performed without template as the negative control. Lower panel Žc. shows a result of the RT-PCR for chicken bactin as the internal control, in which cDNA corresponding to 0.01 mg total RNA was used as template. RT-PCR experiments were repeated five times using independently extracted RNA samples from different animals and gave similar results.
As shown in Fig. 3, the enzymatic activity of 3b-HSD was detected in all of the different brain regions. However, the ability of progesterone formation was different among the brain regions ŽFig. 3.. 3b-HSD activity was greatest in the cerebrum Ž P - 0.01, vs. mesencephalon, cerebellum; P 0.05, vs. diencephalon. and lowest in the mesencephalon Ž P - 0.01, vs. cerebrum, diencephalon; P - 0.05 vs. cerebellum; Fig. 3.. In this study, progesterone concentrations in different brain regions were further measured by a specific RIA using an antibody against progesterone. Progesterone levels in the brain also showed the region-specific distribution. As shown in Fig. 4, progesterone concentration was significantly higher in the diencephalon than in the cerebellum Ž P - 0.05. and mesencephalon Ž P - 0.01.. Progesterone concentrations in the cerebrum also tended to be higher than those in the cerebellum and mesencephalon, but this difference was not statistically significant due to a large variance ŽFig. 4.. Progesterone concentration was minimal in the mesencephalon Ž P - 0.01, vs. diencephalon, cerebellum; Fig. 4.. In plasma, the concentration of progesterone was markedly lower than in several brain
Fig. 2. Typical HPLC chromatograms of steroids extracted from diencephalic slices after different incubation times w0 min Ža., 30 min Žb. and 60 min Žc.x with 3 H-pregnenolone using a reversed-phase column. The column was eluted with a 30-min linear gradient of 40–70% ACN, followed by an isocratic elution of 70% ACN. The eluate was fractionated every 0.5 min from 28 min to 33 min Ž0.35 ml each.. The ordinate indicates the radioactivity measured in each HPLC fraction. The arrows indicate the elution position of standard steroids, i.e., pregnenolone and progesterone. The diencephalic slices were also incubated with trilostane, a specific inhibitor of 3b-HSD, for 60 min and subjected to HPLC analysis Žd..
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Fig. 3. Comparison of 3b-HSD enzymatic activity in different brain regions, i.e., cerebrum, diencephalon, mesencephalon and cerebellum, in the male quails. Each brain region was incubated with 3 H-pregnenolone for 60 min and subjected to HPLC analysis. Progesterone production was calculated from the radioactivity corresponding to progesterone and expressed as pmolrgrh. Each column and the Õertical line represent the mean"S.E.M. Ž ns 5.. Significance of difference: ), P - 0.05; )), P - 0.01 Žvs. mesencephalon.; †, P - 0.05; ††, P - 0.01 Žvs. diencephalon. by Duncan’s multiple range test.
From the present and previous studies, it is probable that 3b-HSD is expressed in the avian brain and is involved in steroid metabolism, such as the conversion of pregnenolone to progesterone. The expression of 3b-HSD andror its enzymatic activity have also been reported in the brain of several mammals w9,10,13,34,42x and an amphibian w24x. Therefore, it may be generally stated that the expression of an enzymatic active form of 3b-HSD is a conserved property of the vertebrate brain. Recently, we have demonstrated that the same avian species also possesses cytochrome P450scc and produces pregnenolone and its sulfate ester from cholesterol, by biochemical and immunochemical approaches w37,38x. Taken together, the results suggest that de novo progesterone biosynthesis from cholesterol occurs in the avian brain. Based on the biochemical analysis, we could detect 3b-HSD activity in all brain regions of the quail used in this study. The most striking observation in the present study was, however, a clear difference in 3b-HSD activity among different brain regions. The enzymatic activity in the cerebrum and diencephalon was much higher than that in the mesencephalon, a regional pattern of 3b-HSD activity which has not yet been reported in birds. On the other hand, it has been demonstrated that the enzymatic activity of 3b-HSD in the rat brain was high in the limbic region and low in the hypothalamus w42x. In addition, we have reported that in the rat the expression of 3b-HSD mRNA
Žisoforms type I plus type II. is comparatively low in the mesencephalon and diencephalon and high in the cerebrum and cerebellum w14x. Accordingly, the expression of 3bHSD and its enzymatic activity in the diencephalon may be different between mammalian and avian species. In this study, we further measured the concentration of progesterone in several brain regions of the quail. Progesterone level was also high in the diencephalon and cerebrum and low in the mesencephalon, although concentration in the cerebrum was not statistically significant due to a large variance. Notwithstanding the large variance in the cerebrum, progesterone levels were in agreement with the result of biochemical analysis, suggesting a regional difference in 3b-HSD activity. Differences in 3b-HSD activity and progesterone concentrations among different brain region suggest that progesterone produced locally by 3b-HSD may contribute to some important events in the corresponding brain regions in birds. Therefore, to understand the physiological role of progesterone in the brain, studies concerning the precise localization of 3b-HSD are essential. According to Guennoun et al. w10x, rat 3b-HSD mRNA is expressed in several brain regions in particular, olfactory bulb, striatum, cortex, thalamus, hypothalamus, habenula, septum, hippocampus, and cerebellum. Although the site showing the 3b-HSD expression is still obscure in birds, regional distribution of 3b-HSD reported in the rat brain may be correlated with that of cytochrome P450scc in the avian brain. Our immunohistochemical studies w38,40x with the quail brain
Fig. 4. Comparison of progesterone concentrations in different brain regions, i.e., cerebrum, diencephalon, mesencephalon and cerebellum in the male quails. The concentrations were measured by a specific RIA using an antibody against progesterone. Each column and the Õertical line represent the mean"S.E.M. Ž ns6.. Significance of difference: )), P - 0.01 Žvs. mesencephalon.; †, P - 0.05; ††, P - 0.01 Žvs. diencephalon. by Kruskal–Wallis test followed by Mann–Whitney’s U-test.
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have shown that clusters of P450scc immunoreactive cells are detected in the hyperstriatum accessorium, ventral portions of the archistriatum and corticoid area, preoptic area, anterior hypothalamus and dorsolateral thalamus. Thus, sites having both steroidogenic enzymes may be able to synthesize progesterone de novo from cholesterol. To draw a firm conclusion in the bird, identification of the cells that express both 3b-HSD and cytochrome P450scc is required. In addition, we need to examine whether other steroidogenic enzymes, such as 17a-hydroxylaserc17, 20-lyase Žcytochrome P450c17., which converts pregnenolone and progesterone to dehydroepiandrosterone and androstenedione, respectively, are expressed in the avian brain. In mammals, progesterone and its metabolites are thought to mediate their actions through ion-gated channel receptors, such as g-aminobutyric acid A and glycine w16,17,20–22,26,29,35x, as well as through classic nuclear steroid receptors. Therefore, further functional studies should contribute to our understanding of the physiological role of neurosteroids including progesterone in the avian brain.
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Acknowledgements
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This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan Ž08454265 and 10874129 to K.T... We are grateful to Dr. Peter J. Sharp ŽRoslin Institute, Edinburgh, UK. and Drs. S. Kominami, T. Yamazaki and S. Takemori ŽHiroshima University, Japan. for their valuable discussions.
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w35x R. Shingai, M.L. Sutherland, E.A. Barnard, Effects of subunit types of the cloned GABA A receptor on the response to a neurosteroid, Eur. J. Pharmacol. 206 Ž1991. 77–80. w36x K. Tsutsui, Pituitary and gonadal hormone-dependent and -independent induction of follicle-stimulating hormone receptors in the developing testis, Endocrinology 128 Ž1991. 477–487. w37x K. Tsutsui, T. Yamazaki, Avian neurosteroids: I. Pregnenolone biosynthesis in the quail brain, Brain Res. 678 Ž1995. 1–9. w38x K. Tsutsui, T. Yamazaki, M. Usui, Y. Furukawa, K. Ukena, C. Kohchi, S. Kominami, P450scc activity in the brain, in: R. Etches, S. Harvey ŽEds.., Perspectives in Avian Endocrinology, J. Endocrinol., Bristol, UK, 1997, pp. 427–436. w39x K. Ukena, M. Usui, C. Kohchi, K. Tsutsui, Cytochrome P450 side-chain cleavage enzyme in the cerebellar Purkinje neuron and its neonatal change in rats, Endocrinology 139 Ž1998. 137–147. w40x M. Usui, T. Yamazaki, S. Kominami, K. Tsutsui, Avian neurosteroids: II. Localization of a cytochrome P450scc-like substance in the quail brain, Brain Res. 678 Ž1995. 10–20. w41x A. Vanson, A.P. Arnold, B.A. Schlinger, 3b-Hydroxysteroid dehydrogenaserisomerase and aromatase activity in primary cultures of developing zebra finch telencephalon: dehydroepiandrosterone as substrate for synthesis of androstenedione and estrogens, Gen. Comp. Endocrinol. 102 Ž1996. 342–350. w42x J. Weidenfeld, R.A. Siegel, I. Chowers, In vitro conversion of pregnenolone to progesterone by discrete brain areas of the male rat, J. Steroid Biochem. 13 Ž1980. 961–963.
Short communication
Expression and activity of 3b-hydroxysteroid dehydrogenaserD5-D4-isomerase in different regions of the avian brain Kazuyoshi Ukena a , Yoko Honda a , Yuto Inai a , Chie Kohchi b , Robert W. Lea c , Kazuyoshi Tsutsui a, ) a
Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima, UniÕersity, Higashi-Hiroshima 739-8521, Japan b Radioisotope Center, Hiroshima UniÕersity, Higashi-Hiroshima 739-8526, Japan c Department of Applied Biology, UniÕersity of Central Lancashire, Preston, PR1 2HE, Lancashire, UK Accepted 24 November 1998
Abstract Recently, we have demonstrated, using biochemical and immunochemical methods, that the quail brain possesses the cholesterol side-chain cleavage enzyme Žcytochrome P450scc. and produces pregnenolone and its sulfate ester. To clarify progesterone biosynthesis in the avian brain, therefore, we examined the expression of messenger RNA ŽmRNA. encoding for the enzyme 3b-hydroxysteroid dehydrogenaserD5-D4-isomerase Ž3b-HSD. and its enzymatic activity using the quail. RT-PCR analysis together with Southern hybridization indicated the expression of 3b-HSD mRNA in the brain of sexually mature birds but with no clear-cut sex difference. Employing biochemical techniques combined with HPLC analysis, the conversion of pregnenolone to progesterone was found in brain slices of mature males. Progesterone biosynthesis was increased in a time dependent manner and completely abolished by trilostane, a specific inhibitor of 3b-HSD. The enzymatic activity of 3b-HSD was greatest in the cerebrum and lowest in the mesencephalon. A specific RIA indicated that progesterone concentrations in the different brain regions closely followed the level of 3b-HSD activity. High levels of progesterone concentration were observed in the diencephalon and cerebrum with lowest values in the mesencephalon. Progesterone levels in the brain regions were significantly higher than those in the plasma. These results suggest that the avian brain possesses not only cytochrome P450scc but also 3b-HSD and produces progesterone. It is also indicated that progesterone biosynthesis in the avian brain may be region-dependent. q 1999 Elsevier Science B.V. All rights reserved. Keywords: 3b-Hydroxysteroid dehydrogenaserD5-D4-isomerase; Gene expression; Enzymatic activity; Progesterone; Quail brain; Reverse transcriptionpolymerase chain reaction
De novo steroidogenesis from cholesterol in the mammalian brain is now well established. For example, pregnenolone and dehydroepiandrosterone, as unconjugated steroids, and their fatty acid or sulfate esters accumulate within the brain in several mammalian species w7,8,12,18,31–33x. The brain content of these steroids is almost constant even after the removal of peripheral steroids. Certain structures in the mammalian brain have the capacity to metabolize cholesterol to pregnenolone w1–3,11,13,28,30x. The cytochrome P450 side-chain cleavage enzyme Ž P450scc. cleaves cholesterol to form pregnenolone, which is a 3b-hydroxy-D5-steroid and a main precursor of steroid hormones secreted by the steroido-
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genic glandular cells Žfor a review, see Ref. w25x.. Recent studies have further indicated that both P450scc protein and its messenger RNA ŽmRNA. are expressed in the rat brain w2,3,6,13,19,23,28,39 x. Considered with these extensive mammalian studies, we have recently demonstrated that the quail brain also possesses cytochrome P450scc and produces pregnenolone and its sulfate ester, using both biochemical and immunochemical techniques w37,38x. In addition, our immunohistochemical studies with this same avian species have shown that P450scc immunoreactive cells are distributed in several telencephalic, diencephalic and mesencephalic regions, such as the hyperstriatum accessorium ŽHA., the ventral portions of the archistriatum ŽA. and the corticoid area ŽC., the preoptic area ŽPOA., the anterior hypothalamus ŽAHy., the dorsolateral thalamus ŽDL. and the cerebellar cortex w38,40x. 3b-Hydroxysteroid dehydrogenaserD5 -D4 -isomerase Ž3b-HSD. is also a key enzyme for steroidogenesis in
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 2 9 6 - 7
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peripheral steroidogenic glands. The enzyme 3b-HSD catalyzes the oxidation and isomerization of 3b-hydroxy-D5steroids, such as pregnenolone and dehydroepiandrosterone. Therefore, the demonstration of the presence of 3b-HSD is essential in order to understand the concept of steroidogenesis in the brain. 3b-HSD mRNA expression andror activity of 3b-HSD have also been observed in the mammalian brain w9,10,13,34,42x. In non-mammalian vertebrates, 3b-HSD activity has recently been found in the brain of both avian w41x and amphibian species w24x. However, the expression of 3b-HSD mRNA and differences in its enzymatic reaction in several brain regions have not been investigated in the non-mammalian brain. In the present study, therefore, we have demonstrated the expression of mRNA encoding for the key steroidogenic enzyme 3b-HSD in the quail brain. 3b-HSD expression was confirmed by biochemical studies combined with HPLC analysis and the measurement of intrabrain progesterone. The second aim of this study was to determine differences in the enzymatic activity of 3b-HSD in different brain regions. Japanese quail Ž Coturnix japonica. was used in these studies. The birds were housed in a temperature-controlled room Ž25 " 28C. under a daily photoperiod of 16-h light and 8-h dark Žlong day; lights on at 0700 h., and given quail food and tap water ad libitum. Sexually mature male and female quails, 3 months of age, were used to examine 3b-HSD mRNA expression. Mature males were used on studies investigating the enzymatic reaction catalyzed by 3b-HSD and for the RIA of progesterone. Birds were isolated in individual cages approved in accordance with the Guide for the Care and Use of Laboratory Animals prepared by Hiroshima University, Japan. To determine the expression of mRNA encoding for 3b-HSD in the brain, RT- PCR analysis was performed using the male and female quails Ž n s 5 in each sex. according to a method described previously w14,39x. All birds were killed between 1000 and 1200 h. Total RNA of the brain was isolated by the guanidium thiocyanate–phenol–chloroform extraction method. Total RNA contains ribosomal RNA and mRNA. Fifteen micrograms of total RNA were reverse transcribed using Oligo dT primer and RT in a 30-ml reaction volume for 2 h at 378C. The reaction mixture contained 15 mg of total RNA, 50 mM Tris–HCl ŽpH 8.3., 75 mM KCl, 3 mM MgCl 2 , 10 mM dithiothreitol, 1 mM deoxynucleoside triphosphate ŽdNTP. mix, 1.5 mg of Oligo dT12 – 18 ŽPharmacia, Uppsala, Sweden., 15 U of ribonuclease inhibitor ŽWako, Osaka, Japan., and 400 U of moloney murine leukemia virus transcriptase ŽPromega, Osaka, Japan.. The reaction was stopped by incubating at 658C for 10 min, and the cDNA ethanol precipitated and redissolved in 15 ml of distilled water. For PCR, an aliquot of the cDNA solution corresponding to 2 mg of initial total RNA was used as a template in a 25-ml reaction mixture. The PCR mixture contained cDNA, 20 mM Tris–HCl ŽpH 8.0., 100 mM KCl, 0.1% Triton X-100,
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1.5 mM MgCl 2 , 0.2 mM dNTP mix, 25 pmol of each primer, and 1 U of Gene Taq polymerase ŽWako, Osaka, Japan.. After denaturation at 958C for 3 min, the mixture was subjected to 30 thermal cycling in a programmed temperature control system ŽPC700; ASTEC, Fukuoka, Japan. as follows: denaturation at 938C for 1 min, primer annealing at 568C for 1 min, and extension at 728C for 1 min. After the thermal cycling, the mixture was additionally incubated at 728C for 10 min. An 8-ml aliquot of each sample was electrophoresed through a 1.5% agarose gel. To confirm the identity of the amplified fragment, the gels were subjected to Southern analysis with a digoxigenin-labeled oligonucleotide probe, corresponding to the internal sequence of the target gene. Digoxigenin DNA labeling and detection were performed according to the manufacturer recommendations ŽBoehringer, Vienna, Austria.. Oligonucleotides used as PCR primer and probe for mRNA detection, were based on cDNA sequences of chicken 3b-HSD w27x and chicken b-actin, and were as follows: 3b-HSD sense primer 5X-TCAACGTGACAGGTACTCAG-3X Žnucleotide number 388–407 in Ref. w27x., 3b-HSD antisense primer 5X-CTCTTTGCCTCTTCCCATGT-3X Žnucleotide number 1125–1144 in Ref. w27x., 3b-HSD probe 5X-TACATCTCAGATGACACTCC-3X Žnucleotide number 855–874 in Ref. w27x., b-actin sense primer 5X-GAGACCTTCAACACCCCAG-3X Žnucleotide number 441–459, gb L08165., and b-actin antisense primer 5X-GACAGAGTACTTGCGCTCAG-3X Žnucleotide number 1066–1085, gb L08165.. The 3b-HSD sense and antisense primers give a 757 bp amplified fragment of chicken 3b-HSD gene. The b-actin primers give a 638 bp amplified fragment. Each RT-PCR analysis was repeated five times using independently extracted RNA samples from different animals. 3b-HSD activity of the quail brain was determined by measuring the conversion of w7 y3 Hx pregnenolone Žspecific activity, 19.9 Cirmmol, New England Nuclear, Boston, MA. to 3 H-progesterone using brain slices of different regions of the mature male. Biochemical analysis in the present study was performed according to a method cited previously w24x. The different brain regions, i.e., cerebrum, diencephalon, mesencephalon and cerebellum, were cut into slices using a razor and preincubated at 408C for 15 min in 1 ml of physiological saline Ž124 mM NaCl, 5 mM KCl, 1.24 mM KH 2 PO4 , 1.3 mM MgSO4 , 2.4 mM CaCl 2 , 26 mM NaHCO 3 , and 10 mM glucose.. Following this, the slices were incubated at 408C for 0, 30 or 60 min in 0.5 ml of physiological saline containing 50 pmol Ž1000,000 cpm. of w7-3 Hxpregnenolone and 4% propylene glycol. The incubation medium was constantly gassed with 95% O 2 and 5% CO 2 . At the end of the incubation period, 2-ml ethyl acetate was added and the slices homogenized with a glass–glass homogenizer. The homogenates were stirred for 15 min and centrifuged at 3000 = g for 5 min. The organic phase was removed and the extraction step repeated twice. The combined organic extracts were dried
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down, re-dissolved in 40% acetonitrile ŽACN., and filtrated through a membrane filter Ž0.45 mm: Ultrafree-MC, Millipore, Bedford, MA.. To detect labeled steroids formed from w7-3 Hxpregnenolone, filtrates were subjected to HPLC analysis using a reversed-phase column, LiCrospher 100 RP-18 Ž4.0 = 250 mm, Kanto Chemical, Tokyo, Japan.. The column was eluted with a 30-min linear gradient of 40–70% ACN at a flow rate of 0.7 mlrmin, followed by an isocratic elution of 70% ACN. The eluate was fractionated every 0.5 min from 28 min to 33 min and counted in a liquid scintillation counter. Reference standards of tritiated pregnenolone and progesterone were chromatographed under similar conditions to the tissue extracts and detected using a liquid scintillation counter. To confirm the involvement of 3b-HSD activity in the formation of the radioactive peak of progesterone, brain slices were incubated with saline containing trilostane ŽMochida, Tokyo, Japan., a specific inhibitor of 3b-HSD, and subjected to HPLC analysis in a similar manner as described earlier. Trilostane Ž2 = 10y2 M; 10 ml. dissolved in acetone, was added to 0.5 ml of the incubation medium and incubated with brain slices for 60 min. These biochemical analyses were repeated independently at least five times with tissue from different animals. To measure progesterone levels in the quail brain, 6 mature males were killed between 1000 and 1200 h and trunk blood collected into heparinized tubes. The blood was centrifuged at 1800 = g for 20 min at 48C and the plasma stored at y808C until assayed for progesterone. Immediately after blood collection, each different brain region, i.e., cerebrum, diencephalon, mesencephalon and cerebellum, was dissected out and weighed and then frozen in liquid nitrogen and stored as a sample at y808C. Extraction of progesterone was performed according to a method described previously w8,37–39x. Each brain sample was homogenized in 5 ml ice-cold phosphate-buffered saline ŽPBS: 10 mM phosphate buffer and 140 mM NaCl, pH 7.3. with a Teflon-glass homogenizer. Plasma Ž200 ml. was diluted with 5 ml cold PBS. Brain and plasma samples were then subjected to steroid extraction. To estimate steroid recovery during extraction, 2000 cpm of w1, 2, 6, 7-3 Hx progesterone Žspecific activity, 115 Cirmmol, New England Nuclear. was added to each sample together with 5-ml ethyl acetate and the tubes stirred for 30 min followed by centrifugation at 3000 = g for 5 min. The organic phase was subsequently removed and the extraction step repeated further twice. The combined organic extracts, which contained progesterone, were dried down and dissolved in 1-ml PBS containing 0.1% gelatin. The aqueous solution was divided into two aliquots: one aliquot for the estimation of recovery, the other for the measurement of progesterone. To measure progesterone concentration, aliquots of organic extracts were assayed in a progesterone RIA w5,8,15,37–39x using an antiserum to progesterone ŽScantibodies Laboratories, Santee, CA. and w1, 2, 6, 7-3 Hx
progesterone. The antiserum used in this assay cross-reacted with deoxycorticosterone at 3.3%, 17a-hydroxyprogesterone at 0.6%, and pregnenolone less than 0.1% and no chromatographic purification was performed. Separation of bound and free steroid was performed by centrifugation after reaction with the IgG SORB ŽThe Enzyme Center, Malden, MA.. The least detectable amount was 0.1 ngrml, and intraassay variation was estimated as less than 7%. The precision index Ž l. of a linear portion of the competition curve computed according to a method described previously w36,37x was 0.037. Results for 3b-HSD activity and progesterone concentration in different brain regions were expressed as the mean " S.E.M. and were analyzed for significance by using Duncan’s multiple range test or the Kruskal–Wallis test followed by Mann–Whitney’s U-test after verification of equality or inequality, respectively, of variances among the groups compared w4x. The expression of mRNA encoding for 3b-HSD in the brains of both male and female quails was examined by RT-PCR analysis. The initial RNA amount used in the RT-PCR was adjusted spectrophotometrically and RT-PCR for b-actin was performed as a control experiment. Gel electrophoresis of the RT-PCR product for the 3b-HSD gene identified a single band of 757 bp corresponding to 3b-HSD mRNA size in both male and female brains ŽFig. 1a.. Serial Southern hybridization confirmed that this band was 3b-HSD mRNA specific ŽFig. 1b.. No clear-cut sex difference in the 3b-HSD mRNA expression was detected in the brain ŽFig. 1.. Although the density of the band corresponding to 3b-HSD mRNA in the brain was less than that in the testis ŽFig. 1., suggesting that 3b-HSD is expressed in the brain of sexually mature quails in both sexes. To demonstrate the enzymatic activity of 3b-HSD in the brain, diencephalic slices of the male quail were incubated with tritiated pregnenolone as a precursor, and radioactive metabolites analyzed by reversed-phase HPLC. As shown in Fig. 2, the radioactive peak was detected approximately 2 min before the elution of the precursor steroid, pregnenolone. Tritiated progesterone used as a reference standard exhibited the same retention time under similar chromatographic conditions. The radioactive peak corresponding to progesterone increased in a time dependent manner from 0 min- to 60 min-incubation ŽFig. 2a, b, c.. In addition, 10y4 M trilostane, a specific inhibitor of 3b-HSD, completely abolished the production of this peak in 60 min-incubation samples ŽFig. 2c, d.. This evidence suggests that the observed radioactive metabolite is progesterone produced by the enzymatic activity of 3b-HSD in the diencephalon. To compare the ability of progesterone biosynthesis among different brain regions, each brain tissue was incubated with tritiated pregnenolone for 60 min. Progesterone formation was calculated from the radioactivity corresponding to progesterone and was expressed as pmolrgrh.
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regions Ž P - 0.01, vs. diencephalon, mesencephalon, cerebellum; Fig. 4.. The data presented in this paper demonstrates the expression of mRNA encoding for 3b-HSD, a key steroidogenic enzyme, in the brain of the Japanese quail in both sexes. To the best of our knowledge, this is the first report showing the expression of 3b-HSD mRNA in the avian brain. Employing biochemical techniques combined with HPLC analysis in the present study, we have observed the conversion of pregnenolone to progesterone in brain slices, suggesting enzymatic activity of 3b-HSD in the quail brain. Considered with this, 3b-HSD activity has previously been reported in primary cultures of the telencephalon in another avian species, i.e., the zebra finch w41x.
Fig. 1. RT-PCR analysis of 3b-HSD mRNA in the brain of the male and female quails. The testis was also used as a positive control. Upper panel Ža. shows a result of the gel electrophoresis of RT-PCR products for chicken 3b-HSD, and middle panel Žb. shows an identification of the band by Southern hybridization using digoxigenin-labeled oligonucleotide probe for chicken 3b-HSD. cDNA corresponding to 2 mg total RNA extracted from the brain or testis was used for a PCR reaction. The lane labeled ‘No cDNA’ was performed without template as the negative control. Lower panel Žc. shows a result of the RT-PCR for chicken bactin as the internal control, in which cDNA corresponding to 0.01 mg total RNA was used as template. RT-PCR experiments were repeated five times using independently extracted RNA samples from different animals and gave similar results.
As shown in Fig. 3, the enzymatic activity of 3b-HSD was detected in all of the different brain regions. However, the ability of progesterone formation was different among the brain regions ŽFig. 3.. 3b-HSD activity was greatest in the cerebrum Ž P - 0.01, vs. mesencephalon, cerebellum; P 0.05, vs. diencephalon. and lowest in the mesencephalon Ž P - 0.01, vs. cerebrum, diencephalon; P - 0.05 vs. cerebellum; Fig. 3.. In this study, progesterone concentrations in different brain regions were further measured by a specific RIA using an antibody against progesterone. Progesterone levels in the brain also showed the region-specific distribution. As shown in Fig. 4, progesterone concentration was significantly higher in the diencephalon than in the cerebellum Ž P - 0.05. and mesencephalon Ž P - 0.01.. Progesterone concentrations in the cerebrum also tended to be higher than those in the cerebellum and mesencephalon, but this difference was not statistically significant due to a large variance ŽFig. 4.. Progesterone concentration was minimal in the mesencephalon Ž P - 0.01, vs. diencephalon, cerebellum; Fig. 4.. In plasma, the concentration of progesterone was markedly lower than in several brain
Fig. 2. Typical HPLC chromatograms of steroids extracted from diencephalic slices after different incubation times w0 min Ža., 30 min Žb. and 60 min Žc.x with 3 H-pregnenolone using a reversed-phase column. The column was eluted with a 30-min linear gradient of 40–70% ACN, followed by an isocratic elution of 70% ACN. The eluate was fractionated every 0.5 min from 28 min to 33 min Ž0.35 ml each.. The ordinate indicates the radioactivity measured in each HPLC fraction. The arrows indicate the elution position of standard steroids, i.e., pregnenolone and progesterone. The diencephalic slices were also incubated with trilostane, a specific inhibitor of 3b-HSD, for 60 min and subjected to HPLC analysis Žd..
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Fig. 3. Comparison of 3b-HSD enzymatic activity in different brain regions, i.e., cerebrum, diencephalon, mesencephalon and cerebellum, in the male quails. Each brain region was incubated with 3 H-pregnenolone for 60 min and subjected to HPLC analysis. Progesterone production was calculated from the radioactivity corresponding to progesterone and expressed as pmolrgrh. Each column and the Õertical line represent the mean"S.E.M. Ž ns 5.. Significance of difference: ), P - 0.05; )), P - 0.01 Žvs. mesencephalon.; †, P - 0.05; ††, P - 0.01 Žvs. diencephalon. by Duncan’s multiple range test.
From the present and previous studies, it is probable that 3b-HSD is expressed in the avian brain and is involved in steroid metabolism, such as the conversion of pregnenolone to progesterone. The expression of 3b-HSD andror its enzymatic activity have also been reported in the brain of several mammals w9,10,13,34,42x and an amphibian w24x. Therefore, it may be generally stated that the expression of an enzymatic active form of 3b-HSD is a conserved property of the vertebrate brain. Recently, we have demonstrated that the same avian species also possesses cytochrome P450scc and produces pregnenolone and its sulfate ester from cholesterol, by biochemical and immunochemical approaches w37,38x. Taken together, the results suggest that de novo progesterone biosynthesis from cholesterol occurs in the avian brain. Based on the biochemical analysis, we could detect 3b-HSD activity in all brain regions of the quail used in this study. The most striking observation in the present study was, however, a clear difference in 3b-HSD activity among different brain regions. The enzymatic activity in the cerebrum and diencephalon was much higher than that in the mesencephalon, a regional pattern of 3b-HSD activity which has not yet been reported in birds. On the other hand, it has been demonstrated that the enzymatic activity of 3b-HSD in the rat brain was high in the limbic region and low in the hypothalamus w42x. In addition, we have reported that in the rat the expression of 3b-HSD mRNA
Žisoforms type I plus type II. is comparatively low in the mesencephalon and diencephalon and high in the cerebrum and cerebellum w14x. Accordingly, the expression of 3bHSD and its enzymatic activity in the diencephalon may be different between mammalian and avian species. In this study, we further measured the concentration of progesterone in several brain regions of the quail. Progesterone level was also high in the diencephalon and cerebrum and low in the mesencephalon, although concentration in the cerebrum was not statistically significant due to a large variance. Notwithstanding the large variance in the cerebrum, progesterone levels were in agreement with the result of biochemical analysis, suggesting a regional difference in 3b-HSD activity. Differences in 3b-HSD activity and progesterone concentrations among different brain region suggest that progesterone produced locally by 3b-HSD may contribute to some important events in the corresponding brain regions in birds. Therefore, to understand the physiological role of progesterone in the brain, studies concerning the precise localization of 3b-HSD are essential. According to Guennoun et al. w10x, rat 3b-HSD mRNA is expressed in several brain regions in particular, olfactory bulb, striatum, cortex, thalamus, hypothalamus, habenula, septum, hippocampus, and cerebellum. Although the site showing the 3b-HSD expression is still obscure in birds, regional distribution of 3b-HSD reported in the rat brain may be correlated with that of cytochrome P450scc in the avian brain. Our immunohistochemical studies w38,40x with the quail brain
Fig. 4. Comparison of progesterone concentrations in different brain regions, i.e., cerebrum, diencephalon, mesencephalon and cerebellum in the male quails. The concentrations were measured by a specific RIA using an antibody against progesterone. Each column and the Õertical line represent the mean"S.E.M. Ž ns6.. Significance of difference: )), P - 0.01 Žvs. mesencephalon.; †, P - 0.05; ††, P - 0.01 Žvs. diencephalon. by Kruskal–Wallis test followed by Mann–Whitney’s U-test.
K. Ukena et al.r Brain Research 818 (1999) 536–542
have shown that clusters of P450scc immunoreactive cells are detected in the hyperstriatum accessorium, ventral portions of the archistriatum and corticoid area, preoptic area, anterior hypothalamus and dorsolateral thalamus. Thus, sites having both steroidogenic enzymes may be able to synthesize progesterone de novo from cholesterol. To draw a firm conclusion in the bird, identification of the cells that express both 3b-HSD and cytochrome P450scc is required. In addition, we need to examine whether other steroidogenic enzymes, such as 17a-hydroxylaserc17, 20-lyase Žcytochrome P450c17., which converts pregnenolone and progesterone to dehydroepiandrosterone and androstenedione, respectively, are expressed in the avian brain. In mammals, progesterone and its metabolites are thought to mediate their actions through ion-gated channel receptors, such as g-aminobutyric acid A and glycine w16,17,20–22,26,29,35x, as well as through classic nuclear steroid receptors. Therefore, further functional studies should contribute to our understanding of the physiological role of neurosteroids including progesterone in the avian brain.
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Acknowledgements
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This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan Ž08454265 and 10874129 to K.T... We are grateful to Dr. Peter J. Sharp ŽRoslin Institute, Edinburgh, UK. and Drs. S. Kominami, T. Yamazaki and S. Takemori ŽHiroshima University, Japan. for their valuable discussions.
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