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Endocrinology Vol. 140, No. 2 805-813
Copyright © 1999 by The Endocrine Society

ARTICLES

Expression and Activity of 3ß-Hydroxysteroid Dehydrogenase/{Delta}5-{Delta}4-Isomerase in the Rat Purkinje Neuron during Neonatal Life1

Kazuyoshi Ukena, Chie Kohchi and Kazuyoshi Tsutsui

Laboratory of Brain Science (K.U., K.T.), Faculty of Integrated Arts and Sciences, and Radioisotope Center (C.K.), Hiroshima University, Higashi-Hiroshima 739-8521, Japan

Address all correspondence and requests for reprints to: Dr. Kazuyoshi Tsutsui, Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan. E-mail: tsutsui{at}ipc.hiroshima-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recently, we demonstrated that cytochrome P450 side-chain cleavage enzyme (P450scc) occurs in the rat cerebellar Purkinje cell after differentiation and remains during neonatal development and into adulthood. 3ß-Hydroxysteroid dehydrogenase/{Delta}5-{Delta}4-isomerase (3ßHSD) is also an essential enzyme for progesterone biosynthesis not only in peripheral steroidogenic glands but also in the nervous system. In the present study, therefore, the expression of 3ßHSD in the rat cerebellum was investigated during neonatal development and in the adult. RT-PCR analysis showed that the expression of 3ßHSD messenger RNA (mRNA) in the cerebellum was higher at 7–14 days of age than at other times. Biochemical studies together with HPLC analysis revealed that cerebellar slices at 10 days of age converted pregnenolone to progesterone, suggesting enzymatic activity of 3ßHSD. This conversion was significantly reduced by trilostane, a specific inhibitor of 3ßHSD. A specific RIA indicated that progesterone concentrations in the cerebellum were higher at 3 and 10 days of age than at 60 days of age. The progesterone level in the cerebellum was significantly higher than that in plasma at 10 days of age. In contrast, the concentrations in both cerebellum and plasma at 3 and 60 days of age were similar. In the present study, the site of 3ßHSD mRNA expression in the cerebellum was further examined in neonatal and adult rats using in situ hybridization. The cerebellar expression of 3ßHSD mRNA was obscure at 3 days of age, whereas intense expression occurred in Purkinje cells and external granule cells throughout the cerebellum at 10 days of age. 3ßHSD mRNA was also expressed in Purkinje cells and granule cells at 60 days of age, but a restricted expression was observed along the cerebellar meninges.

These results suggest that the steroidogenic enzyme 3ßHSD as well as P450scc are expressed at least in the cerebellar Purkinje cell. The expression of 3ßHSD, however, may increase for a limited period around 10 days of age, unlike P450scc.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROSTEROIDS such as pregnenolone and dehydroepiandrosterone are synthesized de novo in the nervous system through mechanisms partly independent of peripheral steroidogenic glands, e.g. the adrenal and gonads (for a review, see Ref. 1). A number of studies in several species of mammals and birds have reported that the brain contains abundant quantities of pregnenolone, dehydroepiandrosterone, and their fatty acid or sulfate esters (2, 3, 4, 5, 6, 7, 8, 9, 10, 11). It has also been demonstrated that certain structures in the brain possess the cytochrome P450 side-chain cleavage enzyme (P450scc), which cleaves cholesterol to form pregnenolone (12, 13, 14, 15, 16, 17, 18, 19). In addition, progesterone and its metabolites, such as 3{alpha}-dehydroxyprogesterone and 3{alpha},5{alpha}-tetrahydroxyprogesterone, are produced and accumulate in the nervous system as neurosteroids (20, 21, 22, 23, 24, 25, 26). Many investigators have reported that progesterone and its metabolites act through ion-gated channel receptors, such as {gamma}-aminobutyric acidA and glycine, to modulate interneuronal communication and excitability as well as through nuclear steroid receptors (27, 28, 29, 30, 31, 32, 33, 34, 35, 36). Furthermore, progesterone has been shown to be involved in myelination of the peripheral nervous system (37).

The biosynthesis of progesterone is performed by 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4-isomerase (3ßHSD) (38). 3ßHSD can catalyze the dehydrogenation and isomerization of the {Delta}5-3ß-hydroxysteroids (pregnenolone and dehydroepiandrosterone) into {Delta}4-ketosteroids (progesterone and androstenedione, respectively) and is highly expressed in the classical steroidogenic glands, i.e. testis, ovary, adrenal, and placenta, as well as in peripheral tissues, such as liver and skin (39). Four different isoforms of rat 3ßHSD complementary DNAs (cDNAs) have been characterized tissue specifically (40, 41, 42). In several brain regions, the expression of both 3ßHSD protein and its messenger RNA (mRNA) has been reported (43, 44, 45). 3ßHSD activity has also been investigated in both brain slice and homogenate as well as in cultured glial cells and neurons in several species (20, 21, 22, 40, 46, 47).

On the other hand, we have recently demonstrated that cytochrome P450scc appears in the rat Purkinje cell, a typical cerebellar neuron, immediately after differentiation (48). In addition, molecular and immunohistochemical studies indicate that the expression of cytochrome P450scc persists during neonatal development through to adulthood (48). Our previous studies strongly suggest a constant production of pregnenolone in the Purkinje cell during postnatal life. With these findings as a background, in this present study we examined the expression of 3ßHSD and its enzymatic activity in the cerebellum of neonatal and adult rats using RT-PCR and biochemical analyses. Subsequently, using in situ hybridization of 3ßHSD mRNA, the site of 3ßHSD expression in the cerebellum was localized.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male rats of the Fisher strain maintained in this laboratory were used. They were housed in a temperature-controlled room (25 ± 2 C) under a daily photoperiod of 14 h of light and 10 h of darkness (lights on at 0600 h) and were given food and tap water ad libitum. Males of 0, 3, 7, 10, 14, and 21 days of age and sexually mature (2 months) were used in this study. The experimental protocol was approved in accordance with the Guide for the Care and Use of Laboratory Animals prepared by Hiroshima University (Higashi-Hiroshima, Japan).

RT-PCR analysis of 3ßHSD mRNA
To determine the expression of mRNA encoding for rat 3ßHSD in the cerebellum, RT-PCR analysis was performed using rats during neonatal development and adults. In this experiment, 24 male rats at 0, 3, 7, 14, 21, and 60 days of age (n = 4 of each age) were killed between 1000–1200 h. Total RNA (including ribosomal RNA and mRNA) from the cerebellum of each rat was isolated by the guanidinium thiocyanate-phenol-chloroform extraction method (49). The average amount of total RNA extracted from one cerebellum was 69 µg on day 0, 97 µg on day 3, 137 µg on day 7, 336 µg on day 14, 349 µg on day 21, and 244 µg on day 60 of age. Thirty micrograms of total RNA were reverse transcribed using oligo(deoxythymidine) primer and RT in a 60-µl reaction volume for 1.5 h at 37 C. The reaction mixture was composed of 30 µg total RNA, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM deoxynucleoside triphosphate mix, 1.5 µg oligo(deoxythymidine)12–18 (Pharmacia, Uppsala, Sweden), 15 U ribonuclease inhibitor (Wako, Osaka, Japan), and 400 U Moloney murine leukemia virus transcriptase (Life Technologies, Burlington, Canada). After the reaction was stopped by incubation at 67 C for 10 min, the cDNA was ethanol precipitated and redissolved in 30 µl distilled water. For PCR, an aliquot of the cDNA solution corresponding to 5 µg of the initial total RNA was used as a template in a 25-µl reaction mixture. The PCR mixture contained cDNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphate mix, 0.5 µM of each primer, and 1 U recombinant Taq DNA polymerase (Toyobo, Osaka, Japan). After denaturation at 95 C for 3 min, the mixture was subjected to 30 thermal cycling in a programmed temperature control system (PC700, ASTEC, Fukuoka, Japan) as follows: denaturation at 93 C for 1 min, primer annealing at 60 C for 1 min, and extension at 72 C for 1 min. After the thermal cycling, the mixture was additionally incubated at 72 C for 10 min. A 10-µl aliquot of each sample was electrophoresed through a 1.8% agarose gel.

To confirm the identity of the amplified fragment, the gels were applied 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’s recommendations (Boehringer Mannheim, Vienna, Austria). Oligonucleotides used as PCR primer and probe for mRNA detection, based on nucleotide sequences of rat 3ßHSD (40) and rat ß-actin (50), were as follows: 3ßHSD sense primer, 5'-GCCCATGTACATTTATGGGG-3' (nucleotides 729–748 of type I in Ref. 40); 3ßHSD antisense primer, 5'-CCCTTTCTGTCACTGAGACT-3' (nucleotides 1302–1283 of type I in Ref. 40); 3ßHSD probe, 5'-TTTTCTGCTTGGCTTCCTCC-3' (nucleotides 1228–1209 of type I in Ref. 40); ß-actin sense primer, 5'-GAGACCTTCAACACCCCAGC-3' (nucleotides 2167–2186 in Ref. 50); and ß-actin antisense primer, 5'-CACAGAGTACTTGCGCTCAG-3' (nucleotides 3023–3004 in Ref. 50). It has been previously reported that rat 3ßHSD has four different isoforms (types I–IV) (40, 41, 42). In this study, the 3ßHSD sense and antisense primers, which are same and complementary to a common sequence of type I and type II, give a 574-bp amplified fragment of the 3ßHSD (isoform type I plus type II) gene. The ß-actin primers give a 645-bp amplified fragment located in exons 3–6. RT-PCR analyses were repeated at least four times with independently extracted RNA samples from different animals.

Biosynthesis of progesterone from pregnenolone
To examine 3ßHSD activity in the cerebellum, biochemical analysis was performed using cerebellar slices from 10-day-old rats. RT-PCR analysis demonstrated that the cerebellum during this particular neonatal period contains the highest expression of 3ßHSD mRNA. The biochemical analysis in this present study was conducted according to a method cited previously (46). Eight 10-day-old male rats were killed between 1000–1200 h, and half (37.2 ± 4.4 mg) of each cerebellum was cut into slices using a razor and preincubated at 37 C for 15 min in 1 ml physiological saline (124 mM NaCl, 5 mM KCl, 1.24 mM KH2PO4, 1.3 mM MgSO4, 2.4 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose). After this, the cerebellar slices were incubated at 37 C for 5 or 15 min in 0.5 ml physiological saline containing 1,000,000 cpm [7-3H]pregnenolone (SA, 19.9 Ci/mmol; New England Nuclear, Boston, MA) and 4% propylene glycol. The incubation medium was constantly gassed with 95% O2 and 5% CO2. At the end of the incubation period, 2 ml ethyl acetate were added, and the slices were homogenized with a glass-glass homogenizer. After stirring the homogenate for 15 min followed by centrifugation at 3,000 x g for 5 min, the organic phase was removed. This extraction step was repeated twice. The combined organic extracts were dried down, redissolved in 70% acetonitrile (ACN), and filtrated through a membrane filter (0.45 µm pore size; Ultrafree-MC, Millipore Corp., Bedford, MA). To detect labeled steroids formed from [7-3H]pregnenolone, filtrates were subjected to HPLC analysis using a reverse phase column, LiCrospher 100 RP-18 (4.0 x 250 mm; Kanto Chemical Co., Inc., Tokyo, Japan). The column was eluted with an isocratic elution of 70% ACN at a flow rate of 0.7 ml/min. The eluate was fractionated every 0.5 min from 10–20 min and counted in a liquid scintillation counter. Reference standards of tritiated pregnenolone and progesterone were chromatographed under conditions similar to those used for the tissue extracts and were detected using a liquid scintillation counter.

To confirm the involvement of 3ßHSD activity in the formation of the radioactive peak of progesterone, cerebellar slices were incubated with saline containing trilostane (Mochida, Tokyo, Japan), a specific inhibitor of 3ßHSD, and subjected to HPLC analysis in a manner similar to that described above. Trilostane (2 x 10-2 M; 10 µl) dissolved in acetone was added to 0.5 ml incubation medium and incubated with cerebellar slices for 15 min. These analyses were repeated independently four times.

To compare the abilities of progesterone biosynthesis in the cerebellum at different ages, the conversion of [3H]pregnenolone to progesterone was further analyzed using cerebellar slices at 3, 10, and 60 days of age, following the same procedure of previous experiments. To adjust the cerebellar weight for the reaction, cerebella at the different ages were pooled as follows; eight cerebella in a reaction at 3 days, two cerebella at 10 days, and half of a cerebellum at 60 days. The cerebellar slices adjusted to 130.0 mg at each age were incubated with [3H]pregnenolone for 15 min. This analysis was also repeated independently four times.

RIA of progesterone
To measure progesterone levels in the cerebellum and plasma during neonatal development and adulthood, 28 male rats of several different ages were killed (n = 16 at 3 days, n = 8 at 10 days, and n = 4 at 60 days). The time lapse between the beginning and the end of the killing did not exceed 2 h, which was always performed between 1000–1200 h. Trunk blood was collected into heparinized tubes and centrifuged at 1800 x g for 20 min at 4 C. Plasma was stored at -80 C until assayed for progesterone. To secure a sufficient volume of plasma for assay from the younger rats, plasma from two to four animals was pooled to form a single sample. Immediately after blood collection, cerebella were removed and weighed. Subsequently, cerebella were pooled from 3-day-old and 10-day-old rats (two to four animals in each pool). Cerebella were frozen in liquid nitrogen and stored at -80 C. There were four plasma and cerebellar samples for each age group.

Extraction of progesterone was performed according to a method described previously (3, 10, 11, 48). Cerebella were homogenized in 5 ml ice-cold PBS (10 mM phosphate buffer and 140 mM NaCl, pH 7.3) with a Teflon-glass homogenizer. Plasma (200 µl) was diluted with 5 ml cold PBS. Cerebellar and plasma samples were then subjected to steroid extraction. To estimate steroid recovery during extraction, 2000 cpm [1,2,6,7-3H]progesterone (SA, 115 Ci/mmol; New England Nuclear) were added to each sample with 5 ml ethyl acetate. The tubes were stirred for 30 min and centrifuged at 3000 x g for 5 min. The organic phase was then removed, and this extraction step was repeated 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 for the estimation of recovery and the other for the measurement of progesterone.

To measure the progesterone concentration, aliquots of organic extracts were assayed in a progesterone RIA (3, 10, 11, 48, 51, 52) using an antiserum to progesterone (Scantibodies Laboratories, Inc., Santee, CA) and [1,2,6,7-3H]progesterone. The antiserum used in this assay cross-reacted with deoxycorticosterone at 3.3%, with 17{alpha}-hydroxyprogesterone at 0.6%, and with pregnenolone at less than 0.1%, and no chromatographic purification of progesterone was performed. Separation of bound and free steroid was performed by centrifugation after reaction with the IgG SORB (The Enzyme Center, Inc., Malden, MA). The least detectable amount was 0.1 ng/ml, and intraassay variation was estimated as less than 7%. The precision index ({lambda}) of a linear portion of the competition curve, computed according to a method described previously (10, 53), was 0.037.

The results of the RIA were expressed as the mean ± SEM. Statistical comparisons of changes in the steroid concentration between different developmental stages were made using Student’s t test.

In situ hybridization of 3ßHSD mRNA
In the present study, the site of 3ßHSD expression in the cerebellum was localized by in situ hybridization. Twelve male rats, 3, 10, and 60 days of age (n = 4 each group), were deeply anesthetized with chloroform before transcardial perfusion with PBS, followed by fixative solution (4% paraformaldehyde in PBS). After dissection from the skull, brains were postfixed for 24 h in a similar fixative solution at 4 C, followed by conversion in cooled sucrose solution (30% sucrose in PBS) until they sank. Six-micron sagittal sections of the cerebella were made using a cryostat at -18 C and were placed onto 3-aminopropyltriethoxysilane-coated slides. Adrenal gland sections served as a positive control for 3ßHSD mRNA expression and were processed under similar conditions as the cerebellar sections.

In situ hybridization was carried out in a manner similar to that described previously (54, 55). In brief, the fixed sections were rehydrated with PBS and treated with 0.2 N HCl for 20 min, followed by 1 µg/ml proteinase K at 37 C for 10 min. After postfixation with 4% paraformaldehyde in PBS for 5 min, the slides were kept in 40% deionized formamide in 4 x SSC (1 x SSC = 150 mM NaCl and 15 mM sodium citrate, pH 7.0) for 30 min. Hybridization was carried out at 37 C for 15–17 h with 50 ng/ml digoxigenin-oligonucleotide probe mixture dissolved in the hybridization medium containing 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.6 M NaCl, 10% dextran sulfate, 1 x Denhardt’s solution, 250 µg/ml yeast transfer RNA, 125 µg/ml salmon sperm DNA, and 40% deionized formamide. After washing six times with 50% formamide-2 x SSC at 42 C for 30 min each time, the sections were treated with 1.5% blocking reagent (Boehringer Mannheim) in PBS and incubated with alkaline phosphatase-labeled sheep antidigoxigenin antibody (1:1000 dilution in the blocking solution; Boehringer Mannheim) for 1 h. After this, the sections were washed four times with 0.075% Brij 35 in PBS for 15 min each time. Immunoreactive products were detected by immersing the sections for 48 h in a substrate solution (0.035% nitro blue tetrazolium and 0.018% 5-bromo-4-chloro-3-indolyl phosphate in 100 mM Tris-HCl, 100 mM NaCl, and 50 mM MgCl2, pH 9.5), and the expression of 3ßHSD mRNA was observed using an Olympus Corp. BH-2 microscope (Melville, NY).

Oligonucleotides used as the 3ßHSD antisense probe mixture were as follows: 5'-TCCAGCAGGAAGGCAAGCCAGTAGAGCAGGGGCA-GAGGAAGGCTCC-3' [nucleotides 1022–1067 of 3ßHSD type I in Ref. 40 , which are complementary to a common sequence of four different isoforms (types I–IV) of 3ßHSD] and 5'-TGTCTCCCTGTGCTGCTCCACTAGTGTCCCGATCCACTCCGAGGT-3' [nucleotides 1228–1272 of 3ßHSD type I in Ref. 40 , which are complementary to a common sequence of four different isoforms (types I–IV) of 3ßHSD except for one base differing from type III]. Digoxigenin 3'-end labelings were performed according to the manufacturer’s instructions (Boehringer Mannheim).

Control for specificity of the in situ hybridization of 3ßHSD mRNA was performed by the addition of an excess amount (4000-fold) of homologous or nonhomologous unlabeled oligonucleotides to the hybridization medium applied to the sections. ß-Actin antisense primer used in the RT-PCR analysis was employed as a nonhomologous competitor.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neonatal change in 3ßHSD mRNA expression in cerebellum
In the present study, the expression of mRNA encoding for 3ßHSD (isoform type I plus type II) in the rat cerebellum was first examined during neonatal development and in the adult by RT-PCR analysis. Five micrograms of total RNA were extracted from the cerebellum of male rats of 0, 3, 7, 14, 21, and 60 days of age. The ovary of the adult female rat was used as a positive control tissue, and the amount of cDNA used in the RT-PCR was reduced to 0.25 µg. The initial amount of RNA used in the RT-PCR was adjusted spectrophotometrically. RT-PCR for ß-actin was performed as a control experiment (Fig. 1cGo). Gel electrophoresis of the RT-PCR product for the 3ßHSD (isoform type I plus type II) gene identified a single band of 574 bp corresponding to 3ßHSD mRNA size, but not 3ßHSD genomic DNA size in the cerebellum (Fig. 1aGo). Interestingly, cerebellar expression was clearly greater at 7 and 14 days of age than at other times, suggesting a clear age-dependent change in 3ßHSD mRNA (Fig. 1aGo). Serial Southern hybridization confirmed that this band was 3ßHSD mRNA specific (Fig. 1bGo). The level of expression in the cerebellum at each age was less than that expressed in the ovary (Fig. 1Go).



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  		Figure 1. RT-PCR analysis of 3ßHSD mRNA in the rat cerebellum at 0, 3, 7, 14, 21, and 60 days of age. The upper panel (a) shows a result of gel electrophoresis of RT-PCR products for rat 3ßHSD, and the middle panel (b) shows an identification of the band by Southern hybridization using digoxigenin-labeled oligonucleotide probe for rat 3ßHSD. cDNA corresponding to 5 µg total RNA extracted from each cerebellar tissue was used for a PCR reaction, and the 2/5 PCR product was applied on one lane. The ovary was used as a positive control tissue, and the amount of cDNA used in the RT-PCR was reduced to 0.25 µg. The lane labeled No cDNA was performed without template as the negative control. The lower panel (c) shows a result of the RT-PCR for ß-actin as the internal control, in which PCR reaction, cDNA corresponding to 0.25 µg total RNA was used as a template. RT-PCR experiments were repeated four times using independently extracted RNA samples from different animals and produced similar results.

 
Enzymatic activity of 3ßHSD in neonatal cerebellum
Subsequently, enzymatic activity of 3ßHSD, which transforms pregnenolone to progesterone, was investigated in the cerebellum during neonatal life. The cerebellum from 10-day-old rats was first chosen because the RT-PCR analysis indicated that the greatest expression of 3ßHSD mRNA occurred in the cerebellum between 7 and 14 days. Cerebellar slices were incubated with tritiated pregnenolone as a precursor, and the radioactive metabolites were analyzed by reverse phase HPLC. As shown in Fig. 2Go, the radioactive peak was detected approximately 2 min before elution of the precursor steroid, pregnenolone. Tritiated progesterone, used as a reference standard, exhibited the same retention time under similar chromatographic conditions, and its elution position is shown as an open arrow in Fig. 2Go. The radioactive peak corresponding to progesterone increased in a time-dependent manner from 5 to 15 min of incubation (Fig. 2Go, a, b, and c). In addition, 10-4 M trilostane, a specific inhibitor of 3ßHSD, significantly reduced the production of this peak in 15-min incubation samples (P < 0.05; Fig. 2Go, c and d). The total radioactivity of combined fractions of this peak was also decreased by trilostane treatment (Fig. 3Go). This evidence suggests that the observed radioactive metabolite is progesterone produced by the enzymatic activity of 3ßHSD in the neonatal cerebellum.



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  		Figure 2. HPLC analysis of steroids extracted from cerebellar slices at 10 days of age after different incubation times [0 min (a), 5 min (b), and 15 min (c)] with tritiated pregnenolone using a reverse phase column. The column was eluted with an isocratic elution of 70% ACN. The eluate was fractionated every 0.5 min from 10–20 min (0.35 ml each). The ordinate indicates radioactivity measured in each HPLC fraction. The open and shaded arrows indicate the elution positions of standard steroids, i.e. progesterone and pregnenolone, respectively, under similar chromatographic conditions. The cerebellar slices were also incubated with 10-4 M trilostane, a specific inhibitor of 3ßHSD, for 15 min and subjected to HPLC analysis (d). HPLC experiments were repeated four times using different animals and produced similar results.

 


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  		Figure 3. Inhibition of the radioactive peak corresponding to progesterone by 10-4 M trilostane in the cerebellar slices at 10 days of age. Each column and the vertical line represent the mean ± SEM radioactivity (n = 4 samples). *, P < 0.05.

 
We then compared the abilities of progesterone biosynthesis in the cerebellum at 3, 10, and 60 days of age using a similar biochemical analysis. As shown in Fig. 4Go, the enzymatic activity of 3ßHSD was detected at all of the observed ages. However, the ability of progesterone formation was very low at 3 and 60 days, unlike that at 10 days, suggesting age-dependent 3ßHSD activity (Fig. 4Go).



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  		Figure 4. Comparison of 3ßHSD enzymatic activity among cerebella at 3, 10, and 60 days of age. Cerebellar slices adjusted to the same weight were incubated with tritiated pregnenolone for 15 min and subjected to HPLC analysis. Each column and the vertical line represent the mean ± SEM radioactivity corresponding to progesterone (n = 4 samples). *, P < 0.05 vs. 3 and 60 days.

 
Change in progesterone in neonatal and adult cerebella
In the present study, progesterone was further measured in the cerebellum of neonatal and adult rats using a specific progesterone RIA. The progesterone concentration in the cerebellum at 3 and 10 days of age was much greater than that at 60 days (P < 0.05; Fig. 5Go). At 10 days of age, the plasma progesterone concentration was significantly lower than that in the cerebellum (P < 0.05; Fig. 5Go). At other ages, the examined concentrations were not significantly different between the cerebellum and plasma (Fig. 5Go).



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  		Figure 5. Changes in progesterone concentrations in the cerebellum and plasma at 3, 10, and 60 days of age. Each column and the vertical line represent the mean ± SEM (n = 4 samples). *, P < 0.05 vs. 60 days); {dagger}, P < 0.05 vs. cerebellum.

 
Cellular localization of 3ßHSD mRNA in neonatal and adult cerebella
In the final experiment, in situ hybridization of 3ßHSD mRNA was examined in the cerebellum of rats at different ages using oligonucleotides with sequences complementary to rat 3ßHSD mRNA. Expression was finally detected by enzyme immunohistochemistry, and rat adrenal gland was used as a positive control tissue. Digoxigenin-labeled oligonucleotide probes for 3ßHSD mRNA gave an intense reaction in the cortical region of the adrenal gland, but not in the medulla (Fig. 6aGo). This staining was completely abolished by the addition of excess homologous unlabeled probes to the hybridization medium (Fig. 6bGo). However, an excess amount of nonhomologous probe failed to reduce the reaction (Fig. 6cGo), suggesting that the reactions with the oligonucleotide probes used in this study were specific for 3ßHSD mRNA.



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  		Figure 6. In situ hybridization using digoxigenin-labeled oligonucleotide probes for 3ßHSD mRNA in the adrenal gland of the adult male rat (a). The probes with an excess amount of homologous (b) or nonhomologous (c) unlabeled oligonucleotides were employed to examine the specificity of 3ßHSD mRNA signals. C, Cortex; M, medulla. Bars, 100 µm. In situ hybridization was repeated independently four times using different animals and produced similar results.

 
3ßHSD mRNA staining in 3-day-old cerebella was not clear, as the reaction was almost similar to that observed in the competition study (Fig. 7Go, a and b). However, an intense expression of 3ßHSD mRNA was detected in the cerebellum at 10 days of age (Fig. 8Go, a, b, and c). 3ßHSD mRNA was highly expressed in Purkinje cells and external granule cells in the cerebellar cortex (Fig. 8Go, a, b, and c). The localization of these reactive cells was confirmed by Nissl staining (Fig. 8Go, d and e). The competition study using an excess amount of homologous unlabeled probes resulted in a complete absence of 3ßHSD mRNA expression in Purkinje cells and external granule cells at 10 days of age (Fig. 8fGo). The expression of 3ßHSD mRNA was also detected in Purkinje cells and granule cells localized in the restricted cerebellar region at 60 days of age (Fig. 9Go, a and b); this was also abolished by the addition of an excess amount of homologous unlabeled probes (Fig. 9dGo). Although no clear-cut difference in the intensity of the 3ßHSD mRNA expression was detected between 10 days (Fig. 8Go, a, b, and c) and 60 days (Fig. 9Go, a and b), the stained Purkinje and granule cells at 60 days (Fig. 9aGo) demonstrated a more restricted localization, i.e. near cerebellar meninges, unlike at 10 days when they were distributed throughout the cerebellum (Fig. 8aGo).



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  		Figure 7. In situ hybridization using digoxigenin-labeled oligonucleotide probes for 3ßHSD mRNA (a) or the probes with an excess amount of homologous unlabeled oligonucleotides (b) in the cerebellar cortex of neonatal rats at 3 days of age. Histology of the cerebellar cortex was shown by Nissl staining (c). EG, External granule cells; M, molecular layer; P, Purkinje cell layer; G, granule cells in the cerebellar cortex. Bars, 100 µm. A similar result was obtained in repeated experiments using four different animals.

 


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  		Figure 8. In situ hybridization using digoxigenin-labeled oligonucleotide probes for 3ßHSD mRNA (a–c) or the probes with an excess amount of homologous unlabeled oligonucleotides (f) in the cerebellar cortex of neonatal rats at 10 days of age. Histology of the cerebellum was shown by Nissl staining (d and e). a and d are of the same low magnification, and b, c, e, and f are of high magnification. The arrowheads show Purkinje cells expressing 3ßHSD mRNA. EG, External granule cells; M, molecular layer; P, Purkinje cell layer; G, granule cells in the cerebellar cortex. Bars, 100 µm. A similar result was obtained in repeated experiments using four different animals.

 


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  		Figure 9. In situ hybridization using digoxigenin-labeled oligonucleotide probes for 3ßHSD mRNA (a and b) or the probes with an excess amount of homologous unlabeled oligonucleotides (d) in the cerebellar cortex of adult rats at 60 days of age. Histology of the cerebellum was shown by Nissl staining (c). a and c are of the same low magnification, and b and d are of high magnification. The arrowheads show Purkinje cells expressing 3ßHSD mRNA. M, Molecular layer; P, Purkinje cell layer; G, granule cells in the cerebellar cortex. Bars, 100 µm. A similar result was obtained in repeated experiments using four different animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have recently demonstrated that both the expression of the steroidogenic enzyme cytochrome P450scc and pregnenolone biosynthesis remain constant in the rat cerebellum during neonatal development and into adulthood (48). In addition, we have further demonstrated the presence of P450scc in the Purkinje cell, an important cerebellar neuron, in the rat (48) as well as in an avian species, the Japanese quail (11, 19). Therefore, this present study sought to examine changes in the expression and activity of 3ßHSD, another key steroidogenic enzyme, in the cerebellum during rat postnatal life and to clarify its cellular localization in the cerebellum.

RT-PCR analysis using common primers to isoforms type I and type II followed by Southern hybridization revealed the expression of mRNA encoding for 3ßHSD in the cerebellum, with a marked increase during the neonatal period, i.e. 7–14 days of age. From previous studies with the rat, four different region-specific isoforms of 3ßHSD cDNA have been characterized in the rat (40, 41, 42). The homology of type I between types II, III, and IV is 94%, 80%, and 91% at the amino acid level, respectively. Guennoun et al. (44) examined the expression of 3ßHSD mRNA by RT-PCR using common primers to all isoforms of 3ßHSD and showed a higher expression of 3ßHSD, which was very closely related to type I by sequencing of the RT-PCR product, in the rat cerebellum. However, there are no reports of expression of 3ßHSD in the rat during neonatal development. Thus, to our knowledge, this is the first observation of an age-dependent expression of 3ßHSD mRNA.

The expression of 3ßHSD in the cerebellum was confirmed by both biochemical and HPLC analysis, indicating progesterone synthesis in the cerebellum at the neonatal period (10 days of age). Although enzymatic activity of 3ßHSD in the brain has been previously investigated using homogenates, slices, and cultured glial and neuronal cells (20, 21, 22, 40, 46, 47), no report exists for the cerebellum. In this study, cerebellar slices at 10 days of age, when 3ßHSD mRNA was highly expressed, were able to produce progesterone from [3H]pregnenolone in a time-dependent manner. This conversion is considered to be 3ßHSD specific, because trilostane, a specific inhibitor of 3ßHSD, significantly reduced the progesterone level. In addition, 3ßHSD activity may be age dependent in the cerebellum, because the ability of progesterone formation was low at 3 and 60 days, unlike that at 10 days.

Taken together, these molecular and biochemical findings suggest that intracerebellar progesterone levels neonatally may be greater than those in adulthood. Subsequently, therefore, we measured progesterone concentrations in the cerebellum as well as plasma of neonatal and adult rats using a specific progesterone RIA. Progesterone concentrations in the cerebellum at both 3 and 10 days of age were much greater than those at 60 days of age. A significant difference in the progesterone level between the cerebellum and plasma at 10 days may reflect the expression of 3ßHSD in the cerebellum. However, progesterone concentrations in the cerebellum and plasma were similar at 3 days of age. A higher progesterone level in the cerebellum at 3 days might be due to the accumulation of peripheral progesterone, as the expression of 3ßHSD mRNA was negligible. On the other hand, we have previously reported changes in the pregnenolone concentration of the cerebellum during neonatal development and in adulthood (48). The profile of intracerebellar change in the concentration of pregnenolone after 3 days of age, when the differentiation of the first Purkinje cells was complete, was similar to that observed at other neonatal stages and in adulthood (48). In addition, the change in the intracerebellar pregnenolone level was correlated with a constant expression of P450scc in the cerebellum (48). Taken together, the results suggest that changes in pregnenolone and progesterone are quite different during neonatal development.

To identify the cellular localization of 3ßHSD mRNA in the cerebellum, in situ hybridization was performed both during development and in adults. Preliminary observations using the adrenal gland support the validity of the in situ hybridization technique, because the staining of 3ßHSD mRNA was reduced by the addition of an excess mixture of homologous unlabeled probes to the hybridization medium, but nonhomologous sequences failed. The cerebellar expression of 3ßHSD mRNA was low at 3 days of age. However, the expression at 10 days of age was intensely detected in both Purkinje cells and external granule cells throughout the cerebellum. These results are consistent with a higher expression of 3ßHSD at the this age obtained previously by RT-PCR analysis. In contrast, mRNA staining at 60 days of age was restricted only to Purkinje cells and granule cells near the meninges of the cerebellum. This limited expression in the cerebellum may be the reason why RT-PCR analysis revealed a lower expression of 3ßHSD mRNA in adult rats. Although there is as yet no report of restricted expression of 3ßHSD mRNA in the cerebellum, the present results obtained for adult rats are partly in agreement with the finding obtained by Guennoun et al. (44), using common oligonucleotide probes to all isoforms, of 3ßHSD mRNA expression in Purkinje cells, granule cells, and some stellate/basket cells. On the other hand, Sanne and Krueger (45) reported, using complementary RNA probes, that in situ localization of 3ßHSD mRNA occurs in the white matter of the adult rat cerebellum. Dupont et al. (43) demonstrated, using cDNA probes, that 3ßHSD mRNA is localized in a restricted area of the medulla ventrally and laterally bordering the fourth ventricle in the rat brain. The reason for such a discrepancy between the findings of this study and that of Guennoun et al. (44) compared with those of Sanne and Krueger (45) and Dupont et al. (43) is unclear. Therefore, to draw a firm conclusion concerning the localization of 3ßHSD in the cerebellum, further molecular experiments together with immunohistochemistry using an antiserum against 3ßHSD protein are needed.

In this study, we have demonstrated that the expression of 3ßHSD and its enzymatic activity increase during neonatal life. It is likely that Purkinje cells may produce not only pregnenolone but also progesterone during the neonatal period. It is well known that drastic morphological changes in the rat cerebellum after birth occur until approximately 21 days of age (56, 57). At 3 days of age, Purkinje cells completely differentiate and are located in a narrow zone between the molecular and granular layers (56, 57). The external granular layer mainly develops at about 10 days of age, followed by a migration of external granule cells into the granular layer through the Purkinje cells, and the external granular layer disappears (56, 57). The formation of the cerebellar cortex is thus almost complete after about 21 days of age (56, 57). Thus, postnatal development in the cerebellum is dramatic during neonatal life, showing a higher expression of 3ßHSD. Therefore, it may be possible that progesterone and/or its metabolites are involved in the formation of the cerebellar neuronal circuit that occurs during neonatal life through promoting neuronal and glial growth and neuronal synaptic contact. It has been demonstrated that progesterone promotes the myelination in the peripheral nervous system (37). In addition, 3{alpha},5{alpha}-tetrahydroxyprogesterone may regulate nerve growth in rat cultured neurons (58). These findings might support the hypothesis postulated here. On the other hand, the limited expression of 3ßHSD mRNA in the Purkinje and granule cells in the adult might be related to the extracellular environment, because a drastic morphological change occurs only during neonatal life, and the cerebellar cortex is completed thereafter. It has been demonstrated that the enzymatic activity of 3ßHSD in cultured cells derived from the nervous system is different in various extracellular environments, such as cell population and coculture of glial cells with neurons (22, 37, 59). Further studies are required to draw a firm conclusion.


   Acknowledgments
 
We are grateful to Drs. S. Kominami, T. Yamazaki, and S. Takemori (Hiroshima University, Higashi-Hiroshima, Japan) for their valuable discussions. We thank Dr. Robert W. Lea (University of Central Lancashire, Preston, UK) for his valuable discussion and reading of the manuscript.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (08454265 and 10874129 to K.T.). Back

Received May 7, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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