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Laboratory of Brain Science (K.U., M.U., K.T.), Faculty of Integrated Arts and Sciences, and Radioisotope Center (C.K.), Hiroshima University, Higashi-Hiroshima 739, Japan
Address all correspondence and requests for reprints to: Kazuyoshi Tsutsui, Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739, Japan. E-mail: tsutsui{at}ue.ipc.hiroshima-u.ac.jp
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These results suggest that steroidogenic enzyme P450scc appears in the rat Purkinje cell immediately after its differentiation. The expression of this enzyme may remain during neonatal development and in adulthood.
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However, new findings have been obtained that pregnenolone and
dehydroepiandrosterone, as unconjugated steroids, and their fatty acid
or sulfate esters accumulate within the brain in several mammalian
species (3, 4, 5, 6, 7, 8, 9, 10) and an avian species (11, 12, 13). The brain content of
these steroids is almost constant even after the removal of peripheral
steroids, e.g. adrenalectomy, castration, and
hypophysectomy, suggesting that the brain can synthesize steroids
de novo (3, 4, 5, 6, 8, 9, 11, 13). Such steroids synthesized in
the brain are called neurosteroids (14). Indeed, it has been
demonstrated that certain structures in the mammalian and avian brain
have the capacity to metabolize cholesterol to pregnenolone (11, 13, 15, 16, 17, 18, 19, 20, 21). The cytochrome P450scc side-chain cleavage enzyme (P450scc)
cleaves cholesterol to form pregnenolone (for a review, see Ref.22).
Recent studies further indicated that both P450scc protein and its
messenger RNA (mRNA) are expressed in the rat brain (14, 16, 17, 19, 20, 23, 24). Neurosteroids are thought to mediate their actions through
ion-gated channel receptors, such as 
-aminobutyric acid A and
N-methyl-D-aspartate (25, 26, 27, 28, 29, 30, 31, 32, 33), rather than
through classic nuclear steroid receptors. Dehydroepiandrosterone
inhibits aggressive behavior of castrated male mice against lactating
female intruders (5, 6, 8, 34), but its activity is probably not
related to the conversion into testosterone and estradiol (34).
In mammals, glial cells are considered to play a major role in neurosteroid formation and metabolism in the brain. P450scc has been found in the white matter throughout the rat brain (14). It has further been shown that both oligodendrocytes and astrocytes are the primary site for pregnenolone synthesis (15, 16, 17, 18, 19, 20). However, the concept of neurosteroidogenesis in neurons is still unclear in the mammalian brain, although neuronal P450scc expression has been reported in the rat nervous system, such as neurons in the retinal ganglion, sensory neurons in the dorsal root ganglia, and motor neurons in the spinal cord (24, 35). On the other hand, we have recently demonstrated that the avian brain also possesses cytochrome P450scc and produces pregnenolone and its sulfate ester, by biochemical and immunochemical approaches (11). In addition, our immunohistochemical studies with avian brain have shown that an intense immunoreaction with the polyclonal antibody directed against the purified bovine adrenal P450scc is present in soma and dendrites of the Purkinje cell, a typical cerebellar neuron (12, 13).
With these findings as a background, we first investigated the presence of P450scc in the cerebellar Purkinje cell using the mammalian species, i.e. rats. The second purpose of this study was to determine neonatal changes in P450scc located in the Purkinje cell. Finally, diurnal changes in the cerebellar P450scc expression were examined as a possible physiological change.
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Immunohistochemical analysis with P450scc antibody
In the present immunohistochemical experiment, 30 male rats at
various ages (n = 5 at each age) were deeply anesthetized with a
chloroform and then perfused transcardially with PBS [0.1
M phosphate buffer (PB); 0.14 M NaCl, pH 7.3]
followed by fixative solution (4% paraformaldehyde in 0.1
M PB). After dissection from the skull, brains were
postfixed for 24–48 h in the same fixative solution at 4 C and then
soaked in a refrigerated sucrose solution (30% sucrose in 0.1
M PB) until they sank. All cerebella were frozen-sectioned
sagittally at 40 µm thickness on a cryostat at -18 C. Every third
section was grouped in a single batch of ice-cold PBS; thus we obtained
three independent series of adjacent sections. Only one of these series
of sections was used for immunohistochemical staining with cytochrome
P450scc, while the remaining two series were used for control staining
of immunohistochemistry and for Nissl-staining, respectively.
The sections were processed according to the avidin-biotin-peroxidase complex (ABC) immunocytochemical technique with the floating method described previously (12, 36, 37). Endogenous peroxidase activity was eliminated from the sections by incubation with 3% H2O2 in absolute methanol. After blocking nonspecific binding components with 5% normal goat serum and 1% BSA in PBS containing 0.3% Triton X-100, the sections were immersed with the primary antiserum directed against the bovine adrenal cytochrome P450scc at a dilution of 1:1,000 for 36–48 h at 4 C. The anti-P450scc serum was raised in a rabbit (38) using purified cytochrome P450scc from bovine adrenocortical mitochondria (39). The details of the characterization of this serum are given elsewhere (11, 12, 38). To block nonspecific binding components, the anti-P450scc serum was also preincubated with PBS containing 0.5% bovine liver acetone powder (Sigma, St. Louis, MO) and 1% BSA for 12–18 h at 4 C as described previously (12, 36, 37). Several concentrations of the antiserum from 1:1,000 to 1:4,000 were examined, and a solution of 1:1,000 proved most satisfactory (12). The primary immunoreaction was followed by a 60 min-incubation with biotinylated antirabbit IgG (10 µg/ml) (Vector Laboratories, Burlingame, CA) and finally by a 60 min-incubation with avidin-biotin complex (Vectastain ABC Elite kit, Vector Laboratories). Immunoreactive products were detected by immersing the sections for 2–7 min in a diaminobenzidine (DAB) solution (0.05% DAB in PBS containing 0.3% H2O2).
The specificity of the anti-P450scc serum was assessed by a substitution of the control serum for the primary antiserum; in this control serum, the antibody (1:1,000 dilution) was preadsorbed by incubation with the purified antigen in a saturating concentration (10 µg P450scc/ml) for 12–18 h before use. The sections were incubated with this control serum, employing the same procedure for the anti-P450scc serum. The localization of immunoreactive cell bodies and fibers in the rat cerebellum was studied using an Olympus BH-2 microscope.
Identification of cell type of immunoreactive cells
To identify the cell type showing P450scc-like immunoreactivity,
immunohistochemical analyses with three kinds of antibodies were
subsequently performed using five adult males. One of these antibodies
was against P450scc, while the remaining two antibodies were prepared
as reference stainings for deciding the cell type: 1) one was against
inositol triphosphate (IP3) receptors that present
abundantly in Purkinje cells, and 2) the other was against glial
fibrillary acidic protein (GFAP) as a specific marker protein of glial
cells. As the IP3 receptor antibody, a purified IgG
fraction of the monoclonal mouse antibody that cross-reacts with
IP3 receptors (Accurate Chemical & Scientific Co., Ltd.,
Westbury, NY) was used in this study. A purified IgG fraction of the
polyclonal rabbit antibody directed against the purified bovine GFAP
(Dako Co., Ltd., Glostrup, Denmark) was used as the GFAP antibody. It
has been previously confirmed that these two reference antibodies
cross-react with each rat antigen.
Fixation and immunohistochemistry were carried out in the same manner mentioned above. In brief, adjacent serial sections (40 µm thickness) were incubated with the anti-P450scc (1:1,000 dilution), the anti-IP3 receptor (1:50 dilution), and the anti-GFAP (1:100 dilution), respectively. After the incubation, immunoreactive products were detected with the avidin-biotin kit (Vectastain Elite kit, Vector Laboratories) followed by DAB reaction.
Western immunoblot analysis with P450scc antibody
To detect cytochrome P450scc in the rat cerebellum, Western
immunoblot analysis with the antibody against bovine P450scc was
conducted after SDS-PAGE of tissue homogenates. Four adult males were
killed between 1000 and 1200 h. In each animal, several brain
regions including the cerebellum were immediately excised and placed on
ice. The testis was used as a control tissue for Western immunoblot
analysis because it was regarded as a classical steroidogenic organ.
The tissues in each animal were separately homogenized in 4 vol of
ice-cold sample buffer containing 0.05% Nonidet P-40, 50
mM Tris-HCl (pH 7.5), 2 mM EDTA, and 1
mM phenylmethylsulfonylfluoride and centrifuged at
15,000 x g for 20 min. The supernatant was
concentrated by precipitation with 30–50% saturation of ammonium
sulfate. Proteins derived from each tissue were subjected to 10%
SDS-PAGE, and then Western immunoblotting was performed according to
our previous methods (11, 12, 13). In brief, after transfer onto
polyvinylidene fluoride membranes (Immobilon-P, Millipore Co., Bedford,
MA), the blot was probed with the anti-P450scc antibody and followed by
incubation with biotinylated goat antirabbit IgG (Vector Laboratories).
Finally, the membrane was incubated with streptavidin-horseradish
peroxidase complex (Amersham International plc, Little Chalfont,
Buckinghamshire, UK). The protein bands were detected by ECL Western
blotting detection reagents (Amersham International plc). Proteins were
measured by the BCA protein assay kit (Pierce, Rockford, IL) with BSA
as a standard.
RT-PCR analysis of P450scc mRNA
To determine expression of the mRNA encoding for rat P450scc in
the cerebellum, RT-PCR analyses were performed using rats in adulthood
and during neonatal development. In this experiment, 24 male rats at
various ages (n = 4 at each age) were also killed between 1000 and
1200 h. Total RNA of each cerebellum (all observed ages) as well
as other brain regions (only 2 months) was isolated by the guanidinium
thiocyanate-phenol-chloroform extraction method (40). Total RNA
contains ribosomal RNA and mRNA. In our experiments, the average amount
of the total RNA extracted from one cerebellum was 69 µg at 0 day, 97
µg at 3 days, 137 µg at 7 days, 336 µg at 14 days, 349 µg at 21
days, and 244 µg at 60 days. Thirty micrograms of total RNA were
reverse transcribed using Oligo dT primer and RT in a 60-µl reaction
volume for 1.5 h at 37 C. The reaction mixture was composed with
30 µg of total RNA, 50 mM Tris-HCl (pH 8.3), 75
mM KCl, 3 mM MgCl2, 10
mM dithiothreitol, 1 mM deoxynucleoside
triphosphate (dNTP) mix, 1.5 µg of Oligo dT12–18
(Pharmacia, Uppsala, Sweden), 15 U of ribonuclease inhibitor (Wako,
Osaka, Japan), and 400 U of moloney murine leukemia virus transcriptase
(GIBCO BRL, Burlington, Canada). After the reaction was stopped by
incubating at 67 C for 10 min, the cDNA was ethanol precipitated and
redissolved in 30 µl of distilled water. For PCR, an aliquot of the
cDNA solution corresponding to 0.5 µg of initial total RNA was used
as 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, 12.5 pmol of each primer, and 1 U of
rTaq 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 1.5% 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 recommendations of the manufacturer (Boehringer, Vienna, Austria). Oligonucleotides used as PCR primer and probe for mRNA detection, which were based on nucleotide sequences of rat P450scc (41) and rat ß-actin (42), were as follows: P450scc sense primer 5'-TCAAAGCCAGCATCAAGGAG-3' (nucleotide number 1141–1160 in Ref.41), P450scc antisense primer 5'-GCAGCCTGCAATTCATACAG-3' (nucleotide number 1594–1613 in Ref.41), P450scc probe 5'-TTCTCAGGCATCAGGATGAG-3' (nucleotide number 1506–1525 in Ref.41), ß-actin sense primer 5'-GAGACCTTCAACACCCCAGC-3' (nucleotide number 2167–2186 in Ref.42), and ß-actin antisense primer 5'-CACAGAGTACTTGCGCTCAG-3' (nucleotide number 3004–3023 in Ref.42). The P450scc sense and antisense primers give 473 bp amplified fragment located in exon 6 to exon 9 of P450scc gene. The ß-actin primers give 645 bp amplified fragment located in exon 3 to 6. RT-PCR analyses were repeated at least four times using independently extracted RNA samples from different animals.
RIAs of pregnenolone and its sulfate ester
To measure levels of pregnenolone and its sulfate ester in the
cerebellum during neonatal development and in adulthood, 64 male rats
at various ages were killed (n = 16 at 0 and 3 days, n = 12
at 7 days, n = 8 at 14 and 21 days, n = 4 at 2 months). The
time lapse between the beginning and the end of the killing did not
exceed 2 h, and this was always performed between 1000 and
1200 h. Trunk blood was collected into heparinized tubes and
centrifuged at 1,800 x g for 20 min at 4 C. Plasma was
stored at -80 C until assayed for pregnenolone and its sulfate ester.
To secure sufficient volume of plasma for assay in younger rats, plasma
from one to four animals was pooled as a sample. The assays of
pregnenolone and pregnenolone sulfate ester were performed on four
pooled samples at each age. Immediately after the blood collection,
cerebella were taken out and weighed. Then, cerebella from one to four
rats were also pooled as a sample, frozen in liquid nitrogen, and
stored at -80 C. The number of cerebellar samples was also four at
each age.
Extraction of unconjugated steroids or steroid sulfates was performed according to the previous method (4, 11, 13). Cerebella were homogenized in 5 ml ice-cold PBS (pH 7.6) with a Teflon-glass homogenizer. Plasma (100–200 µl) was diluted with 5 ml cold PBS. Cerebellar and plasma samples were applied to steroid extraction. To estimate the recovery of the unconjugated steroid during the extraction, 1,500 cpm of [7-3H] pregnenolone was added to the samples with 5 ml ethyl acetate. The tubes were stirred for 30 min and centrifuged at 3,000 x g for 5 min. The organic phase was removed and the extraction step was repeated twice. The combined organic extracts, which contained unconjugated pregnenolone, were put to dryness as the assay samples for pregnenolone. On the other hand, the pH of the water phase was decreased to 1 with 30 µl sulfuric acid, and saturated sodium chloride was added as a final concentration of 20%. To calculate the recovery of the steroid sulfate ester, 1,500 cpm of [7-3H]dehydroepiandrosterone sulfate ester was then added to the mixture. Extraction with ethyl acetate in the water phase was again performed as described above. Steroid sulfates were contained in this extract and solvolyzed in 10 ml 95% ethyl ether at 37 C overnight. The hydrolyzed steroids were washed once with 3 ml of 1 N NaOH and twice with 3 ml of water and put to dryness as the assay samples for pregnenolone sulfate ester. The dried residues were dissolved in 1 ml PBS containing 0.1% gelatin. Each aqueous solution obtained from both extracts of organic and water phases was divided into two aliquots: one aliquot for the recovery measurement, the other for the measurement of pregnenolone or its sulfate ester.
To measure the concentrations of pregnenolone and its sulfate ester,
aliquots from both extracts of organic and water phases were applied to
the pregnenolone RIA (4, 11, 13, 43, 44) using the antiserum to
pregnenolone (Radioassay Systems laboratories, Inc., Immuchem Corp.,
Carson, CA) and [7-3H]pregnenolone (specific
activity, 23.5 Ci/mmol, New England Nuclear, Boston, MA). The
pregnenolone assay was performed without chromatographic purification
of pregnenolone, and the first antiserum used in the present experiment
cross-reacted with pregnenolone sulfate at 50%,
17
-hydroxypregnenolone at 2%, and dehydroepiandrosterone less than
0.01%. Separation of bound and free steroids 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 less than 7%. The precision index (
) of a
linear portion of the competition curve, which was computed according
to the method described previously (11, 45), was 0.037 in the
assay.
Statistical analysis
Results of the RIA were expressed as the mean ±
SEM. Comparisons of changes in steroid concentrations and
total steroid amounts in the cerebellum between different developmental
stages were made by Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). As shown in Fig. 1, a and b, an
intense immunoreaction with the antibody against bovine adrenal P450scc
was restricted to large cell bodies lying at a narrow zone between the
molecular and granular layers and to fibers spreading into the
molecular layer. The distribution of immunoreactive cell bodies and
fibers in the cerebellar cortex was coincident with the location of
somata and dendrites of Purkinje cells, characterized by the
immunohistochemical and Nissl-stainings (Fig. 1, a and c). Furthermore,
in their somata and dendrites, a most intense immunoreaction was
concentrated in a substantial number of granules, which suggests that
some intracellular organelles, such as cytoplasmic mitochondria, may be
the location of P450scc-like protein (Fig. 1b).
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, a and d). Therefore, it is probable that the
observed immunoreaction is due to the antibody recognizing endogenous
cytochrome P450scc-like protein.
Identification of the cell type of P450scc-like immunoreactive
cells in the adult cerebellum
As shown in Fig. 1, P450scc-like immunoreactivity in the
cerebellum was suggested to be located in the somata and dendrites of
Purkinje cells. To confirm this finding, we further performed the
immunolabelings using three kinds of antibodies against P450scc,
IP3 receptor, and GFAP. The antibody against
IP3 receptor, which is considered to be a marker protein of
the Purkinje cell, recognized P450scc-like immunoreactive cells (Fig. 2, a and b). In contrast, the antibody
against GFAP, a specific marker protein of glial cells, stained a
substantial number of small cells in the granular and molecular layers,
but did not stain P450scc-like immunoreactive cells (Fig. 2, a and c).
These results taken together suggest that P450scc-like immunoreactive
cells are not glial cells and are identified as Purkinje cells, a
typical cerebellar neuron.
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, a protein band derived from the
cerebellum as well as other brain regions was recognized by the
antibody. The intensity of immunoreaction in all brain regions was
lower than in the testis (Fig. 3). Electrophoretic mobility of a
protein band in the brain and testis was almost the same as that of the
purified bovine adrenal P450scc (Fig. 3). The molecular mass of this
immunoreactive protein was approximately 54 kDa judging from its
electrophoretic mobility. This value was the same as the molecular mass
of rat P450scc in the previous study (41).
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). As shown in a
result of gel electrophoresis of the RT-PCR product for P450scc gene
(Fig. 4a), a single band corresponding to P450scc mRNA size, but not
P450scc genomic DNA size, was detected in the cerebellum as well as
other brain regions. Serial Southern hybridization confirmed that this
band was P450scc mRNA specific (Fig. 4b). The density of the band in
each brain region was lower than that in the testis (Fig. 4).
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).
Such an immunoreaction at 0 day of age was completely abolished when
the immune serum was immunoadsorbed by the P450scc antigen in a
saturating concentration (10 µg P450scc/ml) (Fig. 5d). At 3 days of
age, P450scc-like immunoreactivity was observed only in somata of
Purkinje cells that were located in a narrow zone between the molecular
and granular layers (Fig. 5b). There was no immunoreaction with P450scc
in either the molecular or granular layers at this age (Fig. 5b). At 7
days of age, a similar immunoreaction with P450scc was found not only
in somata of Purkinje cells but also in the dendrites extending through
the molecular layer (Fig. 5c). Preadsorbing the antibody with an excess
amount of P450scc (10 µg/ml) also resulted in a complete absence of
P450scc-like immunoreactivity in the Purkinje cell at the ages of 3 and
7 days (Fig. 5, e and f).
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, a, c, and e). This
change was associated with a decrease in the external granular layer
(EGL) (Fig. 6, b, d, and f). The length of the immunopositive dendrite
reached an adult level at 21 days of age, when the EGL almost
disappeared (Fig. 6, e and f). Preadsorbing the antibody with P450scc
(10 µg/ml) also resulted in a complete absence of P450scc-like
immunoreactivity during 7–21 days (data not shown). In contrast to
morphological changes in the immunoreactive Purkinje cell, there was no
clear-cut difference in the intensity of the immunoreaction (Fig. 5, a,
b, and c; Fig. 6, a, c, and e). On the other hand, no immunoreactivity
for P450scc was observed in the nucleus of stained Purkinje cells (Fig. 5c; Fig. 6, a, c, and e).
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, a single band corresponding to P450scc
mRNA size, but not P450scc genomic DNA size, was detected at all of the
observed ages. There was no clear-cut difference in the density of each
band during neonatal development (Fig. 7), suggesting constant
expression of the mRNA encoding for P450scc. Although the P450scc mRNA
expression tended to decrease at 60 days of age, the change was not
remarkable (Fig. 7). On the other hand, the ratio of initial total RNA
amount used for RT-PCR reaction out of the total RNA extracted from one
cerebellum was approximately 0.43 at 0 day, 0.31 at 3 days, 0.22 at 7
days, 0.09 at 14 days, 0.09 at 21 days, and 0.12 at 60 days. Therefore,
the total P450scc mRNA level per whole cerebellum may increase at least
during neonatal development.
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). The pregnenolone concentrations
in plasma were significantly lower (P < 0.05, 0.01, or
0.001) than those in the cerebellum during neonatal development and in
adulthood (Fig. 8). In addition to pregnenolone, pregnenolone sulfate
ester was detected in both the cerebellum and plasma, but the level was
low at all of the observed ages (Fig. 8).
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). However, the total cerebellar
pregnenolone level increased significantly at 21 days of age
(P < 0.05 vs.. 0 or 3 days;
P < 0.001 vs.. 7 days) due to the increase
in the cerebellar weight (Fig. 9). The amount of pregnenolone at 21
days of age was almost the same as that at 60 days of age (Fig. 9). In
contrast, the total amount of pregnenolone sulfate ester in the
cerebellum tended to increase during neonatal development, but the
alteration was not significant (Fig. 9).
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, there was no change in the
expression of P450scc mRNA during a diurnal cycle.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, Western immunoblot analysis with the P450scc antibody confirmed the presence of P450scc-like protein in the rat cerebellum. In the cerebellum, the antibody predominantly recognized a protein band showing a similar electrophoretic mobility of testicular P450scc. RT-PCR analysis also indicated the expression of P450scc mRNA in the rat cerebellum. Therefore, it is possible that Purkinje cells possess P450scc, as P450scc-like immunoreactivity was restricted to this neuron in the cerebellum. However, the expressions of both P450scc-like protein and P450scc mRNA in the cerebellum seem to be lower than those in the testis.
It is well known that glial cells produce neurosteroids in the mammalian nervous systems (15, 16, 17, 18, 19, 20). In contrast to glial cells, information on the neurosteroidogenesis in neurons is accumulating slowly in mammals. Some studies with the rat have indicated the neuronal P450scc expression in the nervous system (24, 35). However, to the best of our knowledge, whether neurons located in the mammalian brain produce neurosteroids remains unclear. Yamada and Ochi (48) previously suggested that some immunoreactive neurons were localized in various regions of the rat brain by immunohistochemical analysis with the anti-P450scc serum used in the present study. P450scc-immunoreactive cells were first identified as Purkinje cells in the rat by the present study. This finding is in agreement with our previous finding obtained by the avian species (12, 13). On the other hand, Sanne and Krueger (49) reported a lower expression of P450scc in the rat cerebellar granule layer and white matter. In addition, there is no report showing P450scc-like immunoreactivity in the Purkinje cell using other antisera against P450scc (14, 17, 19, 50). Therefore, to draw a firm conclusion concerning steroidogenesis in this neuron, further experiments with an independent antiserum are needed.
It has previously been reported that in rats the differentiation of Purkinje cells takes place at 3 days of age, when this neuron is located in a narrow zone between the molecular and granular layers (46, 47). Therefore, the question asked in the present study was when the steroidogenic enzyme P450scc appears in the Purkinje cell during cerebellar development. The present immunohistochemical analysis revealed a widespread distribution of the P450scc immunopositive cells throughout the cerebellum of infant male rats at 0 day of age. The immunoreaction examined at this age may be specific for P450scc, as it was inhibited by preincubation of the antibody with P450scc. It is therefore possible that the appearance of cytochrome P450scc occurs in the cerebellum before the differentiation of the first Purkinje cells in the rat. The next important question asked in the present study was to determine neonatal changes of P450scc localized in the Purkinje cell. We observed a steady immunoreaction with the P450scc antibody in soma of the Purkinje cell at 3 days of age, after which immunoreactive dendrites of the Purkinje cell extended into the developing molecular layer at advanced ages. It is well known that in rats the molecular layer is grown longer, as a consequence of the regression of the EGL from 10–21 days, and formation and cellular migration of the cerebellum is almost completed at 21 days (46, 47). In contrast to these morphological changes in the immunoreactive Purkinje cell, there was no clear difference in the intensity of its immunoreactivity during neonatal development. These immunohistochemical findings concur with the finding of the present RT-PCR analysis showing a constant expression of the P450scc mRNA in the cerebellum during the neonatal period. As for widespread immunostaining at 0 day of age, the data obtained by RT-PCR analysis suggest that there is indeed P450scc expression in the cerebellum. The total P450scc mRNA level in the whole cerebellum may increase during neonatal development, due to the increase in the total amount of cerebellar RNA. Unlike neonatal life, the expression of P450scc mRNA might decrease slightly in adulthood, but the change was not remarkable.
In the present study, we further measured pregnenolone and its sulfate
ester in the cerebellum as well as plasma during neonatal life. The
pregnenolone concentration was much higher in the cerebellum than in
plasma during neonatal development as well as in adulthood. Although we
cannot rule out the possibility that pregnenolone produced in the
peripheral steroidogenic glands accumulates in the cerebellum, these
RIA results may reflect the presence of P450scc in the cerebellum.
Pregnenolone concentrations in the cerebellum did not significantly
change during the neonatal period except the rapid decrease just after
birth. A similar decrease in the pregnenolone concentration in the
whole brain has been reported in fetuses and newborn rats (5). A higher
concentration of pregnenolone in the cerebellum of newborn rats is not
consistent with the other results of the present study. Nonbrain
sources of maternal and/or fetal pregnenolone might contribute to its
accumulation in the cerebellum at this period. However, the profiles of
intracerebellar changes in the concentration and the total amount of
pregnenolone after 3 days of age, when the differentiation of the first
Purkinje cells was completed, seem to be correlated with the results of
immunohistochemistry and RT-PCR analysis. The total amount of
pregnenolone in the cerebellum increased, due to the increase in the
cerebellar weight, during 7–21 days of age, when immunoreactive
Purkinje cells developed into the molecular layer without a significant
change in the intensity of P450scc immunoreactivity. However, the
conversion of pregnenolone to progesterone or dehydroepiandrosterone
during neonatal life must be taken into account when studying the
regulation of pregnenolone levels in the cerebellum. Therefore, more
precise experiments, which measure the mRNAs encoding
3ß-hydroxysteroid dehydrogenase and P45017,lyase, are
now in progress.
If pregnenolone and/or its sulfate ester produced in the Purkinje cell contribute to some physiological actions in the cerebellum, the P450scc expression would change under different physiological conditions. To test this hypothesis, therefore, we examined diurnal changes in the mRNA encoding P450scc in the cerebellum of neonatal rats exposed to long day (LD) photoperiod. However, we could not detect any clear-cut diurnal change in the P450scc mRNA expression. In contrast, there is evidence indicating a diurnal rhythm of the pregnenolone level in the rat whole brain (6, 8). Further studies are warranted to determine physiological changes in the expression of P450scc and the pregnenolone level in the cerebellum. On the other hand, it has been recently reported that pregnenolone and/or progesterone play a role in myelination but not axonal growth of the mouse glial cell in peripheral nervous system (51). Conversely, it has been suggested that in mice and rats neurosteroids may function as regulators of nerve growth (52). Therefore, the present finding indicating the presence of P450scc in the Purkinje cell may suggest functional roles of pregnenolone and/or its metabolites in promotion of the growth of neurons and/or glial cells in the cerebellum of neonatal rats.
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Acknowledgments |
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Footnotes |
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Received May 27, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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