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Laboratory for Amphibian Biology, Faculty of Science (M.T.), and Laboratory of Brain Science, Faculty of Integrated Arts and Sciences (K.U., T.Y., S.K., K.T.), 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
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Abstract |
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These results taken together suggest that the amphibian brain possesses steroidogenic enzyme P450scc and produces pregnenolone and its sulfate ester. Pregnenolone sulfate may function well during the breeding and postbreeding active phases of the year in the seasonal breeder.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On the other hand, we have recently demonstrated, using biochemical and immunochemical approaches, that the quail brain also possesses cytochrome P450scc and produces pregnenolone and its sulfate ester (21). In addition, our immunohistochemical studies with the same avian species have shown that P450scc-immunoreactive cells are distributed in several telencephalic, diencephalic, and mesencephalic regions (22, 23). In birds, both glial and neuronal cells also possess P450scc (22, 23).
In contrast to extensive studies with higher vertebrates, limited
information has been available on neurosteroids in lower vertebrates.
Recently, a series of studies using the amphibian brain (24, 25, 26)
indicated that populations of 3ß-hydroxysteroid
dehydrogenase/
5-4-isomerase (3ßHSD)- or
17ßHSD-immunoreactive cell bodies and fibers were observed in the
telencephalon and diencephalon of the brain of Rana
ridibunda. In addition, exogenous pregnenolone was metabolized to
5-3ß-hydroxysteroids and
4-ketosteroids by brain slices of the same amphibian
species (24, 25, 26) and the endozepine triakontatetraneuropeptide
stimulated its 3ßHSD activity (27). However, no study on pregnenolone
biosynthesis from cholesterol, which is an initial step for
steroidogenesis, has been reported to date in the amphibian brain.
With these findings as a background, we measured the intrabrain levels of pregnenolone and its sulfate ester and analyzed the presence of cytochrome P450scc in the brains of amphibians. The second purpose of this study was to determine seasonal changes in neurosteroids formed by P450scc as a possible physiological change using a seasonal breeder.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of plasma and tissue samples
To prepare plasma and brain tissue samples, amphibians were
killed according to the Guide for the Care and Use of Laboratory
Animals prepared by Hiroshima University (Hiroshima, Japan). The time
lapse between the beginning and the end of the killing did not exceed
2 h, and this was always performed between 1000–1200 h. In
X. laevis, trunk blood was collected into heparinized glass
tubes and centrifuged at 1800 x g for 30 min at 4 C.
The plasma samples were stored at -20 C. Immediately after blood
collection, brains and gonads were removed and weighed. Some brains of
X. laevis were carefully dissected into several regions
under a microscope using a reference (28) of the frog brain as follows:
telencephalon, diencephalon, midbrain, and cerebellum. The brain tissue
and plasma samples were also collected in R. nigromaculata.
All tissue samples were snap-frozen in liquid nitrogen and stored at
-80 C.
Steroid extraction
Extraction of unconjugated steroids or their sulfates was
performed according to the method reported previously (3, 18, 21).
Individual frozen brain and gonads were rapidly thawed and separately
homogenized in 5 ml cold PBS (0.14 M NaCl and 0.01
M potassium phosphate buffer, pH 7.6) with a Teflon-glass
homogenizer. Individual plasma (500 µl) was diluted with 5 ml cold
PBS. These tissue and plasma samples were applied to steroid
extraction. To estimate the recovery of the unconjugated steroid during
the extraction, 2000 cpm [7-3H]pregnenolone were added to
each tube with 5 ml ethyl acetate. The tubes were vigorously stirred
for 30 min and centrifuged at 3000 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 dried under a stream of nitrogen. For removal of
steroids conjugated to fatty acid, the dried samples were dissolved in
2 ml 70% methanol by stirring for 5 min and were kept at -20 C
overnight. After centrifugation at 3000 x g for 5 min,
the supernatant was aspirated. The precipitate was washed with 2 ml
cold 70% methanol by centrifugation at 3000 x g for 5
min, and the supernatant was aspirated. The combined supernatants were
dried under a stream of nitrogen as the assay samples for unconjugated
pregnenolone.
On the other hand, the steroid conjugated to sulfate was extracted from the remaining aquatic phase after extraction of ethyl acetate. For estimation of the recovery of the steroid sulfate ester during extraction, 2000 cpm [1,2,6,7-3H]dehydroepiandrosterone sulfate ester were then added to each aquatic phase. The pH of the aquatic phase was decreased to 1 with 30 µl sulfuric acid, and saturated sodium chloride was added at a final concentration of 20%. Extraction of steroid sulfates in the aquatic phase with ethyl acetate was again performed as described above. The steroid sulfate was solvolized in 95% ethyl ether at 37 C overnight with shaking. The hydrolyzed steroid was washed once with 3 ml 1 N NaOH and twice with 3 ml distilled water and dried under a stream of nitrogen as the assay samples of pregnenolone sulfate ester. It might be possible to estimate the concentration of pregnenolone sulfate with RIA using an antipregnenolone serum, but the accuracy is low because of the low cross-reactivity (50%) to pregnenolone sulfate, as described below.
RIAs of pregnenolone and its sulfate ester
The dried residues were dissolved in 1 ml of PBS containing
0.1% gelatin. Each aqueous solution obtained from both extracts of
organic and aquatic phases was divided into two aliquots: one aliquot
for the recovery measurement, and the other for RIA of pregnenolone or
its sulfate ester. To measure the concentrations of pregnenolone and
its sulfate ester, aliquots from both extracts of organic and aquatic
phases were applied to the specific RIA system of pregnenolone (3, 18, 21). In the RIA, [7-3H]pregnenolone (SA, 23.5
Ci/mmol; New England Nuclear, Boston, MA) and the
antiserum to pregnenolone (Radioassay Systems Laboratories, Inc.,
Immuchem Corp., Carson, CA) were used. The antiserum cross-reacted with
pregnenolone sulfate at 50%, 17
-hydroxypregnenolone at 2%, and
dehydroepiandrosterone at less than 0.01%, and the pregnenolone assay
was performed without chromatographic purification of pregnenolone.
Separation of bound and free steroids was performed by centrifugation
after reaction with the IgG SORB (The Enzyme Center, Inc., Malden, MA)
(18). The least detectable amount was 0.1 ng/ml, and interassay
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 (21, 29), was 0.037 in the assay. To verify the
RIA system, competition curves were obtained using the tissue and
plasma samples and were compared with a competition curve of
pregnenolone.
Western immunoblot analysis with P450scc antibody
To detect cytochrome P450scc in the amphibian brain, Western
immunoblot analysis was conducted after SDS-PAGE of tissue homogenates.
The brain and testis of adult male Xenopus were rapidly
thawed and separately homogenized in 4 vol ice-cold sample buffer
containing 0.05% Nonidet P-40, 50 mM Tris-HCl
(pH 7.5), 2 mM EDTA, and 1
mM phenylmethylsulfonylfluoride. The homogenate
was centrifuged at 15,000 x g for 20 min. The
supernatant was concentrated by precipitation with 40% saturation of
ammonium sulfate (18). Proteins derived from each tissue were subjected
to SDS-10% PAGE, and then Western immunoblotting was performed
according to our previous methods (18, 21). In brief, after transfer of
electrophoresed proteins onto polyvinylidene difluoride
membranes (Immobilon-P, Millipore Corp., Bedford, MA), the
blot was probed with a polyclonal antibody (1:1000 dilution) raised
against the bovine adrenal cytochrome P450scc in the rabbit, followed
by incubation with biotinylated goat antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA) and finally by a 30-min
incubation with avidin-biotin complex (Vectastain ABC
Elite kit, Vector Laboratories, Inc.). The details of the
characterization of this serum were reported previously (21, 23, 30).
Immunoreactive bands were detected by immersing the membranes for 2–7
min in a diaminobenzidine (DAB) solution [0.05% DAB and 0.1% NiCl in
0.1 M phosphate buffer (PB), pH 7.4, containing 0.3%
H2O2]. An amount of proteins was measured
using the bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL) with BSA as a standard.
The specificity of the anti-P450scc serum was assessed by substitution of the control serum for the primary antiserum; in this control serum, the antibody (1:1000 dilution) was preabsorbed by incubation with the purified antigen in a saturating concentration (10 µg P450scc/ml) for 16 h before use. The membranes were incubated with this control serum, employing the same procedure as that for the anti-P450scc serum.
Immunohistochemical analysis with P450scc antibody
In the present immunohistochemical experiment, adult males of
X. laevis and R. nigromaculata in the
postbreeding phase (September 1998) were deeply anesthetized with 10%
4-allyl-2-methoxyphenol (FA 100, Tanabe Pharmaceutical Co. Ltd., Osaka,
Japan), and then perfused transcardially with 0.1 M PB
followed by fixative solution (4% paraformaldehyde in 0.1
M PB). After dissection from the skull, brains were
postfixed for 12 h in the same fixative solution at 4 C, and then
soaked in a refrigerated sucrose solution (21% sucrose in 0.1
M PB) until they sank. Whole brains were embedded in OCT
compound (Miles, Inc., Elkhart, IN) and frozen-sectioned sagittally at
8-µm thickness with a cryostat at -18 C. Every third section was
grouped on a slide precoated with 3-aminopropyltriethoxysilane
(Sigma Chemical Co., St. Louis, MO); thus, we obtained
three independent series of adjacent sections. Only one of these series
of sections was used for immunohistochemical staining with the
anti-P450scc serum; the remaining two series were used for
immunohistochemical staining with the antiserum (1:500 dilution)
preincubated with a saturating concentration of the purified antigen
(20 µg P450scc/ml) for 16 h before use as a control and for
hematoxylin staining, respectively.
The sections were processed according to the routine avidin-biotin-peroxidase complex immunohistochemical technique (18, 23). 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 anti-P450scc serum at a dilution of 1:500 for 36 h at 4 C. The primary immunoreaction was followed by a 60-min incubation with biotinylated antirabbit IgG (10 µg/ml; Vector Laboratories, Inc.) and finally by a 60-min incubation with avidin-biotin complex (Vectastain ABC Elite kit, Vector Laboratories, Inc.). Immunoreactive products were detected by immersing the sections for 5–10 min in a DAB solution (0.05% DAB in PBS containing 0.3% H2O2). The localization of immunoreactive cells in the amphibian brain was studied using an Olympus Corp. BH-2 microscope (New Hyde Park, NY).
Statistics
Statistics for linearity and parallelism of competition curves
in the RIA were computed according to the method of Bliss (31). Results
of the RIA were expressed as the mean ± SEM.
Differences in the concentrations of pregnenolone and its sulfate ester
were analyzed by Duncan’s multiple range test or Mann-Whitney U test
after verification of equality or inequality of variances among groups
to be compared (31).
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Results |
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, left panel). Pregnenolone sulfate concentrations in the
brain (0.162 pmol/mg) were significantly (P < 0.05)
higher than those in the testis (0.078 pmol/mg) and plasma (0.035
pmol/µl; Fig. 1, right panel). Also in the female
Xenopus, both concentrations of pregnenolone and its sulfate
ester in the brain (0.257 and 0.087 pmol/mg, respectively) were
significantly (P < 0.05) higher than those in the
ovaries (0.039 and 0.021 pmol/mg, respectively) and plasma (0.006 and
0.014 pmol/µl, respectively; Fig. 2).
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). For these two
steroids, the difference in pregnenolone concentrations between the
cerebellum and other brain regions were statistically significant
(P < 0.05). Also in the female Xenopus,
both concentrations of pregnenolone and its sulfate ester in the
cerebellum (4.51 and 2.44 pmol/mg, respectively) were significantly
(P < 0.05) higher than those in the telencephalon
(0.13 and 0.08 pmol/mg, respectively), diencephalon (1.13 and 0.12
pmol/mg, respectively), and midbrain (0.69 and 0.21 pmol/mg,
respectively; Fig. 4).
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, left panel). Among multiple signals in the brain extract, a
most intense band was detected, and its electrophoretic mobility was in
the proximity of bovine adrenal P450scc (Fig. 5, left
panel). The molecular mass of this immunoreactive protein was
approximately 54 kDa judging from its electrophoretic mobility. The
electrophoretic mobilities of other bands in the brain were much
different from that of bovine adrenal P450scc (Fig. 5, left
panel). Preabsorption of the antibody with an excess of purified
P450scc antigen (10 µg/ml) failed to stain the 54-kDa protein band in
these tissues (Fig. 5, right panel). The P450scc-like
protein band was also detected in females, and the intensity of the
band was similar in males and females (data not shown).
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was suggested to possess P450scc-like protein (Fig. 5), we
examined the localization of P450scc-like immunoreactive cells in the
male Xenopus brain. Although P450scc-like immunoreactive
cells were found in several telencephalic, diencephalic, and
rhombencephalic regions, the striking observation was the distribution
of stained cells in the cerebellar cortex. As shown in Fig. 6, an immunoreaction with the anti-P450
serum was restricted to large cell bodies lying at a zone between the
molecular and granular layers. The distribution of immunoreactive cell
bodies in the cerebellum was coincident with the location of somata of
Purkinje cells, characterized by the immunohistochemical (Fig. 6, b and
e) and hematoxylin stainings (Fig. 6, a and d). Furthermore, in their
somata the 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. 6e). Clusters of immunoreactive cell bodies
were detected in the nucleus preopticus (Fig. 7a) and the nucleus infundibularis
ventralis (Fig. 7b) in the diencephalic region. Some stained cells were
also found in the pallium mediale (Fig. 7c) in the telencephalic
regions. Preabsorption of the antibody with cytochrome P450scc resulted
in no immunoreaction in the cerebellum (Fig. 6, c and f) or other brain
regions (Fig. 7, d–f). Therefore, it is probable that the observed
immunoreaction is due to the antibody recognizing endogenous cytochrome
P450scc-like protein.
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shows immunoreactive cells located in
the cerebellar Purkinje cell (a), the nucleus preopticus (b), and the
nucleus infundibularis ventralis (c) in the diencephalic region of the
male R. nigromaculata in the postbreeding phase
(September).
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, left panel). In contrast,
pregnenolone sulfate concentrations in the male brain were
significantly (P < 0.05) higher during the
postbreeding phase (September 1996) than during the hibernating phase
(January 1997), whereas the plasma pregnenolone sulfate concentration
was low and almost constant throughout the three phases (Fig. 9, right panel). Pregnenolone concentrations in the female
brain were almost constant throughout the different phases (Fig. 10, left panel). Unlike
males, the plasma pregnenolone concentration in females showed a
similar level in the brain, and it was significantly (P
< 0.05) higher during the hibernating (January 1997) and postbreeding
(September 1996) phases than during the breeding phase (April 1996;
Fig. 10, left panel). Pregnenolone sulfate concentrations in
the female brain during the breeding phase (April 1996) were
significantly (P < 0.05) higher than those during the
hibernating phase (January 1997; Fig. 10, right panel).
Similarly, the high level tended to remain during the postbreeding
phase (September 1996; Fig. 10, right panel). In contrast to
those in the brain, preg- nenolone sulfate concentrations in the
female plasma were low and significantly (P < 0.05)
increased during the postbreeding phase (September 1996; Fig. 10, right panel).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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0.12 and 0.038 pmol/mg
tissue, respectively) and posterior (0.07 and 0.014 pmol/mg
tissue, respectively) brains of adult male rats (3) and the brain of
adult male Japanese quails (0.19 and 0.0036 pmol/mg tissue,
respectively) (21) by specific RIAs. Thus, concentrations of these
neurosteroids in the Xenopus brain may be higher than those
in mammalian and avian brains. The present RIA results suggest that the
amphibian brain possesses cytochrome P450scc, although we could not
exclude the possibility that pregnenolone and/or its sulfate produced
in the peripheral steroidogenic glands accumulate in the brain. Western
immunoblot analysis with the antibody against the purified bovine
P450scc also suggests the presence of P450scc-like protein in the
Xenopus brain, because only preabsorption of the antibody
with an excess of purified P450scc antigen resulted in the complete
disappearance of reaction product. In the Xenopus brain, the
antibody predominantly recognized a protein band of electrophoretic
mobility in the proximity of bovine P450scc as well as in the gonad.
Other protein bands showing different electrophoretic mobilities were
also detected in the brain. As the presence of P450scc in the brain has
been established in several mammalian species (10, 11, 13, 15, 16, 17, 18) and
in an avian species (21, 22, 23), the presence of P450scc may be a
conserved property of the vertebrate brain. However, to draw a firm
conclusion, the biochemical demonstration of pregnenolone biosynthesis
in the brain is needed in the amphibian species and other lower
vertebrates.
The Xenopus cerebellum contained much higher
concentrations of pregnenolone and its sulfate ester than did the
telencephalon, diencephalon, and midbrain. The present study further
identified the presence of P450scc-like protein in the cerebellar
Purkinje cell by immunohistochemical analysis. This immunohistochemical
finding is in agreement with our previous findings obtained in the
mammalian (18, 20) and avian (22, 23) species. Interestingly,
Netchitailo et al. (32) reported that atrial natriuretic
factor (ANF)-like immunoreactive fibers innervate the Purkinje cell
layer of the frog Rana ridibunda. In addition, ANF may
contribute to the regulation of adrenal steroidogenesis in amphibians
(33). It may be therefore possible that ANF is involved in the control
of P450scc-containing Purkinje cells in amphibians. On the other hand,
the presence of 3ßHSD and 17ßHSD in the brain of R.
ridibunda has previously been reported (24, 25, 26). However, 3ßHSD-
and 17ßHSD-immunoreactive cells were detected in the telencephalon
and diencephalon, unlike in the cerebellum of R. ridibunda
(24, 25, 26). In addition, the Rana rhombencephalon failed to
metabolize pregnenolone to 17-hydroxypregnenolone, suggesting the
absence of 17-hydroxylase/C17,20-lyase (cytochrome
P450c17) in the amphibian cerebellum (24, 25, 26). Therefore, it may be
considered that pregnenolone and its sulfate ester contribute as major
neurosteroids to some cerebellar function in the amphibian brain. To
identify the corresponding steroid products in the cerebellum as well
as other brain regions, more precise experiments are required in
amphibians.
If pregnenolone and its sulfate ester produced in the amphibian brain are involved in some physiological actions in the brain, these neurosteroids would change under different physiological conditions. To test this hypothesis, therefore, we examined seasonal changes in the concentrations of pregnenolone and its sulfate ester in the brain of R. nigromaculata, a seasonally breeding amphibian. In both sexes, pregnenolone sulfate concentrations in the Rana brain were high during the active season, i.e. breeding phase (female) and postbreeding phase (male), and low during the quiescent season, i.e. hibernating phase, whereas pregnenolone concentrations were almost constant in the brain throughout the year. In addition, such a seasonal change in pregnenolone sulfate observed in the brain may be independent of peripheral steroidogenic glands, because the change in plasma pregnenolone sulfate was clearly different from that in the brain. Further experiments are needed to clarify the significance of different seasonal changes in pregnenolone and pregnenolone sulfate in the amphibian brain.
It has been reported that in the mammalian brain pregnenolone and
its sulfate interact as neurosteroids with the 
-aminobutyric acid A
(GABAA) receptor (34, 35, 36, 37). Modulatory effects of
the neuroactive steroid pregnanolone on GABAA
receptor have been also investigated in amphibian pituitary
melanotrophs (38, 39). GABA is accepted as an inhibitory
neurotransmitter, and pregnenolone sulfate is generally considered to
act as an antagonist of GABAergic neurotransmission (35, 36, 37). Various
regions of the amphibian brain, such as the olfactory bulb,
telencephalic dorsal and medial pallium, diencephalon, and cerebellum,
contained GABA-like immunoreactive neurons (40, 41). The present and
previous results taken together suggest that a higher level of
pregnenolone sulfate during the active season may act as a stimulatory
modulator to increase neuronal activities in the amphibian brain. The
amphibian quiescent phase might change to the active phase by the
action of pregnenolone sulfate in the brain. However, we need to
examine a season-specific change in this neurosteroid in restricted
brain regions with both P450scc and steroid sulfokinase, such as the
cerebellar cortex and some diencephalic areas.
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Footnotes |
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Received June 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-derivatives in the rodent brain. Neurochem Int 7:953–958[CrossRef]
5-derivatives in
rat and monkey brain. J Steroid Biochem 27:649–655[CrossRef][Medline]
5-derivatives in the rat brain. In: Fink G, Harmar AJ,
McKerns KW (eds) Neuroendocrine Molecular Biology. Plenum Press, New
York, pp 367–377
5-4-isomerase in the rat
Purkinje neuron during neonatal life. Endocrinology 140:805–813-aminobutyric acid receptor complex
in the brain? Brain Res 404:355–360[CrossRef][Medline]
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