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ORIGINAL
Effect of peripherally derived steroid hormones on theexpression of steroidogenic enzymes in the rat choroid plexus
Koji Ono1, 2, Akihiko Yano1, 3, 4, and Kazunori Ishimura1, 5
Department of Anatomy and Cell Biology, Institute of Health Biosciences, Tokushima University Graduate School, Tokushima, Japan,
Department of Anatomy, Kawasaki Medical School, Okayama, Japan, 3Student Lab, Tokushima University Faculty of Medicine, Tokushima
Japan, 4Clinical Training Center, University of Yamanashi Hospital, Yamanashi, Japan, 5Department of Nutrition and Health Promotion,
Hiroshima Jogakuin University Faculty of Human Life Studies, Hiroshima, Japan

1
2

Abstract : Peripherally derived steroids affect steroid production in the brain via the blood–brain barrier. However, steroid concentrations are lower in the cerebrospinal fluid than those in the blood, indicating restrictedinflux of steroids because of their metabolization by choroid plexus (CP) epithelial cells. Here, we analyzed thegene expression of steroidogenic enzymes [cholesterol side-chain cleavage enzyme (P450scc), 17α-hydroxylase/
C17-C20 lyase (P450c17), 3β-hydroxysteroid dehydrogenase (3β -HSD), 17β-hydroxysteroid dehydrogenase type 1(17β -HSD1), aromatase (Cyp19a1), and 5α-reductase type 1 (5α -R1)]. These genes were expressed to a lesser extent
in the CP than in the testis and to a similar extent in the cerebral cortex. However, P450scc levels were higher
in the CP than in the cerebral cortex, whereas Cyp19a1 levels showed the opposite trend. We also evaluated theeffects of orchiectomy and testosterone on the expression of these genes. P450c17 and 5α -R1 levels were unaffected by orchiectomy, whereas P450scc and 3β -HSD levels were increased and decreased, respectively. Cyp19a1
expression increased upon testosterone treatment, whereas that of 17β -HSD decreased upon orchiectomy oradministration of testosterone. Immunohistochemistry analysis revealed that 17β -HSD was expressed in the cytoplasm of CP epithelial cells. These results indicate that CP epithelial cells synthesize and convert the certain
types of steroids to contribute to the homeostasis of steroids in the brain. J. Med. Invest. 68 : 238-243, August, 2021
Keywords : steroidogenic enzyme, choroid plexus, orchiectomy, testosterone, immunohistochemistry

INTRODUCTION
Steroid hormones are produced in the adrenal glands, testis,and ovaries and circulate in the blood to act throughout the body.
Neurons in the central nervous system have also been reported
to produce steroid hormones de novo (1, 2). Estrogen protects
estrogen-producing neurons from oxidative stress (3, 4). Steroid
hormones are lipid soluble molecules that cross membranes andaffect the central nervous system. Evidence suggests that thelevels of steroid hormones in the cerebrospinal fluid are lower
compared to those in the blood. The blood–brain and blood–cerebrospinal fluid barriers block the entry of substances into thebrain (5) and may control the inflow of steroid hormones. We predicted that the metabolism of steroid hormones in choroid plexus(CP) epithelial cells is one among the mechanisms responsible forthis barrier function.
The CP plays an important role in the nutritional homeostasisof the whole brain via the cerebrospinal fluid (6). Accordingly,
CP epithelial cells have been shown to constitute the blood–cerebrospinal fluid barrier, with their distal sides facing the cerebrospinal fluid and proximal ends facing the fenestrated capillary,
thereby creating an interface between the cerebrospinal fluid
and peripheral blood (7). The levels of D-amino acid oxidase, an
enzyme involved in the metabolism of D-amino acids, are lower
in the cerebrospinal fluid than those in the blood, similar tosteroid hormones (8).
Abbreviations
P450scc : cholesterol side-chain cleavage enzyme, P450c17 : 17α-hydroxylase/C17-C20 lyase, 3β-HSD : 3β-hydroxysteroid dehydrogenase,17β-HSD1 : 17β-hydroxysteroid dehydrogenase type 1, Cyp19a1 : aromatase, 5α-R1 : 5α-reductase type 1, RT-PCR : reverse transcription-polymerase chain reaction, Tes : testis, Ad : adrenal gland,
Cx : cerebral cortex, CP : choroid plexus

In this study, we conducted gene expression analysis to investigate the presence of steroid-metabolizing enzymes in the
CP. Furthermore, we examined the influence of peripherally
produced steroid hormones on the expression of these genes. We
focused on six enzymes involved in the synthesis and metabolismof sex steroids in the CP of normal and castrated male rats : cholesterol side-chain cleavage enzyme (P450scc), 17α-hydroxylase / C17-C20 lyase (P450c17), 3β-hydroxysteroid dehydrogenase(3β-HSD), 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1),
aromatase (Cyp19a1), and 5α-reductase type 1 (5α-R1).

EXPERIMENTAL PROCEDURES
Animals

Eight-week-old male Wistar rats were subjected to orchiectomy or sham operation. One week after the operation, orchiectomized animals were divided into 2 groups (n = 4). Rats in one
group were subcutaneously administered testosterone (2 mg / kg,
Sigma-Aldrich, St. Louis, MO, USA), whereas those in the othergroup were administered sesame oil (as a solvent) at 10 : 00 a.m.
daily for 7 d. Sham-operated animals were treated in the samemanner. All experiments were conducted in accordance with the
Guidelines for the Care and Use of Laboratory Animals adopted
by the Committee on Animal Research in Tokushima University(No, 11031), and were accredited by the Japanese Ministry ofReceived for publication June 25, 2020 ; accepted March 14, 2021.
Address correspondence and reprint requests to Koji Ono, Department of Anatomy, Kawasaki Medical School, 577, Matsushima, Kurashiki, Okayama 701-0192, Japan and Fax : +81-86-462-1199.

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Education, Culture, Sports, Science, and Technology. Every effort was taken to minimize the number and suffering of animals
used throughout all experimental procedures.

MD, USA). The mRNA levels of steroidogenic enzymes were normalized to those of glyceraldehyde 3-phosphate dehydrogenase,which was used as an internal standard.

Reverse transcription-polymerase chain reaction (RT-PCR)

Immunohistochemistry analysis

Rats administered testosterone or sesame oil were decapitated under anesthesia [0.3 mg / kg medetomidine hydrochloride(Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), 4 mg / kg
midazolam (Astellas Pharma Inc. Tokyo, Japan), and 5 mg / kgbutorphanol tartrate (Meiji Seika Pharma Co., Ltd., Tokyo,
Japan)] at 3 h after treatment. Subsequently, the brains, testes(Tes), and adrenal glands (Ad) were removed. The cerebral
cortices (Cx) of the anterior brain and CP of the fourth ventricle
were used for the experiments, whereas the olfactory bulb, hypothalamus, and thalamus were discarded (Alves et al., 2009).
Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA,
USA). The absorption spectrum (220-350 nm) was measured
with a NanoDrop Spectrophotometer (NanoDrop Technologies,
Wilmington, DE, USA). RNA quantity and quality were calculated by the Beer’s Law equation and determining the ratio
of absorbance at 260 and 280 nm. A 260 / 280 ratio of 1.8-2.0
was accepted as pure for extracted RNA. We used 3 μg of total
RNA for RT with the PrimeScript II 1st Strand cDNA Synthesis
Kit (Takara Bio, Shiga, Japan). The synthesized cDNA was
purified with a GenElute PCR Clean-UP Kit (Sigma-Aldrich).
The concentration of the purified product was measured using
the NanoDrop spectrometer. The PCR mixture contained 200
ng cDNA, 0.5 μM of each primer, and 1× Go Taq Green MasterMix (Promega, Madison, WI, USA) in a total volume of 25 μL.
Amplification was performed using the Gene Amp PCR System
9700 (Applied Biosystems, Foster City, CA, USA). The PCR
cycle parameters were as follows : initial denaturation for 5 min
at 95°C, denaturation for 30 s at 95°C, primer annealing for 45
s at 51-66°C, extension at 74°C for 45 s, and final extension at
74°C for 7 min. The primer sequences, annealing temperature,
PCR cycles, and predicted product size are listed in Table 1.
PCR products were analyzed by electrophoresis on 2% agarosegels and visualized by ethidium bromide staining. Images of
the generated bands were captured using a Gel Doc EZ system(Bio-Rad, Hercules, CA, USA) and analyzed using ImageJ
version 1.44 software (National Institutes of Health, Bethesda,

Orchiectomized and testosterone-administered rats wereanesthetized with pentobarbital and perfused with 4% paraformaldehyde (Merck KGaA, Darmstadt, Germany). The brains of
the rats were removed and fixed overnight, after which the CP of
the fourth ventricle was dissected. Frozen sections of the tissues
were prepared, treated with blocking buffer (phosphate-bufferedsaline (FUJIFILM Wako Pure Chemical Co., Osaka, Japan)
containing 3% bovine serum albumin (FUJIFILM Wako Pure
Chemical Co.), 0.1% Triton X-100 (FUJIFILM Wako Pure
Chemical Co.), and 0.05% sodium azide (FUJIFILM Wako
Pure Chemical Co.) for 30 min, and then incubated with rabbit
anti-17β-HSD1 polyclonal antibody (diluted 1 : 1000) in blocking
buffer overnight at 4°C. All procedures for antigen purification(17β-HSD1), antibody production, and antibody / antigen immunoreactivity assay were performed as previously described (9).
After incubation with the primary antibody, the sections wereincubated with fluorescein-labeled secondary antibody for 2 h at
20°C (Alexa Fluor 488 donkey anti-rabbit IgG, diluted 1 : 1000 ;Invitrogen). The sections were rinsed in phosphate-bufferedsaline and mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). For fluorescence microscopy, the sections wereviewed under a microscope (Eclipse E800 ; Nikon, Tokyo, Japan)
equipped with appropriate filter sets and a digital camera (SDFi1 ; Nikon). For electron microscopy, the frozen sections wereincubated with 3,3′-diaminobenzidine (Dojindo, Kumamoto,Japan) after treatment with the primary antibody and ABC kit(Vector Laboratories) and fixed in 1% osmium tetroxide solution(TAAB Laboratories, Reading, UK). The sections were thenstained with uranyl acetate and embedded in Epon epoxy resin(TAAB Laboratories). Ultrathin sections (80 nm) were cut andobserved without lead staining.

Statistical analysis
All data have are shown as the mean ± standard deviation.
Statistical analysis was performed using Student’s t-test. p < 0.05
was considered to indicate statistically significant results.

Table 1. Primers used for semiquantitative reverse transcription-polymerase chain reaction (PCR)
Enzymename

AccessionNo.

P450scc

NM_017286

3β-HSD

NM_001007719

P450c17

NM_012753

17β-HSD

NM_054007

AromataseNM_017085

5α-reductase

NM_017070

GAPDH

NM_017008

Ta ; Annealing temperature.

PCR
Primer sequence
forward5′-AGAATTGTCCTAAACCAAGAGG-3′

reverse5′-GTGTGCCATTTCATAAAGGTTC-3′

forward5′-GCAGCACAGTTGACGTTGCAG-3′

reverse5′-AAGGCTCCAGCTGGAATCAAGG-3′

forward5′-TTGCCACGGTGGGAGACATC-3′

reverse5′-ACTGATCTGGCTGGTCCCATT-3′

forward5′-GCTGGCCAGACATGGACTCA-3′

reverse5′-AGGTTGGGGAGCATTCCAACA-3′

forward5′-CCATCAAGCAGCATTTGGAC-3′

reverse5′-TCCACGTCTCTCAGCGAA-3′

forward5′-CGCTGTACGAGTACATTCGTC-3′

reverse5′-ACTGGCTCAAGTATCTGCTCT-3′

forward5′-GTGAAGGTCGGTGTGAACG-3′

reverse5′-GGTGAAGACGCCAGTAGACTC-3′

Tacycles

Product
(bp)

60

35

579

62

32

491

66

35

357

59

33

213

53

28

368

55

35

213

55

21

300

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K. Ono, et al. Steroidogenic enzymes in the CP

RESULTSWe performed RT-PCR to evaluate the gene expression of
P450scc, P450c17, 3β-HSD, 17β-HSD1, Cyp19a1, and 5α-R1 in the
rat CP. The Tes expressed all tested genes, whereas the Ad
expressed P450scc, P450c17, 3β-HSD, and 5α-R1 genes but not the
17β-HSD1 and Cyp19a1 genes (Fig. 1). P450scc P450c17, 3β-HSD,
17β-HSD1, Cyp19a1, and 5α-R1 were found to be expressed in the
CP (Fig. 1).
We also examined the influence of peripherally derived testosterone on the expression of steroidogenic enzyme genes by semiquantitative RT-PCR. The levels of P450scc decreased slightly
in rats subjected to orchiectomy but were not significantly different from those in the control group (Fig. 2A). The expression
levels of P450c17 and 3β-HSD were unaffected by orchiectomy
or testosterone replenishment (Fig. 2B, C). Furthermore, thegene expression of 17β-HSD1 was significantly decreased in theoil-treated group compared to in the testosterone-treated groupin sham-operated mice (sham (oil) ; mean ± SD = 1.33 ± 0.19, sham(Tes) ; mean ± SD = 0.86 ± 0.08, p = 0.037, Fig. 2D). In contrast,
the gene expression level of Cyp19a1 was significantly increasedin the testosterone-treated group compared to in the oil-treatedgroup in sham-operated mice (sham (oil) ; mean ± SD = 0.17 ±
0.24, sham (Tes) ; mean ± SD = 0.96 ± 0.32, p = 0.028, Fig. 2E).
Finally, 5α-R1 levels remained unchanged upon orchiectomy ortestosterone replenishment (Fig. 2F).
To investigate whether any of the steroidogenic enzymes isalso present in the CP and to histologically confirm the resultsof gene expression analysis, we performed immunohistochemistry evaluation. We used an anti-17β-HSD1 antibody, which
has been reported to be specific (9). Immunostaining revealed
immunopositive signals in antibody-reacted tissues compared

Fig. 1. The mRNA levels of steroidogenic enzymes in the rat testis (Tes), adrenal gland (Ad), cerebral cortex (Cx), and choroid plexus (CP). (A)RT-PCR products, and histograms of (B) P450scc, (C) P450c17, (D) 3β-HSD, (E) 17β-HSD1, (F) Cyp19a1, and (G) 5α-R1 comparing expression levels(all n = 4). Histograms represent the results of semiquantitative analysis of the expression of steroidogenic enzymes.

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241

to in preimmune serum-reacted tissues. At the optical microscopy level, these signals were observed in the cytoplasm of CP
epithelial cells and not in the connective tissue or blood vesselsthat surrounded by CP cells (Fig. 3). In addition, to observe thesubcellular localization of 17β-HSD1 in the CP by ultrastructure analysis, we applied immunohistochemistry in electron
microscopy. Transmission electron microscopy confirmed that
17β-HSD1 was localized to the cytoplasm (Fig. 4).
Fig. 3. Immunohistochemical analysis of rat CP in the fourth

ventricle using rabbit anti-17β-HSD1 antibody and rabbit serum(preimmune). The immunoreactivity of 17β-HSD1 was detected in CP
epithelial cells. Scale bar = 50 µm.

Fig. 2. mRNA levels of steroidogenic enzymes detected in the rat CP by RT-PCR with (A) P450scc, (B) P450c17, (C) 3β-HSD, (D) 17β-HSD1,(E) Cyp19a1, (F) 5α-R1, and GAPDH-specific primers. Lane 1 : sham operation + sesame oil ; lane 2 : orchiectomy + sesame oil ; lane 3 : shamoperation + testosterone ; lane 4 : orchiectomy + testosterone (all n = 4). Histograms represent the results of semiquantitative analysis of theexpression of steroidogenic enzymes in CP. (A)–(C) and (F) Not significantly different from that of the control group. (D) Significantly decreasedin the oil-treated group compared to the testosterone-treated group in sham-operated mice (Sham (oil) ; mean ± SD = 1.33 ± 0.19, sham (Tes) ; mean
± SD = 0.86 ± 0.08, p = 0.037). (E) Significantly increased in the testosterone-treated group compared to the oil-treated group in sham-operated
mice (Sham (oil) ; mean ± SD = 0.17 ± 0.24, sham (Tes) ; mean ± SD = 0.96 ± 0.32, p = 0.028).

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K. Ono, et al. Steroidogenic enzymes in the CP

Fig. 4. Transmission electron microscopy of rat CP. Immunoelectron
microscopy revealed the cytoplasmic localization of 17β-HSD1. Bar = 5μm.

DISCUSSION
Steroid hormones selectively permeate the blood–brain barrier(10). Aldosterone and cortisol exhibit low levels of permeability,
whereas progesterone, estradiol, and testosterone exhibit highpotential for permeability. Hormones with high permeability
are produced in the gonads and pass through the blood–brain
barrier, and thus may act on neurons or glial cells. However,the correlation between steroid hormones and the CP, which is
responsible for maintaining central nervous system homeostasis
via the cerebrospinal fluid, remains poorly understood. We foundthat genes encoding enzymes involved in the metabolism of steroid hormones were expressed in the CP (Fig. 1 and 2). The levelsof steroidogenic enzymes in the CP were much lower than thosein the Tes (Fig. 1). Thus, we predicted that limited amounts ofmetabolized steroids flow from the blood to the cerebrospinal

fluid. However, the expression of P450scc was higher than thatin the Cx (Fig. 1B). Notably, P450scc, which is known to resultin the production of pregnenolone from cholesterol, was present
in the CP, suggesting that CP epithelial cells not only metabolize
steroid hormones, but also possess the ability to de novo synthesize steroid hormones from cholesterol. Immunostaining (Fig. 3)
and immunoelectron microscopy (Fig. 4) also revealed that 17βHSD1 was expressed in the cytoplasm in CP epithelial cells, inaccordance with previous biochemical results (11).
Taken together, we propose 3 hypotheses for the significanceof steroid hormone-metabolizing enzymes in the CP. 1) Enzymes
present in CP epithelial cells may act as a cerebrospinal fluid
barrier, regulating the influx of steroid hormones from the bloodvessels to the cerebrospinal fluid. 2) Steroid hormones may bedirectly synthesized by CP epithelial cells and secreted into the
cerebrospinal fluid. 3) Steroid hormones discharged from thecerebrospinal fluid into the blood may be metabolized in the CP.
As these hypotheses could not be verified by functional analysis,
we measured the expression of steroid hormone synthesizing
enzymes in primary cultured CP epithelial cells isolated fromthe CP of the lateral ventricle to determine whether steroid hormones were synthesized in these cells.
Androgen receptors are expressed in the brain and the expression levels were altered by steroid hormone levels (12, 13). As
such, changes in the expression of steroid hormone-metabolizing
enzymes in CP epithelial cells in response to administration of
testosterone in this study may have contributed to the variations
observed in the levels of steroid hormones in the peripheral blood.
This phenomenon indicated that CP epithelial cells perceive peripheral steroids via their respective receptors and metabolize
the hormones, thereby providing a potential mechanism for homeostasis of steroid hormones in the cerebrospinal fluid (Fig. 5).

Fig. 5. Graphic abstract of steroidogenic enzymes in the rat choroid plexus. The upper half of the image shows a sagittal section of a rat. The
pink structures in each ventricle represent the choroid plexus. We analyzed the expression of steroidogenic enzymes in the choroid plexus in the
fourth ventricle. Enzymes involved in the steroid synthesis pathway shown in the lower half of the figure were present.

The Journal of Medical Investigation Vol. 68 August 2021
As the expression of steroidogenic enzymes in the CP wasmuch lower than that in Tes, we considered that the metabolismof steroids in the CP was limited upon entry of the hormones
into the cerebrospinal fluid from the blood. However, gene expression in the CP changed in the presence of peripheral steroid
hormones. Although the gene expression profiles in the CP andCx were similar for most genes, the expression of P450scc and
Cyp19a1 in the CP differed from that in the Cx. Thus, among thesteroids required in the brain, some are synthesized as precursors in the CP and may eventually be converted in the brain.

DECLARATION OF INTEREST STATEMENT
The authors declare that they have no conflicts of interest.

ACKNOWLEDGEMENTS
We thank Prof. Kazunori Toida (Department of Anatomy,
Kawasaki Medical School, Okayama, Japan) for providing helpful comments. This study was supported by JSPS KAKENHI[grant number 23590236 (2011-2013)]. K.O. and K.I. designed
the research ; K.O. and A.Y. performed the research ; K.O. and
A.Y. analyzed the data ; K.O. and K.I. wrote the paper. All authors approved the submission and disclosure of the final versionof the article.

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