General and Comparative Endocrinology 194 (2013) 286–294
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Expression profile of the aromatase enzyme in the Xenopus brain and localization of estradiol and estrogen receptors in each tissue Junshin Iwabuchi ⇑, Kouta Koshimizu, Tadahiko Nakagawa Laboratory of Biochemistry, Department of Chemistry, College of Humanities and Sciences, Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan
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Article history: Received 5 June 2013 Revised 2 September 2013 Accepted 28 September 2013 Available online 14 October 2013 Keywords: Aromatase Estradiol Estrogen receptor Brain Choroid plexus Xenopus laevis
a b s t r a c t Estradiol (E2) with the strongest bioactivity of the estrogens, is synthesized by the cytochrome p450 aromatase enzyme and plays a key role in sex differentiation of the vertebrate’s gonads. In Xenopus, aromatase mRNA is highly expressed in the brain rather than in the gonad during sex differentiation. In this study, we analyzed the stage change, tissue specificity, and localization of the aromatase expression in the Xenopus brain. Regardless of the sex difference, expression level of aromatase was remarkably higher in the brain than in other tissues during the early stages of brain morphogenesis and was observed in the formation regions of the choroid plexus of cerebral ventricle and the paleocortex and olfactory bulb of the prosencephalon. However, E2 concentrations in each tissue indicated a different localization of aromatase and were seen in the heart at almost double the level as seen in the brain. In addition, while aromatase expression level in the brain was increasing, E2 in the whole body began to increase at the same stage. Since the expression level of estrogen receptor a also corresponded to localization of E2, these results may imply that the E2 synthesized by the high aromatase expression in the choroid plexus, which generates cerebrospinal fluid, circulates to the heart and acts through ERa. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Estrogens are synthesized by the cytochrome p450 aromatase enzyme encoded on the CYP19a1 gene. Estradiol (E2), with the strongest bioactivity of the estrogens, is a sex steroid that acts on various organs, including the brain, to regulate reproductive behavior (Simpson, 2003). Exposure to estrogenic compounds causes male-to-female sex reversal in Xenopus gonads (Lutz et al., 2008; Pettersson and Berg, 2007). Furthermore, aromatase expression has been reported not only in the gonad of vertebrates but also in the brain (Chow et al., 2009; Harada and Honda, 2005; Kamat et al., 2002; Ramachandran et al., 1999; Urbatzka et al., 2007). Besides its role in neural development, E2 in the brain acts to protect against damage from neuronal injury and disease (Azcoitia et al., 2011a; Duncan and Saldanha, 2011; Melcangi et al., 2011). E2 also is important for sexual differentiation in the rodent’s brain (McCarthy, 2008). However, in mammals, expression level of aromatase mRNA in the brain is not necessarily high as compared to other tissues (Chow et al., 2009; Furbass et al., 1997; Vanselow et al., 1999; Yamada-Mouri et al., 1995). On the other hand, aromatase mRNA in the Xenopus brain is highly expressed as compared to other tissues and aromatase expressions in each tissue are also restricted to the brain and gonads (Nakagawa and Iwabuchi, 2012; ⇑ Corresponding author. Fax: +81 3 5317 9433. E-mail address: [email protected] (J. Iwabuchi). 0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.09.024
Urbatzka et al., 2007). In addition, we previously reported that the aromatase of Xenopus is expressed in the brain at a fivefold higher level than in the gonads at the sex differentiation stage (Iwabuchi et al., 2007). These findings suggest that the high aromatase expression in the brain may have a more important role in Xenopus than in mammals. Therefore, we analyzed when and where the aromatase expression level in each individual with ZZ- or ZW-type sex chromosomes increased in the Xenopus brain and how E2 concentrations changed in the brain or in the whole body along with a change in the expression level of aromatase in the brain. E2 is known to exert its biological effects through genomic and non-genomic pathways. In recent years, E2 signals through the GPR30 of a G protein-coupled receptor (GPCR) have been reported, and it has been suggested that the GPR30 initiates rapid non-genomic signaling events. However, GPR30-mediated signaling remain unclear (Raz et al., 2008; Revankar et al., 2005). While classical estrogen receptors (ERa and ERb), which are ligand-activated transcriptional factors, mediate neuroprotection by regulating the anti-apoptotic members of the Bcl-2 family (Yao et al., 2007), ERs expressed at the plasma membrane may involve rapid membrane-initiated activation of growth factor signaling pathways such as the ERK/MAPK and PI3K/Akt cascades (Lebesgue et al., 2009; Micevych et al., 2010; Morkuniene et al., 2010). In the mammal brain, ERs are expressed throughout the brain with distinct patterns in different brain regions and with differing levels of
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Fig. 1. Expression patterns of aromatase mRNA in Xenopus at the larval stage. Aromatase mRNA levels were quantified in the mixture of ZZ and ZW brains at stages 42, 44, 46, 48, 50, 52, 54, and 56 (A) and in the ZZ or ZW brain, gonads, liver, and heart at stages 50 and 56 (B). The relative transcription levels of aromatase mRNA were normalized to the transcription level of EF-1as mRNA. The expression level in brains at stage 50 was set at 100%. Each real-time RT-PCR analysis was performed at least in triplicate. Error bars represent the standard error (SE) of means. An asterisk indicates a significant difference of mRNA expression level between ZZ and ZW using a Student t-test (⁄P < 0.05).
expression during development in both neurons and glia (Azcoitia et al., 2011b; Wilson et al., 2011). In amphibians, ERa and ERb mRNA levels have been suggested in whole Silurana tropicalis embryos. ERa and ERb mRNA levels increase after neurulation and show different expression patterns in the period of tissue differentiation. The increasing rate of ERa mRNA levels is seen at a tenfold higher level than the increasing rate of ERb mRNA levels. In the brain, ERa and ERb mRNA levels gradually increase in developing from premetamorphosis to beginning of metamorphic climax. The sex differences are only detected at the adult stages for ERb mRNA level (Duarte-Guterman and Trudeau, 2010; Langlois et al., 2010). We previously isolated the ERa and ERb mRNA variants with the differed transcriptional activation function I (AF-1) region from the brain and gonads of the Xenopus embryo (Iwabuchi et al., 2008). Therefore, in order to investigate the relation between aromatase expression level and E2 concentration, the expression levels of the ERs’ mRNA and protein were analyzed in the brain at various stages and in each tissue at tadpole stage. To elucidate why aromatase is highly expressed in the Xenopus brain, we investigated the expression profile of aromatase in the Xenopus brain and the localization of estradiol and estrogen receptors in each tissue. Here, we show that aromatase in the choroid
plexus of the brain of developing Xenopus embryos is expressed regardless of sex and that E2 and ERa expressions are localized in the whole body. We suppose that E2 synthesized by aromatase in the brain will circulate to the whole body. 2. Materials and methods 2.1. Animals Sexually mature individuals of Xenopus laevis were purchased from a commercial supplier in Japan. The frogs were kept in individual containers containing dechlorinated tap water at 22 °C until use. Xenopus tadpoles were generated through in-house breeding by injecting sexually mature males and females with human chorionic gonadotropin (Sigma–Aldrich Inc., St. Louis, Mo, USA) via the dorsal lymph sac. The tadpoles were housed in dechlorinated tap water at 22 °C and were fed once every other day from Nieuwkoop and Faber’s (Nieuwkoop and Faber, 1994) developmental stage 45, which tadpoles starts to feed independently, until stage 56. The tadpoles at the stages indicated in the following experiments were euthanized for RNA isolation, protein extraction, or thin section analysis. The experiments described were performed
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Fig. 2. Morphological change of the Xenopus brain at stages 42–54. Vertical section of the hemisphere (A–D) and cross section of the prosencephalon (E–H) for stage 42 (A and E), stage 46 (B and F), stage 50 (C and G), and stage 54 (D and H) were stained with hematoxylin–eosin (HE). Arrowheads show the formation region at the stage. PSC, prosencephalon; DC, diencephalon; MSC, mesencephalon; MTC, metencephalon; ID, infundibulum; PG, pituitary gland; LV, lateral ventricle; PC, paleocortex; CP; choroid plexus, OB, olfactory bulb; OL, optic lobe. Scale bars; 200 lm.
according to the Guidelines for Care and Use of Animals approved by the Ethics Committees of Nihon University. 2.2. Real-time RT-PCR Determining ZW or ZZ status in individual animals was performed by PCR of genomic DNA isolated from the tail of the tadpole
(Yoshimoto et al., 2008). After the tail was denatured in 180 lL of 50 mM NaOH at 95 °C for 10 min, 20 lL of 1 M Tris–HCl (pH 8.0) was added to the tail lysate, and the tube was vortexed and centrifuged for 5 min at 12,000 rpm. Then, 0.5 lL of supernatant fluid was used to amplify the DM-W gene to determine the ZW type. The DM-W gene was amplified by a KOD FX Neo (Toyobo Co., Osaka, Japan). The PCR was carried out with the primer pairs F, 50 -CCA
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Fig. 3. Expression patterns of aromatase protein in Xenopus at the larval stage. Western blotting analysis was performed using anti-aromatase antibody and antiactin antibody as an internal standard. Mix of nuclear and cytosolic extracts from the mixture of ZZ and ZW brains at stages 42, 44, 46, 48, 50, 52, 54, and 56 (A) and brain, gonads, liver, and heart at stages 50 and 56 (B) were examined by immunoblotting with each primary antibody followed by an HRP-conjugated anti-mouse or rabbit IgG antibody.
CAC CCA GCT CAT GTA AAG-30 and R, 50 -GGG CAG AGT CAC ATA TAC TG-30 for the amplification. The thermal conditions for PCR included a single cycle at 94 °C for 2 min, followed by 32 cycles at 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 1 min. Total RNA was isolated from the brain, gonads, liver, and heart of Xenopus at various stages. After the isolated RNA was treated with DNase I (Invitrogen, Grand Island, NY, USA), first-strand cDNA was synthesized from the 500 ng RNA using a PrimeScript™ RT reagent kit (Takara Bio Inc. Otsu, Japan). Real-time RT-PCR was performed as described previously (Nakagawa and Iwabuchi, 2012). The primers used in the PCR were as follows: F, 50 -AAG CCT TGA ATC CAG TGC AG-30 and R, 50 -CGC CAT TAA TCC AGA CTC TCA C30 for aromatase. As an internal control, EF-1as expression was examined by PCR using specific primers as follows: F, 50 -CCA GAT TGG TGC TGG ATA TG-30 and R, 50 -TTC TGA GCA GAC TTT GTG AC-30 . The primers for additional estrogen receptor genes were designed based on GenBank sequences: ERa (accession number AB435403; F, 50 -AAC AGA AGG CAG AGT GGA AG-30 and R,
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50 -TGT TCT TGT CTA TAG TGC ACT GAT T-30 ; product size: 232 bp) and ERb (accession number AB435407; F, 50 -TCT TAC TTG GAT AGC AGG CAT-30 and R, 50 -CTT CTG ATT TAG CTT CAA ACC AT-30 ; product size: 232 bp). Specificity of every primer set was confirmed by cloning and sequencing the single amplicon obtained. The reaction mixture for real-time RT-PCR using a KAPA SYBRÒ FAST qPCR kit (Kapa Biosystems, South Africa) contained reaction buffer, MgCl2, dNTPs, DNA polymerase and SYBR Green I and was prepared according to the manufacturer’s instructions. PCR amplifications were performed in an ABI PRISMÒ 7700 Sequence Detection System (Bio-Rad, Hercules, CA, USA). Thermal conditions were 95 °C for 30 s and then 40 cycles at 95 °C for 3 s and 60 °C for 30 s. PCR amplifications were performed at least in triplicate. 2.3. Preparation of the brain section Whole bodies of Xenopus at stages 42, 46, 50, and 54 were fixed in 4% paraformaldehyde for 2 h at 4 °C. Following rinsing 3 times in PBS, samples were dehydrated through a 50%, 70%, 80%, 90%, 95%, and 100% ethanol series and remained in absolute ethanol overnight at 4 °C. Then, the samples were embedded with paraffin and were sliced to a thickness of 10 lm. The sections were extended on a glass slide filled with warm water and then were dried. 2.4. Hematoxylin-eosin (HE) staining The sections on glass slides were deparaffinized in xylene and were hydrated through a 100%, 95%, 90%, 80%, 70%, and 50% ethanol series. For HE staining, the sections were stained with Mayer’s Hematoxylin for 1 min, washed briefly with water, stained with eosin for 5 min, washed with 100% ethanol, replaced with xylene, and then air-dried. The sections were covered with Entellan newÒ (Merck Chemicals, Darmstadt, Germany) and were observed under the microscope.
Fig. 4. Localization of aromatase protein in Xenopus brain at stage 50. The brain at stage 50 from the dorsal viewpoint to show orientation of sections in B-P (A); the vertical section (B–H), and the cross section (I–P). Immunohistochemistry (IHC) staining was performed using anti-aromatase antibody followed by an HRP-conjugated anti-mouse IgG antibody. Nuclei were counterstained by hematoxylin. Red arrowheads show parts with strong staining. PSC, prosencephalon; DC, diencephalon; MSC, mesencephalon; PC, paleocortex; CP; choroid plexus, OB, olfactory bulb. Scale bars, 200 lm. (For interpretation of color in this Figure, the reader is referred to the web version of this article.)
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to the precipitates. Ice-cold CER II was added to the tube, and the tube was centrifuged for 5 min at 14,500 rpm. The supernatant was used as a cytosolic protein. In the extraction of nuclear and cytoplasmic extracts, the pellet fractions were resuspended in ice-cold NER, and the suspension was centrifuged for 10 min at 14,500 rpm. Supernatant fluid was added to the cytosolic protein as a nuclear protein. For Western blotting, 10–30 lg of the proteins was loaded on a 10% SDS–PAGE and transferred to a PVDF membrane (GE Healthcare). The membranes were blocked overnight at 4 °C in PBST with 3% membrane blocking agent (GE Healthcare) and were incubated with mouse anti-aromatase antibody or rabbit anti-ERa antibody (Abcam, Cambridge, MA, USA; ab37438) (supplemental Fig. 2) at a 1:500 dilution and anti-mouse IgG or anti-rabbit IgG conjugated with HRP (GE Healthcare) at a 1:10,000 dilution as a secondary antibody. The antibody binding proteins on the membrane were visualized by chemiluminescence using an ECL Plus™ kit (GE Healthcare) according to the manufacturer’s protocol. 2.7. Enzyme-linked immunoassay (EIA)
Fig. 5. Time course and tissue distribution of estradiol (E2) concentration in Xenopus at the larval stage. E2 concentrations were quantified in the mixture of ZZ and ZW brains or the whole body at stages 42, 44, 46, 48, 50, 52, 54, and 56 (A) and brain, gonads, liver, and heart at stage 50 (B). Each point presents mean ± SE (n P 3) of E2 concentration in pg per brain or individual (A) or tissue (B).
2.5. Immunohistochemistry (IHC) staining The sections on glass slides deparaffinized with xylene were rehydrated through the ethanol series. For IHC staining, the sections were activated with 0.03% trypsin (Invitrogen) for 20 min and treated with 3% H2O2 in methanol for 30 min to eliminate endogenous peroxidase. After blocking non-specific IgG binding sites with 5% goat serum in PBST for 60 min, mouse anti-aromatase antibody (ARK Resource Co., Ltd., Kumamoto, Japan) (supplemental Fig. 1) diluted 1:500 in PBS containing 1% BSA and 0.2% Triton X100 was applied (overnight at 4 °C in a humid chamber). After washes in PBST, HRP-labeled goat anti-mouse IgG (GE Healthcare, Little Chalfont, UK) diluted 1:100 in PBS containing 1% BSA and 0.2% Triton X-100 was applied (60 min at 4 °C in a humid chamber). The immunoreactions were visualized with a 0.015% H2O2 substrate and a 0.05% diaminobenzidine (DAB) chromogen. A light counterstain of Mayer’s Hematoxylin also was applied. After washing, the sections were air-dried, covered with Entellan newÒ, and observed under the microscope. 2.6. Western blotting Nuclear and cytoplasm extracts were prepared from tissue at various stage embryos using an NE-PERÒ Nuclear and Cytoplasmic Extraction Kit (Pierce, Rockford, IL, USA). After the tissues were homogenized, Cytoplasmic Extraction Reagent I (CER I) was added
Steroid extraction was performed using the method of Bligh & Dyer (Bligh and Dyer, 1959). After tissues dissected from more than 50 individuals were homogenized, the precipitates were dried in 15-mL glass tubes, and 1 mL of CHCl3 was added. Next, 2.8 mL of 1:2.5 (v/v) AcOH:MeOH was added, and the glass tubes were incubated for 10 min at room temperature. One milliliter of CHCl3 and 1 mL of dH2O were added to the glass tubes, and the tubes were centrifuged for 10 min at 3000 rpm. The steroids contained in the liquid organic phase were collected into additional glass tubes. The tissue content contained in the aqueous phase was added to 1 mL of CHCl3 and centrifuged for 10 min at 3000 rpm. The liquid organic phase was collected into glass tubes, as described above, and dried. The concentration of estradiol (E2) was measured by EIA using an Estradiol EIA Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s protocol. The steroids were redissolved in 0.2–0.5 mL of EIA buffer included in the kit as a sample, and the E2 in the samples was assayed using 50 lL of redissolved steroids along with unextracted standards of equivalent E2 concentration at least in triplicate. Detection limits (80% B/B0) for E2 in samples were 22 pg/mL (supplemental Fig. 3A). Different amounts (25, 50, 250, 1250, and 2500 pg) of known E2 were added to tissue. The extraction efficiency calculated for 25 pg, 50 pg, 250 pg, 1250 pg, and 2500 pg was 86.1%, 69.3%, 107.8%, 69.7%, and 74.3%, respectively (supplemental Fig. 3B). The range of measured E2 concentrations was between 150 and 2352 pg/mL. 2.8. Statistical analysis Data for all the genes and E2 concentrations were analyzed as mean values ± standard error. Statistical analysis was done with Microsoft Excel. Significance among stages was tested by ANOVA followed by Tukey’s test. The measurements in each tissue were subjected to analysis with Student’s t-tests to compare the mRNA expression between ZW and ZZ samples. The criterion for significance was P < 0.05. 3. Results 3.1. Expression changes of aromatase mRNA in the Xenopus brain and the differential expression levels in each tissue between ZZ and ZW individuals The expression patterns of aromatase mRNA in Xenopus were studied by real-time RT-PCR. The RNA extracts were prepared from
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Fig. 6. Expression characteristics of estrogen receptors (ERs) in Xenopus at the larval stage. ER mRNA levels were quantified in the mixture of ZZ and ZW brains at stages 42, 44, 46, 48, 50, 52, 54, and 56 (A) and in ZZ or ZW brain, gonads, liver, and heart at stage 50 (B and C) for ERa (A and B) and ERb (C). The relative transcription levels of ER mRNA were normalized to the transcription level of EF-1as mRNA. The expression level in the stage-50 brains was set at 100%. Each real-time RT-PCR analysis was performed at least in triplicate. Error bars represent the standard error (SE) of means. ⁄P < 0.05. Western blotting analysis was performed using anti-ERa antibody and anti-actin antibody as an internal standard. Mix of nuclear and cytosolic extracts from the mixture of ZZ and ZW brains at stages 42, 44, 46, 48, 50, 52, 54, and 56 (D) and brain, gonads, liver, and heart at stage 50 (E) was examined by immunoblotting with each primary antibody followed by an HRP-conjugated anti-rabbit IgG antibody.
the ZZ and ZW brain mixture of Xenopus embryos at stages 42, 44, 46, 48, 50, 52, 54, and 56 and from the brain, gonads, liver, and heart separated as ZZ and ZW in tissues from stages 50 and 56. Expression levels of aromatase mRNA in the Xenopus brain were higher than they were in other tissues and showed no difference between ZZ and ZW individuals. In the brain, aromatase mRNA level was elevated to stage 48 from stage 42 and was maintained up to stage 56 (Fig. 1A). In the stage 50 and 56 tissues, the expression level in the gonads was significantly higher in ZW than in ZZ. Although the expression level in the brain was detected at a threefold higher level in the brain of ZW and ZZ than in the gonads of ZW, no difference was observed in expression levels by sex. Expressions in the liver and heart were hardly detected (Fig. 1B).
3.2. Development of the Xenopus brain from the tailbud stage Brain sections were analyzed by HE staining in order to investigate development of the brain at the stage in which aromatase mRNA level is increasing. In the development of the Xenopus brain from the tailbud stage to the premetamorphic stage, the formation of the paleocortex, lateral ventricle, choroid plexus, olfactory bulb, and optic lobe was seen. At stage 42 in the brain (Fig. 2A and E), although the prosencephalon, diencephalon, mesencephalon, and metencephalon were formed along the anteroposterior axis, the paleocortex and lateral ventricle were not observed. At the stage 46, the paleocortex and lateral ventricle are beginning to form (Fig. 2B and F, arrowhead). The prosencephalon, diencephalon, mesencephalon,
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and metencephalon were stretched toward the anteroposterior axis, and the paleocortex and lateral ventricle were remarkably formed. Furthermore, at the stage 50, the choroid plexus, which generates cerebrospinal fluid (CSF), was found in the lateral ventricle, and the olfactory bulb and optic lobe were remarkably formed (Fig. 2C and G, arrowhead). At the stage 54, although each brain region was expanded intensely and the nucleus spread, further differentiation of brain regions was not seen (Fig. 2D and H).
brain and whole body were found in similar amounts at stages 42–46, the difference in concentration occurred at stages 48–56. In the tissues at stage 50, despite the high expression level of brain aromatase mRNA and protein, the E2 concentration in the brain was almost equal to that in the gonads and liver and is approximately half the concentration of the heart (Fig. 5B).
3.3. Expression level and localization of the aromatase protein in the Xenopus brain
Finally, expression levels of the ERs mRNA and ERa protein were analyzed using real-time RT-PCR and Western blotting. Expression levels of ERa in each tissue showed results similar to E2 concentrations. The protein levels and the mRNA levels in the brain are not higher than in other tissues. Almost no expression change was detected in the brain. In the stage-50 tissues, although the ERb mRNA level in the gonads was detected specifically and was significantly higher in the ZW gonad than in the ZZ gonads, the expression was hardly detected in the brain, liver and heart (Fig. 6C). The ERa mRNA level was observed in each tissue and was significantly higher in the ZW gonads than in the ZZ gonads (Fig. 6B). Since the ERb mRNA level was barely seen in the brain, only the ERa mRNA level was measured in the brain at each stage. The ERa mRNA levels gradually doubled in stages 42–56 (Fig. 6A). In the analysis of the ERa protein level, although the results partially correlated with the data of the real-time RT-PCR, expression change in the brain at each stage was not observed, and the highest expression level was found in the heart (Fig. 6D and E). As a result, the ERa expression levels at each stage and in each tissue hardly indicated correlation with aromatase expression level. However, ERa expression level was relatively correlated with E2 concentration at the change stages in the brain and in each tissue.
Since the differentiation of the Xenopus brain occurred along with an increase in aromatase mRNA level, expression level and localization of aromatase proteins were detected by a specific antibody (supplemental Fig. 1). The expression patterns of aromatase protein were analyzed from the mixture of ZZ and ZW tissues by Western blotting. The aromatase protein levels in the Xenopus brain showed change similar to the mRNA levels and localizations in the choroid plexus, paleocortex, and olfactory bulb were seen. In the brain, expression levels of aromatase protein were detected at the stage 46 and were gradually elevated to stage 54 (Fig. 3A). Although aromatase protein levels were difficult to detect at stage 50 except in the brain, at stage 56, aromatase protein levels were found in the gonads, liver, and heart in addition to the brain. As compared with other tissues, the high expression level in the brain was notably observed (Fig. 3B). Although the cause by which the aromatase protein level was observed in the liver and heart in the near absence of the mRNA level is unclear, these results may be based on the difference in normalization by total protein or total mRNA. To investigate the regions where estradiol (E2) is generated in the brain, localizations of aromatase protein were analyzed in the brain at stage 50 using IHC. High expression sites of aromatase protein were observed in the choroid plexus of the cerebral ventricle and the paleocortex and olfactory bulb of the prosencephalon which remarkably formed after stage 46 (Fig. 4, arrowhead). However, expression sites in the brain at stages 42 and 46 were not detected (data not shown). 3.4. Quantitative analysis of estradiol (E2) in the Xenopus brain and whole body In order to investigate the association between E2 and aromatase protein level, the E2 concentrations were measured from the mixture of ZZ and ZW tissues using E2-EIA. Total lipid extracts were prepared from the brain and the whole body, including the brain, at stages 42, 44, 46, 48, 50, 52, 54, and 56 and from the brain, gonads, liver, and heart at stage 50. The time course of E2 concentration in the Xenopus whole body has been previously reported by radioimmunoassay (Bogi et al., 2002). Normalization of measured E2 concentrations at 1 g fresh body mass decreased gradually in the whole body at stages 42–56. In this paper, since the brain and body size change in these stages was remarkable, the measured E2 concentrations were normalized by 1 individual or tissue. The increasing rate of E2 concentrations in the whole body is higher than the increasing rate of E2 concentrations in the brain, and the rise of E2 also occurs following the up-regulation of aromatase. At stages 42–46, the E2 concentrations in the brain changed to 3.11 ± 0.61 from 0.92 ± 0.21 pg (per brain), while the E2 concentrations in the whole body changed to 5.40 ± 4.54 from 1.25 ± 0.84 pg (per individual). At stages 48–56, the E2 concentrations in the brain changed to 24.3 ± 2.61 from 3.51 ± 0.22 pg in a gently sloping curve. On the other hand, the concentrations in the whole body began to increase rapidly and changed to 155.7 ± 23.2 from 25.5 ± 2.58 pg (Fig. 5A). Although the E2 concentrations in the
3.5. Expression analysis of estrogen receptors (ERs) mRNA and protein
4. Discussion This study showed that higher aromatase mRNA level in the Xenopus brain than in other tissues occurs irrespective of sex differences, and the protein level is detected in the early stages of brain morphogenesis. Furthermore, it has been reported that at the adult stage, the brain aromatase expression level, which is not related to sex differences, is observed (Urbatzka et al., 2007). In immunohistochemical analysis, we found that aromatase protein began to be expressed in the choroid plexus of cerebral ventricle and in the paleocortex and olfactory bulb of the prosencephalon, which is formed at the stages 42–50. In the mammals, reelin is thought to be essential for the inside-out formation of neocortical layers in the developing neocortex (Yoshida et al., 2006). In the dentate gyrus (DG) and the Purkinje cells (PC) of mice, the expression of ERa is found in Cajal–Retzius (CR) cells, which regulate neuronal migration and synaptogenesis via reelin. E2 increase the reelin expression of CR cells, and inhibition of aromatase activity reduce reelin expression. In addition, it has been reported that E2 regulates a signaling protein involved in neuronal differentiation (Bender et al., 2010; Biamonte et al., 2009). In the amphibian pallium, neurons are aggregated into several periventricular cell rows and have hardly migrated away from the cerebral ventricle (Perez-Garcia et al., 2001). In Xenopus, in contrast to the aromatase expression level, the stage changes in E2 concentration and ERa expression level were not observed so much in the early brain-forming phase. Moreover, E2 and ERa were also remarkable, except in the brain. Actually, we were not able to observe that outgrowth and proliferation of brain cells, such as neurons and astrocytes, were induced by treatment or inhibition of E2 under in vitro circumstance (supplemental Fig. 4). Moreover, reelin mRNA level remained unchanged in the Xenopus brain at stages
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42–54 (supplemental Fig. 5). Therefore, the rise of aromatase expression level may not be associated with morphogenesis, such as a sexual dimorphism and neurogenesis, at least in the Xenopus brain at the larval stage, although consistent expression of ERa may be useful for a protective effect or synapse formation. One possible explanation for why high-level of E2 and ERa were localized in the heart, which has almost no aromatase expression level, is that E2 generated by aromatase in the choroid plexus of the cerebral ventricle may flow to the heart. In this study, we observed that E2 in the whole body is increases greatly, while aromatase protein level in the brain rose at the stage 46, although E2 in the brain changes little. In the cerebral ventricle of mammals, the choroid plexus secretes the cerebrospinal fluid (CSF) that fills the erebral ventricle system and the subarachnoid space. Then, a part of the CSF is absorbed from the arachnoid granule or from the brain capillary, goes into a thick vein, and is carried to the heart (Greitz and Hannerz, 1996). In rats, it has been reported that E2 not only rescues neurons from global ischemia-induced cell death in the brain but also reduces ischemia-induced injury in both male and female hearts (Dai et al., 2007; Lebesgue et al., 2009). In humans, the cardiac protection of E2 is caused mainly by ERa; ERa can exert a rapid transcription-independent effect, which involves membrane-initiated activation of signaling pathways. such as activation of the PI3K–Akt cascade (Deroo and Korach, 2006; Novotny et al., 2009). Moreover, in mammal hearts and gonads, E2 treatment results in up-regulation of ERa and ERb under both in vivo and in vitro circumstances (Bliedtner et al., 2010; Hsieh et al., 2006; Jankowski et al., 2001; Lin et al., 2011; Nakamura et al., 2008). In Xenopus, while aromatase protein level was not seen in the heart at stage 50, ERa protein level was seen at a higher level in the heart than in the brain. These findings may suggest that E2 generated by the choroid plexus in the brain acts in protection of the heart through ERa. In the gonads of the vertebrate, the role of E2 differs in the effect range. In mammals, estrogens play a role in folliculogenesis and in the maintenance of the ovarian somatic cells (Britt and Findlay, 2002). However, in some fish, amphibians, and reptiles, estrogens are not only associated with ovarian development, as in the mammals, but with sex determination (Nakamura, 2010). In this study, aromatase and ERa/b mRNA were both significantly expressed in the gonads of ZW as compared with ZZ. Therefore, ER expression with sex differences at stage 50, presumed to be the sex determination period in Xenopus gonads, may be related to sex determination and differentiation (Yoshimoto and Ito, 2011). 5. Conclusion In this experiment, we revealed three findings: Brain aromatase (1) is expressed remarkably early as compared with other tissues, irrespective of sex differences; (2) begins expression along with the formation of the choroid plexus of the cerebral ventricle and the paleocortex and olfactory bulb of the prosencephalon; (3) shows different expression characteristic from E2 and ERa that have been localized in each tissue. Interestingly, E2 secretion in the heart is considered to result from high aromatase expression in the brain. This finding is also supported by the fact that aromatase expressions in Xenopus are brain-specific expressions that are not tissue-specific as in mammals (Chow et al., 2009; Demura et al., 2008; Nakagawa and Iwabuchi, 2012; Urbatzka et al., 2007). Although the role of the cortical aromatase expression in the Xenopus brain is still unclear, it may work to protect the cells that are developed through differentiation instead of morphogenesis. Disclosure summary The authors have nothing to disclose.
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Acknowledgments We thank K. Masuda, K. Mizuta, K. Yamahana, R. Kitta and Y. Tamura for much help in the laboratory. This work was supported by grants from Nihon University.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ygcen.2013. 09.024.
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General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
Expression profile of the aromatase enzyme in the Xenopus brain and localization of estradiol and estrogen receptors in each tissue Junshin Iwabuchi ⇑, Kouta Koshimizu, Tadahiko Nakagawa Laboratory of Biochemistry, Department of Chemistry, College of Humanities and Sciences, Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan
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Article history: Received 5 June 2013 Revised 2 September 2013 Accepted 28 September 2013 Available online 14 October 2013 Keywords: Aromatase Estradiol Estrogen receptor Brain Choroid plexus Xenopus laevis
a b s t r a c t Estradiol (E2) with the strongest bioactivity of the estrogens, is synthesized by the cytochrome p450 aromatase enzyme and plays a key role in sex differentiation of the vertebrate’s gonads. In Xenopus, aromatase mRNA is highly expressed in the brain rather than in the gonad during sex differentiation. In this study, we analyzed the stage change, tissue specificity, and localization of the aromatase expression in the Xenopus brain. Regardless of the sex difference, expression level of aromatase was remarkably higher in the brain than in other tissues during the early stages of brain morphogenesis and was observed in the formation regions of the choroid plexus of cerebral ventricle and the paleocortex and olfactory bulb of the prosencephalon. However, E2 concentrations in each tissue indicated a different localization of aromatase and were seen in the heart at almost double the level as seen in the brain. In addition, while aromatase expression level in the brain was increasing, E2 in the whole body began to increase at the same stage. Since the expression level of estrogen receptor a also corresponded to localization of E2, these results may imply that the E2 synthesized by the high aromatase expression in the choroid plexus, which generates cerebrospinal fluid, circulates to the heart and acts through ERa. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Estrogens are synthesized by the cytochrome p450 aromatase enzyme encoded on the CYP19a1 gene. Estradiol (E2), with the strongest bioactivity of the estrogens, is a sex steroid that acts on various organs, including the brain, to regulate reproductive behavior (Simpson, 2003). Exposure to estrogenic compounds causes male-to-female sex reversal in Xenopus gonads (Lutz et al., 2008; Pettersson and Berg, 2007). Furthermore, aromatase expression has been reported not only in the gonad of vertebrates but also in the brain (Chow et al., 2009; Harada and Honda, 2005; Kamat et al., 2002; Ramachandran et al., 1999; Urbatzka et al., 2007). Besides its role in neural development, E2 in the brain acts to protect against damage from neuronal injury and disease (Azcoitia et al., 2011a; Duncan and Saldanha, 2011; Melcangi et al., 2011). E2 also is important for sexual differentiation in the rodent’s brain (McCarthy, 2008). However, in mammals, expression level of aromatase mRNA in the brain is not necessarily high as compared to other tissues (Chow et al., 2009; Furbass et al., 1997; Vanselow et al., 1999; Yamada-Mouri et al., 1995). On the other hand, aromatase mRNA in the Xenopus brain is highly expressed as compared to other tissues and aromatase expressions in each tissue are also restricted to the brain and gonads (Nakagawa and Iwabuchi, 2012; ⇑ Corresponding author. Fax: +81 3 5317 9433. E-mail address: [email protected] (J. Iwabuchi). 0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.09.024
Urbatzka et al., 2007). In addition, we previously reported that the aromatase of Xenopus is expressed in the brain at a fivefold higher level than in the gonads at the sex differentiation stage (Iwabuchi et al., 2007). These findings suggest that the high aromatase expression in the brain may have a more important role in Xenopus than in mammals. Therefore, we analyzed when and where the aromatase expression level in each individual with ZZ- or ZW-type sex chromosomes increased in the Xenopus brain and how E2 concentrations changed in the brain or in the whole body along with a change in the expression level of aromatase in the brain. E2 is known to exert its biological effects through genomic and non-genomic pathways. In recent years, E2 signals through the GPR30 of a G protein-coupled receptor (GPCR) have been reported, and it has been suggested that the GPR30 initiates rapid non-genomic signaling events. However, GPR30-mediated signaling remain unclear (Raz et al., 2008; Revankar et al., 2005). While classical estrogen receptors (ERa and ERb), which are ligand-activated transcriptional factors, mediate neuroprotection by regulating the anti-apoptotic members of the Bcl-2 family (Yao et al., 2007), ERs expressed at the plasma membrane may involve rapid membrane-initiated activation of growth factor signaling pathways such as the ERK/MAPK and PI3K/Akt cascades (Lebesgue et al., 2009; Micevych et al., 2010; Morkuniene et al., 2010). In the mammal brain, ERs are expressed throughout the brain with distinct patterns in different brain regions and with differing levels of
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Fig. 1. Expression patterns of aromatase mRNA in Xenopus at the larval stage. Aromatase mRNA levels were quantified in the mixture of ZZ and ZW brains at stages 42, 44, 46, 48, 50, 52, 54, and 56 (A) and in the ZZ or ZW brain, gonads, liver, and heart at stages 50 and 56 (B). The relative transcription levels of aromatase mRNA were normalized to the transcription level of EF-1as mRNA. The expression level in brains at stage 50 was set at 100%. Each real-time RT-PCR analysis was performed at least in triplicate. Error bars represent the standard error (SE) of means. An asterisk indicates a significant difference of mRNA expression level between ZZ and ZW using a Student t-test (⁄P < 0.05).
expression during development in both neurons and glia (Azcoitia et al., 2011b; Wilson et al., 2011). In amphibians, ERa and ERb mRNA levels have been suggested in whole Silurana tropicalis embryos. ERa and ERb mRNA levels increase after neurulation and show different expression patterns in the period of tissue differentiation. The increasing rate of ERa mRNA levels is seen at a tenfold higher level than the increasing rate of ERb mRNA levels. In the brain, ERa and ERb mRNA levels gradually increase in developing from premetamorphosis to beginning of metamorphic climax. The sex differences are only detected at the adult stages for ERb mRNA level (Duarte-Guterman and Trudeau, 2010; Langlois et al., 2010). We previously isolated the ERa and ERb mRNA variants with the differed transcriptional activation function I (AF-1) region from the brain and gonads of the Xenopus embryo (Iwabuchi et al., 2008). Therefore, in order to investigate the relation between aromatase expression level and E2 concentration, the expression levels of the ERs’ mRNA and protein were analyzed in the brain at various stages and in each tissue at tadpole stage. To elucidate why aromatase is highly expressed in the Xenopus brain, we investigated the expression profile of aromatase in the Xenopus brain and the localization of estradiol and estrogen receptors in each tissue. Here, we show that aromatase in the choroid
plexus of the brain of developing Xenopus embryos is expressed regardless of sex and that E2 and ERa expressions are localized in the whole body. We suppose that E2 synthesized by aromatase in the brain will circulate to the whole body. 2. Materials and methods 2.1. Animals Sexually mature individuals of Xenopus laevis were purchased from a commercial supplier in Japan. The frogs were kept in individual containers containing dechlorinated tap water at 22 °C until use. Xenopus tadpoles were generated through in-house breeding by injecting sexually mature males and females with human chorionic gonadotropin (Sigma–Aldrich Inc., St. Louis, Mo, USA) via the dorsal lymph sac. The tadpoles were housed in dechlorinated tap water at 22 °C and were fed once every other day from Nieuwkoop and Faber’s (Nieuwkoop and Faber, 1994) developmental stage 45, which tadpoles starts to feed independently, until stage 56. The tadpoles at the stages indicated in the following experiments were euthanized for RNA isolation, protein extraction, or thin section analysis. The experiments described were performed
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Fig. 2. Morphological change of the Xenopus brain at stages 42–54. Vertical section of the hemisphere (A–D) and cross section of the prosencephalon (E–H) for stage 42 (A and E), stage 46 (B and F), stage 50 (C and G), and stage 54 (D and H) were stained with hematoxylin–eosin (HE). Arrowheads show the formation region at the stage. PSC, prosencephalon; DC, diencephalon; MSC, mesencephalon; MTC, metencephalon; ID, infundibulum; PG, pituitary gland; LV, lateral ventricle; PC, paleocortex; CP; choroid plexus, OB, olfactory bulb; OL, optic lobe. Scale bars; 200 lm.
according to the Guidelines for Care and Use of Animals approved by the Ethics Committees of Nihon University. 2.2. Real-time RT-PCR Determining ZW or ZZ status in individual animals was performed by PCR of genomic DNA isolated from the tail of the tadpole
(Yoshimoto et al., 2008). After the tail was denatured in 180 lL of 50 mM NaOH at 95 °C for 10 min, 20 lL of 1 M Tris–HCl (pH 8.0) was added to the tail lysate, and the tube was vortexed and centrifuged for 5 min at 12,000 rpm. Then, 0.5 lL of supernatant fluid was used to amplify the DM-W gene to determine the ZW type. The DM-W gene was amplified by a KOD FX Neo (Toyobo Co., Osaka, Japan). The PCR was carried out with the primer pairs F, 50 -CCA
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Fig. 3. Expression patterns of aromatase protein in Xenopus at the larval stage. Western blotting analysis was performed using anti-aromatase antibody and antiactin antibody as an internal standard. Mix of nuclear and cytosolic extracts from the mixture of ZZ and ZW brains at stages 42, 44, 46, 48, 50, 52, 54, and 56 (A) and brain, gonads, liver, and heart at stages 50 and 56 (B) were examined by immunoblotting with each primary antibody followed by an HRP-conjugated anti-mouse or rabbit IgG antibody.
CAC CCA GCT CAT GTA AAG-30 and R, 50 -GGG CAG AGT CAC ATA TAC TG-30 for the amplification. The thermal conditions for PCR included a single cycle at 94 °C for 2 min, followed by 32 cycles at 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 1 min. Total RNA was isolated from the brain, gonads, liver, and heart of Xenopus at various stages. After the isolated RNA was treated with DNase I (Invitrogen, Grand Island, NY, USA), first-strand cDNA was synthesized from the 500 ng RNA using a PrimeScript™ RT reagent kit (Takara Bio Inc. Otsu, Japan). Real-time RT-PCR was performed as described previously (Nakagawa and Iwabuchi, 2012). The primers used in the PCR were as follows: F, 50 -AAG CCT TGA ATC CAG TGC AG-30 and R, 50 -CGC CAT TAA TCC AGA CTC TCA C30 for aromatase. As an internal control, EF-1as expression was examined by PCR using specific primers as follows: F, 50 -CCA GAT TGG TGC TGG ATA TG-30 and R, 50 -TTC TGA GCA GAC TTT GTG AC-30 . The primers for additional estrogen receptor genes were designed based on GenBank sequences: ERa (accession number AB435403; F, 50 -AAC AGA AGG CAG AGT GGA AG-30 and R,
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50 -TGT TCT TGT CTA TAG TGC ACT GAT T-30 ; product size: 232 bp) and ERb (accession number AB435407; F, 50 -TCT TAC TTG GAT AGC AGG CAT-30 and R, 50 -CTT CTG ATT TAG CTT CAA ACC AT-30 ; product size: 232 bp). Specificity of every primer set was confirmed by cloning and sequencing the single amplicon obtained. The reaction mixture for real-time RT-PCR using a KAPA SYBRÒ FAST qPCR kit (Kapa Biosystems, South Africa) contained reaction buffer, MgCl2, dNTPs, DNA polymerase and SYBR Green I and was prepared according to the manufacturer’s instructions. PCR amplifications were performed in an ABI PRISMÒ 7700 Sequence Detection System (Bio-Rad, Hercules, CA, USA). Thermal conditions were 95 °C for 30 s and then 40 cycles at 95 °C for 3 s and 60 °C for 30 s. PCR amplifications were performed at least in triplicate. 2.3. Preparation of the brain section Whole bodies of Xenopus at stages 42, 46, 50, and 54 were fixed in 4% paraformaldehyde for 2 h at 4 °C. Following rinsing 3 times in PBS, samples were dehydrated through a 50%, 70%, 80%, 90%, 95%, and 100% ethanol series and remained in absolute ethanol overnight at 4 °C. Then, the samples were embedded with paraffin and were sliced to a thickness of 10 lm. The sections were extended on a glass slide filled with warm water and then were dried. 2.4. Hematoxylin-eosin (HE) staining The sections on glass slides were deparaffinized in xylene and were hydrated through a 100%, 95%, 90%, 80%, 70%, and 50% ethanol series. For HE staining, the sections were stained with Mayer’s Hematoxylin for 1 min, washed briefly with water, stained with eosin for 5 min, washed with 100% ethanol, replaced with xylene, and then air-dried. The sections were covered with Entellan newÒ (Merck Chemicals, Darmstadt, Germany) and were observed under the microscope.
Fig. 4. Localization of aromatase protein in Xenopus brain at stage 50. The brain at stage 50 from the dorsal viewpoint to show orientation of sections in B-P (A); the vertical section (B–H), and the cross section (I–P). Immunohistochemistry (IHC) staining was performed using anti-aromatase antibody followed by an HRP-conjugated anti-mouse IgG antibody. Nuclei were counterstained by hematoxylin. Red arrowheads show parts with strong staining. PSC, prosencephalon; DC, diencephalon; MSC, mesencephalon; PC, paleocortex; CP; choroid plexus, OB, olfactory bulb. Scale bars, 200 lm. (For interpretation of color in this Figure, the reader is referred to the web version of this article.)
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to the precipitates. Ice-cold CER II was added to the tube, and the tube was centrifuged for 5 min at 14,500 rpm. The supernatant was used as a cytosolic protein. In the extraction of nuclear and cytoplasmic extracts, the pellet fractions were resuspended in ice-cold NER, and the suspension was centrifuged for 10 min at 14,500 rpm. Supernatant fluid was added to the cytosolic protein as a nuclear protein. For Western blotting, 10–30 lg of the proteins was loaded on a 10% SDS–PAGE and transferred to a PVDF membrane (GE Healthcare). The membranes were blocked overnight at 4 °C in PBST with 3% membrane blocking agent (GE Healthcare) and were incubated with mouse anti-aromatase antibody or rabbit anti-ERa antibody (Abcam, Cambridge, MA, USA; ab37438) (supplemental Fig. 2) at a 1:500 dilution and anti-mouse IgG or anti-rabbit IgG conjugated with HRP (GE Healthcare) at a 1:10,000 dilution as a secondary antibody. The antibody binding proteins on the membrane were visualized by chemiluminescence using an ECL Plus™ kit (GE Healthcare) according to the manufacturer’s protocol. 2.7. Enzyme-linked immunoassay (EIA)
Fig. 5. Time course and tissue distribution of estradiol (E2) concentration in Xenopus at the larval stage. E2 concentrations were quantified in the mixture of ZZ and ZW brains or the whole body at stages 42, 44, 46, 48, 50, 52, 54, and 56 (A) and brain, gonads, liver, and heart at stage 50 (B). Each point presents mean ± SE (n P 3) of E2 concentration in pg per brain or individual (A) or tissue (B).
2.5. Immunohistochemistry (IHC) staining The sections on glass slides deparaffinized with xylene were rehydrated through the ethanol series. For IHC staining, the sections were activated with 0.03% trypsin (Invitrogen) for 20 min and treated with 3% H2O2 in methanol for 30 min to eliminate endogenous peroxidase. After blocking non-specific IgG binding sites with 5% goat serum in PBST for 60 min, mouse anti-aromatase antibody (ARK Resource Co., Ltd., Kumamoto, Japan) (supplemental Fig. 1) diluted 1:500 in PBS containing 1% BSA and 0.2% Triton X100 was applied (overnight at 4 °C in a humid chamber). After washes in PBST, HRP-labeled goat anti-mouse IgG (GE Healthcare, Little Chalfont, UK) diluted 1:100 in PBS containing 1% BSA and 0.2% Triton X-100 was applied (60 min at 4 °C in a humid chamber). The immunoreactions were visualized with a 0.015% H2O2 substrate and a 0.05% diaminobenzidine (DAB) chromogen. A light counterstain of Mayer’s Hematoxylin also was applied. After washing, the sections were air-dried, covered with Entellan newÒ, and observed under the microscope. 2.6. Western blotting Nuclear and cytoplasm extracts were prepared from tissue at various stage embryos using an NE-PERÒ Nuclear and Cytoplasmic Extraction Kit (Pierce, Rockford, IL, USA). After the tissues were homogenized, Cytoplasmic Extraction Reagent I (CER I) was added
Steroid extraction was performed using the method of Bligh & Dyer (Bligh and Dyer, 1959). After tissues dissected from more than 50 individuals were homogenized, the precipitates were dried in 15-mL glass tubes, and 1 mL of CHCl3 was added. Next, 2.8 mL of 1:2.5 (v/v) AcOH:MeOH was added, and the glass tubes were incubated for 10 min at room temperature. One milliliter of CHCl3 and 1 mL of dH2O were added to the glass tubes, and the tubes were centrifuged for 10 min at 3000 rpm. The steroids contained in the liquid organic phase were collected into additional glass tubes. The tissue content contained in the aqueous phase was added to 1 mL of CHCl3 and centrifuged for 10 min at 3000 rpm. The liquid organic phase was collected into glass tubes, as described above, and dried. The concentration of estradiol (E2) was measured by EIA using an Estradiol EIA Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s protocol. The steroids were redissolved in 0.2–0.5 mL of EIA buffer included in the kit as a sample, and the E2 in the samples was assayed using 50 lL of redissolved steroids along with unextracted standards of equivalent E2 concentration at least in triplicate. Detection limits (80% B/B0) for E2 in samples were 22 pg/mL (supplemental Fig. 3A). Different amounts (25, 50, 250, 1250, and 2500 pg) of known E2 were added to tissue. The extraction efficiency calculated for 25 pg, 50 pg, 250 pg, 1250 pg, and 2500 pg was 86.1%, 69.3%, 107.8%, 69.7%, and 74.3%, respectively (supplemental Fig. 3B). The range of measured E2 concentrations was between 150 and 2352 pg/mL. 2.8. Statistical analysis Data for all the genes and E2 concentrations were analyzed as mean values ± standard error. Statistical analysis was done with Microsoft Excel. Significance among stages was tested by ANOVA followed by Tukey’s test. The measurements in each tissue were subjected to analysis with Student’s t-tests to compare the mRNA expression between ZW and ZZ samples. The criterion for significance was P < 0.05. 3. Results 3.1. Expression changes of aromatase mRNA in the Xenopus brain and the differential expression levels in each tissue between ZZ and ZW individuals The expression patterns of aromatase mRNA in Xenopus were studied by real-time RT-PCR. The RNA extracts were prepared from
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Fig. 6. Expression characteristics of estrogen receptors (ERs) in Xenopus at the larval stage. ER mRNA levels were quantified in the mixture of ZZ and ZW brains at stages 42, 44, 46, 48, 50, 52, 54, and 56 (A) and in ZZ or ZW brain, gonads, liver, and heart at stage 50 (B and C) for ERa (A and B) and ERb (C). The relative transcription levels of ER mRNA were normalized to the transcription level of EF-1as mRNA. The expression level in the stage-50 brains was set at 100%. Each real-time RT-PCR analysis was performed at least in triplicate. Error bars represent the standard error (SE) of means. ⁄P < 0.05. Western blotting analysis was performed using anti-ERa antibody and anti-actin antibody as an internal standard. Mix of nuclear and cytosolic extracts from the mixture of ZZ and ZW brains at stages 42, 44, 46, 48, 50, 52, 54, and 56 (D) and brain, gonads, liver, and heart at stage 50 (E) was examined by immunoblotting with each primary antibody followed by an HRP-conjugated anti-rabbit IgG antibody.
the ZZ and ZW brain mixture of Xenopus embryos at stages 42, 44, 46, 48, 50, 52, 54, and 56 and from the brain, gonads, liver, and heart separated as ZZ and ZW in tissues from stages 50 and 56. Expression levels of aromatase mRNA in the Xenopus brain were higher than they were in other tissues and showed no difference between ZZ and ZW individuals. In the brain, aromatase mRNA level was elevated to stage 48 from stage 42 and was maintained up to stage 56 (Fig. 1A). In the stage 50 and 56 tissues, the expression level in the gonads was significantly higher in ZW than in ZZ. Although the expression level in the brain was detected at a threefold higher level in the brain of ZW and ZZ than in the gonads of ZW, no difference was observed in expression levels by sex. Expressions in the liver and heart were hardly detected (Fig. 1B).
3.2. Development of the Xenopus brain from the tailbud stage Brain sections were analyzed by HE staining in order to investigate development of the brain at the stage in which aromatase mRNA level is increasing. In the development of the Xenopus brain from the tailbud stage to the premetamorphic stage, the formation of the paleocortex, lateral ventricle, choroid plexus, olfactory bulb, and optic lobe was seen. At stage 42 in the brain (Fig. 2A and E), although the prosencephalon, diencephalon, mesencephalon, and metencephalon were formed along the anteroposterior axis, the paleocortex and lateral ventricle were not observed. At the stage 46, the paleocortex and lateral ventricle are beginning to form (Fig. 2B and F, arrowhead). The prosencephalon, diencephalon, mesencephalon,
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and metencephalon were stretched toward the anteroposterior axis, and the paleocortex and lateral ventricle were remarkably formed. Furthermore, at the stage 50, the choroid plexus, which generates cerebrospinal fluid (CSF), was found in the lateral ventricle, and the olfactory bulb and optic lobe were remarkably formed (Fig. 2C and G, arrowhead). At the stage 54, although each brain region was expanded intensely and the nucleus spread, further differentiation of brain regions was not seen (Fig. 2D and H).
brain and whole body were found in similar amounts at stages 42–46, the difference in concentration occurred at stages 48–56. In the tissues at stage 50, despite the high expression level of brain aromatase mRNA and protein, the E2 concentration in the brain was almost equal to that in the gonads and liver and is approximately half the concentration of the heart (Fig. 5B).
3.3. Expression level and localization of the aromatase protein in the Xenopus brain
Finally, expression levels of the ERs mRNA and ERa protein were analyzed using real-time RT-PCR and Western blotting. Expression levels of ERa in each tissue showed results similar to E2 concentrations. The protein levels and the mRNA levels in the brain are not higher than in other tissues. Almost no expression change was detected in the brain. In the stage-50 tissues, although the ERb mRNA level in the gonads was detected specifically and was significantly higher in the ZW gonad than in the ZZ gonads, the expression was hardly detected in the brain, liver and heart (Fig. 6C). The ERa mRNA level was observed in each tissue and was significantly higher in the ZW gonads than in the ZZ gonads (Fig. 6B). Since the ERb mRNA level was barely seen in the brain, only the ERa mRNA level was measured in the brain at each stage. The ERa mRNA levels gradually doubled in stages 42–56 (Fig. 6A). In the analysis of the ERa protein level, although the results partially correlated with the data of the real-time RT-PCR, expression change in the brain at each stage was not observed, and the highest expression level was found in the heart (Fig. 6D and E). As a result, the ERa expression levels at each stage and in each tissue hardly indicated correlation with aromatase expression level. However, ERa expression level was relatively correlated with E2 concentration at the change stages in the brain and in each tissue.
Since the differentiation of the Xenopus brain occurred along with an increase in aromatase mRNA level, expression level and localization of aromatase proteins were detected by a specific antibody (supplemental Fig. 1). The expression patterns of aromatase protein were analyzed from the mixture of ZZ and ZW tissues by Western blotting. The aromatase protein levels in the Xenopus brain showed change similar to the mRNA levels and localizations in the choroid plexus, paleocortex, and olfactory bulb were seen. In the brain, expression levels of aromatase protein were detected at the stage 46 and were gradually elevated to stage 54 (Fig. 3A). Although aromatase protein levels were difficult to detect at stage 50 except in the brain, at stage 56, aromatase protein levels were found in the gonads, liver, and heart in addition to the brain. As compared with other tissues, the high expression level in the brain was notably observed (Fig. 3B). Although the cause by which the aromatase protein level was observed in the liver and heart in the near absence of the mRNA level is unclear, these results may be based on the difference in normalization by total protein or total mRNA. To investigate the regions where estradiol (E2) is generated in the brain, localizations of aromatase protein were analyzed in the brain at stage 50 using IHC. High expression sites of aromatase protein were observed in the choroid plexus of the cerebral ventricle and the paleocortex and olfactory bulb of the prosencephalon which remarkably formed after stage 46 (Fig. 4, arrowhead). However, expression sites in the brain at stages 42 and 46 were not detected (data not shown). 3.4. Quantitative analysis of estradiol (E2) in the Xenopus brain and whole body In order to investigate the association between E2 and aromatase protein level, the E2 concentrations were measured from the mixture of ZZ and ZW tissues using E2-EIA. Total lipid extracts were prepared from the brain and the whole body, including the brain, at stages 42, 44, 46, 48, 50, 52, 54, and 56 and from the brain, gonads, liver, and heart at stage 50. The time course of E2 concentration in the Xenopus whole body has been previously reported by radioimmunoassay (Bogi et al., 2002). Normalization of measured E2 concentrations at 1 g fresh body mass decreased gradually in the whole body at stages 42–56. In this paper, since the brain and body size change in these stages was remarkable, the measured E2 concentrations were normalized by 1 individual or tissue. The increasing rate of E2 concentrations in the whole body is higher than the increasing rate of E2 concentrations in the brain, and the rise of E2 also occurs following the up-regulation of aromatase. At stages 42–46, the E2 concentrations in the brain changed to 3.11 ± 0.61 from 0.92 ± 0.21 pg (per brain), while the E2 concentrations in the whole body changed to 5.40 ± 4.54 from 1.25 ± 0.84 pg (per individual). At stages 48–56, the E2 concentrations in the brain changed to 24.3 ± 2.61 from 3.51 ± 0.22 pg in a gently sloping curve. On the other hand, the concentrations in the whole body began to increase rapidly and changed to 155.7 ± 23.2 from 25.5 ± 2.58 pg (Fig. 5A). Although the E2 concentrations in the
3.5. Expression analysis of estrogen receptors (ERs) mRNA and protein
4. Discussion This study showed that higher aromatase mRNA level in the Xenopus brain than in other tissues occurs irrespective of sex differences, and the protein level is detected in the early stages of brain morphogenesis. Furthermore, it has been reported that at the adult stage, the brain aromatase expression level, which is not related to sex differences, is observed (Urbatzka et al., 2007). In immunohistochemical analysis, we found that aromatase protein began to be expressed in the choroid plexus of cerebral ventricle and in the paleocortex and olfactory bulb of the prosencephalon, which is formed at the stages 42–50. In the mammals, reelin is thought to be essential for the inside-out formation of neocortical layers in the developing neocortex (Yoshida et al., 2006). In the dentate gyrus (DG) and the Purkinje cells (PC) of mice, the expression of ERa is found in Cajal–Retzius (CR) cells, which regulate neuronal migration and synaptogenesis via reelin. E2 increase the reelin expression of CR cells, and inhibition of aromatase activity reduce reelin expression. In addition, it has been reported that E2 regulates a signaling protein involved in neuronal differentiation (Bender et al., 2010; Biamonte et al., 2009). In the amphibian pallium, neurons are aggregated into several periventricular cell rows and have hardly migrated away from the cerebral ventricle (Perez-Garcia et al., 2001). In Xenopus, in contrast to the aromatase expression level, the stage changes in E2 concentration and ERa expression level were not observed so much in the early brain-forming phase. Moreover, E2 and ERa were also remarkable, except in the brain. Actually, we were not able to observe that outgrowth and proliferation of brain cells, such as neurons and astrocytes, were induced by treatment or inhibition of E2 under in vitro circumstance (supplemental Fig. 4). Moreover, reelin mRNA level remained unchanged in the Xenopus brain at stages
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42–54 (supplemental Fig. 5). Therefore, the rise of aromatase expression level may not be associated with morphogenesis, such as a sexual dimorphism and neurogenesis, at least in the Xenopus brain at the larval stage, although consistent expression of ERa may be useful for a protective effect or synapse formation. One possible explanation for why high-level of E2 and ERa were localized in the heart, which has almost no aromatase expression level, is that E2 generated by aromatase in the choroid plexus of the cerebral ventricle may flow to the heart. In this study, we observed that E2 in the whole body is increases greatly, while aromatase protein level in the brain rose at the stage 46, although E2 in the brain changes little. In the cerebral ventricle of mammals, the choroid plexus secretes the cerebrospinal fluid (CSF) that fills the erebral ventricle system and the subarachnoid space. Then, a part of the CSF is absorbed from the arachnoid granule or from the brain capillary, goes into a thick vein, and is carried to the heart (Greitz and Hannerz, 1996). In rats, it has been reported that E2 not only rescues neurons from global ischemia-induced cell death in the brain but also reduces ischemia-induced injury in both male and female hearts (Dai et al., 2007; Lebesgue et al., 2009). In humans, the cardiac protection of E2 is caused mainly by ERa; ERa can exert a rapid transcription-independent effect, which involves membrane-initiated activation of signaling pathways. such as activation of the PI3K–Akt cascade (Deroo and Korach, 2006; Novotny et al., 2009). Moreover, in mammal hearts and gonads, E2 treatment results in up-regulation of ERa and ERb under both in vivo and in vitro circumstances (Bliedtner et al., 2010; Hsieh et al., 2006; Jankowski et al., 2001; Lin et al., 2011; Nakamura et al., 2008). In Xenopus, while aromatase protein level was not seen in the heart at stage 50, ERa protein level was seen at a higher level in the heart than in the brain. These findings may suggest that E2 generated by the choroid plexus in the brain acts in protection of the heart through ERa. In the gonads of the vertebrate, the role of E2 differs in the effect range. In mammals, estrogens play a role in folliculogenesis and in the maintenance of the ovarian somatic cells (Britt and Findlay, 2002). However, in some fish, amphibians, and reptiles, estrogens are not only associated with ovarian development, as in the mammals, but with sex determination (Nakamura, 2010). In this study, aromatase and ERa/b mRNA were both significantly expressed in the gonads of ZW as compared with ZZ. Therefore, ER expression with sex differences at stage 50, presumed to be the sex determination period in Xenopus gonads, may be related to sex determination and differentiation (Yoshimoto and Ito, 2011). 5. Conclusion In this experiment, we revealed three findings: Brain aromatase (1) is expressed remarkably early as compared with other tissues, irrespective of sex differences; (2) begins expression along with the formation of the choroid plexus of the cerebral ventricle and the paleocortex and olfactory bulb of the prosencephalon; (3) shows different expression characteristic from E2 and ERa that have been localized in each tissue. Interestingly, E2 secretion in the heart is considered to result from high aromatase expression in the brain. This finding is also supported by the fact that aromatase expressions in Xenopus are brain-specific expressions that are not tissue-specific as in mammals (Chow et al., 2009; Demura et al., 2008; Nakagawa and Iwabuchi, 2012; Urbatzka et al., 2007). Although the role of the cortical aromatase expression in the Xenopus brain is still unclear, it may work to protect the cells that are developed through differentiation instead of morphogenesis. Disclosure summary The authors have nothing to disclose.
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Acknowledgments We thank K. Masuda, K. Mizuta, K. Yamahana, R. Kitta and Y. Tamura for much help in the laboratory. This work was supported by grants from Nihon University.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ygcen.2013. 09.024.
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