Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Medical Association of Nippon Medical School  

DOI： 10.1272/jnms.jnms.2023_90-507

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.peptides.2023.171064

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society of Histochemistry & Cytochemistry  

DOI： 10.1267/ahc.23-00024

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Medical Association of Nippon Medical School  

DOI： 10.1272/jnms.jnms.2023_90-209

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Authorship：Last author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Medical Association of Nippon Medical School  

DOI： 10.1272/manms.19.84

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.peptides.2022.170929

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Bioscientifica  

The nutritional environment during development periods induces metabolic programming, leading to metabolic disorders and detrimental influences on human reproductive health. This study aimed to determine the long-term adverse effect of intrauterine malnutrition on the reproductive center kisspeptin-neurokinin B-dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus (ARC) of female offspring. Twelve pregnant rats were divided into ad-lib-fed (control, n  = 6) and 50% undernutrition (UN, n  = 6) groups. The UN group was restricted to 50% daily food intake of the control dams from gestation day 9 until term delivery. Differences between the two groups in terms of various maternal parameters, including body weight (BW), pregnancy duration, and litter size, as well as birth weight, puberty onset, estrous cyclicity, pulsatile luteinizing hormone (LH) secretion, and hypothalamic gene expression of offspring, were determined. Female offspring of UN dams exhibited low BW from birth to 3 weeks, whereas UN offspring showed signs of precocious puberty; hypothalamic Tac3 (a neurokinin B gene) expression was increased in prepubertal UN offspring, and the BW at the virginal opening was lower in UN offspring than that in the control group. Interestingly, the UN offspring showed significant decreases in the number of KNDy gene-expressing cells after 29 weeks of age, but the number of ARC kisspeptin-immunoreactive cells, pulsatile LH secretions, and estrous cyclicity were comparable between the groups. In conclusion, intrauterine undernutrition induced various changes in KNDy gene expression depending on the life stage. Thus, intrauterine undernutrition affected hypothalamic developmental programming in female rats.

DOI： 10.1530/ec-22-0307

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Other Link： https://ec.bioscientifica.com/downloadpdf/journals/ec/12/1/EC-22-0307.xml

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society of Histochemistry & Cytochemistry  

Despite the physiological significance of ESR2, a lack of well-validated detection systems for ESR2 proteins has hindered progress in ESR2 research. Thus, recent identification of a specific anti-human ESR2 monoclonal antibody (PPZ0506) and its specific cross-reactivity against mouse and rat ESR2 proteins heightened momenta toward development of appropriate immunohistochemical detection systems for rodent ESR2 proteins. Building upon our previous optimization of ESR2 immunohistochemical detection in rats using PPZ0506, in this study, we further aimed to optimize mouse-on-mouse immunohistochemical detection using PPZ0506. Our assessment of several staining conditions using paraffin-embedded ovary sections revealed that intense heat-induced antigen retrieval, appropriate blocking, and appropriate antibody dilutions were necessary for optimization of mouse-on-mouse immunohistochemistry. Subsequently, we applied the optimized immunostaining method to determine expression profiles of mouse ESR2 proteins in peripheral tissues and brain subregions. Our analyses revealed more localized distribution of mouse ESR2 proteins than previously assumed. Moreover, comparison of these results with those obtained in humans and rats using PPZ0506 revealed interspecies differences in ESR2 expression. We expect that our optimized methodology for immunohistochemical staining of mouse ESR2 proteins will help researchers to solve multiple lines of controversial evidence concerning ESR2 expression.

DOI： 10.1267/ahc.22-00043

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Medical Association of Nippon Medical School  

BACKGROUND: The nuclear receptor genes, including estrogen receptor β (ERβ), contain non-conventional internal and terminal exons, and alternative choice of the exons yields multiple mRNA and protein variants with unique structures and functions. However, the genomic structure of the intronic and 3'-downstream regions of the human ERβ gene and the presence of novel ERβ variants with non-conventional sequences have not been re-examined in about 20 years. Therefore, we attempted to re-characterize the structure of the human ERβ gene and identify novel non-conventional exons and distinct splice variants. METHODS: Rapid amplification of cDNA 3'-end and RT-PCR cloning were used to isolate human ERβ mRNA variants from the testis. The identified cDNA sequences were mapped on the human genome assembly. Expression profiles of the variants were assessed by RT-PCR analysis. RESULTS: We cloned multiple ERβ mRNA variants with novel nucleotide sequences from the testis and identified several alternative splice sites, 3'-elongation of conventional coding exons, and novel terminal exons in the human ERβ gene. The variants encode C-terminally truncated ERβ proteins termed ERβ6, ERβ7, ERβEx. 4L, and ERβEx. 6L. Furthermore, we identified exon 7-defective forms of ERβ2/βcx, ERβ4, ERβ6, and ERβ7. Subsequently, we noted distinct expression patterns of the variants in human peripheral organs and brain subregions. CONCLUSION: This study clarified complicated genomic organization and splicing patterns of the human ERβ gene that contribute to the distinct heterogeneity of human ERβ mRNAs and proteins.

DOI： 10.1272/jnms.jnms.2021_88-105

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Although there are few well-validated antibodies against ESR2 proteins, a recent validation assessment identified a specific monoclonal antibody against human ESR2 proteins (PPZ0506). Furthermore, our previous study confirmed its cross-reactivity and specificity against rodent ESR2 proteins, enabling the determination of true ESR2 distribution profiles in rodents. Therefore, we aimed to determine optimal conditions for ESR2 detection by PPZ0506 immunostaining and analyze ESR2 distribution in rats. We evaluated several staining conditions using paraffin-embedded and frozen ovary sections. Immunohistochemical staining with PPZ0506 antibody required strong antigen retrieval and appropriate antibody dilution. Subsequent immunohistochemical analysis in multiple tissues under optimized conditions revealed that rat ESR2 proteins are expressed in a more localized manner than previously assumed. Our results suggest that previous immunohistochemical studies using inadequately validated antibodies against ESR2 proteins overestimated their distribution profiles. We expect that optimized immunohistochemical detection with PPZ0506 antibody can help researchers solve several conflicting problems in ESR2 research. (150 words).

DOI： 10.1016/j.mce.2020.111145

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Expression profiles of gonadal steroid receptor variants have been reportedly associated with malignancy in breast and prostate cancers [1,2]. However, such associations with pituitary tumors remain unclear. Therefore, the expression levels of the wild-type ESR1 (ERα66) and the ESR1 variants (ERαi34, ERαi45c, and ERαΔ5) transcripts encoding constitutively active ERα proteins with C-terminal truncation in non-functioning pituitary adenomas (NFPAs) were evaluated using reverse transcription-digital polymerase chain reaction. The results revealed that the expression levels of the variants were approximately two orders of magnitude lower than that of ERα66 in NFPAs. These data were based on our previous article entitled "Accurate assessment of estrogen receptor profiles in non-functioning pituitary adenomas using RT-digital PCR and immunohistochemistry" [3].

DOI： 10.1016/j.dib.2020.106452

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BACKGROUND: Non-functioning pituitary adenomas (NFPAs) are common pituitary tumors, and surgery is generally the only treatment option. Few attempts have been made to explore target molecules for the development of NFPA pharmacological treatments. METHOD: We quantitatively assessed the expression profiles of estrogen receptor (ER) transcripts and proteins in NFPA samples, using reverse transcription-digital polymerase chain reaction (RT-dPCR) and immunohistochemistry, and further investigated the correlations between the expression levels of ER and those of downstream responsive genes. All patients had undergone surgery at the same high-volume hospital. A total of 20 patients with NFPAs were included. All patients were new-onset, and none were diagnosed with intratumoral hemorrhages or cysts. RESULTS: NFPA samples exhibited a bimodal ESR1 expression pattern and were categorized into significantly different high- and low-ESR1 expression level groups (P < 0.05). In contrast, expression levels of ESR1 variants and ESR2 could barely be detected. Similar results were obtained through the immunohistochemical staining of NFPAs, using well-validated antibodies against ERs. The expression levels of ESR1 positively correlated with those of GREB1, an estrogen-responsive gene [correlation coefficient (r) = 0.623, P = 0.003]. CONCLUSIONS: ESR1 expression levels in NFPAs exhibited a bimodal pattern and were positively correlated with GREB1 expression levels. The accurate assessment of ER expression levels may further advance future NFPA-related research.

DOI： 10.1016/j.lfs.2020.118416

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Accumulating evidence suggests that kisspeptin neurons in the arcuate nucleus (ARC), which coexpress neurokinin B and dynorphin, are involved in gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulse generation, while the anteroventral periventricular nucleus (AVPV) kisspeptin neurons are responsible for GnRH/LH surge generation. The present study aims to examine whether GnRH(1-5), a GnRH metabolite, regulates LH release via kisspeptin neurons. GnRH(1-5) was intracerebroventricularly injected to ovariectomized and estrogen-treated Wistar-Imamichi female rats. Immediately after the central GnRH(1-5) administration at 2 nmol, plasma LH concentration increased, resulting in significantly higher levels of the area under the curve and baseline of plasma LH concentrations compared to vehicle-injected controls. On the other hand, in Kiss1 knockout rats, GnRH(1-5) administration failed to affect LH secretion, suggesting that the facilitatory effect of GnRH(1-5) on LH release is mediated by kisspeptin neurons. Double in situ hybridization (ISH) for Kiss1 and Gpr101, a GnRH(1-5) receptor gene, revealed that few Kiss1-expressing cells coexpress Gpr101 in both ARC and AVPV. On the other hand, double ISH for Gpr101 and Slc17a6, a glutamatergic marker gene, revealed that 29.2% of ARC Gpr101-expressing cells coexpress Slc17a6. Further, most of the AVPV and ARC Kiss1-expressing cells coexpress Grin1, a gene encoding a subunit of NMDA receptor. Taken together, these results suggest that the GnRH(1-5)-GPR101 signaling facilitates LH release via indirect activation of kisspeptin neurons and that glutamatergic neurons may mediate the signaling. This provides a new aspect of kisspeptin- and GnRH-neuronal communication with the presence of stimulation from GnRH to kisspeptin neurons in female rats.

DOI： 10.1507/endocrj.EJ19-0444

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Constitutively active estrogen receptor α (ERα) variants with C-terminal truncation are candidate molecules for gain of both endocrine- and chemotherapy-resistance in estrogen-sensitive tumors. Our previous reports using artificially truncated ERα constructs demonstrated that ERα variants encoded in 1-2-3-cryptic exon- and 1-2-3-4-cryptic exon-types of transcripts have potentials to display constitutive transactivation of an estrogen response element reporter. However, naturally occurring 1-2-3-cryptic exon-type ERα variants have not been cloned yet. Therefore, the present study was designed to identify naturally occurring ERα variants encoded in 1-2-3-cryptic exon-type ERα transcripts. We cloned a novel C-terminally truncated ERα variant (ERαi34) encoded in a 1-2-3-i34 transcript from MCF-7 cells and characterized its constitutive and ER antagonist-resistant transactivation in transfected cells. Stable transfection of the variant into MCF-7 cells augmented basal cell proliferation insensitive to fulvestrant. Collectively, we validated the structure-based mechanisms underlying constitutive and ER antagonist-resistant transactivation by C-terminally truncated ERα variants.

DOI： 10.1016/j.mce.2019.110693

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Several lines of controversial evidence concerning estrogen receptor β (ERβ) remain to be solved because of the unavailability of specific antibodies against ERβ. The recent validation analysis identified a monoclonal antibody (PPZ0506) with sufficient specificity against human ERβ. However, the specificity and cross-reactivity of PPZ0506 antibody against ERβ proteins from laboratory animals have not been confirmed. In the present study, we aimed to validate the applicability of PPZ0506 to rodent studies. The antibody exhibited specific cross-reactivity against mouse and rat ERβ proteins in immunoblot and immunocytochemical experiments using transfected cells. In immunohistochemistry for rat tissue sections, PPZ0506 showed immunoreactive signals in the ovary, prostate, and brain. These immunohistochemical profiles of rat ERβ proteins in rat tissues accord well with its mRNA expression patterns. Although the antibody was reported to show the moderate signals in human testis, no immunoreactive signals were observed in rat testis. Subsequent RT-PCR analysis revealed that this species difference in ERβ expression resulted from different expression profiles related to the alternative promoter usage between humans and rats. In conclusion, we confirmed applicability of PPZ0506 for rodent ERβ studies, and our results provide a fundamental basis for further examination of ERβ functions.

DOI： 10.3390/ijms20246312

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Other Link： http://orcid.org/0000-0003-2044-316X

Authorship：Corresponding author   Language：Japanese  

DOI： 10.1272/manms.15.24

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publisher：日本医科大学医学会  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

Osteoarthritis (OA) is the leading cause of musculoskeletal disability in the elderly. Insights into the biological features of OA are obtained by characterization of the molecular features by gene expression profiling using reverse transcription-quantitative PCR (RT-qPCR). However, it has recently become evident that the use of suitable reference genes is required for appropriate normalization of this technique. Here total RNA was isolated from the synovium of 18 men and 20 women who underwent total knee arthroplasty for knee OA (KOA). We validated the expression stability of 7 candidate housekeeping genes (ACTB, B2M, GAPDH, HPRT1, RPL13A, SDHA, and YWHAZ) in the synovium of KOA with 3 commonly used algorithms (geNorm, NormFinder, and BestKeeper). Additionally, we evaluated expression profiles of the steroid hormone receptor (AR, ESR1, ESR2, GR, MR, and PR) and proinflammatory cytokines (IL1B and IL6) genes in the synovium and their correlations with the risk factors of KOA, using the most and least stable housekeeping genes for comparison. Results showed that HPRT1 was the most stable gene, whereas B2M was the least stable. RT-qPCR analysis revealed sexually dimorphic expression of AR, IL1B, and IL6; intercorrelations between steroid hormone receptor expression levels and female-specific correlations of IL1B expression with ESR1 and PR expression, IL6 expression with ESR1 and GR expression, and body mass index with AR and PR expression; and the choice of the least stable reference gene altered several correlations and statistical significances. In conclusion, HPRT1 was identified as the suitable reference gene for normalization in the OA synovium.

DOI： 10.1080/03008207.2017.1391234

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Other Link： http://orcid.org/0000-0003-2044-316X

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC HISTOCHEMISTRY & CYTOCHEMISTRY  

Liposomes, artificial phospholipid vesicles, have been developed as a non-viral drug delivery system to allow contained agents to be efficiently delivered to target sites via systemic circulation. Liposomes have been used as a gene transfer tool with cultured cells; however, their precise trafficking and processing remain uncertain. Furthermore, liposomes with different surface charges are known to exhibit distinct properties. The purpose of the current study was to elucidate the intracellular trafficking and processing of liposomes with anionic and cationic surface charges from a morphological view point. We found that cationic liposomes (CLs) were more effectively taken by the cells than anionic liposomes (ALs). Confocal laser scanning microscopy and transmission electron microscopy demonstrated distinct intracellular localization and processing patterns of ALs and CLs. ALs and their contents were localized in lysosomes but not in cytosol, indicating that ALs are subjected to the endosome-lysosome system. In contrast, contents of CLs were distributed mainly in the cytosol. CLs appear to disturb the cell membrane and then collapse to release their contents into the cytosol. It is feasible that the contents of CLs enter the cytosol directly rather than via the endosome-lysosome system.

DOI： 10.1267/ahc.17021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley-Blackwell  

Purpose: Hypothalamic kisspeptin neurons are considered to play a critical role in regulating mammalian reproduction and integrating humoral and neuronal inputs that control gonadotropin-releasing hormone (GnRH)/gonadotropin release. The present study aimed to investigate the upstream regulator candidates for kisspeptin neurons. Methods: Visualized kisspeptin neurons that were taken from the arcuate nucleus (ARC) of Kiss1-tdTomato rats were subjected to next-generation sequencing (NGS) analysis. In situ hybridization (ISH) for the calcitonin receptor gene (Calcr) was performed throughout the whole forebrain of ovariectomized wild-type female rats that had been implanted with a negative feedback level of estrogen, because the Calcr expression was evident in the ARC kisspeptin neurons from the NGS analysis. Then, a double ISH was performed for the Calcr and kisspeptin gene (Kiss1) in the brain regions, containing either the anteroventral periventricular nucleus (AVPV) or ARC of the female rats. Results: The NGS analysis revealed that the Calcr was highly expressed in the ARC kisspeptin neurons. It was found that the Calcr was co-expressed in 12% and 22% of the Kiss1-expressing cells in the ARC and AVPV, respectively. Conclusion: The present study suggests that calcitonin receptor signaling could be involved in the regulation of reproductive function through the direct control of the ARC and/or AVPV kisspeptin neurons, and then GnRH/gonadotropin release.

DOI： 10.1002/rmb2.12085

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Estrogen receptor α (ERα) mRNAs exhibit remarkable heterogeneity owing to complicated alternative splicing. Some encode C-terminally-truncated ERα proteins, which display ligand-independent transactivation or dominant-negative activity. We previously characterized C-terminally-truncated ERα mRNA variants with cryptic sequences in humans and mice, and demonstrated that helices in the ligand-binding domains (LBDs) of ERα variants contribute to ligand-independent transcriptional activity. However, existence of non-conventional coding exons and generation of constitutively active ERα variants have remained to be examined in rats. To comparatively analyze modular organization and splicing profiles of the ERα genes, the range of C-terminally-truncated ERα variants was explored in rats and mice using rapid amplification of cDNA ends and RT-PCR cloning. Furthermore, their functions were determined in transiently transfected cells using expression constructs and luciferase reporter assays. Multiple cryptic exons and C-terminally-truncated ERα variant mRNAs were identified in rats and mice. Naturally occurring and artificially truncated variants/constructs lacking helix 5 potentially exhibited gain-of-function in transfected cells. Although cryptic exons and splicing profiles were poorly conserved among humans, mice, and rats, constitutively active variants were generated from the ERα genes. Moreover, the primary mechanism of ligand-independent activation by C-terminally-truncated ERα variants is C-terminus to helix 5 deletion in the LBD. This comparative study documented the complexity of ERα genes, mRNAs, and proteins, and further determined the underlying structural basis of ligand-independent activation by C-terminally-truncated ERα variants.

DOI： 10.1016/j.ygcen.2017.04.009

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

GnRH neurons form a final common pathway for the central regulation of reproduction. Although the involvement of acetylcholine in GnRH secretion has been reported, direct effects of acetylcholine and expression profiles of acetylcholine receptors (AChRs) still remain to be studied. Using immortalized GnRH neurons (GT1-7 cells), we analyzed molecular expression and functionality of AChRs. Expression of the mRNAs were identified in the order α7 > β2 = β1 ≧ α4 ≧ α5 = β4 = δ > α3 for nicotinic acetylcholine receptor (nAChR) subunits and m4 > m2 for muscarinic acetylcholine receptor (mAChR) subtypes. Furthermore, this study revealed that α7 nAChRs contributed to Ca2+ influx and GnRH release and that m2 and m4 mAChRs inhibited forskolin-induced cAMP production and isobutylmethylxanthine-induced GnRH secretion. These findings demonstrate the molecular profiles of AChRs, which directly contribute to GnRH secretion in GT1-7 cells, and provide one possible regulatory action of acetylcholine in GnRH neurons.

DOI： 10.1007/s12576-016-0464-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

The inhalation anesthetic sevoflurane suppresses Per2 expression in the suprachiasmatic nucleus (SCN) in rodents. Here, we investigated the intra-SCN regional specificity, time-dependency, and pharmacological basis of sevoflurane-effects. Bioluminescence image was taken from the SCN explants of mPer2 promoter-destabilized luciferase transgenic rats, and each small regions of interest (ROI) of the image was analyzed. Sevoflurane suppressed bioluminescence in all ROIs, suggesting that all regions in the SCN are sensitive to sevoflurane. Clear time-dependency in sevoflurane effects were also observed; application during the trough phase of the bioluminescence cycle suppressed the subsequent increase in bioluminescence and resulted in a phase delay of the cycle; sevoflurane applied during the middle of the ascending phase induced a phase advance; sevoflurane on the descending phase showed no effect. These results indicate that the sevoflurane effect may depend on the intrinsic state of circadian machinery. Finally, we examined the involvement of GABAergic signal transduction in the sevoflurane effect. Co-application of both GABAA and GABAB receptor antagonists completely blocked the effect of sevoflurane on the bioluminescence rhythm, suggesting that sevoflurane inhibits Per2 expression via GABAergic signal transduction. Current study elucidated the anesthetic effects on the molecular mechanisms of circadian rhythm.

DOI： 10.1016/j.neures.2015.11.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

General anesthesia affects the expression of clock genes in various organs. Expression of Per2, a core component of the circadian clock, is markedly and reversibly suppressed by sevoflurane in the suprachiasmatic nucleus (SCN), and is considered to be a biochemical marker of anesthetic effect in the brain. The SCN contains various types of neurons, and this complexity makes it difficult to investigate the molecular mechanisms of anesthesia. Here, we established an in vitro experimental system using a cell line to investigate the mechanisms underlying anesthetic action. Development of the system comprised two steps: first, we developed a system for application of inhalational anesthetics and incubation; next, we established cultures of anesthetic-responsive cells expressing mPer2 promoter-dLuc. GT1-7 cells, derived from the mouse hypothalamus, responded to sevoflurane by reversibly decreasing mPer2-promoter-driven bioluminescence. Interestingly, the suppression of bioluminescence was found only in the serum-starved GT1-7 cells, which showed neuron-like morphology, but not in growing cells, suggesting that neuron-like characteristics are required for anesthetic effects in GT1-7 cells.

DOI： 10.1016/j.neulet.2016.04.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

The nuclear receptor genes contain alternative internal and terminal exons, with alternative exon incorporation yielding mRNA variants that encode various receptor types, including some with C-terminal truncation that exhibit constitutive activation or dominant-negative transcriptional transactivation. However, C-terminally truncated estrogen receptor α (ERα) variants with alternative sequences have rarely been reported in humans. Therefore, we assessed human ERα genomic organization and alternative splicing profiles, and identified both alternative exons and C-terminally truncated ERα variants. These naturally occurring C-terminally truncated ERα proteins were localized in the nuclei of transfected cells. In addition, ERαi45c and ERαΔ5 variants exhibited constitutive transactivation of an estrogen responsive element-driven promoter in transfected cells. We manufactured expression vectors encoding artificially truncated ERα constructs and evaluated their transactivation abilities to establish mechanisms determining the constitutive activity and dominant-negative properties of truncated variants. Lack of the region encoded in exon 8 eliminated basal and ligand-induced transcriptional transactivation. The C-terminally truncated ERα variants/constructs containing the helices 5 in their ligand-binding domains did not exhibit constitutive transactivation. Furthermore, we demonstrated that truncation from C-termini to helices 5 in the variant ligand-binding domains was required for constitutive activation and found that the remnant regions of the ligand-binding domains and variant-specific sequences influenced transcriptional transactivation efficiency. In conclusion, we elucidated the structural and functional features of novel C-terminally truncated ERα variants and revealed the mechanisms underlying constitutive transactivation by C-terminally truncated nuclear receptor variants.

DOI： 10.1016/j.mce.2016.01.026

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.joca.2016.01.150

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

In the rat, induction of maternal behavior depends on the parity of the female. For example, nulliparous (NP) females need longer exposure to pups than multiparous (MP) or lactating (L) females to exhibit similar maternal behavior. In this study, we investigated the role of brain oxytocin in the approaching behavior of these female rats. Olfactory preferences for pup odors were examined for 8 consecutive days. Each preference test was followed by direct overnight exposure to pups. On the 8th day, MP and L, but not NP females showed robust pup-odor preferences. After the behavioral test, half of the females were exposed to pups for 2 h, whereas the other half were not. The females were then sacrificed to analyze brain oxytocin (OXT) and vasopressin (AVP) activities by cFos immunohistochemistry and to quantify their receptor mRNA expression using real-time PCR. In the paraventricular nucleus (PVN), the percentage of cFos-positive OXT neurons was significantly larger in MP and L females than in NP females after pup exposure. No significant differences were found in cFos expression in OXT neurons of the supraoptic nucleus (SON) or in AVP neurons of either the PVN or SON. Expression of OXT receptor mRNA in the medial preoptic area and amygdala of the control groups was also higher in MP females than in NP females. Finally, we demonstrated that infusion of OXT into the lateral ventricle of NP females promoted preferences for pup odors. These results indicate that puerperal and parental experiences enhance the responsiveness of OXT neurons in the PVN to pup stimuli and establish olfactory preferences for these odors in a parity-dependent manner.

DOI： 10.1262/jrd.2015-046

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The estrogen receptor α (ERα) directs transactivation of target genes, and splice variants have been shown to exhibit altered activation properties. We previously documented the complicated alternative promoter usage and splicing patterns of the rat ERα gene; however, the information was restricted to a few specific organs. Therefore, we re-examined the rat mRNA profiles of ERα, including the generation of the exon 1-skipping, ERα46 transcript in a wider variety of rat organs and further characterized the fundamental functional properties of rat ERα46 variants. With the use of RT-PCR, we discovered unique distribution and splicing patterns for promoter-specific ERα isoforms, as well as the extensive expression of the Δ exon 1 variant in the rat. Similar to wild-type ERα, an immunocytochemical analysis showed a predominant localization of ERα46 proteins in the nuclei of transfected cells. Luciferase reporter assays revealed that ERα46 variants stimulated the transcriptional activity of an estrogen response element-driven promoter in response to estrogen. In addition, the variants exhibited distinct transactivation and reactivity to 4-hydroxytamoxifen in different cell types. Although the alternative splicing patterns are species-specific, the profiles of the alternative use of promoters, and the fundamental properties of the rat ERα46 variant are similar to those of human and mouse homologs. Therefore, the present study provides fundamental and useful information for further research into the regulation and functions of ERα gene variants.

DOI： 10.1016/j.gene.2015.06.086

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Other Link： http://orcid.org/0000-0003-2044-316X

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Expression of the estrogen receptor α (ERα) gene is subject to complex regulation. To elucidate the mechanisms of this regulation, the genomic organization and the physiological role of the multiple 5'-untranslated regions (5'-UTRs) must be determined. Here, we investigated the expression and splicing patterns of the human ERα E isoforms. We identified two novel untranslated exons, N1 and N2, in the 5'-region of the human ERα gene and multiple E isoform mRNA variants generated by alternative usage of non-coding internal exons. Expression of the N1-containing variants was observed only in the human breast adenocarcinoma cell line, MCF7, while the N2-containing variants were expressed in the adult liver and MCF7 cells. We examined post-transcriptional regulation of the variant mRNAs using luciferase reporter assays and quantitative PCR. The insertion of untranslated internal exons into the 5'-UTRs of the E isoforms reduced their translation efficiency, but barely influenced mRNA turnover. Our results indicate that the genomic organization of the human ERα gene and the splicing profiles of the human ERα E isoforms are more complicated than previously reported. Furthermore, the 5'-UTRs of the E isoforms post-transcriptionally control human ERα expression mainly through translational repression.

DOI： 10.1016/j.jsbmb.2012.09.027

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ENDOCRINE SOC  

In rodents, GnRH neurons are diffusely distributed from the medial septum through to the medial preoptic area and control gonadal functions through the pituitary. The activity of GnRH neurons is regulated by a variety of bioactive substances, including the inhibitory peptide somatostatin. In the present study, we focused on somatostatin because intracerebroventricular injection of somatostatin inhibits the LH surge in rats and reduces LH secretion in ewes. Somatostatin also decreases GnRH release from rat hypothalamic slices. In mice, somatostatin is also thought to suppress GnRH neuronal activity through contact on the soma of GnRH neurons. However, similar data are missing in rats. Moreover, rat GnRH neurons receive only a few synaptic inputs. In this study, we assessed the morphological relationship between GnRH and somatostatin neurons. Confocal microscopy on the sections from the medial septum through medial preoptic area revealed about 35 close contacts per rat between the GnRH and somatostatin neuronal fibers in the organum vasculosum of the lamina terminalis region. No contact of somatostatin fibers on the GnRH neuronal somata was observed. Multicell RT-PCR for somatostatin receptor mRNA in rat GnRH neurons was also performed, which revealed moderate expression of somatostatin receptor subtypes 1-5. In addition, patch clamp experiments were carried out in acute slice preparations. Somatostatin suppressed neuronal firing in cells recorded in a cell-attached configuration and also induced whole-cell outward currents in GnRH neurons. These findings suggest that somatostatin directly inhibits the activity of rat GnRH neurons through volume transmission in the organum vasculosum of the lamina terminalis region.

DOI： 10.1210/en.2011-1374

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The 5'-untranslated region (5'-UTR) of the estrogen receptor α (ERα) gene plays an important role in determining its tissue-specific expression. We examined the 5'-UTRs of the mouse ERα mRNA variants in depth using the Basic Local Alignment Search Tool (BLAST), rapid amplification of 5'-cDNA ends (5'-RACE) and RT-PCR. We demonstrated the presence of multiple variants containing unique 5'-UTRs. We mapped the cDNA sequences onto the mouse genome, and found that both alternative splicing from four different leader exons (A, C, F1, and H) to exon 1, and combinations of 12 internal exons (X1, X2, X3, X4, F2/X5, X6, X7, X8, X9, X10, X11, and B) generate multiple ERα transcripts. Mouse exon B, that has homologies with human exon B and rat exon 0T, was used as an internal exon, not as a leader exon. RT-PCR analysis revealed distinct expression patterns of the variants, suggesting that the alternative promoter usage and alternative splicing are regulated in a tissue-specific manner. Our results indicate that the genomic organization of the mouse ERα gene is complicated as previously shown in the rat ERα gene. In addition, both alternative promoter usage and alternative splicing contribute to the remarkable mRNA diversity of the mouse ERα gene.

DOI： 10.1016/j.jsbmb.2011.03.004

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We re-examined mouse ERα mRNA variants using rapid amplification of cDNA ends (RACE) and RT-PCR. Our analysis showed the presence of several mRNA variants containing unique 5'- or 3'-nucleotide sequences. We mapped the cDNA sequences on the mouse genome, and identified four novel 3'-terminal and 5'-leader exons in the intronic region between exons 4 and 5. RT-PCR analysis revealed that the expression patterns of the C-terminally truncated ERα products (CTERPs) were similar to that of Wild-type ERα and that the N-terminally truncated ERα products (NTERPs) appeared to have different expression profiles. Moreover, we constructed expression vectors and analyzed the subcellular localization and the transcriptional activation abilities of the variant proteins in transfected HEK293 cells using immunocytochemistry and luciferase reporter assay. The CTERP variants localized in the nuclei and constitutively activated estrogen response element (ERE)-driven promoters, while the NTERP variant was located in the extra-nuclear regions and had no ability to activate the ERE promoters in the presence or absence of 10 nM estradiol. Our results indicate that the mouse ERα gene is more complex than previously thought in terms of genomic organization and that alternative splicing and alternative usage of intronic promoters contribute to the remarkable diversity of ERα mRNAs and proteins.

DOI： 10.1016/j.jsbmb.2011.01.003

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

The 5'-untranslated regions (5'-UTRs) of the estrogen receptor α (ERα) mRNAs play important roles in the modulation of translation. To elucidate the mechanisms regulating human ERα gene expression, it is necessary to determine its genomic organization and the roles of the multiple 5'-UTRs. We therefore examined the splicing and expression profiles of the human ERα F isoforms. A novel non-coding exon E3 was found in the 5'-region of the human ERα gene and six F isoform mRNA variants were identified. The variants revealed the preferred expression patterns. Post-transcriptional regulation was also investigated. The 5'-UTRs of the F isoforms decreased the translation efficiency but had no effect on mRNA stability. The results indicate that the organization and splicing patterns of the human ERα gene are more complex than previously reported, and that the 5'-UTRs of the F isoforms contribute to the post-transcriptional control of human ERα gene expression through translation repression.

DOI： 10.1016/j.mce.2010.12.003

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Language：Japanese   Publisher：公益社団法人 日本薬学会  

DOI： 10.14894/faruawpsj.47.3_213

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BACKGROUND: Brain synthesis of steroids including sex-steroids is attracting much attention. The endogenous synthesis of corticosteroids in the hippocampus, however, has been doubted because of the inability to detect deoxycorticosterone (DOC) synthase, cytochrome P450(c21). METHODOLOGY/PRINCIPAL FINDINGS: The expression of P450(c21) was demonstrated using mRNA analysis and immmunogold electron microscopic analysis in the adult male rat hippocampus. DOC production from progesterone (PROG) was demonstrated by metabolism analysis of (3)H-steroids. All the enzymes required for corticosteroid synthesis including P450(c21), P450(2D4), P450(11β1) and 3β-hydroxysteroid dehydrogenase (3β-HSD) were localized in the hippocampal principal neurons as shown via in situ hybridization and immunoelectron microscopic analysis. Accurate corticosteroid concentrations in rat hippocampus were determined by liquid chromatography-tandem mass spectrometry. In adrenalectomized rats, net hippocampus-synthesized corticosterone (CORT) and DOC were determined to 6.9 and 5.8 nM, respectively. Enhanced spinogenesis was observed in the hippocampus following application of low nanomolar (10 nM) doses of CORT for 1 h. CONCLUSIONS/SIGNIFICANCE: These results imply the complete pathway of corticosteroid synthesis of 'pregnenolone →PROG→DOC→CORT' in the hippocampal neurons. Both P450(c21) and P450(2D4) can catalyze conversion of PROG to DOC. The low nanomolar level of CORT synthesized in hippocampal neurons may play a role in modulation of synaptic plasticity, in contrast to the stress effects by micromolar CORT from adrenal glands.

DOI： 10.1371/journal.pone.0021631

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Sex steroids play essential roles in the modulation of synaptic plasticity and neuroprotection in the hippocampus. Accumulating evidence shows that hippocampal neurons synthesize both estrogen and androgen. Recently, we also revealed the hippocampal synthesis of corticosteroids. The accurate concentrations of these hippocampus-synthesized steroids are determined by liquid chromatography-tandem mass-spectrometry in combination with novel derivatization. The hippocampal levels of 17β-estradiol (E2), testosterone (T), dihydrotestosterone (DHT), and corticosterone (CORT), are 5-15 nM, and these levels are sufficient to modulate synaptic plasticity. Hippocampal E2 modulates memory-related synaptic plasticity not only slowly/genomically but also rapidly/non-genomically. Slow actions of E2 occur via classical nuclear receptors (ERα or ERβ), while rapid E2 actions occur via synapse-localized or extranuclear ERα or ERβ. Nanomolar concentrations of E2 change rapidly the density and morphology of spines in hippocampal neurons. ERα, but not ERβ, drives this enhancement/suppression of spinogenesis in adult animals. Nanomolar concentrations of androgens (T and DHT) and CORT also increase the spine density. Kinase networks are involved downstream of ERα and androgen receptor. Newly developed Spiso-3D mathematical analysis is useful to distinguish these complex effects by sex steroids and kinases. Significant advance has been achieved in investigations of rapid modulation by E2 of the long-term depression or the long-term potentiation.

DOI： 10.3389/fendo.2011.00043

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ENDOCRINE SOC  

Although sex steroids play a crucial role in the postnatal brain development, the age-related changes in the hippocampal steroidogenesis remain largely unknown. We performed comprehensive investigations for the mRNA expressions of 26 sex steroidogenic enzymes/proteins and three sex steroid receptors in the male rat hippocampus, at the ages of postnatal day (PD) 1, PD4, PD7, PD10, PD14, 4 wk, and 12 wk (adult), by RT-PCR/Southern blotting analysis. The relative expression levels of these enzymes/receptors at PD1 were Srd5a1 > Star > Ar ∼ Hsd17b4 ∼ Hsd17b1 ∼ Hsd17b7 ∼ Esr1 ∼ Srd5a2 > Hsd17b3 > Esr2 > Cyp11a1 > Cyp17a1 > Cyp19a1 ∼ Hsd17b2 > 3β-hydroxysteroid dehydrogenase I. The mRNA levels of essential enzymes for progesterone/testosterone/estradiol metabolisms (Cyp17a1, Hsd17b7, and Cyp19a1) were approximately constant between PD1 and PD14 and then declined toward the adult levels. Cyp11a1 increased during PD4-PD14 and then considerably decreased toward the adult level (∼8% of PD1). Hsd17b1, Hsd17b2, and 3β-hydroxysteroid dehydrogenase I mRNA decreased approximately monotonously. Hsd17b3 increased to approximately 200% of PD1 during PD4-PD14 and was maintained at this high level. The 5α-reductase mRNA was maintained constant (Srd5a1) or decreased monotonically (Srd5a2) toward the adult level. The Esr1 level peaked at PD4 and decreased toward the adult level, whereas Ar greatly increased during PD1-PD14 and was maintained at this high level. The Star and Hsd17b4 levels were maintained constant from neonate to adult. These results suggest that the hippocampal sex steroidogenic properties are substantially altered during the postnatal development processes, which might contribute to brain sexual maturation.

DOI： 10.1210/en.2010-0581

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

Gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in the neuroendocrine regulation of reproduction. We have previously reported that rat GnRH neurons exhibit voltage-gated Ca(2+) currents. In this study, oligo-cell RT-PCR was carried out to identify subtypes of the alpha(1) subunit of voltage-gated Ca(2+) channels in adult rat GnRH neurons. GnRH neurons expressed mRNAs for all five types of voltage-gated Ca(2+) channels. For T-type Ca(2+) channels, alpha(1H) was dominantly expressed in GnRH neurons. Electrophysiological analysis in acute slice preparations revealed that GnRH neurons from adult rats exhibited T-type Ca(2+) currents with fast inactivation kinetics (~20 ms at -30 mV) and a time constant of recovery from inactivation of ~0.6 s. These results indicate that rat GnRH neurons express subtypes of the alpha(1) subunit for all five types of voltage-gated Ca(2+) channel, and that alpha(1H) was the dominant subtype in T-type Ca(2+) channels.

DOI： 10.1007/s12576-010-0085-z

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The 5'-untranslated region (UTR) of the estrogen receptor alpha (ERalpha) gene plays an important role in determining tissue-specific expression. To elucidate the regulatory mechanisms of rat ERalpha gene expression, the genomic organization must be investigated. We therefore analyzed the structure of the rat ERalpha mRNA 5'-UTR using rapid amplification of 5'-cDNA ends (5'-RACE) and RT-PCR. The analysis showed the presence of multiple variants containing unique 5'-UTRs. We mapped the cDNA sequences on the rat genome, and newly identified one leader exon (exon 0U) and ten untranslated internal exons (exons I1-10). Both splicing from four different leader exons (exons 0S, 0N, 0U, and 0/B) onto exon 1 and alternative splicing in combination with eleven internal exons (exons I1-10, and 0T) produce multiple transcripts. RT-PCR analysis revealed that each variant had preferred expression sites, suggesting that promoter usage and splicing are regulated in tissue-specific manners. Moreover, we determined a splicing event to yield Deltaexon 1 variants (0S-2-3-4-5-6-7-8), which are translated into rat 46 kDa ERalpha proteins. Our results indicate that the rat ERalpha gene is more complex than previously thought in terms of genomic organization and that both alternative promoter usage and alternative splicing contribute to the remarkable diversity of ERalpha mRNAs.

DOI： 10.1016/j.jsbmb.2009.10.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ENDOCRINE SOC  

Estradiol (E2) and other sex steroids play essential roles in the modulation of synaptic plasticity and neuroprotection in the hippocampus. To clarify the mechanisms for these events, it is important to determine the respective role of circulating vs. locally produced sex steroids in the male hippocampus. Liquid chromatography-tandem mass spectrometry in combination with novel derivatization was employed to determine the concentration of sex steroids in adult male rat hippocampus. The hippocampal levels of 17beta-E2, testosterone (T), and dihydrotestosterone (DHT) were 8.4, 16.9, and 6.6 nm, respectively, and these levels were significantly higher than circulating levels. The hippocampal estrone (E1) level was, in contrast, very low around 0.015 nm. After castration to deplete circulating high level T, hippocampal levels of T and DHT decreased considerably to 18 and 3%, respectively, whereas E2 level only slightly decreased to 83%. The strong reduction in hippocampal DHT resulting from castration implies that circulating T may be a main origin of DHT. In combination with results obtained from metabolism analysis of [(3)H]steroids, we suggest that male hippocampal E2 synthesis pathway may be androstenedione --> T --> E2 or dehydroepiandrosterone --> androstenediol --> T --> E2 but not androstenedione --> E1 --> E2.

DOI： 10.1210/en.2009-0305

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The hippocampus is essentially involved in learning and memory processes. Its functions are affected by various neuromodulators, including 17beta-estradiol, testosterone, and retinoid. Brain-synthesized steroid hormones act as autocrine and paracrine modulators. The regulatory mechanism underlying brain steroidogenesis has not been fully elucidated. Synthesis of sex steroids in the gonads is stimulated by retinoic acids. Therefore, we examined the effects of retinoic acids on estradiol and testosterone biosynthesis in the rat hippocampus. We used cultured hippocampal slices from 10- to 12-d-old male rats to investigate de novo steroidogenesis. The infant rat hippocampus possesses mRNAs for steroidogenic enzymes and retinoid receptors. Slices were used after 24 h of preculture to obtain maximal steroidogenic activity because steroidogenesis in cultured slices decreases with time. The mRNA levels for P450(17alpha), P450 aromatase and estrogen receptor-beta in the slices were increased by treatment with 9-cis-retinoic acid but not by all-trans-isomer. The magnitude of stimulation and the shape of the dose-response curve for the mRNA level for P450(17alpha) were similar to those for cellular retinoid binding protein type 2, the transcription of which is activated by retinoid X receptor signaling. 9-cis-Retinoic acid also induced a 1.7-fold increase in the protein content of P450(17alpha) and a 2-fold increase in de novo synthesis of 17beta-estradiol and testosterone. These steroids may be synthesized from a steroid precursor(s), such as pregnenolone or other steroids, or from cholesterol, as so-called neurosteroids. The stimulation of estradiol and testosterone synthesis by 9-cis-retinoic acid might be caused by activation of P450(17alpha) transcription via retinoid X receptor signaling.

DOI： 10.1210/en.2008-1644

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Sex-steroid synthesis in the hippocampus had been thought to be much more active at the neonatal stage than at the adult stage. However, the detailed comparison between these two stages had not been demonstrated yet. Here we performed the comparison about the mRNA level of steroidogenic enzymes and the rate of steroid metabolism between these two stages of the hippocampus. The relative expression level of P450(17alpha), 17beta- or 3beta-hydroxysteroid dehydrogenase, or P450arom was approximately 1.3-1.5-fold higher at the neonatal than at the adult stage. The rate of sex-steroid metabolism (from dehydroepiandrosterone to estradiol) was 2-7-fold (depending on different steps) more rapid at the neonatal than at the adult stage. Taken together, neonatal steroidogenesis is moderately more active than adult steroidogenesis.

DOI： 10.1016/j.bbrc.2009.05.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Gonadotrophin-releasing hormone (GnRH) neurones represent the final output neurones in the neuroendocrine system for the control of reproduction. To understand the reproductive neuroendocrine system, an investigation of the intrinsic and extrinsic properties of GnRH neurones is essential. In this review, we focus on the intrinsic properties and summarise our recent findings of ion channels expressed in rat GnRH neurones. Rat GnRH neurones express all four types of high voltage-activated Ca(2+) channel (L, N, P/Q, R) and the low voltage-activated Ca(2+) channel (T). GnRH neurones also express two types of Ca(2+)-activated K(+) [K(Ca)] channel: the small conductance Ca(2+)-activated K(+) (SK) channel and the large conductance Ca(2+)- and voltage-activated K(+) (BK) channel. The activities of these Ca(2+) and K(Ca) channels regulate cell excitability and cellular calcium homeostasis.

DOI： 10.1111/j.1365-2826.2009.01849.x

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

G-protein coupled receptors for the pineal hormone melatonin have been partially cloned from rats. However, insufficient information about their cDNA sequences has hindered studies of their distribution and physiological responses to melatonin using rats as an animal model. We have cloned cDNAs of two rat membrane melatonin receptor subtypes, melatonin receptor 1a (MT1) and melatonin receptor 1b (MT2), using a rapid amplification of cDNA end (RACE) method. The rat MT1 and MT2 cDNAs encode proteins of 353 and 364 amino acids, respectively, and show 78-93% identities with the human and mouse counterparts. Stable expression of either rat MT1 or MT2 in NIH3T3 cells resulted in high affinity 2-[(125)I]-iodomelatonin ((125)I-Mel) binding (K (d) = 73.2 +/- 9.0 and 73.7 +/- 2.9 pM, respectively), and exhibited a similar rank order of inhibition of specific (125)I-Mel binding by five ligands (2-iodomelatonin > melatonin > 6-hydroxymelatonin > luzindole > N-acetyl-5-hydroxytryptamine). RT-PCR analysis showed that MT1 is highly expressed in the hypothalamus, lung, kidney, adrenal gland, stomach, and ovary, while MT2 is highly expressed in the hippocampus, kidney, and ovary. We also performed multi-cell RT-PCR to examine the expression of mRNAs encoding MT1 and MT2 in adult GnRH neurons. MT1 was weakly expressed in male GnRH neurons, and was less expressed in the female neurons. MT2 expression was undetectable in GnRH neurons from either sex. This study delineates the gene structures, fundamental properties, and distribution of both rat melatonin receptor subtypes, and may offer opportunities to assess the physiological significance of melatonin in rats.

DOI： 10.1007/s12576-008-0003-9

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Melatonin has been implicated in the control of the reproductive system, and the modulatory actions of melatonin on gonadotropin-releasing hormone (GnRH) neurons have been assumed to be indirectly mediated through afferent neurons. However, our previous studies demonstrate sexually dimorphic modulation of A-type gamma-aminobutyric acid (GABA) receptor (GABA(A)R) currents by melatonin in adult rat GnRH neurons and a preferential expression of melatonin 1a receptor (MT1) in male GnRH neurons. Using immortalized GnRH neurons (GT1-7 cells), the present study investigated the mechanism by which the expression of melatonin receptors is regulated in GnRH neurons. Like endogenous GnRH neurons, GT1-7 cells express both GnRH and GnRH receptor mRNAs, indicating that the cells have a self-stimulatory system. A 2-iodomelatonin binding assay and RT-PCR analysis demonstrated that the cells expressed neither MT1 nor MT2. However, treatment of GT1-7 cells with the GnRH antagonist cetrorelix significantly increased 2-iodomelatonin binding and induced a time- and concentration-dependent MT1 mRNA expression. The GABA(A)R currents were then measured using a perforated patch-clamp technique to examine whether the treatment with cetrorelix changed the responses to melatonin. Melatonin augmented the GABA(A)R currents in GT1-7 cells treated with 1 muM cetrorelix for 24 h, while melatonin decreased the currents in the cells not treated with cetrorelix, probably via receptor-independent processes. The present results suggest that GnRH downregulates the expression of MT1 via an autocrine-paracrine mechanism in GT1-7 cells, and modifies the melatonin-induced modulation of GABA(A)R currents. These findings may provide one possible mechanism for the sexually dimorphic responses to melatonin in adult rat GnRH neurons.

DOI： 10.1159/000231993

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Estrogen and androgen are synthesized from cholesterol locally in hippocampal neurons of adult animals. These neurosteroids are synthesized by cytochrome P450s and hydroxysteroid dehydrogenases (HSDs) and 5alpha-reductase. The expression levels of enzymes are as low as 1/200-1/50,000 of those in endocrine organs, however these numbers are high enough for local synthesis. Localization of P450(17alpha), P450arom, 17beta-HSD and 5alpha-reductase is observed in principal glutamatergic neurons in CA1, CA3 and the dendate gyrus. Several nanomolar levels of estrogen and androgen are observed in the hippocampus. Estrogen modulates memory-related synaptic plasticity not only slowly but also rapidly in the hippocampus. Rapid action of 17beta-estradiol via membrane receptors is demonstrated for spinogenesis and long-term depression (LTD). The enhancement of LTD by 1-10nM estradiol occurs within 1 h. The density of spine is increased in CA1 pyramidal neurons within 2h after application of estradiol. The density of spine-like structure is, however, decreased by estradiol in CA3 pyramidal neurons. ERalpha, but not ERbeta, induces the same enhancement/suppression effects on both spinogenesis and LTD.

DOI： 10.1016/j.mce.2008.04.017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Gonadotrophin-releasing hormone (GnRH) neurones represent the final output neurones in the neuroendocrine control of reproduction, and gamma-amino butyric acid (GABA) is one of the major players in the regulation of GnRH neurones. GABA inhibits a large proportion of brain neurones in adult animals by acting on A-type GABA receptors (GABA(A)Rs). Two contradictory reports on the action of GABA in the GnRH neurones of adult mice have been published. DeFazio et al. (Mol Endocrinol 2002; 16: 2872) demonstrated that activation of GABA(A)Rs excites the GnRH neurones of adult mice, whereas Han et al. (Endocrinology 2002; 143: 1459) showed that the response to GABA on GnRH neurones switches from depolarisation to hyperpolarisation around puberty in female mice. Therefore, we examined the reversal potential of GABA(A)R currents by means of perforated patch-clamp recording with gramicidin in overnight-cultured GnRH neurones isolated from adult GnRH-enhanced green fluorescent protein transgenic rats. The reversal potential was -26 +/- 1.4 mV (mean +/- SEM, n = 42) in GnRH neurones, whereas it was -57 +/- 2.7 mV (n = 34) in unidentified neurones, and GABA depolarised the GnRH neurones in current-clamp condition. The GABA(A)R currents in rat GnRH neurones were augmented by neurosteroids, allopregnanolone and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one, at submicromolar concentrations. In addition, the expression patterns of GABA(A)R subunit mRNAs were determined by multi-cell reverse transcription-polymerase chain reaction, which revealed that the alpha2, beta 3, gamma 1 and gamma 2 subunits were dominant and the alpha 6 and gamma 3 subunits were negative in rat GnRH neurones. These results indicate that GABA(A)Rs in the soma of rat GnRH neurones are comprised mainly of alpha2, beta 3 and gamma 1 or gamma 2 subunits and that they are sensitive to neurosteroids; moreover, they suggest that activation of these receptors depolarises GnRH neurones. Thus, GABA and neurosteroids influence the electrical activity of GnRH neurones.

DOI： 10.1111/j.1365-2826.2008.01697.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ENDOCRINE SOC  

Estrogens play essential roles in the neuroendocrine control of reproduction. In the present study, we focused on the effects of 17beta-estradiol (E2) on the K(+) currents that regulate neuronal cell excitability and carried out perforated patch-clamp experiments with the GnRH-secreting neuronal cell line GT1-7. We revealed that a 3-d incubation with E2 at physiological concentrations (100 pm to 1 nm) augmented Ca(2+)-activated K(+) [K(Ca)] currents without influencing Ca(2+)-insensitive voltage-gated K(+) currents in GT1-7 cells. Acute application of E2 (1 nm) had no effect on the either type of K(+) current. The augmentation was completely blocked by an estrogen receptor (ER) antagonist, ICI-182,780. An ERbeta-selective agonist, 2,3-bis(4-hydroxyphenyl)-propionitrile, augmented the K(Ca) currents, although an ERalpha-selective agonist, 4,4',4''-[4-propyl-(1H)-pyrazole-1,3,5-triyl]tris-phenol, had no effect. Knockdown of ERbeta by means of RNA interference blocked the effect of E2 on the K(Ca) currents. Furthermore, semiquantitative RT-PCR analysis revealed that the levels of BK channel subunit mRNAs for alpha and beta4 were significantly increased by incubating cells with 300 pm E2 for 3 d. In conclusion, E2 at physiological concentrations augments K(Ca) currents through ERbeta in the GT1-7 GnRH neuronal cell line and increases the expression of the BK channel subunit mRNAs, alpha and beta4.

DOI： 10.1210/en.2007-0759

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHYSIOLOGICAL SOC JAPAN  

Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction. As in other neurons, the discharge pattern of action potentials is important for these neurons to function properly. Therefore it is important to elucidate the expression patterns of various types of ion channels in these neurons because they determine cell excitability. To date, voltage-gated Ca2+ channels and SK channels have been reported to be expressed in rat GnRH neurons. In this study, we focused on K+ channels and analyzed their expression in primary cultured GnRH neurons, prepared from GnRH-EGFP transgenic rats, by means of perforated patch-clamp recordings. GnRH neurons exhibited delayed-rectifier K+ currents and large conductance voltage- and Ca2+-activated K+ (BK) currents. Moreover, multicell RT-PCR (reverse transcriptase-polymerase chain reaction) experiments revealed the expression of BK channel mRNAs (alpha, beta1, beta2, and beta4). The results show the presence of delayed-rectifier K+ currents and BK currents besides previously reported slow afterhyperpolarization currents. These currents control the action potential repolarization and probably also the firing pattern, thereby regulating the cell excitability of GnRH neurons.

DOI： 10.2170/physiolsci.RP013207

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer Netherlands  

It has long been a common sense that sex hormones are synthesized in the gonads, and reach the brain via the blood circulation. In contrast with this view, the authors demonstrate that estrogen and androgen are also synthesized locally in the hippocampus of adult animals, from cholesterol through dehydroepiandrosterone in hippocampal neurons. These neurosteroids are synthesized by cytochrome P450s and hydroxysteroid dehydrogenases and 5α-reductase. The expression levels of enzymes are as low as 1/200-1/50,000 of those in endocrine organs, preventing quantitative investigations. Localization of P450(17α) and P450arom is observed in synapses of principal glutamatergic neurons, in addition to endoplasmic reticulum, suggesting synaptocrine machanisms. Because several nanomolar estrogen and androgen are observed in the hippocampus, they are expected to have physiological functions. Estrogen modulates memory-related synaptic plasticity not only slowly, but also rapidly in the hippocampus. Molecular mechanisms of rapid action via membrane receptors have not been well elucidated in comparison with those of delayed action via genomic processes. We here describe rapid modulation of representative synaptic plasticity, i.e., long-term depression (LTD), long-term potentiation (LTP) and spinogenesis, by 17β-estradiol, selective estrogen agonists. We demonstrate that 1-10 nM estradiol induced rapid enhancement of LTD within 1 h in CA1, CA3 and dentate gyrus (DG). On the other hand, the modulation of LTP by estradiol is not statistically significant. The total density of spines is increased in CA1 pyramidal neurons, within 2 h after application of estradiol. The total density of thorns (postsynaptic spine-like structure) is, however, decreased by estradiol in CA3 pyramidal neurons. Both the increase of spines in CA1 and the decrease of thorns in CA3 are driven by Erk MAP kinase. Only agonist of estrogen receptor ERalpha induces the same enhancement/suppression effect as estradiol on both LTD and spinogenesis in CA1 and CA3. ERbeta agonist induces completely different results. Estrogen receptor ERalpha localizes in spines and presynapses of principal glutamatergic neurons in CA1, CA3 and DG. The same ERalpha is also located in nuclei. Identification of ERalpha is successfully performed using purified RC-19 antibody. Attention must be paid to the fact that non-purified ERalpha antisera often react significantly with unknown proteins, resulting in wrong staining different from real ERalpha distribution. Identification of kinases/phosphatases in downstream of ERalpha as well as other synaptic estrogen receptors is essential to advance the field. © 2008 Springer Netherlands.

DOI： 10.1007/978-1-4020-6854-6_7

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS INC  

It is believed that sex hormones are synthesized in the gonads and reach the brain via the blood circulation. In contrast with this view, the authors have demonstrated that sex hormones are also synthesized locally in the hippocampus and that these steroids act rapidly to modulate neuronal synaptic plasticity. The authors demonstrated that estrogens are locally synthesized from cholesterol through dehydroepiandrosterone and testosterone in adult hippocampal neurons. Significant expression of mRNA for P450(17alpha), P450arom, and other steroidogenic enzymes was demonstrated. Localization of P450(17alpha) and P450arom was observed in synapses of principal neurons. In contrast to the slow action of gonadal estradiol, hippocampal neuron-derived estradiol may act locally and rapidly within the neurons. For example, 1 to 10 nM estradiol rapidly enhances long-term depression (LTD). The density of thin spines is selectively increased within two hours upon application of estradiol in pyramidal neurons. Estrogen receptor ERalpha agonist has the same enhancing effect as estradiol on both LTD and spinogenesis. Localization of ERalpha in spines in addition to nuclei of principal neurons implies that synaptic ERalpha is responsible for rapid modulation of synaptic plasticity by endogenous estradiol. Activin A, a peptide sex hormone, may also play a role as a local endogenous modulator of synaptic plasticity.

DOI： 10.1177/10738584070130040601

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Rapid modulation of hippocampal synaptic plasticity by estrogen has long been a hot topic, but analysis of molecular mechanisms via synaptic estrogen receptors has been seriously difficult. Here, two types of independent synaptic plasticity, long-term depression (LTD) and spinogenesis, were investigated, in response to 17beta-estradiol and agonists of estrogen receptors using hippocampal slices from adult male rats. Multi-electrode investigations demonstrated that estradiol rapidly enhanced LTD not only in CA1 but also in CA3 and dentate gyrus. Dendritic spine morphology analysis demonstrated that the density of thin type spines was selectively increased in CA1 pyramidal neurons within 2 h after application of 1 nm estradiol. This enhancement of spinogenesis was completely suppressed by mitogen-activated protein (MAP) kinase inhibitor. Only the estrogen receptor (ER) alpha agonist, (propyl-pyrazole-trinyl)tris-phenol (PPT), induced the same enhancing effect as estradiol on both LTD and spinogenesis in the CA1. The ERbeta agonist, (4-hydroxyphenyl)-propionitrile (DPN), suppressed LTD and did not affect spinogenesis. Because the mode of synaptic modulations by estradiol was mostly the same as that by the ERalpha agonist, a search was made for synaptic ERalpha using purified RC-19 antibody qualified using ERalpha knockout (KO) mice. Localization of ERalpha in spines of principal glutamatergic neurons was demonstrated using immunogold electron microscopy and immunohistochemistry. ERalpha was also located in nuclei, cytoplasm and presynapses.

DOI： 10.1111/j.1471-4159.2006.04264.x

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

The current quantitative study demonstrates that the recruitment of neuronal nitric oxide synthase (nNOS) beneath N-methyl-D-aspartate (NMDA) receptors, via postsynaptic density 95 (PSD-95) proteins significantly enhances nitric oxide (NO) production. Real-time single-cell fluorescence imaging was applied to measure both NO production and Ca(2+) influx in Chinese hamster ovary (CHO) cells expressing recombinant NMDA receptors (NMDA-R), nNOS, and PSD-95. We examined the relationship between the rate of NO production and Ca(2+) influx via NMDA receptors using the NO-reactive fluorescent dye, diaminofluorescein-FM (DAF-FM) and the Ca(2+)-sensitive yellow cameleon 3.1 (YC3.1), conjugated with PSD-95 (PSD-95-YC3.1). The presence of PSD-95 enhanced the rate of NO production by 2.3-fold upon stimulation with 100 microm NMDA in CHO1(+) cells (expressing NMDA-R, nNOS and PSD-95) when compared with CHO1(-) cells (expressing NMDA-R and nNOS lacking PSD-95). The presence of nNOS inhibitor or NMDA-R blocker almost completely suppressed this NMDA-stimulated NO production. The Ca(2+) concentration beneath the NMDA-R, [Ca(2+)](NR), was determined to be 5.4 microm by stimulating CHO2 cells (expressing NMDA-R and PSD-95-YC3.1) with 100 microm NMDA. By completely permealizing CHO1 cells with ionomycin, a general relationship curve of the rate of NO production versus the Ca(2+) concentration around nNOS, [Ca(2+)](NOS), was obtained over the wide range of [Ca(2+)](NOS). This sigmoidal curve had an EC(50) of approximately 1.2 microm of [Ca(2+)](NOS), implying that [Ca(2+)](NR) = 5.4 microm can activate nNOS effectively.

DOI： 10.1111/j.1471-4159.2006.03656.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

In neuroendocrinology, it is believed that steroid hormones are synthesized in the gonads and/or adrenal glands, and reach the brain via the blood circulation. In contrast to this view, we are in progress of demonstrating that estrogens and androgens are also synthesized locally by cytochrome P450s in the hippocampus, and that these steroids act rapidly to modulate neuronal synaptic plasticity. We demonstrated that estrogens were locally synthesized in the adult hippocampal neurons. In the pathway of steroidogenesis, cholesterol is converted to pregnenolone (by P450scc), dehydroepiandrosterone [by P450(17alpha)], androstenediol (by 17beta-hydroxysteroid dehydrogenase, 17beta-HSD), testosterone (by 3beta-HSD) and finally to estradiol (by P450arom) and dihydrotestosterone (by 5alpha-reductase). The basal concentration of estradiol in the hippocampus was approximately 1 nM, which was greater than that in blood plasma. Significant expression of mRNA for P450scc, P450(17alpha), P450arom, 17beta-HSD, 3beta-HSD and 5alpha-reductase was demonstrated by RT-PCR. Their mRNA levels in the hippocampus were 1/200-1/5,000 of those in the endocrine organs. Localization of P450(17alpha) and P450arom was observed in synapses in addition to endoplasmic reticulum of principal neurons using immunoelectron microscopy. Different from slow action of gonadal estradiol which reaches the brain via the blood circulation, hippocampal neuron-derived estradiol may act locally and rapidly within the neurons. For example, 1 nM 17beta-estradiol rapidly enhanced the long-term depression (LTD) not only in CA1 but also in CA3 and dentate gyrus. The density of thin spines was selectively increased within 2 h upon application of 1 nM estradiol in CA1 pyramidal neurons. Only ERalpha agonist propyl-pyrazole-trinyl-phenol induced the same enhancing effect as estradiol on both LTD and spinogenesis in the CA1. ERbeta agonist hydroxyphenyl-propionitrile suppressed LTD and did not affect spinogenesis. Localization of estrogen receptor ERalpha in spines in addition to nuclei of principal neurons implies that synaptic ERalpha can drive rapid modulation of synaptic plasticity by endogenous estradiol.

DOI： 10.1159/000097747

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Hippocampal pyramidal neurons and granule neurons of adult male rats are equipped with a complete machinery for the synthesis of pregnenolone, dehydroepiandrosterone, testosterone, dihydrotestosterone and 17 beta-estradiol. Both estrogens and androgens are synthesized in male hippocampus. These brain steroids are synthesized by cytochrome P450s (P450scc, P45017 alpha and P450arom), hydroxysteroid dehydrogenases and reductases from endogenous cholesterol. The expression levels of enzymes are as low as 1/300-1/1000 of those in endocrine organs. Synthesis is dependent on the acute Ca2+ influx upon neuron-neuron communication via NMDA receptors. Estradiol is particularly important because estradiol rapidly modulates neuronal synaptic transmission such as long-term potentiation via synaptic estrogen receptors. Xenoestrogens may also act via estrogen-driven signaling pathways. (C) 2005 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2005.09.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

In the hippocampus, the center for learning and memory, cytochrome P450s (P450scc, P450(17 alpha), and P450arom) as well as 17 beta-, 3 beta-hydroxysteroid dehydrogenases, and 5 alpha-reductase participate in the synthesis of brain steroids from endogenous cholesterol. These brain steroids include pregnenolone, dehydroepiandrosterone, testosterone, dihydrotestosterone, and 17 beta-estradiol. Both estrogens and androgens are synthesized in the adult male hippocampal neurons. Although the expression levels of steroidogenic enzymes are as low as 1/200 to 1/50,000 of those in testis or ovary, the levels of synthesized steroids are sufficient for the local usage within small neurons (i.e., intracrine system). This intracrine system contrasts with the endocrine system in which high expression levels of steroidogenic enzymes are necessary in endocrine organs in order to supply steroids to many other organs via blood circulation. Endogenous synthesis of sex steroids in the hypothalamus is also discussed.
Rapid modulation by estrogens and xenoestrogens is discussed concerning synaptic plasticity such as the long-term potentiation, the long-term depression, or spinogenesis. Synaptic expression of P450(17 alpha), P450arom, and estrogen receptors suggests "synaptocrine mechanisms of brain steroids, which are synthesized at synapses and act as synaptic modulators.

DOI： 10.1080/03602530600724068

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

In adult mammalian brain, occurrence of the synthesis of estradiol from endogenous cholesterol has been doubted because of the inability to detect dehydroepiandrosterone synthase, P45017alpha. In adult male rat hippocampal formation, significant localization was demonstrated for both cytochromes P45017alpha and P450 aromatase, in pyramidal neurons in the CA1-CA3 regions, as well as in the granule cells in the dentate gyrus, by means of immunohistochemical staining of slices. Only a weak immunoreaction of these P450s was observed in astrocytes and oligodendrocytes. ImmunoGold electron microscopy revealed that P45017a and P450 aromatase were localized in pre- and postsynaptic compartments as well as in the endoplasmic reticulum in principal neurons. The expression of these cytochromes was further verified by using Western blot analysis and RT-PCR. Stimulation of hippocampal neurons with N-methyl-D-aspartate induced a significant net production of estradiol. Analysis of radioactive metabolites demonstrated the conversion from [H-3]pregnenolone to [H-3]estradiol through dehydroepiandrosterone and testosterone. This activity was abolished by the application of specific inhibitors of cytochrome P450s. Interestingly, estradiol was not significantly converted to other steroid metabolites. Taken together with our previous finding of a P450scc-containing neuronal system for pregnenolone synthesis, these results imply that 17beta-estradiol is synthesized by P45017alpha and P450 aromatase localized in hippocampal neurons from endogenous cholesterol. This synthesis may be regulated by a glutamate-mediated synaptic communication that evokes Ca2+ signals.

DOI： 10.1073/pnas.2630225100

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Language：English   Publishing type：Research paper (international conference proceedings)  

Hippocampal pyramidal neurons and granule neurons of adult male rats are equipped with a complete machinery for the synthesis of pregnenolone, dehydroepiandrosterone, 17β-estradiol and testosterone as well as their sulfate esters. These brain neurosteroids are synthesized by cytochrome P450s (P450scc, P45017α and P450arom) from endogenous cholesterol. Synthesis is acutely dependent on the Ca2+ influx attendant upon neuron-neuron communication via N-methyl-D-aspartate (NMDA) receptors. Pregnenolone sulfate, estradiol and corticosterone rapidly modulate neuronal signal transduction and the induction of long-term potentiation via NMDA receptors and putative membrane steroid receptors. Brain neurosteroids are therefore promising neuromodulators that may either activate or inactivate neuron-neuron communication, thereby mediating learning and memory in the hippocampus. © 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0304-4165(02)00489-0

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Language：Japanese   Publisher：日本組織細胞化学会  

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当院で経鼻的下垂体手術を施行した非機能性下垂体腺腫(NFPA)20例(男性15例、女性5例、平均年齢60.7歳)を対象に、デジタルPCR法(dPCR)と免疫組織化学染色法を用いてエストロゲン受容体(ESR)発現の定量解析を行った。その結果、dPCRによる遺伝子の絶対定量と特異的抗体を用いた免疫組織化学染色法を組み合わせることで、NFPAにおけるESR発現の正確な定量系を確立することができた。また、本研究ではESR1遺伝子発現と術前の臨床像との間に相関性を見い出すことはできなかったが、ESR1発現がGREB1を介してNFPAの臨床像に関与する可能性が示唆された。

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DOI： 10.2142/biophys.45.S92_4

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