中尾 暢宏

Abstract Imidazole dipeptides (ID) are abundant in skeletal muscle and the brain and have various functions, such as antioxidant, pH-buffering, metal-ion chelation. However, the physiological significance of ID has not been fully elucidated. In this study, we orally administered ID to conventional carnosine synthase gene-deficient mice (Carns-KO mice) to investigate the pharmacokinetics. Carnosine or anserine was administered at a dose of 500 mg (approximately 2 mmol) per kilogram of mouse body weight, and ID contents in the tissues were measured. No ID were detected in untreated Carns-KO mice. In the ID treatment groups, the ID concentrations in the tissues increased in a time-dependent manner in the gastrocnemius muscle, soleus muscle, and cerebrum after ID administration. Our findings suggest that the Carns-KO mice are a valuable animal model for directly evaluate the effects of dietary ID and for elucidating the physiological functions of oral ID administration.

Prolactin (PRL) is a hormone mainly secreted by the anterior pituitary gland. In birds, PRL exerts a variety of physiological functions in target tissues expressing the PRL receptor (PRLR). In chicken, the PRLR mRNA is abundant in the anterior pituitary gland, but its regional and cellular localization are unknown. In the present study, we investigated the expression of the PRLR mRNA in cephalic and caudal lobes of the chicken anterior pituitary gland. Real-time polymerase chain reaction (PCR) revealed high levels of PRLR mRNA in both cephalic and caudal lobes. In situ hybridization revealed that the PRLR mRNA was distributed in a wide area of both lobes, and co-localized with the PRL and growth hormone (GH) mRNAs in the cephalic and caudal lobes, respectively. These results suggest that PRL exerts autocrine/paracrine effects through PRLR on PRL-producing lactotrophs and GH-producing somatotrophs in the chicken anterior pituitary gland.

Background: The pathological condition of canine prostate cancer resembles that of human androgen-independent prostate cancer. Both canine and human androgen receptor (AR) signalling are inhibited by overexpression of the dimerized co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein a (SGTA), which is considered to cause the development of androgen-independency. Reduced expression in immortalised cells (REIC/Dkk-3) interferes with SGTA dimerization and rescues AR signalling. This study aimed to assess the effects of REIC/Dkk-3 and SGTA interactions on AR signalling in the canine androgen-independent prostate cancer cell line CHP-1. Results: Mammalian two-hybrid and Halo-tagged pull-down assays showed that canine REIC/Dkk-3 interacted with SGTA and interfered with SGTA dimerization. Additionally, reporter assays revealed that canine REIC/Dkk-3 restored AR signalling in both human and canine androgen-independent prostate cancer cells. Therefore, we confirmed the interaction between canine SGTA and REIC/Dkk-3, as well as their role in AR signalling. Conclusions: Our results suggest that this interaction might contribute to the development of a novel strategy for androgen-independent prostate cancer treatment. Moreover, we established the canine androgen-independent prostate cancer model as a suitable animal model for the study of this type of treatment-refractory human cancer.

Acetoacetyl-CoA thiolase (EC 2.3.1.9) catalyzes the cleavage of acetoacetyl-CoA into acetylCoA and its reverse reaction, the synthesis of acetoacetyl-CoA. Cytosolic acetoacetyl-CoA thiolase (CT) is a key enzyme in the initial step of the cholesterol synthesis pathway. In the present study, we characterized the amino acid sequence of chicken CT and the tissue distribution of its mRNA and protein, together with their developmental changes in the liver. The amino acid sequence encoded by the nucleotide sequence of chicken CT cDNA showed a higher overall identity with those of human (74.3%) and rat (74.6%) CTs. Amino acid residues known to participate in enzymatic activity in human CT are conserved in chicken CT. Real-time PCR analysis revealed the expression of CT mRNA in the liver, kidney, adrenal gland, jejunum and ovary of adult hens, with higher levels in the liver, kidney, adrenal gland and ovary. Western blot analysis detected an immunoreactive protein of 41 kDa from cytoplasmic fraction but not particulate fractions of adult chicken liver. The immunoreactive protein was detected in all the tissues. The mRNA levels in the liver rapidly increased after hatching, with a maximum on d 5 post-hatching, after which they gradually decreased to adult levels. A similar change was observed in the protein levels. The increase in transcription and protein synthesis of CT suggests that the synthetic pathway of cholesterol from acetyl-CoA produced by CT replaces the hydrolysis of accumulated cholesteryl ester in the liver, in response to a change in the nutrient source from the lipid-rich yolk to a lower-lipid diet during the early post-hatching period.

TSH secreted from the pars distalis (PD) of the pituitary gland stimulates the thyroid gland. In contrast, TSH secreted from the pars tuberalis (PT) of the pituitary gland regulates seasonal reproduction. The ontogeny of thyrotrophs and the regulatory mechanisms of TSH are apparently different between the PD and the PT. Interestingly, fish do not have an anatomically distinct PT, and the saccus vasculosus (SV) of fish is suggested to act as a seasonal sensor. Thus, it is possible that the SV is analogous to the PT. Here we examined the ontogeny of the pituitary gland and SV using rainbow trout. A histological analysis demonstrated the development of the pituitary anlage followed by that of the SV. Lhx3 and Pit-1, which are required for the development of PD thyrotrophs, clearly labeled the pituitary anlage. The common glycoprotein alpha-subunit (CGA) and TSH alpha-subunit (TSHB) genes were also detected in the pituitary anlage. In contrast, none of these genes were detected in the SV anlage. We then performed a microarray analysis and identified parvalbumin (Pvalb) as a marker for SV development. Because Pvalb expression was not detected in the pituitary anlage, no relationship was observed between the development of the SV and the pituitary gland. In contrast to embryos, Lhx3, Pit-1, CGA, and TSHB were all expressed in the adult SV. These results suggest that the morphological differentiation of SV occurs during the embryonic stage but that the functional differentiation into a seasonal sensor occurs in a later developmental stage.

The ileal apical sodium-dependent bile acid cotransporter (ASBT) plays an essential role in the absorption of bile acids from intestinal lumina. ASBT cDNA has been cloned from mammalian and fish species, and the primary structure of the protein and expression properties of the mRNA have been characterized. In this study, we identified chicken ASBT mRNA by cDNA cloning. Chicken ASBT cDNA consisted of 91 bp of the 5'-untranslated region, 1,083 bp of the coding region, and 1,896 bp of the 3'-untranslated region. The cDNA encoded a protein of 360 amino acids showing significant sequence identity with mammalian and fish ASBT. The amino acid residues known to participate in the functions of mammalian ASBT were conserved in chicken ASBT. Real-time polymerase chain reaction analysis revealed that chicken ASBT mRNA was expressed at markedly higher levels in the ileum and proximal colon/rectum, relatively lower levels in the kidney, and very low levels in the jejunum and cecum. Expression levels in the ileum markedly increased after hatching, reached the highest levels on day 7 posthatching, and then decreased to adult levels. A similar expression pattern was observed in the proximal colon/rectum except for the significant decrease from day 7 posthatching to day 21 posthatching. These results suggest that chicken ASBT functions as a bile acid transporter in the ileum and proximal colon/rectum, particularly during the early posthatching period.

G-protein-coupled receptors (GPRs) 40 and 120 are members of the Free Fatty Acid (FFA) receptor group and are termed FFAR1 and FFAR4, respectively. The aim of this study was to clone cat GPR40 and GPR120 cDNAs in several tissues. There was high sequence homology to other mammalian GPR40 and GPR120, with encoding 320 and 361 amino acid residues, respectively. Cat GPR40 encoded extra 21 amino acid residues in the C-terminal cytoplasmic region. Quantitative RT-PCR revealed expression of GPR40 mRNA in the duodenum, liver and pancreas. The GPR120 mRNA was expressed in adipose tissues, cerebral cortex and colon. In conclusion, GPR40 and GPR120 were well conserved and were expressed in cat tissues with different distribution patterns.

We isolated a high-purity carp glycophorin from carp erythrocyte membranes following extraction using the lithium diiodosalicylate (LIS)-phenol method and streptomycin treatment. The main carp glycophorin was observed to locate at the position of the carp and human band-3 proteins on an SDS-polyacrylamide gel. Only the N-glycolylneuraminic acid (NeuGc) form of sialic acid was detected in the carp glycophorin. The oligosaccharide fraction was separated into two components (P-1 and P-2) using a Glyco-Pak DEAE column. We observed bacteriostatic activity against five strains of bacteria, including two known fish pathogens. Fractions from the carp erythrocyte membrane, the glycophorin oligosaccharide and the P-1 also exhibited bacteriostatic activity whereas the glycolipid fraction and the glycophorin fraction without sialic acid did not show the activity. The carp glycophorin molecules attach to the flagellum of V. anguillarum or the cell surface of M. luteus and inhibited bacterial growth. © 2014 by the authors licensee MDPI, Basel, Switzerland.

Neurotensin is a tridecapeptide that has multiple functions as a neurotransmitter and as a circulating hormone. Neurotensin and its related peptide, LANT6, have been isolated in the chicken, but the mRNA encoding these peptides has not been identified. In this study, we first cloned the cDNA for the chicken neurotensin precursor mRNA from the duodenum and characterized its primary structure and then investigated tissue expression patterns of neurotensin precursor and receptor mRNA. The cDNA encoded a protein of 495 amino acids that contains the sequences of chicken neurotensin and LANT6 in the C-terminal region. Real-time PCR analysis showed that the neurotensin precursor mRNA is preferentially expressed in intestinal tissues, such as the duodenum, jejunum, ileum, and colon/rectum, with temporal increases during the hatching period. The expression levels of neurotensin receptor 1 mRNA were relatively higher during the late embryonic period compared with the posthatching period in the duodenum and jejunum, whereas the expression levels were higher in the colon/rectum during the posthatching period. In the liver, the expression levels of neurotensin receptor 1 were markedly increased during the early posthatching period. These results suggest that chicken neurotensin is involved in the regulation of gastrointestinal and hepatic functions, especially during the hatching period.

Puberty onset in mammals is tightly coupled to the animal's nutritional and metabolic state. In the present study, we evaluated the effects of a high-fat diet on leptin and adiponectin levels, leptin mRNA expression and puberty onset in female rats. On day 21, female rats were divided into 2 groups, normal food (NF) and high-fat food (HF). The HF group showed a significantly earlier (P<0.001) date of vaginal opening and lower body weight (P<0.001) than the NF group. The rats fed the HF food had a significantly heavier uterus (P<0.05) than those fed the NF food, whereas the serum leptin and adiponectin levels and leptin mRNA expression were not significantly different between the NF and HF groups. We speculate that the fat-induced nutritional imbalance in young females may lead to neuroendocrine dysfunction during adolescence.

The aim of this study was to investigate food intake, serum leptin levels, and leptin mRNA expression during the sexual cycle in rats. Female Wistar-Imamichi rats aged 8-10 weeks were used in this experiment. Food intake was measured during the light and dark phases (light on at 07:00 and off at 19:00) of the 4-day estrous cycle in female rats. Serum leptin levels were measured by ELISA, and leptin mRNA expression levels were analyzed using real-time PCR on diestrous- and proestrous-stage rats. Our results revealed that during the sexual cycle, food intake was significantly higher in the dark phase compared with the light phase. Food intake in proestrous females was significantly lower in the light and dark phases compared with the other groups. Serum leptin concentrations were significantly higher in both phases in proestrous rats compared with diestrous rats. There was a significant increase in leptin mRNA expression in adipose tissue during the proestrous period compared with the diestrous period. These findings suggest that increased leptin mRNA expression and serum leptin levels, which are induced by estrogen during the proestrous stage, may play a role in regulating appetitive behavior.

Prolactin (PRL) has numerous physiological functions that are mediated by its receptors in target cells. Expression of the rat PRL receptor (PRLR) gene is regulated in a tissue-specific manner via the transcriptional activation of five distinct first exons, i.e., E1(1), E1(2), E1(3), E1(4), and E1(5). In the present study, we investigated the expression profiles of these first exon variants of PRLR mRNA in the rat choroid plexus, which is considered to be a site of receptor-mediated PRL transport from the blood to cerebrospinal fluid. Real-time PCR analysis revealed that E1(3)-, E1(4)-, and E1(5)-PRLR mRNA expression levels increased in the choroid plexus in male and female rats during postnatal development, with markedly higher level of E1(4)-PRLR mRNA. In female rats, the E1(4)-PRLR mRNA expression levels increased markedly during lactation compared with the diestrus state, whereas there was no increase in the E1(3)- and E1(5)-PRLR mRNA levels. The E1(4)-PRLR mRNA expression pattern was similar to that of the total PRLR mRNA. The PRL plasma concentration generally correlated with the E1(4)-PRLR mRNA expression levels in both sexes. These findings suggest that PRLR gene expression in the choroid plexus is upregulated mainly via the transcriptional activation of the E1(4)-first exon.

Prolactin (Prl) receptor (Prlr) gene is expressed in various brain regions, with the highest level present in the choroid plexus, a site for receptor-mediated PRL transport from the blood to cerebrospinal fluid. We investigated the regulatory mechanism of Prlr gene expression by PRL in the murine choroid plexus. We first examined the organization of the alternative first exons in murine Prlr gene. In addition to the three known first exons, mE1(1), mE1(2), and mE1(3), two first exons, mE1(4) and mE1(5), were newly identified by cDNA cloning. Each first exon variant of Prlr mRNA exhibited tissue-specific or generic expression. In the choroid plexus of mice, the expression levels of mE1(3)(-), mE1(4)(-), and mE1(5)(-)Prlr mRNAs were increased in the lactating mice compared with those in the diestrus mice. Furthermore, the expression level of mE1(4)-Prlr mRNA was decreased in the PRL-deficient (Prl (/) ) mice compared with the PRL-normal (Prl (+/-/+/-) and Prl (+/-/-)) mice. In the ovariectomized Prl(-/-) mice, the expression level of mE1(4)-Prlr mRNA was significantly increased by PRL administration but not by 17 beta-estradiol administration. The expression levels of the two last exon variants of Prlr mRNAs, encoding the long and short cytoplasmic regions of PRLR, were also increased in the lactating mice and decreased in the Prl(-/-) mice. These findings suggest that PRL stimulates the Prlr gene expression through the transcriptional activation of mE1(4) first exon, leading to increases in the long-and short-form variants of Prlr mRNA in the murine choroid plexus. Journal of Molecular Endocrinology (2012) 48, 169-176

Neuromedin U (NMU) is a bioactive peptide that is involved in a variety of physiological functions. Two of its receptors, NMUR1 and NMUR2, have been identified and characterized in mammals. In this study, we performed cDNA cloning of chicken NMUR1 and NMUR2, and characterized their primary structure, biological activity, and expression patterns in chicken tissues. The chicken NMUR1 and NMUR2 cDNAs encoded 438 and 395 amino acid sequences, respectively. Chicken NMUR1 showed 54.8%-56.5% sequence identity with human, rat, and mouse NMUR1, and NMUR2 shared 67.3%-70.1% sequence identity with mammalian orthologs. Both chicken receptors have typical characteristics of G-protein-coupled receptors with seven transmembrane domains and the D/ERY motif. An increase in intracellular Ca(2+) mobilization was observed in HEK293 cells transfected with chicken NMUR1 or NMUR2 cDNA and treated with chicken or rat NMU. Real-time PCR analysis revealed that NMUR1 mRNA was preferentially expressed in the intestinal tissues such as the duodenum, jejunum, ileum, cecum, and colon/rectum, and brain regions such as the midbrain and optic lobe, and the ovary in adult hens. NMUR2 mRNA was exclusively expressed in the brain regions such as the cerebrum and midbrain. These results indicate that NMUR1 and NMUR2 mRNAs, which encode functional receptor proteins, are expressed in chicken tissues with different distribution patterns. (C) 2011 Elsevier Inc. All rights reserved.

Neurotensin, a tridecapeptide, is distributed in a wide range of tissues and exhibits multiple functions through its receptors. Hitherto molecular characterization of the neurotensin receptor has been reported in mammalian, amphibian, and fish species but not in avian species. In this study, we cloned the cDNA encoding chicken neurotensin receptor from the duodenum and characterized its primary structure, biological activity and distribution in the gastrointestinal tract. The cDNA encoded a protein consisting of 399 amino acids that had significant overall sequence homology to other vertebrate neurotensin receptor 1 with higher extent in the seven transmembrane domains. Chicken neurotensin increased intracellular Ca(2+) concentrations in human embryonic kidney 293 cells transiently expressing the chicken neurotensin receptor 1. Real-time PCR analysis showed that chicken neurotensin receptor 1 mRNA is expressed throughout the gastrointestinal tract with markedly higher level in the colon/rectum. These results indicate that the chicken neurotensin receptor 1 is involved in gastrointestinal functions through an intracellular signaling pathway accompanied by an increase in Ca(2+) levels. (C) 2011 Elsevier Inc. All rights reserved.

Circadian rhythms in physiology and behavior are known to be influenced by the estrous cycle in female rodents. The clock genes responsible for the generation of circadian oscillations are widely expressed both within the central nervous system and peripheral tissues, including those that comprise the reproductive system. To address whether the estrous cycle affects rhythms of clock gene expression in peripheral tissues, we first examined rhythms of clock gene expression (Per1, Per2, Bmal1) in reproductive (uterus, ovary) and non-reproductive (liver) tissues of cycling rats using quantitative real-time PCR (in vivo) and luminescent recording methods to measure circadian rhythms of PER2 expression in tissue explant cultures from cycling PER2:: LUCIFERASE (PER2::LUC) knockin mice (ex vivo). We found significant estrous variations of clock gene expression in all three tissues in vivo, and in the uterus ex Vivo. We also found that exogenous application of estrogen and progesterone altered rhythms of PER2::LUC expression in the uterus. In addition, we measured the effects of ovarian steroids on clock gene expression in a human breast cancer cell line (MCF-7 cells) as a model for endocrine cells that contain both the steroid hormone receptors and clock genes, We found that progesterone, but not estrogen, acutely up-regulated Per1, Per2, and Binal1 expression in MCF-7 cells. Together, our findings demonstrate that the timing of the circadian clock in reproductive tissues is influenced by the estrous cycle and suggest that fluctuating steroid hormone levels may be responsible, in part, through direct effects on the timing of clock gene expression, (C) 2010 Elsevier Inc. All rights reserved.

Animals measure photoperiod (daylength) and adapt to seasonal changes in the environment by altering their physiology and behavior accordingly. Although this photoperiodic response has long been of interest, the underlying mechanism has only recently begun to be uncovered at the molecular level. Japanese quail provide an excellent model to study the molecular mechanism underlying the vertebrate photoperiodic response. The recent sequencing of the chicken genome allowed a system-level analysis of photoperiodic time measurement in quail, and this approach uncovered the key event in the photoperiodic signaling cascade that regulates seasonal reproduction. Long photoperiod-induced expression of thyrotropin in the pars tuberalis of the pituitary gland was found to trigger local thyroid hormone catabolism in the mediobasal hypothalamus, which increases the activity of the reproductive neuroendocrine system resulting in gonadal development. Since thyrotropin was only known to stimulate the thyroid gland, a traditional hypothesi s-d riven approach would not have been expected to predict this discovery. Thus, a functional genomics approach, which is a discovery-driven approach, provides new insights in the field of endocrinology. (C) 2008 Elsevier Inc. All rights reserved.

Photoperiodism is an adaptation mechanism that enables animals to predict seasonal changes in the environment. Japanese quail is the best model organism for studying photoperiodism. Although the recent availability of chicken genome sequences has permitted the expansion from single gene to genome-wide transcriptional analysis in this organism, the photoperiodic response of the domestic chicken is less robust than that of the quail. Therefore, in the present study, we examined the photoperiodic response of the red jungle fowl (Gallus gallus), a predecessor of the domestic chicken, to test whether this animal could be developed as an ideal model for studying the molecular mechanisms of seasonal reproduction. When red jungle fowls were transferred from short-day- to long-day conditions, gonadal development and an increase in plasma LH concentration were observed. Furthermore, rapid induction of thyrotropin beta subunit, a master regulator of photoperiodism, was observed at 16 h after dawn on the first long day. In addition, the long-day condition induced the expression of type 2 deiodinase, the key output gene of photoperiodism. These results were consistent with the results obtained in quail and suggest that the red jungle fowl could be an ideal model animal for the genome-wide transcriptional analysis of photoperiodism.

GPR39 is an orphan G protein-coupled receptor that is thought to be involved in gastrointestinal and metabolic function. In this study, we cloned bovine GPR39 cDNA that encoded 462 amino acids showing high sequence homology to other mammalian GPR39 proteins. Real-time PCR showed expression of GPR39 mRNA in the liver, kidney, abomasums, small intestine, colon, rectum and uterus, with the highest level in the abomasums. Significant promoter activity was observed within the -2.3 kb 5'-upstream region of bovine GPR39 gene with human colon carcinoma-derived CACO-2 cells. These findings suggest that GPR39 may have important roles in gastrointestinal and metabolic functions in bovines as in other mammals.<br>

Many seasonal breeding animals can estimate the day length (photoperiod) and prepare for breeding in the optimal season. The Japanese quail (Coturnix japonica) is an excellent model for studying photoperiodism because of its rapid and dramatic response to the photoperiod. Recent molecular analysis using the quail has revealed that local thyroid hormone activation by thyroid hormone deiodinases (DIO2 and DIO3) in the mediobasal hypothalamus (MBH) plays a critical role in the regulation of seasonal reproduction in birds. However, the molecular dynamics of gene expression that regulates photoperiodic thyroid hormone activation in the MBH during the photoinduction process remains unclear. The chicken genome project has enabled the analysis of the conservation of the genetic interaction networks for studying photoperiodism. This review focuses on genome-wide transcriptional Studies of avian photoperiodism.

A new circadian variant was isolated by screening the intercross offspring of wild-caught mice (Mus musculus castaneus). This variant was characterized by an initial maintenance of damped oscillations and subsequent loss of rhythmicity after being transferred from light-dark (LD) cycles to constant darkness (DD). To map the genes responsible for the persistence of rhythmicity (circadian ratio) and the length of free-running period (tau), quantitative trait locus (QTL) analysis was performed using F-2 mice obtained from an F-1 cross between the circadian variant and C57BL/6J mice. As a result, a significant QTL with a main effect for circadian ratio (Arrhythmicity; Arrh-1) was mapped on Chromosome (Chr) 8. For tau, four significant QTLs, Short free-running period (Sfp-1) (Chr 1), Sfp-2 (Chr 6), Sfp-3 (Chr 8), Sfp-4 (Chr 11) were determined. An epistatic interaction was detected between Chr 3 (Arrh-2) and Chr 5 (Arrh-3). An in situ hybridization study of clock genes and mouse Period1::luciferase (mPer1::luc) real-time monitoring analysis in the suprachiasmatic nucleus (SCN) suggested that arrhythmicity in this variant might not be attributed to core circadian mechanisms in the SCN neurons. Our strategy using wild-derived variant mice may provide a novel opportunity to evaluate circadian and its related disorders in human that arise from the interaction between multiple variant genes.

Many animals that breed seasonally measure the day length (photoperiod) and use these measurements as predictive information to prepare themselves for annual breeding. For several decades, thyroid hormones have been known to be involved in this biological process; however, their precise roles remain unknown. Recent molecular analyses have revealed that local thyroid hormone activation in the hypothalamus plays a critical role in the regulation of the neuroendocrine axis involved in seasonal reproduction in both birds and mammals. Furthermore, functional genomics analyses have revealed a novel function of the hormone thyrotropin. This hormone plays a key role in signaling day-length changes to the brain and thus triggers seasonal breeding. This review aims to summarize the currently available knowledge on the interactions between elements of the thyroid hormone axis and the neuroendocrine system involved in seasonal reproduction.

Molecular mechanisms regulating animal seasonal breeding in response to changing photoperiod are not well understood. Rapid induction of gene expression of thyroid- hormone- activating enzyme ( type 2 deiodinase, DIO2) in the mediobasal hypothalamus ( MBH) of the Japanese quail ( Coturnix japonica) is the earliest event yet recorded in the photoperiodic signal transduction pathway. Here we show cascades of gene expression in the quail MBH associated with the initiation of photoinduced secretion of luteinizing hormone. We identified two waves of gene expression. The first was initiated about 14 h after dawn of the first long day and included increased thyrotrophin ( TSH) beta- subunit expression in the pars tuberalis; the second occurred approximately 4 h later and included increased expression of DIO2. Intracerebroventricular ( ICV) administration of TSH to short- day quail stimulated gonadal growth and expression of DIO2 which was shown to be mediated through a TSH receptor - cyclic AMP ( cAMP) signalling pathway. Increased TSH in the pars tuberalis therefore seems to trigger long- day photoinduced seasonal breeding.

In order to adapt to seasonal changes, animals exhibit robust changes in their reproductive status, body weight, and molt. However, the molecular mechanisms regulating such seasonal changes in physiology and behavior are not fully understood. Here, we report the photoperiodic regulation of the insulin receptor (IR) gene in the infundibular nucleus (anatomically homologous to the mammalian arcuate nucleus) of the Japanese quail. When the birds were transferred from short-day to long-day conditions, a significant increase in the level of IR mRNA was observed on the 10th long day, whereas that in testicular length was observed on the 5th long day. Castration abolished IR mRNA expression induced by long-day conditions, whereas the testosterone administration mimicked induction of IR mRNA expression induced by long-day conditions. These results suggested that the photoperiodic regulation of the IR mRNA in the infundibular nucleus is mediated by testosterone from the testes. It has been known that the central administration of insulin increases luteinizing hormone (LH) secretion, and neuron-specific disruption of IR gene causes impaired gonadal function due to the dysregulation of LH and increased food intake and body weight. Together with these results, the photoperiodic regulation of the IR mRNA in the hypothalamus may enhance the effect of long days in the seasonal response of reproduction and body weight changes. (c) 2007 Elsevier B.V. All rights reserved.

It is now known that circadian clocks are localized not only in the central pacemaker but also in peripheral organs. An example of a clock-dependent peripheral organ is the ovary of domestic poultry in which ovulation is induced by the positive feedback action of ovarian progesterone on the neuroendocrine system to generate a preovulatory release of LH during a daily 6-10 h "open period" of the ovulatory cycle. It has been assumed previously that the timing of ovulation in poultry is controlled solely by a clock-dependent mechanism within the neuroendocrine system. Here, we question this assumption by demonstrating the expression of the clock genes, Per2 (Period 2) and Per3, Clock, and Bmal1 (brain and muscle Arnt-like protein 1), in preovulatory follicles in laying quail. Diurnal changes in Per2 and Per3 expression were seen in the largest preovulatory follicle (F1) but not in smaller follicles. We sought to identify clock-driven genes in preovulatory follicles focusing on those involved in the synthesis of progesterone. One such gene was identified, encoding steroidogenic acute regulatory protein (StAR), which showed 24-h changes in expression in the F1 follicle coinciding with those of Per2. Evidence that StAR gene expression is clock driven was obtained by showing that its 5' flanking region contains E-box enhancers that bind to CLOCK/BMAL1 heterodimers to activate gene transcription. We also showed that LH administration increased the promoter activity of chicken StAR. We therefore suggest that the timing of ovulation in poultry involves an LH-responsive F1 follicular clock that is involved in the timing of the preovulatory release of progesterone.

The molecular mechanism underlying photoperiodism is not well understood in any organism. Long-day-induced conversion of prohormone T-4 to bioactive T-3 within the mediobasal hypothalamus (MBH) is critical for the photoperiodic regulation of reproduction. However, because thyroidectomy does not completely block the photoperiodic response in some species, the existence of a thyroid hormone-independent regulatory mechanism appears certain. To identify this novel mechanism, differential subtractive hybridization analysis was performed using MBH of quail kept under short-day and longday conditions. This analysis identified a gene encoding TGF alpha. Expression of TGF alpha mRNA was induced in the median eminence by the stimulus of long days, and this induction was observed at dusk on the first long day. This rapid induction of TGF alpha mRNA was similar to induction of the thyroid hormone-activating enzyme gene [Dio2 (type 2 iodothyronine deiodinase)], which is the earliest event yet determined in the photoinduction process. Expression analysis of epidermal growth factor receptors revealed strong expression of erbB4 and weak expression of erbB1 and erbB2 in the median eminence. Intracerebroventricular infusion of physiological dose of TGF alpha induced LH secretion and testicular growth under short-day conditions. Finally, we demonstrate that T-3 implantation and TGF alpha infusion into the MBH, either of which causes testicular growth, do not affect the expression of TGF alpha and Dio2, respectively. Thus, long-day-induced activation of the TGF alpha signaling pathway appears to mediate a thyroid hormone-independent pathway for the photoperiodic regulation of reproduction.

Using the mPer1::luc real-time monitoring technique, the authors observed the bimodal patterns of mPer1 bioluminescence on each side of the SCN, in parallel with maintaining synchronization between the left and right sides of the SCN under an artificial light:dark:light:dark (LDLD) 7:5:7:5 condition. In situ hybridization analysis of mPer1 and mBmal1 mRNA distribution in the SCN showed that in I photophase (morning photophase; M) of LDLD, the mPer1 level in the ventrolateral-like (VL-like) subdivision of the SCN was higher than that in the dorsomedial-like (DM-like) subdivision, and this regional distribution pattern was reversed in another photophase (evening photophase; E). In contrast, the mBmal1 level was higher in the DM-like subdivision than in the VL-like subdivision in the M phase, and this distribution changed in the E phase. The prokineticin 2 (PK2) mRNA that encodes an SCN output molecule that is thought to transmit the circadian locomotor rhythms was reduced in both the DM-like and VL-like SCN and did not clearly correlate with the activity under the LDLD condition. The expression of mPer1 and mPer2 in the liver was clearly bimodal, whereas the expressions of other clock genes were not synchronized to the LDLD condition. These results may provide important insights into the mechanism underlying the splitting or bimodal rhythms that may in turn facilitate the understanding of the ability to measure the seasonal day length in mammals.

Photorefractoriness is the insensitivity of gonadal development to the stimulatory effects of long photoperiods in birds and to the inhibitory effects of short photoperiods in small mammals. Its molecular mechanism remains unknown. Recently, it has been shown that reciprocal expression of thyroid hormone-activating enzyme [type 2 deiodinase (Dio2)] and-inactivating enzyme [type 3 deiodinase (Dio3)] genes in the mediobasal hypothalamus is critical for photoperiodically induced gonadal growth. Since thyroid hormones are required not only for photoinduction, but also for the induction of photorefractoriness, we examined the expression of these genes in relation to photorefractoriness in birds and mammals. Transfer of birds to long photoperiods induced strong expression of Dio2. This was maintained in tree sparrow when they later became photorefractory, but decreased somewhat in quail. In hamsters, transfer to long photoperiods also induced strong expression of Dio2. High values were not maintained under long photoperiods, and, indeed, expression decreased at the same rate as in animals transferred to short photoperiods. There was no renewed expression of Dio2 associated with testicular growth as animals became refractory to short photoperiods. Expression of Dio3 was high under short photoperiods and low under long photoperiods in all the animals examined, except for the short photoperiod-refractory hamsters. Our present study revealed complex regulation of deiodinase genes in the photoinduction and photorefractory processes in birds and mammals. These gene changes may be involved in the regulation of photorefractoriness, as well as photoinduction.

CS mice show unique properties of circadian rhythms: unstable free-running periods and distinct bimodal rhythms (similar to rhythm splitting, but hereafter referred to as bimodal rhythms) under constant darkness. In the present study, we compared clock-related gene expression (mPer1, mBmal1 and Dbp) in the SCN and peripheral tissues (liver, adrenal gland and heart) between CS and C57BL/6J mice. In spite of normal robust oscillation in the SCN of both mice, behavioral rhythms and peripheral rhythms of clock-related genes were significantly different between these mice. However, when daytime restricted feeding was given, no essential differences between the two strains were observed. These results indicate that unusual circadian behaviors and peripheral gene expression in CS mice do not depend on the SCN but rather mechanisms outside of the SCN. (c) 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

The photoperiodic response of the gonads requires T-3, which is generated photoperiodically from T-4 by type 2 iodothyronine deiodinase in the hypothalamus. Although thyroid hormones were long thought to traverse the plasma membrane by passive diffusion due to their lipophilic nature, it is now known that several organic anion transporting polypeptides (Oatp) transport thyroid hormones into target cells. In this study, we have used database searches to isolate DNA sequences encoding members of the chicken Oatp family and constructed a molecular phylogenetic tree. Comprehensive expression analyses using in situ hybridization revealed strong expression of cOatp1c1 and weak expression of cOatp1b1 in the ventro-lateral walls of basal tuberal hypothalamus, whereas expression of four genes (cOatp1a1, cOatp1b1, cOatp1c1, and cOatp3a2) was observed in the choroid plexus. Expression levels of all these genes in both regions were not different between short-day and long-day conditions. Functional expression of cOatp1c1 in Chinese hamster ovary cells revealed that cOatp1c1 is a highly specific transporter for T-4 with an apparent Km of 6.8 nM and a Vmax of 1.50 pmol per milligram of protein per minute. These results suggest that cOatp1c1 could be involved in the thyroxine transport necessary for the avian photoperiodic response of the gonads.

In most animals that live in temperate regions, reproduction is under photoperiodic control. In long-day breeders such as Japanese quail and Djungarian hamsters, type 2 deiodinase (Dio2) plays an important role in the mediobasal hypothalamus, catalyzing the conversion of prohormone T-4 to bioactive T-3 to regulate the photoperiodic response of the gonads. However, the molecular basis for seasonal reproduction in shortday breeders remains unclear. Because thyroid hormones are also known to be involved in short- day breeders, we examined the effect of an artificial long-day stimulus on Dio2 expression in the male Saanen goat (Capra hircus), a short-day breeder. Dio2 expression was observed in the caudal continuation of the arcuate nucleus, known as the target site for both melatonin and T-4 action. In addition, expression of Dio2 and T-3 content in the mediobasal hypothalamus was suppressed by artificial long-day conditions, which is the opposite of the results of long-day breeders. Thyroid hormone action on the development of neuroendocrine anestrus is known to be limited to a specific seasonal window. This long-day suppression of Dio2 may provide a mechanism that accounts for the lack of responsiveness to thyroxine during the mid to late anestrus.

In most organisms living in temperate zones, reproduction is under photoperiodic control. Although photoperiodic time measurement has been studied in organisms ranging from plants to vertebrates, the underlying molecular mechanism is not well understood. The Japanese quail (Coturnix japonica) represents an excellent model to study this problem because of the rapid and dramatic photoperiodic response of its hypothalamic-pituitary-gonadal axis. Recent investigations of Japanese quail show that long-day-induced type 2 deiodinase (Dio2) expression in the mediobasal hypothalamus (MBH) plays an important role in the photoperiodic gonadal regulation by catalyzing the conversion of the prohormone thyroxine (T-4) to bioactive 3,5,3'-triiodothyronine (T-3). The T-3 content in the MBH is approximately 10-fold higher under long than short days and conditions, and the intracerebroventricular infusion of T-3 under short days and conditions mimics the photoperiodic gonadal response. While Dio2 generates active T-3 from T-4 by outer ring deiodination, type 3 deiodinase (Dio3) catalyzes the conversion of both T-3 and T-4 into inactive forms by inner ring deiodination. In contrast to Dio2 expression, Dio3 expression in the MBH is suppressed under the long-day condition. Photoperiodic changes in the expression of both genes during the photoinduction process occur before the changes in the level of luteinizing hormone (LH) secretion, suggesting that the reciprocal changes in Dio2 and Dio3 expression act as gene switches of the photoperiodic molecular cascade to trigger induction of LH secretion.

The molecular mechanisms underlying photoperiodic time measurement are not well understood in any organism. Relatively recently, however, it has become clear that thyroid hormones play an important role in photoperiodism, and in a previous study we reported that long daylengths in Japanese quail increase hypothalamic levels of T-3 and of the thyroid hormone-activating enzyme, type 2 iodothyronine deiodinase. The present study extends these observations to measure gene levels of the thyroid hormone-inactivating enzyme, type 3 deiodinase. Levels decreased after exposure to long days, but increased under short days. Changes in the two genes were then analyzed during the precisely timed photoinduction that occurs in quail exposed to a single long day. The two gene switches are the earliest events yet recorded in the photoinduction process, and overall, these reciprocal changes offer the potential to regulate active brain thyroid hormone concentrations rather precisely at the site in the brain where photoinduction is triggered.

Ghrelin, an endogenous ligand for growth hormone (GH) secretagogue receptor, stimulates GH secretion. The ghrelin gene is expressed most abundantly in stomach. The mRNA is also detected in other tissues and cell lines. However, the mechanism of the transcriptional regulation of the ghrelin gene has not yet been clarified. In the present study, we have investigated the regulatory region of the ghrelin gene expression in the human medullary thyroid carcinoma cell line (TT cells). PCR analysis of the 5'-region for human ghrelin gene revealed the presence of the first exon corresponding to the short non-coding first exon of the mouse ghrelin gene. The first exon is located at the 502 by upstream from the 5'-end of the formerly reported human ghrelin gene. RT PCR analysis showed the expression of the first exon in the stomach and TT cells. The expression of the first exon in the human stomach was confirmed by 5'-RACE method. Significant level of promoter activity was observed in the 1225-1107 by up-stream region of the translation initiation site by luciferase assay. Specific protein binding to the promoter region of -1129 to -1100 was detected by electrophoretic mobility shift assay with nuclear extract from TT cells. These results suggest that the ghrelin gene expression in TT cells might be regulated by the upstream region of the first exon. (C) 2004 Elsevier Inc. All rights reserved.

Growth hormone receptor (GHR) cDNA and gene of the Japanese flounder (Paralicthys olivaceus) were cloned and their molecular structures were characterized. The 641 amino acid sequence predicted from the cDNA sequence showed more than 75% overall sequence similarity with GHRs of other teleosts such as turbot and goldfish, and contained common structural features of vertebrate GHRs. The extracellular domain of flounder GHR had three pairs of cysteines and an FGEFS motif with a replacement E to D. The cytoplasmic domain contained two conserved motifs referred to as box 1 and box 2. The flounder GHR gene was cloned by PCR using primers designed from the sequence of the GHR cDNA. The GHR gene was composed of 10 exons. The sequence of exon 1 corresponded to the 5'-untranslated region of the cDNA, and exons 2-6 encoded most parts of the extracellular domain. The transmembrane domain was found in exon 7, and the intracellular domain was encoded in exons 8-10. Exon 10 also encoded the 3'-untranslated region. Comparison of the flounder GHR gene with the human GHR gene shows that the flounder gene contains no exons corresponding to exon 3 of the human GHR gene, and that the region corresponding to exon 10 in the human GHR gene is encoded by exons 9 and 10 in the flounder GHR gene. These findings indicate that the flounder GHR gene diverged from those of mammalian and avian GHR genes, especially in the organization of the exons encoding the cytoplasmic domain. In addition to the regular form of GHR mRNA, a 3'-truncated form lacking the region derived from exons 9 and 10 was detected as a minor species in the liver by RT-PCR and by RNase protection assay. RT-PCR analysis showed that both the regular and the 3'-truncated GHR mRNAs are expressed in a wide range of flounder tissues with the highest levels being found in the liver. The 5'-flanking region of the flounder GHR gene was cloned by inverse PCR, and three transcription start points were identified with similar frequency by RNase protection assay.

The molecular mechanisms responsible for seasonal time measurement have yet to be fully described. Recently, we used differential analysis to identify that the type 2 iodothyronine deiodinase (Dio2) gene is responsible for the photoperiodic response of gonads in Japanese quail. It was found that expression of Dio2 in the mediobasal hypothalamus is induced by light and that T-3 content in the mediobasal hypothalamus increased under long day conditions. In addition, we showed that intracerebroventricular infusion of T-3 mimics photoperiodically induced testicular growth. Because it is well known that thyroid hormone is also essential for the maintenance of the seasonal reproductive changes in a number of mammals, we examined expression of Dio2 in Djungarian hamsters and found expression in the ependymal cell layer lining the infralateral walls of the third ventricle and the cell-clear zone overlying the tuberoinfundibular sulcus. Signal intensity was high under long days and weak under short days. Although light pulse did not affect Dio2 expression, melatonin injections decreased Dio2 expression under long days. These results indicate that Dio2 may be involved in the regulation of seasonal reproduction in mammals in the same way as observed in birds.

The signaling pathway of GH-stimulated IGF-I gene expression is still unclear, although it has been reported that the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) 5b pathway plays an important role in liver IGF-I expression. In this study, the GH-dependent IGF-I gene expression and its intracellular signaling mechanism have been examined in mouse pro-B, Ba/F3 cells stably expressing human GH receptor (Ba/F3-hGHR). The IGF-I gene expression was stimulated by human GH(0.01-10 nM) in a dose-dependent fashion in Ba/F3-hGHR cells. The specific inhibitors for JAK2 remarkably suppressed the GH-induced IGF-I gene expression, but MAPK or phosphatidylinositol 3 kinase-specific inhibitors failed to block the GH stimulation of the IGF-I gene expression. However, genistein, a nonspecific tyrosine kinase inhibitor that does not inhibit JAK2 and STAT5 phosphorylation, significantly suppressed the GH-induced IGF-I gene expression. Additionally, a Ba/F3-hGHR mutant that contained the truncated C-terminal hGHR up to D351 showed no IGF-I gene expression in response to human GH. The D351 form normally has the GH-induced JAK/STAT5 tyrosine phosphorylation. These results suggest that the JAK-STAT5 pathway and the novel tyrosine phosphorylation pathway, dependent on signaling from the C-terminal region of hGHR, might be involved in the GH-stimulated IGF-I gene expression in Ba/F3 cells.

Toluene is widely used as an organic solvent in various industries and commercial products. Recent investigations have shown that toluene may induce male reproductive dysfunctions and carcinogenicity. To clarify whether the toxicity results from the interference of endocrine systems or direct damage to reproductive organs, we examined the effects of toluene on the male reproductive system in rats, comparing to those of diethylstilbestrol (DES), a potent synthetic estrogen. Toluene (50, 500 mg/kg) or DES (2 mg/kg) injected subcutaneously to male Sprague-Dawley rats once a day for 10 days decreased the epididymal sperm counts and the serum concentrations of testosterone. The mRNA level for gonadotropin-releasing hormone receptor in the pituitary was decreased by DES, but not by toluene. On the contrary, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in testes, the biological marker for oxidative DNA damage, was increased by toluene but not by DES. These results suggest that toluene induces reproductive toxicity via direct oxidative damage of spermatozoa, whereas DES affects endocrine systems via the hypothalamo-pituitary-gonadal axis. Morphological findings supported the idea. To determine the mechanism of 8-oxodG formation in vivo , we examined DNA damage induced by toluene metabolic products in vitro . Minor toluene metabolites, methylhydroquinone and methylcatechols, induced oxidative DNA damage, and the methylcatechols induced NADH-mediated 8-oxodG formation more efficiently than methylhydroquinone did. We propose that oxidative DNA damage in the testis plays a role in reproductive toxicity induced by toluene.

A novel first exon, E1(4), whose sequence was distinct from those of the three known first exons, E1(1), E1(2), and E1(3), of the rat PRL receptor (PRL-R) gene was identified by cDNA cloning for the 5'-end region of PRL-R mRNA expressed in the rat brain. Sequence analysis revealed the presence of two different length E14 cDNAs. The longer cDNA contained the 243-bp E14 sequence, and the shorter cDNA lacked the 139-bp sequence at the 5'-end of the longer one. Neither El. cDNA has a second exon sequence, indicating that the E14 first exon is extensively spliced to the third exon. E1(4)-containing PRL-R mRNAs were detected only in the brain by RT-PCR and ribonuclease protection assay. The longer E14 mRNA was expressed as the major PRL-R mRNA species in the brain and was greatly increased in pregnant (d 18) and lactating (d 5) rats. A genomic clone containing the El. first exon together with its 5'- and 3'-flanking regions was isolated from a rat kidney genomic library. Ribonuclease protection assay revealed that the position corresponding to the 5'-end of the shorter El. cDNA is the major transcription start point for the E1(4)exon. The 5'-flanking region of E1(4) contained a TATA box-like element 23 bp upstream of the major transcription start point. Other putative transcription factor-binding sites, such as CCAAT, Sp1, and glucocorticoid-responsive elements, were observed at further upstream regions. These results suggest that PRL-R gene expression in rat brain is controlled by the promoter for the E1(4) first exon.