Central injection of neuropeptide B induces luteinizing hormone release in male and female rats.

Neuropeptide B (NPB) has been identified as an endogenous peptide ligand for the orphan receptor NPBWR1. However, the effect of NPB on the central regulatory mechanisms of reproductive functions remains unclear. Our findings indicated the presence of Npb, Npw (which is another ligand for NPBWR1), and Npbwr1 mRNA in the hypothalamus of male and female rats at each stage of the estrous cycle. Npb mRNA expression was found to be significantly higher in diestrus compared to estrus. The expression of Npw mRNA was one order of magnitude lower than that of Npb mRNA, and Npw mRNA expression in diestrus was significantly higher than that in the other stages of the estrous cycle. Furthermore, Npbwr1 mRNA expression was found to be significantly higher in diestrus compared to the other stages of the estrous cycle and intact males. Notably, estrogen did not alter the expression of Npb, Npw, and Npbwr1 mRNAs in the hypothalamus of females. Central injection of NPB increased plasma luteinizing hormone (LH) levels in both intact males and estrogen-primed ovariectomized females but not in ovariectomized females. These results suggest that NPB-NPBWR1 signaling would be a facilitatory regulatory mechanism in the reproductive function of male and female rats. To the best of our knowledge, this study is the first report to describe the central role of NPB-NPBWR1 signaling in LH regulation in mammals.

Immunoglobulin G4-related Disease with Marked Eosinophilia: A Case Report and Literature Review.

We encountered a 78-year-old Japanese man with IgG4-related sialoadenitis complicated with marked eosinophilia. We diagnosed him with IgG4-RD (related disease) with a submandibular gland tumor, serum IgG4 elevation, IgG4-positive plasma cell infiltration, and storiform fibrosis. During follow-up after total incision of the submandibular gland, the peripheral eosinophil count was markedly elevated to 29,480/µL. The differential diagnosis of severe eosinophilia without IgG4-RD was excluded. The patient exhibited a prompt response to corticosteroid therapy. His peripheral blood eosinophil count was the highest ever reported among similar cases. We also review previous cases of IgG4-RD with severe eosinophilia.

Pharmacokinetics and tissue distribution of orally administrated imidazole dipeptides in carnosine synthase gene knockout mice.

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 (∼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 evaluating the effects of dietary ID and for elucidating the physiological functions of oral ID administration.

Photoperiod-Specific Expression of Eyes Absent 3 Splice Variant in the Pars Tuberalis of the Japanese Quail.

The molecular mechanism underlying photoperiodic response in seasonal breeding animals such as the Japanese quail, red jungle fowl, sheep, mouse, and hamster involves thyroid-stimulating hormone beta subunit (TSHß) mRNA expression in the pars tuberalis stimulated by the extension in day length. Furthermore, this mechanism is regulated by eyes absent 3 (Eya3) in mammals. Even in birds, the expression of both TSHß and EYA3 is induced in the pars tuberalis by the extension in day length; however, the relationship between the two genes is unknown. To clarify the function of EYA3 in quail photoperiodism, in the present study, we performed mRNA structure analysis of the Japanese quail EYA3 mRNA using reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis. The results revealed that there are four types of splice variants within regions of exons 7, 8, and 9 of quail EYA3 mRNA. Among the four splice variants of quail EYA3, the splice variant containing exon 7 was expressed in the pars tuberalis on the first long day, when quails were transferred from the short-day condition to the long-day condition. The results indicate that EYA3 splice variant containing exon 7 is involved in the photoperiodic response of the pars tuberalis in the Japanese quail.

Cholecystokinin induces crowing in chickens.

Animals that communicate using sound are found throughout the animal kingdom. Interestingly, in contrast to human vocal learning, most animals can produce species-specific patterns of vocalization without learning them from their parents. This phenomenon is called innate vocalization. The underlying molecular basis of both vocal learning in humans and innate vocalization in animals remains unknown. The crowing of a rooster is also innately controlled, and the upstream center is thought to be localized in the nucleus intercollicularis (ICo) of the midbrain. Here, we show that the cholecystokinin B receptor (CCKBR) is a regulatory gene involved in inducing crowing in roosters. Crowing is known to be a testosterone (T)-dependent behavior, and it follows that roosters crow but not hens. Similarly, T-administration induces chicks to crow. By using RNA-sequencing to compare gene expression in the ICo between the two comparison groups that either crow or do not crow, we found that CCKBR expression was upregulated in T-containing groups. The expression of CCKBR and its ligand, cholecystokinin (CCK), a neurotransmitter, was observed in the ICo. We also showed that crowing was induced by intracerebroventricular administration of an agonist specific for CCKBR. Our findings therefore suggest that the CCK system induces innate vocalization in roosters.

Molecular characterization of feline melanocortin 4 receptor and melanocortin 2 receptor accessory protein 2.

Melanocortin 4 receptor (MC4R), which is a member of the G protein-coupled receptor (GPCR) family, mediates regulation of energy homeostasis upon the binding of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system (CNS). Melanocortin 2 receptor accessory protein 2 (MRAP2) modulates the function of MC4R. We performed cDNA cloning of cat MC4R and MRAP2 and characterized their amino acid sequences, mRNA expression patterns in cat tissues, protein-protein interactions, and functions. We found high sequence homology (>88%) with other mammalian MC4R and MRAP2 encoding 332 and 206 amino acid residues, respectively. Reverse transcription-polymerase chain reaction analysis revealed that cat MC4R and MRAP2 mRNA were expressed highly in the CNS. In CHO-K1 cells transfected with cat MC4R, stimulation with α-MSH increased intracellular cyclic adenosine monophosphate (cAMP) concentration in a dose-dependent manner. Furthermore, the presence of MRAP2 enhanced the cat MC4R-mediated cAMP production. These results suggested that cat MC4R acts as a neuronal mediator in the CNS and that its function is modulated by MRAP2. In addition, our NanoBiT study showed the dynamics of their interactions in living cells; stimulation with α-MSH slightly affected the interaction between MC4R and MRAP2, and did not affect MC4R homodimerization, suggesting that they interact in the basal state and that structural change of MC4R by activation may affect the interaction between MC4R and MRAP2.

Expression of Prolactin Receptor mRNA in Lactotrophs and Somatotrophs of the Chicken Anterior Pituitary Gland.

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.

Canine REIC/Dkk-3 interacts with SGTA and restores androgen receptor signalling in androgen-independent prostate cancer cell lines.

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 α (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.

Ontogeny of the Saccus Vasculosus, a Seasonal Sensor in Fish.

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 α-subunit (CGA) and TSH ß-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.

Isolation and characterization of glycophorin from carp red blood cell membranes.

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.

Serum leptin concentrations, leptin mRNA expression, and food intake during the estrous cycle in rats.

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.

Regulation of prolactin receptor gene expression in the rat choroid plexus via transcriptional activation of multiple first exons during postnatal development and lactation.

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.

Characterization of multiple first exons in murine prolactin receptor gene and the effect of prolactin on their expression in the choroid plexus.

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 17ß-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.

cDNA cloning and mRNA expression of cat and dog Cdkal1.

The cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1 (CDKAL1) gene encodes methylthiotransferase, and the gene contains risk variants for type 2 diabetes in humans. In this study, we performed complementary DNA cloning for Cdkal1 in the cat and dog and characterized the tissue expression profiles of its messenger RNA. Cat and dog Cdkal1 complementary DNA encoded 576 and 578 amino acids, showing very high sequence homology to mammalian CDKAL1 (>88.4%). Real-time polymerase chain reaction analyses revealed that Cdkal1 messenger RNA is highly expressed in smooth muscle and that tissue distribution of Cdkal1 is similar in cats and dogs. Genotyping analysis of single-nucleotide polymorphism for cat Cdkal1 revealed that obese cats had different tendencies from normal cats. These findings suggest that the cat and dog Cdkal1 gene is highly conserved among mammals and that cat Cdkal1 may be a candidate marker for genetic diagnosis of obesity.

Two chicken neuromedin U receptors: characterization of primary structure, biological activity and tissue distribution.

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.

Molecular characterization of structure and tissue distribution of chicken neurotensin receptor.

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.

Influence of the estrous cycle on clock gene expression in reproductive tissues: effects of fluctuating ovarian steroid hormone levels.

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 Bmal1 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.

Genetic and molecular analysis of wild-derived arrhythmic mice.

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.

Identification of the photoperiodic signaling pathway regulating seasonal reproduction using the functional genomics approach.

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 hypothesis-driven 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.

Red jungle fowl (Gallus gallus) as a model for studying the molecular mechanism of seasonal reproduction.

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.