中田 友明

© 2019 Elsevier Inc. In this review article, information about the development of the hypothalamo–hypophyseal axis, endocrine control of metamorphosis, and hormonal and pheromonal involvements in reproductive behavior in some amphibian species is assembled from the works conducted mainly by our research group. The hypothalamic and pituitary development was studied using Bufo embryos and larvae. The primordium of the epithelial hypophysis originates at the anterior neural ridge and migrates underneath the brain to form a Rathke's pouch-like structure. The hypothalamo–hypophyseal axis develops under the influence of thyroid hormone (TH). For the morphological and functional development of the median eminence, which is a key structure in the transport of regulatory hormones to the pituitary, contact of the adenohypophysis with the undeveloped median eminence is necessary. For the development of proopiomelanocortin-producing cells, contact of the pituitary primordium with the infundibulum is required. The significance of avascularization in terms of the function of the intermediate lobe of the pituitary was evidenced with transgenic Xenopus frogs expressing a vascular endothelial growth factor in melanotropes. Metamorphosis progresses via the interaction of TH, adrenal corticosteroids, and prolactin (PRL). We emphasize that PRL has a dual role: modulation of the speed of metamorphic changes and functional development of organs for adult life. A brief description about a novel type of PRL (1B) that was detected was made. A possible reason why the main hypothalamic factor that stimulates the release of thyrotropin is not thyrotropin-releasing hormone, but corticotropin-releasing factor is considered in light of the fact that amphibians are poikilotherms. As regards the reproductive behavior in amphibians, studies were focused on the courtship behavior of the newt, Cynops pyrrhogaster. Male newts exhibit a unique courtship behavior toward sexually developed conspecific females. Hormonal interactions eliciting this behavior and hormonal control of the courtship pheromone secretion are discussed on the basis of our experimental results.

The newt, a group of urodele amphibians, has outstanding ability to repeatedly regenerate various body parts, even in the terrestrial life-stage. In this animal, when the limb is amputated, a cell mass named the blastema appears on the stump and eventually gives rise to a new functional limb. Erythrocytes (red blood cells) in most non-mammalian vertebrates, including the newt, preserve their nucleus throughout their life-span, although physiological roles of such nucleated erythrocytes, other than oxygen delivery, are not known. Here we report novel behavior of erythrocytes in the newt. We identified an orphan gene Newtic1, whose transcripts significantly increased in the blastema. Newtic1 was expressed in a subset of erythrocytes that formed a novel clump (EryC). EryC formed a complex with monocytes and was circulating throughout the body. When the limb was amputated, EryCs were newly generated in the stump and accumulated into a distal portion of the growing blastema. Our data suggested that the newt erythrocytes carried multiple secretory molecules including growth factors and matrix metalloproteases, and were capable of delivering these molecules into the blastema as a form of EryCs. This study provides insight into regulations and roles of nucleated erythrocytes, that are independent of oxygen delivery.

Objectives: On computed tomography (CT), sinonasal schwannoma displays as a soft-tissue mass without any distinctive features. Our aim was to define the radiological criteria for distinguishing schwannoma from other sinonasal benign tumours. Methods: We retrospectively identified consecutive patients who were pathologically diagnosed with benign sinonasal tumours between 2007 and 2016. CT attenuation values were compared between benign tumours and the brainstem. The utilities of demographic factors, clinical factors, and CT parameters for predicting the CT attenuation values of the brainstem were analysed by univariate and multivariate regression. Results: Of the 111 identified cases of benign tumours, the CT attenuation values of tumours and the brainstem were analysed in 36 cases (schwannoma, 4 cases inverted papilloma, 26 juvenile nasopharyngeal angiofibroma, 3 cavernous haemangioma, 3). The CT attenuation values of the schwannomas were significantly lower than in the brainstem, while those of the other tumours were significantly higher than in the brainstem. No factors affected the CT attenuation values of the brainstem. Conclusion: Low CT attenuation values of sinonasal benign tumours relative to the brainstem could distinguish schwannomas from other benign tumours.

The male red-bellied newt (Cynops pyrrhogaster) approaches the female's cloaca prior to performing any courtship behaviour, as if he is using some released substance to gauge whether she is sexually receptive. Therefore, we investigated whether such a female sexual attractiveness pheromone exists. We found that a tripeptide with amino acid sequence Ala-Glu-Phe is secreted by the ciliary cells in the epithelium of the proximal portion of the oviduct of sexually developed newts and confirmed that this is the major active substance in water in which sexually developed female newts have been kept. This substance only attracted sexually developed male newts and acted by stimulating the vomeronasal epithelial cells. This is the first female sexual attractiveness peptide pheromone to be identified in a vertebrate.

Reproductive behavior in amphibians, as in other vertebrate animals, is under the control of multiple hormonal substances. Prolactin (PRL), arginine vasotocin (AVT), androgen, and 7 alpha-hydroxypregnenolone (7 alpha-OH PREG), four such substances with hormonal activity, are known to be involved in the expression of the tail vibration behavior which is the initial step of courtship performed by the male newt, Cynops pyrrhogaster. As current information on the interaction(s) between these hormones in terms of eliciting tail vibration behavior is limited, we have investigated whether the decline of expression of tail vibration behavior due to suppression of the activity of any one of these hormones can be restored by supplying any one of the other three hormones exogenously. Expression of the behavior was determined in terms of incidence (% of test animals exhibiting the behavior) and frequency (number of times that the behavior was repeated during the test period). Neither PRL nor androgen restored the decline in the incidence and frequency of the tail vibration behavior caused by the suppression of the activity of any one of other three hormones. AVT completely restored both the anti-PRL antibody-induced and flutamide (an androgen receptor antagonist)-induced, but not ketoconazole (an inhibitor of the steroidogenic CYP enzymes)-induced decline in the incidence and frequency of the tail vibration behavior. The neurosteroid, 7 alpha-OH PREG, failed to restore flutamide-induced decline in the incidence and frequency of the behavior. However, it was able to restore both anti-PRL antibody-induced and AVT receptor antagonist-induced decline in the incidence, but not in the frequency of the behavior. In another experiment designed to see the activity of hormones enhancing the frequency of the tail vibration behavior, AVT was revealed to be more potent than 7 alpha-OH PREG. The role of each hormonal substance in determining the expression of the tail vibration behavior was discussed based on the results. (C) 2015 Elsevier Inc. All rights reserved.

We analyzed the expression of G protein a subunits and the axonal projection into the brain in the olfactory system of the semiaquatic newt Cynops pyrrhogaster by immunostaining with antibodies against G alpha(olf) and G alpha(o), by in situ hybridization using probes for G alpha(olf), G alpha(o), and G alpha(i2), and by neuronal tracing with DiI and DiA. The main olfactory epithelium (OE) consists of two parts, the ventral OE and dorsal OE. In the ventral OE, the G alpha(olf)-and G alpha(o)-expressing neurons are located in the apical and basal zone of the OE, respectively. This zonal expression was similar to that of the OE in the middle cavity of the fully aquatic toad Xenopus laevis. However, the G alpha(olf)-and G alpha(o)-expressing neurons in the newt ventral OE project their axons toward the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively, whereas in Xenopus, the axons of both neurons project solely toward the MOB. In the dorsal OE of the newt, as in the principal cavity of Xenopus, the majority of the neurons express G alpha(olf) and extend their axons into the MOB. In the vomeronasal organ (VNO), the neurons mostly express G alpha(o). These neurons and quite a few G alpha(olf)-expressing neurons project their axons toward the AOB. This feature is similar to that in the terrestrial toad Bufo japonicus and is different from that in Xenopus, in which VNO neurons express solely G alpha(o), although their axons invariably project toward the AOB. We discuss the findings in the light of diversification and evolution of the vertebrate olfactory system. (C) 2014 Wiley Periodicals, Inc.

© Medwell Journals, 2014.The purpose of this study is to reveal the effect of estrogen-inducing SOD activity on the endometrium of domestic rabbits. Researchers measured Superoxide Dismutase (SOD) activity to investigate the reason of occurrence of popular uterine adenocarcinoma in rabbits in estradiol-17β-stimulated endometrium epithelial cell culture. Then, we also examined the distribution of cells expressing Estrogen Receptor α (ERα)-like Immunoreactivites (ERα-like-IR) in the uterus prone to developing adenocarcinoma. The addition of estradiol-17β to the primary culture of endometrial epithelial cells obviously decreased SOD activity. Moreover, the areas of endometrium in which uterine glands were confined in control rabbits and notably larger in rabbits with glandular hyperplasia of the uterus compared in normal dog uterus that the occurrence of uterine adenocarcinoma is rare. ERα-like-IR cells were found in all proliferating glands in rabbits with hyperplasia.

A peptide pheromone of the red-bellied male newt, sodefrin was tested for its ability to increase intracellular concentrations of Ca2+ ([Ca2+](i)) in the dissociated vomeronasal (VN) cells of females by means of calcium imaging system. The pheromone elicited a marked elevation of [Ca2+](i) in a small population of VN cells from sexually developed females. The population of cells exhibiting sodefrin-induced elevation of [Ca2+](i) increased concentration-dependently. A pheromone of a different species was ineffective in this respect. The VN cells from non-reproductive females or from reproductive males scarcely responded to sodefrin in terms of elevating [Ca2+](i). In the cells from hypophysectomized and ovariectomized females, the sodefrin-inducible increase of [Ca2+](i) never occurred. The cells from the operated newts supplemented with prolactin and estradiol exhibited [Ca2+](i) responses to sodefrin with a high incidence. Thus, sex- and hormone-dependency as well as species-specificity of the responsiveness of the VN cells to sodefrin was evidenced at the cellular level. Subsequently, possibility of involvement of phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3) and/or PLC-diacylglycerol (DAG)-protein kinase C (PKC) pathways in increasing [Ca2+](i) in VN cells in response to sodefrin was explored using pharmacological approaches. The results indicated that PLC is involved in generating the Ca2+ signal in all sodefrin-responsive VN cells, whereas IP3 in approximately 50% of the cells and DAG-PKC in the remaining cells. In the latter case, the increase of [Ca2+](i) was postulated to be induced by the influx of Ca2+ through the L-type channel. The significance of the finding is discussed. (c) 2013 Elsevier Inc. All rights reserved.

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.

Reproductive behavior in amphibians, as in other vertebrate animals, is controlled by multiple hormones. A neurosteroid, 7α-hydroxypregnenolone, has recently been found to enhance locomotor activity in the male newt, Cynops pyrrhogaster. Here, we show that this neurosteroid is also involved in enhancing the expression of courtship behavior. Intracerebroventricular (ICV) injection of 7α-hydroxypregnenolone enhanced courtship behavior dose-dependently in the sexually undeveloped males that had been pretreated with prolactin and gonadotropin, which is known to bring the males to a sexually developed state. But, unlike the case in the locomotion activity, 7α-hydroxypregnenolone did not elicit the behavior in males receiving no prior injections of these hormones. ICV administration of ketoconazole, an inhibitor of the steroidogenic enzyme cytochrome P450s, suppressed the spontaneously occurring courtship behavior in sexually active males. Supplementation with 7α-hydroxypregnenolone reversed the effect of ketoconazole in these animals. It was also demonstrated that the effect of the neurosteroid on the courtship behavior was blocked by a dopamine D2-like, but not by a D1-like, receptor antagonist. These results indicate that endogenous 7α-hydroxypregnenolone enhances the expression of the male courtship behavior through a dopaminergic system mediated by a D2-like receptor in the brain. © 2012 Elsevier Inc.

We investigated the effects of a bioartificial endocrine pancreas (Bio-AEP) produced by mouse β cells on sexual dysfunction of streptozotocin (STZ)-induced diabetic female rats. Female rats were administered STZ (60 mg/kg BW, i.v.) at the age of 10 weeks and transplanted with a Bio-AEP including mouse β cells at the age of 14 weeks (STZ+Bio-AEP group). Lordosis and proceptive sexual behavior of female rats were observed. The results showed that after the Bio-AEP transplant blood glucose recovered from 380-450 mg/dl induced by streptozotocin to 140-230 mg/dl and suppressed lordosis and proceptive behavior also recovered. These results suggest that it is possible to reverse sexual dysfunction by continuous administration of mouse insulin.

Gonadotropin-releasing hormone (GnRH) neurons in the basal forebrain are the final common pathway through which the brain regulates reproduction. GnRH secretion occurs in a pulsatile manner, and indirect evidence suggests the kisspeptin neurons in the arcuate nucleus (ARC) serve as the central pacemaker that drives pulsatile GnRH secretion. The purpose of this study was to investigate the possible coexpression of kisspeptin, neurokinin B (NKB), and dynorphin A (Dyn) in neurons of the ARC of the goat and evaluate their potential roles in generating GnRH pulses. Using double and triple labeling, we confirmed that all three neuropeptides are coexpressed in the same population of neurons. Using electrophysiological techniques to record multiple-unit activity (MUA) in the medial basal hypothalamus, we found that bursts of MUA occurred at regular intervals in ovariectomized animals and that these repetitive bursts (volleys) were invariably associated with discrete pulses of luteinizing hormone (LH) (and by inference GnRH). Moreover, the frequency of MUA volleys was reduced by gonadal steroids, suggesting that the volleys reflect the rhythmic discharge of steroid-sensitive neurons that regulate GnRH secretion. Finally, we observed that central administration of Dyn-inhibit MUA volleys and pulsatile LH secretion, whereas NKB induced MUA volleys. These observations are consistent with the hypothesis that kisspeptin neurons in the ARC drive pulsatile GnRH and LH secretion, and suggest that NKB and Dyn expressed in those neurons are involved in the process of generating the rhythmic discharge of kisspeptin.

Purpose: The dopamine-derived endogenous compound, R-salsolinol (SAL), was recently identified as a putative endogenous prolactin (PRL)-releasing factor. However, how SAL influences copulatory behavior is unknown. In this study, we examined the relationship between SAL and copulatory behavior in male rats. Methods: Male Sprague-Dawley rats administered SAL were exposed to female rats in estrus, the plasma PRL concentration was measured, and the behavioral frequency and time during copulatory behavior were noted. Results: In the control and SAL groups, plasma PRL concentrations at 15 min before exposure to the female were 7.3 ± 2.0 and 8.0 ± 1.5 ng/ml, respectively. Moreover, plasma PRL concentrations in males immediately after exposure to the female were 7.4 ± 1.2 and 68.0 ± 5.9 ng/ml, respectively (P &lt 0.05). All (8/8) of the control animals ejaculated in the presence of the female, whereas only 33% (2/6) of the SAL group ejaculated. An increasing tendency for mount latency and intromission latency and a decreasing tendency for intromission frequency were observed in the SAL group. Conclusions: Copulatory behavior was inhibited in male rats after SAL injection, suggesting that SAL is a copulatory behavior inhibiting factor. © 2010 Japan Society for Reproductive Medicine.

Three types of cDNA encoding the arginine vasotocin (AVT) receptors from the newt, Cynops pyrrhogaster were cloned and the gene expression of each receptor analyzed in the organs and tissues of the newt. The deduced amino acid sequence of one type of AVT receptor, consisting of 418 amino acid residues, showed a high degree of sequence identity with the mammalian arginine vasopressin (AVP) V1a receptors (61-68%). The second type of cDNA, encoding an amino acid sequence consisting of 367 amino acid residues, exhibited a relatively high sequence identity with mammalian AVP V2 receptors (50-51%). The third cDNA, encoding a sequence of 415 amino acid residues, possessed high sequence identity with mammalian AVP V3/V1b receptors (59-63%). Phylogenetic analysis revealed that the first, second and third types of receptor were close to mammalian AVP V1a, V2 and V3/V1b receptors, respectively, and RT-PCR using gene specific primers for each type of receptor indicated that the first and second types of receptor mRNA were expressed in various organs and tissues, including the circulatory, osmoregulatory, and reproductive organs of both male and female newts. In contrast, mRNA expression of the third cDNA was mainly detected in the brain and pituitary, and its expression pattern was distinctly different from that of the other two. We suggest that the first, second and third types of newt AVT receptor obtained in the present study are counterparts of mammalian AVP V1a, V2 and V3/V1b receptors, respectively. (c) 2007 Elsevier Inc. All rights reserved.

Sodefrin (Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys) is a female-attracting peptide pheromone secreted by the abdominal gland of the male red-bellied newt, Cynops pyrrhogaster. Sequence analysis of a cDNA encoding sodefrin revealed that the peptide is located in the C-terminal region of its precursor protein (residues 177-186 of preprosodefrin) and extended from its C-terminus by the tripeptide sequence Ile(187)-Ser(188)-Ala(189) and flanked at its N-terminus by Leu(174)-Gly(175)-Arg-(176). This suggests that sodefrin is generated by enzymatic cleavage at monobasic (Lys and Arg) sites within the precursor molecule. To demonstrate the presence in the abdominal gland of proteolytic enzymes capable of generating sodefrin, an enzymatic assay was developed using t-butoxycarbonyl (Boc)-Leu-Gly-Arg--4methylcoumaryl-7-amide (MCA) and Boc-Leu-Leu-Lys-MCA as synthetic substrates. A crude extract of the abdominal gland hydrolyzed both substrates to liberate 7-amino-4-methylcoumarin, suggesting that enzymes that generate sodefrin from its precursor molecule are present in the gland. The activity in the extract for cleaving Boc-Leu-Gly-Arg-MCA was optimal at pH 9.0 and 45 degrees C and for Boc-Leu-Leu-Lys-MCA at pH 9.0 and 40 degrees C. The effects of a range of specific inhibitors on activities in the extract suggest an involvement of enzymes belonging to the serine protease family. It was also demonstrated that enzymatic activity in an extract of the abdominal glands of sexually developed males was significantly (three- to six-fold; p < 0.01) higher than that of sexually undeveloped males.

Previous analysis of PCR products derived from total RNA from the abdominal gland of the male newt, Cynops pyrrhogaster, inhabiting the Nara area of Japan led to the identification of a gene encoding [Val(8)]sodefrin, as well as the female-attracting peptide pheromone, sodefrin. In this study, purification of this sodefrin variant from the abdominal glands of male newts from the Nara area was accomplished using gel-filtration chromatography and reversed-phase HPLC. Amino acid sequence analysis and mass spectrometry confirmed that the final product was [Val(8)]sodefrin. A full-length cDNA encoding the biosynthetic precursor of [Val(8)]sodefrin was cloned and characterized. The deduced amino acid sequence of prepro[Val(8)]sodefrin showed 86.2% identity with that of the sodefrin precursor. The [Val(8)]sodefrin variant potently attracted females from the Nara area, but the variant was much less or not effective in attracting females captured in the Niigata and Chiba areas. The term aonirin ("aoni" from "aoni-yoshi", the conventional epithet of Nara) is proposed to designate this region-specific pheromone. It is speculated that the coevolution of a novel pheromone and its complementary receptor in the Nara newts may lead to reproductive isolation and eventual differentiation into a separate species. (c) 2006 Elsevier Inc. All rights reserved.

Sodefrin (SIPSKDALLK) is a female-attracting pheromone that is secreted by the abdominal gland of the male red-bellied newt. We found that mRNA encoding a sodefrin variant, [Val(8)] sodefrin, is expressed exclusively in specimens captured in the Nara area of Japan. The synthetic peptide was tested for its activity. It attracted females from Nara, but not those from other regions, suggesting that there is a geographic variation in the pheromone molecule and in the responsiveness to the pheromone. Employing an abdominal gland extract and synthetic substrates, the possibility of generation of the putative pheromone, [Val(8)] sodefrin, from the precursor molecule was demonstrated.

Amphibian sex pheromones of 3 urodele (Cynops pyrrhogaster, C. ensicauda, and Plethodon jordani) and 1 anuran (Litoria splendida) species have been isolated and characterized and found to be either small peptides or larger proteins. Each pheromone secreted by the male acts on conspecific females. Endocrine control of pheromone secretion has been best studied in Cynops. The C. pyrrhogaster pheromone, sodefrin, and the C. ensicauda pheromone, silefrin, are generated from their precursor proteins. The sodefrin and silefrin precursor mRNA levels in the abdominal gland of the cloaca are elevated by prolactin and androgen. An increase in the level of both immunoassayable pheromones caused by treatment with these hormones has also been demonstrated. Receptors for both of these hormones have been localized in the abdominal gland. The discharge of sodefrin into the water is elicited by arginine vasotocin. The responsiveness of the female vomeronasal epithelial cells to sodefrin, as estimated by electro-olfactography, is enhanced markedly by a combination of prolactin and estrogen. Sodefrin elevates intracellular calcium levels in vomeronasal epithelial cells. The population of the sodefrin-responsive cells increases during the breeding period.

Peptides derived from the post-translational processing of preprosodefrin were isolated from an extract of the abdominal glands of male red-bellied newts Cynops pyrrhogaster obtained 5 months prior to the onset of the breeding season. Structural characterization of the peptides showed that the pheromone sodefrin (SIPSKDALLK) is stored in a biologically inactive COOH-terminally extended form (SIPSKDALLKISA). It follows, therefore. that the activation of a protease that cleaves at a Lys-Ile bond to generate the active pheromone must occur by the time of onset of reproductive behavior. Additional peptides (representing preprosodefrin-(146-175)-peptide and preprosodefrin-(159-173)-peptide), that are derived from the precursor by cleavage at monobasic and dibasic processing sites, were also purified from the extract. The isolation of paralogs of these peptides, including an inactive COOH-terminally extended form of [Asn(10)]sodefrin, provides evidence for the expression of multiple genes encoding preprosodefrin. PCR products derived from total RNAs from the abdominal gland of individual newts collected from three different regions of Japan were analyzed. The data confirm the existence of multiple genes encoding sodefrin and its variants whose expression varied according to the individuals and the regions. However, genes encoding sodefrin were found to be expressed in all the specimens sampled. (C) 2004 Elsevier Inc. All rights reserved.