The significance of Ciona intestinalis as a stem organism in integrative studies of functional evolution of the chordate endocrine, neuroendocrine, and nervous systems.

Ascidians are the closest phylogenetic neighbors to vertebrates and are believed to conserve the evolutionary origin in chordates of the endocrine, neuroendocrine, and nervous systems involving neuropeptides and peptide hormones. Ciona intestinalis harbors various homologs or prototypes of vertebrate neuropeptides and peptide hormones including gonadotropin-releasing hormones (GnRHs), tachykinins (TKs), and calcitonin, as well as Ciona-specific neuropeptides such as Ciona vasopressin, LF, and YFV/L peptides. Moreover, molecular and functional studies on Ciona tachykinin (Ci-TK) have revealed the novel molecular mechanism of inducing oocyte growth via up-regulation of vitellogenesis-associated protease activity, which is expected to be conserved in vertebrates. Furthermore, a series of studies on Ciona GnRH receptor paralogs have verified the species-specific regulation of GnRHergic signaling including unique signaling control via heterodimerization among multiple GnRH receptors. These findings confirm the remarkable significance of ascidians in investigations of the evolutionary processes of the peptidergic systems in chordates, leading to the promising advance in the research on Ciona peptides in the next stage based on the recent development of emerging technologies including genome-editing techniques, peptidomics-based multi-color staining, machine-learning prediction, and next-generation sequencing. These technologies and bioinformatic integration of the resultant "multi-omics" data will provide unprecedented insights into the comprehensive understanding of molecular and functional regulatory mechanisms of the Ciona peptides, and will eventually enable the exploration of both conserved and diversified endocrine, neuroendocrine, and nervous systems in the evolutionary lineage of chordates.

Nasal oxytocin administration reduces food intake without affecting locomotor activity and glycemia with c-Fos induction in limited brain areas.

Recent studies have considered oxytocin (Oxt) as a possible medicine to treat obesity and hyperphagia. To find the effective and safe route for Oxt treatment, we compared the effects of its nasal and intraperitoneal (IP) administration on food intake, locomotor activity, and glucose tolerance in mice. Nasal Oxt administration decreased food intake without altering locomotor activity and increased the number of c-Fos-immunoreactive (ir) neurons in the paraventricular nucleus (PVN) of the hypothalamus, the area postrema (AP), and the dorsal motor nucleus of vagus (DMNV) of the medulla. IP Oxt administration decreased food intake and locomotor activity and increased the number of c-Fos-ir neurons not only in the PVN, AP, and DMNV but also in the nucleus of solitary tract of the medulla and in the arcuate nucleus of the hypothalamus. In IP glucose tolerance tests, IP Oxt injection attenuated the rise of blood glucose, whereas neither nasal nor intracerebroventricular Oxt affected blood glucose. In isolated islets, Oxt administration potentiated glucose-induced insulin secretion. These results indicate that both nasal and IP Oxt injections reduce food intake to a similar extent and increase the number of c-Fos-ir neurons in common brain regions. IP Oxt administration, in addition, activates broader brain regions, reduces locomotor activity, and affects glucose tolerance possibly by promoting insulin secretion from pancreatic islets. In comparison with IP administration, the nasal route of Oxt administration could exert a similar anorexigenic effect with a lesser effect on peripheral organs.

Transportation-induced nausea-like behavior in goats and the effects of anti-motion sickness medication.

This study investigated the nausea-like behavior induced by road transportation in goats, and the effects of an anti-motion sickness (MS) medication on this behavior. In the first experiment, 11 adult Shiba goats were road transported twice with either a saline (control) or a commercial anti-MS medication (Travelmin) injection at the first or second transportation. Almost all goats showed nausea-like behavior, which was defined as pointing their heads downward, closing their eyes, and staying relatively still. These goats did not respond when they were touched during blood collection. The anti-MS medication significantly reduced the total time spent in nausea-like behavior (P < 0.05) and tended to increase the frequency of escape attempts during blood collection (P < 0.1). In a second experiment, the effects of the anti-MS medication were examined in goats held under normal housing. The anti-MS medication increased the time spent feeding (P < 0.01) and reduced the time spent in rumination (P < 0.05) but did not change the frequency of lying down nor plasma cortisol concentrations. Our results indicate that the nausea-like behavior in transported goats might be induced, at least in part, by regulatory mechanisms similar to the MS.

Traditional Japanese medicine Kamikihito ameliorates sucrose preference, chronic inflammation and obesity induced by a high fat diet in middle-aged mice.

The high prevalence of obesity has become a pressing global public health problem and there exists a strong association between increased BMI and mortality at a BMI of 25 kg/m2 or higher. The prevalence of obesity is higher among middle-aged adults than among younger groups and the combination of aging and obesity exacerbate systemic inflammation. Increased inflammatory cytokines such as interleukin 6 and tumor necrosis factor alpha (TNFα) are hallmarks of obesity, and promote the secretion of hepatic C-reactive protein (CRP) which further induces systematic inflammation. The neuropeptide oxytocin has been shown to have anti-obesity and anti-inflammation effects, and also suppress sweet-tasting carbohydrate consumption in mammals. Previously, we have shown that the Japanese herbal medicine Kamikihito (KKT), which is used to treat neuropsychological stress disorders in Japan, functions as an oxytocin receptors agonist. In the present study, we further investigated the effect of KKT on body weight (BW), food intake, inflammation, and sweet preferences in middle-aged obese mice. KKT oral administration for 12 days decreased the expression of pro-inflammatory cytokines in the liver, and the plasma CRP and TNFα levels in obese mice. The effect of KKT administration was found to be different between male and female mice. In the absence of sucrose, KKT administration decreased food intake only in male mice. However, while having access to a 30% sucrose solution, both BW and food intake was decreased by KKT administration in male and female mice; but sucrose intake was decreased in female mice alone. In addition, KKT administration decreased sucrose intake in oxytocin deficient lean mice, but not in the WT lean mice. The present study demonstrates that KKT ameliorates chronic inflammation, which is strongly associated with aging and obesity, and decreases food intake in male mice as well as sucrose intake in female mice; in an oxytocin receptor dependent manner.

Angiotensin AT(1)-receptor blockers enhance cardiac responses to parasympathetic nerve stimulation via presynaptic AT(1) receptors in pithed rats.

In the present study, we investigated the effects of angiotensin AT1-receptor blockers, KT3-671 and losartan, on the cardiac vagal neurotransmission in pithed rats. The bradycardia induced by vagal nerve stimulation (VNS, at 5 Hz) was potentiated significantly and dose-dependently by KT3-671 and also losartan. This enhancement effect of KT3-671 (10 mg/kg) was slightly potent than that of losartan (10 mg/kg). On the other hand, an angiotensin AT2-receptor blocker, PD123319 (10 mg/kg), did not affect VNS-induced bradycardia. KT3-671 and losartan did not affect the exogenous acetylcholine-evoked bradycardia. Intravenous infusion of AngII (100 ng/kg per min) attenuated the VNS-induced bradycardia. This inhibitory effect of AngII on bradycardia was restored by both KT3-671 and losartan. These results suggest that endogenous AngII can have a tonic inhibitory effect on cardiac vagal transmission by stimulating the presynaptic AT1 receptors not AT2 receptors. Suppression of this mechanism by the AT1-receptor blockers causes the facilitation of acetylcholine release from vagal nerve endings. This acceleratory effect of AT1-receptor blockers on cardiac vagal neurotransmission may contribute to the lack of reflex tachycardia following hypotension.

Radial polarity in the first cranial neuromast of selected teleost fishes.

The neuromast is a sensory structure of the lateral line system in aquatic vertebrates, which consists of hair cells and supporting cells. Hair cells are mechanosensory cells, generally arranged with bidirectional polarity. Here, we describe a neuromast with hair cells arranged radially instead of bidirectionally in the first cranial neuromast of four teleost species: red seabream (Pagrus major), spotted halibut (Verasper variegatus), brown sole (Pseudopleuronectes herzensteini), and marbled sole (Pseudopleuronectes yokohamae). In these four species, this polarity was identified only in the first cranial neuromast, where it appeared at the rostral edge of the otic vesicle before hatching. We investigated the initial appearance and fate of this unique neuromast using scanning electron microscopy. We also assessed characteristics of radial neuromast pertaining to morphogenesis, development, and innervation using a vital fluorescent marker and immunohistochemistry in V. variegatus. The kinocilium initially appears at the center of each hair cell, then moves to its outer perimeter to form radial polarity by around 7 days postfertilization. However, hair cells arranged radially disappear about 15 days after hatching. This is followed by the appearance of bidirectionally arranged hair cells, indicating that polarity replacement from radial to bidirectional has occurred. In P. herzensteini, both afferent and efferent synapses between the nerve fibers and hair cells were observed by transmission electron microscopy, suggesting that radial neuromast is functional. Our discovery suggests that neuromasts with radial polarity could enable larval fish to assimilate multiaxial stimuli during this life stage, potentially assisting them in detecting small water vibrations or water pressure changes.

Functional diversity of signaling pathways through G protein-coupled receptor heterodimerization with a species-specific orphan receptor subtype.

Gonadotropin-releasing hormones (GnRHs) play pivotal roles in control of reproduction via a hypothalamic-pituitary-periphery endocrine system and nervous systems of not only vertebrates but also invertebrates. GnRHs trigger several signal transduction cascades via GnRH receptors (GnRHRs), members of the G protein-coupled receptor (GPCR) family. Recently, six GnRHs (tunicate GnRH [tGnRH]-3 to tGnRH-8) and four GnRHRs (Ciona intestinalis [Ci]-GnRHR1 to GnRHR-4), including a species-specific paralog, Ci-GnRHR4 (R4) regarded as an orphan receptor or nonfunctional receptor, were identified in the protochordate, C. intestinalis, which lacks the hypothalamic-pituitary system. Here, we show novel functional modulation of GnRH signaling pathways via GPCR heterodimerization. Immunohistochemical analysis showed colocalization of R1 and R4 in test cells of the ascidian ovary. The native R1-R4 heterodimerization was detected in the Ciona ovary by coimmunoprecipitation analysis. The heterodimerization in HEK293 cells cotransfected with R1 and R4 was also observed by coimmunoprecipitation and fluorescent energy transfer analyses. Binding assay revealed that R4 had no affinity for tGnRHs, and the heterodimerization did not alter the binding affinity of R1 to the ligands. The R1-R4 elicited 10-fold more potent Ca2+ mobilization than R1 exclusively by tGnRH-6, although R1-mediated cyclic AMP production was not affected by any of tGnRHs via the R1-R4 heterodimer. Moreover, the R1-R4 heterodimer potentiated translocation of both Ca2+-dependent protein kinase C-alpha (PKCalpha) by tGnRH-6 and Ca2+-independent PKCzeta by tGnRH-5 and tGnRH-6, eventually leading to the upregulation of extracellular signal-regulated kinase (ERK) phosphorylation compared with R1 alone. These results provide evidence that the species-specific GnRHR orphan paralog, R4, serves as an endogenous modulator for the fine-tuning of activation of PKC subtype-selective signal transduction via heterodimerization with R1 and that the species-specific GPCR heterodimerization, in concert with multiplication of tGnRHs and Ci-GnRHRs, participates in functional evolution of neuropeptidergic GnRH signaling pathways highly conserved throughout the animal kingdom.

[A case of castration-refractory prostate cancer showing marked decrease of serum PSA level after zoledronic acid treatment with estramustine phosphate and prednisolone].

A 66-year-old man was referred to our outpatient clinic for an elevated serum prostatic-specific antigen (PSA 4,319 ng/ mL). Magnetic resonance imaging (MRI) showed multiple metastatic lesions in the bones. The patient had received androgen deprivation therapy, but six months after treatment, he was diagnosed as having prostate cancer refractory to hormones. Combined treatment with docetaxel (DOC 30 mg/m²/week )and estramustine phosphate (EMP 560 mg/day) was initiated as first-line chemotherapy, but the treatment was discontinued because of side effects. Then, treatment with zoledronic acid was started(4 mg/4 weeks)and the PSA level decreased dramatically from 457.2 ng/mL to 5.5 ng/mL. Seven months after the diagnosis of CRPC, MRI showed a decrease ofbone metastases, and the PSA levels continued to decrease, eventually reaching 0.3 ng/mL. Zoledronic acid appears to not only show efficacy in preventing skeletal-related events, but has a potential antitumor effect in patients with metastatic CRPC.

The effects of cisplatin, an emetic agent, on behavior and plasma cortisol levels in goats.

Ruminants are not considered to experience nausea because they do not possess the emetic reflex. This study examined the effects of administration of cisplatin (cis-diamminedichloro platinum (II): CDDP), a common emetic agent, on the behavior of goats. In Experiment 1, adult Shiba goats received intravenous (IV) administration of CDDP. CDDP-administered goats spent a shorter time feeding (P < 0.01), lied down earlier (P < 0.01), and spent a longer period lying down (P < 0.01) than the saline-administered control goats, and sometimes, they directed their face downward and close their eyes. These behavioral responses were followed by a significant (P < 0.01) increase in plasma cortisol (Cor) levels, which indicated that goats experienced stress. The dose dependency was found in the extent of the CDDP effects. In Experiment 2, the effects of pretreatment of ondansetron (Ond), an antiemetic agent, were examined. Pretreatment of Ond extended the latency of lying (P < 0.01), shortened the time spent lying (P < 0.05), and reduced the extent of the increase in plasma Cor levels (P < 0.01). These results suggested that CDDP administration generated some state of stress in goats via the similar physiological mechanisms as emesis-caused stress in emetic species.

Neuropeptides, hormone peptides, and their receptors in Ciona intestinalis: an update.

The critical phylogenetic position of ascidians leads to the presumption that neuropeptides and hormones in vertebrates are highly likely to be evolutionarily conserved in ascidians, and the cosmopolitan species Ciona intestinalis is expected to be an excellent deuterostome Invertebrate model for studies on neuropeptides and hormones. Nevertheless, molecular and functional characterization of Ciona neuropeptides and hormone peptides was restricted to a few peptides such as a cholecystokinin (CCK)/gastrin peptide, cionin, and gonadotropin-releasing hormones (GnRHs). In the past few years, mass spectrometric analyses and database searches have detected Ciona orthologs or prototypes of vertebrate peptides and their receptors, including tachykinin, insulin/relaxin, calcitonin, and vasopressin. Furthermore, studies have shown that several Ciona peptides, including vasopressin and a novel GnRH-related peptide, have acquired ascidian-specific molecular forms and/or biological functions. These findings provided indisputable evidence that ascidians, unlike other invertebrates (including the traditional protostome model animals), possess neuropeptides and hormone peptides structurally and functionally related to vertebrate counterparts, and that several peptides have uniquely diverged in ascidian evolutionary lineages. Moreover, recent functional analyses of Ciona tachykinin in the ovary substantiated the novel tachykininergic protease-assoclated oocyte growth pathway, which could not have been revealed in studies on vertebrates. These findings confirm the outstanding advantages of ascidians in understanding the neuroscience, endocrinology, and evolution of vertebrate neuropeptides and hormone peptides. This article provides an overview of basic findings and reviews new knowledge on ascidian neuropeptides and hormone peptides.

Light and electron microscopy study of the pecten oculi of the jungle crow (Corvus macrorhynchos).

In this study, the pecten oculi of a diurnally active bird, the Japanese jungle crow (Corvus macrorhynchos), was examined using light and electron microscopy. In this species, the pecten consisted of 24-25 highly vascularized pleats held together apically by a heavily pigmented 'bridge' and projected freely into the vitreous body in the ventral part of the eye cup. Ascending and descending blood vessels of varying caliber, together with a profuse network of capillaries, essentially constituted the vascular framework of the pecten. A distinct distribution of melanosomes was discernible on the pecten, the concentration being highest at its apical end, moderate at the crest of the pleats and lowest at the basal and lateral margins. Overlying and within the vascular network, a close association between blood vessels and melanocytes was evident. It is conjectured that such an association may have evolved to augment the structural reinforcement of this nutritive organ in order to keep it firmly erectile within the gel-like vitreous. Such erectility may be an essential prerequisite for its optimal functioning as well as in its overt use as a protective shield against the effects of ultraviolet light, which otherwise might lead to damage of the pectineal vessels.

[Female case of successful engraftment after PBSCT from her HLA-haploidentical son for severe aplastic anemia during maintenance peritoneal dialysis].

A 51-year-old woman developed very severe aplastic anemia complicated by chronic renal failure. She underwent ATG therapy, resulting in a transient effect. Thereafter, renal insufficiency progressed, followed by the induction of CAPD therapy. Although rabbit ATG (rATG) was administered as the next immunotherapy for aplastic anemia, pancytopenia persisted and she experienced repeated episodes of severe infection including MRSA and fungal infections. She was transplanted with peripheral blood stem cells from her HLA-haploidentical son after a reduced-intensity conditioning regimen that included cyclophosphamide (three days of 30 mg/kg), fludarabine (two days of 20 mg/m(2)) and rATG (three days of 2.5 mg/kg). FK506 (0.03 mg/kg/24H) was administered for the prophylaxis of GVHD. Prompt trilineage engraftment occurred, resulting in the improvement of infections. Three months after grafting, she succumbed to cerebrovascular disease, although there was no apparent GVHD and she had remained well with stable hematopoiesis.

Histological properties of the nasal cavity and olfactory bulb of the Japanese jungle crow Corvus macrorhynchos.

The nasal cavity and olfactory bulb (OB) of the Japanese jungle crow (Corvus macrorhynchos) were studied using computed tomography (CT) and histochemical staining. The nasal septum divided the nasal cavity in half. The anterior and maxillary conchae were present on both sides of the nasal cavity, but the posterior concha was indistinct. A small OB was present on the ventral surface of the periphery of the cerebrum. The OB-brain ratio--the ratio of the size of the OB to that of the cerebral hemisphere--was 6.13. The olfactory nerve bundles projected independently to the OB, which appeared fused on gross examination. Histochemical analysis confirmed the fusion of all OB layers. Using a neural tracer, we found that the olfactory nerve bundles independently projected to the olfactory nerve layer (ONL) and glomerular layer (GL) of the left and right halves of the fused OB. Only 4 of 21 lectins bound to the ONL and GL. Thus, compared with mammals and other birds, the jungle crow may have a poorly developed olfactory system and an inferior sense of olfaction. However, it has been contended recently that the olfactory abilities of birds cannot be judged from anatomical findings alone. Our results indicate that the olfactory system of the jungle crow is an interesting research model to evaluate the development and functions of vertebrate olfactory systems.

Morphological and histochemical study of the nasal cavity and fused olfactory bulb of the brown-eared bulbul, Hysipetes amaurotis.

The brown-eared bulbul (Hysipetes amaurotis) is commonly found in Japan where it is regarded as a harmful bird that causes damage to agricultural products. Few studies have investigated the sensory apparatus of this bird, and consequently little is known of the sensory modalities it uses. Here we analyzed the anatomical and histological properties of the nasal cavity and olfactory bulb (OB) of the bulbul in order to investigate the functional level of olfaction in this species. Although both anterior and maxillary conchae were observed in the bulbul nasal cavity, there was no structure equivalent to the posterior concha. The OB located on the ventral side of the anterior extremity of the cerebrum and the ratio of olfactory bulb size to that of the cerebral hemisphere were very small. Interestingly, the left and right OBs were completely fused at the midline of the cerebrum. Furthermore, certain types of lectins that bind to the olfactory nerve of vertebrates with a well-developed sense of smell also bound positively to the olfactory nerve and glomerular layers of the bulbul OB. These findings suggest that the brown-eared bulbul has an anatomically and functionally less well developed sense of smell compared to other avian species. Although the molecular and developmental mechanisms underlying the fusion of the OB remain unknown, we suggest that the fused OB may offer a unique model for studying the evolution and development of the central olfactory nervous system in vertebrates.

An animal study of a newly developed skin-penetrating pad and covering material for catheters to prevent exit-site infection in continuous ambulatory peritoneal dialysis.

Because currently available peritoneal dialysis catheters are not sufficiently biocompatible with the skin and subcutaneous tissue at the site of penetration, exit-site infection due to pericatheter pocket formation caused by epidermal downgrowth over a long period of time has increasingly become a problem. We developed a new, biocompatible, segmented polyurethane porous material and devised a novel skin-penetrating pad, the form and material of which we optimized for application in peritoneal dialysis catheters. For the extent of tissue ingrowth into this porous material to be examined, test materials with different pore diameters were inserted into hollow silicone tubes and implanted in the subcutaneous tissue of a goat. Four weeks later, the tubes were extracted, and, after the extent of granulation tissue ingrowth was measured, histopathological evaluation was made. Our novel skin-penetrating pad has three disklike layers of the segmented polyurethane material with different pore sizes, into the center of which a polyurethane catheter is inserted. These pads were implanted in the skin of a goat and clinically observed over a 2-year period, after which they were extracted and histopathologically analyzed. In accordance with actual clinical procedures, a commercial CAPD catheter equipped with our skin-penetrating pad was left indwelling in a goat for 4 months, and the performance of the pad was evaluated after repeated periodic infusion and drainage of the dialysate in and out of the abdominal cavity. There was no inflammation of the ingrown tissue in the pores of the segmented polyurethane material as well as the surrounding tissue, which indicated favorable tissue biocompatibility. The extent of tissue ingrowth was greater as the pore size of the material was larger, and the tissue tended to be mature, mainly consisting of collagenous fibers. The skin-penetrating pad using the porous material, of which tissue ingrowth was thus optimized, tightly adhered to the goat skin throughout the 2-year experimental period without any special wound care such as cleansing or disinfection. The performance of the skin-penetrating pad was similarly favorable when attached to a commercial continuous ambulatory peritoneal dialysis catheter. The newly developed segmented polyurethane porous material had excellent tissue biocompatibility and tissue ingrowth. The skin-penetrating pad devised by using this porous material did not cause epidermal downgrowth, suggesting that it may be effective for the prevention of exit-site infection.

Distribution of the androgen receptor in the diencephalon and the pituitary gland in goats: Co-localisation with corticotrophin releasing hormone, arginine vasopressin and corticotrophs.

Previously it has been shown that androgen suppresses transportation-induced increases in plasma adrenocorticotropic hormone (ACTH), possibly by suppressing the secretion of corticotrophin releasing hormone (CRH) or arginine vasopressin (AVP) from the hypothalamus, or secretion of ACTH from the pituitary gland. The aim of the present study was to examine androgen target sites in the caprine diencephalon and pituitary gland using immunohistochemical methods. The androgen receptor (AR) was expressed strongly in the bed nucleus of the stria terminalis, the medial preoptic area, the arcuate nucleus, the ventromedial hypothalamic nucleus and the suprachiasmatic nucleus in the diencephalon. Between 8% and 11% of CRH and AVP neurons in the paraventricular hypothalamic nucleus (PVN) expressed AR. In the pituitary gland, 7.1% of corticotrophs expressed AR. The results are consistent with the proposal that androgen acts directly and indirectly on CRH and/or AVP neurons in the PVN. The possibility of a direct action of androgen on the corticotrophs in the pituitary gland was also considered.

Androgen suppresses corticotropin-induced increase in plasma cortisol level but enhances the increase in plasma aldosterone level in goats.

Previously we reported that androgen treatment reduced the extent of the increase in plasma cortisol (Cor) levels induced by adrenocorticotropic hormone (ACTH) administration in goats. In this study, we investigated the effect of androgen on the plasma levels of androstenedione and aldosterone. Four castrated male goats, which were treated with either 5alpha-dihydrotestosterone (DHT) or cholesterol (cho), were injected intravenously with 0.005, 0.02 or 0.1 mg of ACTH(1-24). Plasma Cor levels were increased significantly by all doses of ACTH injection, and these extents were lower in DHT-treated goats. Plasma androstenedione levels were also increased by ACTH injection, but DHT treatment seemed to little affect. Plasma aldosterone levels were also increased by ACTH injection, and there were no differences between cho- and DHT treated goats at 15 and 30 min after the ACTH injection. However, when goats were given the lower doses of ACTH (0.02 and 0.005 mg), plasma aldosterone levels were restored rapidly only in cho-treated goats, whereas those in DHT-treated goats were maintained throughout the 60 min experimental period. Consequently, plasma aldosterone levels in DHT-treated goats were higher than those in cho-treated goats at 45 and 60 min. One possible mechanism of the effect of DHT on the ACTH-induced increase in aldosterone synthesis may be the reduction of the activity of P450-17alpha, that is the enzyme to convert pregnenolone to 17alpha-OH-pregnenolone, and this mechanism may also be responsible to the suppressive effect of DHT on the ACTH-induced Cor synthesis.

The effect of avertin anesthesia and a mixture of three types of anesthetic agents on food intakeand body weight in high fat-induced obese maleand female mice.

Basic research on obesity is becoming more important due to an increasing number of obese people. Experiments using obesity-model animals often require surgical interventions, such as gastric operation, and proper selection of anesthesia is important. Avertin, an agent mainly composed of 2,2,2-Tribromoethanol, has been used as general anesthesia for a long time, without the use of narcotic drugs. In the current study, we found that a single injection of avertin can decrease body weight (BW) in male and female C57BL/6J and ICR mice with high fat-diet (HFD)-induced obesity, but not in standard diet-fed nonobese males and females. Because the BW-reducing effect was more prominent in the female mice, we compared the effects of avertin and a mixture of three types of anesthetic agents (3MIX), which was developed in 2011, on BW reduction in HFD-induced obese female mice. Although both avertin and 3MIX decreased food intake and BW, the effects of avertin were significantly more potent than those of 3MIX. C-Fos expression, a neural activation marker, was dramatically increased in the brain regions related to the regulation of both food intake and the autonomic nervous system after avertin injection, but not after 3MIX injection. This suggests that avertin strongly stimulates the center of feeding regulation and the autonomic nervous system and therefore decreases BW. The current study suggests the advantages of using 3MIX for surgical interventions in mice in obesity research, as it is ideal to prevent anesthesia-induced BW decline.

Identification of a New Theca/Interstitial Cell-Specific Gene and Its Biological Role in Growth of Mouse Ovarian Follicles at the Gonadotropin-Independent Stage.

Theca/interstitial cells are responsible for the growth and maturation of ovarian follicles. However, little is known about the theca/interstitial cell-specific genes and their functions. In this study, we explored transcriptomes of theca/interstitial cells by RNA-seq, and the novel biological roles of a theca cell marker, asporin (Aspn)/periodontal ligament-associated protein 1 (PLAP-1). RNA-seq detected 432 and 62 genes expressed specifically in theca/interstitial cells and granulosa cells isolated from 3-weeks old mouse ovaries. Gene ontology analysis demonstrated that these genes were largely categorized into four major groups: extracellular matrix organization-related terms, chemotaxis-related terms, the angiogenesis-related terms, and morphogenesis-related terms. In situ hybridization demonstrated that the newly detected representative gene, Aspn/PLAP-1, was detected specifically in the outer layer of theca cells in contrast with the expression of the basal lamina-specific gene, Nidgen-1. Intriguingly, an Aspn/PLAP-1 antibody completely arrested the growth of secondary follicles that is the gonadotropin-independent follicle developmental stage. Furthermore, transforming growth factor-ß (TGF-ß)-triggered signaling was induced by the Aspn/PLAP-1 antibody treatment, which is consistent with the inhibitory effect of Aspn/PLAP-1 on TGF-ß. Altogether, these results suggest that theca cells are classified into subpopulations on the basis of new marker genes and their biological functions, and provide evidence that Aspn/PLAP-1 is expressed exclusively in the outer layer of theca cells and plays a pivotal role in the growth of secondary follicles via downregulation of the canonical TGF-ß signaling cascade.

A novel biological role of tachykinins as an up-regulator of oocyte growth: identification of an evolutionary origin of tachykininergic functions in the ovary of the ascidian, Ciona intestinalis.

Tachykinins (TKs) and their receptors have been shown to be expressed in the mammalian ovary. However, the biological roles of ovarian TKs have yet to be verified. Ci-TK-I and Ci-TK-R, characterized from the protochordate (ascidian), Ciona intestinalis, are prototypes of vertebrate TKs and their receptors. In the present study, we show a novel biological function of TKs as an inducible factor for oocyte growth using C. intestinalis as a model organism. Immunostaining demonstrated the specific expression of Ci-TK-R in test cells residing in oocytes at the vitellogenic stage. DNA microarray and real-time PCR revealed that Ci-TK-I induced gene expression of several proteases, including cathepsin D, chymotrypsin, and carboxy-peptidase B1, in the ovary. The enzymatic activities of these proteases in the ovary were also shown to be enhanced by Ci-TK-I. Of particular significance is that the treatment of Ciona oocytes with Ci-TK-I resulted in progression of growth from the vitellogenic stage to the post-vitellogenic stage. The Ci-TK-I-induced oocyte growth was blocked by a TK antagonist or by protease inhibitors. These results led to the conclusion that Ci-TK-I enhances growth of the vitellogenic oocytes via up-regulation of gene expression and enzymatic activities of the proteases. This is the first clarification of the biological roles of TKs in the ovary and the underlying essential molecular mechanism. Furthermore, considering the phylogenetic position of ascidians as basal chordates, we suggest that the novel TK-regulated oocyte growth is an "evolutionary origin" of the tachykininergic functions in the ovary.