Publications by authors named "Shinji Kanda"

24 Publications

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Divalent metal transporter-related protein restricts animals to marine habitats.

Commun Biol 2021 Apr 12;4(1):463. Epub 2021 Apr 12.

Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa-shi, Chiba, Japan.

Utilization and regulation of metals from seawater by marine organisms are important physiological processes. To better understand metal regulation, we searched the crown-of-thorns starfish genome for the divalent metal transporter (DMT) gene, a membrane protein responsible for uptake of divalent cations. We found two DMT-like sequences. One is an ortholog of vertebrate DMT, but the other is an unknown protein, which we named DMT-related protein (DMTRP). Functional analysis using a yeast expression system demonstrated that DMT transports various metals, like known DMTs, but DMTRP does not. In contrast, DMTRP reduced the intracellular concentration of some metals, especially zinc, suggesting its involvement in negative regulation of metal uptake. Phylogenetic distribution of the DMTRP gene in various metazoans, including sponges, protostomes, and deuterostomes, indicates that it originated early in metazoan evolution. However, the DMTRP gene is only retained in marine species, and its loss seems to have occurred independently in ecdysozoan and vertebrate lineages from which major freshwater and land animals appeared. DMTRP may be an evolutionary and ecological limitation, restricting organisms that possess it to marine habitats, whereas its loss may have allowed other organisms to invade freshwater and terrestrial habitats.
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http://dx.doi.org/10.1038/s42003-021-01984-8DOI Listing
April 2021

Roles of the ClC chloride channel CLH-1 in food-associated salt chemotaxis behavior of .

Elife 2021 Jan 25;10. Epub 2021 Jan 25.

Department of Biological Sciences, School of Science, The University of Tokyo, Tokyo, Japan.

The ability of animals to process dynamic sensory information facilitates foraging in an ever-changing environment. However, molecular and neural mechanisms underlying such ability remain elusive. The ClC anion channels/transporters play a pivotal role in cellular ion homeostasis across all phyla. Here, we find a ClC chloride channel is involved in salt concentration chemotaxis of . Genetic screening identified two altered-function mutations of that disrupt experience-dependent salt chemotaxis. Using genetically encoded fluorescent sensors, we demonstrate that CLH-1 contributes to regulation of intracellular anion and calcium dynamics of salt-sensing neuron, ASER. The mutant CLH-1 reduced responsiveness of ASER to salt stimuli in terms of both temporal resolution and intensity, which disrupted navigation strategies for approaching preferred salt concentrations. Furthermore, other ClC genes appeared to act redundantly in salt chemotaxis. These findings provide insights into the regulatory mechanism of neuronal responsivity by ClCs that contribute to modulation of navigation behavior.
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http://dx.doi.org/10.7554/eLife.55701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7834019PMC
January 2021

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes).

J Vis Exp 2020 12 11(166). Epub 2020 Dec 11.

Physiology Unit, Faculty of Veterinary Medicine, Norwegian University of Life Sciences;

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.
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http://dx.doi.org/10.3791/62006DOI Listing
December 2020

Examination of methods for manipulating serum 17β-Estradiol (E2) levels by analysis of blood E2 concentration in medaka (Oryzias latipes).

Gen Comp Endocrinol 2020 01 13;285:113272. Epub 2019 Sep 13.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan; Present address: Laboratory of Physiology, Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan. Electronic address:

It is widely known that reproduction in vertebrates is regulated by the hypothalamus-pituitary-gonadal (HPG) axis. Although the mechanism of the HPG axis has been well documented in mammals, it cannot be always applied to that in non-mammalian species, which is a great disadvantage in understanding reproduction of vertebrates in general. Recently, transgenic and genome editing tools have rapidly been developed in small teleosts, and thus these species are expected to be useful for the understanding of general mechanism of reproduction in vertebrates. One of the major sex steroid hormones in female vertebrates 17β-Estradiol (E2) plays crucial roles in the formation of sexual dimorphism and the HPG axis regulation. In spite of the importance of E2 in reproductive regulation, only a few studies have analyzed blood E2 levels in small teleosts that are easily amenable to genetic manipulation. In the present study, we analyzed blood E2 concentration in medaka and demonstrated that female medaka show diurnal changes in blood E2 concentration. We then examined the best method for manipulating the circulating E2. First, we found that ovariectomy (OVX) drastically removes endogenous E2 in a day in female medaka. We examined different methods for E2 administration and revealed that feeding administration of E2-containing food is the most convenient and physiological method for mimicking the diurnal E2 changes of female medaka. On the other hand, the medaka exposed to E2 containing water showed high blood E2 concentrations, which exceeds those of environmental water, suggesting that E2 may cause bioconcentration.
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http://dx.doi.org/10.1016/j.ygcen.2019.113272DOI Listing
January 2020

Gene knockout analysis reveals essentiality of estrogen receptor β1 (Esr2a) for female reproduction in medaka.

Sci Rep 2019 06 20;9(1):8868. Epub 2019 Jun 20.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo, 113-0033, Japan.

In vertebrates, sex steroids play crucial roles in multiple systems related to reproduction. In females, estrogens and their receptor estrogen receptor (ER or Esr) play indispensable roles in the negative sex steroid feedback regulation of pituitary gonadotropin secretion, which prevents excessive development of ovarian follicles. However, the mechanism of this feedback regulation of a gonadotropin, follicle stimulating hormone (FSH), which is essential for folliculogenesis throughout vertebrates, is poorly understood. In the present study, we generated knockouts of all subtypes of nuclear estrogen receptors in a model teleost medaka, which is suitable for the study of endocrine control and behavioral assays, and analyzed fertility, behavior and functionality of estrogen feedback in each knockout line. Among the estrogen receptors, we revealed that an estrogen receptor Esr2a plays an essential role in this feedback regulation. In addition to this, we also found that esr2a females showed oviduct atresia, which causes complete infertility. Interestingly, esr2a females showed apparently normal sexual behavior but without oviposition in response to male courtship. This phenotype indicates that physical readiness and motivation of sexual behavior is independently controlled.
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http://dx.doi.org/10.1038/s41598-019-45373-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586646PMC
June 2019

Sexually Dimorphic Neuropeptide B Neurons in Medaka Exhibit Activated Cellular Phenotypes Dependent on Estrogen.

Endocrinology 2019 04;160(4):827-839

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo, Japan.

Brain and behavior of teleosts are highly sexually plastic throughout life, yet the underlying neural mechanisms are largely unknown. On examining brain morphology in the teleost medaka (Oryzias latipes), we identified distinctively large neurons in the magnocellular preoptic nucleus that occurred much more abundantly in females than in males. Examination of sex-reversed medaka showed that the sexually dimorphic abundance of these neurons is dependent on gonadal phenotype, but independent of sex chromosome complement. Most of these neurons in females, but none in males, produced neuropeptide B (Npb), whose expression is known to be estrogen-dependent and associated with female sexual receptivity. In phenotypic analysis, the female-specific Npb neurons had a large euchromatic nucleus with an abundant cytoplasm containing plentiful rough endoplasmic reticulum, exhibited increased overall transcriptional activity, and typically displayed a spontaneous regular firing pattern. These phenotypes, which are probably indicative of cellular activation, were attenuated by ovariectomy and restored by estrogen replacement. Furthermore, the population of Npb-expressing neurons emerged in adult males treated with estrogen, not through frequently occurring neurogenesis in the adult teleost brain, but through the activation of preexisting, quiescent male counterpart neurons. Collectively, our results demonstrate that the morphological, transcriptional, and electrophysiological phenotypes of sexually dimorphic preoptic Npb neurons are highly dependent on estrogen and can be switched between female and male patterns. These properties of the preoptic Npb neurons presumably underpin the neural mechanism for sexual differentiation and plasticity of brain and behavior in teleosts.
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http://dx.doi.org/10.1210/en.2019-00030DOI Listing
April 2019

Evolution of the regulatory mechanisms for the hypothalamic-pituitary-gonadal axis in vertebrates-hypothesis from a comparative view.

Authors:
Shinji Kanda

Gen Comp Endocrinol 2019 12 27;284:113075. Epub 2018 Nov 27.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan. Electronic address:

Reproduction is regulated by the hypothalamic-pituitary-gonadal (HPG) axis in vertebrates. In addition to wealth of knowledge in mammals, recent studies in non-mammalian species, especially teleosts, have provided evidence that some of the components in the HPG axis are conserved in bony vertebrates. On the other hand, from the comparisons of the recent accumulating knowledge between mammals and teleosts, unique characteristics of the regulatory system in each group have been unveiled. A hypophysiotropic neurotransmitter/hormone, gonadotropin releasing hormone (GnRH), pituitary gonadotropins, follicle stimulating hormone (FSH), and luteinizing hormone (LH) were proven to be common important elements of the HPG axis in teleosts and mammals, although the roles of each vary. Conversely, there are some modulators of GnRH or gonadotropins that are not common to all vertebrates. In this review, I will introduce the mechanism for HPG axis regulation in mammals and teleosts, and describe their evolution from a hypothetical common ancestor.
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http://dx.doi.org/10.1016/j.ygcen.2018.11.014DOI Listing
December 2019

Morphological Analysis of the Axonal Projections of EGFP-Labeled Esr1-Expressing Neurons in Transgenic Female Medaka.

Endocrinology 2018 02;159(2):1228-1241

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.

Some hypothalamic neurons expressing estrogen receptor α (Esr1) are thought to transmit a gonadal estrogen feedback signal to gonadotropin-releasing hormone 1 (GnRH1) neurons, which is the final common pathway for feedback regulation of reproductive functions. Moreover, estrogen-sensitive neurons are suggested to control sexual behaviors in coordination with reproduction. In mammals, hypothalamic estrogen-sensitive neurons release the peptide kisspeptin and regulate GnRH1 neurons. However, a growing body of evidence in nonmammalian species casts doubt on the regulation of GnRH1 neurons by kisspeptin neurons. As a step toward understanding how estrogen regulates neuronal circuits for reproduction and sex behavior in vertebrates in general, we generated a transgenic (Tg) medaka that expresses enhanced green fluorescent protein (EGFP) specifically in esr1-expressing neurons (esr1 neurons) and analyzed their axonal projections. We found that esr1 neurons in the preoptic area (POA) project to the gnrh1 neurons. We also demonstrated by transcriptome and histological analyses that these esr1 neurons are glutamatergic or γ-aminobutyric acidergic (GABAergic) but not kisspeptinergic. We therefore suggest that glutamatergic and GABAergic esr1 neurons in the POA regulate gnrh1 neurons. This hypothesis is consistent with previous studies in mice that found that glutamatergic and GABAergic transmission is critical for estrogen-dependent changes in GnRH1 neuron firing. Thus, we propose that this neuronal circuit may provide an evolutionarily conserved mechanism for regulation of reproduction. In addition, we showed that telencephalic esr1 neurons project to medulla, which may control sexual behavior. Moreover, we found that some POA-esr1 neurons coexpress progesterone receptors. These neurons may form the neuronal circuits that regulate reproduction and sex behavior in response to the serum estrogen/progesterone.
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http://dx.doi.org/10.1210/en.2017-00873DOI Listing
February 2018

Evolutionally Conserved Function of Kisspeptin Neuronal System Is Nonreproductive Regulation as Revealed by Nonmammalian Study.

Endocrinology 2018 01;159(1):163-183

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan.

The kisspeptin neuronal system, which consists of a neuropeptide kisspeptin and its receptor Gpr54, is considered in mammals a key factor of reproductive regulation, the so-called hypothalamic-pituitary-gonadal (HPG) axis. However, in nonmammalian vertebrates, especially in teleosts, existence of kisspeptin regulation on the HPG axis is still controversial. In this study, we applied multidisciplinary techniques to a teleost fish, medaka, and examined possible kisspeptin regulation on the HPG axis. First, we generated knockout medaka for kisspeptin-related genes and found that they show normal fertility, gonadal maturation, and expression of gonadotropins. Moreover, the firing activity of GnRH1 neurons recorded by the patch clamp technique was not altered by kisspeptin application. Furthermore, in goldfish, in vivo kisspeptin administration did not show any positive effect on HPG axis regulation. However, as kisspeptin genes are completely conserved among vertebrates except birds, we surmised that kisspeptin should have some important nonreproductive functions in vertebrates. Therefore, to discover novel functions of kisspeptin, we generated a gpr54-1:enhanced green fluorescent protein (EGFP) transgenic medaka, whose gpr54-1-expressing cells are specifically labeled by EGFP. Analysis of neuronal projection of gpr54-1:EGFP-expressing neurons showed that these neurons in the ventrolateral preoptic area project to the pituitary and are probably involved in endocrine regulation other than gonadotropin release. Furthermore, combination of deep sequencing, histological, and electrophysiological analyses revealed various novel neural systems that are under control of kisspeptin neurons-that is, those expressing neuropeptide Yb, cholecystokinin, isotocin, vasotocin, and neuropeptide B. Thus, our new strategy to genetically label receptor-expressing neurons gives insights into various kisspeptin-dependent neuronal systems that may be conserved in vertebrates.
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http://dx.doi.org/10.1210/en.2017-00808DOI Listing
January 2018

Female-Specific Glucose Sensitivity of GnRH1 Neurons Leads to Sexually Dimorphic Inhibition of Reproduction in Medaka.

Endocrinology 2016 Nov 8;157(11):4318-4329. Epub 2016 Sep 8.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.

Close interaction exists between energy-consuming reproduction and nutritional status. However, there are differences in costs and priority for reproduction among species and even between sexes, which leads to diversification of interactions between reproduction and nutritional status. Despite such diversified interactions among species and sexes, most of the analysis of the nutritional status-dependent regulation of reproduction has been limited to an endothermic vertebrate, mammalian species of either sex. Therefore, the mechanisms underlying the diversified interactions remain elusive. In the present study, we demonstrated the effects of malnutritional status on reproduction at both organismal and cellular levels in an ectothermic vertebrate, a teleost medaka of both sexes. First, we analyzed the effects of malnutrition by fasting on gonadosomatic index, number of spawned/fertilized eggs, and courtship behavior. Fasting strongly suppressed reproduction in females but, surprisingly, not in males. Next, we analyzed the effects of fasting on firing activity of hypothalamic GnRH1 neurons, which form the final common pathway for the control of reproduction. An electrophysiological analysis showed that low glucose, which is induced by fasting, directly suppresses the firing activity of GnRH1 neurons specifically in females through intracellular ATP-sensitive potassium channels and AMP-activated protein kinase pathways. Based on the fact that such suppressions occurred only in females, we conclude that nutritional status-dependent, glucose-sensing in GnRH1 neurons may contribute to the most fitted reproductive regulation for each sex.
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http://dx.doi.org/10.1210/en.2016-1352DOI Listing
November 2016

Evolution of the Hypothalamic-Pituitary-Gonadal Axis Regulation in Vertebrates Revealed by Knockout Medaka.

Endocrinology 2016 Oct 25;157(10):3994-4002. Epub 2016 Aug 25.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.

Reproduction is essential for life, but its regulatory mechanism is diverse. The analysis of this diversity should lead us to understand the evolutionary process of the regulation of reproduction. In mammals, the hypothalamic-pituitary-gonadal axis plays an essential role in such regulation, and each component, hypothalamic GnRH, and pituitary gonadotropins, LH, and FSH, is indispensable. However, the common principle of the hypothalamic-pituitary-gonadal axis regulation among vertebrates remains unclear. Here, we used a teleost medaka, which is phylogenetically distant from mammals, and analyzed phenotypes of gene knockouts (KOs) for GnRH, LH, and FSH. We showed that LH release, which we previously showed to be directly triggered by GnRH, is essential for ovulation in females, because KO medaka of GnRH and LH were anovulatory in spite of the full follicular growth and normal gonadosomatic index, and spawning could be induced by a medaka LH receptor agonist. On the other hand, we showed that FSH is necessary for the folliculogenesis, because the follicular growth of FSH KO medaka was halted at the previtellogenic stage, but FSH release does not necessarily require GnRH. By comparing these results with the previous studies in mammals that both GnRH and LH are necessary for folliculogenesis, we propose a hypothesis as follows. During evolution, LH was originally specialized for ovulation, and regulation of folliculogenesis by GnRH-LH (pulsatile release) was newly acquired in mammals, which enabled fine tuning of reproduction through hypothalamus.
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http://dx.doi.org/10.1210/en.2016-1356DOI Listing
October 2016

Kiss1 neurons drastically change their firing activity in accordance with the reproductive state: insights from a seasonal breeder.

Endocrinology 2014 Dec 23;155(12):4868-80. Epub 2014 Sep 23.

Department of Biological Sciences (M.H., S.K., H.S., Y.A., Y.O.), Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan; and Laboratory of Fish Biology (H.A.), Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.

Kisspeptin (Kiss) neurons show drastic changes in kisspeptin expression in response to the serum sex steroid concentration in various vertebrate species. Thus, according to the reproductive states, kisspeptin neurons are suggested to modulate various neuronal activities, including the regulation of GnRH neurons in mammals. However, despite their reproductive state-dependent regulation, there is no physiological analysis of kisspeptin neurons in seasonal breeders. Here we generated the first kiss1-enhanced green fluorescent protein transgenic line of a seasonal breeder, medaka, for histological and electrophysiological analyses using a whole-brain in vitro preparation in which most synaptic connections are intact. We found histologically that Kiss1 neurons in the nucleus ventralis tuberis (NVT) projected to the preoptic area, hypothalamus, pituitary, and ventral telencephalon. Therefore, NVT Kiss1 neurons may regulate various homeostatic functions and innate behaviors. Electrophysiological analyses revealed that they show various firing patterns, including bursting. Furthermore, we found that their firings are regulated by the resting membrane potential. However, bursting was not induced from the other firing patterns with a current injection, suggesting that it requires some chronic modulations of intrinsic properties such as channel expression. Finally, we found that NVT Kiss1 neurons drastically change their neuronal activities according to the reproductive state and the estradiol levels. Taken together with the previous reports, we here conclude that the breeding condition drastically alters the Kiss1 neuron activities in both gene expression and firing activities, the latter of which is strongly related to Kiss1 release, and the Kiss1 peptides regulate the activities of various neural circuits through their axonal projections.
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http://dx.doi.org/10.1210/en.2014-1472DOI Listing
December 2014

Whole brain-pituitary in vitro preparation of the transgenic medaka (Oryzias latipes) as a tool for analyzing the differential regulatory mechanisms of LH and FSH release.

Endocrinology 2014 Feb 18;155(2):536-47. Epub 2013 Nov 18.

Department of Biological Sciences (T.K., M.A., C.K., H.A., S.K., Y.O.), Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan; and Laboratory of Fish Biology (H.A.), Graduate School of Bioagricultural Sciences, Nagoya University, Aichi 464-8601, Japan.

Two types of gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), are important pituitary hormones for sexual maturation and reproduction, and both of them are centrally regulated by gonadotropin-releasing hormone (GnRH) from the hypothalamus. In mammals, these two gonadotropins are secreted from a single type of gonadotrope. The mechanisms of differential regulation by GnRH of the release of two types of gonadotropins with different secretory profiles are still unknown. In teleosts, however, LH and FSH are secreted from separate cellular populations, unlike in mammals. This feature makes them useful for studying the regulatory mechanisms of LH and FSH secretions independently. Here, we generated transgenic medaka lines that express Ca(2+) indicator protein, inverse-pericam, specifically in the LH or FSH cells. We performed cell-type-specific Ca(2+) imaging of LH and FSH cells, respectively, using the whole brain-pituitary preparations of these transgenic fish in which all neural circuits and GnRH neuronal projection to the pituitary are kept intact. LH and FSH cells showed different Ca(2+) responses to GnRH. The results suggest differential regulation mechanisms for LH and FSH release by GnRH. Moreover, we also succeeded in detecting the effect on LH cells of endogenous GnRH peptide, which was released by electrical stimulation of the axons of GnRH1 neurons. Thus, our newly developed experimental model system using the whole brain-pituitary in vitro preparation of the transgenic medaka is a powerful tool for analyzing the differential regulatory mechanisms of the release of LH and FSH by multisynaptic neural inputs to the pituitary.
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http://dx.doi.org/10.1210/en.2013-1642DOI Listing
February 2014

Neuroanatomical evidence that kisspeptin directly regulates isotocin and vasotocin neurons.

PLoS One 2013 25;8(4):e62776. Epub 2013 Apr 25.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.

Neuropeptide kisspeptin has been suggested to be an essential central regulator of reproduction in response to changes in serum gonadal steroid concentrations. However, in spite of wide kisspeptin receptor distribution in the brain, especially in the preoptic area and hypothalamus, the research focus has mostly been confined to the kisspeptin regulation on GnRH neurons. Here, by using medaka whose kisspeptin (kiss1) neurons have been clearly demonstrated to be regulated by sex steroids, we analyzed the anatomical distribution of kisspeptin receptors Gpr54-1 and Gpr54-2. Because the both receptors were shown to be activated by kisspeptins (Kiss1 and Kiss2), we analyzed the anatomical distribution of the both receptors by in situ hybridization. They were mainly expressed in the ventral telencephalon, preoptic area, and hypothalamus, which have been suggested to be involved in homeostatic functions including reproduction. First, we found gpr54-2 mRNA expression in nucleus preopticus pars magnocellularis and demonstrated that vasotocin and isotocin (Vasopressin and Oxytocin ortholog, respectively) neurons express gpr54-2 by dual in situ hybridization. Given that kisspeptin administration increases serum oxytocin and vasopressin concentration in mammals, the present finding are likely to be vertebrate-wide phenomenon, although direct regulation has not yet been demonstrated in mammals. We then analyzed co-expression of kisspeptin receptors in three types of GnRH neurons. It was clearly demonstrated that gpr54-expressing cells were located adjacent to GnRH1 neurons, although they were not GnRH1 neurons themselves. In contrast, there was no gpr54-expressing cell in the vicinities of neuromodulatory GnRH2 or GnRH3 neurons. From these results, we suggest that medaka kisspeptin neurons directly regulate some behavioral and neuroendocrine functions via vasotocin/isotocin neurons, whereas they do not regulate hypophysiotropic GnRH1 neurons at least in a direct manner. Thus, direct kisspeptin regulation of GnRH1 neurons proposed in mammals may not be the universal feature of vertebrate kisspeptin system in general.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062776PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636218PMC
November 2013

Structure, synthesis, and phylogeny of kisspeptin and its receptor.

Adv Exp Med Biol 2013 ;784:9-26

Department of Biological Sciences, University of Tokyo, Bunkyo, Tokyo, Japan.

The kisspeptin system is considered to be essential for successful mammalian reproduction. In addition to the Kiss1 peptide, Kiss2, the product of kiss2 (the kiss1 paralogue), has also been shown to activate kisspeptin receptor signaling pathways in nonmammalian species. Furthermore, in nonmammalian species, there are two subtypes of receptors, Gpr54-1 (known as GPR54 or Kiss1R in mammals) and Gpr54-2. Although complete understanding of the two kisspeptin-two kisspeptin receptor systems in vertebrates is not so simple, a careful examination of the phylogeny of their genes may provide insights into the functional generality and differences among the kisspeptin systems in different animal phyla. In this chapter, we first discuss the structure of kisspeptin ligands, Kiss1 and Kiss2, and their characteristics as physiologically active peptides. Then, we discuss the evolutionary traits of kiss1 and kiss2 genes and their receptor genes, gpr54-1 and gpr54-2. It appears that each animal species has selected either kiss1 or kiss2 rather randomly, leading us to propose that some of the important characteristics of kisspeptin neurons, such as steroid sensitivity and the anatomical relationship with the hypophysiotropic GnRH1 neurons, may be the keys to understanding the general functions of different kisspeptin neuronal populations throughout vertebrates. Species differences in kiss1/kiss2 may also provide insights into the evolutionary mechanisms of paralogous gene-expressing neuronal systems. Finally, because kisspeptins belong to one of the members of the RFamide peptide families, we discuss the functional divergence of kisspeptins from the other RFamide peptides, which may be explained from phylogenetic viewpoints.
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http://dx.doi.org/10.1007/978-1-4614-6199-9_2DOI Listing
August 2013

Anatomical distribution of sex steroid hormone receptors in the brain of female medaka.

J Comp Neurol 2013 Jun;521(8):1760-80

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.

Estrogen and androgen play crucial roles in coordinating reproductive functions through estrogen receptors (ERs) and androgen receptors (ARs), respectively. These receptors are considered important for regulation of the hypothalamo-pituitary-gonadal (HPG) axis. Despite their biological importance, the distribution of sex steroid receptors has not been fully analyzed anatomically in the teleost brain. The teleosts have many characteristic features, which allow unique approaches toward an understanding of the regulatory mechanisms of reproductive functions. Medaka serves as a good model system for studying the mechanisms by which steroid receptor-mediated systems are regulated, because (1) their breeding conditions can be easily manipulated; (2) we can take advantage of the genome database; and 3) molecular genetic tools, such as transgenic techniques, are applicable. We analyzed the distribution of ERα, ERβ1, ERβ2, ARα, and ARβ mRNA by in situ hybridization in the brain of female medaka. We found that all subtypes of ERs and ARs were expressed in the following nuclei: the dorsal part of the ventral telencephalic area (Vd), supracommissural part of the ventral telencephalic area (Vs), postcommissural part of the ventral telencephalic area (Vp), preoptic area (POA), and nucleus ventralis tuberis (NVT). These regions are known to be involved in the regulation of sexual behavior (Vd, Vs, Vp, POA) or the HPG axis (NVT). These ER- and/or AR-expressing neurons may regulate sexual behavior or the HPG axis according to their axonal projections. Future analysis should be targeted to the neurons described in the present study to extend our understanding of the central regulatory mechanisms of reproduction.
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http://dx.doi.org/10.1002/cne.23255DOI Listing
June 2013

Female-specific target sites for both oestrogen and androgen in the teleost brain.

Proc Biol Sci 2012 Dec 17;279(1749):5014-23. Epub 2012 Oct 17.

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan.

To dissect the molecular and cellular basis of sexual differentiation of the teleost brain, which maintains marked sexual plasticity throughout life, we examined sex differences in neural expression of all subtypes of nuclear oestrogen and androgen receptors (ER and AR) in medaka. All receptors were differentially expressed between the sexes in specific nuclei in the forebrain. The most pronounced sex differences were found in several nuclei in the ventral telencephalic and preoptic areas, where ER and AR expression were prominent in females but almost completely absent in males, indicating that these nuclei represent female-specific target sites for both oestrogen and androgen in the brain. Subsequent analyses revealed that the female-specific expression of ER and AR is not under the direct control of sex-linked genes but is instead regulated positively by oestrogen and negatively by androgen in a transient and reversible manner. Taken together, the present study demonstrates that sex-specific target sites for both oestrogen and androgen occur in the brain as a result of the activational effects of gonadal steroids. The consequent sex-specific but reversible steroid sensitivity of the adult brain probably contributes substantially to the process of sexual differentiation and the persistent sexual plasticity of the teleost brain.
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http://dx.doi.org/10.1098/rspb.2012.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497244PMC
December 2012

Evolutionary Insights into the Steroid Sensitive kiss1 and kiss2 Neurons in the Vertebrate Brain.

Front Endocrinol (Lausanne) 2012 22;3:28. Epub 2012 Feb 22.

Department of Biological Science, Graduate School of Sciences, The University of Tokyo Tokyo, Japan.

Kisspeptin was originally found as a peptide product of Kiss1 gene and is now supposed to be an essential central regulator of reproduction in mammals. However, there is now a growing body of evidence to suggest that kiss2, the paralogous gene for kiss1, evolved in parallel during vertebrate lineage, and the kiss2 product also activates the GPR54 (kisspeptin receptor) signaling pathways. Therefore, it is now widely accepted that both kiss1 and kiss2 are the kisspeptin genes. Interestingly, either kiss1 or kiss2 or both have been lost during evolution in many vertebrate species, and the functional significance of kiss1 or kiss2 for the central regulation of reproduction is suggested to vary according to the species. Here, we argue that the steroid sensitivity of the kiss1 or kiss2 neurons has been well conserved during evolution among tetrapods and teleosts, and thus it may be the key to understanding the functional homologies of certain populations of kisspeptin (kiss1 or kiss2) neurons among different species of vertebrates. In the present review, we will first introduce recent advances in the study of steroid sensitive kiss1 and kiss2 systems in vertebrates and effects of peptide administrations in vivo. By comparing the similarities and differences between kiss1 and kiss2 of neuronal localization and sensitivity to gonadal steroids in various tetrapods and teleosts, we discuss the evolution of kisspeptin neuronal systems after gene duplication of ancestral kisspeptin genes to give rise to kiss1 and kiss2.
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http://dx.doi.org/10.3389/fendo.2012.00028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356069PMC
August 2012

Time-of-day-dependent changes in GnRH1 neuronal activities and gonadotropin mRNA expression in a daily spawning fish, medaka.

Endocrinology 2012 Jul 27;153(7):3394-404. Epub 2012 Apr 27.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

GnRH neurons in the preoptic area and hypothalamus control the secretion of GnRH and form the final common pathway for hypothalamic-pituitary-gonadal axis regulation in vertebrates. Temporal regulation of reproduction by coordinating endogenous physiological conditions and behaviors is important for successful reproduction. Here, we examined the temporal regulation of reproduction by measuring time-of-day-dependent changes in the electrical activity of GnRH1 neurons and in levels of expression of pituitary gonadotropin mRNA using a daily spawning teleost, medaka (Oryzias latipes). First, we performed on-cell patch-clamp recordings from GnRH1 neurons that directly project to the pituitary, using gnrh1-green fluorescent protein transgenic medaka. The spontaneous firing activity of GnRH1 neurons showed time-of-day-dependent changes: overall, the firing activity in the afternoon was higher than in the morning. Next, we examined the daily changes in the pituitary gonadotropin transcription level. The expression levels of lhb and fshb mRNA also showed changes related to time of day, peaking during the lights-off period. Finally, we analyzed effects of GnRH on the pituitary. We demonstrated that incubation of isolated pituitary with GnRH increases lhb mRNA transcription several hours after GnRH stimulation, unlike the well-known immediate LH releasing effect of GnRH. From these results, we propose a working hypothesis concerning the temporal regulation of the ovulatory cycle in the brain and pituitary of female medaka.
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http://dx.doi.org/10.1210/en.2011-2022DOI Listing
July 2012

Central distribution of kiss2 neurons and peri-pubertal changes in their expression in the brain of male and female red seabream Pagrus major.

Gen Comp Endocrinol 2012 Feb 7;175(3):432-42. Epub 2011 Dec 7.

Laboratory of Fish Biology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.

kisspeptins that are encoded by kiss1 gene are now considered the key regulator of reproduction from a number of studies in mammals. In most vertebrates, a paralogue of kiss1, called kiss2, is also present, and the functional significance of kisspeptins is not known precisely. In the present study, we have cloned kiss2 from a perciform teleost, the red seabream Pagrus major. The amino acid sequence deduced from the red seabream kiss2 contained a highly conserved 10-amino-acid residue, Kiss2(10) or kp-10. A kiss1-like transcript was also identified, but it appears to be non-functional due to the presence of a "premature" stop codon. Neurons that express kiss2 mRNA were distributed in the dorsal (NRLd) and ventral (NRLv) parts of nucleus recessi lateralis in the hypothalamus. In some fish a few kiss2-expressing neurons were detected in the preoptic area and nucleus ventralis tuberis. The number of kiss2-expressing neurons in the NRLd was larger during the first spawning season in both males and females compared with fish in the post-spawning periods. In males the number of kiss2 neurons in the NRLd of maturing fish was also larger than those in the post-spawning periods. In males the number of kiss2 neurons in the NRLv showed a similar pattern of changes to that of NRLd, while significant changes were not detected for females. The numbers of gonadotropin-releasing hormone 1 (GnRH1)-immunoreactive neurons in the preoptic area showed a similar pattern of change as those of kiss2 cells of the NRLd in both males and females (and also the NRLv in males). These results are in good agreement with the hypothesis that kiss2 neurons are involved in pubertal processes via regulatory influences on GnRH1 neurons in red seabream.
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http://dx.doi.org/10.1016/j.ygcen.2011.11.038DOI Listing
February 2012

Differential regulation of the luteinizing hormone genes in teleosts and tetrapods due to their distinct genomic environments--insights into gonadotropin beta subunit evolution.

Gen Comp Endocrinol 2011 Sep 30;173(2):253-8. Epub 2011 May 30.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.

The pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are essential for the control of vertebrate reproduction. Although the molecular structures of these two hormones are well conserved from teleosts to mammals, some studies report differences in their regulatory mechanisms of gene expression between teleosts and tetrapods. In the present study, we examined the molecular evolution of the gonadotropin gene loci in vertebrates and found that there is a syntenic conservation among the teleost fshb and tetrapod fshb and lhb loci. However, the teleost lhb locus has no syntenic homology to either tetrapod lhb or teleost fshb; this fact suggests that an extensive genome-wide rearrangement of the lhb locus, caused by an accelerated genome evolution speed after the third round of genome-wide duplication, occurred in the teleost lineage. We subsequently demonstrated by double labeling in situ hybridization using a teleost medaka that the fshb and lhb genes in teleosts are expressed in completely separate cellular populations in the pituitary, which is different in tetrapods. Furthermore, the expression analysis in ovariectomized and steroid-treated medaka revealed that, under breeding conditions, the expression of the medaka LHβ was down-regulated by ovariectomy and recovered by treatment with gonadal steroids; this result is also completely opposite in mammals, where the steroids have negative-feedback effects on LHβ expression. We suggest that these differences between teleosts and mammals in the cellular expression pattern and dynamic expressional changes of the lhb gene are the result of the drastic changes in the genomic environment of the lhb gene that occurred early in teleost evolution.
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http://dx.doi.org/10.1016/j.ygcen.2011.05.015DOI Listing
September 2011

Hypothalamic Kiss1 but not Kiss2 neurons are involved in estrogen feedback in medaka (Oryzias latipes).

Endocrinology 2010 Apr 5;151(4):1751-9. Epub 2010 Mar 5.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.

Kiss2, a paralogous gene for kiss1, has recently been identified in several vertebrates. However, their relative potencies for the regulation of reproductive functions appear to differ among species. Here we used medaka as a model animal to examine the kiss1 and kiss2 expression dynamics by in situ hybridization under different conditions: breeding or nonbreeding and ovariectomized or sham operated. Medaka kiss1-expressing neurons and kiss2-expressing neurons were mainly localized in two hypothalamic nuclei, nucleus ventralis tuberis (NVT) and nucleus recessus lateralis (NRL), respectively. NRL kiss2 expression did not change according to differences in breeding condition, whereas NVT kiss1 expression was strongly correlated with breeding condition. In addition, ovariectomy did not change kiss2 expression but significantly decreased the kiss1 expression. Moreover, double-label in situ hybridization revealed that NVT Kiss1 neurons coexpress estrogen receptor-alpha, whereas NRL Kiss2 neurons do not. From these results, we conclude that the NVT Kiss1 neurons are positively regulated by ovarian estrogen via their coexpressed estrogen receptor-alpha and are directly involved in the central regulation of reproduction in medaka. In contrast, we argue that the NRL Kiss2 neurons in medaka may serve nonreproductive functions. These functional differences between Kiss1 and Kiss2 neurons are discussed from a phylogenetic viewpoint.
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http://dx.doi.org/10.1210/en.2009-1174DOI Listing
April 2010

Regular pacemaker activity characterizes gonadotropin-releasing hormone 2 neurons recorded from green fluorescent protein-transgenic medaka.

Endocrinology 2010 Feb 23;151(2):695-701. Epub 2009 Dec 23.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.

GnRH2 is a molecule conserved from fish to humans, suggesting its important functions. However, recent studies have shown that GnRH2 neurons project widely in the brain but not to the pituitary, which suggests their functions other than stimulation of gonadotropin secretion. In contrast to the wealth of knowledge in GnRH1 and GnRH3 neuronal systems, the GnRH2 neuronal system remains to be studied, and there has been no single cell approach so far, partly because of the lack of GnRH2 system in rodents. Here, we generated GnRH2-green fluorescent protein (GFP) transgenic medaka for the first single cell electrophysiological recording from GnRH2 neurons in vertebrates. Whole-cell and on-cell patch clamp analyses revealed their regular pacemaker activities that are intrinsic to the GnRH2 neurons. Pacemaker activities of GnRH2 neurons were not peculiar to medaka because dwarf gourami GnRH2 neurons also showed similar pacemaker activities. By comparing with spontaneous action currents from GFP-expressing GnRH1 and GnRH3 neurons in the adult transgenic medaka, which were already in our hands, we have demonstrated that GnRH2 neurons show pacemaker activity similar to nonhypophysiotropic GnRH3 neurons but not to hypophysiotropic GnRH1 neurons. Thus, by taking advantage of medaka brain, which has all three GnRH neuronal systems with different axonal projection patterns and thus different functions, we have gained insights into the close relationship between the pattern of spontaneous electrical activity and the functions of the three. Moreover, the three types of GnRH-GFP transgenic medaka will provide useful models for studying multifunctional GnRH systems in future.
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http://dx.doi.org/10.1210/en.2009-0842DOI Listing
February 2010

Identification of KiSS-1 product kisspeptin and steroid-sensitive sexually dimorphic kisspeptin neurons in medaka (oryzias latipes).

Endocrinology 2008 May 17;149(5):2467-76. Epub 2008 Jan 17.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.

Recently, a novel physiologically active peptide, kisspeptin (metastin), has been reported to facilitate sexual maturation and ovulation by directly stimulating GnRH neurons in several mammalian species. Despite its importance in the neuroendocrine regulation of reproduction, kisspeptin neurons have only been studied in mammals, and there has been no report on the kisspeptin or kisspeptin neuronal systems in nonmammalian vertebrates. We used medaka for the initial identification of the KiSS-1 gene and the anatomical distribution of KiSS-1 mRNA expressing neurons (KiSS-1 neurons) in the brain of nonmammalian species. In situ hybridization for the medaka KiSS-1 gene cloned here proved that two kisspeptin neuronal populations are localized in the hypothalamic nuclei, the nucleus posterioris periventricularis and the nucleus ventral tuberis (NVT). Furthermore, NVT KiSS-1 neurons were sexually dimorphic in number (male neurons > female neurons) under the breeding conditions. We also found that the number of KiSS-1 neurons in the NVT but not that in the nucleus posterioris periventricularis was positively regulated by ovarian estrogens. The fact that there were clear differences in the number of NVT KiSS-1 neurons between the fish under the breeding and nonbreeding conditions strongly suggests that the steroid-sensitive changes in the KiSS-1 mRNA expression in the NVT occur physiologically, according to the changes in the reproductive state. From the present results, we conclude that the medaka KiSS-1 neuronal system is involved in the central regulation of reproductive functions, and, given many experimental advantages, the medaka brain may serve as a good model system to study its physiology.
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http://dx.doi.org/10.1210/en.2007-1503DOI Listing
May 2008