Identification of the FSH-RH as the other gonadotropin-releasing hormone.

In vertebrates, folliculogenesis and ovulation are regulated by two distinct pituitary gonadotropins: follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Currently, there is an intriguing consensus that a single hypothalamic neurohormone, gonadotropin-releasing hormone (GnRH), regulates the secretion of both FSH and LH, although the required timing and functions of FSH and LH are different. However, recent studies in many non-mammalian vertebrates indicated that GnRH is dispensable for FSH function. Here, by using medaka as a model teleost, we successfully identify cholecystokinin as the other gonadotropin regulator, FSH-releasing hormone (FSH-RH). Our histological and in vitro analyses demonstrate that hypothalamic cholecystokinin-expressing neurons directly affect FSH cells through the cholecystokinin receptor, Cck2rb, thereby increasing the expression and release of FSH. Remarkably, the knockout of this pathway minimizes FSH expression and results in a failure of folliculogenesis. Here, we propose the existence of the "dual GnRH model" in vertebrates that utilize both FSH-RH and LH-RH.

Long-lasting redundant gnrh1/3 expression in GnRH neurons enabled apparent switching of paralog usage during evolution.

Expressed subtype of paralogous genes in functionally homologous cells sometimes show differences across species, the reasons for which have not been explained. The present study examined hypophysiotropic gonadotropin-releasing hormone (GnRH) neurons in vertebrates to investigate this mechanism. These neurons express either gnrh1 or gnrh3 paralogs, depending on the species, and apparent switching of the expressed paralogs in them occurred at least four times in vertebrate evolution. First, we found redundant expression of gnrh1 and gnrh3 in a single neuron in piranha and hypothesized that it may represent an ancestral GnRH system. Moreover, the gnrh1/gnrh3 enhancer of piranha induced reporter RFP/GFP co-expression in a single hypophysiotropic GnRH neuron in both zebrafish and medaka, whose GnRH neurons only express either gnrh3 or gnrh1. Thus, we propose that redundant expression of gnrh1/3 of relatively recent common ancestors may be the key to apparent switching of the paralog usage among present-day species.

Involvement of the kisspeptin system in regulation of sexual behaviors in medaka.

In mammals, kisspeptin (Kiss1) neurons are generally considered as a sex steroid-dependent key regulator of hypothalamic-pituitary-gonadal (HPG) axis. In contrast, previous studies in non-mammalian species, especially in teleosts, propose that Kiss1 is not directly involved in the HPG axis regulation, which suggests some sex-steroid-dependent functions of kisspeptin(s) other than the HPG axis regulation in non-mammals. Here, we used knockout (KO) medaka of kisspeptin receptor-coding genes (gpr54-1 and gpr54-2) and examined possible roles of kisspeptin in the regulation of sexual behaviors. We found that the KO pairs of gpr54-1, but not gpr54-2, spawned fewer eggs and exhibited delayed spawning than wild type pairs. Detailed behavior analysis suggested that the KO females are responsible for the delayed spawning and that the KO males showed hyper-motivation for courtship. Taken together, the present finding suggests that one of the reproductive-state-dependent functions of the Kiss1 may be the control of successful sexual behaviors.

Male-specific vasotocin expression in the medaka tuberal hypothalamus: Androgen dependence and probable role in aggression.

Terrestrial vertebrates have a population of androgen-dependent vasotocin (VT)-expressing neurons in the extended amygdala that are more abundant in males and mediate male-typical social behaviors, including aggression. Teleosts lack these neurons but instead have novel male-specific VT-expressing neurons in the tuberal hypothalamus. Here we found in medaka that vt expression in these neurons is dependent on post-pubertal gonadal androgens and that androgens can act on these neurons to directly stimulate vt transcription via the androgen receptor subtype Ara. Furthermore, administration of exogenous VT induced aggression in females and alterations in the androgen milieu led to correlated changes in the levels of tuberal hypothalamic vt expression and aggression in both sexes. However, genetic ablation of vt failed to prevent androgen-induced aggression in females. Collectively, our results demonstrate a marked androgen dependence of male-specific vt expression in the teleost tuberal hypothalamus, although its relevance to male-typical aggression needs to be further validated.

Egg envelope formation of medaka Oryzias latipes requires ZP proteins originating from both the liver and ovary.

Teleost oocytes are surrounded by a structure called chorion or egg envelopes, which is composed of zona pellucida (ZP) proteins. As a result of the gene duplication in teleost, the expression site of the zp genes, coding the major component protein of egg envelopes, changed from the ovary to the maternal liver. In Euteleostei, there are three liver-expressed zp genes, named choriogenin (chg) h, chg hm, and chg l, and the composition of the egg envelope is mostly made up of these Chgs. In addition, ovary-expressed zp genes are also conserved in the medaka genomes, and their proteins have also been found to be minor components of the egg envelopes. However, the specific role of liver-expressed versus ovary-expressed zp genes was unclear. In the present study, we showed that ovary-synthesized ZP proteins first form the base layer of the egg envelope and then Chgs polymerize inwardly to thicken the egg envelope. To analyze the effects of dysfunction of the chg gene, we generated some chg knockout medaka. All knockout females failed to produce normally fertilized eggs by the natural spawning. The egg envelopes lacking Chgs were significantly thinner, but layers formed by ZP proteins synthesized in the ovary were found in the thin egg envelope of knockout as well as wildtype eggs. These results suggest that the ovary-expressed zp gene is well conserved in all teleosts, including those species in which liver-derived ZP proteins are the major component, because it is essential for the initiation of egg envelope formation.

In vitro and in vivo gene transfer in the cloudy catshark Scyliorhinus torazame.

Cartilaginous fishes have various unique physiological features such as a cartilaginous skeleton and a urea-based osmoregulation strategy for adaptation to their marine environment. Also, because they are a sister group of bony vertebrates, understanding their unique features is important from an evolutionary perspective. However, genetic engineering based on gene functions as well as cellular behavior has not been effectively utilized in cartilaginous fishes. This is partly because their reproductive strategy involves internal fertilization, which results in difficulty in microinjection into fertilized eggs at the early developmental stage. Here, to identify efficient gene transfer methods in cartilaginous fishes, we examined the effects of various methods both in vitro and in vivo using the cloudy catshark, a candidate model cartilaginous fish species. In all methods, green fluorescent protein (GFP) expression was used to evaluate exogenous gene transfer. First, we examined gene transfer into primary cultured cells from cloudy catshark embryos by lipofection, polyethylenimine (PEI) transfection, adenovirus infection, baculovirus infection, and electroporation. Among the methods tested, lipofection, electroporation, and baculovirus infection enabled the successful transfer of exogenous genes into primary cultured cells. We then attempted in vivo transfection into cloudy catshark embryos by electroporation and baculovirus infection. Although baculovirus-injected groups did not show GFP fluorescence, electroporation successfully introduced GFP into muscle cells. Furthermore, we succeeded in GFP transfer into adult tissues by electroporation. The in vitro and in vivo gene transfer methods that worked in this study may open ways for genetic manipulation including knockout experiments and cellular lineage analysis in cartilaginous fishes.

Retinal Cone Mosaic in sws1-Mutant Medaka (Oryzias latipes), A Teleost.

Purpose: Ablation of short single cones (SSCs) expressing short-wavelength-sensitive opsin (SWS1) is well analyzed in the field of regenerative retinal cells. In contrast with ablation studies, the phenomena caused by the complete deletion of SWS1 are less well-understood. To assess the effects of SWS1 deficiency on retinal structure, we established and analyzed sws1-mutant medaka. Methods: To visualize SWS1, a monoclonal anti-SWS1 antibody and transgenic reporter fish (Tg(sws1:mem-egfp)) were generated. We also developed a CRISPR/Cas-driven sws1-mutant line. Retinal structure of sws1 mutant was visualized using anti-SWS1, 1D4, and ZPR1 antibodies and coumarin derivatives and compared with wild type, Tg(sws1:mem-egfp), and another opsin (lws) mutant. Results: Our rat monoclonal antibody specifically recognized medaka SWS1. Sws1 mutant retained regularly arranged cone mosaic as lws mutant and its SSCs had neither SWS1 nor long wavelength sensitive opsin. Depletion of sws1 did not affect the expression of long wavelength sensitive opsin, and vice versa. ZPR1 antibody recognized arrestin spread throughout double cones and long single cones in wild-type, transgenic, and sws1-mutant lines. Conclusions: Comparative observation of sws1-mutant and wild-type retinas revealed that ZPR1 negativity is not a marker for SSCs with SWS1, but SSCs themselves. Loss of functional sws1 did not cause retinal degeneration, indicating that sws1 is not essential for cone mosaic development in medaka. Our two fish lines, one with visualized SWS1 and the other lacking functional SWS1, offer an opportunity to study neural network synapsing with SSCs and to clarify the role of SWS1 in vision.

Allogeneic testes transplanted into partially castrated adult medaka (Oryzias latipes) can produce donor-derived offspring by natural mating over a prolonged period.

Generally, successful testis transplantation has been considered to require immune suppression in the recipient to avoid rejection of the transplanted tissue. In the present study, we demonstrate in medaka that allogeneic adult testicular tissue will engraft in adult recipients immediately after partial castration without the use of immunosuppressive drugs. The allografted testes are retained in the recipient's body for at least 3 months and are able to produce viable sperm that yield offspring after natural mating. Some recipients showed a high frequency (over 60%) of offspring derived from spermatozoa produced by the transplanted testicular tissue. Histological analyses showed that allografted testicular tissues included both germ cells and somatic cells that had become established within an immunocompetent recipient testis. The relative simplicity of this testis transplantation approach will benefit investigations of the basic processes of reproductive immunology and will improve the technique of gonadal tissue transplantation.

Molecular encoding and synaptic decoding of context during salt chemotaxis in C. elegans.

Animals navigate toward favorable locations using various environmental cues. However, the mechanism of how the goal information is encoded and decoded to generate migration toward the appropriate direction has not been clarified. Here, we describe the mechanism of migration towards a learned concentration of NaCl in Caenorhabditis elegans. In the salt-sensing neuron ASER, the difference between the experienced and currently perceived NaCl concentration is encoded as phosphorylation at Ser65 of UNC-64/Syntaxin 1 A through the protein kinase C(PKC-1) signaling pathway. The phosphorylation affects basal glutamate transmission from ASER, inducing the reversal of the postsynaptic response of reorientation-initiating neurons (i.e., from inhibitory to excitatory), guiding the animals toward the experienced concentration. This process, the decoding of the context, is achieved through the differential sensitivity of postsynaptic excitatory and inhibitory receptors. Our results reveal the mechanism of migration based on the synaptic plasticity that conceptually differs from the classical ones.

Estrogen upregulates the firing activity of hypothalamic gonadotropin-releasing hormone (GnRH1) neurons in the evening in female medaka.

The reproductive function of vertebrates is regulated by the hypothalamic-pituitary-gonadal axis. In sexually mature females, gonadotropin-releasing hormone (GnRH) neurons in the preoptic area (POA) are assumed to be responsible for a cyclic large increase in GnRH release, the GnRH surge, triggering a luteinizing hormone (LH) surge, which leads to ovulation. Precise temporal regulation of the preovulatory GnRH/LH surge is important for successful reproduction because ovulation should occur after follicular development. The time course of the circulating level of estrogen is correlated with the ovulatory cycle throughout vertebrates. However, the neural mechanisms underlying estrogen-induced preovulatory GnRH surge after folliculogenesis still remain unclear, especially in non-mammals. Here, we used a versatile non-mammalian model medaka for the analysis of the involvement of estrogen in the regulation of POA-GnRH (GnRH1) neurons. Electrophysiological analysis using a whole brain-pituitary in vitro preparation, which maintains the hypophysiotropic function of GnRH1 neurons intact, revealed that 17ß-estradiol (E2 ) administration recovers the ovariectomy-induced lowered GnRH1 neuronal activity in the evening, indicating the importance of E2 for upregulation of GnRH1 neuronal activity. The importance of E2 was also confirmed by the fact that GnRH1 neuronal activity was low in short-day photoperiod-conditioned females (low E2 model). However, E2 failed to upregulate the firing activity of GnRH1 neurons in the morning, suggesting the involvement of additional time-of-day signal(s) for triggering GnRH/LH surges at an appropriate timing. We also provide morphological evidence for the localization of estrogen receptor subtypes in GnRH1 neurons. In conclusion, we propose a working hypothesis in which both estrogenic and time-of-day signals act in concert to timely upregulate the firing activity of GnRH1 neurons that trigger the GnRH surge at an appropriate timing in a female-specific manner. This neuroendocrinological mechanism is suggested to be responsible for the generation of ovulatory cycles in female teleosts in general.

Co-existing Neuropeptide FF and Gonadotropin-Releasing Hormone 3 Coordinately Modulate Male Sexual Behavior.

Animals properly perform sexual behaviors by using multiple sensory cues. However, neural mechanisms integrating multiple sensory cues and regulating motivation for sexual behaviors remain unclear. Here, we focused on peptidergic neurons, terminal nerve gonadotropin-releasing hormone (TN-GnRH) neurons, which receive inputs from various sensory systems and co-express neuropeptide FF (NPFF) in addition to GnRH. Our behavioral analyses using knockout medaka of GnRH (gnrh3) and/or NPFF (npff) demonstrated that some sexual behavioral repertoires were delayed, not disrupted, in gnrh3 and npff single knockout males, while the double knockout appeared to alleviate the significant defects that were observed in single knockouts. We also found anatomical evidence to show that both neuropeptides modulate the sexual behavior-controlling brain areas. Furthermore, we demonstrated that NPFF activates neurons in the preoptic area via indirect pathway, which is considered to induce the increase in motivation for male sexual behaviors. Considering these results, we propose a novel mechanism by which co-existing peptides of the TN-GnRH neurons, NPFF, and GnRH3 coordinately modulate certain neuronal circuit for the control of behavioral motivation. Our results may go a long way toward understanding the functional significance of peptidergic neuromodulation in response to sensory information from the external environments.

Transmembrane channel-like 4 is involved in pH and temperature-dependent modulation of salty taste.

Human susceptibility to NaCl varies depending on temperature and pH, the molecular mechanisms of which remain unclear. The voltage-dependent chloride channel, transmembrane channel-like 4 (TMC4), is activated at approximately 40 °C and is suppressed at pH 5.5. As these are similar in character to human sensory evaluations, human TMC4 may be involved in human salt taste reception.

TMC4 is a novel chloride channel involved in high-concentration salt taste sensation.

"Salty taste" sensation is evoked when sodium and chloride ions are present together in the oral cavity. The presence of an epithelial cation channel that receives Na+ has previously been reported. However, no molecular entity involving Cl- receptors has been elucidated. We report the strong expression of transmembrane channel-like 4 (TMC4) in the circumvallate and foliate papillae projected to the glossopharyngeal nerve, mediating a high-concentration of NaCl. Electrophysiological analysis using HEK293T cells revealed that TMC4 was a voltage-dependent Cl- channel and the consequent currents were completely inhibited by NPPB, an anion channel blocker. TMC4 allowed permeation of organic anions including gluconate, but their current amplitudes at positive potentials were less than that of Cl-. Tmc4-deficient mice showed significantly weaker glossopharyngeal nerve response to high-concentration of NaCl than the wild-type littermates. These results indicated that TMC4 is a novel chloride channel that responds to high-concentration of NaCl.

Establishment of open-source semi-automated behavioral analysis system and quantification of the difference of sexual motivation between laboratory and wild strains.

Behavioral analysis plays an important role in wide variety of biological studies, but behavioral recordings often tend to be laborious and are associated with inevitable human-errors. It also takes much time to perform manual behavioral analyses while replaying the videos. On the other hand, presently available automated recording/analysis systems are often specialized for certain types of behavior of specific animals. Here, we established an open-source behavioral recording system using Raspberry Pi, which automatically performs video-recording and systematic file-sorting, and the behavioral recording can be performed more efficiently, without unintentional human operational errors. We also developed an Excel macro that enables us to easily perform behavioral annotation with simple manipulation. Thus, we succeeded in developing an analysis suite that mitigates human tasks and thus reduces human errors. By using this suite, we analyzed the sexual behavior of a laboratory and a wild medaka strain and found a difference in sexual motivation presumably resulting from domestication.

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

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.

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

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 Caenorhabditis elegans. Genetic screening identified two altered-function mutations of clh-1 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.

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

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.

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

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 17ß-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.

Gene knockout analysis reveals essentiality of estrogen receptor ß1 (Esr2a) for female reproduction in medaka.

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.