Satoshi Ogawa

Cloning and Expression of kiss2 in the Zebrafish and Medaka

Newly discovered kisspeptin (metastin), encoded by the Kiss1/KISS1 gene, is considered as a major gatekeeper of puberty through the regulation of GnRH. In the present study, we cloned a novel kisspeptin gene (kiss2) in the zebrafish Danio rerio and the medaka Oryzias latipes, which encodes a sequence of 125 and 115 amino acids, respectively, and its core sequence (FNLNPFGLRF, F-F form) is different from the previously characterized kiss1 (YNLNSFGLRY, Y-Y form). Our in silico data mining shows kiss1 and kiss2 are highly conserved across nonmammalian vertebrate species, and we have identified two putative kisspeptins in the platypus and three forms in Xenopus. In the brain of zebrafish and medaka, in situ hybridization and laser capture microdissection coupled with real-time PCR showed kiss1 mRNA expression in the ventromedial habenula and the periventricular hypothalamic nucleus. The kiss2 mRNA expression was observed in the posterior tuberal nucleus and the periventricular hypothalamic nucleus. Quantitative real-time PCR analysis during zebrafish development showed a significant increase in zebrafish kiss1, kiss2 (P < 0.002), gnrh2, and gnrh3 (P < 0.001) mRNA levels at the start of the pubertal phase and remained high in adulthood. In sexually mature female zebrafish, Kiss2 but not Kiss1 administration significantly increased FSH-beta (2.7-fold, P < 0.05) and LH-beta (8-fold, P < 0.01) mRNA levels in the pituitary. These results suggest that the habenular Kiss1 and the hypothalamic Kiss2 are potential regulators of reproduction including puberty and that Kiss2 is the predominant regulator of gonadotropin synthesis in fish.

Kisspeptin cells in the ewe brain respond to leptin and communicate with neuropeptide Y and proopiomelanocortin cells.

Kisspeptin stimulates reproduction, and kisspeptin cells in the arcuate nucleus (ARC) express Ob-Rb in the mouse. Herein we report studies in ewes to determine whether kisspeptin cells express Ob-Rb and respond to leptin and whether reciprocal connections exist between kisspeptin cells and proopiomelanocortin (POMC) or neuropeptide Y (NPY) cells to modulate reproduction and metabolic function. Kiss1 mRNA was measured by in situ hybridization in ovariectomized ewes that were normal body weight, lean, or lean with leptin treatment by intracerebroventricular (icv) infusion (4 microg/h, 3 d). Kiss1 expression in the ARC and the preoptic area was lower in hypogonadotropic lean animals than animals of normal weight, and icv infusion of leptin partially restored Kiss1 expression in lean animals. Single-cell laser capture microdissection coupled with real-time PCR showed that Kiss1 cells in the preoptic area and ARC express Ob-Rb. Double-label fluorescent immunohistochemistry showed that reciprocal connections exist between kisspeptin cells and NPY and POMC cells. Accordingly, we treated ovariectomized ewes with kisspeptin (5 microg/h, icv) or vehicle for 20 h and examined POMC and NPY gene expression by in situ hybridization. Kisspeptin treatment reduced POMC and increased NPY gene expression. Thus, kisspeptin neurons respond to leptin and expression of Kiss1 mRNA is affected by leptin status. Kisspeptin cells communicate with NPY and POMC cells, altering expression of the relevant genes in the target cells; reciprocal connections also exist. This network between the three cell types could coordinate brain control of reproduction and metabolic homeostatic systems.

Characteristics of patients with right bundle branch block and ST-segment elevation in right precordial leads

To elucidate the clinical characteristics of patients with right bundle branch block and ST elevation in the right precordial leads, a prospective follow-up study was made in 63 registered patients, including 17 with a history of ventricular fibrillation (VF) and 14 with a history of syncope. The prevalence of coved type ST elevation was significantly higher in patients who had had cardiac events, and during the initial 15-month follow-up, 2 patients in the VF group died suddenly.

RFamide peptides as mediators in environmental control of GnRH neurons

Hypothalamic gonadotropin-releasing hormone (GnRH) is a key hormone for reproductive functions in vertebrates and non-vertebrates. Although GnRH neuronal system is regulated by several factors such as steroids, neurotransmitters and neuropeptides, it is not fully understood how environmental signals control the GnRH neuronal system. RFamide peptides, members of peptides possessing an Arg-Phe-NH(2) motif at their C-terminus, have recently been characterized as major regulators of GnRH neurons. In particular, two key RFamide peptides, kisspeptin and gonadotropin-inhibitory hormone (GnIH), are emerging as important regulators of the reproductive axis. Kisspeptin acts as the accelerator, directly driving GnRH neurons, whereas GnIH acts as the restraint. In addition, other RFamide peptides such as prolactin-releasing peptide (PrRP), PQRFa peptide, 26RFa/QRFP are also known to control reproduction. These RFamide peptides are regulated by environmental factors such as photoperiods, steroid hormones, metabolic signals, and stress. How environmental signals are integrated by RFamide peptides to regulate reproduction through the GnRH neurons?

Localization of the three GnRH types and GnRH receptors in the brain of a cichlid fish: Insights into their neuroendocrine and neuromodulator functions.

The cognate receptor for any of the known gonadotropin‐releasing hormones (GnRHs) has not been directly demonstrated. In order to establish this and shed light on the functions of GnRH types, we analyzed the neuroanatomical location and time of initial expression of three distinct GnRH receptors (GnRH‐Rs) and the three endogenous GnRHs in the brain of developing and sexually mature tilapia Oreochromis niloticus using immunocytochemistry. In all age groups, including males and females, GnRH‐RIA was seen specifically in gonadotropes (Parhar et al. [2002] J Neuroendocrinol 14:657–665) but was undetectable in the brain. On day 8 after fertilization, GnRH‐RIB was first seen in the periventricular hypothalamus (lateral recess nucleus, posterior recess nucleus, posterior tuberal nucleus) and GnRH‐RIII in the olfactory epithelium, olfactory bulb, telencephalon, preoptic region, mediobasal hypothalamus, thalamus, mesencephalon, and in the hindbrain. Double‐label immunocytochemistry showed GnRH1 (Ser8 GnRH)‐immunoreactive neuronal processes projecting mainly to the proximal pars distalis of the pituitary, while GnRH2 (His5, Trp7, Tyr8 GnRH) and GnRH3 (Trp7, Leu8 GnRH) fibers were observed in close association with cells containing GnRH‐RIB and GnRH‐RIII in the brain. These results suggest that GnRH‐RIA might be hypophysiotropic in nature, whereas GnRH‐RIB and GnRH‐RIII could have additional neuromodulatory functions. Further, evidence of close proximity of GnRH‐R‐containing cells and neuronal processes of multiple GnRH types suggests complex cross‐talk between several GnRH ligands and GnRH‐Rs. J. Comp. Neurol. 487:28–41, 2005.

Immunoneutralization of gonadotropin-releasing hormone type-III suppresses male reproductive behavior of cichlids

To investigate the roles of gonadotropin-releasing hormone (GnRH) types in reproductive behaviors, antisera against GnRH1, GnRH2 and GnRH3 were stereotaxically administered into the intracerebroventricular region to neutralize the three native GnRH types in the brain of male tilapia Oreochromis niloticus. Reproductive behaviors (nest-building and aggressive behaviors), and morphological changes of the three GnRH systems were investigated by immunocytochemistry. GnRH1, GnRH2 and GnRH3 immunoreactive fibers were significantly decreased following injections of GnRH antisera indicating successful neutralization of their respective endogenous GnRH peptides. GnRH1- and GnRH2-immunoneutralization did not inhibit reproductive behaviors but GnRH3-immunoneutralization significantly decreased nest-building ability (Saline: 26.5 +/- 3.7%/day versus GnRH3: 6.1 +/- 2.9%/day, P < 0.001), nest size (Saline: 0.67 +/- 0.09 points versus GnRH3: 0.10 +/- 0.05 points, P < 0.0002) and aggressive behavior (Saline: 2.34 +/- 0.19 points versus GnRH3 1.06 +/- 0.12 points, P < 0.0001). These observations provide evidence that GnRH3 is a potent neuromodulator of reproductive behaviors in male tilapia.

The kiss/kissr Systems Are Dispensable for Zebrafish Reproduction: Evidence From Gene Knockout Studies

The kiss1/gpr54 signaling system is considered to be a critical regulator of reproduction in most vertebrates. However, this presumption has not been tested vigorously in nonmammalian vertebrates. Distinct from mammals, multiple kiss1/gpr54 paralogous genes (kiss/kissr) have been identified in nonmammalian vertebrates, raising the possibility of functional redundancy among these genes. In this study, we have systematically generated the zebrafish kiss1(-/-), kiss2(-/-), and kiss1(-/-);kiss2(-/-) mutant lines as well as the kissr1(-/-), kissr2(-/-), and kissr1(-/-);kissr2(-/-) mutant lines using transcription activator-like effector nucleases. We have demonstrated that spermatogenesis and folliculogenesis as well as reproductive capability are not impaired in all of these 6 mutant lines. Collectively, our results indicate that kiss/kissr signaling is not absolutely required for zebrafish reproduction, suggesting that the kiss/kissr systems play nonessential roles for reproduction in certain nonmammalian vertebrates. These findings also demonstrated that fish and mammals have evolved different strategies for neuroendocrine control of reproduction.

FSH and LH-β subunits in the preoptic nucleus: ontogenic expression in teleost

In the present study we cloned, sequenced, and confirmed the presence of mRNAs of gonadotropins (FSH-β, LH-β subunits) from the brain and pituitary of tilapia, Oreochromis niloticus. Further, we examined the spatio-temporal expression pattern of FSH-β and LH-β in the brain and pituitary of two species of teleost (tilapia, O. niloticus; sockeye salmon, Oncorhynchus nerka), using in situ hybridization and immunological methods. The expression of FSH and LH immunoreactivity appeared simultaneously in the brain and pituitary (tilapia, 14 days; sockeye, 51 days after fertilization). In the pituitary, FSH mRNA and peptide expressing cells were distinct from LH expressing cells located in the ventral proximal pars distalis. In the brain, FSH and LH immunoreactivity was co-localized in cells of the preoptic nucleus parvocellularis, magnocellularis, and gigantocellularis. Fibers immunoreactive to FSH and LH antisera were seen along the forebrain-hypothalamus and in the neurohypophysis of the pituitary. Double-label immunofluorescence revealed FSH and LH immunoreactivity co-localized in arginine vasotocin synthesizing preoptic neurons. Our results show that FSH and LH-producing cells have developmental origins in the brain as well as in the pituitary. In addition, we propose that the brain-derived gonadotropins may function as hypophysiotropic hormones that regulate pituitary cells and along with arginine vasotocin could act as neuromodulators of reproductive behaviors.

Habenular Kiss1 Neurons Modulate the Serotonergic System in the Brain of Zebrafish

The Kiss1/KISS1 gene has recently been implicated as a potent hypothalamic regulator of reproductive functions, in particular, the onset of puberty in mammals. In zebrafish (Danio rerio), there are two kiss1 homologues (kiss1 and kiss2) expressed in the brain: Kiss2-expressing neurons in the hypothalamic nuclei are considered potent regulators of reproduction, whereas the role of Kiss1-expressing neurons in the habenula remains unknown. We first analyzed the expression of kiss1 mRNA in a transgenic zebrafish, in which the habenula-interpeduncular nucleus (IPN) pathway is labelled with green fluorescent protein, and our application of a biocytin neural tracer into the habenula showed the presence of neuronal projections of Kiss1 neurons to the ventral IPN. Therefore, we speculated that kiss1 neurons might regulate the serotonergic system in the raphe. However, laser microdissection followed by real-time PCR revealed the expression of Kiss1 receptor (kissr1) mRNA in the habenula and the ventral IPN but not in the dorsal IPN or the serotonergic neurons in the raphe nuclei. Dual-fluorescent in situ hybridization revealed the coexpression of kiss1 and kissr1 mRNA in the habenula. Administration of Kiss1 significantly decreased the level of kiss1 mRNA (0.3- to 0.5-fold, P < 0.001), but the level of c-fos mRNA was increased (≈ 3-fold, P < 0.05) in the ventral habenula, suggesting that there is autocrine regulation of the kiss1 gene. Kiss1 administration significantly increased the c-fos mRNA levels in the raphe nuclei (2.5-fold, P < 0.001) and genes involved in the regulation of serotonin levels (pet1 and slc6a4a; 3.3- and 2.2-fold, P < 0.01). These findings suggest that the autocrine-regulated habenular Kiss1 neurons indirectly regulate the serotonergic system in the raphe nuclei through the IPN in the zebrafish.

Habenular kisspeptin modulates fear in the zebrafish.

Significance Kisspeptin, a hypothalamic neuropeptide, plays a key role in vertebrate reproduction. In the zebrafish, kisspeptin and its receptor are predominantly expressed in the habenula, a highly evolutionarily conserved brain region mediating behavioral responses to stressful conditions. However, the physiological significance of kisspeptin expressed in the habenula remains unknown. Here we demonstrate a unique role for the kisspeptin system in inhibiting fear response in the zebrafish that extends beyond the control of reproduction. Kisspeptin, a neuropeptide encoded by the KISS1/Kiss1, and its cognate G protein-coupled receptor, GPR54 (kisspeptin receptor, Kiss-R), are critical for the control of reproduction in vertebrates. We have previously identified two kisspeptin genes (kiss1 and kiss2) in the zebrafish, of which kiss1 neurons are located in the habenula, which project to the median raphe. kiss2 neurons are located in the hypothalamic nucleus and send axonal projections to gonadotropin-releasing hormone neurons and regulate reproductive functions. However, the physiological significance of the Kiss1 expressed in the habenula remains unknown. Here we demonstrate the role of habenular Kiss1 in alarm substance (AS)-induced fear response in the zebrafish. We found that AS-evoked fear experience significantly reduces kiss1 and serotonin-related genes (plasmacytoma expressed transcript 1 and solute carrier family 6, member 4) in the zebrafish. Furthermore, Kiss1 administration suppressed the AS-evoked fear response. To further evaluate the role of Kiss1 in fear response, zebrafish Kiss1 peptide was conjugated to saporin (SAP) to selectively inactivate Kiss-R1-expressing neurons. The Kiss1-SAP injection significantly reduced Kiss1 immunoreactivity and c-fos mRNA in the habenula and the raphe compared with control. Furthermore, 3 d after Kiss1-SAP injection, the fish had a significantly reduced AS-evoked fear response. These findings provide an insight into the role of the habenular kisspeptin system in inhibiting fear.