Ishwar S. Parhar

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.

Organization of two independent kisspeptin systems derived from evolutionary-ancient kiss genes in the brain of zebrafish.

Kisspeptins are new actors in the neuroendocrine regulation of reproduction. In vertebrates, the number of kiss genes varies from none to three. Zebrafish have two kiss genes, kiss1 and kiss2, and two kiss receptors (GPR54), kiss1r and kiss2r. To provide detailed information on the organization of the kiss systems in zebrafish, antibodies were raised against the C terminus of zebrafish preproKiss1 and preproKiss2. Immunohistochemistry fully confirmed in situ hybridization data, showing that kiss1-expressing neurons are only located in the habenular nucleus, while kiss2-expressing neurons are found in the dorsal and ventral hypothalamus. Kiss1-expressing cells project only to the interpeduncular and raphe nuclei and strongly expressed the kiss1r receptor. In contrast, kiss2-expressing cells are mostly present in the dorsal and ventral hypothalamus and project widely into the subpallium, the preoptic area, the thalamus, the ventral and caudal hypothalamus, and the mesencephalon. All these regions strongly expressed the kiss2r messengers. Kiss2 fibers profusely innervate the ventral forebrain and notably made close apposition with GnRH3 neurons. Estrogen treatment of juvenile fish with estradiol causes increase in kiss2 and kiss2r expression. In the pituitary gland, no proKiss2- positive fibers were detected, while positive cells were observed in the pars intermedia. In addition to proposing a successful strategy to develop antibodies to kisspeptins, these data indicate that the kiss2 systems of zebrafish are implicated in reproductive events, while the kiss1 gene would play other functions that remain to be established.

Generation of polyclonal antiserum against the growth hormone secretagogue receptor (GHS-R): evidence that the GHS-R exists in the hypothalamus, pituitary and stomach of rats.

Growth hormone (GH) secretagogues (GHSs), which stimulate GH secretion, are synthetic compounds that act through the GHS receptor (GHS-R) which has been recently cloned. We raised an antiserum in a rabbit against a synthetic peptide corresponding to amino acid residues 248-260 of the third intracellular loop of the rat GHS-R. A competitive immunoassay showed that the antiserum had a specific affinity for the target peptide. To confirm the specificity of the antiserum, the GHS-R cDNA was stably expressed in COS-7 cells. In Western blot analysis, the band was detected at 44 kDa in the extracts from COS-7 cells expressing GHS-R (COS-7/tf3-2) but not in those from wild-type COS-7 cells. Furthermore, while COS-7/tf3-2 cells were strongly immunostained for GHS-R, no GHS-R-like immunoreactivity was observed in wild-type COS-7 cells. Immunoreactive bands were also observed at approximately 46 kDa in the extracts from rat hypothalamus, pituitary and stomach by Western blot analysis. These studies are the first to show the existence of GHS-R protein in the stomach. The antiserum for the GHS-R is sensitive and specific, and it would be useful for clarifying the roles of GHS/ghrelin.

Neurons synthesizing gonadotropin-releasing hormone mRNA subtypes have multiple developmental origins in the medaka

The origins of the different populations of gonadotropin‐releasing hormone (GnRH)‐containing neurons in the brains of two genotypes (HO4C; HNI‐II) of medaka Oryzias latipes were analyzed at different stages of development (day 1 after fertilization through adulthood), by using oligonucleotide probes specific to salmon‐, seabream‐, and chicken II‐GnRH mRNA and antisera against specific GnRH peptides. Between the two genotypes, there was no difference in the site and time of GnRH expression or the final pattern of GnRH neuronal organization. In the adult fish of both sexes, salmon GnRH mRNA and peptide‐containing neurons were seen in the terminal nerve ganglia (nucleus olfactoretinalis; NOR) and chicken II‐GnRH mRNA and peptide‐containing neurons in the midbrain tegmentum. GnRH cells at the base of the olfactory placode (1–2 cells) and in the midbrain tegmentum were first seen in 1‐day‐old fish of both genotypes. On day 15, lightly immunoreactive GnRH cells were seen in the NOR of only HNI genotype. By day 30, GnRH expression in the NOR and in the midbrain was prominent. GnRH cells along the basal olfactory bulb and basal telencephalon were occasionally seen in animals 30 days or older. This developmental study shows differential distribution of salmon and chicken II‐GnRH mRNA subtypes and emphasizes their separate embryonic origins from the olfactory apparatus (salmon‐GnRH) and the ependymal cells of the third ventricle (chicken II‐GnRH). The absence of preoptic GnRH hybridization signals, immunoreactivity and the lack of GnRH fibers in the pituitary suggests that the preoptic GnRH neurons are distinct from the olfactory derived‐terminal nerve GnRH neurons, and that the GnRH neurites reported in the pituitary of teleost must be of preoptic origin. J. Comp. Neurol. 401:217–226, 1998.

Preoptic gonadotropin-releasing hormone(GnRH) neurons innervate the pituitary in teleosts

In most teleosts, there are three groups of gonadotropin-releasing hormone (GnRH) neurons. In this study we addressed the question of GnRH neuronal innervation of the pituitary in the dwarf gourami and the tilapia using immunocytochemistry combined with biocytin tract tracing. Biocytin was applied to the pituitary attached to the brain in vitro. Similar results were obtained in both species. GnRH neurons retrogradely labeled with biocytin were observed only in the preoptic area. These results indicate that preoptic GnRH neurons innervate the pituitary. Negative labeling of biocytin in the terminal-nerve and midbrain GnRH neurons suggests that these two GnRH neuronal populations do not project to the pituitary. Biocytin-positive but GnRH-negative neurons were also observed in the preoptic area and the ventromedial parts of the hypothalamus, suggesting neuropeptidergic and aminergic innervation of the pituitary besides GnRH.

SEX DIFFERENCES IN THE BRAIN OF GOLDFISH: GONADOTROPIN-RELEASING HORMONE AND VASOTOCINERGIC NEURONS

The differences between male and female behaviors are reflected in sexual dimorphism of brain structures and are found throughout the nervous system in a variety of vertebrates. The present study examined neurons immunolabeled for gonadotropin-releasing hormone and arginine vasotocin in the brain of the goldfish Carassius auratus to determine if these neurons are sexually dimorphic. There was no sex difference or influence of sex steroids on the neuronal volume and optical density of staining of arginine vasotocin neurons. Similarly, gonadotropin-releasing hormone neurons of the terminal nerve and midbrain tegmentum did not differ between sexually mature males, females and maturing females replaced with sex steroids with respect to distribution, numbers, optical density of staining, or gross morphology. In maturing females, testosterone specifically recruited additional preoptic gonadotropin-releasing hormone neurons to equal those in sexually mature individuals. Since estrogen had no effect, the influence of testosterone on gonadotropin-releasing hormone neuronal numbers appears to be independent of aromatization. Specifically, the preoptic gonadotropin-releasing hormone neuronal size was significantly larger in sexually mature males than females. 11-Ketotestosterone-replacement to ovariectomized maturing females induced male-typical secondary characters and male-type courtship behavior but did not masculinize the preoptic gonadotropin-releasing hormone neuronal size. Our results show that the sexually dimorphic preoptic gonadotropin-releasing hormone neuronal size is determined by factors (genetic) other than gonadal steroids. Further, we propose the hypothesis that phenotypic and behavioral sex differences need not be accompanied by structural differences in gonadotropin-releasing hormone and arginine vasotocin in the brain.

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?

Spatio‐Temporal Expression of Gonadotropin‐Releasing Hormone Receptor Subtypes in Gonadotropes, Somatotropes and Lactotropes in the Cichlid Fish

The description of two or more forms of gonadotropin‐releasing hormone (GnRH) in most vertebrates suggests multiple roles for this family of peptide hormones. In order to verify these functions, we analysed the anatomical location, time of initial expression and ontogenic changes in three distinct GnRH receptors (GnRH‐Rs) in developing and sexually mature tilapia, using antisera raised against the extracellular loop three of the receptor, which is a determinant in ligand‐selectivity and receptor coupling to signalling pathways. In all age groups, including males and females, using in situ hybridization and double‐label immunological methods, GnRH‐R type IA was colocalized in cells containing luteinizing hormone (LH) β‐subunit in the pituitary. GnRH‐R type IB was visualized in prolactin cells and LH cells. The type III GnRH‐R was expressed in growth hormone cells. On day 8 after fertilization, GnRH‐R type III was first seen in growth hormone cells and, subsequently, on day 15, GnRH‐Rs type IA and type IB were first seen in LH and prolactin cells, respectively. On day 25, the receptor occupied area per pituitary and the staining intensity of GnRH‐R type IA increased significantly, consistent with the hypothesis that differentiation of GnRH neurones and their inputs to the pituitary coincide precisely with gonadal sex differentiation and steroidogenesis in tilapia. The differential distribution of GnRH‐Rs in the pituitary provides the first clear evidence that the three native GnRH variants in tilapia have cognate receptors, each capable of regulating different pituitary endocrine cells.

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.