Yoshitaka Oka

Identification of KiSS-1 Product Kisspeptin and Steroid-Sensitive Sexually Dimorphic Kisspeptin Neurons in Medaka (Oryzias latipes)

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.

Lesions of gonadotropin-releasing hormone-immunoreactive terminal nerve cells: effects on the reproductive behavior of male dwarf gouramis.

Functions of the terminal nerve (TN) are largely unknown. To examine whether gonadotropin-releasing hormone (GnRH)-immunoreactive TN cells (TN-GnRH cells) are involved in the control of reproductive behavior, effects of lesions of TN-GnRH cells were studied in male dwarf gouramis, Colisa lalia. After bilateral electrolytic lesion, a characteristic impairment was observed in one of the repertoires of male reproductive behavior, nest-building. The occurrence of mating trials in which males showed no nest-building was increased. However, the incidence of nest-building behavior during postoperative trials was not affected by the lesion. No impairment was observed in other reproductive repertoires. These results suggest that (1) TN-GnRH cells are involved in the control of the threshold for nest-building behavior initiation and (2) TN-GnRH cells are not a prerequisite for other aspects of reproductive behavior in the male gouramis.

Neurobiological mechanisms underlying GnRH pulse generation by the hypothalamus.

Gonadotropin-releasing hormone (GnRH) secretion has two modes of release in mammalian species; the surge mode and the pulse mode. The surge mode, which is required for the induction of the preovulatory gonadotropin discharge in most species, is induced by the positive feedback of estrogen secreted by the mature ovarian follicle. The pulse mode of GnRH secretion stimulates tonic luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion which drives folliculogenesis, spermatogenesis and steroidogenesis and is negatively fine-tuned by estrogen or androgen. The GnRH pulse-generating mechanism is sensitive to environmental cues, such as photoperiod, nutrition and stress surge-generating mechanism is relatively emancipated from these environmental cues. The present article first provides a brief historical background to the work that led to the concept of the GnRH pulse generator: a hypothalamic network that is central to our understanding of the regulation of reproduction. We then discuss possible neurobiological mechanisms underlying GnRH pulse generation, and conclude by proposing that kisspeptin neurons in the arcuate nucleus are key players in this regard.

Possible role of oestrogen in pubertal increase of Kiss1/kisspeptin expression in discrete hypothalamic areas of female rats.

Kisspeptin, a peptide encoded by the Kiss1 gene, has been considered as a potential candidate for a factor triggering the onset of puberty, and its expression in the hypothalamus was found to increase during peripubertal period in rodent models. The present study aimed to clarify the oestrogenic regulation of peripubertal changes in Kiss1 mRNA expression in the anteroventral periventricular nucleus (AVPV) and hypothalamic arcuate nucleus (ARC), and to determine which population of kisspeptin neurones shows a change in kisspeptin expression parallel to that in luteinising hormone (LH) pulses at the peripubertal period. Quantitative reverse transcriptase‐polymerase chain reaction and immunohistochemistry revealed an apparent increase in the ARC Kiss1 mRNA expression and kisspeptin immunoreactivity around the time of vaginal opening in intact female rats. The AVPV Kiss1 mRNA levels also increased at day 26, but decreased at day 31, and then increased at day 36/41. In ovariectomised (OVX) rats, ARC Kiss1 mRNA expression did not show peripubertal changes and was kept at a high level throughout peripubertal periods. Apparent LH pulses were found in these prepubertal OVX rats. Oestradiol replacement suppressed ARC Kiss1 mRNA expression in OVX prepubertal rats, but not in adults. Similarly, LH pulses were suppressed by oestradiol in the prepubertal period (days 21 and 26), but regular pulses were found in adulthood. The present study suggests that a pubertal increase of Kiss1/kisspeptin expression both in the ARC and AVPV is involved in the onset of puberty. These results also suggest that both LH pulses and ARC Kiss1 expression are more negatively regulated by oestrogen in prepubertal female rats compared to adult rats.

Telencephalic and preoptic areas integrate sexual behavior in hime salmon (landlocked red salmon, Oncorhynchus nerka): results of electrical brain stimulation experiments

Various patterns of sexual behavior were evoked in freely swimming hime salmon by electrical stimulation of specific loci in the telencephalon and the preoptic area (POA) using chronically implanted electrodes. Furthermore, co-ordinated sexual behavior corresponding to stages of the natural spawning sequence was elicited from some of these brain regions. These results suggest that (1) sexual behavior is integrated in specific parts of the telencephalon and POA, and (2) within these regions there is a hierarchy of neural systems which mediate progressively more complete components of normal sexual behavior.

Descending pathways to the spinal cord in the himé salmon (landlocked red salmon, Oncorhynchus nerka)

Distribution and morphology of the cells of origin of the descending spinal pathways and their axonal courses were studied in the himé salmon, using retrograde labelling with cobaltic lysine and horseradish peroxidase (HRP). Following application of the tracers to the cut end of the spinal cord or injection of the tracers at the 10th to 15th spinal segment, neurons mainly labelled via the axons of passage were distributed in the mesencephalon and the rhombencephalon. Mesencephalic cell groups consisted of the nucleus pretectalis, the nucleus fasciculi longitudinalis medialis, and the nucleus ruber. The former two cell groups sent their axons to the fasciculus longitudinalis medialis. The axons of the nucleus ruber formed a separate loose bundle, the “tractus rubrospinalis.” The rhombencephalic cell groups consisted of the rhombencephalic reticular formation, the Mauthner cells (one cell for each side), and the octavolateral area. The rhombencephalic reticular formation could be further subdivided into the nucleus reticularis superior, nucleus reticularis medius, and nucleus reticularis inferior. The axons of these cell groups joined the fasciculus longitudinalis medialis and the “tractus bulbospinalis.” The Mauthner cell had two main gigantic dendrites, and its giant axons formed a conspicuous fiber of Mauthner throughout the rhombencephalon down to the spinal cord. The octavolateral area could be subdivided into the nucleus vestibularis magnocellularis, nucleus tangentialis, nucleus vestibularis descendens and nucleus intermedius. The axons of the nucleus vestibularis magnocellularis and nucleus intermedius entered the fasciculus longitudinalis medialis and/or the tractus bulbospinalis. Those of the nucleus vestibularis descendens and nucleus tangentialis formed the “tractus vestibulospinalis”. The descending spinal pathways of the himé salmon were compared with those of other fishes and other vertebrates. The significance of these descending spinal pathways in the control of locomotion and sexual behavior is also discussed.

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.

Hypothalamic Kiss1 but Not Kiss2 Neurons Are Involved in Estrogen Feedback in Medaka (Oryzias latipes)

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.

Gonadotropin-releasing hormone (GnRH) cells of the terminal nerve as a model neuromodulator system

Modulation of ionic channel properties by neurotransmitters and hormones is called neuromodulation and may be the basis for many long-lasting changes in animal behavior, e.g. changes in the arousal or motivational states. Gonadotropin-releasing hormone (GnRH), originally identified as a hypophysiotropic hormone, is now believed to act also as a neuromodulator. From studies of electrical activities and morphology of terminal nerve cells (major source of GnRH) of a fish brain, a general hypothesis regarding modulator neurons is proposed; modulator neurons have endogenous oscillatory activities which vary according to the animals hormonal or environmental conditions. These modulator neurons, in turn, regulate neuronal excitabilities in a wide variety of brain regions simultaneously via multiple axonal branches.

Involvement of the telencephalic hemispheres and the preoptic area in sexual behavior of the male goldfish, Carassius auratus: a brain-lesion study.

To locate the brain areas involved in sexual behavior, the effects on male sexual behavior of localized electrolytic lesions in various parts of the telencephalic hemispheres and the preoptic area and of gross ablations of parts of the telencephalic hemispheres were examined in goldfish. All three patterns of male sexual behavior examined (following, butting , and spawning) were severely impaired after bilateral lesions confined to the area ventralis telencephali pars supracommissuralis and/or posterior parts of the area ventralis telencephali pars ventralis (Vs- pVv ), the nucleus preopticus periventricularis (NPP), the lateral forebrain bundle ( LFB ), or the medial forebrain bundle (MFB). On the other hand, sexual behavior was almost unaffected after bilateral ablations of the anterior part or lateral and dorsal parts of the telencephalic hemispheres (AT or LT-DT). Bilateral lesions confined to the area ventralis telencephali pars dorsalis (Vd), the area ventralis telencephali pars postcommissuralis (Vp), the area dorsalis telencephali pars centralis (Dc), the nucleus preopticus (NPO), or the lateral preoptic area (LPA) also had almost no effect on sexual behavior. These results indicate that a particular site in the ventral part of the telencephalic hemispheres (Vs- pVv ) and the anterior part of the preoptic area (NPP) play important roles in the sexual behavior of male goldfish. It was suggested that facilitatory influences from these areas are transmitted through the LFB and/or the MFB.