Shinji Kanda

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.

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.

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.

Neuroanatomical Evidence That Kisspeptin Directly Regulates Isotocin and Vasotocin Neurons

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.

Sex differences in aromatase gene expression in the medaka brain.

The brain of teleost fish exhibits a significant degree of sexual plasticity, even in adulthood. This unique feature is almost certainly attributable to a teleost‐specific sexual differentiation process of the brain, which remains largely unknown. To dissect the molecular basis of sexual differentiation of the teleost brain, we searched for genes differentially expressed between both sexes in the medaka brain. One gene identified in the screen, cyp19a1b, which encodes the steroidogenic enzyme aromatase, was selected for further analysis. As opposed to the situation in most vertebrates, medaka cyp19a1b is expressed at higher levels in the adult female brain than the male brain. The female‐biased expression in the brain is consistent regardless of reproductive or diurnal cycle. Medaka cyp19a1b is expressed throughout the ventricular zones in wide areas of the brain, where, in most regions, females have a greater degree of expression compared to males, with the optic tectum exhibiting the most conspicuous predominance in females. Contrary to what is known in mammals, cyp19a1b expression exhibits neither a transient elevation nor a sex difference in medaka embryos. It is not until just before the onset of puberty that cyp19a1b expression in the medaka brain is sexually differentiated. Finally, cyp19a1b expression in the medaka brain is not under the direct control of sex chromosome genes but relies mostly, if not solely, on oestrogen derived from the gonad. These unique properties of aromatase expression in the brain probably contribute substantially to the less rigid sexual differentiation process, thus ensuring remarkable sexual plasticity in the teleost brain.

Time-of-Day-Dependent Changes in GnRH1 Neuronal Activities and Gonadotropin mRNA Expression in a Daily Spawning Fish, Medaka

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.

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

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.

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

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.

Regular Pacemaker Activity Characterizes Gonadotropin-Releasing Hormone 2 Neurons Recorded from Green Fluorescent Protein-Transgenic Medaka

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.

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

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.