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.

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.

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.

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.

Sexually Dimorphic Neuropeptide B Neurons in Medaka Exhibit Activated Cellular Phenotypes Dependent on Estrogen.

Brain and behavior of teleosts are highly sexually plastic throughout life, yet the underlying neural mechanisms are largely unknown. On examining brain morphology in the teleost medaka (Oryzias latipes), we identified distinctively large neurons in the magnocellular preoptic nucleus that occurred much more abundantly in females than in males. Examination of sex-reversed medaka showed that the sexually dimorphic abundance of these neurons is dependent on gonadal phenotype, but independent of sex chromosome complement. Most of these neurons in females, but none in males, produced neuropeptide B (Npb), whose expression is known to be estrogen-dependent and associated with female sexual receptivity. In phenotypic analysis, the female-specific Npb neurons had a large euchromatic nucleus with an abundant cytoplasm containing plentiful rough endoplasmic reticulum, exhibited increased overall transcriptional activity, and typically displayed a spontaneous regular firing pattern. These phenotypes, which are probably indicative of cellular activation, were attenuated by ovariectomy and restored by estrogen replacement. Furthermore, the population of Npb-expressing neurons emerged in adult males treated with estrogen, not through frequently occurring neurogenesis in the adult teleost brain, but through the activation of preexisting, quiescent male counterpart neurons. Collectively, our results demonstrate that the morphological, transcriptional, and electrophysiological phenotypes of sexually dimorphic preoptic Npb neurons are highly dependent on estrogen and can be switched between female and male patterns. These properties of the preoptic Npb neurons presumably underpin the neural mechanism for sexual differentiation and plasticity of brain and behavior in teleosts.

Morphological Analysis of the Axonal Projections of EGFP-Labeled Esr1-Expressing Neurons in Transgenic Female Medaka.

Some hypothalamic neurons expressing estrogen receptor α (Esr1) are thought to transmit a gonadal estrogen feedback signal to gonadotropin-releasing hormone 1 (GnRH1) neurons, which is the final common pathway for feedback regulation of reproductive functions. Moreover, estrogen-sensitive neurons are suggested to control sexual behaviors in coordination with reproduction. In mammals, hypothalamic estrogen-sensitive neurons release the peptide kisspeptin and regulate GnRH1 neurons. However, a growing body of evidence in nonmammalian species casts doubt on the regulation of GnRH1 neurons by kisspeptin neurons. As a step toward understanding how estrogen regulates neuronal circuits for reproduction and sex behavior in vertebrates in general, we generated a transgenic (Tg) medaka that expresses enhanced green fluorescent protein (EGFP) specifically in esr1-expressing neurons (esr1 neurons) and analyzed their axonal projections. We found that esr1 neurons in the preoptic area (POA) project to the gnrh1 neurons. We also demonstrated by transcriptome and histological analyses that these esr1 neurons are glutamatergic or γ-aminobutyric acidergic (GABAergic) but not kisspeptinergic. We therefore suggest that glutamatergic and GABAergic esr1 neurons in the POA regulate gnrh1 neurons. This hypothesis is consistent with previous studies in mice that found that glutamatergic and GABAergic transmission is critical for estrogen-dependent changes in GnRH1 neuron firing. Thus, we propose that this neuronal circuit may provide an evolutionarily conserved mechanism for regulation of reproduction. In addition, we showed that telencephalic esr1 neurons project to medulla, which may control sexual behavior. Moreover, we found that some POA-esr1 neurons coexpress progesterone receptors. These neurons may form the neuronal circuits that regulate reproduction and sex behavior in response to the serum estrogen/progesterone.

Evolutionally Conserved Function of Kisspeptin Neuronal System Is Nonreproductive Regulation as Revealed by Nonmammalian Study.

The kisspeptin neuronal system, which consists of a neuropeptide kisspeptin and its receptor Gpr54, is considered in mammals a key factor of reproductive regulation, the so-called hypothalamic-pituitary-gonadal (HPG) axis. However, in nonmammalian vertebrates, especially in teleosts, existence of kisspeptin regulation on the HPG axis is still controversial. In this study, we applied multidisciplinary techniques to a teleost fish, medaka, and examined possible kisspeptin regulation on the HPG axis. First, we generated knockout medaka for kisspeptin-related genes and found that they show normal fertility, gonadal maturation, and expression of gonadotropins. Moreover, the firing activity of GnRH1 neurons recorded by the patch clamp technique was not altered by kisspeptin application. Furthermore, in goldfish, in vivo kisspeptin administration did not show any positive effect on HPG axis regulation. However, as kisspeptin genes are completely conserved among vertebrates except birds, we surmised that kisspeptin should have some important nonreproductive functions in vertebrates. Therefore, to discover novel functions of kisspeptin, we generated a gpr54-1:enhanced green fluorescent protein (EGFP) transgenic medaka, whose gpr54-1-expressing cells are specifically labeled by EGFP. Analysis of neuronal projection of gpr54-1:EGFP-expressing neurons showed that these neurons in the ventrolateral preoptic area project to the pituitary and are probably involved in endocrine regulation other than gonadotropin release. Furthermore, combination of deep sequencing, histological, and electrophysiological analyses revealed various novel neural systems that are under control of kisspeptin neurons-that is, those expressing neuropeptide Yb, cholecystokinin, isotocin, vasotocin, and neuropeptide B. Thus, our new strategy to genetically label receptor-expressing neurons gives insights into various kisspeptin-dependent neuronal systems that may be conserved in vertebrates.

Female-Specific Glucose Sensitivity of GnRH1 Neurons Leads to Sexually Dimorphic Inhibition of Reproduction in Medaka.

Close interaction exists between energy-consuming reproduction and nutritional status. However, there are differences in costs and priority for reproduction among species and even between sexes, which leads to diversification of interactions between reproduction and nutritional status. Despite such diversified interactions among species and sexes, most of the analysis of the nutritional status-dependent regulation of reproduction has been limited to an endothermic vertebrate, mammalian species of either sex. Therefore, the mechanisms underlying the diversified interactions remain elusive. In the present study, we demonstrated the effects of malnutritional status on reproduction at both organismal and cellular levels in an ectothermic vertebrate, a teleost medaka of both sexes. First, we analyzed the effects of malnutrition by fasting on gonadosomatic index, number of spawned/fertilized eggs, and courtship behavior. Fasting strongly suppressed reproduction in females but, surprisingly, not in males. Next, we analyzed the effects of fasting on firing activity of hypothalamic GnRH1 neurons, which form the final common pathway for the control of reproduction. An electrophysiological analysis showed that low glucose, which is induced by fasting, directly suppresses the firing activity of GnRH1 neurons specifically in females through intracellular ATP-sensitive potassium channels and AMP-activated protein kinase pathways. Based on the fact that such suppressions occurred only in females, we conclude that nutritional status-dependent, glucose-sensing in GnRH1 neurons may contribute to the most fitted reproductive regulation for each sex.

Evolution of the Hypothalamic-Pituitary-Gonadal Axis Regulation in Vertebrates Revealed by Knockout Medaka.

Reproduction is essential for life, but its regulatory mechanism is diverse. The analysis of this diversity should lead us to understand the evolutionary process of the regulation of reproduction. In mammals, the hypothalamic-pituitary-gonadal axis plays an essential role in such regulation, and each component, hypothalamic GnRH, and pituitary gonadotropins, LH, and FSH, is indispensable. However, the common principle of the hypothalamic-pituitary-gonadal axis regulation among vertebrates remains unclear. Here, we used a teleost medaka, which is phylogenetically distant from mammals, and analyzed phenotypes of gene knockouts (KOs) for GnRH, LH, and FSH. We showed that LH release, which we previously showed to be directly triggered by GnRH, is essential for ovulation in females, because KO medaka of GnRH and LH were anovulatory in spite of the full follicular growth and normal gonadosomatic index, and spawning could be induced by a medaka LH receptor agonist. On the other hand, we showed that FSH is necessary for the folliculogenesis, because the follicular growth of FSH KO medaka was halted at the previtellogenic stage, but FSH release does not necessarily require GnRH. By comparing these results with the previous studies in mammals that both GnRH and LH are necessary for folliculogenesis, we propose a hypothesis as follows. During evolution, LH was originally specialized for ovulation, and regulation of folliculogenesis by GnRH-LH (pulsatile release) was newly acquired in mammals, which enabled fine tuning of reproduction through hypothalamus.

Kiss1 neurons drastically change their firing activity in accordance with the reproductive state: insights from a seasonal breeder.

Kisspeptin (Kiss) neurons show drastic changes in kisspeptin expression in response to the serum sex steroid concentration in various vertebrate species. Thus, according to the reproductive states, kisspeptin neurons are suggested to modulate various neuronal activities, including the regulation of GnRH neurons in mammals. However, despite their reproductive state-dependent regulation, there is no physiological analysis of kisspeptin neurons in seasonal breeders. Here we generated the first kiss1-enhanced green fluorescent protein transgenic line of a seasonal breeder, medaka, for histological and electrophysiological analyses using a whole-brain in vitro preparation in which most synaptic connections are intact. We found histologically that Kiss1 neurons in the nucleus ventralis tuberis (NVT) projected to the preoptic area, hypothalamus, pituitary, and ventral telencephalon. Therefore, NVT Kiss1 neurons may regulate various homeostatic functions and innate behaviors. Electrophysiological analyses revealed that they show various firing patterns, including bursting. Furthermore, we found that their firings are regulated by the resting membrane potential. However, bursting was not induced from the other firing patterns with a current injection, suggesting that it requires some chronic modulations of intrinsic properties such as channel expression. Finally, we found that NVT Kiss1 neurons drastically change their neuronal activities according to the reproductive state and the estradiol levels. Taken together with the previous reports, we here conclude that the breeding condition drastically alters the Kiss1 neuron activities in both gene expression and firing activities, the latter of which is strongly related to Kiss1 release, and the Kiss1 peptides regulate the activities of various neural circuits through their axonal projections.

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.

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.

Structure, synthesis, and phylogeny of kisspeptin and its receptor.

The kisspeptin system is considered to be essential for successful mammalian reproduction. In addition to the Kiss1 peptide, Kiss2, the product of kiss2 (the kiss1 paralogue), has also been shown to activate kisspeptin receptor signaling pathways in nonmammalian species. Furthermore, in nonmammalian species, there are two subtypes of receptors, Gpr54-1 (known as GPR54 or Kiss1R in mammals) and Gpr54-2. Although complete understanding of the two kisspeptin-two kisspeptin receptor systems in vertebrates is not so simple, a careful examination of the phylogeny of their genes may provide insights into the functional generality and differences among the kisspeptin systems in different animal phyla. In this chapter, we first discuss the structure of kisspeptin ligands, Kiss1 and Kiss2, and their characteristics as physiologically active peptides. Then, we discuss the evolutionary traits of kiss1 and kiss2 genes and their receptor genes, gpr54-1 and gpr54-2. It appears that each animal species has selected either kiss1 or kiss2 rather randomly, leading us to propose that some of the important characteristics of kisspeptin neurons, such as steroid sensitivity and the anatomical relationship with the hypophysiotropic GnRH1 neurons, may be the keys to understanding the general functions of different kisspeptin neuronal populations throughout vertebrates. Species differences in kiss1/kiss2 may also provide insights into the evolutionary mechanisms of paralogous gene-expressing neuronal systems. Finally, because kisspeptins belong to one of the members of the RFamide peptide families, we discuss the functional divergence of kisspeptins from the other RFamide peptides, which may be explained from phylogenetic viewpoints.

Anatomical distribution of sex steroid hormone receptors in the brain of female medaka.

Estrogen and androgen play crucial roles in coordinating reproductive functions through estrogen receptors (ERs) and androgen receptors (ARs), respectively. These receptors are considered important for regulation of the hypothalamo-pituitary-gonadal (HPG) axis. Despite their biological importance, the distribution of sex steroid receptors has not been fully analyzed anatomically in the teleost brain. The teleosts have many characteristic features, which allow unique approaches toward an understanding of the regulatory mechanisms of reproductive functions. Medaka serves as a good model system for studying the mechanisms by which steroid receptor-mediated systems are regulated, because (1) their breeding conditions can be easily manipulated; (2) we can take advantage of the genome database; and 3) molecular genetic tools, such as transgenic techniques, are applicable. We analyzed the distribution of ERα, ERß1, ERß2, ARα, and ARß mRNA by in situ hybridization in the brain of female medaka. We found that all subtypes of ERs and ARs were expressed in the following nuclei: the dorsal part of the ventral telencephalic area (Vd), supracommissural part of the ventral telencephalic area (Vs), postcommissural part of the ventral telencephalic area (Vp), preoptic area (POA), and nucleus ventralis tuberis (NVT). These regions are known to be involved in the regulation of sexual behavior (Vd, Vs, Vp, POA) or the HPG axis (NVT). These ER- and/or AR-expressing neurons may regulate sexual behavior or the HPG axis according to their axonal projections. Future analysis should be targeted to the neurons described in the present study to extend our understanding of the central regulatory mechanisms of reproduction.

Female-specific target sites for both oestrogen and androgen in the teleost brain.

To dissect the molecular and cellular basis of sexual differentiation of the teleost brain, which maintains marked sexual plasticity throughout life, we examined sex differences in neural expression of all subtypes of nuclear oestrogen and androgen receptors (ER and AR) in medaka. All receptors were differentially expressed between the sexes in specific nuclei in the forebrain. The most pronounced sex differences were found in several nuclei in the ventral telencephalic and preoptic areas, where ER and AR expression were prominent in females but almost completely absent in males, indicating that these nuclei represent female-specific target sites for both oestrogen and androgen in the brain. Subsequent analyses revealed that the female-specific expression of ER and AR is not under the direct control of sex-linked genes but is instead regulated positively by oestrogen and negatively by androgen in a transient and reversible manner. Taken together, the present study demonstrates that sex-specific target sites for both oestrogen and androgen occur in the brain as a result of the activational effects of gonadal steroids. The consequent sex-specific but reversible steroid sensitivity of the adult brain probably contributes substantially to the process of sexual differentiation and the persistent sexual plasticity of the teleost brain.

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.

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.