Kataaki Okubo

Structural and functional evolution of gonadotropin-releasing hormone in vertebrates

The neuropeptide gonadotropin‐releasing hormone (GnRH) has a central role in the neural control of vertebrate reproduction. This review describes an overview of what is currently known about GnRH in vertebrates in the context of its structural and functional evolution. A large body of evidence has demonstrated the existence of three paralogous genes for GnRH (GnRH1, GnRH2 and GnRH3) in the vertebrate lineage. They are most probably the products of whole‐genome duplications that occurred early in vertebrate evolution. Although GnRH3 has been identified only in teleosts, comparative genomic analyses indicated that GnRH3 has not arisen from a teleost‐specific genome duplication, but has been derived from an earlier genome duplication in an ancestral vertebrate, followed by its loss in the tetrapod lineage. A loss of other paralogous genes has also occurred independently in different vertebrate lineages, leading to species‐specific differences in the organization of the GnRH system. In addition to the GnRH3 gene, the GnRH2 gene has been deleted or silenced in certain mammalian species, while some teleosts seem to have lost the GnRH1 or GnRH3 gene. The duplicated GnRH genes have undergone subfunctionalization during the evolution of vertebrates; GnRH1 has become the major stimulator of gonadotropins and probably other pituitary hormones as well, whereas GnRH2 and GnRH3 would have functioned as neuromodulators, affecting reproductive behaviour. Conversely, in cases where a paralogous gene for GnRH has been lost, one of the remaining paralogues appears to have adopted its role.

A Neural Mechanism Underlying Mating Preferences for Familiar Individuals in Medaka Fish

Familiarity Does Not Breed Contempt Female mating preference is influenced by social familiarity in various species from fish to primates. Okuyama et al. (p. 91) showed in Japanese rice fish that females prefer to mate with visually familiarized males over unfamiliar males and that this preference is mediated by specific neuromodulatory neurons in the female brain. A particular class of neurons regulates female fish mating preference based on social familiarity. Social familiarity affects mating preference among various vertebrates. Here, we show that visual contact of a potential mating partner before mating (visual familiarization) enhances female preference for the familiarized male, but not for an unfamiliarized male, in medaka fish. Terminal-nerve gonadotropin-releasing hormone 3 (TN-GnRH3) neurons, an extrahypothalamic neuromodulatory system, function as a gate for activating mating preferences based on familiarity. Basal levels of TN-GnRH3 neuronal activity suppress female receptivity for any male (default mode). Visual familiarization facilitates TN-GnRH3 neuron activity (preference mode), which correlates with female preference for the familiarized male. GnRH3 peptides, which are synthesized specifically in TN-GnRH3 neurons, are required for the mode-switching via self-facilitation. Our study demonstrates the central neural mechanisms underlying the regulation of medaka female mating preference based on visual social familiarity.

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.

Expression of Two Gonadotropin-Releasing Hormone (GnRH) Precursor Genes in Various Tissues of the Japanese Eel and Evolution of GnRH

Abstract We isolated and characterized two distinct cDNAs for mammalian gonadotropin-releasing hormone (mGnRH) and chicken GnRH-II (cGnRH-II) precursors from the Japanese eel by rapid amplification of cDNA ends. Each GnRH precursors were composed of a signal peptide, a GnRH decapeptide, a processing site and a GnRH-associated peptide. Northern blot and reverse transcription-polymerase chain reaction analysis revealed that the mGnRH precursor gene is expressed in all tissues tested including the brain, pituitary, eye, olfactory epithelium, ovary, testis, liver, kidney, spleen, heart, gill, intestine, pancreas, muscle, skin, fin and peripheral blood leukocyte. In contrast, the cGnRH-II precursor gene expression was detected only in the brain, pituitary, olfactory epithelium, ovary and testis. These findings suggest unknown physiological function(s) for mGnRH besides the well-documented role in the pituitary gonadotropin synthesis and release. The eel mGnRH and cGnRH-II precursors have high amino acid homologies with seabream GnRH (sbGnRH) precursors of the Perciforms and cGnRH-II precursors of other teleosts, respectively. Phylogenetic analysis showed the existence of three distinct evolutionary arms of GnRHs; multiple GnRH forms (mGnRH, guinea pig GnRH, chicken GnRH-I, sbGnRH and catfish GnRH (cfGnRH)) on the first, cGnRH-II on the second, and salmon GnRH (sGnRH) on the third arm. This analysis suggests that mGnRH progenitor has undergone sequence divergence to give rise to sbGnRH and cfGnRH, whereas sGnRH represents a separate evolutionary line.

A novel third gonadotropin-releasing hormone receptor in the medaka Oryzias latipes: evolutionary and functional implications

Gonadotropin-releasing hormone (GnRH) plays pivotal roles in the regulation of vertebrate reproduction through binding to its specific membrane receptor. Within the past few years, substantial evidence has accumulated that more than one GnRH receptor (GnRH-R) is expressed in individual vertebrate species. Two GnRH-Rs, termed GnRH-R1 and GnRH-R2, have been identified in a teleost, the medaka Oryzias latipes. Here we describe the identification and characterization of a novel third member of GnRH-R, designated GnRH-R3, in the medaka. GnRH-R3 share high sequence homology (77% amino acid identity in the transmembrane domain) with GnRH-R1. Phylogenetic analysis and genetic mapping demonstrated that both GnRH-R1 and GnRH-R3 were orthologous to the type 2 GnRH-R in primates and that these two medaka receptors were duplicates resulting from the genome-wide duplication within the teleost lineage. GnRH-R3, however, contained three introns, whereas GnRH-R1 had only two. Moreover, unlike GnRH-R1, GnRH-R3 exhibited an approximately equal selectivity for two of three native GnRH forms in the medaka, chicken-II-type GnRH (cGnRH-II) and salmon-type GnRH (sGnRH), and a less sensitivity for the other form, medaka-type GnRH. GnRH-R3 was found to be expressed throughout the brain, and thus appeared to mediate the neuromodulatory functions of both cGnRH-II and sGnRH. These data identify GnRH-R3 as a new member of GnRH-R that arose in a recent genome duplication but has distinctive genomic structure and functional characteristic.

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.

Screening estrogenic activity of environmental contaminants and water samples using a transgenic medaka embryo bioassay.

Many natural or synthetic chemicals may act as exogenous estrogens and affect the reproductive health of humans and wildlife. Since these xenoestrogens are ubiquitous, it is essential to monitor their presence in the environment. Hence, we developed a bioassay using the transgenic medaka (Oryzias latipes) embryo, in which the green fluorescent protein (GFP) was placed under the control of the gnrh3 promoter, one of the three paralogous gonadotropin-releasing hormone (GnRH) genes that regulate reproductive function and behavior. As medaka embryos are transparent, the fluorescent expression of GFP can be easily observed in vivo during development. We exposed newly fertilized medaka embryos to varying solutions of bisphenol A (BPA), nonylphenol (NP), 17β-estradiol (E2), or a river water sample, and monitored their development. During embryonic development, the mRNA levels of GnRHs, GnRH receptors, and estrogen receptors (ERs) were measured with quantitative real-time reverse transcription-PCR. Our results showed that the chemicals and the river water significantly decreased the fluorescent intensity of the GnRH3 neurons, postponed the eye development, and retarded the growth of the embryos. The three xenoestrogens also lowered the heart rate, lengthened the time to hatch, suppressed the expression of the three GnRH genes, and up-regulated the ERα mRNA level. In addition, the GnRH3 mRNA level was significantly correlated with the fluorescence intensity of the GnRH neurons. We concluded that the transgenic medaka embryo is a rapid and sensitive bioassay for screening environmental water samples. We also found that xenoestrogens had significant effects on GnRH gene expression and embryonic development.

Sex differences in the expression of vasotocin/isotocin, gonadotropin-releasing hormone, and tyrosine and tryptophan hydroxylase family genes in the medaka brain.

In teleost fish, sex differences in several behavioral and physiological traits have been assumed to reflect underlying sex differences in the central expression of neurotransmitter/neuromodulator-related molecules, including vasotocin (VT)/isotocin (IT), gonadotropin-releasing hormone (GnRH), and tyrosine and tryptophan hydroxylases (TH and TPH). However, the sex-dependent expression patterns of these molecules have not been fully characterized in the teleost brain. In the present study, we therefore systematically evaluated sex differences in their expression in the medaka (Oryzias latipes) brain. The most prominent sex difference was observed in vt expression in the nucleus posterior tuberis (NPT) and the posterior part of the nucleus ventral tuberis (NVT) in the hypothalamus, where the expression was completely male-specific. Male-biased expression of gnrh1, tph1, and tph2 was also evident in the supracommissural and posterior nuclei of the ventral telencephalic area (Vs/Vp), medial nucleus of the dorsal telencephalic area (Dm), and thalamic dorsal posterior nucleus (DP), respectively. In contrast, the overall expression levels of it and gnrh3 were higher in the female brain than in the male brain. Equally importantly, no conspicuous sex differences were observed in the expression of gnrh2, th1, and th2, despite several previous reports of their sex-biased expression in the brains of other teleost species. Taken together, these data have uncovered previously unidentified sex differences in the expression of VT/IT, GnRH, and TPH in the teleost brain, which may possibly be relevant to sexual dimorphism in some behavioral and/or physiological traits, and have simultaneously highlighted potential species differences in the roles of these molecules.

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.

Molecular cloning of three cDNAs encoding different gnrhs in the brain of barfin flounder

To examine the reproductive endocrinology of a large pleuronectiform fish, barfin flounder, Verasper moseri, a promising candidate for aquaculture and resource enhancement in northern Japan due to its high commercial value, three gonadotropin-releasing hormones (GnRHs) in the brain was identified by isolation of their cDNAs. This species had three molecular forms of GnRH; salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and seabream GnRH (sbGnRH). Each GnRH cDNA encoded a signal peptide (SP), GnRH, and a GnRH-associated peptide (GAP), which was connected to GnRH by a Gly-Lys-Arg sequence. The sGnRH cDNA encoded an SP composed of 23 amino acids and a GAP composed of 54 amino acids. The cGnRH-II cDNA encoded an SP of 23 amino acids and a GAP of 49 amino acids. The sbGnRH cDNA encoded an SP of 26 amino acids and a GAP of 57 amino acids. In situ hybridization showed that the genes for sGnRH, cGnRH-II, and sbGnRH are expressed in the ventromedial olfactory bulbs and the terminal nerve ganglion, the midbrain tegmentum, and the preoptic area, respectively. These results indicate that sbGnRH neurons in the preoptic area are involved in gonadotropin secretion in barfin flounder.