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Endocrinology, doi:10.1210/en.2007-1503
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Endocrinology Vol. 149, No. 5 2467-2476
Copyright © 2008 by The Endocrine Society

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

Shinji Kanda, Yasuhisa Akazome, Takuya Matsunaga, Naoyuki Yamamoto, Shunji Yamada, Hiroko Tsukamura, Kei-ichiro Maeda and Yoshitaka Oka

Department of Biological Sciences (S.K., Y.A., T.M., Y.O.), Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan; and Laboratories of Fish Biology (N.Y.) and Reproductive Science (S.Y., H.T., K.-i.M.), Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

Address all correspondence and requests for reprints to: Yoshitaka Oka, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan. E-mail: okay{at}biol.s.u-tokyo.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A GROWING BODY of evidence suggests that the KiSS-1 gene product kisspeptin (metastin) plays a critical role in reproduction by inducing gonadotropin release via the G protein-coupled receptor 54 (GPR54), which is suggested in mammals to be expressed in hypothalamic [preoptic area (POA)] GnRH neurons (1, 2, 3, 4, 5, 6, 7, 8, 9). The lack of GPR54 leads to abnormal sexual development in mice and to hypogonadotropic hypogonadism in humans because of low circulating gonadotropin concentrations (10, 11, 12). The kisspeptin neurons have been localized in several hypothalamic nuclei, including the arcuate nucleus and anteroventral periventricular nucleus (AVPV) in rodents and sheep (1, 13, 14, 15), which have been suggested to be a negative/positive feedback regulator of the hypophysiotropic GnRH neurons, respectively (15, 16, 17, 18).

Despite its importance in the neuroendocrine regulation of reproduction, kisspeptin neurons have only been studied in mammals, and there has been no report in nonmammalian vertebrates except for a few studies about its receptor, GPR54 (19, 20, 21, 22). This is of a great disadvantage to the understanding of neuroendocrine control of reproduction and reproductive behaviors because the nonmammalian vertebrates, especially teleosts, have greatly contributed to our understanding of the GnRH neurons, especially at the cellular level (for review, see Refs. 23 and 24). The hypothalamo-pituitary-gonadal (HPG) axis is critical for reproduction and is well conserved among the vertebrate species. However, teleosts have an interesting characteristic among the vertebrates in that the hypothalamic GnRH neurons directly project their axons to the pituitary, which enabled direct amperometric measurement of GnRH secretion in the pituitary (25). Moreover, the nature of extrahypothalamic GnRH neuronal systems, which generally exist in the brains of various vertebrate species, has been most intensively studied anatomically as well as electrophysiologically in the terminal nerve-GnRH neurons of the teleosts (23, 26, 27, 28, 29, 30, 31, 32, 33, 34). Therefore, the teleosts should provide a good model system for studying the mechanisms of possible kisspeptin regulation of the extrahypothalamic as well as hypothalamic GnRH neurons. Among the teleosts, medaka may be the best model system for the study of the kisspeptin neuronal system because the reproductive biology of medaka has been well documented, and medaka enables the use of various molecular genetic tools such as transgenic technologies. Here, we report on the initial identification and characterization of kisspeptin and the kisspeptin neuronal system in nonmammalian species.

We first cloned the KiSS-1 gene of medaka (Oryzias latipes). We then analyzed the anatomical distribution of KiSS-1 mRNA expression by in situ hybridization and found two distinctive neuronal populations in the hypothalamus. One of them was further found to be sexually dimorphic, sensitive to ovarian estrogens, and show higher KiSS-1 expression in the breeding condition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male and female d-rR strain medaka (O. latipes) were maintained under a 14-h light, 10-h dark photoperiod at a temperature of 27 C. The fish were fed twice daily with live brine shrimp and flake food. The animals were maintained and used in accordance with the guidelines of The Physiological Society of Japan and the University of Tokyo for the Use and Care of Experimental Animals.

Isolation of the KiSS-1 candidate gene and determination by synteny analysis
Blasting the human kisspeptin core sequence (kp-10: YNWNSFGLRF) against the Medaka Genome Project database (http://dolphin.lab.nig.ac.jp/medaka/, TBLASTN) gave one candidate sequence in scaffold 45 of the whole genome library.

From this candidate sequence, the gene-specific primers for 5' RACE and 3' RACE were designed (Kiss1RACE-R1–3, Kiss1RACE-F1,2; Table 1Go).


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  		TABLE 1. Sequences of PCR primers and probes used for PCR

 
The total RNA isolated from the 12 medaka brains using ISOGEN (Nippon Gene, Tokyo, Japan) was applied to the SMART RACE kit (Clontech Laboratories, Inc., Palo Alto, CA) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed into cDNA with Powerscript Reverse Transcriptase (Clontech) according to the manufacturer’s protocol for 5' and 3' first-strand cDNA synthesis. The PCR conditions were as follows.

The 5'- and 3'- ends were isolated with two rounds of 5' and 3' RACE-PCRs, respectively, following the SMART-RACE PCR protocol. Sequences of the primers used in the present experiments are shown in Table 1Go. The primers Kiss1RACE-R1–3 were used for the 5' RACE-PCR with the adaptor primers UPM and NUP, for first and secondary (nested) reactions, respectively. The primers Kiss1RACE-F1 and 2 were for the 3' RACE-PCR.

Ten microliters of reaction contained 1x UPM (first PCR) or 1x NUP (secondary PCR), 1 µM gene specific primers, 1x ExTaq buffer (TaKaRa, Shiga, Japan), 200 µM each of deoxynucleotide, 0.5 U ExTaq polymerase (TaKaRa), and 1 µl cDNA solution. The reaction condition was as follows: 94 C for 5 min, five cycles of 94 C for 30 sec and 72 C for 2 min 30 sec, five cycles of 94 C 30 sec, 70 C 30 sec and 72 C 2 min, followed by nine cycles of 94 C 30 sec, 64 C 30 sec, and 72 C 2 min. Two hundred nanoliters of first-round PCR solutions were applied to the nested PCRs. The reaction condition was as follows: 94 C for 5 min, five cycles of 94 C for 30 sec and 72 C for 2min 30 sec, five cycles of 94 C 30 sec, 70 C 30 sec and 72 C 2 min, followed by 10 cycles of 94 C 30 sec, 68 C 30 sec and 72 C 2 min and 10 cycles of 94 C 30 sec, 64 C 30 sec and 72 C 2 min, followed by 72 C 5 min. The PCR product was gel purified (1.5% agarose gel) and sequenced using an ABI prism 310 system (Perkin-Elmer-Applied Biosystems, Foster City, CA).

Synteny analysis of the candidate KiSS-1 gene
To determine the syntenic relationships of KiSS-1 between medaka and human genomes, the mapped human genes flanking a particular human KiSS-1 gene were identified. The protein sequences of the flanking genes were used to identify medaka KiSS-1 candidates by BLAST searches. The chromosomal location of the KiSS-1 orthologue in medaka was determined by searching University of Tokyo Genome Browser (Medaka, http://medaka.utgenome.org/).

Expression analysis of the KiSS-1 gene
RT-PCR was performed to identify the possible source of KiSS-1 in the adult medaka. Total RNA was extracted using ISOGEN from male and female medaka pairs that underwent oviposition of fertilized eggs every day. The cDNAs used as templates for RT-PCR were synthesized from denatured total RNA using 100 pmol oligo(dT) primer and 100 U ReverTra Ace (TOYOBO, Osaka, Japan) in a 20-µl reaction volume with incubation at 42 C for 0.5 h. The KiSS-1 cDNA fragments were then amplified with GeneTaq (Nippon Gene) using specific primer sets (Table 1Go; KiSS-1, KiSS1-F, KiSS1-R, β-actin, actin-F, and actin-R) from 1 µl cDNAs from each tissue. β-Actin was used for an internal control of successful synthesis of cDNAs. The PCR conditions were as follows: 94 C for 5 min, 35 cycles (30 cycles for β-actin) of 94 C for 40 sec, 64 C for 20 sec, and 72 C for 1 min, and final extension step of 72 C for 7 min. The amplified products were electrophoresed on 2.0% agarose gel and stained with ethidium bromide.

In situ hybridization
In the in situ hybridization analysis, we used male and female medaka that had oviposited fertilized eggs every day [at least for 3 d; 0.22- to 0.26-g body weight; male gonadosomatic index (GSI), 0.8–1.4, n = 6; female GSI, 8.0–14.0, n = 5].

The medaka was deeply anesthetized with MS-222 (Sigma, St. Louis, MO) and perfused with 4% paraformaldehyde in 0.05 M PBS from the conus arteriosus. The brain was postfixed with the same fixative for at least 6 h at 4 C. The fixed specimens were immersed in diethyl pyrocarbonate-treated PBS with 30% sucrose for 3–6 h, embedded in 5% agarose (type IX-A; Sigma) and 20% sucrose in diethyl pyrocarbonate-treated PBS, frozen in n-hexane (–60 C), and serial frontal sections were cut at 20-µm thick on a cryostat.

To detect KiSS-1 mRNA, we made a KiSS-1-specific digoxigenin (DIG)-labeled probe and performed nonradioactive slide-embedded in situ hybridization as previously reported (15, 35). Briefly, every section through the olfactory bulb to the spinal cord was washed with PBS and treated with 1 µg/ml protease K for 15 min at 37 C and then incubated with 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min. Finally, the sections were hybridized with 1 µg/ml DIG-labeled antisense cRNA probes (position 21–365 in Fig. 1AGo) synthesized from the medaka brain using a labeling kit (Roche Molecular Biochemicals GmbH, Mannheim, Germany) overnight at 60 C. A sense RNA probe was used as a negative control. After hybridization, the sections were washed twice with 2x saline sodium citrate (SSC) containing 50% formamide for 15 min at 60 C. The sections were then treated with 20 µg/ml RNase A for 30 min at 37 C and immersed sequentially with 2x SSC, 0.5x SSC, and DIG-1 buffer [100 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] for 15 min, twice each. The sections were then immersed with 1.5% blocking reagent (Roche Molecular Biochemicals) in DIG-1 buffer for 1 h at 37 C and incubated with an alkaline phosphatase-conjugated anti-DIG antibody (1:1000; Roche Diagnostics Corp., Indianapolis, IN) for 2 h at 37 C. Then the sections were washed with DIG-1 buffer and treated with DIG-3 buffer [100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2]. After this the sections were treated with a chromogen solution (337 µg/ml 4-nitroblue tetrazolium chloride, 175 µg/ml 5-bromo-4-chloro-3-indoyl-phosphate in DIG-3 buffer) until a visible signal was detected. The reaction was stopped by adding a reaction stop solution [10 mM Tris-HCl (pH 7.6) and 1 mM EDTA (pH 8.0)]. The sections were observed by light microscopy. For the nomenclature of the medaka brain nuclei, we followed the Medaka Histological Atlas (Wakamatsu et al., Medaka Histological Atlas, edited by the Editorial Board of Medaka Histological Atlas of National BioResource Project Medaka, http://www.shigen.nig.ac.jp/medaka/medaka_atlas). After the histological identification of the KiSS-1-expressing neurons, their number was counted. Furthermore, the diameters of randomly selected neurons were measured in each nucleus.


Figure 1
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  		FIG. 1. cDNA and deduced amino acid sequence of the medaka KiSS-1 gene. A, The predicted cDNA sequence of the medaka KiSS-1. Arrows indicate predicted intron-exon boundaries. B, Comparison of KiSS-1 precursor protein sequences among different mammalian species, medaka, and zebra fish. The C-terminal 10 amino acid residues (kisspeptin-10) are well conserved among various vertebrate species, including teleosts. C, Synteny analysis of the KiSS-1 gene between the medaka and rat. The medaka KiSS-1 gene was located next to GOLT1A, which was also located adjoining the KiSS-1 in humans. It is strongly suggested that the predicted gene sequence shown here represents the medaka KiSS-1 gene. D, Expression of KiSS-1 in different tissues assessed by RT-PCR. Amplifications of β-actin transcripts were performed in parallel as controls (β-actin). Reactions without reverse transcriptase served as negative controls (w/o RTase).

 
Ovariectomy (OVX) and estrogen administration
The fish were deeply anesthetized with 0.01% MS222. For the OVX the ovaries of female medaka (0.11- to 0.13-g body weight) were excised through a 2- to 3-mm incision made along the abdomen of the fish. At the end of the operation, the incision was sutured with a nylon thread. For sham-operated fish (sham), all the aforementioned steps were followed except for the removal of the ovaries. The operated fish were allowed to survive for at least 1 wk. After that the OVX fish were fed with estrogen (β-estradiol; E) (Sigma)-containing flake food for estrogen treatment (0.1 µg E/d) or normal food for 1 wk. This dose of estrogen was high enough to induce sex reversal in embryos and was determined as follows. According to the literature (36, 37), 100% of male medaka embryos developed into sex-reversed females with a 12.5-µg E/g diet, whereas 51% of them did so with 5-µg/g diet. We converted the total amount of E assuming that it is administered to the adult fish. We used the value for the standard amount of food intake of medaka [10 mg/d (38)] to calculate the total amount of E in a day (0.1 µg/d). The in situ hybridization protocol described previously was performed using the brains from completely OVX fish and OVX + E, and the brains from fish with intact ovaries (sham). The KiSS-1 mRNA-expressing cells in each nucleus in sham, OVX, and OVX + E were counted (short-day, SD).

Comparison of KiSS-1 expressing neurons between the fish under the breeding and nonbreeding conditions
Male and female medaka (male, 0.18- to 0.24-g body weight; female, 0.13- to 0.20-g body weight) under the breeding and nonbreeding conditions were placed in tanks at 27 C. Considering that the medaka is a long-day (LD) breeder, the tank was set at a constant 14-h light, 10-h dark photoperiod for the breeding conditions. For the nonbreeding condition, the tank was set at a 10-h light, 14-h dark photoperiod.

Statistical analysis
All the data were expressed as means ± SEM. The presence of sexual dimorphism and variations according to the day length were tested statistically using Mann-Whitney’s U test, whereas the results of the experiments involving three groups in the sham, OVX, and OVX + E were analyzed by the Kruskal-Wallis test, followed by post hoc Mann-Whitney’s U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of the medaka KiSS-1 gene and the amino acid sequence of its product kisspeptin
The PCR using total RNA isolated from the medaka brain resulted in the production of about a 400-bp nucleotide sequence composed of three exons (Fig. 1AGo; DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank with the accession no. AB272755).1 The medaka kisspeptin precursor predicted from the cDNA sequence obtained here was 100 amino acid residues, including N-terminal 20 amino acid residues of the signal peptide. Compared with the human kisspeptin precursor [145 amino acid residues (39)], the medaka kisspeptin precursor is shorter, and the amino acid homology is only 34%. Furthermore, the position of the dibasic sequence, which is predicted to be cleaved by subtilisin-like convertase, is different from that of the human kisspeptin precursor. However, the core sequence, kp-10 was highly conserved (80% against human, 90% against mouse or rat; Fig. 1BGo). Medaka kisspeptin as well as mammalian kisspeptin is predicted to be amidated by carboxypeptidase in their C terminals. Thus, we predicted that the medaka kp-10 is YNLNSFGLRY-NH2. The synteny analysis proved that the gene identified here is the orthologue of human KiSS-1 because it was located next to Golgi transport 1 homolog A (GOLT1A), which is also located next to GOLT1A in human, although it has an inversion (Fig. 1CGo). We also found another candidate for medaka KiSS-1, whose deduced C-terminal amino acid sequence is FNYNPFGLRF and is also similar to the human kisspeptin core sequence. However, it had no syntenic relationship with the KiSS-1 gene. Thus, we concluded that the core sequence of the medaka kisspeptin, medaka kp-10, is YNLNSFGLRY-NH2.

Tissue-specific expression of KiSS-1
Expression of KiSS-1 in various tissues was examined by RT-PCR. The PCR primers were designed to identify cDNA and genomic DNA by flanking an intron. Thus, the effects of genomic DNA contamination of cDNA could be excluded. As shown in Fig. 1DGo, KiSS-1 signals were detected in the brain, testis, and stomach, but not in the ovary, liver, intestine, and retina. Although KiSS-1 mRNA expression in the brain and the testis was detected in all fish, it was detected only in the stomach of two out of six fish.

Localization of KiSS-1-expressing neurons in the brain by in situ hybridization
Localization of neurons expressing the KiSS-1 gene was analyzed by using in situ hybridization using RNA probes complementary to the position 21–365 of KiSS-1 cDNA (Fig. 1AGo). KiSS-1-expressing neuronal cell bodies were found in two hypothalamic nuclei, the nucleus posterioris periventricularis (NPPv) and the nucleus ventral tuberis (NVT) (Fig. 2Go). No labeled cell was detected in the control sections using the sense probe.


Figure 2
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  		FIG. 2. Light photomicrographs and schematic illustrations showing the representative sections of the NVT and the NPPv KiSS-1 mRNA-expressing cells (KiSS-1 neurons) in the male and female medaka brains. KiSS-1 neurons labeled by KiSS-1 in situ hybridization in the male NVT (A), those in the male NPPv (B), those in the female NVT (C), and those in the female NPPv (D). E, Outline drawing of parasagittal section showing the plane of sectioning corresponding to F. F, Schematic illustration showing the distribution of the NVT and the NPPv KiSS-1 neurons plotted on a representative frontal section of the hypothalamus. The dots illustrate the distribution and density of KiSS-1 neurons in the two nuclei but do not reflect the correct numbers. See Fig. 3Go for quantitative data on the number of cells. Scale bars: A–D, 50 µm; and E and F, 200 µm. CP, Commissura posterior; DM, nucleus dorsomedialis thalami; fr, fasciculus retroflexus; NAT, nucleus anterior tuberis; NDTL, nucleus diffusus tori lateralis; NVT, nucleus ventralis tuberis; PG, nucleus preglomerulosus; PIT, pituitary; TL, torus longitudinalis; VM, nucleus ventromedialis thalami.

 
We found a distinctive sexual dimorphism in the number of KiSS-1-expressing neurons specifically in one of the nuclei NVT but not in NPPv, with male neurons being more than female ones (Fig. 3Go). We found 367.2 ± 16.7 KiSS-1 neurons in males (n = 6) and 113.0 ± 13.0 in females (n = 5) in the NVT (mean ± SEM; P < 0.01). On the other hand, there were no significant differences in the cell number in NPPv (male, 7.8 ± 2.24; female, 7.0 ± 0.89; P > 0.05). There was no significant difference in the size of the KiSS-1-expressing cells in either population (in NPPv, diameter of the male soma = 13.8 ± 0.5 µm, n = 11, that of female soma = 13.3 ± 0.3 µm, n = 10; in NVT, diameter of the male soma = 11.1 ± 0.1 µm, n = 142, that of female soma = 11.0 ± 0.1 µm, n = 63; P > 0.05 for both nuclei).


Figure 3
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  		FIG. 3. The expression of KiSS-1 mRNA in the NVT (A) and NPPv (B) of male and female medaka in the breeding condition. **, Significant difference between the males and females (P < 0.01). The bars represent means (n = 6 males; n = 5 females), and all the values are presented as the mean ± SEM.

 
The effects of OVX and estrogen replacement on KiSS-1 mRNA expression in the hypothalamus
We examined whether estrogens regulate KiSS-1 expression in each nucleus by comparing the number of KiSS-1 mRNA-expressing cells among the groups of sham-operated (n = 4), OVX (n = 4), and OVX estrogen-treated fish (OVX + E, n = 4). Unfortunately, we could not examine the effects of castration in males because of technical difficulties. In the NVT the number of KiSS-1-expressing neurons decreased after OVX (Fig. 4Go, A vs. B; P < 0.05) and recovered with estrogen treatment (Fig. 4Go, B vs. C; P < 0.05). There was no significant difference in the number of KiSS-1 neurons between sham and OVX + E (Fig. 4Go, A vs. C; P > 0.05). On the other hand, we did not find any significant changes after OVX or estrogen treatment in the NPPv (Fig. 4Go, D–H). From these results we conclude that KiSS-1 mRNA expression in the NVT, but not that in the NPPv, is regulated by ovarian estrogens.


Figure 4
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  		FIG. 4. Bright-field photomicrographs of KiSS-1 neurons in the NVT and NPPv. The photographs show representative sections before (A and D) and after (B and E) OVX and after estrogen treatment (C and F). Sham-operated (A and D), OVX (B and E), and OVX + E-fish (C and F) in the NVT (A–C) or NPPv (D–F). KiSS-1 neurons decreased in number after OVX and recovered after E treatment in the NVT, but no significant changes occurred in the NPPv. G and H, Graphs showing the number of KiSS-1 neurons among different experimental groups in two different populations of kisspeptin neurons. G, The number of KiSS-1 neurons in the NVT was significantly less in OVX animals (n = 4) than sham (n = 4). E treatment (OVX + E, n = 4) completely reversed the effects of OVX. H, In the NPPv there was no significant change in cell number. Scale bars, 50 µm. *, Significant difference (P < 0.05). All values are presented as the mean ± SEM. Note that all the female fish were kept in isolation for the purpose of experimental procedures, which led to the generally decreased number of NVT KiSS-1 neurons compared with the fully breeding conditions in Fig. 3Go.

 
Comparison of KiSS-1 expressing neurons between the fish under the breeding and nonbreeding conditions
To determine if the KiSS-1 neuronal populations change their mRNA expressions according to the physiological changes in the breeding conditions, we compared the expressions of KiSS-1 mRNA between the fish under the breeding and nonbreeding conditions by in situ hybridization (Fig. 5Go). We used LD conditioned male and female medaka that had well-developed gonads (breeding condition; male GSI = 1.1 ± 0.06, female GSI = 5.5 ± 0.4) and SD conditioned medaka that had regressed gonads (nonbreeding condition; male GSI = 0.54 ± 0.1, female GSI = 1.0 ± 0.2). All of the LD conditioned females in this experiment had fully matured eggs. The GSI of the LD conditioned medaka was significantly higher than that of the SD conditioned ones (P < 0.05, in both males and females). The number of NVT KiSS-1 neurons in the breeding condition was larger than that in the nonbreeding condition (breeding male, 226.5 ± 40.2; nonbreeding male, 0 ± 0; P < 0.05; Fig. 5EGo: breeding female, 54.8 ± 12.8; nonbreeding female, 0 ± 0; P < 0.05; Fig. 5KGo), whereas there was no such difference in the NPPv (breeding male, 3.8 ± 0.7; nonbreeding male, 3.8 ± 2.2; P > 0.1; Fig 5FGo: breeding female, 5.0 ± 1.1; nonbreeding female, 4.0 ± 0.6; P > 0.1; Fig. 5LGo).


Figure 5
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  		FIG. 5. Bright-field photomicrographs of KiSS-1 neurons in the male and female NVT and NPPv. The photographs show representative sections of NVT in breeding condition (A) and nonbreeding condition (B), and that of NPPv in breeding condition (C) and nonbreeding condition (D) in male. The graphs show the number of female KiSS-1 neurons in breeding and nonbreeding conditions in NVT (E) and NPPv (F). The photographs show representative sections of female NVT in breeding condition (G) and nonbreeding condition (H), and those of female NPPv in breeding condition (I) and nonbreeding condition (J) in female. The graphs show the number of female KiSS-1 neurons in breeding and nonbreeding conditions in NVT (K) and NPPv (L) in female. Scale bars, 50 µm. *, Significant difference (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here, we report the initial identification of the KiSS-1 gene and prediction of the amino acid sequence of its gene product kisspeptin, and the anatomical distribution of KiSS-1 mRNA-expressing neurons in the brain of nonmammalian species. Distribution of KiSS-1 mRNA-expressing cells was restricted to two regions in the hypothalamus, NVT and NPPv. It should be noted that the number of KiSS-1 expressing neurons in the NVT changed dramatically according to the differences in the breeding states, which was evidenced by the changes in the GSI. The results strongly suggest the significant involvement of the kisspeptin neuronal system in the central regulation of reproductive functions and provide an insight into the general significance of kisspeptin in vertebrate reproduction. Furthermore, we noted the presence of steroid-sensitive sexually dimorphic KiSS-1 neurons; the KiSS-1 neurons in the NVT proved to be sexually dimorphic in number, the male neurons being more than in females, both of which were under the breeding conditions. We also found that KiSS-1 mRNA expression in the NVT but not that in the NPPv was positively regulated by ovarian estrogens, suggesting that NVT KiSS-1 neurons may be involved in the positive feedback regulation of the HPG axis.

Conserved amino acid sequence of kisspeptin in the C-terminal 10 residues
The amino acid sequence of the medaka kisspeptin predicted from the KiSS-1 gene proved to be conserved in C-terminal 10 residues (kissepeptin-10, kp-10) after comparing mammalian and teleost kisspeptin sequences. This conservation of kp-10 suggests that it is essential for the biological activity, and its function is essential for reproduction in vertebrates in general, including teleosts. Intrinsic kisspeptin peptides have been purified from human placenta, which are composed of 54, 14, and 13 amino acid residues (40). We have not determined the endogenous forms of mature kisspeptin peptides in medaka, and it is an immediate future problem to identify it and assay for the biological activities.

Anatomy of KiSS-1 neurons in the medaka brain
We found two neuronal populations of KiSS-1 mRNA-expressing neurons in the NPPv and in the NVT. Many reports have also shown that KiSS-1 mRNA-expressing neurons or kisspeptin neurons in mammals have multiple populations, including arcuate nucleus and AVPV, which is suggested to be involved in the negative/positive feedback regulation of the HPG axis, respectively (15, 16, 17, 18). Although it is premature to discuss the homology of each medaka kisspeptin neuronal population with the mammalian counterpart, we should soon be able to discuss it by accumulating experimental evidence as shown here. We also found KiSS-1 mRNA expression in the testis as reported in human tissues (39) but did not in the ovary. Although the sensitivity of RT-PCR depends on the number of PCR cycles, it is evident that the testis shows a higher KiSS-1 expression level than the ovary. We also found KiSS-1 mRNA expression in the stomach of some fish. Functions of KiSS-1 and its mature peptides in the peripheral organs of vertebrates in general are still unclear and are worth future investigation.

Steroid-sensitive sexually dimorphic KiSS-1 mRNA expression in the NVT, which changes according to the reproductive states
We have shown that the NVT KiSS-1 neurons are positively regulated by ovarian estrogens in the OVX and estrogen replacement experiments, which suggested their involvement in the positive feedback of the HPG axis. The KiSS-1 expressions in the NVT were also found to change dramatically between breeding and nonbreeding conditions in accordance with changes in the GSI. The GSI of the LD conditioned medaka was significantly higher than that of the SD conditioned medaka in both males and females, which agrees well with the fact that medaka is a LD breeder. These results are also consistent with the results that NVT KiSS-1 neurons decreased in number after OVX. It is strongly suggested that KiSS-1 neurons, at least those in the NVT, play important roles in the central regulation of reproductive functions. On the other hand, we found a prominent sexual difference in cell number (male >> female) in the NVT KiSS-1 neurons. However, recent studies in mammals have shown that the female AVPV KiSS-1 neurons, which are also positively regulated by ovarian estrogens (15, 41, 42), are more numerous than the male ones in mice and rats (16, 43). The fact that KiSS-1 expression is higher in female than in male mice is evidence for the positive feedback hypothesis of kisspeptin neurons in the AVPV (42). On the other hand, KiSS-1 neurons in medaka NVT, which are suggested to be up-regulated by sex steroid, were more numerous in males than females. This inconsistency may be explained by the presence of a positive feedback, which has been reported in the male goldfish to be induced by favorable breeding conditions, especially in the presence of a female (44). The castrated human (45) as well as the castrated monkeys show LH surges equivalent to those of the females (reviewed in Ref. 46). In this regard, the teleost may be a good model for the study of LH surge generation mechanisms in male brains. Thus, the origin and physiological effects of this sexually dimorphic KiSS-1 expression in medaka NVT are worth studying in detail. However, it is difficult at present to determine if the present result is only due to the effects of sex steroids, regardless of the genetic sex or to the sex determination in the early stages or both. It may be necessary in the future to examine the effects of acute administration of sex steroids on the KiSS-1 neurons or sex reversal by exposing the fish to the sex steroids in the early stages.

In the present experiments, it should be noted that the numbers of NVT KiSS-1 neurons in intact or sham-operated fish among the series of experiments were different (Figs. 2–5GoGoGoGo). We used the fish under different conditions in Figs. 2–5GoGoGoGo. Thus, it is quite natural that the reproductive conditions of the fish were different among the sham-operated fish, which were kept in isolation (Fig. 4Go), in one male-one female-paired tank (Figs. 2Go and 3Go), and in a mixed population of some males and females (Fig. 5Go). However, it should be stressed that we used the fish under the same conditions for each category of experiment, e.g. all the fish for the sham, OVX, and OVX + E in Fig. 4Go were kept in isolation. All the tissue preparations for each figure were processed for in situ hybridization at the same time under the identical conditions. Therefore, the comparisons within each category of experiment are valid.

Unfortunately, we could not examine the sensitivity of kisspeptin neurons to androgens in the male kisspeptin neurons because of the technical difficulty of male castration in medaka. This problem should be solved in future studies, possibly in some other fish species. In relation to the arguments on the possible physiological functions of kisspeptin other than the regulation of ovulation, it is noteworthy that the kisspeptin receptor GPR54 has been expressed in all three types of GnRH neuronal systems (20), including the extrahypothalamic nonhypophysiotropic GnRH systems, which are suggested to regulate certain aspects of sexual behaviors, e.g. the terminal nerve GnRH neurons (47, 48, 49). Therefore, it is possible that kisspeptin regulates certain aspects of sexual behaviors by acting on these extrahypothalamic GnRH neurons. Such a physiological function of kisspeptin in the regulation of sexual behaviors via extrahypothalamic nonhypophysiotropic GnRH systems, in addition to its facilitatory function of LH release via the hypothalamic hypophysiotropic GnRH system, is definitely one of the most interesting topics in future studies.

Previous studies on peptidergic neurons have shown that orexin neuronal cell bodies (50) and melanin-concentrating hormone (MCH) cell bodies are localized in the teleost NVT (51). The orexin neurons are generally supposed to be involved in energy homeostasis, arousal conditions, etc. (50, 52, 53, 54), whereas the MCH neurons are suggested to regulate appetite (55, 56, 57). Considering the proximity of kisspeptin neurons, orexin neurons, and MCH neurons in the NVT, it is an interesting future topic to study the neural relationships among these neurons, and how the arousal states, energy homeostasis, and reproduction are cooperatively regulated by these neurons.

Steroid insensitive stable KiSS-1 mRNA expression in the small number of NPPv KiSS-1 neurons
In contrast to the NVT kisspeptin neurons, we found neither sex difference nor sex steroid sensitivity of NPPv kisspeptin neurons, which were less than 10 in total. This result is different from that in mice and rats in which both the KiSS-1 expressing neuronal populations showed steroid sensitivities (41, 42, 58). Interestingly, in the ewe it has been reported that KiSS-1 mRNA expression in the POA is not regulated by either estrogens or progesterone, despite the fact that 50% of kisspeptin-ir cells in the POA express estrogen receptor {alpha} (14, 59). Further studies on the physiological functions of steroid insensitive kisspeptin neurons such as those in NPPv in the present paper are necessary, considering the possibility that they are engaged in the functions other than the regulation of the HPG axis. Although the number of NPPv KiSS-1 neurons was small, they showed stable expression of KiSS-1, even in the absence of ovary. Considering their steroid insensitivity and nonsexual difference in number, we suggest that the functions of NPPv KiSS-1 neurons are different from those in the NVT.

There are some reports that peptidergic neurons, such as neuropeptide Y (NPY) neurons and pancreatic polypeptide (PPY) neurons, are localized in the NPPv of cyprinid teleosts or sea bass (60, 61). Furthermore, NPY and PPY neurons were shown to regulate food intake (62, 63). Therefore, it is interesting to elucidate the functional relationship between food intake and reproduction by studying the neural relationships between NPY/PPY neurons and kisspeptin neurons.

To summarize, we report on the initial identification and characterization of the nonmammalian KiSS-1 neurons and the presence of two distinctive populations of KiSS-1 mRNA expressing neurons in two hypothalamic nuclei, the NVT and the NPPv. The NVT KiSS-1 neurons increased their expressions under the breeding conditions and showed conspicuous sexual difference (the number of NVT Kiss-1 neurons in males >> females) and steroid sensitivity, whereas the NPPv KiSS-1 neurons were neither sexually dimorphic nor steroid sensitive. Because the teleost brains have well-developed hypothalamic and extrahypothalamic GnRH neuronal systems, which are suited for the cellular and molecular neurobiological analyses (26, 35), the present study should provide important bases for the general study of physiological properties and functions of kisspeptin neurons of the vertebrate brains, using the teleosts as a good model. The medaka may prove to be especially promising because of its advantages for the application of various molecular genetic tools such as transgenic technologies combined with various electrophysiological and imaging techniques.


   Acknowledgments
 
We thank Drs. Hiroyuki Takeda, Atsuko Shimada, and Daisuke Kobayashi, and Ms. Yasuko Ozawa (University of Tokyo, Tokyo, Japan) for providing the d-rR strain medaka and helpful technical advice. We also thank Drs. M. K. Park, Hideki Abe, Tadahiro Ikemoto (University of Tokyo, Tokyo, Japan), Kataaki Okubo (National Institute for Basic Biology, Okazaki, Japan), Masafumi Amano (Kitasato University, Sagamihara, Japan), and Makito Kobayashi (International Christian University, Tokyo, Japan) for helpful technical advice and discussion.


   Footnotes
 
This work was supported by Grants-in-Aid from the Japan Society for the Promotion of Science (18370029), Ministry of Education, Culture, Sports, Science and Technology (18021009), and the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) of Japan (to Y.O.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 17, 2008

Abbreviations: AVPV, Anteroventral periventricular nucleus; DIG, digoxigenin; E, β-estradiol; GOLT1A, Golgi transport 1 homolog A; GPR54, G protein-coupled receptor 54; GSI, gonadosomatic index; HPG, hypothalamo-pituitary-gonadal; LD, long-day; MCH, melanin-concentrating hormone; NPPv, nucleus posterioris periventricularis; NPY, neuropeptide Y; NVT, nucleus ventral tuberis; OVX, ovariectomy; POA, preoptic area; PPY, pancreatic polypeptide; SD, short-day; SSC, saline sodium citrate.

1 These sequence data have been submitted to the DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank databases under accession no. AB272755. Back

Received November 1, 2007.

Accepted for publication January 7, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals