Neuroscience Letters 561 (2014) 203–207
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Functional analysis of kisspeptin peptides in adult immature chub mackerel (Scomber japonicus) using an intracerebroventricular administration method Hirofumi Ohga, Sethu Selvaraj, Hayato Adachi, Yui Imanaga, Mitsuo Nyuji, Akihiko Yamaguchi, Michiya Matsuyama ∗ Laboratory of Marine Biology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581 Japan
h i g h l i g h t s • Synthetic Kisspeptin peptides were administrated into the ICV region. • gnrh1 levels decreased by both Kisspeptin peptides in POA at 12 h post-injection. • fshˇ and lhˇ levels increased by Kiss2-12 peptides in pituitary at 12 h post-injection.
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Article history: Received 29 October 2013 Received in revised form 16 December 2013 Accepted 31 December 2013 Keywords: Kiss1-15 Kiss2-12 Perciformes
a b s t r a c t In vertebrates (including teleosts), the pivotal hierarchical factor in the control of gonadotropin secretion is the hypothalamic gonadotropin-releasing hormone (GnRH) decapeptide, which regulates the release of pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Recently, kisspeptins encoded by the Kiss1 gene have been shown to act as upstream endogenous regulators of GnRH neurons in mammals. The chub mackerel (Scomber japonicus) brain expresses two kiss genes (kiss1 and kiss2) that show sexually dimorphic expression profiles during the seasonal gonadal cycle. In the present study, we evaluated the biological potency of kisspeptin peptides to induce transcriptional changes in gnrh1 (hypophysiotropic GnRH form in this species), fshˇ and lhˇ during the immature stage of adult chub mackerel (2+ years old). Synthetic Kiss1 pentadecapeptide (Kiss1-15) or Kiss2 dodecapeptide (Kiss2-12) at a dose of 100 ng were administered into the intracerebroventricular (ICV) region, and brains were sampled at 6 and 12 h post-injection. In female fish, gnrh1 levels decreased in the presence of both kisspeptin peptides at 12 h post-injection. No significant variation was observed in male fish. In contrast, ICV administration of Kiss2-12 (but not Kiss1-15) significantly increased fshˇ and lhˇ mRNAs at 12 h post-injection compared to a saline injected control in both sexes. These results suggested that synthetic Kiss2-12 could induce transcriptional changes in gnrh1 and gths. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In teleosts, like other vertebrates, reproductive processes are regulated by the precise coordination of neuroendocrine hormones acting through the brain–pituitary–gonad (BPG) axis. Gonadotropin-releasing hormone (GnRH) from the hypothalamus stimulates the synthesis and release of pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH); these hormones act on the gonads to induce oogenesis and spermatogenesis by stimulating the production of sex steroids.
∗ Corresponding author. Tel.: +81 92 642 2888; fax: +81 92 642 2888. E-mail address: rinya [email protected] (M. Matsuyama). 0304-3940/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.12.072
Kisspeptins (Kiss1), a member of the RFamide peptide family, are known to be potent upstream regulators of the reproductive BPG axis in vertebrates, including teleosts [32]. This fact is supported by studies demonstrating that both Kiss1 receptor (Kiss1R)-knockout mice and mutations in Kiss1R in patients cause reproductive/pubertal failure and autosomal recessive idiopathic hypogonadotropic hypogonadism [3,31]. Subsequent studies in mammals indicated that Kiss1/Kiss1R act as major upstream regulators of gonadotropin secretion, mediated by controlling GnRH secretion. Endogenous fragments of mammalian kisspeptin peptides; namely, Kiss-10, -13, -14, and -54 amino acids (aa) in length share a common core 10-aa sequence at their C-terminus, and all possess equal binding affinity to Kiss1R [12]. The sequence of Kiss-10 is highly conserved across vertebrate species. In contrast to the
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Fig. 1. Schematic illustration of coordinates for injection into the intracerebroventricular region of chub mackerel. (A) Stereotaxic coordinates for X and Y axis. (B) Crosssection view after injection of PBS with 1% Evans blue (Wako) to validation of coordinates for insert of needle into the third ventricle and stereotaxic coordinates for Z axis.
presence of a single Kiss1 and Kiss1R genes in placental mammals, multiple kiss ligand (kiss1 and kiss2) and receptor (kissr1 and kissr2) genes have been identified in non-mammalian species, including teleosts [32]. Recent studies have shown that the fish Kiss1 precursor contains a conserved dibasic five amino acid site upstream of the decapeptide, indicating that the fish kiss1 gene produces a mature 15-aa peptide (Kiss1-15) [13]. Similarly, a conserved Arg at position 13 is present in all available fish Kiss2 sequences, indicating that a putative cleavage site exists that produces mature Kiss2-12 peptide [13]. Interestingly, retention of two functional kiss and kissr genes in teleosts has provided a unique opportunity to explore how the duplicated Kiss/KissR system may have evolved its specialized functions. In teleosts, previous studies have reported that administration of Kiss2-10 up-regulates the expression of pituitary fshˇ and lhˇ in zebrafish (Danio rerio) [11] and promotes LH secretion in sea bass (Dicentrarchus labrax) [5]. On the contrary, Kiss1-10 administration in goldfish (Carassius auratus) up-regulated plasma LH levels [14]. These studies suggest that the two forms of kisspeptins may play different biological roles in fish species. We have previously identified the key molecular elements of the chub mackerel (Scomber japonicus) BPG axis; namely, kisspeptins [27], kisspeptin receptors [22], GnRHs [28], GtHs [17,21], and GtH receptors [18]. The chub mackerel brain expresses two kiss and receptor genes and shows sexually dimorphic changes during the seasonal gonadal cycle [22,27]. Recently, we characterized the signal transduction pathways using in vitro reporter gene assays. Synthetic chub mackerel Kiss1-15 and Kiss2-12 peptides showed the highest activity towards KissR1 and KissR2, respectively, which was stronger than their corresponding Kiss-10 peptides [22]. Furthermore, pharmacological analyses of kisspeptin peptides indicated that peripheral administration of chub mackerel Kiss1-15 induces spermiation in pre-pubertal and sexually immature adult male chub mackerel [29,30]. Based on these facts, the present study evaluated the role of synthetic chub mackerel Kiss1-15 and Kiss2-12 in immature adult chub mackerel (2+ years old) using an intracerebroventricular (ICV) administration method. 2. Materials and methods 2.1. Animals and experimental set up Sexually immature adult chub mackerel (2+ years old) were caught with a purse seine and reared in sea pens at a fish farm in Oita
prefecture, Kyushu Island. The fish was transferred to experimental tanks (three ton capacity) prior to the experiment in November 2012 and stocked with running sea water. The experiment was performed 6 times during 15–28 November 2012. Injection was started on 10:00 h and one experimental plot was conducted at each time. At the time of sampling, the fish were carefully treated and in agreement with the laws (No. 105) and declaration (No. 6) of the Japanese Government. 2.2. Synthetic peptides Synthetic peptides corresponding to chub mackerel (cm) Kiss115 (QDMSSYNFNSFGLRY-NH2 ; GenBank accession No. GU731672) and Kiss2-12 (SNFNFNPFGLRF-NH2 ; GenBank accession No. GU731673) were purchased from Genenet Co. (Fukuoka, Japan). The cmKiss1-15 and cmKiss2-12 had a purity level of 88.54% and 100%, as determined by analytical HPLC, respectively. Each stock peptide (1 mg) was initially diluted with saline solution (0.8% NaCl containing 0.042% KCl, 0.025% CaCl2 and 0.02% MgCl2 6H2 O) and stored at −80 ◦ C until use. A fresh preparation was used on each day of injection. These stock peptides were previously used for pharmacological analyses and found to possess biological activities under in vivo and in vitro conditions [22,29,30]. 2.3. Administration procedure Intracerebroventricular (ICV) injections were performed based on the ratio of the head region of adult chub mackerel. We determined that if head length (rostral end to upper end of gill) was assumed to be X and length of the rostral end to immediately above the third ventricle was assumed to be Y, the ratio of X to Y converged from 1.4 to 1.0. In our preliminary analyses, we measured the above values using adult chub mackerel (n = 290), and the X: Y ratio was found to be = 1.404 ± 0.001994: 1 (means ±SEM) (Fig. 1A). Peptide administration was performed as described previously [2,20,23]. Fish were netted from the holding tank and anesthetized with 2-phenoxyethanol (100 mg/l) until they lost equilibrium. The fish was held in a stereotaxic apparatus and head length (X) was immediately measured using calipers. The injection position was midline of the head Y mm (X divided by 1.4 equals Y), posterior to the rostal end (Fig. 1A). The coordinates for implantation of the needle into the third ventricle was validated by injecting PBS with 1% Evans blue (Wako) and observing the dye distribution pattern, as described below (Fig. 1B). Using a 10-l glass syringe with a 26-G needle (Hamilton #701, Hamilton Co., Reno, NV, USA), 5 l
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Fig. 2. Analysis of the effect of ICV administration of cmKiss1-15 and cmKiss2-12 peptides on gnrh1 mRNA levels in the preoptic region of adult male (A) and female (B) immature chub mackerel. Transcription levels are the mean ± SEM of 9–16 independent determinations. Different letters above the bars represent significant differences (P < 0.05).
of solution containing peptides (20 ng/l) were administered into the third ventricle to a depth of 13 mm below the tissue surface. To prevent excess leakage of the chemicals, the needle was held in the same position for 5 s after infusion to allow for clearance. The ICV injection procedure took approximately 30 s per fish. Accuracy of the peptide delivery into the third ventricle and fish recovery rate after ICV injections were 95% and 97.5%, respectively (n = 135). Groups of fish (n = 21–25) were sampled 0, 6 and 12 h after ICV injection. During all sampling periods, the brain and pituitary were stored in RNA later (Qiagen, Hilden, Germany) solution until use.
4. Results
2.4. Real-time PCR
4.2. Effect of administration of kisspeptins on fshˇ and lhˇ mRNA expression
Quantitative real-time PCR analysis was performed on an Mx 3000P quantitative PCR system (Stratagene, USA). The brain tissue was divided into seven regions according to Selvaraj et al. [29], and only, the preoptic area (POA) region was used for gnrh1 mRNA expression analysis. Total RNA was extracted using ISOGENE (Nippon Gene, Japan), according to the manufacturer’s protocol. The RNA concentrations were estimated using UltrospecTM 3000 pro spectrophotometer at 260 nm. Two microgram of total RNA from each tissue sample was digested with DNase I (Invitrogen) according to the manufacturer’s protocol and used as template for reverse transcription reaction, as described previously [22]. All transcripts were quantified using a standard curve method and previously validated qRT-PCR for gnrh1, fshˇ and lhˇ mRNAs [17,28]. PCR mixture (10 l) contained 1.5 l of sample or standard cDNA, 0.1 M of sense and antisense primers, 3.75 l of PCR-grade water, 0.05 l of ROX dye and 5 l of Brilliant III Ultra-Fast SYBR Green QPCR master mix (Agilent, CA, USA). For negative control, cDNA sample was replaced with PCR-grade water. Duplicate reactions were performed for standards, target and reference genes. PCR conditions were set as follow: 95 ◦ C (5 min); 40 cycles at 95 ◦ C for 10 s, 60 ◦ C for 30 s. Melting curve analysis was also included at 1 cycle of 94 ◦ C for 1 min, 60 ◦ C for 30 s, 95 ◦ C for 30 s. These data were analyzed using MaxPro-Mx3000P software version 3.00 (Stratagene, USA). All qRT-PCR assays were conducted where practically possible according to the MIQE (Minimum Information for Publication of qRT-PCR experiments) guidelines by Bustin et al. [1]. 3. Statistical analysis Data were expressed as means ±SEM (standard errors of the mean), and analyzed by one-way ANOVA followed by a Tukey’s Multiple Comparison Test using Prism 4 (GraphPad Software, San Diego, CA, USA).
4.1. Effect of administration of kisspeptins on gnrh1 mRNA expression There was no variation in gnrh1 mRNA levels in the POA region of male fish injected with cmKiss1-15 and cmKiss2-12 compared to the saline-injected control (Fig. 2A). In female fish, injected with kisspeptin peptides, gnrh1 mRNA levels decreased at 12 h postinjection compared to saline-injected control (Fig. 2B).
ICV administration of cmKiss2-12 (but not cmKiss1-15) significantly increased fshˇ and lhˇ mRNA levels at 12 h post-injection compared to the saline-injected control in both sexes (Fig. 3). In contrast, at 6 h post-injection, fshˇ and lhˇ mRNA levels showed significant increase compared to 0 h control; however, no significant difference was observed in comparison with the saline-injected control in both sexes. Accordingly, there remains a possibility that a daily fluctuation of target genes. 5. Discussion In most mammalian species, kisspeptin has a strong stimulatory effect on gonadotropin secretion. This is mostly mediated by the stimulatory action on GnRH release. Indeed, ICV administration of kisspeptin peptide (Kiss-10) in sheep induced a large and sustained release of GnRH into the cerebrospinal fluid [15]. Similarly, ICV administration of Kiss-10 rapidly increased plasma LH levels in rodents; this effect was blocked by pretreatment with GnRH antagonist [6,16]. However, few studies have assessed the role of kisspeptin in non-mammalian species due to the technical difficulties in central administration. In this study, the acute effects of ICV administration of highly active chub mackerel peptides Kiss1-15 and Kiss2-12 in 2-year-old adult immature male and female fish were examined. The third ventricle was chosen as the site of injection because of its proximity to brain centers controlling reproductive activity. Therefore, injections into the third ventricle are most likely to affect the areas of the diencephalon, including the preoptic nucleus, nucleus preopticus magnocelluraris, and nucleus preopticus parvocellularis [2]. The experimental fish chub mackerel expresses three GnRH forms in the brain, and GnRH1 synthesizing neurons, which are specifically distributed in the preoptic nucleus, are known to send
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Fig. 3. Analysis of the effect of ICV administration of cmKiss1-15 and cmKiss2-12 peptides on fshˇ (A and B) and lhˇ (C and D) mRNA levels in the pituitary, each together in adult male (A and C) or female (B and D) immature chub mackerel. Transcription levels are the mean ± SEM of 9–16 independent determinations. Different letters above the bars represent significant differences (P < 0.05).
axonal projections to the anterior pituitary regions where FSH and LH producing cells are localized [26]. Thus, it is possible that ICV injected kisspeptin peptides modulate transcriptional changes in GnRH1 neurons. We observed a sexual dimorphic expression change in gnrh1. In the present results, levels of gnrh1 mRNA in the POA region of male fish did not show a response to both Kiss1-15 and Kiss2-12 peptides at 12 h post-injection (Fig. 2A). However, for female fish, administration of either Kiss1-15 or Kiss2-12 peptide decreased gnrh1 mRNA levels at 12 h post-injection (Fig. 2B). In mammals, it is known that GnRH1 neurons co-express Kiss1R to mediate downstream effects [15,19], and Kiss1 immunoreactivity levels play a role in sexual dimorphism since the number of Kiss-synthesizing neurons are higher in females than males [8]. Our previous studies demonstrated that kiss1 and kissr2 transcription levels in the brain are higher in females than males [22,27]. Based on this information, sex-specific action of the kisspeptin system on regulation of the BPG axis in chub mackerel is possible. Indeed, in medaka (Oryzias latipes), a sex difference in the number of nucleus ventral tuberiskiss1 neurons has been demonstrated [9]. Presently, in teleosts, the Kiss-KissR-GnRH relationship remains unclear. Teleost fish brain expresses multiple GnRH forms, and in the cichlid fish Nile tilapia (Oreochromis niloticus), all three types of GnRH neurons (gnrh1, gnrh2 and gnrh3) are known to express the kisspeptin receptor [24]. A recent study in striped bass (Morone saxatilis) identified two different modes of GnRH1 regulation, where kissr2 was co-localized in GnRH1 neurons and kissr1 was expressed in cells attached to GnRH1 fibers in the POA [34]. Levels of gnrh1 mRNA in the brains of prepubertal fish increased in response to Kiss1-15 and Kiss2-12 peptides at 24 h post-injection [34]. In contrast to the above findings, Grone et al. [7] in an African cichlid fish (Astatotilapia burtoni) reported that some cells in the POA expressing gnrh1 do not co-express kisspeptin receptor. Likewise, in medaka it was clearly demonstrated that kissr-expressing cells
were located adjacent to GnRH1 neurons, although they were not GnRH1 neurons themselves [10]. For chub mackerel, there is no anatomical evidence to suggest that the kisspeptin system is directly or indirectly involved in the regulation of GnRH neurons. A recent study in 3-year-old spermiating male sea bass found that ICV injection of Kiss2-12 evoked GnRH1 release into the pituitary and increased serum FSH and LH levels [4]. Furthermore, only Kiss2-12 increased the plasma levels of sex steroids and milt volume, suggesting that the ICV administration of Kiss2-12 potently stimulates the BPG-axis via the hypothalamic GnRH system [4]. Similarly, in the present study, a single ICV injection of synthetic Kiss2-12 increased pituitary fshˇ and lhˇ mRNA levels at 12 h post-injection compared to the saline-injected control (Fig. 3). Our analyses suggest that chub mackerel kisspeptin peptides play a central role in the control of gonadotropin secretion with differential potencies. When the relative potencies of these peptides were compared, it is clear that the cmKiss2-12 peptide is a potent elicitor of gonadotropin synthesis compared to cmKiss1-15 in this species. However, activation of gonadotropins by Kiss2-12 through preoptic GnRH neurons or KissR at the level of the pituitary requires future investigation, because it is possible that kisspeptin directly stimulates gonadotropin secretion through KissR in the pituitary of mammals [25] and goldfish [33] in vitro. Indeed, our recent study demonstrated that chub mackerel expresses kissr2 gene in the pituitary, and KissR2 is the intrinsic receptor for Kiss2-12 peptides in this species [22]. Future studies on the localization of the Kisspeptin–GnRH–GtH system and in vitro analyses on the effect of synthetic kisspeptin peptides to stimulate gonadotropin secretion in the pituitary will increase our understanding of the role of the kisspeptin system in this species. In conclusion, this study revealed that ICV administration of Kiss2-12 stimulates pituitary fshˇ and lhˇ mRNA synthesis, suggesting that kisspeptin peptides play a central role in control of the reproductive BPG axis in this species.
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Acknowledgment We are also grateful to Sanny David Lumayno, Naoki Sugihara, Shugo Igarashi, Ryoko Kodama, and Kayo Yashiki for their assistance during fish sampling. This work was supported by a grant for scientific research (23658163) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), and through a subproject on studies on the prediction and application of fish species alternation (SUPRFISH) financed by the Agriculture, Forestry, and Fisheries Research Council (AFFRC) of Japan, as part of the Population Outbreak of Marine Life (POMAL) Project. References [1] S. Bustin, V. Benes, J. Garson, J. Hellemans, J. Huggett, M. Kubista, R. Mueller, T. Nolan, M. Pfaffl, G. Shipley, J. Vandesompele, C. Wittwer, The MIQE guidelines: minimal information for publication of quantitative real-time PCR experiments, Clin. Chem. 55 (2009) 611–622. [2] S. Clements, C.B. Schreck, The GABAa agonist muscimol enhances locomotor activity, but does not alter the behavioural effects of CRH in juvenile spring Chinook salmon (Oncorhynchus tshawytscha), Fish Physiol. Biochem. 24 (2001) 41–48. [3] N. de Roux, E. Genin, J.C. Carel, F. Matsuda, J.L. Chaussain, E. Milgrom, Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 10972–10976. [4] F. Espigares, M. Carrillo, A. Gómez, S. Zanuy, Central administration of kiss1 and kiss2 peptides stimulates the hypothalamic-pituitary-gonadal (HPG) axis in male European sea bass (Dicentrarchus labrax), 17th ICCE 2013 Barcelona Congress P-040. [5] A. Felip, S. Zauny, R. Pineda, L. Pinilla, M. Carrillo, M. Tena-Sempere, A. Gomez, Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals, Mol. Cell. Endocrinol. 312 (2009) 61–71. [6] M.L. Gottsch, M.J. Cunningham, J.T. Smith, S.M. Popa, B.V. Acohido, W.F. Crowley, S. Seminara, D.K. Clifton, R.A. Steiner, A role for kisspeptins in the regulation of gonadotropin secretion in the mouse, Endocrinology 145 (2004) 4073–4077. [7] B.P. Grone, K.P. Maruska, W.J. Korzan, R.D. Fernald, Social status regulates kisspeptin receptor mRNA in the brain of Astatotilapia burtoni, Gen. Comp. Endocrinol. 169 (2010) 98–107. [8] E. Hrabovszky, P. Ciofi, B. Vida, M.C. Horvath, E. Keller, A. Caraty, S.R. Bloom, M.A. Ghatei, W.S. Dhillo, Z. Liposits, I. Kallo, The kisspeptin system of the human hypothalamus: sexual dimorphism and relationship with gonadotropin-releasing hormone and neurokinin B neurons, Eur. J. Neurosci. 31 (2010) 1984–1998. [9] S. Kanda, Y. Akazome, T. Matsunaga, N. Yamamoto, S. Yamada, H. Tsukamura, K. Maeda, Y. Oka, Identification of KiSS-1 product kisspeptin and steroid-sensitive sexually dimorphic kisspeptin neurons in medaka (Oryzias latipes), Endocrinology 149 (2008) 2467–2476. [10] S. Kanda, Y. Akazome, Y. Mitani, K. Okubo, Y. Oka, Neuroanatomical evidence that kisspeptin directly regulates isotocin and vasotocin neurons, Plos One 8 (2013) e62776. [11] T. Kitahashi, S. Ogawa, I.S. Parhar, Cloning and expression of kiss2 in the zebrafish and medaka, Endocrinology 150 (2009) 821–831. [12] M. Kotani, M. Detheux, A. Vandenbogaerde, D. Communi, J.M. Vanderwinden, E. Le Poul, S. Brezillon, R. Tyldesley, N. Suarez-Huerta, F. Vandeput, C. Blanpain, S.N. Schiffmann, G. Vassart, M. Parmentier, The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54, J. Biol. Chem. 276 (2001) 34631–34636. [13] Y.R. Lee, K. Tsunekawa, M.J. Moon, H.N. Um, J.I. Hwang, T. Osugi, N. Otaki, Y. Sunakawa, K. Kim, H. Vaudry, H.B. Kwon, J.Y. Seong, K. Tsutsui, Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates, Endocrinology 150 (2009) 2837–2846. [14] S. Li, Y. Zhang, Y. Liu, X. Huang, W. Huang, D. Lu, P. Zhu, Y. Shi, C.H. Cheng, X. Liu, H. Lin, Structural and functional multiplicity of the kisspeptin/GPR54 system in goldfish (Carassius auratus), J. Endocrinol. 201 (2009) 407–418. [15] S. Messager, E.E. Chatzidaki, D. Ma, A.G. Hendrick, D. Zahn, J. Dixon, R.R. Thresher, I. Malinge, D. Lomet, M.B.L. Carlton, W.H. Colledge, A. Caraty, S.A.J.R. Aparicio, Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein-coupled receptor 54, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 1761–1766.
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Functional analysis of kisspeptin peptides in adult immature chub mackerel (Scomber japonicus) using an intracerebroventricular administration method Hirofumi Ohga, Sethu Selvaraj, Hayato Adachi, Yui Imanaga, Mitsuo Nyuji, Akihiko Yamaguchi, Michiya Matsuyama ∗ Laboratory of Marine Biology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581 Japan
h i g h l i g h t s • Synthetic Kisspeptin peptides were administrated into the ICV region. • gnrh1 levels decreased by both Kisspeptin peptides in POA at 12 h post-injection. • fshˇ and lhˇ levels increased by Kiss2-12 peptides in pituitary at 12 h post-injection.
a r t i c l e
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Article history: Received 29 October 2013 Received in revised form 16 December 2013 Accepted 31 December 2013 Keywords: Kiss1-15 Kiss2-12 Perciformes
a b s t r a c t In vertebrates (including teleosts), the pivotal hierarchical factor in the control of gonadotropin secretion is the hypothalamic gonadotropin-releasing hormone (GnRH) decapeptide, which regulates the release of pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Recently, kisspeptins encoded by the Kiss1 gene have been shown to act as upstream endogenous regulators of GnRH neurons in mammals. The chub mackerel (Scomber japonicus) brain expresses two kiss genes (kiss1 and kiss2) that show sexually dimorphic expression profiles during the seasonal gonadal cycle. In the present study, we evaluated the biological potency of kisspeptin peptides to induce transcriptional changes in gnrh1 (hypophysiotropic GnRH form in this species), fshˇ and lhˇ during the immature stage of adult chub mackerel (2+ years old). Synthetic Kiss1 pentadecapeptide (Kiss1-15) or Kiss2 dodecapeptide (Kiss2-12) at a dose of 100 ng were administered into the intracerebroventricular (ICV) region, and brains were sampled at 6 and 12 h post-injection. In female fish, gnrh1 levels decreased in the presence of both kisspeptin peptides at 12 h post-injection. No significant variation was observed in male fish. In contrast, ICV administration of Kiss2-12 (but not Kiss1-15) significantly increased fshˇ and lhˇ mRNAs at 12 h post-injection compared to a saline injected control in both sexes. These results suggested that synthetic Kiss2-12 could induce transcriptional changes in gnrh1 and gths. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In teleosts, like other vertebrates, reproductive processes are regulated by the precise coordination of neuroendocrine hormones acting through the brain–pituitary–gonad (BPG) axis. Gonadotropin-releasing hormone (GnRH) from the hypothalamus stimulates the synthesis and release of pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH); these hormones act on the gonads to induce oogenesis and spermatogenesis by stimulating the production of sex steroids.
∗ Corresponding author. Tel.: +81 92 642 2888; fax: +81 92 642 2888. E-mail address: rinya [email protected] (M. Matsuyama). 0304-3940/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.12.072
Kisspeptins (Kiss1), a member of the RFamide peptide family, are known to be potent upstream regulators of the reproductive BPG axis in vertebrates, including teleosts [32]. This fact is supported by studies demonstrating that both Kiss1 receptor (Kiss1R)-knockout mice and mutations in Kiss1R in patients cause reproductive/pubertal failure and autosomal recessive idiopathic hypogonadotropic hypogonadism [3,31]. Subsequent studies in mammals indicated that Kiss1/Kiss1R act as major upstream regulators of gonadotropin secretion, mediated by controlling GnRH secretion. Endogenous fragments of mammalian kisspeptin peptides; namely, Kiss-10, -13, -14, and -54 amino acids (aa) in length share a common core 10-aa sequence at their C-terminus, and all possess equal binding affinity to Kiss1R [12]. The sequence of Kiss-10 is highly conserved across vertebrate species. In contrast to the
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Fig. 1. Schematic illustration of coordinates for injection into the intracerebroventricular region of chub mackerel. (A) Stereotaxic coordinates for X and Y axis. (B) Crosssection view after injection of PBS with 1% Evans blue (Wako) to validation of coordinates for insert of needle into the third ventricle and stereotaxic coordinates for Z axis.
presence of a single Kiss1 and Kiss1R genes in placental mammals, multiple kiss ligand (kiss1 and kiss2) and receptor (kissr1 and kissr2) genes have been identified in non-mammalian species, including teleosts [32]. Recent studies have shown that the fish Kiss1 precursor contains a conserved dibasic five amino acid site upstream of the decapeptide, indicating that the fish kiss1 gene produces a mature 15-aa peptide (Kiss1-15) [13]. Similarly, a conserved Arg at position 13 is present in all available fish Kiss2 sequences, indicating that a putative cleavage site exists that produces mature Kiss2-12 peptide [13]. Interestingly, retention of two functional kiss and kissr genes in teleosts has provided a unique opportunity to explore how the duplicated Kiss/KissR system may have evolved its specialized functions. In teleosts, previous studies have reported that administration of Kiss2-10 up-regulates the expression of pituitary fshˇ and lhˇ in zebrafish (Danio rerio) [11] and promotes LH secretion in sea bass (Dicentrarchus labrax) [5]. On the contrary, Kiss1-10 administration in goldfish (Carassius auratus) up-regulated plasma LH levels [14]. These studies suggest that the two forms of kisspeptins may play different biological roles in fish species. We have previously identified the key molecular elements of the chub mackerel (Scomber japonicus) BPG axis; namely, kisspeptins [27], kisspeptin receptors [22], GnRHs [28], GtHs [17,21], and GtH receptors [18]. The chub mackerel brain expresses two kiss and receptor genes and shows sexually dimorphic changes during the seasonal gonadal cycle [22,27]. Recently, we characterized the signal transduction pathways using in vitro reporter gene assays. Synthetic chub mackerel Kiss1-15 and Kiss2-12 peptides showed the highest activity towards KissR1 and KissR2, respectively, which was stronger than their corresponding Kiss-10 peptides [22]. Furthermore, pharmacological analyses of kisspeptin peptides indicated that peripheral administration of chub mackerel Kiss1-15 induces spermiation in pre-pubertal and sexually immature adult male chub mackerel [29,30]. Based on these facts, the present study evaluated the role of synthetic chub mackerel Kiss1-15 and Kiss2-12 in immature adult chub mackerel (2+ years old) using an intracerebroventricular (ICV) administration method. 2. Materials and methods 2.1. Animals and experimental set up Sexually immature adult chub mackerel (2+ years old) were caught with a purse seine and reared in sea pens at a fish farm in Oita
prefecture, Kyushu Island. The fish was transferred to experimental tanks (three ton capacity) prior to the experiment in November 2012 and stocked with running sea water. The experiment was performed 6 times during 15–28 November 2012. Injection was started on 10:00 h and one experimental plot was conducted at each time. At the time of sampling, the fish were carefully treated and in agreement with the laws (No. 105) and declaration (No. 6) of the Japanese Government. 2.2. Synthetic peptides Synthetic peptides corresponding to chub mackerel (cm) Kiss115 (QDMSSYNFNSFGLRY-NH2 ; GenBank accession No. GU731672) and Kiss2-12 (SNFNFNPFGLRF-NH2 ; GenBank accession No. GU731673) were purchased from Genenet Co. (Fukuoka, Japan). The cmKiss1-15 and cmKiss2-12 had a purity level of 88.54% and 100%, as determined by analytical HPLC, respectively. Each stock peptide (1 mg) was initially diluted with saline solution (0.8% NaCl containing 0.042% KCl, 0.025% CaCl2 and 0.02% MgCl2 6H2 O) and stored at −80 ◦ C until use. A fresh preparation was used on each day of injection. These stock peptides were previously used for pharmacological analyses and found to possess biological activities under in vivo and in vitro conditions [22,29,30]. 2.3. Administration procedure Intracerebroventricular (ICV) injections were performed based on the ratio of the head region of adult chub mackerel. We determined that if head length (rostral end to upper end of gill) was assumed to be X and length of the rostral end to immediately above the third ventricle was assumed to be Y, the ratio of X to Y converged from 1.4 to 1.0. In our preliminary analyses, we measured the above values using adult chub mackerel (n = 290), and the X: Y ratio was found to be = 1.404 ± 0.001994: 1 (means ±SEM) (Fig. 1A). Peptide administration was performed as described previously [2,20,23]. Fish were netted from the holding tank and anesthetized with 2-phenoxyethanol (100 mg/l) until they lost equilibrium. The fish was held in a stereotaxic apparatus and head length (X) was immediately measured using calipers. The injection position was midline of the head Y mm (X divided by 1.4 equals Y), posterior to the rostal end (Fig. 1A). The coordinates for implantation of the needle into the third ventricle was validated by injecting PBS with 1% Evans blue (Wako) and observing the dye distribution pattern, as described below (Fig. 1B). Using a 10-l glass syringe with a 26-G needle (Hamilton #701, Hamilton Co., Reno, NV, USA), 5 l
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Fig. 2. Analysis of the effect of ICV administration of cmKiss1-15 and cmKiss2-12 peptides on gnrh1 mRNA levels in the preoptic region of adult male (A) and female (B) immature chub mackerel. Transcription levels are the mean ± SEM of 9–16 independent determinations. Different letters above the bars represent significant differences (P < 0.05).
of solution containing peptides (20 ng/l) were administered into the third ventricle to a depth of 13 mm below the tissue surface. To prevent excess leakage of the chemicals, the needle was held in the same position for 5 s after infusion to allow for clearance. The ICV injection procedure took approximately 30 s per fish. Accuracy of the peptide delivery into the third ventricle and fish recovery rate after ICV injections were 95% and 97.5%, respectively (n = 135). Groups of fish (n = 21–25) were sampled 0, 6 and 12 h after ICV injection. During all sampling periods, the brain and pituitary were stored in RNA later (Qiagen, Hilden, Germany) solution until use.
4. Results
2.4. Real-time PCR
4.2. Effect of administration of kisspeptins on fshˇ and lhˇ mRNA expression
Quantitative real-time PCR analysis was performed on an Mx 3000P quantitative PCR system (Stratagene, USA). The brain tissue was divided into seven regions according to Selvaraj et al. [29], and only, the preoptic area (POA) region was used for gnrh1 mRNA expression analysis. Total RNA was extracted using ISOGENE (Nippon Gene, Japan), according to the manufacturer’s protocol. The RNA concentrations were estimated using UltrospecTM 3000 pro spectrophotometer at 260 nm. Two microgram of total RNA from each tissue sample was digested with DNase I (Invitrogen) according to the manufacturer’s protocol and used as template for reverse transcription reaction, as described previously [22]. All transcripts were quantified using a standard curve method and previously validated qRT-PCR for gnrh1, fshˇ and lhˇ mRNAs [17,28]. PCR mixture (10 l) contained 1.5 l of sample or standard cDNA, 0.1 M of sense and antisense primers, 3.75 l of PCR-grade water, 0.05 l of ROX dye and 5 l of Brilliant III Ultra-Fast SYBR Green QPCR master mix (Agilent, CA, USA). For negative control, cDNA sample was replaced with PCR-grade water. Duplicate reactions were performed for standards, target and reference genes. PCR conditions were set as follow: 95 ◦ C (5 min); 40 cycles at 95 ◦ C for 10 s, 60 ◦ C for 30 s. Melting curve analysis was also included at 1 cycle of 94 ◦ C for 1 min, 60 ◦ C for 30 s, 95 ◦ C for 30 s. These data were analyzed using MaxPro-Mx3000P software version 3.00 (Stratagene, USA). All qRT-PCR assays were conducted where practically possible according to the MIQE (Minimum Information for Publication of qRT-PCR experiments) guidelines by Bustin et al. [1]. 3. Statistical analysis Data were expressed as means ±SEM (standard errors of the mean), and analyzed by one-way ANOVA followed by a Tukey’s Multiple Comparison Test using Prism 4 (GraphPad Software, San Diego, CA, USA).
4.1. Effect of administration of kisspeptins on gnrh1 mRNA expression There was no variation in gnrh1 mRNA levels in the POA region of male fish injected with cmKiss1-15 and cmKiss2-12 compared to the saline-injected control (Fig. 2A). In female fish, injected with kisspeptin peptides, gnrh1 mRNA levels decreased at 12 h postinjection compared to saline-injected control (Fig. 2B).
ICV administration of cmKiss2-12 (but not cmKiss1-15) significantly increased fshˇ and lhˇ mRNA levels at 12 h post-injection compared to the saline-injected control in both sexes (Fig. 3). In contrast, at 6 h post-injection, fshˇ and lhˇ mRNA levels showed significant increase compared to 0 h control; however, no significant difference was observed in comparison with the saline-injected control in both sexes. Accordingly, there remains a possibility that a daily fluctuation of target genes. 5. Discussion In most mammalian species, kisspeptin has a strong stimulatory effect on gonadotropin secretion. This is mostly mediated by the stimulatory action on GnRH release. Indeed, ICV administration of kisspeptin peptide (Kiss-10) in sheep induced a large and sustained release of GnRH into the cerebrospinal fluid [15]. Similarly, ICV administration of Kiss-10 rapidly increased plasma LH levels in rodents; this effect was blocked by pretreatment with GnRH antagonist [6,16]. However, few studies have assessed the role of kisspeptin in non-mammalian species due to the technical difficulties in central administration. In this study, the acute effects of ICV administration of highly active chub mackerel peptides Kiss1-15 and Kiss2-12 in 2-year-old adult immature male and female fish were examined. The third ventricle was chosen as the site of injection because of its proximity to brain centers controlling reproductive activity. Therefore, injections into the third ventricle are most likely to affect the areas of the diencephalon, including the preoptic nucleus, nucleus preopticus magnocelluraris, and nucleus preopticus parvocellularis [2]. The experimental fish chub mackerel expresses three GnRH forms in the brain, and GnRH1 synthesizing neurons, which are specifically distributed in the preoptic nucleus, are known to send
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Fig. 3. Analysis of the effect of ICV administration of cmKiss1-15 and cmKiss2-12 peptides on fshˇ (A and B) and lhˇ (C and D) mRNA levels in the pituitary, each together in adult male (A and C) or female (B and D) immature chub mackerel. Transcription levels are the mean ± SEM of 9–16 independent determinations. Different letters above the bars represent significant differences (P < 0.05).
axonal projections to the anterior pituitary regions where FSH and LH producing cells are localized [26]. Thus, it is possible that ICV injected kisspeptin peptides modulate transcriptional changes in GnRH1 neurons. We observed a sexual dimorphic expression change in gnrh1. In the present results, levels of gnrh1 mRNA in the POA region of male fish did not show a response to both Kiss1-15 and Kiss2-12 peptides at 12 h post-injection (Fig. 2A). However, for female fish, administration of either Kiss1-15 or Kiss2-12 peptide decreased gnrh1 mRNA levels at 12 h post-injection (Fig. 2B). In mammals, it is known that GnRH1 neurons co-express Kiss1R to mediate downstream effects [15,19], and Kiss1 immunoreactivity levels play a role in sexual dimorphism since the number of Kiss-synthesizing neurons are higher in females than males [8]. Our previous studies demonstrated that kiss1 and kissr2 transcription levels in the brain are higher in females than males [22,27]. Based on this information, sex-specific action of the kisspeptin system on regulation of the BPG axis in chub mackerel is possible. Indeed, in medaka (Oryzias latipes), a sex difference in the number of nucleus ventral tuberiskiss1 neurons has been demonstrated [9]. Presently, in teleosts, the Kiss-KissR-GnRH relationship remains unclear. Teleost fish brain expresses multiple GnRH forms, and in the cichlid fish Nile tilapia (Oreochromis niloticus), all three types of GnRH neurons (gnrh1, gnrh2 and gnrh3) are known to express the kisspeptin receptor [24]. A recent study in striped bass (Morone saxatilis) identified two different modes of GnRH1 regulation, where kissr2 was co-localized in GnRH1 neurons and kissr1 was expressed in cells attached to GnRH1 fibers in the POA [34]. Levels of gnrh1 mRNA in the brains of prepubertal fish increased in response to Kiss1-15 and Kiss2-12 peptides at 24 h post-injection [34]. In contrast to the above findings, Grone et al. [7] in an African cichlid fish (Astatotilapia burtoni) reported that some cells in the POA expressing gnrh1 do not co-express kisspeptin receptor. Likewise, in medaka it was clearly demonstrated that kissr-expressing cells
were located adjacent to GnRH1 neurons, although they were not GnRH1 neurons themselves [10]. For chub mackerel, there is no anatomical evidence to suggest that the kisspeptin system is directly or indirectly involved in the regulation of GnRH neurons. A recent study in 3-year-old spermiating male sea bass found that ICV injection of Kiss2-12 evoked GnRH1 release into the pituitary and increased serum FSH and LH levels [4]. Furthermore, only Kiss2-12 increased the plasma levels of sex steroids and milt volume, suggesting that the ICV administration of Kiss2-12 potently stimulates the BPG-axis via the hypothalamic GnRH system [4]. Similarly, in the present study, a single ICV injection of synthetic Kiss2-12 increased pituitary fshˇ and lhˇ mRNA levels at 12 h post-injection compared to the saline-injected control (Fig. 3). Our analyses suggest that chub mackerel kisspeptin peptides play a central role in the control of gonadotropin secretion with differential potencies. When the relative potencies of these peptides were compared, it is clear that the cmKiss2-12 peptide is a potent elicitor of gonadotropin synthesis compared to cmKiss1-15 in this species. However, activation of gonadotropins by Kiss2-12 through preoptic GnRH neurons or KissR at the level of the pituitary requires future investigation, because it is possible that kisspeptin directly stimulates gonadotropin secretion through KissR in the pituitary of mammals [25] and goldfish [33] in vitro. Indeed, our recent study demonstrated that chub mackerel expresses kissr2 gene in the pituitary, and KissR2 is the intrinsic receptor for Kiss2-12 peptides in this species [22]. Future studies on the localization of the Kisspeptin–GnRH–GtH system and in vitro analyses on the effect of synthetic kisspeptin peptides to stimulate gonadotropin secretion in the pituitary will increase our understanding of the role of the kisspeptin system in this species. In conclusion, this study revealed that ICV administration of Kiss2-12 stimulates pituitary fshˇ and lhˇ mRNA synthesis, suggesting that kisspeptin peptides play a central role in control of the reproductive BPG axis in this species.
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