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The Expression Pattern of Melatonin Receptor 1a Gene during Early Life Stages in the Nile tilapia (Oreochromis niloticus)

The action of melatonin within the body of animals is known to be mediated by melatonin receptors. Three different types of melatonin receptors have been identified so far in fish. However, which of these are specifically involved in puberty onset is not known in fish. We cloned and analyzed the sequence of melatonin receptor 1a (mel 1a) gene in Nile tilapia Oreochromis niloticus. In addition, we examined the tissue distribution of gene expressions for three types of receptors, mel 1a, 1b and lc and investigated which of them is involved in the onset of puberty by comparing their expression with that of gonadotropin-releasing hormone receptor I (GnRHr I) gene using quantitative real-time PCR from 1 week post hatch (wph) to 24 wph. The mel 1a gene of Nile tilapia consisted of two exons and one bulky intron between them. Mel 1a gene was found to be highly conserved gene showing high homology with the corresponding genes from different teleost. All three types of melatonin receptor genes were expressed in the brain, eyes and ovary in common. Expression of mel 1a gene was the most abundant and ubiquitous among 3 receptors in the brain, liver, gill, ovary, muscle, eye, heart, intestine, spleen and kidney. Mel 1b and mel 1c genes were, however, expressed in fewer tissues at low level. During the development post hatch, expressions of both mel 1a and GnRHr I genes significantly increased at 13 wph which was close to the putative timing of puberty onset in this species. These results suggest that among three types of receptors mel 1a is most likely associated with the action of melatonin in the onset of puberty in Nile tilapia.


INTRODUCTION
Melatonin mediates environmental cues such as light and photoperiod and consequently influences important physiological processes including reproduction in mammals (Malpaux et al., 2001;Pevet, 2003) and in teleosts (Mayer et al., 1997;Falcón et al., 2007). However, the role of melatonin in the process of teleost reproduction seems less clear due to the descrepancies observed in the previous studies. In European eel, Anguilla anguilla, the effect of melatonin treatment on reproduction was inhibitory (Sébert et al., 2008) while the same treatments were stimulatory in carp, Cyprinus catla (Bhattacharya et al., 2007), seabass, Dicentrarchus labrax (Bayarri et al., 2004) and zebrafish, Danio rerio (Carnevali et al., 2011). Even within the same species, the effect of melatonin administration was dependent on gonadal stage or sex of subjects (Bhattacharya et al., 2007). This indicates that the action of melatonin on fish reproduction is far more complicated than we thought.
The effects of melatonin within the body of animals are known to be mediated through melatonin receptors that belong to the G protein-coupled receptor (GPCR) superfamily (Dubocovich, 1995;Reppert et al., 1996). Supporting this, in one of teleost fish, seabass, the effects of melatonin rely not only on its rhythmic production, but also on the rhythmic expression of its receptors (Bayarri et al., 2010). There are three different subtypes of melatonin receptors, mel 1a, mel 1b and mel 1c. While the mel 1a and mel 1b subtypes are found in all vertebrates investigated, the mel 1c subtype has been found only in non-mammalian so far (Ebisawa et reproduction including puberty is not known. Puberty is the developmental period when an individual matures sexually for the first time, and activation of brainpituitary-gonadal axis (BPG axis) is prerequisite for puberty.
This activation of BPG axis is initiated by an increase of gonadotropin-releasing hormone (GnRH). GnRH released from hypothalamus promotes the maturation of gonad by inducing release of luteinizing hormone (LH) and folliclestimulating hormone (FSH) via its cognate receptor located in pituitary gonadotropic cells. GnRHs are key regulators in sexual development and reproduction in vertebrates and there are two or three piscine GnRH genes encoding GnRH peptides of different structures (Parhar, 2002). Among three types of GnRH identified in Nile tilapia Oreochromis niloticus (Parhar et al., 2003), GnRH I is located in the preoptic area (POA) and pituitary showing a close relationship with the activation of BPG axis (White et al., 1995;Parhar et al., 1996;Gothilf et al., 1996). Thus, the expression of GnRH receptor I (GnRHr I) gene could be indicative of the activation of BPG axis in this species.
In this study, we cloned and analyzed the sequence of melatonin receptor 1a gene in Nile tilapia and examined the tissue distribution of three receptors, mel 1a, mel 1b and mel lc genes. Finally, we investigated which receptor is involved in the onset of puberty by comparing their gene expression with GnRHr I gene expression using quantitative real-time PCR.    (Tamura et al., 2011) and Neighbor-Joining method (Saitou and Nei, 1987).

Tissue distribution of melatonin receptor genes (mel 1a, mel lb and mel lc)
To investigate the tissue specific expressions of mel 1a, mel 1b and mel 1c, total RNA was isolated from whole brain, liver, gill, ovary, muscle, eye, heart, intestine, spleen and kidney of an adult female tilapia (body length: 15.0 cm, body weight: 57.9 g) and subjected to RT-PCR using primers for mel 1a, mel 1b and mel 1c genes (Table 2).  of the fish, their heads (1-7 wph) or brains (10-24 wph) were sampled (n=4). Protocols of total RNA extraction and cDNA synthesis are the same as above. We examined the expression of GnRHr I and mel 1a genes together with the expression of β-actin gene for normalization. Quantitative

Mel 1a gene sequence
The sequence of mel 1a mRNA cloned from the brain of

Tissue distribution of melatonin receptor genes (mel 1a, lb and lc)
Tissue specific expression of melatonin receptors analyzed by RT-PCR was shown in Fig. 4. Mel 1a gene was expressed abundantly and ubiquitously in all tissues investigated including the brain and various peripheral tissues: liver, gill, ovary, muscle, eye, heart, intestine, spleen and kidney. In contrast, mel 1b and mel 1c genes were faintly detected in only several tissues. Mel 1b transcript existed in the brain, gill, ovary, eye and kidney, and mel 1c transcript was distributed in the brain, gill, ovary, eye, heart and intestine. Three different melatonin receptors were commonly detected in the brain, gill, ovary and eye.    (Kokkola et al., 2005). Anterior two residues of N-R-Y motif are known to be involved directly in the activation of rhodopsin and β-adrenergic receptors (Ballesteros et al., 2001;Gether et al., 2002). The mel 1a gene structure of O. niloticus that had two exons and one bulky intron (35 kilobases) was similar to that of mouse (Roca et al., 1996). In addition, the mel 1a gene in tilapia showed high similarity to the same gene from different teleost species.

Tissue distribution of melatonin receptor genes
(mel 1a, lb and lc) All three types of Nile tilapia melatonin receptor genes were expressed in the brain, eye and ovary in common.
Especially, they were abundant in the brain and eye. Even though these receptors were expressed abundantly in the brain and eye in common, mel 1a mRNA was the most abundant suggesting that this receptor mediate most of melatonin actions at the level of central nervous system.
Mel 1a gene was also prominently expressed in all peripheral tissues investigated in this study, including liver, gill, ovary, muscle, heart, intestine, spleen and kidney while mel 1b and mel 1c genes were expressed in fewer Dev. Reprod. Vol. 17, No. 1 (2013) Mel 1a (Vera et al., 2007).
In summary, we have cloned melatonin receptor 1a gene from the brain of Nile tilapia. This gene was highly conserved among species based on the phylogenetic and sequence analysis. Mel 1a mRNA was most abundant in all tissues investigated in this study, suggesting that the considerable melatonin actions are mediated by mel 1a in these peripheral tissues. Gene expression profiles of GnRHr I revealed a putative timing of puberty onset at 13 wph, and expression of mel 1a gene showed a close relationship with puberty onset in this species. Further studies should clarify the exact function of mel 1a gene that was concomitantly increased with GnRHr I gene during the putative puberty onset.