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

Abstract

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.

Keywords: Mel 1a; Melatonin receptors; Nile tilapia; Oreochromis niloticus; Puberty.

Fig. 1.

Nucleotide and deduced amino acid sequence of O. niloticus mel 1a. The length of ORF is 1053 nt and encodes a protein of 350 amino acids, leaving a 82 nt 5'-UTR and a 49 nt 3'-UTR. Start and stop (*) codons are boxed. Predicted trans-membrane (TM1-7) domains are underlined. The gray background shows signal peptide. The dotted box is the conserved NRY motif.

Fig. 2.

Mel 1a gene structure of O. niloticus. It is comprised of 2 exons of 265 bp and 936 bp, respectively. Between exon1 and exon2, there is one bulky intron of 35,231 bp.

Fig. 3.

Phylogenetic analysis of mel 1a genes from various vertebrates. This analysis was performed with multiple alignments from amino acid sequences using the ClustalW program and neighbor joining method. GenBank accession Numbers for the respective animals: human (Homo sapiens) NP005949.1; mouse (Mus musculus) NP032665.1; chicken (Gallus gallus) AAA92498.1; zebrafish (Danio rerio) NP571468.1; rabbitfish (Siganus guttatus) ABG77572.1; Mozambique tilapia (O. mossambicus) ACL78767.1; European seabass (Dicentrarchus labrax) ACB13280.1; rainbow trout (Oncorhynchus mykiss) AAF00191.1; sweetfish (Plecoglossus altivelis) BAI65858.1

Fig. 4.

Expression of mel 1a, mel 1b and mel 1c gene from various tissues (B: brain, L: liver, G: gill, O: ovary, M: muscle, E: eye, H: heart, I: intestine, S: spleen and K: kidney) of Nile tilapia. The expressions of melatonin receptor genes and β-actin gene (as a control) were examined by using RT-PCR. PCR products were run on 1% agarose gels.

Fig. 5.

Relative expression pattern of mel 1a mRNA (A) and GnRHr I mRNA (B) in the brain of Nile tilapia from 1 wph to 24 wph. Differences in the mRNA levels were estimated by quantitative real-time PCR. The relative abundance of the mRNAs was normalized to the amount of β-actin mRNA by the comparative threshold cycle method. Each value represents mean ± SEM (n = 4). Asterisks (*) indicate significant difference from the immediately previous observation within this study (P<0.05).