Temporal expression of G-protein-coupled receptor 54 (GPR54), gonadotropin-releasing hormones (GnRH), and dopamine receptor D2 (drd2) in pubertal female grey mullet, Mugil cephalus☆
Introduction
As in other vertebrates, puberty in fish is a complex developmental process by which an immature individual develops into a mature reproductive-competent adult (Okuzawa, 2002). It is characterized by achievement of the brain–pituitary–gonadal (BPG) axis of full hormonal and gametogenetic capacity (reviewed by Weltzien et al., 2004, Schulz and Goos, 1999). Among teleosts that undergo periodic gonadal recrudescence and quiescence after sexual maturation, puberty refers to the stage when the fish undergoes its first reproductive cycle.
The BPG axis regulates the reproductive function in fish (Weltzien et al., 2004). Stimulatory and inhibitory signals are integrated in the brain, primarily by the hypothalamic neuroendocrine neurons that produce gonadotropin-releasing hormone (GnRH), which turns on the downstream hormonal cascade that ultimately stimulates gonadal activity (Peter and Yu, 1997). Since the discovery of the novel seabream GnRH (GnRH1) in the gilthead seabream, Sparus aurata (Powell et al., 1994), it is now established that most modern teleosts express three forms of GnRH in the brain that includes cGnRH-II (GnRH2) and sGnRH (GnRH3) (Lethimonier et al., 2004). GnRH1 is predominant in the preoptic area and the hypothalamus while GnRH2 and GnRH3 are expressed in the midbrain neurons and terminal nerve ganglion, respectively (reviewed by Somoza et al., 2002). Only GnRH1 mRNA and peptide levels fluctuate in the brain with gonadal development, as reported in the barfin flounder, Verasper moseri (Amano et al., 2004), tilapia, Oreochromis niloticus (Parhar et al., 2003), turbot, Scophthalmus maximus (Andersson et al., 2001), red seabream, Pagrus major (Senthilkumaran et al., 1999) and gilthead seabream (Holland et al., 1998). The changes in hypothalamic GnRH1 mRNA levels are accompanied by corresponding changes in GnRH1 levels in the pituitary, as observed in the European seabass, Dicentrarchus labrax (Rodríguez et al., 2000), red seabream (Senthilkumaran et al., 1999) and grass rockfish, Sebastes rastrelliger (Collins et al., 2001). GnRH2 and GnRH3 have been proposed as neuromodulators for reproductive and non-reproductive functions (Parhar et al., 2004).
In fish, neurotransmitters, such as γ-amino butyric acid (GABA), neuropeptide Y (NPY), norepinephrine and serotonin regulate, through their stimulatory effects, the GnRH system and the action of GnRH on the pituitary (Amano et al., 1997, Senthilkumaran et al., 2001). On the other hand, dopamine (DA), acting through the dopamine D2 receptor (drd2), inhibits GnRH activity by downregulating the synthesis of GnRH receptors (Levavi-Sivan et al., 2004) and inhibiting gonadotropin release from the pituitary (reviewed by Yaron et al., 2003).
As in mammals, activation of the GnRH system and the subsequent synthesis and release of pituitary gonadotropins that stimulate gonadal development are considered key events during puberty (Weltzien et al., 2004, Dufuor et al., 2000). The mechanisms underlying the activation of the BPG axis during pubertal development are not as yet fully understood. The missing link hypothesis suggests that one or more components of the axis is/are not functional prior to puberty in fish (Schulz and Goos, 1999). For instance, the pituitary GnRH receptors and ovarian gonadotropin receptors were identified as the nonfunctional components of the BPG axis in immature black carp, Mylopharyngodon piceus (Gur et al., 2000).
Recently, a signaling mechanism, mediated by the G-protein-coupled receptor 54 (GPR54), has been demonstrated as a potent stimulator of pulsatile GnRH secretion in mammals and has been proposed as a key factor for the onset of puberty (reviewed by Seminara, 2005, Murphy, 2005, Dungan et al., 2006). So far, GPR54 has been studied in only one teleost species, the male cichlid fish tilapia, where it was found to be expressed in all three GnRH neuron subtypes, the number of neurons expressing GPR54, as well as the level of GPR54 expression, increased with gonadal maturation (Parhar et al., 2004).
In a step towards elucidating some of the molecular aspects of the regulation of puberty in fish, we investigated the levels of brain, pituitary and ovarian gene expression of GPR54, GnRH1, GnRH2, GnRH3, and drd2 in pubertal female grey mullet, a species that matures in 3–5 years (de Monbrison et al., 1997) and where DA strongly inhibits its reproductive function (Aizen et al., 2005).
Section snippets
Molecular cloning of mullet GPR54 (muGPR54)
Whole brain was obtained from maturing female grey mullet held in captivity at the Bribie Island Aquaculture Research Centre, following the procedures approved by the Department of Primary Industries and Fisheries Animal Ethics Committee (approval number Bribie/032/07/02). The dissected tissues were first stored in RNALater (Ambion, Austin, TX) for 24 h at 4 °C and subsequently at −80 °C until used. Total RNA was extracted with TriZOL Reagent (Invitrogen, Carlsbad, CA) and treated with DNAse
Isolation of muGPR54 cDNA and structural analysis
The nucleotide sequence of the isolated muGPR54 cDNA included an open reading frame of 1140 bp encoding a predicted 380 amino acid peptide, a 442 bp 5′UTR and 1.2 kb 3′UTR (DQ683737). Fig. 1 shows the homology of the deduced amino acid residues of muGPR54 to other vertebrate GPR54 cDNAs. The muGPR54 is highly homologous to the tilapia GPR54 (94.7%) while it only shares 56–57% homology to its human, mouse and rat homologues. It is 37–48% homologous to the related galanin receptors (GalR1, GalR2
Discussion
We report the isolation of the muGPR54 cDNA from the brain of grey mullet and the characterization of its expression levels in pubertal female fish, compared with the expression levels of GnRH1, GnRH2, GnRH3 and drd2. We focused on the expression profile of these genes because of their unique relationship in modulating the reproductive function in a species like the grey mullet. DA inhibits the reproductive function in this species via the drd2 (Aizen et al., 2005). DA downregulates the
Acknowledgments
We thank Anna Kuballa for advice during the development of the QPCR assays, Dr. David Mayer for statistical analysis, Hazra Thaggard for expert laboratory assistance, and Prof. Zvi Yaron for constructive comments on the manuscript.
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Cited by (0)
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Funded by the Aquaculture Industry Development Initiative (AIDI), Queensland, to A. Elizur.
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Supported by AIDI and UQ Graduate School Scholarship.