Identification and gene expression analysis of three GnRH genes in female Atlantic cod during puberty provides insight into GnRH variant gene loss in fish

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Abstract

Gonadotropin releasing hormone (GnRH) is a key regulator of sexual development and reproduction in vertebrates. Fish have either two or three pre-pro-GnRH genes, encoding structurally distinct peptides. We identified three pre-pro-GnRH genes in Atlantic cod (Gadus morhua, gmGnRH) using RT-PCR, RACE-PCR and BAC DNA library clone sequencing based on synteny searching. Gene identity was confirmed by sequence alignment and subsequent phylogenetic analysis. The expression of these genes was measured by quantitative PCR in the brain and pituitary of female cod throughout their reproductive cycle and in peripheral tissues. All three gmGnRH genes have highly conserved deduced decapeptide sequences, but sequence and phylogenetic data for gmGnRH1 suggest that this is a pseudogene. gmGnRH1 shares low identity with all fish GnRH variants and grouped with the GnRH3 clade. Although gmGnRH1 is a putative pseudogene, it is transcribed in multiple tissues but at low levels in the brain, indicating the loss of conserved hypophysiotrophic function. Phylogenetic analysis reveals that gmGnRH2 and gmGnRH3 variants are located in variant-specific clades. Both gmGnRH2 and gmGnRH3 transcripts are most abundant in the brain, with lower expression in pituitaries and ovaries. Brain gmGnRH3 gene expression increases in spawning fish and is expressed in the pituitary during puberty. Brain gmGnRH2 transcripts are highly expressed relative to gmGnRH3 before and during spawning. Sequence and expression data suggest that gmGnRH1 is a pseudogene and that gmGnRH3 is likely the hypophysiotrophic form of GnRH in Atlantic cod.

Highlights

► We have identified three pre-pro-gonadotropin releasing hormone (GnRH) genes in female Atlantic cod (Gadus morhua). ► One variant, gmGnRH1, appears to be a pseudogene based on sequence and phylogenetic analysis. ► The putative gmGnRH1 pseudogene is expressed in multiple tissues but at low levels in the brain. ► gmGnRH2 and gmGnRH3 are highly expressed in female brains and to a lesser extent in the pituitary and ovaries. ► gmGnRH3 is upregulated during spawning, suggesting that a functional shift has led to gmGnRH3 acting as the hypophysiotrophic form in Atlantic cod.

Introduction

Sexual maturation in vertebrates is largely controlled by the brain-pituitary-gonad axis [43], [59]. A key regulator of this system is the gonadotropin releasing hormone (GnRH), which stimulates synthesis and release of pituitary hormones [9], most notably follicle stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH, in turn, stimulate gametogenesis and steroidgenesis in the gonad. This process involves a complex interplay of neuroendocrine and endocrine inputs with multiple receptors, local paracrine and autocrine regulation and feedback controls [59], [54].

The pre-pro-GnRH genes are highly conserved throughout vertebrate evolution and are composed of four exons and three introns. Exon 1 and 2 encode the 5′ untranslated region (UTR). Exon 2 also encodes a signal peptide, the GnRH decapeptide, a 3 amino acid (aa) proteolytic cleavage site and part of a GnRH associated peptide (GAP). Exon 3 and 4 encode the remainder of GAP and the 3′ UTR [2], [46]. GnRH was first discovered to play a critical role in the neuroendocrine control of reproduction in mammals, by stimulating the synthesis and secretion of gonadotropic hormones from the pituitary [10]. Additional variants were later discovered in non-mammalian vertebrates and found to be involved in the control of reproductive behavior [42], [55], [56], [57], [62] in addition to neuromodulation [28].

The majority of tetrapods possess two GnRH genes, but one variant has been lost in some mammalian species such as mice, rats and chimpanzee [25], [38]. Teleost fish possess two or three GnRH genes, which display distinct expression patterns in the brain [43], [64], [25]. These hormones were originally named by the first species in which they were identified, but subsequently a universal nomenclature has been suggested [14], [61], which will be used in this paper. GnRH1 is a species specific form of GnRH expressed and localized primarily in olfactory bulb, ventral telencephalon and pre-optic area [17], [18]. GnRH2, a form conserved from fish to mammals, was first identified in chicken (cGnRH-II) [37] and is primarily found in the midbrain tegmentum. GnRH3, which is a fish specific form of GnRH, was originally identified in salmon (sGnRH) [50] and has partially overlapping distribution with GnRH1 in the olfactory bulb, ventral telencephalon and pre-optic area. The functional diversification of these hormones is not fully understood, but the main hypophysiotropic hormone in most teleosts is considered to be GnRH1 due to its expression in the ventral forebrain and well established stimulation of gonadotropins [8], [63]. GnRH1 has also been found to stimulate other pituitary hormones in some species, namely growth hormone [33], prolactin [58] and somatolactin [26]. In teleosts that possess only two forms of GnRH, GnRH1 (in eel and catfish) or GnRH3 (in cyprinid and salmonid fish) appear to functionally compensate for the missing variant [43]. For example, the hypophysiotrophic function of GnRH1 is compensated for by GnRH3 in goldfish (Carassius auratus) that lack GnRH1 [29].

Pseudogenes are remnants of genes that have lost their protein-coding ability or are otherwise no longer expressed in cells. These can occur by mutation leading to a non-processed gene, a retrotransposition mechanism leading to a processed pseudogene (no introns) or by integration of mitochondrial DNA [11]. These are relatively common in mammalian genomes, and in the human genome alone it is estimated that there are 8000 processed pseudogenes [11]. Pseudogenes are often considered “junk DNA” with no function, although various alternative functions have been proposed in recent years [52]. Pseudogenes have been identified for members of the GnRH system in mammals, namely GnRH2 ligand and GnRH2 receptors [51], [39].

Atlantic cod (Gadus morhua) is an economically important marine fish species for both farmed and wild fisheries. For this reason, there is considerable interest in understanding the control of its sexual maturation to improve broodstock gamete production quality while avoiding early reproductive development in the production fish as this leads to a significant reduction in body weight [12], [21], [27], [53]. It was recently shown that Atlantic cod FSH and LH genes (fshβ and lhβ) have increased expression during spawning with an additional pronounced fshβ expression peak just prior to the spawning period [36]. In accordance, LH receptor (LHR) mRNA expression increases during spawning while FSH receptor (FSHR) mRNA expression increases before and during the spawning period when corrected for GSI [35]. Further, GnRH1 and GnRH2 analogs stimulate ovulation in Atlantic cod [16]. Little information, however, exists concerning the regulation of GnRH in Atlantic cod. In a parallel study to this one, four GnRH receptors were identified in Atlantic cod and shown to be differentially regulated in the brain and pituitary during puberty (Hildahl et al. submitted for publication).

Although there is considerable data published on the developmental expression of GnRH variants in fish, there is relatively little quantitative gene expression data, especially during reproduction. Therefore, in this paper, we aim to characterize genes for all the GnRH variants in Atlantic cod and to quantify transcript expression in females throughout sexual development to gain further insight into the regulation of sexual maturation in this species. In addition, phylogenetic analysis and tissue gene expression profiles provide novel insight into the evolution and functional diversification of multiple forms of GnRH.

Section snippets

Animal and tissue sampling

Details of the rearing conditions and sampling procedure for the Atlantic cod used for real-time reverse transcriptase quantitative PCR (qPCR) analysis during sexual maturation have been described previously [36], [35]. Briefly, Norwegian coastal cod larvae were hatched in the laboratory and received natural zooplankton from first feeding until being transferred to experimental tanks or net pens where they were fed ad lib with commercial dry pellets. For qPCR analysis, fish were kept at natural

Cloning and identification

Complete coding sequences for three pre-pro-GnRH genes were cloned and sequenced in Atlantic cod (Fig. 1B) and named according to the species (gm for G. morhua): gmGnRH1 (of the sbGnRH type), gmGnRH2 (cGnRH-II) and gmGnRH3 (sGnRH). The gmGnRH2 and gmGnRH3 genes are composed of conserved regions that include a 23–24 aa signal peptide, 10 aa GnRH peptide, a 3 aa cleavage site and 50 (GnRH2) or 54 (GnRH3) aa GAP (Fig. 1A and B). Whereas the gmGnRH1 deduced decapeptide sequence has a completely

Discussion

In the present study, we aimed to elucidate the molecular basis driving the functional diversification of the GnRH system in Atlantic cod by characterizing the genes that encode GnRH variants and analyzing their expression during reproductive development. We have identified three forms of pre-pro-GnRH in Atlantic cod, similar to what is found in many other fish species. However, our data indicate that one variant, gmGnRH1, is likely a pseudogene. While this probably renders gmGnRH1 without

Acknowledgments

We thank Olav Sand, Jonathan Colman, Kirsten Ore, Ida G Lunde, and Helene Kile Larsen for various help and discussions. We’d like to give special thanks to Kamran Shalchian-Tabrizi for his kind assistance with the phylogenetic analysis. This study was supported by the Research Council of Norway (Grant 139630/140 to BN; 165120/S40 and 184851 to FAW) and a Marie Curie Reintegration Grant to FAW.

References (64)

  • K. Matsuda et al.

    Inhibitory effect of chicken gonadotropin-releasing hormone II on food intake in the goldfish, Carassius auratus

    Horm. Behav.

    (2008)
  • C. Mittelholzer et al.

    Molecular characterization and quantification of the gonadotropin receptors FSH-R and LH-R from Atlantic cod (Gadus morhua)

    Gen. Comp. Endocrinol.

    (2009)
  • C. Mittelholzer et al.

    Quantification of gonadotropin subunits GPalpha, FSHbeta, and LHbeta mRNA expression from Atlantic cod (Gadus morhua) throughout a reproductive cycle

    Comp. Biochem. Physiol. B Biochem. Mol. Biol.

    (2009)
  • K. Miyamoto et al.

    Isolation and characterization of chicken hypothalamic luteinizing hormone-releasing hormone

    Biochem. Biophys. Res. Commun.

    (1982)
  • K. Morgan et al.

    Evolution of GnRH ligand precursors and GnRH receptors in protochordate and vertebrate species

    Gen. Comp. Endocrinol.

    (2004)
  • J.N. Nocillado et al.

    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

    Gen. Comp. Endocrinol.

    (2007)
  • S. Ogawa et al.

    Immunoneutralization of gonadotropin-releasing hormone type-III suppresses male reproductive behavior of cichlids

    Neurosci. Lett.

    (2006)
  • P.H. Seeburg et al.

    The mammalian GnRH gene and its pivotal role in reproduction

    Recent Prog. Horm. Res.

    (1987)
  • M. Shahjahan et al.

    Differential expression of three types of gonadotropin-releasing hormone genes during the spawning season in grass puffer, Takifugu niphobles

    Gen. Comp. Endocrinol.

    (2010)
  • G.L. Taranger et al.

    Control of puberty in farmed fish

    Gen. Comp. Endocrinol.

    (2010)
  • H. Volkoff et al.

    Actions of two forms of gonadotropin releasing hormone and a GnRH antagonist on spawning behavior of the goldfish Carassius auratus

    Gen. Comp. Endocrinol.

    (1999)
  • H. Volkoff et al.

    Aspects of the hormonal regulation of appetite in fish with emphasis on goldfish, Atlantic cod and winter flounder: notes on actions and responses to nutritional, environmental and reproductive changes

    Comp. Biochem. Physiol. A Mol. Integr. Physiol.

    (2009)
  • F.A. Weltzien et al.

    The brain–pituitary–gonad axis in male teleosts, with special emphasis on flatfish (Pleuronectiformes)

    Comp. Biochem. Physiol. A Mol. Integr. Physiol.

    (2004)
  • F.A. Weltzien et al.

    A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase

    Gen. Comp. Endocrinol.

    (2005)
  • Y. Zohar et al.

    Gonadotropin-releasing activities of the three native forms of gonadotropin-releasing hormone present in the brain of gilthead seabream, Sparus aurata

    Gen. Comp. Endocrinol.

    (1995)
  • F. Abascal et al.

    ProtTest: selection of best-fit models of protein evolution

    Bioinformatics

    (2005)
  • J.P. Adelman et al.

    Isolation of the gene and hypothalamic cDNA for the common precursor of gonadotropin-releasing hormone and prolactin release-inhibiting factor in human and rat

    Proc. Natl. Acad. Sci. USA

    (1986)
  • R. Baertsch et al.

    Retrocopy contributions to the evolution of the human genome

    BMC Genomics

    (2008)
  • J. Bogerd et al.

    Two gonadotropin-releasing hormone receptors in the African catfish: no differences in ligand selectivity, but differences in tissue distribution

    Endocrinology

    (2002)
  • L.F. Canosa et al.

    Changes in brain mRNA levels of gonadotropin-releasing hormone, pituitary adenylate cyclase activating polypeptide, and somatostatin during ovulatory luteinizing hormone and growth hormone surges in goldfish

    Am. J. Physiol. Regul. Integr. Comp. Physiol.

    (2008)
  • J. Carolsfeld et al.

    Primary structure and function of three gonadotropin-releasing hormones, including a novel form, from an ancient teleost, herring

    Endocrinology

    (2000)
  • R. Counis et al.

    Gonadotropin-releasing hormone and the control of gonadotrope function

    Reprod. Nutr. Dev.

    (2005)
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