Gnrh receptor gnrhr2bbα is expressed exclusively in lhb-expressing cells in Atlantic salmon male parr

Gonadotropin-releasing hormone (Gnrh) plays a major role in the regulation of physiological and behavioural processes related to reproduction. In the pituitary, it stimulates gonadotropin synthesis and release via activation of Gnrh receptors (Gnrhr), belonging to the G protein-coupled receptor superfamily. Evidence suggests that differential regulation of the two gonadotropins (Fsh and Lh) is achieved through activation of distinct intracellular pathways and, probably, through the action of distinct receptors. However, the roles of the different Gnrhr isoforms in teleosts are still not well understood. This study investigates the gene expression of Gnrhr in the pituitary gland of precociously maturing Atlantic salmon (Salmo salar) male parr. A total of six Gnrhr paralogs were identified in the Atlantic salmon genome and named according to phylogenetic relationship; gnrhr1caα, gnrhr1caβ, gnrhr1cbα, gnrhr1cbβ, gnrhr2bbα, gnrhr2bbβ. All paralogs, except gnrhr1caα, were expressed in male parr pituitary during gonadal maturation as evidenced by qPCR analysis. Only one gene, gnrhr2bbα, was differentially expressed depending on maturational stage (yearly cycle), with high expression levels in maturing fish, increasing in parallel with gonadotropin subunit gene expression. Additionally, a correlation in daily expression levels was detected between gnrhr2bbα and lhb (daily cycle) in immature fish in mid-April. Double fluorescence in situ hybridization showed that gnrhr2bbα was expressed exclusively in lhb gonadotropes in the pituitary, with no expression detected in fshb cells. These results suggest the involvement of receptor paralog gnrhr2bbα in the regulation of lhb cells, and not fshb cells, in sexually maturing Atlantic salmon male parr.publishedVersio

Differential expression of gonadotropin and estrogen receptors and oocyte cytology during follicular maturation associated with egg viability in European eel (Anguilla anguilla)

acceptedVersio

Endocrine regulation of early sexual maturation in male Atlantic salmon parr

This thesis deals with changes in gene expression during activation of the brain-pituitary-gonadal axis at puberty in early maturing male Atlantic salmon (Salmo salar) parr. To help elucidate the physiological roles of gonadotropins and their receptors in the regulation of puberty, cDNAs encoding FSH and LH receptors (FSHR and LHR, respectively) of Atlantic salmon were cloned and characterized. Gene expression of the receptors in the testes was analyzed in parallel with pituitary expression of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) β-subunit genes by RT-PCR. In addition, functional genes encoding proteins involved in the steroidogenic pathway and anti-Müllerian hormone (AMH) were studied in the testes, and plasma 11-ketostestosterone levels were measured. One-summer-old male Atlantic salmon parr were sampled from the prepubertal stage in December until spermiation in October. Sequence analysis of the Atlantic salmon FSHR and LHR showed the typical structure features of glycoprotein receptors including a large extracellular domain connected to a G protein-coupled transmembrane domain. Both of these gonadotropin receptors were expressed in immature testis, FSHR more abundantly than LHR. FSHR transcript levels increased in parallel with FSHβ levels from early spermatogenesis onwards while LHR mRNA levels started to increase prior to any major changes in LHβ expression. De novo transcription of genes encoding steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, cytochrome P450 17α-hydroxylase/17,20-lyase, and 11β-hydroxysteroid dehydrogenase was observed during the initiation of spermatogenesis in parallel with the changes in FSHβ levels. In contrast, AMH expression was downregulated and AMH levels were lowest during spermiogenesis. During spermatogenesis, large increases in the expression of LHR and all of the steroidogenic genes studied occurred concomitantly with the rise in LHβ transcripts. These findings suggest that FSH is involved in regulation of the expression of several testicular genes during the initiation of puberty and LH during the later stages of spermatogenesis. In addition, results of in vitro studies using serum-free primary cultures of pituitary cells indicate that IGF-I differentially modulates gonadotropin expression in the pituitary cells. IGF-I may stimulate FSHβ expression levels though interactions with gonadotropin-releasing hormone (GnRH) in immature males while it directly activates LHβ expression

Demonstration of the Coexistence of Duplicated LH Receptors in Teleosts, and Their Origin in Ancestral Actinopterygians

International audiencePituitary gonadotropins, FSH and LH, control gonad activity in vertebrates, via binding to their respective receptors, FSHR and LHR, members of GPCR superfamily. Until recently, it was accepted that gnathostomes possess a single FSHR and a single LHR, encoded by fshr and lhcgr genes. We reinvestigated this question, focusing on vertebrate species of key-phylogenetical positions. Genome analyses supported the presence of a single fshr and a single lhcgr in chondrichthyans, and in sarcopterygians including mammals, birds, amphibians and coelacanth. In contrast, we identified a single fshr but two lhgcr in basal tel-eosts, the eels. We further showed the coexistence of duplicated lhgcr in other actinoptery-gians, including a non-teleost, the gar, and other teleosts, e.g. Mexican tetra, platyfish, or tilapia. Phylogeny and synteny analyses supported the existence in actinopterygians of two lhgcr paralogs (lhgcr1/ lhgcr2), which do not result from the teleost-specific whole-genome duplication (3R), but likely from a local gene duplication that occurred early in the actinopter-ygian lineage. Due to gene losses, there was no impact of 3R on the number of gonadotro-pin receptors in extant teleosts. Additional gene losses during teleost radiation, led to a single lhgcr (lhgcr1 or lhgcr2) in some species, e.g. medaka and zebrafish. Sequence comparison highlighted divergences in the extracellular and intracellular domains of the duplicated lhgcr, suggesting differential properties such as ligand binding and activation mechanisms. Comparison of tissue distribution in the European eel, revealed that fshr and both lhgcr transcripts are expressed in the ovary and testis, but are differentially expressed in non-gonadal tissues such as brain or eye. Differences in structure-activity relationships and tissue expression may have contributed as selective drives in the conservation of the duplicated lhgcr. This study revises the evolutionary scenario and nomenclature of gonado-tropin receptors, and opens new research avenues on the roles of duplicated LHR in actinopterygians

Tissue distribution of <i>fshr</i>, <i>lhcgr1</i> and <i>lhcgr2</i> transcripts in European eel.

<p>Messenger RNA levels for <i>fshr</i> (B), <i>lhcgr1</i> (C) and <i>lhcgr2</i> (D) were assayed by qPCR in various tissues of silver female European eels: Ovary (Ov); brain dissected in five parts: olfactory bulbs (Ob) telencephalon (Tel), di- and mes-encephalon (Di/Mes), corpus cerebellum (Cb), and medulla oblongata (Mo); pituitary (Pit); eyes; liver (Liv); intestine (Int); muscle (Mus); adipose tissue (AT); gills and thyroid follicles (TF). Receptor mRNA levels in testis of silver male European eels were also assayed. Data are normalized to total RNA and the expression level in the ovary was set as 1. Means are given ± SEM (n = 8 eels).</p

Origin and evolution of duplicated <i>lhcgr</i> in actinopterygians.

<p><i>Fshr</i>-<i>lhcgr</i> tandem was inherited by actinopterygians and sarcopterygians from a commun osteichthyan ancestor. The <i>fshr</i>-<i>lhcgr</i> tandem was conserved in sarcopterygians including tetrapod [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135184#pone.0135184.ref052" target="_blank">52</a>] and actinistian (coelacanth) lineages. In actinopterygians, a gene duplication event occurred before the emergence of teleosts, leading to two <i>lhcgr</i> genes, one maintained in tandem position with <i>fshr</i>, and named <i>lhcgr1</i>, and the other one named <i>lhcgr2</i>. The tandem <i>fshr</i>- <i>lhcgr1</i> and the <i>lhcgr2</i> were conserved on the same chromosome after the emergence of the holostean (spotted gar) lineage. Teleost whole-genome duplication (3R) potentially generated duplicated <i>fshr</i>, <i>lhcgr1</i> and <i>lhcgr2</i>. Early after the 3R, multiple and selective gene losses led to the conservation of only single genes for <i>fshr</i>, <i>lhcgr1</i> and <i>lhcgr2</i>, and to the separation of the <i>fshr</i>-<i>lhcgr(1)</i> tandem. Additional loss events of <i>lhcgr1</i> or <i>lhcgr2</i> have occurred independently throughout teleost radiation, so that some extant teleosts have conserved only a single <i>lhcgr</i>, <i>lhcgr1</i> or <i>lhcgr2</i>, according to species.</p

Phylogeny relationships of the gonadotropin receptors.

<p>Phylogram of maximum likelihood relationships between LHR and FSHR amino acid sequences of representative gnathostome species. Bootstrap values over 500 replicates (%) are given next to each node in red and SH-like aLRT values (%) are given in black (when different from the boostrap value). Asterisks indicate partial sequences. The actinopterigyan FSHR and LHR clades are highlighted to facilitate the phylogram examination. The sequence of the glycoprotein hormone receptor (GpHR) of an early chordate, the lancelet, was designated to root the tree.</p

Syntenic analysis of gonadotropin receptor genomic region.

<p>Genomic regions flanking <i>fshr</i> and <i>lhcgr</i> genes were analysed in representative vertebrate species including sarcopterygians (human, chicken, coelacanth) and actinopterygians (spotted gar, tilapia, zebrafish, medaka) by using the region overview on the Ensembl or NCBI genome browsers. The chromosome number is indicated beside the species name. <i>Fshr</i> genes are indicated in red, <i>lhcgr/lhcgr1</i> in light blue and <i>lhcgr2</i> in darker blue. The symbols of the genes of interest present on another genomic region in some species are indicated in green. Genes are named according to the Ensembl nomenclature. Gene positions are given in Mega base below the symbol of the genes.</p

Current status and evolutionary scenario of gonadotropin receptors in gnathostomes.

<p>The presence of gonadotropin receptors (<i>fshr</i>, <i>lhcgr</i>, <i>lhcgr1</i>, <i>lhcgr2</i>) in representative extant species of gnathostome lineages is shown on the right. In some species, the presence or absence of some gonadotropin receptors could not be assessed, due to the lack of genomic data, and these cases are indicated by a question mark. Two gonadotropin receptors, <i>fshr</i> and <i>lhcgr</i> are present in gnathostomes including chondrichthyans and osteichthyans. They were positioned in tandem in the early osteichthyes. The <i>fshr</i>-<i>lhcgr</i> tandem was maintained after the split between sarcopterygians and actinopterygians. Gene loss of <i>lhcgr</i> in various sauropsid groups led to the presence of only <i>fshr</i> in some extant sauropsids. A duplicated <i>lhcgr</i> was generated by a local gene duplication event in the actinopterygian lineage before the emergence of teleosts. Teleost whole-genome duplication (3R) generated duplicated <i>fshr</i>, <i>lhcgr1</i> and <i>lhcgr2</i>. Multiple and selective gene losses after the 3R led to the maintenance of only a single copy of <i>fshr</i>, <i>lhcgr1</i> and <i>lhcgr2</i> and to the physical separation of the tandem <i>fshr</i>-<i>lhcgr</i>. Additional <i>lhr</i> losses occurred independently through the teleost radiation, <i>i</i>.<i>e</i>. <i>lhcgr1</i> was lost in cyprinidae and <i>lhcgr2</i> in some percomorphs. Recent tetraploidization (4R) event has generated additional copies of <i>lhcgr1</i> in salmonids.</p