Anne-Gaëlle Lafont

First evidence for a direct inhibitory effect of kisspeptins on LH expression in the eel, Anguilla anguilla

The kisspeptin system has emerged as one of the main puberty gatekeepers among vertebrates. The European eel (Anguilla anguilla) is a remarkable model due to its phylogenetical position at the basis of teleosts, and its unique life cycle with a blockade of puberty before reproductive migration. We cloned the full-length coding sequence of a kisspeptin receptor (Kissr) in the eel. Comparison of Kissr sequences assigned the eel Kissr to a basal position in a clade including most of the known teleost Kissr, in agreement with the eel phylogenetical position. Eel Kissr tissue distribution was analyzed by quantitative real-time PCR. Eel Kissr was highly expressed in the brain, especially in the telencephalon and di-/mes-encephalon, while a very low or undetectable expression was observed in various peripheral organs. A high expression of Kissr was also found in the pituitary indicating a possible direct pituitary role of kisspeptin. Primary cultures of eel pituitary cells were performed to investigate the direct effects of kisspeptin on pituitary hormone expression. Human/lamprey kisspeptin exerted a time- and dose-dependent inhibitory effect on LHβ expression. All other tested kisspeptins had a similar inhibitory effect on LHβ expression. The inhibitory effect of kisspeptins was exerted specifically on LHβ as no change was induced on the expression of other glycoprotein hormone subunits (GPα, FSHβ and TSHβ) nor of growth hormone. These data provide the first evidence for the existence, in the European eel, of a kisspeptin system, which may play a direct inhibitory role on pituitary LHβ expression.

Comparative Evolutionary Histories of Kisspeptins and Kisspeptin Receptors in Vertebrates Reveal Both Parallel and Divergent Features

During the past decade, the kisspeptin system has been identified in various vertebrates, leading to the discovery of multiple genes encoding both peptides (Kiss) and receptors (Kissr). The investigation of recently published genomes from species of phylogenetic interest, such as a chondrichthyan, the elephant shark, an early sarcopterygian, the coelacanth, a non-teleost actinopterygian, the spotted gar, and an early teleost, the European eel, allowed us to get new insights into the molecular diversity and evolution of both Kiss and Kissr families. We identified four Kissr in the spotted gar and coelacanth genomes, providing the first evidence of four Kissr genes in vertebrates. We also found three Kiss in the coelacanth and elephant shark genomes revealing two new species, in addition to Xenopus, presenting three Kiss genes. Considering the increasing diversity of kisspeptin system, phylogenetic, and synteny analyses enabled us to clarify both Kiss and Kissr classifications. We also could trace back the evolution of both gene families from the early steps of vertebrate history. Four Kissr and four Kiss paralogs may have arisen via the two whole genome duplication rounds (1R and 2R) in early vertebrates. This would have been followed by multiple independent Kiss and Kissr gene losses in the sarcopterygian and actinopterygian lineages. In particular, no impact of the teleost-specific 3R could be recorded on the numbers of teleost Kissr or Kiss paralogs. The origin of their diversity via 1R and 2R, as well as the subsequent occurrence of multiple gene losses, represent common features of the evolutionary histories of Kiss and Kissr families in vertebrates. In contrast, comparisons also revealed un-matching numbers of Kiss and Kissr genes in some species, as well as a large variability of Kiss/Kissr couples according to species. These discrepancies support independent features of the Kiss and Kissr evolutionary histories across vertebrate radiation.

Possible role of calcitonin gene-related peptide in osmoregulation via the endocrine control of the gill in a teleost, the eel, Anguilla anguilla

Osmoregulation is a major challenge in aquatic animals involving a complex endocrine control. We investigated the potential role of calcitonin gene-related peptide (CGRP, a neuromediator in mammals) in the endocrine control of the gill in a teleost, the eel. Transfer from freshwater to seawater induced an hyperosmolality and a concomitant large increase in plasma CGRP levels. Specific CGRP binding sites were characterized in the gill and their number was up-regulated after seawater transfer. This study suggests that the endocrine control of gill function during osmoregulation may represent an ancient role of CGRP in vertebrates.

Characterisation and distribution of calcitonin gene-related peptide in a primitive teleost, the eel, Anguilla anguilla and comparison with calcitonin

Radioimmunoassay (RIA), radioreceptor assay and chromatography were used to study the occurrence of calcitonin gene-related peptide (CGRP) in a primitive teleost, the eel, Anguilla anguilla. Immunologically and biologically active CGRP-like molecules were found in brain, heart, kidney, liver, spleen and ultimobranchial body with the higher concentrations in brain, spleen and heart. Gel exclusion chromatography of heart and spleen extracts followed by SDS-PAGE showed that the eel CGRP-like molecules presented a molecular weight between 3.30 and 3.95 kDa similar to that of human CGRP. The wide distribution of CGRP reflects its multiple role as brain neuromediator and peripheral paracrine effector as described in mammals. In comparison, the distribution of calcitonin (CT) was much more restricted, immunologically and biologically active CT-like molecules being localised in the ultimobranchial bodies (UBB) that is the site of CT synthesis in non-mammalian vertebrates. In plasma, CGRP-like concentrations were 10 to 100 higher than those of CT. These high concentrations in a primitive teleost strengthen the possible endocrine role of CGRP in early vertebrates and emphasise the important role of this hormone in evolution.

Vg mRNA induction in an endangered fish species (Anguilla anguilla) from the Loire estuary (France)

Estuarine zones are extremely fragile due to increasing stress from anthropogenic activities. Among those, the Loire estuary (France) is potentially exposed to various contaminants including Endocrine Disruptors Compounds (EDCs) able to impact the reproduction physiology of fish. The European eel (Anguilla anguilla), endangered fish species, is apparently not relevant, in its yellow stage, to monitor the effects of endocrine disruption. Despite this weakly responsiveness, this study aimed to investigate whether European eel from the Loire estuary may still be the subject of estrogenic disruption quantifying the hepatic Vg gene expression according to gender and sexual stage. Vitellogenin (Vg) appears as a valuable biomarker of EDCs, as well as for exposure and effects. Quantitative real-time Reverse Transcription Polymerase Chain Reaction (q RT PCR) was used in this study to amplify responses of hepatic Vg transcripts. European eels were sampled in May 2009 (N=57) and November 2010 (during the downstream migration, N=10) in two sites of the Loire estuary with different ecological conditions and contamination pressures (upstream: Varades; downstream: Nantes). Reproductive (gender, sexual maturity stage) and biometric parameters of collected eels were determined. A laboratory exposure of silver male to steroid hormones (Testosterone (T), 11-KetoTestosterone (11-KT), Estradiol (E2)) was conducted in parallel to validate the q RT PCR approach on hepatic Vg mRNA. Results demonstrated the responsiveness of exposed silver male eels, since hepatic mRNA Vg induction was observed in E2 treated males compared to control specimens. In the field, results of female silver eels reflected large inter-individual differences in the activation of hepatic Vg at silvering. However, while only female silver eels should express hepatic Vg mRNA, quantifiable levels were also detected in a proportion of 38% of the other individuals sampled, normally not inclined to express it, those being undifferentiated eels, yellow females, yellow and silver males. According to each sexual stage, no difference of expression was observed between eels from the two sampling sites. Histological results as well as low Vg mRNA levels detected do not permit a conclusion as to a potential effect of endocrine disruption.

Recurrent DCC gene losses during bird evolution

During development, midline crossing by axons brings into play highly conserved families of receptors and ligands. The interaction between the secreted ligand Netrin-1 and its receptor Deleted in Colorectal Carcinoma (DCC) is thought to control midline attraction of crossing axons. Here, we studied the evolution of this ligand/receptor couple in birds taking advantage of a wealth of newly sequenced genomes. From phylogeny and synteny analyses we can infer that the DCC gene has been conserved in most extant bird species, while two independent events have led to its loss in two avian groups, passeriformes and galliformes. These convergent accidental gene loss events are likely related to chromosome Z rearrangement. We show, using whole-mount immunostaining and 3Disco clearing, that in the nervous system of all birds that have a DCC gene, DCC protein expression pattern is similar to other vertebrates. Surprisingly, we show that the early developmental pattern of commissural tracts is comparable in all birds, whether or not they have a DCC receptor. Interestingly, only 4 of the 5 genes encoding secreted netrins, the DCC ligands in vertebrates, were found in birds, but Netrin-5 was absent. Together, these results support a remarkable plasticity of commissural axon guidance mechanisms in birds.

Evidence for the Presence of Molecules Related to the Neuropeptide CGRP in Two Cephalopods, Sepia officinalis and Nautilus macromphalus: Comparison with Its Target Organ Distribution

Calcitonin gene-related peptide (CGRP) is a neuropeptide mainly involved in brain and cardiovascular functions in mammals. We investigated its presence and potential roles in two cephalopods, Sepia officinalis and Nautilus macromphalus. CGRP-like, but not calcitonin (CT)-like, molecules were detected by specific radioimmuno- and radioreceptor assays in the brain, optic lobes, branchial heart or afferent branchial vein and kidney. Gel exclusion chromatography of cephalopod brain extracts, followed by SDS-PAGE, indicated that CGRP-like molecules had a molecular weight of around 3 kDa, close to that of human CGRP. The distribution of CGRP target organs was characterized by binding studies in cuttlefish. Specific CGRP binding sites were detected in the brain, optic lobes, and kidney, indicating potential autocrine/paracrine roles of CGRP. Specific CGRP binding sites were also detected in the gills and shell sac that do not contain the peptide itself, indicating potential endocrine roles of CGRP. Accordingly, high circulating levels of CGRP-like molecules were detected in hemolymph of both cuttlefish and nautilus, unlike the situation in mammals. CGRP binding sites were further characterized in the cuttlefish gills by the Scatchard method. Our study indicates that the brain neurotransmitter role of CGRP could represent an ancient role in metazoa, already present in cephalopods and conserved among vertebrates. In contrast, the endocrine role of CGRP, which was suggested in cephalopods and also present in teleosts, may have been lost during the evolution of the tetrapod lineage. Our data support the hypothesis that CGRP represents the ancestral molecule of the CT/CGRP family appeared in metazoa before the vertebrate emergence.

Eel Kisspeptins: Identification, Functional Activity, and Inhibition on both Pituitary LH and GnRH Receptor Expression

The European eel (Anguilla anguilla) presents a blockade of sexual maturation at a prepubertal stage due to a deficient production of gonadotropins. We previously initiated, in the eel, the investigation of the kisspeptin system, one of the major gatekeepers of puberty in mammals, and we predicted the sequence of two Kiss genes. In the present study, we cloned and sequenced Kiss1 and Kiss2 cDNAs from the eel brain. The tissue distributions of Kiss1 and Kiss2 transcripts, as investigated by quantitative real-time PCR, showed that both genes are primarily expressed in the eel brain and pituitary. The two 10-residue long sequences characteristic of kisspeptin, eel Kp1(10) and Kp2(10), as well as two longer sequences, predicted as mature peptides, eel Kp1(15) and Kp2(12), were synthesized and functionally analyzed. Using rat Kiss1 receptor-transfected Chinese hamster ovary cells, we found that the four synthesized eel peptides were able to induce [Ca2+]i responses, indicating their ability to bind mammalian KissR-1 and to activate second messenger pathways. In primary culture of eel pituitary cells, all four peptides were able to specifically and dose-dependently inhibit lhβ expression, without any effect on fshβ, confirming our previous data with heterologous kisspeptins. Furthermore, in this eel in vitro system, all four peptides inhibited the expression of the type 2 GnRH receptor (gnrh-r2). Our data revealed a dual inhibitory effect of homologous kisspeptins on both pituitary lhβ and gnrh-r2 expression in the European eel.