Molecular markers of oocyte differentiation in European eel during hormonally induced oogenesis

[EN] Reproduction in captivity is a key study issue in Anguilla anguilla as a possible solution for its dwindling population. Understanding the mechanisms controlling the production of ribosomal building blocks during artificially induced oocyte maturation could be particularly interesting. Transcription levels of ribosomal biogenesis associated genes could be used as markers to monitor oogenesis. Eels from the Albufera Lagoon were injected with carp pituitary extract for 15 weeks and ovaries in previtellogenic (PV) stage (non-injected), in early-, mid-, late-vitellogenesis (EV, MV, LV), as well as in migratory nucleus stage (MN) were analysed. 5S rRNA and related genes were highly transcribed in ovaries with PV oocytes. As oocytes developed, transcriptional levels of genes related to 5S rRNA production (gtf3a), accumulation (gtf3a, 42sp43) and nucleocytoplasmic transport (tp15, tp111) and the 5S/18S rRNA index decreased (PV > EV > MV > LV > MN). On the contrary, 18S rRNA was at its highest at MN stage while ubtf1 in charge of activating RNA-polymerase I and synthesising 18S rRNA behaved as 5S related genes. Individuals that did not respond (NR) to the treatment showed 5S/18S index values similar to PV females, while studied genes showed EV/LV-like transcription levels. Therefore, NR females fail to express the largest rRNAs, which could thus be taken as markers of successful vitellogenesis progression. In conclusion, we have proved that the transcriptional dynamics of ribosomal genes provides useful tools to characterize induced ovarian development in European eels. In the future, such markers should be studied as putative indicators of response to hormonal treatments and of the quality of obtained eel oocytes.This work was supported by EU 7th Framework Programme (PRO EEL, grant agreement no. 245257 & AQUAGAMETE COST Action FA1205), Spanish MINECO (AGL2012-33477 and AGL2015-63936-R), Basque Government (S-PE12UN086 & IT810-13) & UPV/EHU (UFI 11/37). Some fish were supplied by the Hunting and Continental Fishing Service of Generalitat Valenciana. I.R.B holds a PhD fellowship of the Basque Government.Rojo-Bartolomé, I.; Martínez-Miguel, L.; Lafont, A.; Vilchez Olivencia, MC.; Asturiano Nemesio, JF.; Pérez Igualada, LM.; Cancio, I. (2017). Molecular markers of oocyte differentiation in European eel during hormonally induced oogenesis. Comparative Biochemistry and Physiology Part A Molecular & Integrative Physiology. 211:17-25. https://doi.org/10.1016/j.cbpa.2017.05.018S172521

Duplicated leptin receptors in two species of eel bring new insights into the evolution of the leptin system in vertebrates

Since its discovery in mammals as a key-hormone in reproduction and metabolism, leptin has been identified in an increasing number of tetrapods and teleosts. Tetrapods possess only one leptin gene, while most teleosts possess two leptin genes, as a result of the teleost third whole genome duplication event (3R). Leptin acts through a specific receptor (LEPR). In the European and Japanese eels, we identified two leptin genes, and for the first time in vertebrates, two LEPR genes. Synteny analyses indicated that eel LEPRa and LEPRb result from teleost 3R. LEPRb seems to have been lost in the teleost lineage shortly after the elopomorph divergence. Quantitative PCRs revealed a wide distribution of leptins and LEPRs in the European eel, including tissues involved in metabolism and reproduction. Noticeably, leptin1 was expressed in fat tissue, while leptin2 in the liver, reflecting subfunctionalization. Four-month fasting had no impact on the expression of leptins and LEPRs in control European eels. This might be related to the remarkable adaptation of silver eel metabolism to long-term fasting throughout the reproductive oceanic migration. In contrast, sexual maturation induced differential increases in the expression of leptins and LEPRs in the BPG-liver axis. Leptin2 was strikingly upregulated in the liver, the central organ of the reproductive metabolic challenge in teleosts. LEPRs were differentially regulated during sexual maturation, which may have contributed to the conservation of the duplicated LEPRs in this species. This suggests an ancient and positive role of the leptin system in the vertebrate reproductive function. This study brings new insights on the evolutionary history of the leptin system in vertebrates. Among extant vertebrates, the eel represents a unique case of duplicated leptins and leptin receptors as a result of 3R

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

acceptedVersio

The expression of nuclear and membrane estrogen receptors in the European eel throughout spermatogenesis

[EN] Estradiol (E-2) can bind to nuclear estrogen receptors (ESR) or membrane estrogen receptors (GPER). While mammals possess two nuclear ESRs and one membrane GPER, the European eel, like most other teleosts, has three nuclear ESRs and two membrane GPERs, as the result of a teleost specific genome duplication. In the current study, the expression of the three nuclear ESRs (ESR1, ESR2a and ESR2b) and the two membrane GPERs (GPERa and GPERb) in the brain-pituitary-gonad (BPG) axis of the European eel was measured, throughout spermatogenesis. The eels were first transferred from freshwater (FW) to seawater (SW), inducing parallel increases in E2 plasma levels and the expression of ESRs. This indicates that salinity has a stimulatory effect on the E-2 signalling pathway along the BPG axis. Stimulation of sexual maturation by weekly injections of human chorionic gonadotropin (hCG) induced a progressive decrease in E-2 plasma levels, and different patterns of expression of ESRs and GPERs in the BPG axis. The expression of nuclear ESRs increased in some parts of the brain, suggesting a possible upregulation due to a local production of E-2. In the testis, the highest expression levels of the nuclear ESRs were observed at the beginning of spermatogenesis, possibly mediating the role of E2 as spermatogonia renewal factor, followed by a sharply decrease in the expression of ESRs. Conversely, there was a marked increase observed in the expression of both membrane GPERs throughout spermatogenesis, suggesting they play a major role in the final stages of spermatogenesis.Funded by the Spanish Ministry of Science and Innovation (REPRO-TEMP project; AGL2013-41646-R) and IMPRESS (Marie Sklodowska-Curie Actions; Grant agreement no: 642893). M.C. Vilchez has a predoctoral grant from UPV PAID Programme (2011-S2-02-6521), M. Morini has a predoctoral grant from Generalitat Valenciana (Programa Grisolia). D.S. Penaranda was supported by MICINN and UPV (PTA2011-4948-1).Morini, M.; Peñaranda, D.; Vilchez Olivencia, MC.; Tveiten, H.; Lafont, A.; Dufour, S.; Pérez Igualada, LM.... (2017). The expression of nuclear and membrane estrogen receptors in the European eel throughout spermatogenesis. Comparative Biochemistry and Physiology Part A Molecular & Integrative Physiology. 203:91-99. doi:10.1016/j.cbpa.2016.08.020S919920

Caractérisation et rôle fonctionnel d'une famille de peptides calciotropes (CT et CGRP) (nouvelles données chez un téléostéen et deux mollusques céphalopodes)

Afin de mieux comprendre leur histoire évolutive, nous avons recherché la présence de la CT, du CGRP et de leurs organes cibles, chez un téléostéen, Anguilla anguilla, et deux céphalopodes, Nautilus macromphalus et Sepia officinalis. Contrairement à l anguille, aucune molécule biologiquement apparentée à la CT n a été détectée chez les céphalopodes. La co-localisation du CGRP et de son récepteur dans le système nerveux central de l anguille et de la seiche, suggère que ce peptide pourrait jouer un rôle de neuromédiateur ou neurotransmetteur comme décrit chez les mammifères. L implication du CGRP dans les mécanismes de régulation ionique, de façon endocrine dans les branchies et autocrine/paracrine dans le système rénal, représenterait une fonction ancienne, partagée par les téléostéens et les céphalopodes. Il semble que le CGRP ait une origine ancestrale intervenue avant l émergence des deutérostomiens. La CT constituerait un peptide apparu plus tardivement au cours de l évolution.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Three nuclear and two membrane estrogen receptors in basal teleosts, Anguilla sp.: Identification, evolutionary history and differential expression regulation

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Looking for the bird Kiss: evolutionary scenario in sauropsids.

International audienceBACKGROUND: The neuropeptide Kiss and its receptor KissR are key-actors in the brain control of reproduction in mammals, where they are responsible for the stimulation of the activity of GnRH neurones. Investigation in other vertebrates revealed up to 3 Kiss and 4 KissR paralogs, originating from the two rounds of whole genome duplication in early vertebrates. In contrast, the absence of Kiss and KissR has been suggested in birds, as no homologs of these genes could be found in current genomic databases. This study aims at addressing the question of the existence, from an evolutionary perspective, of the Kisspeptin system in birds. It provides the first large-scale investigation of the Kisspeptin system in the sauropsid lineage, including ophidian, chelonian, crocodilian, and avian lineages. RESULTS: Sauropsid Kiss and KissR genes were predicted from multiple genome and transcriptome databases by TBLASTN. Phylogenetic and syntenic analyses were performed to classify predicted sauropsid Kiss and KissR genes and to re-construct the evolutionary scenarios of both gene families across the sauropsid radiation.Genome search, phylogenetic and synteny analyses, demonstrated the presence of two Kiss genes (Kiss1 and Kiss2 types) and of two KissR genes (KissR1 and KissR4 types) in the sauropsid lineage. These four genes, also present in the mammalian lineage, would have been inherited from their common amniote ancestor. In contrast, synteny analyses supported that the other Kiss and KissR paralogs are missing in sauropsids as in mammals, indicating their absence in the amniote lineage. Among sauropsids, in the avian lineage, we demonstrated the existence of a Kiss2-like gene in three bird genomes. The divergence of these avian Kiss2-like sequences from those of other vertebrates, as well as their absence in the genomes of some other birds, revealed the processes of Kiss2 gene degeneration and loss in the avian lineage. CONCLUSION: These findings contribute to trace back the evolutionary history of the Kisspeptin system in amniotes and sauropsids, and provide the first molecular evidence of the existence and fate of a Kiss gene in birds

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

International audienceDuring 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, anon-teleost actinopterygian, the spotted gar, and an early teleost, the European eel, allowedus to get new insights into the molecular diversity and evolution of both Kiss and Kissrfamilies.We identified four Kissr in the spotted gar and coelacanth genomes, providing thefirst evidence of four Kissr genes in vertebrates. We also found three Kiss in the coelacanthand 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 ofvertebrate history. Four Kissr and four Kiss paralogs may have arisen via the two wholegenome duplication rounds (1R and 2R) in early vertebrates.Thiswould have been followedby multiple independent Kiss and Kissr gene losses in the sarcopterygian and actinopterygianlineages. In particular, no impact of the teleost-specific 3R could be recorded on thenumbers of teleost Kissr or Kiss paralogs. The origin of their diversity via 1R and 2R, aswell as the subsequent occurrence of multiple gene losses, represent common featuresof the evolutionary histories of Kiss and Kissr families in vertebrates. In contrast, comparisonsalso revealed un-matching numbers of Kiss and Kissr genes in some species, aswell as a large variability of Kiss/Kissr couples according to species. These discrepanciessupport independent features of the Kiss and Kissr evolutionary histories across vertebrateradiation

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

International audienceThe 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