Structural and Functional Characterization of Orcokinin B-like Neuropeptides in the Cuttlefish (Sepia officinalis).

The cuttlefish (Sepia officinalis) is a Cephalopod mollusk that lives in the English Channel and breeds in coastal spawning grounds in spring. A previous work showed that the control of egg-laying is monitored by different types of regulators, among which neuropeptides play a major role. They are involved in the integration of environmental cues, and participate in the transport of oocytes in the genital tract and in the secretion of capsular products. This study addresses a family of neuropeptides recently identified and suspected to be involved in the control of the reproduction processes. Detected by mass spectrometry and immunocytochemistry in the nerve endings of the accessory sex glands of the females and ovary, these neuropeptides are also identified in the hemolymph of egg-laying females demonstrating that they also have a hormone-like role. Released in the hemolymph by the sub-esophageal mass, a region that innervates the genital tract and the neurohemal area of the vena cava, in in vitro conditions these neuropeptides modulated oocyte transport and capsular secretion. Finally, in silico analyses indicated that these neuropeptides, initially called FLGamide, had extensive structural homology with orcokinin B, which motivated their name change.

Basal teleosts provide new insights into the evolutionary history of teleost-duplicated aromatase.

Duplicated cyp19a1 genes (cyp19a1a encoding aromatase a and cyp19a1b encoding aromatase b) have been identified in an increasing number of teleost species. Cyp19a1a is mainly expressed in the gonads, while cyp19a1b is mainly expressed in the brain, specifically in radial glial cells, as largely investigated by Kah and collaborators. The third round of whole-genome duplication that specifically occurred in the teleost lineage (TWGD or 3R) is likely at the origin of the duplicated cyp19a1 paralogs. In contrast to the situation in other teleosts, our previous studies identified a single cyp19a1 in eels (Anguilla), which are representative species of a basal group of teleosts, Elopomorpha. In the present study, using genome data mining and phylogenetic and synteny analyses, we confirmed that the whole aromatase genomic region was duplicated in eels, with most aromatase-neighboring genes being conserved in duplicate in eels, as in other teleosts. These findings suggest that specific gene loss of one of the 3R-duplicated cyp19a1 paralogs occurred in Elopomorpha after TWGD. Similarly, a single cyp19a1 gene was found in the arowana, which is a representative species of another basal group of teleosts, Osteoglossomorpha. In eels, the single cyp19a1 is expressed in both the brain and the gonads, as observed for the single CYP19A1 gene present in other vertebrates. The results of phylogenetic, synteny, closest neighboring gene, and promoter structure analyses showed that the single cyp19a1 of the basal teleosts shared conserved properties with both teleost cyp19a1a and cyp19a1b paralogs, which did not allow us to conclude which of the 3R-duplicated paralogs (cyp19a1a or cyp19a1b) was lost in Elopomorpha. Elopomorpha and Osteoglossomorpha cyp19a1 genes exhibited preserved ancestral functions, including expression in both the gonad and brain. We propose that the subfunctionalization of the 3R-duplicated cyp19a1 paralogs expressed specifically in the gonad or brain occurred in Clupeocephala, after the split of Clupeocephala from Elopomorpha and Osteoglossomorpha, which represented a driving force for the conservation of both 3R-duplicated paralogs in all extant Clupeocephala. In contrast, the functional redundancy of the undifferentiated 3R-duplicated cyp19a1 paralogs in elopomorphs and osteoglossomorphs would have favored the loss of one 3R paralog in basal teleosts.

Crustacean cardioactive peptides: Expression, localization, structure, and a possible involvement in regulation of egg-laying in the cuttlefish Sepia officinalis.

The cuttlefish (Sepia officinalis) is a cephalopod mollusk distributed on the western European coast, in the West African Ocean and in the Mediterranean Sea. On the Normandy coast (France), cuttlefish is a target species of professional fishermen, so its reproduction strategy is of particular interest in the context of stock management. Egg-laying, which is coastal, is controlled by several types of regulators among which neuropeptides. The cuttlefish neuropeptidome was recently identified by Zatylny-Gaudin et al. (2016). Among the 38 neuropeptide families identified, some were significantly overexpressed in egg-laying females as compared to mature males. This study is focused on crustacean cardioactive peptides (CCAPs), a highly expressed neuropeptide family strongly suspected of being involved in the control of egg-laying. We investigated the functional and structural characterization and tissue mapping of CCAPs, as well as the expression patterns of their receptors. CCAPs appeared to be involved in oocyte transport through the oviduct and in mechanical secretion of capsular products. Immunocytochemistry revealed that the neuropeptides were localized throughout the central nervous system (CNS) and in the nerve endings of the glands involved in egg-capsule synthesis and secretion, i.e. the oviduct gland and the main nidamental glands. The CCAP receptor was expressed in these glands and in the subesophageal mass of the CNS. Multiple sequence alignments revealed a high level of conservation of CCAP protein precursors in Sepia officinalis and Loligo pealei, two cephalopod decapods. Primary sequences of CCAPs from the two species were fully conserved, and cryptic peptides detected in the nerve endings were also partially conserved, suggesting biological activity that remains unknown for the time being.

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

In captivity, oogenesis and ovarian follicle maturation in European eel can be induced experimentally using hormonal therapy. The follicle's ability to respond effectively to the induction of maturation and ovulation, resulting in viable eggs, depends on the oocyte stage at the time of induction. We hypothesized that variation in the expression of key hormone receptors in the ovary and size of oocyte lipid droplets are associated with changes in oocyte stage. Thus, we induced ovarian follicle maturation using a priming dose of fish pituitary extract followed by the administration of a 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) injection. Females were then strip-spawned, the eggs were fertilized in vitro, incubated and larval survival was recorded at 3 days post hatch (dph). The expression of gonadotropin receptors (fshr, lhcgr1 and lhcgr2) and estrogen receptors (esr1, esr2a, esr2b, gpera and gperb) was quantified and the size of oocyte lipid droplets measured. Larval survival at 3 dph was used to differentiate high- and low-quality egg batches. Results showed significantly higher abundance of lhcgr1 and esr2a at priming for high-quality egg batches whereas fshr and gperb transcripts were significantly higher at DHP injection for low-quality egg batches. Therefore, high levels of lhcgr1 and esr2a may be important for attaining follicular maturational competence, while high fshr and gperb mRNA levels may indicate inadequate maturational competence. Furthermore, lipid droplet size at DHP and in ovulated eggs was significantly smaller in high-quality egg batches than in low-quality, which indicates that droplet size may be a useful marker of follicular maturational stage.

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

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 15weeks 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 (rpl5, rpl11) 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.

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

Estradiol (E2) 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 E2 signalling pathway along the BPG axis. Stimulation of sexual maturation by weekly injections of human chorionic gonadotropin (hCG) induced a progressive decrease in E2 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 E2. 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.

Nuclear and membrane progestin receptors in the European eel: Characterization and expression in vivo through spermatogenesis.

Characterization of all the progestin receptor genes (PRs) found in the European eel has been performed. There were five membrane PRs (mPRs): mPRα (alpha), mPRAL1 (alpha-like1), mPRAL2 (alpha-like2), mPRγ (gamma), mPRδ (delta) and two nuclear PRs (nPRs or PGRs): pgr1 and pgr2. In silico studies showed that the C and E(F) domains of Pgr are well conserved among vertebrates whereas the A/B domain is not. Phylogeny and synteny analyses suggest that eel duplicated pgr (pgr1 and pgr2) originated from the teleost-specific third whole genome duplication (3R). mPR phylogeny placed three eel mPRs together with the mPRα clade, being termed mPRα, mPRAL1 and mPRAL2, while the other two eel mPRs clustered with mPRγ and mPRδ clades, respectively. The in vivo study showed differential expression patterns along the brain-pituitary-gonad axis. An increase in nPR transcripts was observed in brain (in pgr1) and pituitary (in pgr1 and pgr2) through the spermatogenesis, from the spermatogonia B/spermatocyte stage to the spermiation stage. In the testis, mPRγ, mPRδ and pgr2 transcripts showed the highest levels in testis with A spermatogonia as dominant germ cell, while the highest mPRα, mPRAL1 and mPRAL2 transcripts were observed in testis from spermiating males, where the dominant germ cell were spermatozoa. Further studies should elucidate the role of both nuclear and membrane progestin receptors on eel spermatogenesis.

A novel function of calcitonin gene-related peptide in body fluid Cl- homeostasis.

Vertebrates need to maintain extracellular chloride (Cl(-)) concentrations to ensure the normal operation of physiological processes; the transition from aquatic to terrestrial environments necessitated the development of sophisticated mechanisms to ensure Cl(-) homeostasis in the face of fluctuating Cl(-) levels. Zebrafish calcitonin gene-related peptide (CGRP), unlike its splice variant calcitonin, does not respond to environmental Ca(2+) levels. This study aimed to test the hypothesis that CGRP is involved in the control of body fluid Cl(-) homeostasis. Acclimation to high-Cl(-) artificial water stimulated the mRNA expression of cgrp and the receptor (crlr1) when compared with low-Cl(-) CGRP knockdown induced upregulation of the Na(+)-Cl(-) co-transporter (ncc2b), while overexpression of CGRP resulted in the downregulation of ncc2b mRNA synthesis and a simultaneous decrease in Cl(-) uptake in embryos. Consistent with these findings, knockdown of either cgrp or crlr1 was found to increase the density of NCC2b-expressing cells in embryos. This is the first demonstration that CGRP acts as a hypochloremic hormone through suppressing NCC2b expression and the differentiation of NCC-expressing ionocytes. Elucidation of this novel function of CGRP in fish body fluid Cl(-) homeostasis promises to enhance our understanding of the related physiology in vertebrates.

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

Estrogens interact with classical intracellular nuclear receptors (ESR), and with G-coupled membrane receptors (GPER). In the eel, we identified three nuclear (ESR1, ESR2a, ESR2b) and two membrane (GPERa, GPERb) estrogen receptors. Duplicated ESR2 and GPER were also retrieved in most extant teleosts. Phylogeny and synteny analyses suggest that they result from teleost whole genome duplication (3R). In contrast to conserved 3R-duplicated ESR2 and GPER, one of 3R-duplicated ESR1 has been lost shortly after teleost emergence. Quantitative PCRs revealed that the five receptors are all widely expressed in the eel, but with differential patterns of tissue expression and regulation. ESR1 only is consistently up-regulated in vivo in female eel BPG-liver axis during induced sexual maturation, and also up-regulated in vitro by estradiol in eel hepatocyte primary cultures. This first comparative study of the five teleost estradiol receptors provides bases for future investigations on differential roles that may have contributed to the conservation of multiple estrogen receptors.

Looking for the bird Kiss: evolutionary scenario in sauropsids.

BACKGROUND: 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.

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.

The evolution of milk casein genes from tooth genes before the origin of mammals.

Caseins are among cardinal proteins that evolved in the lineage leading to mammals. In milk, caseins and calcium phosphate (CaP) form a huge complex called casein micelle. By forming the micelle, milk maintains high CaP concentrations, which help altricial mammalian neonates to grow bone and teeth. Two types of caseins are known. Ca-sensitive caseins (α(s)- and ß-caseins) bind Ca but precipitate at high Ca concentrations, whereas Ca-insensitive casein (κ-casein) does not usually interact with Ca but instead stabilizes the micelle. Thus, it is thought that these two types of caseins are both necessary for stable micelle formation. Both types of caseins show high substitution rates, which make it difficult to elucidate the evolution of caseins. Yet, recent studies have revealed that all casein genes belong to the secretory calcium-binding phosphoprotein (SCPP) gene family that arose by gene duplication. In the present study, we investigated exon-intron structures and phylogenetic distributions of casein and other SCPP genes, particularly the odontogenic ameloblast-associated (ODAM) gene, the SCPP-Pro-Gln-rich 1 (SCPPPQ1) gene, and the follicular dendritic cell secreted peptide (FDCSP) gene. The results suggest that contemporary Ca-sensitive casein genes arose from a putative common ancestor, which we refer to as CSN1/2. The six putative exons comprising CSN1/2 are all found in SCPPPQ1, although ODAM also shares four of these exons. By contrast, the five exons of the Ca-insensitive casein gene are all reminiscent of FDCSP. The phylogenetic distribution of these genes suggests that both SCPPPQ1 and FDCSP arose from ODAM. We thus argue that all casein genes evolved from ODAM via two different pathways; Ca-sensitive casein genes likely originated directly from SCPPPQ1, whereas the Ca-insensitive casein genes directly differentiated from FDCSP. Further, expression of ODAM, SCPPPQ1, and FDCSP was detected in dental tissues, supporting the idea that both types of caseins evolved as Ca-binding proteins. Based on these findings, we propose two alternative hypotheses for micelle formation in primitive milk. The conserved biochemical characteristics in caseins and their immediate ancestors also suggest that many slight genetic modifications have created modern caseins, proteins vital to the sustained success of mammals.

The enamelin genes in lizard, crocodile, and frog and the pseudogene in the chicken provide new insights on enamelin evolution in tetrapods.

Enamelin (ENAM) has been shown to be a crucial protein for enamel formation and mineralization. Previous molecular analyses have indicated a probable origin early in vertebrate evolution, which is supported by the presence of enamel/enameloid tissues in early vertebrates. In contrast to these hypotheses, ENAM was only characterized in mammals. Our aims were to 1) look for ENAM in representatives of nonmammalian tetrapods, 2) search for a pseudogene in the chicken genome, and 3) see whether the new sequences could bring new information on ENAM evolution. Using in silico approach and polymerase chain reaction, we obtained and characterized the messenger RNA sequences of ENAM in a frog, a lizard, and a crocodile; the genomic DNA sequences of ENAM in a frog and a lizard; and the putative sequence of chicken ENAM pseudogene. The comparison with mammalian ENAM sequences has revealed 1) the presence of an additional coding exon, named exon 8b, in sauropsids and marsupials, 2) a simpler 5'-untranslated region in nonmammalian ENAMs, 3) many sequence variations in the large exons while there are a few conserved regions in small exons, and 4) 25 amino acids that have been conserved during 350 million years of tetrapod evolution and hence of crucial biological importance. The chicken pseudogene was identified in a region that was not expected when considering the gene synteny in mammals. Together with the location of lizard ENAM in a homologous region, this result indicates that enamel genes were probably translocated in an ancestor of the sauropsid lineage. This study supports the origin of ENAM earlier in vertebrate evolution, confirms that tooth loss in modern birds led to the invalidation of enamel genes, and adds information on the important role played by, for example, the phosphorylated serines and the glycosylated asparagines for correct ENAM functions.

First identification of dopamine receptors in pikeperch, Sander lucioperca, during the pre-ovulatory period.

Dopamine (DA) is a ubiquitous neurotransmitter exerting a range of pleiotropic actions through two DA receptor families, the D1 and the D2. To date in vertebrates, a maximum of four receptor subtypes have been identified within the D1 family, D1 (former D1A), D5 (former D1B), D6 (former D1C and D1D) and D7 (former D1E), while the D2 family encloses five subtypes, D2, D3, D4, D8 (former D2like or D2l) and D9 (former D4-related sequence or D4-rs). In teleosts, no study has investigated in parallel all the DA receptors to identify and localize the whole receptor repertoire from both families. In pikeperch, Sander lucioperca, a species of interest for aquaculture development, the existence, number and location of the DA receptors are totally unknown. To address these questions, RNA-seq with de novo transcriptome reconstruction, functional annotation and phylogenetic analysis were performed to characterize the transcript repertoire of DA receptors in the brain of female pikeperch at the pre-ovulatory period. Ten different cDNA were identified and showed to belong to the D1 family: two D1, one D5a, one D6a and one D6b and to the D2 family: two spliced variants of D2, one D3, one D8 and one D9. Unlike zebrafish, the subtypes D4 and D7 have not yet been isolated in pikeperch. As expected D1, D3, D8 and D9 are mostly expressed in brain parts except for the cerebellum (D1 and D3). The inter-species differences in the number of DA receptors and the inter-organ differences in the gene expression of all receptors support the complexity of the dopaminergic actions in vertebrate.

Functional divergence of thyrotropin beta-subunit paralogs gives new insights into salmon smoltification metamorphosis.

Smoltification is a metamorphic event in salmon life history, which initiates downstream migration and pre-adapts juvenile salmon for seawater entry. While a number of reports concern thyroid hormones and smoltification, few and inconclusive studies have addressed the potential role of thyrotropin (TSH). TSH is composed of a α-subunit common to gonadotropins, and a ß-subunit conferring hormone specificity. We report the presence and functional divergence of duplicated TSH ß-subunit paralogs (tshßa and tshßb) in Atlantic salmon. Phylogeny and synteny analyses allowed us to infer that they originated from teleost-specific whole genome duplication. Expression profiles of both paralogs in the pituitary were measured by qPCR throughout smoltification in Atlantic salmon from the endangered Loire-Allier population raised in a conservation hatchery. This revealed a striking peak of tshßb expression in April, concomitant with downstream migration initiation, while tshßa expression remained relatively constant. In situ hybridization showed two distinct pituitary cell populations, tshßa cells in the anterior adenohypophysis, and tshßb cells near to the pituitary stalk, a location comparable to the pars tuberalis TSH cells involved in seasonal physiology and behaviour in birds and mammals. Functional divergence of tshß paralogs in Atlantic salmon supports a specific role of tshßb in smoltification.

Tachykinin-3 Genes and Peptides Characterized in a Basal Teleost, the European Eel: Evolutionary Perspective and Pituitary Role.

In mammals, neurokinin B (NKB) is a short peptide encoded by the gene tac3. It is involved in the brain control of reproduction by stimulating gonadotropin-releasing hormone (GnRH) neurons, mainly via kisspeptin. We investigated tac3 genes and peptides in a basal teleost, the European eel, which shows an atypical blockade of the sexual maturation at a prepubertal stage. Two tac3 paralogous genes (tac3a and tac3b) were identified in the eel genome, each encoding two peptides (NKBa or b and NKB-related peptide NKB-RPa or b). Amino acid sequence of eel NKBa is identical to human NKB, and the three others are novel peptide sequences. The four eel peptides present the characteristic C-terminal tachykinin sequence, as well as a similar alpha helix 3D structure. Tac3 genes were identified in silico in 52 species of vertebrates, and a phylogeny analysis was performed on the predicted TAC3 pre-pro-peptide sequences. A synteny analysis was also done to further assess the evolutionary history of tac3 genes. Duplicated tac3 genes in teleosts likely result from the teleost-specific whole genome duplication (3R). Among teleosts, TAC3b precursor sequences are more divergent than TAC3a, and a loss of tac3b gene would have even occurred in some teleost lineages. NKB-RP peptide, encoded beside NKB by tac3 gene in actinopterygians and basal sarcopterygians, would have been lost in ancestral amniotes. Tissue distribution of eel tac3a and tac3b mRNAs showed major expression of both transcripts in the brain especially in the diencephalon, as analyzed by specific qPCRs. Human NKB has been tested in vitro on primary culture of eel pituitary cells. Human NKB dose-dependently inhibited the expression of lhß, while having no effect on other glycoprotein hormone subunits (fshß, tshß, and gpα) nor on gh. Human NKB also dose-dependently inhibited the expression of GnRH receptor (gnrh-r2). The four eel peptides have been synthesized and also tested in vitro. They all inhibited the expression of both lhß and of gnrh-r2. This reveals a potential dual inhibitory role of the four peptides encoded by the two tac3 genes in eel reproduction, exerted at the pituitary level on both luteinizing hormone and GnRH receptor.

Characterization of Gonadotropin-Releasing Hormone (GnRH) Genes From Cartilaginous Fish: Evolutionary Perspectives.

The neuropeptide gonadotropin-releasing hormone (GnRH) plays an important role in the control of reproductive functions. Vertebrates possess multiple GnRH forms that are classified into three main groups, namely GnRH1, GnRH2, and GnRH3. In order to gain more insights into the GnRH gene family in vertebrates, we sought to identify which paralogs of this family are present in cartilaginous fish. For this purpose, we searched the genomes and/or transcriptomes of three representative species of this group, the small-spotted catshark, Scyliorhinus canicula, the whale shark, Rhincodon typus and the elephant shark Callorhinchus milii. In each species, we report the identification of three GnRH genes. In catshark and whale shark, phylogenetic and synteny analysis showed that these three genes correspond to GnRH1, GnRH2, and GnRH3. In both species, GnRH1 was found to encode a novel form of GnRH whose primary structure was determined as follows: QHWSFDLRPG. In elephant shark, the three genes correspond to GnRH1a and GnRH1b, two copies of the GnRH1 gene, plus GnRH2. 3D structure prediction of the chondrichthyan GnRH-associated peptides (GAPs) revealed that catshark GAP1, GAP2, and elephant shark GAP2 peptides exhibit a helix-loop-helix (HLH) structure. This structure observed for many osteichthyan GAP1 and GAP2, may convey GAP biological activity. This HLH structure could not be observed for elephant shark GAP1a and GAP1b. As for all other GAP3 described so far, no typical 3D HLH structure was observed for catshark nor whale shark GAP3. RT-PCR analysis revealed that GnRH1, GnRH2, and GnRH3 genes are differentially expressed in the catshark brain. GnRH1 mRNA appeared predominant in the diencephalon while GnRH2 and GnRH3 mRNAs seemed to be most abundant in the mesencephalon and telencephalon, respectively. Taken together, our results show that the GnRH gene repertoire of the vertebrate ancestor was entirely conserved in the chondrichthyan lineage but that the GnRH3 gene was probably lost in holocephali. They also suggest that the three GnRH neuronal systems previously described in the brain of bony vertebrates are also present in cartilaginous fish.

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.

Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide.

GnRH-associated peptide (GAP) is the C-terminal portion of the gonadotropin-releasing hormone (GnRH) preprohormone. Although it was reported in mammals that GAP may act as a prolactin-inhibiting factor and can be co-secreted with GnRH into the hypophyseal portal blood, GAP has been practically out of the research circuit for about 20 years. Comparative studies highlighted the low conservation of GAP primary amino acid sequences among vertebrates, contributing to consider that this peptide only participates in the folding or carrying process of GnRH. Considering that the three-dimensional (3D) structure of a protein may define its function, the aim of this study was to evaluate if GAP sequences and 3D structures are conserved in the vertebrate lineage. GAP sequences from various vertebrates were retrieved from databases. Analysis of primary amino acid sequence identity and similarity, molecular phylogeny, and prediction of 3D structures were performed. Amino acid sequence comparison and phylogeny analyses confirmed the large variation of GAP sequences throughout vertebrate radiation. In contrast, prediction of the 3D structure revealed a striking conservation of the 3D structure of GAP1 (GAP associated with the hypophysiotropic type 1 GnRH), despite low amino acid sequence conservation. This GAP1 peptide presented a typical helix-loop-helix (HLH) structure in all the vertebrate species analyzed. This HLH structure could also be predicted for GAP2 in some but not all vertebrate species and in none of the GAP3 analyzed. These results allowed us to infer that selective pressures have maintained GAP1 HLH structure throughout the vertebrate lineage. The conservation of the HLH motif, known to confer biological activity to various proteins, suggests that GAP1 peptides may exert some hypophysiotropic biological functions across vertebrate radiation.

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 lhß expression, without any effect on fshß, 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 lhß and gnrh-r2 expression in the European eel.