Publications by authors named "Roderick Nigel Finn"

35 Publications

A multiplier peroxiporin signal transduction pathway powers piscine spermatozoa.

Proc Natl Acad Sci U S A 2021 Mar;118(10)

Institute of Agrifood Research and Technology, Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain;

The primary task of a spermatozoon is to deliver its nuclear payload to the egg to form the next-generation zygote. With polyandry repeatedly evolving in the animal kingdom, however, sperm competition has become widespread, with the highest known intensities occurring in fish. Yet, the molecular controls regulating spermatozoon swimming performance in these organisms are largely unknown. Here, we show that the kinematic properties of postactivated piscine spermatozoa are regulated through a conserved trafficking mechanism whereby a peroxiporin ortholog of mammalian aquaporin-8 (Aqp8bb) is inserted into the inner mitochondrial membrane to facilitate HO efflux in order to maintain ATP production. In teleosts from more ancestral lineages, such as the zebrafish () and the Atlantic salmon (), in which spermatozoa are activated in freshwater, an intracellular Ca-signaling directly regulates this mechanism through monophosphorylation of the Aqp8bb N terminus. In contrast, in more recently evolved marine teleosts, such the gilthead seabream (), in which spermatozoa activation occurs in seawater, a cross-talk between Ca- and oxidative stress-activated pathways generate a multiplier regulation of channel trafficking via dual N-terminal phosphorylation. These findings reveal that teleost spermatozoa evolved increasingly sophisticated detoxification pathways to maintain swimming performance under a high osmotic stress, and provide insight into molecular traits that are advantageous for postcopulatory sexual selection.
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http://dx.doi.org/10.1073/pnas.2019346118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958246PMC
March 2021

A transcriptomic analysis of diploid and triploid Atlantic salmon lenses with and without cataracts.

Exp Eye Res 2020 10 28;199:108150. Epub 2020 Jul 28.

Institute of Marine Research, Nordnes, Bergen, Norway; Department of Biological Sciences, University of Bergen, Bergen, Norway.

To avoid negative environmental impacts of escapees and potential inter-breeding with wild populations, the Atlantic salmon farming industry has and continues to extensively test triploid fish that are sterile. However, they often show differences in performance, physiology, behavior and morphology compared to diploid fish, with increased prevalence of vertebral deformities and ocular cataracts as two of the most severe disorders. Here, we investigated the mechanisms behind the higher prevalence of cataracts in triploid salmon, by comparing the transcriptional patterns in lenses of diploid and triploid Atlantic salmon, with and without cataracts. We assembled and characterized the Atlantic salmon lens transcriptome and used RNA-seq to search for the molecular basis for cataract development in triploid fish. Transcriptional screening showed only modest differences in lens mRNA levels in diploid and triploid fish, with few uniquely expressed genes. In total, there were 165 differentially expressed genes (DEGs) between the cataractous diploid and triploid lens. Of these, most were expressed at lower levels in triploid fish. Differential expression was observed for genes encoding proteins with known function in the retina (phototransduction) and proteins associated with repair and compensation mechanisms. The results suggest a higher susceptibility to oxidative stress in triploid lenses, and that mechanisms connected to the ability to handle damaged proteins are differentially affected in cataractous lenses from diploid and triploid salmon.
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http://dx.doi.org/10.1016/j.exer.2020.108150DOI Listing
October 2020

Unravelling the Complex Duplication History of Deuterostome Glycerol Transporters.

Cells 2020 07 10;9(7). Epub 2020 Jul 10.

Department of Biological Sciences, Bergen High Technology Centre, University of Bergen, 5020 Bergen, Norway.

Transmembrane glycerol transport is an ancient biophysical property that evolved in selected subfamilies of water channel (aquaporin) proteins. Here, we conducted broad level genome (>550) and transcriptome (>300) analyses to unravel the duplication history of the glycerol-transporting channels () in Deuterostomia. We found that tandem duplication (TD) was the major mechanism of gene expansion in echinoderms and hemichordates, which, together with whole genome duplications (WGD) in the chordate lineage, continued to shape the genomic repertoires in craniates. Molecular phylogenies indicated that -like and -like channels were the probable stem subfamilies in craniates, with WGD generating and in gnathostomes but arising through TD in Osteichthyes. We uncovered separate examples of gene translocations, gene conversion, and concerted evolution in humans, teleosts, and starfishes, with DNA transposons the likely drivers of gene rearrangements in paleotetraploid salmonids. Currently, gene copy numbers and BLAST are poor predictors of orthologous relationships due to asymmetric gene evolution in the different lineages. Such asymmetries can impact estimations of divergence times by millions of years. Experimental investigations of the salmonid channels demonstrated that approximately half of the 20 ancestral paralogs are functional, with neofunctionalization occurring at the transcriptional level rather than the protein transport properties. The combined findings resolve the origins and diversification of over >800 million years old and thus form the novel basis for proposing a pandeuterostome gene nomenclature.
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http://dx.doi.org/10.3390/cells9071663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408487PMC
July 2020

The vertebrate Aqp14 water channel is a neuropeptide-regulated polytransporter.

Commun Biol 2019 11;2:462. Epub 2019 Dec 11.

1IRTA-Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, 08193 Bellaterra, (Cerdanyola del Vallès) Spain.

Water channels (aquaporins) were originally discovered in mammals with fourteen subfamilies now identified (AQP0-13). Here we show that a functional Aqp14 subfamily phylogenetically related to AQP4-type channels exists in all vertebrate lineages except hagfishes and eutherian mammals. In contrast to the water-selective classical aquaporins, which have four aromatic-arginine constriction residues, Aqp14 proteins present five non-aromatic constriction residues and facilitate the permeation of water, urea, ammonia, HO and glycerol. Immunocytochemical assays suggest that Aqp14 channels play important osmoregulatory roles in piscine seawater adaptation. Our data indicate that Aqp14 intracellular trafficking is tightly regulated by the vasotocinergic/isotocinergic neuropeptide and receptor systems, whereby protein kinase C and A transduction pathways phosphorylate highly conserved C-terminal residues to control channel plasma membrane insertion. The neuropeptide regulation of Aqp14 channels thus predates the vasotocin/vasopressin regulation of AQP2-5-6 orthologs observed in tetrapods. These findings demonstrate that vertebrate Aqp14 channels represent an ancient subfamily of neuropeptide-regulated polytransporters.
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http://dx.doi.org/10.1038/s42003-019-0713-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906440PMC
July 2020

The cellular localization and redistribution of multiple aquaporin paralogs in the spermatic duct epithelium of a maturing marine teleost.

J Anat 2018 08 27;233(2):177-192. Epub 2018 May 27.

Group of Comparative Molecular Physiology, IRTA-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain.

Aquaporin-mediated fluid transport in the mammalian efferent duct and epididymis is believed to play a role in sperm maturation and concentration. In fish, such as the marine teleost gilthead seabream (Sparus aurata), the control of fluid homeostasis in the spermatic duct seems also to be crucial for male fertility, but no information exists on the expression and distribution of aquaporins. In this study, reverse transcriptase-polymerase chain reaction and immunoblotting analyses, employing available and newly raised paralog-specific antibodies for seabream aquaporins, indicate that up to nine functional aquaporins, Aqp0a, -1aa, -1ab, -3a, -4a, -7, -8bb, -9b and -10b, are expressed in the spermatic duct. Immunolocalization of the channels in the resting spermatic duct reveals that Aqp0a, -1aa, -4a, -7 and -10b are expressed in the monolayered luminal epithelium, Aqp8b and -9b in smooth muscle fibers, and Aqp1ab and -3a in different interstitial lamina cells. In the epithelial cells, Aqp0a and -1aa are localized in the short apical microvilli, and Aqp4a and -10b show apical and basolateral staining, whereas Aqp7 is solely detected in vesicular compartments. Upon spermiation, an elongation of the epithelial cells sterocilia, as well as the folding of the epithelium, is observed. At this stage, single- and double-immunostaining, using two aquaporin paralogs or the Na /K -ATPase membrane marker, indicate that Aqp1ab, -3a, -7, -8bb and -9b staining remains unchanged, whereas in epithelial cells Aqp1aa translation is supressed, Aqp4a internalizes, and Aqp0a and -10b accumulate in the apical, lateral and basal plasma membrane. These findings uncover a cell type- and region-specific distribution of multiple aquaporins in the piscine spermatic duct, which shares conserved features of the mammalian system. The data therefore suggest that aquaporins may play different roles in the regulation of fluid homeostasis and sperm maturation in the male reproductive tract of fish.
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http://dx.doi.org/10.1111/joa.12829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6036930PMC
August 2018

The Physiological Role and Regulation of Aquaporins in Teleost Germ Cells.

Adv Exp Med Biol 2017 ;969:149-171

Department of Biology, Bergen High Technology Centre, University of Bergen, 5020, Bergen, Norway.

The unicellular germ cells and gametes of oviparous teleosts lack the osmoregulatory organs present in juveniles and adults, yet during development and particularly at spawning, they face tremendous osmotic challenges when released into the external aquatic environment. Increasing evidence suggests that transmembrane water channels (aquaporins) evolved to play vital adaptive roles that mitigate the osmotic and oxidative stress problems of the developing oocytes , embryos and spermatozoa. In this chapter, we provide a short overview of the diversity of the aquaporin superfamily in teleosts, and summarize the findings that uncovered a highly specific molecular regulation of aquaporins during oogenesis and spermatogenesis. We further review the multiple functions that these channels play during the establishment of egg buoyancy and the activation and detoxification of spermatozoa in the marine environment.
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http://dx.doi.org/10.1007/978-94-024-1057-0_10DOI Listing
September 2017

Molecular and functional characterization of Bemisia tabaci aquaporins reveals the water channel diversity of hemipteran insects.

Insect Biochem Mol Biol 2016 10 2;77:39-51. Epub 2016 Aug 2.

USDA-ARS, U.S. Arid Land Agricultural Research Center, Maricopa, AZ 85138, USA. Electronic address:

The Middle East-Asia Minor 1 (MEAM1) whitefly, Bemisia tabaci (Gennadius) is an economically important pest of food, fiber, and ornamental crops. This pest has evolved a number of adaptations to overcome physiological challenges, including 1) the ability to regulate osmotic stress between gut lumen and hemolymph after imbibing large quantities of a low nitrogen, sugar-rich liquid diet; 2) the ability to avoid or prevent dehydration and desiccation, particularly during egg hatching and molting; and 3) to be adapted for survival at elevated temperatures. One superfamily of proteins involved in the maintenance of fluid homeostasis in many organisms includes the aquaporins, which are integral membrane channel proteins that aid in the rapid flux of water and other small solutes across biological membranes. Here, we show that B. tabaci has eight aquaporins (BtAqps), of which seven belong to the classical aquaporin 4-related grade of channels, including Bib, Drip, Prip, and Eglps and one that belongs to the unorthodox grade of aquaporin 12-like channels. B. tabaci has further expanded its repertoire of water channels through the expression of three BtDrip2 amino-terminal splice variants, while other hemipteran species express amino- or carboxyl-terminal isoforms of Drip, Prip, and Eglps. Each BtAqp has unique transcript expression profiles, cellular localization, and/or substrate preference. Our phylogenetic and functional data reveal that hemipteran insects lost the classical glp genes, but have compensated for this by duplicating the eglp genes early in their evolution to comprise at least three separate clades of glycerol transporters.
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http://dx.doi.org/10.1016/j.ibmb.2016.07.010DOI Listing
October 2016

Auto-Adhesion Potential of Extraocular Aqp0 during Teleost Development.

PLoS One 2016 6;11(5):e0154592. Epub 2016 May 6.

Department of Biology, Bergen High Technology Centre, University of Bergen, 5020 Bergen, Norway.

AQP0 water channels are the most abundant proteins expressed in the mammalian lens fiber membranes where they are essential for lens development and transparency. Unlike other aquaporin paralogs, mammalian AQP0 has a low intrinsic water permeability, but can form cell-to-cell junctions between the lens fibers. It is not known whether the adhesive properties of AQP0 is a derived feature found only in mammals, or exists as a conserved ancestral trait in non-mammalian vertebrates. Here we show that a tetraploid teleost, the Atlantic salmon, expresses four Aqp0 paralogs in the developing lens, but also expresses significant levels of aqp0 mRNAs and proteins in the epithelia of the pronephros, presumptive enterocytes, gill filament and epidermis. Quantitative PCR reveals that aqp0 mRNA titres increase by three orders of magnitude between the onset of somitogenesis and pigmentation of the eye. Using in situ hybridization and specific antisera, we show that at least two of the channels (Aqp0a1, -0b1 and/or -0b2) are localized in the extraocular basolateral and apical membranes, while Aqp0a2 is lens-specific. Heterologous expression of the Aqp0 paralogs in adhesion-deficient mouse fibolast L-cells reveals that, as for human AQP0, each intact salmon channel retains cell-to-cell adhesive properties. The strongest Aqp0 interactions are auto-adhesion, suggesting that homo-octamers likely form the intercellular junctions of the developing lens and epithelial tissues. The present data are thus the first to show the adhesion potential of Aqp0 channels in a non-mammalian vertebrate, and further uncover a novel extraocular role of the channels during vertebrate development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154592PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4859563PMC
July 2017

Evolution and functional diversity of aquaporins.

Biol Bull 2015 Aug;229(1):6-23

Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), 08003 Barcelona, Spain.

In this review, we provide a brief synopsis of the evolution and functional diversity of the aquaporin gene superfamily in prokaryotic and eukaryotic organisms. Based upon the latest data, we discuss the expanding list of molecules shown to permeate the central pore of aquaporins, and the unexpected diversity of water channel genes in Archaea and Bacteria. We further provide new insight into the origin by horizontal gene transfer of plant glycerol-transporting aquaporins (NIPs), and the functional co-option and gene replacement of insect glycerol transporters. Finally, we discuss the origins of four major grades of aquaporins in Eukaryota, together with the increasing repertoires of aquaporins in vertebrates.
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http://dx.doi.org/10.1086/BBLv229n1p6DOI Listing
August 2015

Phylogenomic and functional analyses of salmon lice aquaporins uncover the molecular diversity of the superfamily in Arthropoda.

BMC Genomics 2015 Aug 19;16:618. Epub 2015 Aug 19.

Sea Lice Research Centre, Department of Biology, Bergen High Technology Centre, University of Bergen, 5020, Bergen, Norway.

Background: An emerging field in biomedical research is focusing on the roles of aquaporin water channels in parasites that cause debilitating or lethal diseases to their vertebrate hosts. The primary vectorial agents are hematophagous arthropods, including mosquitoes, flies, ticks and lice, however very little is known concerning the functional diversity of aquaporins in non-insect members of the Arthropoda. Here we conducted phylogenomic and functional analyses of aquaporins in the salmon louse, a marine ectoparasitic copepod that feeds on the skin and body fluids of salmonids, and used the primary structures of the isolated channels to uncover the genomic repertoires in Arthropoda.

Results: Genomic screening identified 7 aquaporin paralogs in the louse in contrast to 42 in its host the Atlantic salmon. Phylogenetic inference of the louse nucleotides and proteins in relation to orthologs identified in Chelicerata, Myriapoda, Crustacea and Hexapoda revealed that the arthropod aquaporin superfamily can be classified into three major grades (1) classical aquaporins including Big brain (Bib) and Prip-like (PripL) channels (2) aquaglyceroporins (Glp) and (3) unorthodox aquaporins (Aqp12-like). In Hexapoda, two additional subfamilies exist as Drip and a recently classified entomoglyceroporin (Eglp) group. Cloning and remapping the louse cDNAs to the genomic DNA revealed that they are encoded by 1-7 exons, with two of the Glps being expressed as N-terminal splice variants (Glp1_v1, -1_v2, -3_v1, -3_v2). Heterologous expression of the cRNAs in amphibian oocytes demonstrated that PripL transports water and urea, while Bib does not. Glp1_v1, -2, -3_v1 and -3_v2 each transport water, glycerol and urea, while Glp1_v2 and the Aqp12-like channels were retained intracellularly. Transcript abundance analyses revealed expression of each louse paralog at all developmental stages, except for glp1_v1, which is specific to preadult and adult males.

Conclusions: Our data suggest that the aquaporin repertoires of extant arthropods have expanded independently in the different lineages, but can be phylogenetically classified into three major grades as opposed to four present in deuterostome animals. While the aquaporin repertoire of Atlantic salmon represents a 6-fold redundancy compared to the louse, the functional assays reveal that the permeation properties of the different crustacean grades of aquaporin are largely conserved to the vertebrate counterparts.
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http://dx.doi.org/10.1186/s12864-015-1814-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539701PMC
August 2015

Insect glycerol transporters evolved by functional co-option and gene replacement.

Nat Commun 2015 Jul 17;6:7814. Epub 2015 Jul 17.

Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Passeig Marítim 37-49, 08003 Barcelona, Spain.

Transmembrane glycerol transport is typically facilitated by aquaglyceroporins in Prokaryota and Eukaryota. In holometabolan insects however, aquaglyceroporins are absent, yet several species possess polyol permeable aquaporins. It thus remains unknown how glycerol transport evolved in the Holometabola. By combining phylogenetic and functional studies, here we show that a more efficient form of glycerol transporter related to the water-selective channel AQP4 specifically evolved and multiplied in the insect lineage, resulting in the replacement of the ancestral branch of aquaglyceroporins in holometabolan insects. To recapitulate this evolutionary process, we generate specific mutants in distantly related insect aquaporins and human AQP4 and show that a single mutation in the selectivity filter converted a water-selective channel into a glycerol transporter at the root of the crown clade of hexapod insects. Integration of phanerozoic climate models suggests that these events were associated with the emergence of complete metamorphosis and the unparalleled radiation of insects.
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http://dx.doi.org/10.1038/ncomms8814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4518291PMC
July 2015

The pH sensitivity of Aqp0 channels in tetraploid and diploid teleosts.

FASEB J 2015 May 9;29(5):2172-84. Epub 2015 Feb 9.

*Department of Biology, Bergen High Technology Centre, University of Bergen, Bergen, Norway; Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain; and Institute of Marine Research, Nordnes, Bergen, Norway

Water homeostasis and the structural integrity of the vertebrate lens is partially mediated by AQP0 channels. Emerging evidence indicates that external pH may be involved in channel gating. Here we show that a tetraploid teleost, the Atlantic salmon, retains 4 aqp0 genes (aqp0a1, -0a2, -0b1, and -0b2), which are highly, but not exclusively, expressed in the lens. Functional characterization reveals that, although each paralog permeates water efficiently, the permeability is respectively shifted to the neutral, alkaline, or acidic pH in Aqp0a1, -0a2, and -0b1, whereas that of Aqp0b2 is not regulated by external pH. Mutagenesis studies demonstrate that Ser(38), His(39), and His(40) residues in the extracellular transmembrane domain of α-helix 2 facing the water pore are critical for the pH modulation of water transport. To validate these findings, we show that both zebrafish Aqp0a and -0b are functional water channels with respective pH sensitivities toward alkaline or acid pH ranges and that an N-terminal allelic variant (Ser(19)) of Aqp0b exists that abolishes water transport in Xenopus laevis oocytes. The data suggest that the alkaline pH sensitivity is a conserved trait in teleost Aqp0 a-type channels, whereas mammalian AQP0 and some teleost Aqp0 b-type channels display an acidic pH permeation preference.
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http://dx.doi.org/10.1096/fj.14-267625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4423293PMC
May 2015

Mitochondrial aquaporin-8-mediated hydrogen peroxide transport is essential for teleost spermatozoon motility.

Sci Rep 2015 Jan 14;5:7789. Epub 2015 Jan 14.

Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), 08003 Barcelona, Spain.

Reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2), cause oxidative cell damage and inhibit sperm function. In most oviparous fishes that spawn in seawater (SW), spermatozoa may be exposed to harmful ROS loads associated with the hyperosmotic stress of axonemal activation and ATP synthesis from mitochondrial oxidative phosphorylation. However, it is not known how marine spermatozoa can cope with the increased ROS levels to maintain flagellar motility. Here, we show that a marine teleost orthologue of human aquaporin-8, termed Aqp8b, is rapidly phosphorylated and inserted into the inner mitochondrial membrane of SW-activated spermatozoa, where it facilitates H2O2 efflux from this compartment. When Aqp8b intracellular trafficking and mitochondrial channel activity are immunologically blocked in activated spermatozoa, ROS levels accumulate in the mitochondria leading to mitochondrial membrane depolarisation, the reduction of ATP production, and the progressive arrest of sperm motility. However, the decreased sperm vitality underlying Aqp8b loss of function is fully reversed in the presence of a mitochondria-targeted antioxidant. These findings reveal a previously unknown detoxification mechanism in spermatozoa under hypertonic conditions, whereby mitochondrial Aqp8b-mediated H2O2 efflux permits fuel production and the maintenance of flagellar motility.
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http://dx.doi.org/10.1038/srep07789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4293619PMC
January 2015

The lineage-specific evolution of aquaporin gene clusters facilitated tetrapod terrestrial adaptation.

PLoS One 2014 26;9(11):e113686. Epub 2014 Nov 26.

Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Institut de Ciències del Mar, CSIC, Barcelona, Spain.

A major physiological barrier for aquatic organisms adapting to terrestrial life is dessication in the aerial environment. This barrier was nevertheless overcome by the Devonian ancestors of extant Tetrapoda, but the origin of specific molecular mechanisms that solved this water problem remains largely unknown. Here we show that an ancient aquaporin gene cluster evolved specifically in the sarcopterygian lineage, and subsequently diverged into paralogous forms of AQP2, -5, or -6 to mediate water conservation in extant Tetrapoda. To determine the origin of these apomorphic genomic traits, we combined aquaporin sequencing from jawless and jawed vertebrates with broad taxon assembly of >2,000 transcripts amongst 131 deuterostome genomes and developed a model based upon Bayesian inference that traces their convergent roots to stem subfamilies in basal Metazoa and Prokaryota. This approach uncovered an unexpected diversity of aquaporins in every lineage investigated, and revealed that the vertebrate superfamily consists of 17 classes of aquaporins (Aqp0 - Aqp16). The oldest orthologs associated with water conservation in modern Tetrapoda are traced to a cluster of three aqp2-like genes in Actinistia that likely arose >500 Ma through duplication of an aqp0-like gene present in a jawless ancestor. In sea lamprey, we show that aqp0 first arose in a protocluster comprised of a novel aqp14 paralog and a fused aqp01 gene. To corroborate these findings, we conducted phylogenetic analyses of five syntenic nuclear receptor subfamilies, which, together with observations of extensive genome rearrangements, support the coincident loss of ancestral aqp2-like orthologs in Actinopterygii. We thus conclude that the divergence of sarcopterygian-specific aquaporin gene clusters was permissive for the evolution of water conservation mechanisms that facilitated tetrapod terrestrial adaptation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0113686PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4245216PMC
December 2015

Thermal stress alters expression of genes involved in one carbon and DNA methylation pathways in Atlantic cod embryos.

Comp Biochem Physiol A Mol Integr Physiol 2014 07 16;173C:17-27. Epub 2014 Mar 16.

National Institute of Nutrition and Seafood Research (NIFES), PO Box 2029 Nordnes, 5817 Bergen, Norway.

One-carbon (1-C) metabolism is essential for normal embryonic development through its regulation of DNA methylation and cell proliferation. With consideration to the potential future anthropogenic oceanic warming, we studied the effects of both acute thermal stress and continuous thermal stress (10°C) during Atlantic cod embryo development on the expression levels of genes associated with the 1-C metabolism, including DNA methyltransferases. We conducted a phylogenetic analysis of vertebrate DNA methyltransferases to determine the number and similarity of DNMT found in Atlantic cod. This analysis revealed that Atlantic cod have one maintenance dnmt (dnmt1) and five de novo DNMTs (dnmt4, dnmt3, dnmt3b, dnmt3aa, dnmt3ab). Stage specific changes in expression levels occurred for all genes analyzed. The effect of acute thermal stress was evaluated during early development. Compared to controls these experiments showed significant alterations in expression levels of several genes, that in some instances were reversed at later stages of development. A significant effect of continuous thermal stress was found in gastrula embryos where lower mRNA expression levels of 1-C metabolism, de novo DNMTs and cell proliferation genes were detected. One exception was the maintenance DNMT, which was only sensitive to acute and not continuous thermal stress. DNA methylation status indicated that blastula embryos were hypomethylated compared to spermatozoa and late gastrula stages. In post-gastrula stage, however, continuous thermal stress resulted in a higher degree of DNA methylation compared to controls. These data reveal that the regulation of epigenetically important transcripts in the 1-C metabolism of Atlantic cod embryos is sensitive to thermal stress.
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http://dx.doi.org/10.1016/j.cbpa.2014.03.003DOI Listing
July 2014

Differential expression and novel permeability properties of three aquaporin 8 paralogs from seawater-challenged Atlantic salmon smolts.

J Exp Biol 2013 Oct 18;216(Pt 20):3873-85. Epub 2013 Jul 18.

Institute of Biology, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark.

Aquaporins may facilitate transepithelial water absorption in the intestine of seawater (SW)-acclimated fish. Here we have characterized three full-length aqp8 paralogs from Atlantic salmon (Salmo salar). Bayesian inference revealed that each paralog is a representative of the three major classes of aqp8aa, aqp8ab and aqp8b genes found in other teleosts. The permeability properties were studied by heterologous expression in Xenopus laevis oocytes, and the expression levels examined by qPCR, immunofluorescence and immunoelectron microscopy, and immunoblotting of membrane fractions from intestines of SW-challenged smolts. All three Aqp8 paralogs were permeable to water and urea, whereas Aqp8ab and -8b were, surprisingly, also permeable to glycerol. The mRNA tissue distribution of each paralog was distinct, although some tissues such as the intestine showed redundant expression of more than one paralog. Immunofluorescence microscopy localized Aqp8aa(1+2) to intracellular compartments of the liver and intestine, and Aqp8ab and Aqp8b to apical plasma membrane domains of the intestinal epithelium, with Aqp8b also in goblet cells. In a control experiment with rainbow trout, immunoelectron microscopy confirmed abundant labeling of Aqp8ab and -8b at apical plasma membranes of enterocytes in the middle intestine and also in subapical vesicular structures. During SW challenge, Aqp8ab showed significantly increased levels of protein expression in plasma-membrane-enriched fractions of the intestine. These data indicate that the Atlantic salmon Aqp8 paralogs have neofunctionalized on a transcriptional as well as a functional level, and that Aqp8ab may play a central role in the intestinal transcellular uptake of water during SW acclimation.
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http://dx.doi.org/10.1242/jeb.087890DOI Listing
October 2013

Subcellular localization of selectively permeable aquaporins in the male germ line of a marine teleost reveals spatial redistribution in activated spermatozoa.

Biol Reprod 2013 Aug 15;89(2):37. Epub 2013 Aug 15.

Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain.

In oviparous vertebrates such as the marine teleost gilthead seabream, water and fluid homeostasis associated with testicular physiology and the external activation of spermatozoa is potentially mediated by multiple aquaporins. To test this hypothesis, we isolated five novel members of the aquaporin superfamily from gilthead seabream and developed paralog-specific antibodies to localize the cellular sites of protein expression in the male reproductive tract. Together with phylogenetic classification, functional characterization of four of the newly isolated paralogs, Aqp0a, -7, -8b, and -9b, demonstrated that they were water permeable, while Aqp8b was also permeable to urea, and Aqp7 and -9b were permeable to glycerol and urea. Immunolocalization experiments indicated that up to seven paralogous aquaporins are differentially expressed in the seabream testis: Aqp0a and -9b in Sertoli and Leydig cells, respectively; Aqp1ab, -7, and -10b from spermatogonia to spermatozoa; and Aqp1aa and -8b in spermatids and sperm. In the efferent duct, only Aqp10b was found in the luminal epithelium. Ejaculated spermatozoa showed a segregated spatial distribution of five aquaporins: Aqp1aa and -7 in the entire flagellum or the head, respectively, and Aqp1ab, -8b, and -10b both in the head and the anterior tail. The combination of immunofluorescence microscopy and biochemical fractionation of spermatozoa indicated that Aqp10b and phosphorylated Aqp1ab are rapidly translocated to the head plasma membrane upon activation, whereas Aqp8b accumulates in the mitochondrion of the spermatozoa. In contrast, Aqp1aa and -7 remained unchanged. These data reveal that aquaporin expression in the teleost testis shares conserved features of the mammalian system, and they suggest that the piscine channels may play different roles in water and solute transport during spermatogenesis, sperm maturation and nutrition, and the initiation and maintenance of sperm motility.
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http://dx.doi.org/10.1095/biolreprod.113.110783DOI Listing
August 2013

Primary oocyte transcriptional activation of aqp1ab by the nuclear progestin receptor determines the pelagic egg phenotype of marine teleosts.

Dev Biol 2013 May 13;377(2):345-62. Epub 2013 Mar 13.

IRTA-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), 08003 Barcelona, Spain.

In marine teleosts, the aqp1ab water channel plays a vital role in the development of the pelagic egg phenotype. However, the developmental control of aqp1ab activation during oogenesis remains to be established. Here, we report the isolation of the 5'-flanking region of the teleost gilthead seabream aqp1ab gene, in which we identify conserved cis-regulatory elements for the binding of the nuclear progestin receptor (Pgr) and members of the Sox family of transcription factors. Subcellular localization studies indicated that the Pgr, as well as sox3 and -8b transcripts, are co-expressed in seabream oogonia, whereas in meiosis-arrested primary growth (pre-vitellogenic) oocytes, when aqp1ab mRNA and protein are first synthesized, the Pgr appears to be completely translocated from the ooplasm into the nucleus. By contrast, sox9b is highly expressed in more advanced oocytes, coinciding with a strong depletion of aqp1ab transcripts in the oocyte. Functional characterization of wild-type and mutated aqp1ab promoter constructs, using mammalian cells and Xenopus laevis oocytes, demonstrated that aqp1ab transcription is initiated by the Pgr, which is activated by the progestin 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P), the natural ligand of the seabream Pgr. In vitro incubation of seabream primary ovarian explants with the follicle-stimulating hormone or 17,20β-P confirmed that progestin-activated Pgr enhanced Aqp1ab synthesis via the aqp1ab promoter. However, transactivation assays in heterologous systems showed that Sox transcription factors can potentially modulate this mechanism. These data uncover the existence of an endocrine pathway involved in the early activation of a water channel necessary for egg formation in marine teleosts.
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http://dx.doi.org/10.1016/j.ydbio.2013.03.001DOI Listing
May 2013

Alternative splicing of the nuclear progestin receptor in a perciform teleost generates novel mechanisms of dominant-negative transcriptional regulation.

Gen Comp Endocrinol 2013 Feb 4;182:24-40. Epub 2012 Dec 4.

IRTA-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas, 08003 Barcelona, Spain.

In mammals, downstream function of the nuclear progestin receptor (PGR) can be differentially regulated in each target tissue by altering the expression levels of PGR mRNA variants. Such PGR isoforms have also been identified in birds and reptiles, but not in non-amniote vertebrates. Based upon extensive phylogenetic, syntenic and functional analyses, here we show that higher orders of Teleostei retain a single pgr gene, and that four different pgr transcript variants of the extant gene are expressed in the ovary of an evolutionary advanced perciform teleost, the gilthead seabream (Sparus aurata). Three of the isoforms (pgr_tv2, pgr_tv3 and pgr_tv4) arise from alternative pre-mRNA splicing resulting in different N-terminally truncated receptors, whereas one isoform (pgr_tv1) is a deletion variant. Seabream wild-type Pgr shows the highest transactivational response to native euteleostean progestins, 17α,20β-dihydroxy-4-pregnen-3-one and 17α,20β,21-trihydroxy-4-pregnen-3-one, whereas the Pgr_tv3 and Pgr_tv4 isoforms independently regulate novel nuclear and cytosolic mechanisms of dominant-negative repression of Pgr-mediated transcription. In the seabream ovary, the wild-type Pgr protein is localized in oogonia, in the nuclei of primary (previtellogenic) oocytes, as well as in follicular (granulosa) cells and the oocyte cytoplasm of early and late vitellogenic ovarian follicles. Expression of wild-type pgr, pgr_tv3 and pgr_tv4 was the highest in seabream primary ovaries, while expression of both inhibitory receptor isoforms, but not of pgr, decreased during vitellogenesis. Stimulation of primary ovarian explants in vitro with recombinant piscine follicle-stimulating hormone and estrogen differentially regulated the temporal expression of pgr, pgr_tv3 and pgr_tv4. These findings suggest that, as in mammals, ovarian progestin responsiveness in the seabream, particularly during early oogenesis, may be regulated through alternative splicing of the nuclear pgr mRNA. Thus, the dominant-negative mechanism of PGR transcriptional regulation likely evolved prior to the separation of Actinopterygii (ray-finned fishes) from Sarcopterygii (lobe-finned fishes).
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http://dx.doi.org/10.1016/j.ygcen.2012.11.015DOI Listing
February 2013

Short-term, but not long-term feed restriction causes differential expression of leptins in Atlantic salmon.

Gen Comp Endocrinol 2013 Mar 27;183:83-8. Epub 2012 Nov 27.

Department of Biology, University of Bergen, Bergen High Technology Centre, Bergen, Norway.

Atlantic salmon was used to investigate the effect of long- and short-term dietary ration on the tissue expression levels of leptins. Compared to ad libitum fed fish (0.8-3kg), 6months of dietary restriction (60%) resulted in significantly lower body mass and adiposity, but did not produce a clear effect on the expression levels of either lepa1 or lepa2. For visceral adipose tissue, however, the long-term data indicated that season appeared to influence the levels of lepa1 expression of ad libitum fed fish, but not feed-restricted fish. By comparing the total levels of leptin mRNA expression to the tissue lipid contents, we found that only white muscle lepa1 showed the positive relation reported in mammals. The existence of a postprandial leptin response in Atlantic salmon parr was determined in fed and unfed parr over a 24h period. In contrast to other animals, lepa1 peaked in the unfed fish, initially in the white muscle at 6h, and subsequently in belly flap, liver and visceral adipose tissue at 9h. Only lepa2 in the visceral adipose tissue of fed fish showed a similar 9h peak, but at an order of magnitude lower than lepa1 in the unfed fish. These data reveal that short-term feed restriction causes a latent (6-9h) upregulation of lepa-type genes in the fatty tissues of Atlantic salmon, a finding that contrasts the mammalian response.
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http://dx.doi.org/10.1016/j.ygcen.2012.09.027DOI Listing
March 2013

Developmental expression, differential hormonal regulation and evolution of thyroid and glucocorticoid receptor variants in a marine acanthomorph teleost (Sciaenops ocellatus).

Gen Comp Endocrinol 2012 Mar 28;176(1):39-51. Epub 2011 Dec 28.

The University of Texas at Austin, Marine Science Institute, 750 Channel View Drive, Port Aransas, TX 78373, USA.

Interactions between the thyroid hormone (TH) and corticosteroid (CS) hormone axes are suggested to regulate developmental processes in vertebrates with a larval phase. To investigate this hypothesis, we isolated three nuclear receptors from a larval acanthomorph teleost, the red drum (Sciaenops ocellatus), and established their orthologies as thraa, thrb-L and gra-L using phylogenomic and functional analyses. Functional characterization of the TH receptors in COS-1 cells revealed that Thraa and Thrb-L exhibit dose-dependent transactivation of a luciferase reporter in response to T3, while SoThraa is constitutively active at a low level in the absence of ligand. To test whether interactions between the TH and CS systems occur during development, we initially quantified the in vivo receptor transcript expression levels, and then examined their response to treatment with triiodothyronine (T3) or cortisol. We find that sothraa and sothrb-L are autoregulated in response to exogenous T3 only during early larval development. T3 did not affect sogra-L expression levels, nor did cortisol alter levels of sothraa or sothrb-L at any stage. While differential expression of the receptors in response to non-canonical ligand hormone was not observed under the conditions in this study, the correlation between sothraa and sogra-L transcript abundance during development suggests a coordinated function of the TH and CS systems. By comparing the findings in the present study to earlier investigations, we suggest that the up-regulation of thraa may be a specific feature of metamorphosis in acanthomorph teleosts.
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http://dx.doi.org/10.1016/j.ygcen.2011.12.026DOI Listing
March 2012

Aquaporin evolution in fishes.

Front Physiol 2011 26;2:44. Epub 2011 Jul 26.

Institute of Biology, Bergen High Technology Center, University of Bergen Bergen, Norway.

Aquaporins represent a primordial group of transmembrane solvent channels that have been documented throughout the living biota. This facet alone emphasizes the positive selection pressure for proteins associated with intracellular fluid homeostasis. Amongst extant Eukaryota the highest gene copy number can be found in plants and teleosts, a feature that reflects the genomic duplication history in both groups. In this minireview we discuss the discovery, structure, duplication, and diversification of the aquaporin superfamily. We focus on teleosts as the main models, but include data available for other organisms to provide a broader perspective.
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http://dx.doi.org/10.3389/fphys.2011.00044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145251PMC
November 2011

Dual neofunctionalization of a rapidly evolving aquaporin-1 paralog resulted in constrained and relaxed traits controlling channel function during meiosis resumption in teleosts.

Mol Biol Evol 2011 Nov 8;28(11):3151-69. Epub 2011 Jun 8.

Laboratory of Institut de Recerca i Tecnologia Agroalimentàries, Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas, Barcelona, Spain.

The preovulatory hydration of teleost oocytes is a unique process among vertebrates. The hydration mechanism is most pronounced in marine acanthomorph teleosts that spawn pelagic (floating) eggs; however, the molecular pathway for water influx remains poorly understood. Recently, we revealed that whole-genome duplication (WGD) resulted in teleosts harboring the largest repertoire of molecular water channels in the vertebrate lineage and that a duplicated aquaporin-1 paralog is implicated in the oocyte hydration process. However, the origin and function of the aquaporin-1 paralogs remain equivocal. By integrating the molecular phylogeny with synteny and structural analyses, we show here that the teleost aqp1aa and -1ab paralogs (previously annotated as aqp1a and -1b, respectively) arose by tandem duplication rather than WGD and that the Aqp1ab C-terminus is the most rapidly evolving subdomain within the vertebrate aquaporin superfamily. The functional role of Aqp1ab was investigated in Atlantic halibut, a marine acanthomorph teleost that spawns one of the largest pelagic eggs known. We demonstrate that Aqp1ab is required for full hydration of oocytes undergoing meiotic maturation. We further show that the rapid structural divergence of the C-terminal regulatory domain causes ex vivo loss of function of halibut Aqp1ab when expressed in amphibian oocytes but not in zebrafish or native oocytes. However, by using chimeric constructs of halibut Aqp1aa and -1ab and antisera specifically raised against the C-terminus of Aqp1ab, we found that this cytoplasmic domain regulates in vivo trafficking to the microvillar portion of the oocyte plasma membrane when intraoocytic osmotic pressure is at a maximum. Interestingly, by coinjecting polyA(+) mRNA from postvitellogenic halibut follicles, ex vivo intracellular trafficking of Aqp1ab is rescued in amphibian oocytes. These data reveal that the physiological role of Aqp1ab during meiosis resumption is conserved in teleosts, but the remarkable degeneracy of the cytoplasmic domain has resulted in alternative regulation of the trafficking mechanism.
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http://dx.doi.org/10.1093/molbev/msr146DOI Listing
November 2011

Ontogenetic expression of maternal and zygotic genes in Atlantic cod embryos under ambient and thermally stressed conditions.

Comp Biochem Physiol A Mol Integr Physiol 2011 Jun 3;159(2):196-205. Epub 2011 Mar 3.

National Institute of Nutrition and Seafood Research (NIFES), PO Box 2029 Nordnes, 5817 Bergen, Norway.

The embryonic stages of Atlantic cod (Gadus morhua) are especially sensitive to incubation temperature. The purpose of the present study was to follow the ontogenetic expression of selected genes of maternal (pou2 and nanog) and zygotic origin (hsp70, hsp90α and stip1), in Atlantic cod embryos under ambient and thermally stressed conditions. The study also investigated how reference genes can be applied to studies on embryonic development, when maternal genes are degraded and the zygotic transcription stabilizes. Three batches of eggs were reared and gene expression profiles from the reference and target genes were determined. The embryos were reared at ambient 6 °C, and 10 °C for continuous long-term and acute short-term heat exposure. Both pou2 and nanog showed reduced expression whereas the zygotic and reference genes showed increased expression until stabilizing at gastrulation, when a normalized ontogenetic expression profile of target genes could be generated. pou2 and nanog were not affected by thermal stress. In contrast, hsp70 and hsp90α were upregulated after short-term heat exposure at the early blastula (hsp70 only), late blastula, 50% epiboly and 90% epiboly stages (hsp90α only). Long-term heat exposure of Atlantic cod embryos upregulated both hsp70 (90% epiboly) and hsp90α (90% epiboly and 20-somites). The results suggest that a cellular defense mechanism is activated even in the earliest stages of embryonic development, a period critical to developmental temperature.
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http://dx.doi.org/10.1016/j.cbpa.2011.02.026DOI Listing
June 2011

Piscine aquaporins: an overview of recent advances.

J Exp Zool A Ecol Genet Physiol 2010 Dec 17;313(10):623-50. Epub 2010 Aug 17.

Laboratory of Institut de Recerca i Tecnologia Agroalimentàries (IRTA)- Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain.

Aquaporins are a superfamily of integral membrane proteins that facilitate the rapid and yet highly selective flux of water and other small solutes across biological membranes. Since their discovery, they have been documented throughout the living biota, with the majority of research focusing on mammals and plants. Here, we review available data for piscine aquaporins, including Agnatha (jawless fish), Chondrichthyes (chimaeras, sharks, and rays), Dipnoi (lungfishes), and Teleostei (ray-finned bony fishes). Recent evidence suggests that the aquaporin superfamily has specifically expanded in the chordate lineage consequent to serial rounds of whole genome duplication, with teleost genomes harboring the largest number of paralogs. The selective retention and dichotomous clustering of most duplicated paralogs in Teleostei, with differential tissue expression profiles, implies that novel or specialized physiological functions may have evolved in this clade. The recently proposed new nomenclature of the piscine aquaporin superfamily is discussed in relation to the phylogenetic signal and genomic synteny, with the teleost aquaporin-8 paralogs used as a case study to illustrate disparities between the underlying codons, molecular phylogeny, and physical locus. Structural data indicate that piscine aquaporins display similar channel restriction residues found in the tetrapod counterparts, and hence their functional properties seem to be conserved. However, emerging evidence suggests that regulation of aquaporin function in teleosts may have diverged in some cases. Cell localization and experimental studies imply that the physiological roles of piscine aquaporins extend at least to osmoregulation, reproduction, and early development, although in most cases their specific functions remain to be elucidated.
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http://dx.doi.org/10.1002/jez.634DOI Listing
December 2010

Ontogeny of energy homeostatic pathways via neuroendocrine signaling in Atlantic salmon.

Dev Neurobiol 2010 Aug;70(9):649-58

Department of Biology, University of Bergen, Bergen High Technology Center, Norway.

Leptin and ghrelin are known to regulate energy homeostasis via hypothalamic neuropeptide signaling in mammals. Recent studies have discovered that these hormones exist in teleosts, however, very little is known concerning their role during teleost ontogeny. Here, we have examined the steady state levels of leptins, ghrelins, their target neuropetides and several growth factors during Atlantic salmon development. Initial experiments revealed differential expression of leptin genes and ghrelin isoforms during embryogenesis. In larvae, equal upregulation of ghrl1 and ghrl2 was observed just prior to exogenous feeding while a surge of lepa1 occurred one week after first-feeding. Subsequent dissection of the embryos and larvae showed that lepa1, cart, pomca1, and agrp are supplied as maternal transcripts. The earliest zygotic expression was observed for lepa1 and cart at 320 day degrees. By 400 day degrees, this expression was localized to the head and coincided with upregulation of ghrl2 and npy. Over the hatching period growth factor signaling predominated. The ghrelin surge prior to first-feeding was exclusively localized in the internal organs and coincided with upregulation of npy and agrp in the head and agrp in the trunk. One week after exogenous feeding was established major peaks were detected in the head for lepa1 and pomca1 with increasing levels of cart, while lepa1 was also significantly expressed in the trunk. By integrating theses data into an ontogenetic model, we suggest that the mediation of Atlantic salmon energy homeostatic pathways via endocrine and neuropeptide signaling retains putative features of the mammalian system.
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http://dx.doi.org/10.1002/dneu.20803DOI Listing
August 2010

Functional and evolutionary analysis of flatfish gonadotropin receptors reveals cladal- and lineage-level divergence of the teleost glycoprotein receptor family.

Biol Reprod 2010 Jun 3;82(6):1088-102. Epub 2010 Mar 3.

Laboratory of Institut de Recerca i Tecnologia Agroalimentàries-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas, Barcelona, Spain.

Pituitary gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) act via their cognate glycoprotein hormone receptors (GpHRs), FSH receptor (FSHR), and LH/choriogonadotropin receptor (LHCGR) to regulate gonad physiology. Here, we show that the flatfish Senegalese sole (Solea senegalensis) expresses functional isoforms of fshr and lhcgr, but the genomic origin, ligand activation, and tissue distribution of the receptor transcripts are more complex than expected. By integrating the molecular phylogeny of GpHRs with the syntenic loci of vertebrate orthologs, and by subsequently characterizing the physical maps with the phylogeny of flanking genes, we found that vertebrate GpHRs have undergone a divergent evolution. In Teleostei, fshr genes have a common descent and can be classified as fshra, whereas lhcgrb genes exist as alternatively coded genes even in closely related species. Structural analyses of the receptors revealed that Fshra has an elongated ligand-binding domain, containing an extra leucine-rich repeat that specifically arose in the Acanthomorpha because of exon duplication. Ectopic expression in Xenopus laevis oocytes demonstrated that sole Fshra responded to piscine Fsh and Lh, whereas Lhcgrba was preferentially activated by its cognate hormone. The expression pattern of sole fshra and lhcgrba in gonads during the reproductive cycle was consistent with earlier observations wherein Fshra regulates ovarian growth and spermatogenesis and Lhcgrb triggers gamete maturation, respectively. However, contrary to observations in other teleosts, fshra was localized exclusively in Sertoli cells of the testis, whereas lhcgrba was expressed in Leydig cells as well as in spermatids. These results demonstrate the presence of alternatively coded lhcgr isoforms (lhcgrba and lhcgrbb) in teleosts and suggest a role of the lhcgrba receptor in the differentiation of spermatids into spermatozoa in Senegalese sole.
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http://dx.doi.org/10.1095/biolreprod.109.082289DOI Listing
June 2010

Genomic and proteomic analyses reveal non-neofunctionalized vitellogenins in a basal clupeocephalan, the Atlantic herring, and point to the origin of maturational yolk proteolysis in marine teleosts.

Mol Biol Evol 2009 May 27;26(5):1029-44. Epub 2009 Jan 27.

Department of Biology, University of Bergen, Bergen High Technology Center, Bergen, Norway.

Oocyte hydration is a unique event in oviparous marine teleosts that provides the single-celled egg with an essential pool of water for survival during early development in the saline oceanic environment. A conserved mechanism of maturational yolk proteolysis of a neofunctionalized vitellogenin (VtgAa) has been shown to underlie the hydration event in all teleosts that spawn pelagic eggs (pelagophils), and is argued to be a key adaptation for teleost radiation in the oceanic environment 55 Ma. We have recently shown that a small pool of free amino acids (FAAs) significantly contributes to the osmolarity of the ovulated egg in an ancestral marine teleost, the Atlantic herring that spawns benthic eggs (benthophil). To determine whether multiple forms of vtg exist and whether neofunctionalization of the gene products are related to the egg FAA pool in this species, genomic sequences conserved between the exons of Atlantic herring and zebrafish were amplified. This approach identified a small polymorphic intron between exons 9 and 10 in Atlantic herring and demonstrated that two closely related major vtg transcripts (chvtgAc1 and chvtgAc2) are expressed during oogenesis. A separate polymerase chain reaction-based approach identified a more ancestral phosvitinless transcript (chvtgC). Proteomic analyses of the translated products of the major vtg forms demonstrated that the yolk proteins are similarly processed during deposition, and oocyte maturation and reveal that vtgs have duplicated but not neofunctionalized in this species. Phylogenetic analyses consistently clustered the transcripts and proteins as the basal sister group to the Ostariophysi in full congruence with the Clupeocephalan rank, and suggest that expansion of ostariophysan vtgAo1 and vtgAo2 genes occurred in a lineage-specific manner after separation from the Clupeiformes. Three-dimensional modeling of the ChvtgAc1 sequence against the resolved lamprey lipovitellin module revealed that the tertiary structure is highly conserved, with most substitutions occurring on the outside of the molecule. The data indicate that the phosvitin domain, the smallest yet reported for teleosts, and an N-terminal fragment of the lipovitellin light chain contribute to the FAA pool. The present findings thus show that yolk proteolysis and the generation of an organic osmolyte pool of FAAs was an adaptive response to spawning in seawater also for the Clupeiformes, but that this process was not evolutionarily successful in terms of biodiversity until vtg gene neofunctionalization occurred in the Acanthomorpha.
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http://dx.doi.org/10.1093/molbev/msp014DOI Listing
May 2009

Evolution and differential expression of a vertebrate vitellogenin gene cluster.

BMC Evol Biol 2009 Jan 5;9. Epub 2009 Jan 5.

Department of Biology, University of Bergen, Bergen High Technology Center, Postbox 7803, N-5020, Bergen, Norway.

Background: The multiplicity or loss of the vitellogenin (vtg) gene family in vertebrates has been argued to have broad implications for the mode of reproduction (placental or non-placental), cleavage pattern (meroblastic or holoblastic) and character of the egg (pelagic or benthic). Earlier proposals for the existence of three forms of vertebrate vtgs present conflicting models for their origin and subsequent duplication.

Results: By integrating phylogenetics of novel vtg transcripts from old and modern teleosts with syntenic analyses of all available genomic variants of non-metatherian vertebrates we identify the gene orthologies between the Sarcopterygii (tetrapod branch) and Actinopterygii (fish branch). We argue that the vertebrate vtg gene cluster originated in proto-chromosome m, but that vtg genes have subsequently duplicated and rearranged following whole genome duplications. Sequencing of a novel fourth vtg transcript in labrid species, and the presence of duplicated paralogs in certain model organisms supports the notion that lineage-specific gene duplications frequently occur in teleosts. The data show that the vtg gene cluster is more conserved between acanthomorph teleosts and tetrapods, than in ostariophysan teleosts such as the zebrafish. The differential expression of the labrid vtg genes are further consistent with the notion that neofunctionalized Aa-type vtgs are important determinants of the pelagic or benthic character of the eggs in acanthomorph teleosts.

Conclusion: The vertebrate vtg gene cluster existed prior to the separation of Sarcopterygii from Actinopterygii >450 million years ago, a period associated with the second round of whole genome duplication. The presence of higher copy numbers in a more highly expressed subcluster is particularly prevalent in teleosts. The differential expression and latent neofunctionalization of vtg genes in acanthomorph teleosts is an adaptive feature associated with oocyte hydration and spawning in the marine environment.
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http://dx.doi.org/10.1186/1471-2148-9-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2632621PMC
January 2009

Goldsinny wrasse (Ctenolabrus rupestris) is an extreme vtgAa-type pelagophil teleost.

Mol Reprod Dev 2008 Jun;75(6):1011-20

Department of Biology, University of Bergen, Allégaten, Bergen, Norway.

During oocyte maturation in the goldsinny wrasse (Ctenolabrus rupestris) extensive proteolysis of yolk proteins generates a large pool of free amino acids that drive hydration of the pelagic egg. By cloning hepatic vitellogenins (vtg) and using mass spectrometry, N-terminal microsequencing, and Western-immunoblotting to identify the yolk proteins (Yp), we show that multiple forms of vitellogenin mRNAs (vtgAa, vtgAb, and vtgC) are expressed in the liver, but only a single major class of the Yps derived from vtgAa predominates in the oocytes. Some Yps derived from vtgAb and vtgC appear also to be incorporated in the oocytes and eggs, but only at background levels. During oocyte hydration the vtgAa-derived lipovitellin heavy chain (LvH-Aa) and its cleavage variants are completely degraded leaving only a processed lipovitellin light chain (LvL-Aa) fragment as the major yolk protein for embryonic development. The maturational cleavage site of the LvL-Aa is identified as two amino acids downstream from the conserved Tyr(1168) of VtgAa in Atlantic halibut. In addition, although a beta'-component (approximately 18 kDa) is present in the oocytes, it is not fully degraded during the hydration process.
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http://dx.doi.org/10.1002/mrd.20845DOI Listing
June 2008