Publications by authors named "Frederick W Goetz"

42 Publications

RADSex: A computational workflow to study sex determination using restriction site-associated DNA sequencing data.

Mol Ecol Resour 2021 Jul 9;21(5):1715-1731. Epub 2021 Mar 9.

Physiological Chemistry, Biocenter, University of Wuerzburg, Wuerzburg, Germany.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.
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http://dx.doi.org/10.1111/1755-0998.13360DOI Listing
July 2021

The rise and fall of the ancient northern pike master sex-determining gene.

Elife 2021 Jan 28;10. Epub 2021 Jan 28.

INRAE, Sigenae, Genotoul Bioinfo, Toulouse, France.

The understanding of the evolution of variable sex determination mechanisms across taxa requires comparative studies among closely related species. Following the fate of a known master sex-determining gene, we traced the evolution of sex determination in an entire teleost order (Esociformes). We discovered that the northern pike () master sex-determining gene originated from a 65 to 90 million-year-old gene duplication event and that it remained sex linked on undifferentiated sex chromosomes for at least 56 million years in multiple species. We identified several independent species- or population-specific sex determination transitions, including a recent loss of a Y chromosome. These findings highlight the diversity of evolutionary fates of master sex-determining genes and the importance of population demographic history in sex determination studies. We hypothesize that occasional sex reversals and genetic bottlenecks provide a non-adaptive explanation for sex determination transitions.
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http://dx.doi.org/10.7554/eLife.62858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870143PMC
January 2021

Seasonal variation of pituitary gonadotropin subunit, brain-type aromatase and sex steroid receptor mRNAs, and plasma steroids during gametogenesis in wild sablefish.

Comp Biochem Physiol A Mol Integr Physiol 2018 05 26;219-220:48-57. Epub 2018 Feb 26.

Environmental and Fisheries Sciences Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd East, Seattle, WA 98112, USA; Center for Reproductive Biology, Washington State University, PO Box 647521, Pullman, WA 99164, USA.

Pituitary-hormone signaling plays critical roles in the onset and progression of gametogenesis in vertebrates. This study characterized expression patterns of pituitary gonadotropin beta-subunits (fshb and lhb), brain-type aromatase (cyp19a1b), androgen (ar1, ar2) and estrogen receptors (esr1, esr2a, esr2b), and changes in plasma steroid levels by liquid chromatography/tandem mass spectrometry in wild sablefish (Anoplopoma fimbria, order Scorpaeniformes) during a complete reproductive cycle. Transcripts for fshb increased during early gametogenesis and peaked in late vitellogenic females and late recrudescent males, while expression of lhb reached maximum levels in periovulatory and spermiating fish. Pituitary levels of cyp19a1b and ar1 were strongly correlated with those of lhb in females and males, increasing during gametogenesis and reaching maximum levels prior to spawning. By contrast, expression of ar2, and the three estrogen receptors differed between female and male sablefish. 17β-estradiol (E2) was the dominant steroid in females during vitellogenesis, while a range of at least 6 steroids (11β-hydroxyandrostenedione, testosterone [T], E2, 11-ketotestosterone [11KT], 11-deoxycortisol, and 17α,20β,21-trihydroxyprogesterone) were detected at similar levels in males during testicular development. Prior to spawning, a marked increase in 4-androstenedione, T, 11KT and E2 was found in both periovulatory females and spermiating males. In conclusion, the concomitant changes in plasma androgen levels and pituitary ar1 expression during gametogenesis suggest a specific role for androgens in pituitary hormone regulation of reproduction in sablefish. Further, our data highlight the importance of E2 during final stages of maturation in this species, which may regulate the transcription of pituitary lhb in a paracrine fashion.
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http://dx.doi.org/10.1016/j.cbpa.2018.02.010DOI Listing
May 2018

Reproductive life history of sablefish (Anoplopoma fimbria) from the U.S. Washington coast.

PLoS One 2017 8;12(9):e0184413. Epub 2017 Sep 8.

Environmental and Fisheries Sciences Division, Northwest Fisheries Science Center, National Marine Fisheries Service, NOAA, Seattle, WA, United States of America.

Sablefish (Anoplopoma fimbria) is a marine groundfish that supports valuable fisheries in the North Pacific Ocean and holds promise for marine aquaculture. Limited information is available, however, about its reproductive biology. This study aimed to characterize the complete reproductive cycle, including seasonal changes in gonadal development (macroscopic and histological), plasma sex steroid levels (17β-estradiol -E2-, and 11-ketotestosterone -11KT-), gonadosomatic and hepatosomatic indices (GSI, and HSI), and condition factor (K) of female and male sablefish captured off the Washington coast. Adult fish (209 females, 159 males) were caught by longline monthly from August 2012 to August 2013. Early signs of recruitment of ovarian follicles into secondary growth, indicated by oocytes containing small yolk granules and cortical alveoli, were first observed in March. Oogenesis progressed during spring and summer, and fully vitellogenic follicles were first observed in July. Vitellogenic growth was correlated with increases in plasma E2, GSI, HSI and K. Periovulatory females, indicated by fully-grown oocytes with migrating germinal vesicles and hydrated oocytes, were found from November to February. At this stage, plasma E2 and GSI reached maximal levels. In males, proliferating cysts containing spermatocytes were first observed in April. Testicular development proceeded during spring and summer, a period during which all types of male germ cells were found. The first clusters of spermatozoa appeared in July, concomitant with a 5.2-fold increase in GSI. Spermiating males were observed from November to April; at this time, spermatids were absent or greatly reduced, and testis lobules were filled with spermatozoa. The highest levels of plasma 11KT were found in males at this stage. Postspawning ovaries and testes, and basal steroids levels were found in fish captured from February to April. These results suggest that sablefish in coastal Washington initiate their reproductive cycle in March/April and spawn primarily in January/February.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184413PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590928PMC
October 2017

Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs.

Int J Mol Sci 2017 Feb 3;18(2). Epub 2017 Feb 3.

Institute of Biotechnology and Biomedicine, University Autónoma de Barcelona, Barcelona 08193, Spain.

This study describes the development and validation of an enriched oligonucleotide-microarray platform for (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in with an emphasis upon immunity and the immune response.
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http://dx.doi.org/10.3390/ijms18020317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343853PMC
February 2017

Dimethylsulfoniopropionate (DMSP) Increases Survival of Larval Sablefish, Anoplopoma fimbria.

J Chem Ecol 2016 Jun 15;42(6):533-6. Epub 2016 Jun 15.

Environmental and Fisheries Sciences Division, Northwest Fisheries Science Center, National Marine Fisheries Service, NOAA, 7305 Beach Dr E, Port Orchard, WA, 98366, USA.

High concentrations of dimethylsulfoniopropionate (DMSP), a chemical compound released by lysed phytoplankton, may indicate high rates of grazing by zooplankton and may thus be a foraging cue for planktivorous fishes. Previous studies have shown that some planktivorous fishes and birds aggregate or alter locomotory behavior in response to this chemical cue, which is likely adaptive because it helps them locate prey. These behavioral responses have been demonstrated in juveniles and adults, but no studies have tested for effects on larval fish. Larvae suffer from high mortality rates and are vulnerable to starvation. While larvae are generally thought to be visual predators, they actually have poor vision and cryptic prey. Thus, larval fish should benefit from a chemical cue that provides information on prey abundance. We reared larval sablefish, Anoplopoma fimbria, for one week and supplemented feedings with varying concentrations of DMSP to test the hypothesis that DMSP affects larval survival. Ecologically relevant DMSP concentrations increased larval survival by up to 70 %, which has implications for production in aquaculture and recruitment in nature. These results provide a new tool for increasing larval production in aquaculture and also suggest that larvae may use DMSP as an olfactory cue. The release of DMSP may be a previously unappreciated mechanism through which phytoplankton affect larval survival and recruitment.
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http://dx.doi.org/10.1007/s10886-016-0713-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4947478PMC
June 2016

Luteinizing hormone induces ovulation via tumor necrosis factor α-dependent increases in prostaglandin F2α in a nonmammalian vertebrate.

Sci Rep 2015 Sep 16;5:14210. Epub 2015 Sep 16.

Departament de Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona and Institut de Biomedicina de la Universitat de Barcelona (IBUB), Avinguda Diagonal 643, 08028 Barcelona, Spain.

Ovulation is induced by the preovulatory surge of luteinizing hormone (LH) that acts on the ovary and triggers the rupture of the preovulatory ovarian follicle by stimulating proteolysis and apoptosis in the follicle wall, causing the release of the mature oocyte. The pro-inflammatory cytokine tumor necrosis factor α (TNFα) and prostaglandin (PG) F2α (PGF2α) are involved in the control of ovulation but their role mediating the pro-ovulatory actions of LH is not well established. Here we show that Lh induces PGF2α synthesis through its stimulation of Tnfα production in trout, a primitive teleost fish. Recombinant trout Tnfα (rTnfα) and PGF2α recapitulate the stimulatory in vitro effects of salmon Lh (sLh) on contraction, proteolysis and loss of cell viability in the preovulatory follicle wall and, finally, ovulation. Furthermore, all pro-ovulatory actions of sLh are blocked by inhibition of Tnfα secretion or PG synthesis and all actions of rTnfα are blocked by PG synthesis inhibitors. Therefore, we provide evidence that the Tnfα-dependent increase in PGF2α production is necessary for the pro-ovulatory actions of Lh. The results from this study shed light onto the mechanisms underlying the pro-ovulatory actions of LH in vertebrates and may prove important in clinical assessments of female infertility.
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http://dx.doi.org/10.1038/srep14210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4570979PMC
September 2015

An Enriched European Eel Transcriptome Sheds Light upon Host-Pathogen Interactions with Vibrio vulnificus.

PLoS One 2015 24;10(7):e0133328. Epub 2015 Jul 24.

Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Spain; Institute of Aquaculture, University of Stirling, Stirling, United Kingdom.

Infectious diseases are one of the principal bottlenecks for the European eel recovery. The aim of this study was to develop a new molecular tool to be used in host-pathogen interaction experiments in the eel. To this end, we first stimulated adult eels with different pathogen-associated molecular patterns (PAMPs), extracted RNA from the immune-related tissues and sequenced the transcriptome. We obtained more than 2 x 10(6) reads that were assembled and annotated into 45,067 new descriptions with a notable representation of novel transcripts related with pathogen recognition, signal transduction and the immune response. Then, we designed a DNA-microarray that was used to analyze the early immune response against Vibrio vulnificus, a septicemic pathogen that uses the gills as the portal of entry into the blood, as well as the role of the main toxin of this species (RtxA13) on this early interaction. The gill transcriptomic profiles obtained after bath infecting eels with the wild type strain or with a mutant deficient in rtxA13 were analyzed and compared. Results demonstrate that eels react rapidly and locally against the pathogen and that this immune-response is rtxA13-dependent as transcripts related with cell destruction were highly up-regulated only in the gills from eels infected with the wild-type strain. Furthermore, significant differences in the immune response against the wild type and the mutant strain also suggest that host survival after V. vulnificus infection could depend on an efficient local phagocytic activity. Finally, we also found evidence of the presence of an interbranchial lymphoid tissue in European eel gills although further experiments will be necessary to identify such tissue.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133328PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4514713PMC
May 2016

High-throughput sequencing and pathway analysis reveal alteration of the pituitary transcriptome by 17α-ethynylestradiol (EE2) in female coho salmon, Oncorhynchus kisutch.

Aquat Toxicol 2013 Oct 9;142-143:146-63. Epub 2013 Aug 9.

School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA 98195, USA.

Considerable research has been done on the effects of endocrine disrupting chemicals (EDCs) on reproduction and gene expression in the brain, liver and gonads of teleost fish, but information on impacts to the pituitary gland are still limited despite its central role in regulating reproduction. The aim of this study was to further our understanding of the potential effects of natural and synthetic estrogens on the brain-pituitary-gonad axis in fish by determining the effects of 17α-ethynylestradiol (EE2) on the pituitary transcriptome. We exposed sub-adult coho salmon (Oncorhynchus kisutch) to 0 or 12 ng EE2/L for up to 6 weeks and effects on the pituitary transcriptome of females were assessed using high-throughput Illumina(®) sequencing, RNA-Seq and pathway analysis. After 1 or 6 weeks, 218 and 670 contiguous sequences (contigs) respectively, were differentially expressed in pituitaries of EE2-exposed fish relative to control. Two of the most highly up- and down-regulated contigs were luteinizing hormone β subunit (241-fold and 395-fold at 1 and 6 weeks, respectively) and follicle-stimulating hormone β subunit (-3.4-fold at 6 weeks). Additional contigs related to gonadotropin synthesis and release were differentially expressed in EE2-exposed fish relative to controls. These included contigs involved in gonadotropin releasing hormone (GNRH) and transforming growth factor-β signaling. There was an over-representation of significantly affected contigs in 33 and 18 canonical pathways at 1 and 6 weeks, respectively, including circadian rhythm signaling, calcium signaling, peroxisome proliferator-activated receptor (PPAR) signaling, PPARα/retinoid x receptor α activation, and netrin signaling. Network analysis identified potential interactions between genes involved in circadian rhythm and GNRH signaling, suggesting possible effects of EE2 on timing of reproductive events.
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http://dx.doi.org/10.1016/j.aquatox.2013.07.020DOI Listing
October 2013

Luteinizing hormone stimulation of in vitro ovulation in brook trout (Salvelinus fontinalis) involves follicle contraction and activation of proteolytic genes.

Gen Comp Endocrinol 2013 Jul 14;188:175-82. Epub 2013 Mar 14.

Departament de Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona and Institut de Biomedicina de la Universitat de Barcelona (IBUB), 08028 Barcelona, Spain.

Luteinizing hormone (LH) is an essential hormone for the stimulation of the ovulatory process in vertebrates. However, little is known in fish regarding the different mechanisms induced by LH during ovulation that facilitate the rupture of the follicle wall and the subsequent expulsion of the mature oocyte. In this study, the effects of salmon LH (sLH) on in vitro ovulation were investigated in brook trout (Salvelinus fontinalis) isolated follicles. sLH significantly stimulated in vitro ovulation and contraction of brook trout preovulatory follicles. In order to investigate the possible involvement of proteolytic events in the ovulatory action of LH, the expression of genes known to have a crucial role in the degradation of follicle wall structure was examined. Our results show that sLH clearly stimulated the mRNA expression levels of matrix metalloproteinases (MMPs; including mmp2 and mmp19) and other enzymes with proteolytic action during ovulation, such as a disintegrin and metalloproteinase with thrombospondin-like motifs 1 (adamts1) and plasminogen (plg), in brook trout preovulatory follicles. In addition, the expression of mmp2, adamts1 and plg increased in brook trout follicles during the progression of LH-induced ovulation. Interestingly, the expression of tissue inhibitor of matrix metalloproteinase 2 (timp2), a known regulator of MMP2 activity, paralleled that of mmp2, suggesting the existence of a controlled mechanism of MMP2 action. Therefore, the known increase in proteolytic activity during ovulation in fish could be the result of the stimulation of the expression of proteolytic enzymes by LH in preovulatory follicles. We propose that LH may stimulate ovulation in brook trout follicles by stimulating proteolysis of the follicle wall and by stimulating follicle contraction.
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http://dx.doi.org/10.1016/j.ygcen.2013.02.031DOI Listing
July 2013

Effects of methylmercury on epigenetic markers in three model species: mink, chicken and yellow perch.

Comp Biochem Physiol C Toxicol Pharmacol 2013 Apr 26;157(3):322-7. Epub 2013 Feb 26.

Department of Environmental Health Sciences, University of Michigan, 1415 Washington Heights, Ann Arbor, MI 48109, USA.

We previously reported that methylmercury (MeHg) exposure is associated with DNA hypomethylation in the brain stem of male polar bears. Here, we conveniently use archived tissues obtained from controlled laboratory exposure studies to look for evidence that MeHg can disrupt DNA methylation across taxa. Brain (cerebrum) tissues from MeHg-exposed mink (Neovison vison), chicken (Gallus gallus) and yellow perch (Perca flavescens) were analyzed for total Hg levels and global DNA methylation. Tissues from chicken and mink, but not perch, were also analyzed for DNA methyltransferase (DNMT) activity. In mink we observed significant reductions in global DNA methylation in an environmentally-relevant dietary exposure group (1 ppm MeHg), but not in a higher group (2 ppm MeHg). DNMT activity was significantly reduced in all treatment groups. In chicken or yellow perch, no statistically significant effects of MeHg were observed. Dose-dependent trends were observed in the chicken data but the direction of the change was not consistent between the two endpoints. Our results suggest that MeHg can be epigenetically active in that it has the capacity to affect DNA methylation in mammals. The variability in results across species may suggest inter-taxa differences in epigenetic responses to MeHg, or may be related to differences among the exposure scenarios used as animals were exposed to MeHg through different routes (dietary, egg injection), for different periods of time (19-89 days) and at different life stages (embryonic, juvenile, adult).
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http://dx.doi.org/10.1016/j.cbpc.2013.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4346372PMC
April 2013

Identification of ovarian gene expression patterns during vitellogenesis in Atlantic cod (Gadus morhua).

Gen Comp Endocrinol 2012 Nov 13;179(2):296-304. Epub 2012 Sep 13.

Department of Biological Sciences, University of New Hampshire, 38 College Road, Durham, NH 03824, USA.

Follicular maturational competence and ovulatory competence in teleost fish refer to the ability of the ovarian follicle to undergo final oocyte maturation and ovulation, respectively, in response to gonadotropin stimulation and other external cues. Some gene products related to competence acquisition are likely synthesized during vitellogenic growth, as these follicles gain in vivo responsiveness to exogenous gonadotropin stimulation and can be induced to undergo maturation and ovulation. In Atlantic cod (Gadus morhua), gonadotropin responsiveness has been shown to be oocyte size-dependent, and only ovaries containing late-stage vitellogenic follicles can be induced to ovulate. The purpose of the present study was to compare gene expression patterns between mid (unresponsive) and late (responsive) vitellogenic ovaries to identify genes involved in gonadotropin responsiveness and the acquisition of maturational and ovulatory competencies. Representational difference analysis was conducted in two reciprocal comparisons using intact ovarian fragments and follicle wall-enriched tissues, and genes of interest were used in real time quantitative PCR to confirm differential expression. Few differences were detected in intact ovarian fragments, but type IV ice-structuring protein and gephyrin were upregulated later in development and may be involved in lipid and sulfur metabolism, respectively. Candidate gene assays for luteinizing hormone receptor and aromatase also exhibited significant upregulation during vitellogenesis. Many genes were differentially expressed in follicle wall-enriched tissues, including endocrine maturational regulators and smooth muscle genes. Overall, maturational and ovulatory competencies during vitellogenesis in Atlantic cod are associated with up- and downregulation of many genes involved in lipid metabolism, endocrine regulation, and ovulatory preparation.
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http://dx.doi.org/10.1016/j.ygcen.2012.09.003DOI Listing
November 2012

Characterization and evaluation of sex-specific expression of suppressors of cytokine signaling (SOCS)-1 and -3 in juvenile yellow perch (Perca flavescens) treated with lipopolysaccharide.

Fish Shellfish Immunol 2012 Sep 24;33(3):468-81. Epub 2012 May 24.

USDA/ARS/School of Freshwater Sciences, University of Wisconsin at Milwaukee, 600 E. Greenfield Avenue, Milwaukee, WI 53204, USA.

The suppressor of cytokine signaling (SOCS) proteins are a family of intracellular proteins that are centrally involved with vertebrate growth, development and immunity via their effects as negative feed-back regulators of cytokine (and hormone) signaling. The genes for SOCS-1 & -3 were cloned, sequences analyzed and expression patterns examined in the commercially-important teleost, yellow perch (Perca flavescens). The deduced (mature) proteins for yellow perch (yp)SOCS-1 and (yp)SOCS-3 consist of 211 and 205 amino acids, respectively. Functional domains such as the Src homology-2 (SH2) and SOCS-box were present in ypSOCS-1 and ypSOCS-3 and these domains were well conserved between teleost species. Sequence analysis showed that ypSOCS-1 & -3 share highest homology (among similar teleost sequences), to the stickleback (Gasterosteus aculatus) SOCS-1 & -3 protein homologs. To investigate sex-specific expression of the ypSOCS-1 and ypSOCS-3 mRNAs, juvenile male and female yellow perch were immunologically challenged with a single injection (10 μg/g bw) of lipopolysaccharide (LPS) and tissues (gill, head kidney, kidney, liver and spleen) were sampled over a 48-h time-course. Quantitative real-time PCR analysis showed that ypSOCS-1 & -3 were expressed in all tissues examined and at all sampling time-points. LPS injection significantly induced ypSOCS-1 & -3 mRNA levels in gill, head kidney, liver, kidney and spleen, with maximal induction occurring at 6 h post-injection in each tissue. By 48-h post-injection, expression levels for ypSOCS-1 & -3 mRNAs approached, or reached, control levels in all tissues examined. While there were statistical interactions among variables (treatment, time and sex) for ypSOCS-1, we only found a main effect of sex on SOCS-3 mRNA expression in head kidney with higher copy numbers occurring in males than in females treated with LPS. Sexually-dimorphic expression of SOCS-1 or -3 mRNA has not been examined, or described, in a teleost. Our findings suggest the involvement of the SOCS genes in the yellow perch immune response and that differences among the sexes are evident and should be explored further.
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http://dx.doi.org/10.1016/j.fsi.2012.05.026DOI Listing
September 2012

RNA-Seq reveals an integrated immune response in nucleated erythrocytes.

PLoS One 2011 27;6(10):e26998. Epub 2011 Oct 27.

Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Barcelona, Spain.

Background: Throughout the primary literature and within textbooks, the erythrocyte has been tacitly accepted to have maintained a unique physiological role; namely gas transport and exchange. In non-mammalian vertebrates, nucleated erythrocytes are present in circulation throughout the life cycle and a fragmented series of observations in mammals support a potential role in non-respiratory biological processes. We hypothesised that nucleated erythrocytes could actively participate via ligand-induced transcriptional re-programming in the immune response.

Methodology/principal Findings: Nucleated erythrocytes from both fish and birds express and regulate specific pattern recognition receptor (PRR) mRNAs and, thus, are capable of specific pathogen associated molecular pattern (PAMP) detection that is central to the innate immune response. In vitro challenge with diverse PAMPs led to de novo specific mRNA synthesis of both receptors and response factors including interferon-alpha (IFNα) that exhibit a stimulus-specific polysomal shift supporting active translation. RNA-Seq analysis of the PAMP (Poly (I:C), polyinosinic:polycytidylic acid)-erythrocyte response uncovered diverse cohorts of differentially expressed mRNA transcripts related to multiple physiological systems including the endocrine, reproductive and immune. Moreover, erythrocyte-derived conditioned mediums induced a type-1 interferon response in macrophages thus supporting an integrative role for the erythrocytes in the immune response.

Conclusions/significance: We demonstrate that nucleated erythrocytes in non-mammalian vertebrates spanning significant phylogenetic distance participate in the immune response. RNA-Seq studies highlight a mRNA repertoire that suggests a previously unrecognized integrative role for the erythrocytes in other physiological systems.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026998PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203173PMC
March 2012

Isolation and molecular characterization of Rem2 isoforms in the rainbow trout (Oncorhynchus mykiss): Tissue and central nervous system expression.

Comp Biochem Physiol B Biochem Mol Biol 2012 Feb 29;161(2):93-101. Epub 2011 Sep 29.

Department of Biology, Furman University, Greenville, SC, USA.

REM2 is a member of the REM, RAD, and GEM/KIR (RGK) subfamily of RAS superfamily proteins and plays an important role in brain development and function. In this study, two Rem2 isoforms were isolated from the rainbow trout (Oncorhynchus mykiss). The two genes, designated O. mykiss rem2a and rem2b, both encode 304 amino acid proteins with 61% and 62% identities to zebrafish (Danio rerio) Rem2, respectively, and each with 43% identity to mammalian (human) REM2. To our knowledge, this is the first incidence of Rem2 isoforms in a species that are the result of gene duplication. Both isoforms possessed similar tissue expression profiles with the highest levels in the brain. The rem2a gene has significantly higher expression levels than rem2b in all tissues assayed except the brain and head kidney. In the central nervous system, both isoforms showed similar expression levels with the highest levels occurring in the olfactory bulb, cerebrum, and midbrain, though rem2a expression is significantly higher in the spinal cord. Based on known functional roles of Rem2 in synapse development and stem cell proliferation, the characterization of Rem2 in rainbow trout could shed light on its role in adult vertebrate neurogenesis and brain regeneration.
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http://dx.doi.org/10.1016/j.cbpb.2011.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3242864PMC
February 2012

PAMPs, PRRs and the genomics of gram negative bacterial recognition in fish.

Dev Comp Immunol 2011 Dec 29;35(12):1195-203. Epub 2011 Mar 29.

Institute of Biotechnology and Biomedicine, Dep. Biologia Cel·lular, Immunologia i Fisiologia Animal, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain.

Understanding the mechanisms that underpin pathogen recognition and subsequent orchestration of the immune response in fish is an area of significant importance for both basic research and management of health in aquaculture. In recent years much attention has been given to the identification of pattern recognition receptors (PRRs) in fish, however, characterisation of interactions with specific pathogen-associated molecular patterns (PAMPs) is still incomplete. Microarray studies have significantly contributed to functional studies and early descriptions of PAMP-PRR driven activation of specific response cassettes in the genome have been obtained although much is left to be done. In this review we will address gram negative (G-negative) bacterial recognition in fish addressing contributing factors such as structure-function relationships between G-negative PAMPs, current knowledge of fish PRRs and the input achieved by microarray-based studies ranging from in vivo infection studies to directed in vitro PAMP-cell studies. Finally we revisit the endotoxic recognition paradigm in fish and suggest a series of future perspectives that could contribute toward the further elucidation of G-negative bacterial recognition across the highly diverse group of vertebrates that encompass the fishes.
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http://dx.doi.org/10.1016/j.dci.2011.02.010DOI Listing
December 2011

Disruption of the salmon reproductive endocrine axis through prolonged nutritional stress: changes in circulating hormone levels and transcripts for ovarian genes involved in steroidogenesis and apoptosis.

Gen Comp Endocrinol 2011 Jul 3;172(3):331-43. Epub 2011 Apr 3.

School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA 98195, USA.

Mechanisms regulating the normal progression of ovarian follicular growth versus onset of atresia in fishes are poorly understood. To gain a better understanding of these processes, we exposed immature female coho salmon (Oncorhynchus kisutch) to prolonged fasting to induce follicular atresia and monitored body growth, development of the ovarian follicles, changes in reproductive hormones, and transcripts for ovarian genes. Prolonged fasting reduced body and ovary weight and increased the appearance of atretic follicles relative to normally fed controls. Endocrine analyses showed that fasting reduced plasma insulin-like growth factor 1 (IGF1), estradiol-17β (E2), and pituitary, but not plasma, levels of follicle-stimulating hormone (FSH). Transcripts for ovarian fsh receptor (fshr) and steroidogenesis-related genes, such as steroidogenic acute regulatory protein (star), 3β-hydroxysteroid dehydrogenase (hsd3b), and P450 aromatase (cyp19a1a) were significantly lower in fasted fish. Ovarian expression of apoptosis-related genes, such as Fas-associated death domain (fadd), caspase 8 (casp8), caspase 3 (casp3), and caspase 9 (casp9) were significantly elevated in fasted fish compared to fed fish, indicating that apoptosis is involved in the process of atresia in this species. Interestingly, some genes such as fadd, casp8, casp3, and hsd3b, were differentially expressed prior to increases in the number of atretic follicles and reductions in hormone levels induced by fasting, and may therefore have potential as early indicators of atresia. Together these results suggest that prolonged nutritional stress may disrupt the reproductive system and induce follicular atresia in part via reductions in ovarian IGF and FSH signaling, and downstream effects on steroidogenesis-related genes and E2 production.
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http://dx.doi.org/10.1016/j.ygcen.2011.03.017DOI Listing
July 2011

Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout.

Reprod Biol Endocrinol 2010 Apr 12;8:34. Epub 2010 Apr 12.

Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona and Institut de Biomedicina de la Universitat de Barcelona (IBUB), 08028 Barcelona, Spain.

Background: The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor alpha (TNF alpha) and other immune factors, are produced and act on the reproductive system. However, TNF alpha is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNF alpha in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta).

Methods: To determine the in vivo regulation of TNF alpha expression in the ovary, preovulatory brook trout (Salvelinus fontinalis) were injected intraperitoneally with either saline or bacterial lipopolysaccharide (LPS). In control and recombinant trout TNF alpha (rtTNF alpha)-treated brown trout granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNF alpha on follicle contraction and testosterone production in preovulatory brown trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNF alpha-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses.

Results: LPS administration in vivo causes a significant induction of the ovarian expression of TNF alpha. Treatment with rtTNF alpha induces granulosa cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNF alpha causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNF alpha induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. Furthermore, the expression of kallikrein, TOP-2, serine protease 23 and ADAM 22, genes that have been postulated to be involved in proteolytic and tissue remodeling processes during ovulation in trout, increases in follicles incubated in the presence of rtTNF alpha.

Conclusions: In view of these results, we propose that TNF alpha could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction.
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http://dx.doi.org/10.1186/1477-7827-8-34DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2873445PMC
April 2010

Peptidoglycan, not endotoxin, is the key mediator of cytokine gene expression induced in rainbow trout macrophages by crude LPS.

Mol Immunol 2010 Apr 20;47(7-8):1450-7. Epub 2010 Mar 20.

Institute of Biotechnology and Biomedicine, Dep. Biologia Cellular, Immunologia i Fisiologia Animal, Universitat Autónoma de Barcelona, 08193 Barcelona, Spain.

In rainbow trout macrophages, phenol-extracted lipopolysaccharide (LPS) preparations stimulate proinflammatory cytokine gene expression but ultrapure preparations of LPS are inactive. Crude LPS preparations could potentially have a number of contaminants including peptidoglycans (PGNs), nucleic acids and lipoproteins. Thus, in the current study we individually tested potentially contaminating pathogen associated molecular patterns (PAMPs) on rainbow trout (Oncorhynchus mykiss) macrophages to determine which ones could induce proinflammatory cytokine expression. We found that PGNs derived from Gram-negative bacteria (Escherichia coli 0111:B4 and K12), are potent inducers of IL-1beta and IL-6 gene expression and were equal to, or more potent than, crude LPS. On the other hand, PGNs of Gram-positive bacteria, DNA, RNA and lipoteichoic acid were weak stimulators, and lipid A, lipoprotein (Pam3CSK4) and ultrapure LPS were nonstimulatory. More importantly, crude LPS treated with lysozyme to degrade PGNs, exhibited greatly reduced activity in stimulating IL-1beta and IL-6 gene expression, indicating that PGNs in the crude LPS are responsible for a significant amount of the proinflammatory activity. Finally, we showed that PGN treatment induces expression of COX-2 and the subsequent synthesis and release of prostaglandin E(2) (PGE(2)), an important mediator of inflammatory processes. The strong stimulatory effect of E. coli PGNs by themselves on trout macrophages suggests that the recognition of Gram-negative bacteria in trout is through PGNs in the bacterial wall, and indicates that the systems responsible for bacterial recognition in invertebrates (e.g., Drosophila) may also be conserved in some vertebrates.
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http://dx.doi.org/10.1016/j.molimm.2010.02.009DOI Listing
April 2010

Endotoxin recognition in fish results in inflammatory cytokine secretion not gene expression.

Innate Immun 2011 Feb 18;17(1):16-28. Epub 2010 Jan 18.

Departament Biologia Cellular, Immunologia i Fisiologia Animal Universitat Autónoma de Barcelona, Barcelona, Spain.

Macrophages are phagocytes that have a central role in the organization of the immune system after an infection. These cells can recognize specific molecular components of micro-organisms (pathogen-associated molecular patterns, PAMPs) via specific receptors (PRRs) and elicit specific cellular responses. In the past, the expression of immune genes in response to different PAMPs has been characterized in different fish species. However, little is known about actual cytokine release. We characterized the secretion of tumour necrosis factor (TNF)-α in primary macrophage cultures of rainbow trout (Oncorhynchus mykiss) in response to several PAMPs by Western blot and compared this to the induction of TNF-α gene expression as well as other pro-inflammatory cytokines such as interleukin (IL)-1β and IL-6 and anti-viral molecules such as INF-α and Mx protein (Mx). We show that lipopolysaccharide (LPS) and zymosan are major inducers of TNF-α secretion, which is not initially linked to the induction of TNF-α mRNA expression. The secretion of TNF-α, but intriguingly not the expression, is also stimulated by ultrapure LPS meaning that, in fish, contaminants of commercial LPS preparations are better inducers of the inflammatory response. Moreover, we have characterized the signaling pathways that are activated by different PAMPs and the link between those pathways and the final step of TNF-α secretion: TNF-α shedding by TNF-α converting enzyme (TACE/ ADAM17). For the first time, we show that, in fish macrophages, TNF-α is processed by TACE-like activity and this cleavage is dependent upon the activation of ERK, p38MAPK and JNK signaling pathways by LPS.
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http://dx.doi.org/10.1177/1753425909348232DOI Listing
February 2011

Stimulation of growth and changes in the hepatic transcriptome by 17beta-estradiol in the yellow perch (Perca flavescens).

Physiol Genomics 2009 Aug 23;38(3):261-80. Epub 2009 Jun 23.

Great Lakes WATER Institute, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, USA.

The effects of dietary 17beta-estradiol (E(2)) on growth and liver transcriptomics were investigated in the yellow perch (Perca flavescens). After a 3-mo treatment, E(2) significantly stimulated an increase in length and weight of juvenile male and female perch relative to control animals. The increase was significantly greater in females compared with males. Separate, unnormalized cDNA libraries were constructed from equal quantities of RNA from 6 male and 6 female livers of E(2)-treated and control perch, and 3,546 and 3,719 expressed sequence tags (ESTs) were obtained, respectively. To characterize E(2)-regulated transcripts, EST frequencies between libraries were calculated within contiguous sequences that were assembled from the combined ESTs of both libraries. Frequencies were also determined in EST transcript groupings produced by aligning all of the ESTs from both libraries at the nucleotide level. From these analyses, there were 28 annotated transcripts that were regulated by 75% between libraries and for which there were at least 5 ESTs of the same transcript between libraries. Regulation of a subset (14) of these transcripts was confirmed by quantitative reverse transcription-polymerase chain reaction (QPCR). Transcripts that were upregulated by E(2) included reproduction-related proteins, binding proteins, and proteases and protease inhibitors. While not part of the transcript frequency analysis, QPCR showed significant upregulation of estrogen receptor esr1 and of insulin-like growth factor I (IGF-I) in E(2) livers. E(2)-downregulated transcripts represented a variety of functional categories including components of the respiratory chain, lipid transport and metabolism, glycolysis, amino acid and nitrogen metabolism, binding proteins, a hydrolytic enzyme, and a transcriptional regulator. In perch it appears that exogenous estrogen drastically shifts liver metabolism toward the production of lipoproteins and carbohydrate binding proteins, and that the growth-promoting action may involve an increase in hepatic IGF-I production.
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http://dx.doi.org/10.1152/physiolgenomics.00069.2009DOI Listing
August 2009

Characterization and expression of NADPH oxidase in LPS-, poly(I:C)- and zymosan-stimulated trout (Oncorhynchus mykiss W.) macrophages.

Fish Shellfish Immunol 2009 Apr 28;26(4):651-61. Epub 2008 Nov 28.

Unitat de Fisiologia Animal, Departament de Biologia Cellular, Fisiologia i d'Immunologia, Facultat de Biociencies, Universitat Autònoma de Barcelona, Barcelona, Spain.

In vertebrates, the generation of superoxide reactive oxygen species (ROS) via activation of the Nox/Duox family of NADPH oxidases is a prototypical feature of the pathogen-induced defensive responses of activated professional phagocytes. To understand the role of the rainbow trout (Oncorhynchus mykiss) Phox oxidase from a phylogenetic and functional perspective we describe the cloning, sequencing and expression analysis of multiple NADPH components in cultured macrophages. Phylogenetic analyses support the notion of the emergence of Phox-related components before the diversification of basal euteleosts and add to the limited collection of teleost NADPH oxidases. Expression studies using lipopolysaccharide, polyinosine-polycytidylic acid and zymosan to mimic the onset of inflammatory responses in trout macrophages suggest differences in regulation of the NADPH complex throughout the maturation/differentiation period of culture and between different treatments.
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http://dx.doi.org/10.1016/j.fsi.2008.11.011DOI Listing
April 2009

Molecular characterization of interleukin-6 in the gilthead seabream (Sparus aurata).

Mol Immunol 2008 Jul 2;45(12):3363-70. Epub 2008 Jun 2.

Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona and Institut de Biomedicina de la Universitat de Barcelona (IBUB), 08028 Barcelona, Spain.

A cDNA clone, designated sbIL-6 (seabream interleukin-6), was obtained from a cDNA library of enriched immune-stimulated sequences from gilthead seabream. The deduced sbIL-6 protein corresponds to a 225-amino acid protein with a putative 24-amino acid signal peptide, four conserved alpha helices and one N-linked glycosylation site. At the amino acid level sbIL-6 shares 23-26% identity with mammalian IL-6 sequences and 30-51% identity with other fish IL-6 sequences. The structure of the sbIL-6 gene consisted of 5 exons and 4 introns, spanning 2.4 kb. Healthy fish expressed sbIL-6 in white muscle, skin, spleen, anterior intestine and stomach, while no expression was detected in brain, gill, head kidney, posterior intestine and adipose tissue. A significant up-regulation of sbIL-6 expression was observed after lipopolysaccharide (LPS), Vibrio anguillarum DNA (VaDNA) and peptidoglycan treatment in cultured seabream head kidney leukocytes. Using purified immune cells, sbIL-6 expression was induced similarly in macrophages and acidophilic granulocytes by VaDNA but LPS was more effective in inducing sbIL-6 expression in acidophilic granulocytes than in macrophages. Furthermore, in vivo infection of seabream with live V. anguillarum caused significant increases in sbIL-6 mRNA expression in the thymus, peritoneal exudate, head kidney and gills. In summary, our study provides further evidence for the existence of distinct IL-6 genes in lower vertebrates and for the strong induction of their expression by immune stimuli, supporting the notion of a potentially important role for this cytokine in fish.
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http://dx.doi.org/10.1016/j.molimm.2008.04.012DOI Listing
July 2008

Stress-induced regulation of steroidogenic acute regulatory protein expression in head kidney of Gilthead seabream (Sparus aurata).

J Endocrinol 2008 Feb;196(2):313-22

Unitat de Fisiologia Animal, Departament de Biologia Cel.lular, Fisiologia i d'Immunologia, Facultat de Biociències Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona Spain.

Steroidogenic acute regulatory protein (StAR) transfers cholesterol over the inner mitochondrial membrane. In mammals, StAR controls this rate-limiting step of steroidogenesis, but its expression and regulation has not been well explored in fish. The present work investigates StAR mRNA expression in the head kidney of the gilthead seabream (Sparus aurata) under different stressors. We have cloned the StAR cDNA (1461 bp) in seabream (accession number EF640987), which has an open reading frame of 861 nucleotides encoding a polypeptide of 286 aa, and displays high sequence identity with StAR of other fish and mammalian counterparts. Seabream StAR transcripts were found to be expressed exclusively in head kidneys and gonads. In fish under acute stress (chased with a net), plasma cortisol levels peaked within 1 h, were still high after 6 h, and decreased after 16 h, although no increases in head kidney StAR expression were observed at any time post-stressor. Fish under chronic high-density stress showed cortisol levels 90-fold higher than controls and StAR mRNA levels increased threefold. Lipopolysaccharide (LPS) injection increased head kidney StAR mRNA levels after 6 h, reached a maximum at 12 h, and decreased until 72 h. When the head kidney cells were incubated in vitro and treated with ACTH or LPS, ACTH induced an increase in StAR expression as expected, but LPS induced a reduction in StAR expression. In conclusion, StAR expression in seabream head kidneys is highly regulated by different stressors.
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http://dx.doi.org/10.1677/JOE-07-0440DOI Listing
February 2008

Identification of differentially expressed ovarian genes during primary and early secondary oocyte growth in coho salmon, Oncorhynchus kisutch.

Reprod Biol Endocrinol 2008 Jan 18;6. Epub 2008 Jan 18.

School of Aquatic and Fishery Sciences, University of Washington, Seattle, Washington 98195, USA.

Background: The aim of this study was to identify differentially expressed ovarian genes during primary and early secondary oocyte growth in coho salmon, a semelparous teleost that exhibits synchronous follicle development.

Methods: Reciprocal suppression subtractive hybridization (SSH) libraries were generated from ovaries with perinucleolus (P) or cortical alveolus (CA) stage follicles and selected genes were assessed with quantitative PCR (qPCR). An assessment of changes in RNA composition during oocyte growth and its relationship to transcript levels was also conducted.

Results: SSH revealed several differentially expressed genes during early oogenesis, some which will not likely be utilized until 1-3 years later in salmon. Zona pellucida glycoprotein (zp) genes, vitellogenin receptor (vldlr) isoforms, cathepsin B (ctsba), cyclin E (ccne), a DnaJ transcript (dnaja2), and a ferritin subunit (fth3) were significantly elevated at the P stage, while a C-type lectin, retinol dehydrogenase (rdh1), and a coatomer protein subunit (cope) were upregulated at the CA stage. Putative follicle cell transcripts such as anti-Müllerian hormone (amh), lipoprotein lipase (lpl), apolipoprotein E (apoe), gonadal soma-derived growth factor (gsdf) and follicle-stimulating hormone receptor (fshr) also increased significantly at the CA stage. The analysis of RNA composition during oocyte growth showed that the total RNA yield and proportion of messenger RNA relative to non-polyadenylated RNAs declined as oogenesis progressed. This influenced apparent transcript levels depending on the type of RNA template used and normalization method.

Conclusion: In coho salmon, which exhibit a dramatic change in oocyte size and RNA composition during oogenesis, use of messenger RNA as template and normalization of qPCR data to a housekeeping gene, ef1a, yielded results that best reflected transcript abundance within the ovarian follicle. Synthesis of zp transcripts and proteins involved in yolk incorporation and processing occurred during primary growth, while increased expression of a CA component and genes related to lipid incorporation occurred concomitant with the appearance of CA, but prior to lipid accumulation. Significant increases in transcripts for fshr, gsdf, and amh at the CA stage suggest a role of FSH and TGFbeta peptides in previtellogenic oocyte growth and puberty onset in female salmon.
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http://dx.doi.org/10.1186/1477-7827-6-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2262088PMC
January 2008

Characterization and expression of the transcription factor PU.1 during LPS-induced inflammation in the rainbow trout (Oncorhynchus mykiss).

Fish Shellfish Immunol 2008 Jan 5;24(1):35-45. Epub 2007 Oct 5.

Departament de Biologia Cellular, Fisiologia i d'Immunologia, Facultat de Ciencies, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.

The transcription factor PU.1 plays a key role in hematopoietic lineage development and therefore in determining immune cell fate. A full length cDNA transcript of 1237 nucleotides encoding a highly conserved putative protein of 293 amino acids was identified by EST analysis in lipopolysaccharide (LPS) activated trout macrophages. Phylogenetic analyses highlight the significant level of structural conservation of the PU.1 transcription factor reinforcing the importance of this molecule in animal immunity. In trout, the PU.1 mRNA shows a tissue-specific expression pattern and is induced in vivo by LPS in muscle, liver, intestine and brain. Furthermore PU.1 is highly expressed in trout macrophages in primary culture. In situ expression analysis in the head kidney describes a large number of PU.1+ve cells distributed through the tissue in both LPS-treated and control animals. Cellular proliferation examined by BrdU immunohistochemistry (IHC) shows that LPS regulates hematopoietic processes in adult fish by stimulating cellular proliferation 3 days after treatment. These studies provide initial insights into hematopoietic/cellular processes in the head kidney of rainbow trout after in vivo LPS challenge.
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http://dx.doi.org/10.1016/j.fsi.2007.07.009DOI Listing
January 2008

Pathogen-associated gene expression profiles in rainbow trout macrophages.

Comp Biochem Physiol Part D Genomics Proteomics 2006 Dec 20;1(4):416-22. Epub 2006 Oct 20.

Great Lakes WATER Institute, University of Wisconsin-Milwaukee, 600 E. Greenfield Ave., Milwaukee, Wisconsin 53204, USA; University of Notre Dame, Department of Biological Sciences, Notre Dame, Indiana 46556, USA.

Pathogens can be distinctively recognized by the cells of the immune system through interactions between the Pathogen-Associated Molecular Patterns (PAMPs) that they produce and the innate immune receptors of leukocytes. The present paper reports on the PAMP-modulated expression of a group of genes expressed in trout macrophages. The genes were identified in subtracted libraries from lipopolysaccharide (LPS)-stimulated macrophages and their expression was analyzed using quantitative real time PCR following stimulation of the cells with E. coli LPS, poly (I:C) and zymosan; representing Gram-negative bacteria, viruses and fungi, respectively. Genes (SPINT1L, DDIT4L, STEAP4, and TNFAIP3), the expression of which was induced by LPS and zymosan, were not significantly up-regulated by poly(I:C) and the opposite was found for transcripts (HMGB1 and PSMB9) up-regulated by poly(I:C). Overall, the differences in gene expression were greater at a later stage of macrophage activation (24 h) at a time when stimulation with poly(I:C) resulted in substantially different responses as compared to LPS and, to a lesser extent, zymosan.
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http://dx.doi.org/10.1016/j.cbd.2006.10.003DOI Listing
December 2006

Cloning and expression analysis of an IL-6 homolog in rainbow trout (Oncorhynchus mykiss).

Mol Immunol 2007 Mar 12;44(7):1803-7. Epub 2006 Oct 12.

Great Lakes WATER Institute, University of Wisconsin Milwaukee, 600 E. Greenfield Avenue, Milwaukee, WI 53204, USA.

A partial cDNA with significant similarity to IL-6 was identified in rainbow trout. Rapid amplification of cDNA ends was used to obtain the full sequence of the trout IL-6 homolog which contains 1180 nucleotides. The transcript encodes a predicted protein of 219 amino acids and eight instability motifs in the 3'UTR. While the complete sequence of the trout IL-6 is poorly conserved, the protein contains a distinct IL-6/G-CSF/MGF family consensus pattern and predicted characteristic alpha-helical tertiary structure. However, like in fugu, trout IL-6 lacks a pair of cysteine residues, which in mammals are involved in formation of a disulphide bond. The expression of the IL-6 homolog in trout mononuclear phagocytes was highly up-regulated by LPS but not poly(I:C) as demonstrated by Northern analysis. Using RT-PCR the IL-6 expression was detected in trout spleen, gill, gastrointestinal tract, ovary and brain. The highest transcript levels were detected in the ovary suggesting that IL-6 may perform specific functions within this organ.
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http://dx.doi.org/10.1016/j.molimm.2006.07.297DOI Listing
March 2007

A proposed nomenclature consensus for the myostatin gene family.

Am J Physiol Endocrinol Metab 2007 Feb 26;292(2):E371-2. Epub 2006 Sep 26.

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http://dx.doi.org/10.1152/ajpendo.00395.2006DOI Listing
February 2007

Bacterial lipopolysaccharide induces apoptosis in the trout ovary.

Reprod Biol Endocrinol 2006 Aug 31;4:46. Epub 2006 Aug 31.

Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.

Background: In mammals it is well known that infections can lead to alterations in reproductive function. As part of the innate immune response, a number of cytokines and other immune factors is produced during bacterial infection or after treatment with lipopolysaccharide (LPS) and acts on the reproductive system. In fish, LPS can also induce an innate immune response but little is known about the activation of the immune system by LPS on reproduction in fish. Therefore, we conducted studies to examine the in vivo and in vitro effects of lipopolysaccharide (LPS) on the reproductive function of sexually mature female trout.

Methods: In saline- and LPS -injected brook trout, we measured the concentration of plasma steroids as well as the in vitro steroidogenic response (testosterone and 17alpha-hydroxyprogesterone) of ovarian follicles to luteinizing hormone (LH), the ability of 17alpha,20beta-dihydroxy-4-pregnen-3-one to induce germinal vesicle breakdown (GVBD) in vitro, and that of epinephrine to stimulate follicular contraction in vitro. We also examined the direct effects of LPS in vitro on steroid production, GVBD and contraction in brook trout ovarian follicles. The incidence of apoptosis was evaluated by TUNEL analysis. Furthermore, we examined the gene expression pattern in the ovary of saline- and LPS-injected rainbow trout by microarray analysis.

Results: LPS treatment in vivo did not affect plasma testosterone concentration or the basal in vitro production of steroids, although a small but significant potentiation of the effects of LH on testosterone production in vitro was observed in ovarian follicles from LPS-treated fish. In addition, LPS increased the plasma concentration of cortisol. LPS treatment in vitro did not affect the basal or LH-stimulated steroid production in brook trout ovarian follicles. In addition, we did not observe any effects of LPS in vivo or in vitro on GVBD or follicular contraction. Therefore, LPS did not appear to impair ovarian steroid production, oocyte final maturation or follicular contraction under the present experimental conditions. Interestingly, LPS administration in vivo induced apoptosis in follicular cells, an observation that correlated with changes in the expression of genes involved in apoptosis, as evidenced by microarray analysis.

Conclusion: These results indicate that female trout are particularly resistant to an acute administration of LPS in terms of ovarian hormone responsiveness. However, LPS caused a marked increase in apoptosis in follicular cells, suggesting that the trout ovary could be sensitive to the pro-apoptotic effects of LPS-induced inflammatory cytokines.
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http://dx.doi.org/10.1186/1477-7827-4-46DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1570353PMC
August 2006