Immunological stimuli change expression of genes and neutrophil function in fathead minnow Pimephales promelas Rafinesque.

Fathead minnows Pimephales promelas were exposed to lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid [poly(I:C)] to observe immunological responses during simulated bacterial and viral challenge at the level of gene expression and granulocyte function. Complementary DNA libraries were created from LPS- and poly(I:C)-treated fish and c. 5000 expressed sequence tags (ESTs) were sequenced. The ESTs were subjected to BLASTx analysis and 1500 genes were annotated, grouped by function and 20 immune genes were selected for expression studies by real-time PCR. Lipopolysaccharide treatment significantly downregulated expression of interferon regulatory factor 2 binding protein 1 (nine-fold), Chemokine (C-X-C motif) ligand 12a (three-fold) and TNF-related apoptosis-inducing ligand, TRAIL (two-fold). In poly(I:C)-treated fish, a significant upregulation was observed for IFN-inducible and antiviral proteins belonging to the family of Mx proteins (73-fold) and chemokine CCL-C5a (28-fold). Blood neutrophil count was significantly increased in poly(I:C)-treated fish at 24 and 48 h post-injection. Neutrophil extracellular trap release and respiratory burst of kidney granulocytes were suppressed in poly(I:C)-treated fish, while degranulation of primary granules was not affected significantly by the treatment. The changes in gene expression and neutrophil function in P. promelas exposed to LPS and poly(I:C) support the use of this species as an alternative model for studies of pathogen effects on the innate immune system of fishes.

CD83 expression in sea bream macrophages is a marker for the LPS-induced inflammatory response.

CD83, a cell surface membrane glycoprotein member of the Ig superfamily which is commonly used as standard surface marker for dendritic cells, was cloned from gilthead sea bream macrophages using degenerate primers against conserved motifs of known CD83 sequences. The obtained cDNA contains an open reading frame of 669 nucleotides that translate into a 222 amino acid putative peptide. The deduced protein sequence shows conservation of features shared by vertebrate CD83 and multiple alignment with fish CD83 sequences reveals high homology. In cultured sea bream macrophages CD83 mRNA expression was significantly enhanced in a dose- and time-dependent fashion after stimulation with Escherichia coli LPS. These results indicate that in fish, macrophages express high levels of CD83 mRNA after LPS exposure and CD83 is therefore a good marker for activated mature myeloid cells in fish.

Zebrafish and giant danio as models for muscle growth: determinate vs. indeterminate growth as determined by morphometric analysis.

The zebrafish has become an important genetic model, but their small size makes them impractical for traditional physiological studies. In contrast, the closely related giant danio is larger and can be utilized for physiological studies that can also make use of the extensive zebrafish genomic resources. In addition, the giant danio and zebrafish appear to exhibit different growth types, indicating the potential for developing a comparative muscle growth model system. Therefore, the present study was conducted to compare and characterize the muscle growth pattern of zebrafish and giant danio. Morphometric analyses demonstrated that giant danio exhibit an increased growth rate compared with zebrafish, starting as early as 2 wk posthatch. Total myotome area, mean fiber area, and total fiber number all exhibited positive correlations with larvae length in giant danio but not in zebrafish. Morphometric analysis of giant danio and zebrafish larvae demonstrated faster, more efficient growth in giant danio larvae. Similar to larger teleosts, adult giant danio exhibited increased growth rates in response to growth hormone, suggesting that giant danio exhibit indeterminate growth. In contrast, adult zebrafish do not exhibit mosaic hyperplasia, nor do they respond to growth hormone, suggesting they exhibit determinate growth like mammals. These results demonstrate that giant danio and zebrafish can be utilized as a direct comparative model system for muscle growth studies, with zebrafish serving as a model organism for determinate growth and giant danio for indeterminate growth.

Transcriptional analysis of LPS-stimulated activation of trout (Oncorhynchus mykiss) monocyte/macrophage cells in primary culture treated with cortisol.

Primary immune responses to pathogen invasion are mediated by the innate immune system in which tissue macrophages play a key role. During infectious processes glucocorticoids generally may function to dampen inflammatory responses. In this study, the ability of cortisol to directly modulate the transcriptional response of rainbow trout macrophages to the cellular activator lipopolysaccharide (LPS) was investigated. The results indicate that cortisol significantly inhibits the well-described LPS-dependent induction of the expression of TNF-alpha2, a pro-inflammatory cytokine. In order to further characterize the molecular effects of LPS and the immunomodulatory role of cortisol, the in vitro macrophage response to LPS in the absence or presence of 12-h cortisol exposure was analyzed utilizing a salmonid-specific microarray platform. Genes that were stimulated or inhibited with LPS plus cortisol fell into several major functional groups. The first, a general "response" group comprising genes within ontology classes including the response to external stimuli, stress, humoral immunity and apoptosis, exhibited a significant increase after LPS stimulation, whereas suppression of this response was observed in the presence of cortisol. LPS stimulated other genes in a second group involved in cell signalling and also genes in a third group involved in the activation of transcription. Categories activated with cortisol were mainly related to various aspects of metabolism (including protein biosynthesis, binding and transport of ions) and structural proteins (mainly cytoskeleton and microtubules). The immunomodulatory action of cortisol on LPS-stimulated macrophages therefore appears more complex than simply the antagonism of LPS-induced transcriptional responses.

Characterization of a highly inducible novel CC chemokine from differentiated rainbow trout (Oncorhynchus mykiss) macrophages.

A full-length cDNA clone encoding a novel trout CC chemokine was identified in expressed sequence tags generated from lipopolysaccharide (LPS)-stimulated in vitro differentiated macrophages isolated from the head kidney of the rainbow trout (Oncorhynchus mykiss). The putative 101-amino-acid protein is 38% similar to Macaca mulatta CCL4 (macrophage inflammatory protein 1beta) but is also similar to several other related mammalian CC chemokines, including human Act-2. Real-time PCR and conventional RT-PCR revealed significant up-regulation of transcript levels of the trout CCL4-like mRNA in LPS-stimulated in vitro differentiated macrophages. In unstimulated trout, CCL4-like mRNA expression was detected at different levels in all tissues tested, whereas in LPS-challenged animals (6 mg/kg), CCL4-like mRNA increased in intestine, ovary and spleen at both 24 h and 72 h post-injection. In gills, CCL4-like mRNA expression was inhibited after LPS administration. Based on the highly regulated expression pattern exhibited by the trout CCL4-like mRNA, it is likely that this chemokine plays an important regulatory role in the immune response of trout.

Acute neurosteroid modulation and subunit isolation of the gamma-aminobutyric acidA receptor in the bullfrog, Rana catesbeiana.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) has multiple receptors. In mammals, the GABA(A) receptor subtype is modulated by neurosteroids. However, whether steroid interaction with the GABA(A) receptor is unique to mammals or a conserved feature in vertebrates is unknown. Thus, neurosteroid modulation of the GABA(A) receptor was investigated in the brain of the bullfrog (Rana catesbeiana) using the mammalian GABA(A) receptor agonist [(3)H]muscimol. Two neurosteroids, allopregnanolone and pregnenolone sulfate, affected [(3)H]muscimol specific binding in bullfrog brain membrane preparations. Allopregnanolone significantly increased [(3)H]muscimol specific binding in a dose- and time-dependent manner. The pattern of allopregnanolone modulation supports the hypothesis that the bullfrog brain possesses both high-affinity and low-affinity [(3)H]muscimol binding sites. Unlike allopregnanolone, pregnenolone sulfate showed biphasic modulation with increased [(3)H]muscimol specific binding at low nanomolar concentrations and decreased specific binding at micromolar concentrations. Additionally, three cDNA fragments with significant homology to mammalian GABA(A) receptor subunits were isolated from the bullfrog brain. These fragments belong to the alpha1, beta1, and gamma2 subunit families. In mammals, GABA(A) receptors composed of these specific subunit isoforms are effectively modulated by neurosteroids, including allopregnanolone. Neurosteroid modulation of the amphibian brain GABA(A) receptor is therefore supported by both [(3)H]muscimol binding studies and subunit sequences. Allopregnanolone and pregnenolone sulfate modulation of this receptor may thus represent a significant mechanism for steroid influence on amphibian brain and behavior.

Cysteine protease inhibitor is specifically expressed in pre- and early-vitellogenic oocytes from the brook trout periovulatory ovary.

A cDNA fragment hybridizing with a transcript abundant in the periovulatory ovary was obtained while performing subtractive cloning on brook trout ovulatory and postovulatory ovarian tissue. Using this fragment as a probe, a 478 bp full-length cDNA was obtained by screening an ovulatory ovarian cDNA library. This cDNA presumably codes for an 88 amino acid protein that is structurally related to a new family of cysteine protease inhibitors characterized by the presence of a type I thyroglobulin motif in the amino acid sequence. Therefore, the protein was tentatively named an oocyte cysteine protease inhibitor (OCPI). On Northern blots, the OCPI cDNA hybridizes with a 0.5 kb transcript present in the ovary during the periovulatory period. The OCPI transcript and protein were localized to the cytoplasm of pre- and early-vitellogenic oocytes. On Northern blots of RNA from other tissues, the OCPI transcript was detected only in the ovary. On Western blots, OCPI was detected in the ovarian tissue at all periovulatory stages tested. The specific localization of both OCPI transcript and protein to pre- and early-vitellogenic oocytes and the structural similarity to protease inhibitors, suggest that OCPI might be involved in the protection of oocytes during the periovulatory period or in the regulation of yolk formation and degradation.

Molecular cloning and expression of a TNF receptor and two TNF ligands in the fish ovary.

Using degenerative primers, partial cDNAs of a TNF (tumor necrosis factor) receptor and two TNF ligands were obtained by PCR of zebrafish and trout cDNAs, or cDNA libraries. These fragments were then used to screen cDNA libraries of appropriate tissues to obtain clones containing full coding sequences. A zebrafish cDNA was obtained that presumably codes for a 438 amino acid ovarian TNF receptor (OTR) that was identified as a death-domain-containing member of the TNF receptor family. On Northern blots, the OTR cDNA hybridized with a 3.4-kb transcript that is abundant in the zebrafish ovary but lightly detected in all other tissues tested. A zebrafish cDNA presumably coding for a 214 amino acid protein with sequence similarity to mammalian TRAIL (TNF-related apoptosis inducing ligand), was also isolated. In addition, a fragment of the brook trout TRAIL homologue was obtained. Finally, a full-length brook trout cDNA, that presumably codes for a 255 amino acid protein with sequence similarity to mammalian TNF-alpha and lymphotoxin-alpha, was isolated. This study is the first report of a death-domain-containing TNF receptor and the first published report of a TNF ligand in fish.

Conservation of death receptor-6 in avian and piscine vertebrates.

One of the most recently identified members of the tumor necrosis factor receptor family, death receptor-6 (DR6), has been shown to mediate apoptosis following overexpression in HeLa cells. The avian and piscine orthologs of DR6 have now been identified, and the deduced amino acid sequence for each demonstrates a high level of conservation compared to the mammalian sequence. Expression of dr6 mRNA occurs widely across tissues of both the mature chicken and brook trout. It is now well-established that ovarian follicular atresia occurs via apoptosis originating within the granulosa cell layer. Accordingly, DR6 expression within the ovary was examined to assess the relationship between stage of follicle development and relative levels of this death receptor. Of particular interest was the finding that elevated levels of dr6 mRNA, as well as the translated protein, are expressed in atretic compared to healthy follicles of the hen ovary, thus providing the first association between DR6 expression and apoptosis, in vivo. We conclude that DR6 is a highly conserved and widely expressed death-domain-containing receptor and may be implicated in regulating follicle atresia within the vertebrate ovary.

Characterization and distribution of neuropeptide Y in the brain of a caecilian amphibian.

Neuropeptide Y (NPY) from the brain of an amphibian from the order Gymnophiona (the caecilian, Typhlonectes natans) was characterized. We cloned a 790 base pair cDNA encoding the caecilian NPY precursor. The open reading frame consisted of 291 bases, indicating an NPY precursor of 97 amino acids. Both deduced and isolated NPY primary structures were Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu(10)-Asp-Ala-Pro-Ala-Glu-Asp-Met-Ala-Lys-Tyr(20)-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu(30)-Ile-Thr-Arg-Gln-Arg-Tyr. NH2. In caecilian brain, we observed NPY immunoreactive cells within the medial pallium, basal forebrain, preoptic area, midbrain tegmentum and trigeminal nucleus. The prevalence of preoptic and hypothalamic terminal field staining supports the hypothesis that NPY controls pituitary function in this caecilian.

An ovarian progastricsin is present in the trout coelomic fluid after ovulation.

An up-regulated cDNA fragment was isolated using a differential display polymerase chain reaction between ovulatory and postovulatory brook trout ovarian tissues. Using this fragment as a probe, a full-length cDNA of 1783 base pairs was obtained from an ovarian cDNA library. The cDNA presumably codes for a 383-amino acid protein with strong sequence similarity to an aspartic protease, progastricsin (EC 3.4.23.3), also known as pepsinogen C. On Northern blots of ovarian tissue, the trout progastricsin cDNA hybridized with a 1.8-kilobase transcript that was strongly up-regulated 4-6 days after ovulation. Of all other tissues tested, a transcript was only detected in the stomach. A recombinant trout progastricsin protein was produced and used to raise an antibody. On Western blots of ovarian tissue, the progastricsin antibody recognized a single 39-kDa protein that was present in the ovary only following ovulation. On Western blots of coelomic fluid, the 39-kDa protein was strongly detected 4-10 days after ovulation. The trout progastricsin was immunocytochemically localized to the granulosa cells of postovulatory follicles, suggesting that it is released from this tissue into the coelomic fluid following ovulation. Progastricsin has been found in the stomach, prostate, seminal vesicle, seminal fluid, and pancreas of vertebrates; however, this is the first report of a progastricsin in an animal ovary.

Differential skeletal muscle expression of myostatin across teleost species, and the isolation of multiple myostatin isoforms.

Two myostatin (MSTN) isoforms were isolated from brook trout with 92% identity in corresponding regions at the nucleotide level. One isoform was isolated from muscle and brain and the second from ovarian tissue. To our knowledge this is the first time two MSTN isoforms have been isolated from a given vertebrate species. Within the brain, MSTN transcripts were localized to the optic lobes, hindbrain, and hypothalamus. In the trout ovary, MSTN transcripts were upregulated at ovulation in several females. MSTN cDNA fragments were also isolated from several other fish species and differential expression of MSTN among muscle fiber types was observed.

Isolation of gonadal mutations in adult zebrafish from a chemical mutagenesis screen.

A mutagenesis screen was conducted on zebrafish using N:-ethyl N:-nitrosourea as a mutagen and an F2 crossing scheme to obtain homozygous mutants in the F3 generation. Whole abdomens of 3-mo-old F3 zebrafish progeny were fixed and mass-embedded in paraffin blocks. Blocks were cut with a microtome to obtain cross-sections of the entire body cavity that included the ovaries and testes. Slides of the cross-sections were analyzed for alterations in gonadal structure and gametogenesis and were compared with gonads of wild-type fish. A total of 125 mutagenized genomes in 81 families were screened and 11 mutations were observed that produced visible phenotypes in only one sex per family. Male mutations included testes without mature sperm that contained either predominantly spermatocytes or spermatogonia. Female mutations included ovaries containing 1) degenerating oocytes surrounded by hypertrophied follicle walls or stroma, 2) extrafollicular tissue proliferation, 3) proliferating postovulatory follicle walls, and 4) large numbers of degenerating preovulatory and postovulatory oocytes. While past screens on zebrafish have concentrated on early developmental mutations, the results of this study demonstrate for the first time that mutagenesis can be used with zebrafish to study reproduction in adult animals.

A novel osteopontin-like protein is expressed in the trout ovary during ovulation.

Using suppression subtraction hybridization between ovulatory and postovulatory trout ovaries, a down-regulated cDNA was obtained that presumably encodes a novel ovarian protein ('NOP'). NOP mRNA is present in the ovary during ovulation and down-regulated by 48 h postovulation, suggesting an important role for NOP during ovulation. Besides the ovary, NOP is also strongly expressed in the testis and at lower levels in the skin, gills, kidney and gastrointestinal tract. While the overall identity is not high, NOP shares several sequence similarities with mammalian and chicken osteopontins, including the percentage of aspartate, serine and alanine residues and the presence of a cell attachment motif.

A S100 homologue mRNA isolated by differential display PCR is down-regulated in the brook trout (Salvelinus fontinalis) post-ovulatory ovary.

A down-regulated cDNA fragment was isolated using differential display PCR between ovulatory and post-ovulatory brook trout ovarian tissue. Using this cDNA as a probe, a full-length cDNA of 538bp was obtained by screening an ovarian cDNA library. The cDNA isolated presumably encodes for a 101 amino acid protein that is similar in sequence to a group of calcium binding proteins called S100 proteins. Within the S100 family, the differentially expressed trout protein is more similar to S100 A1 and A10 proteins. A comparison of the trout S100 protein with mammalian S100 A1 proteins and a S100 protein previously isolated from the loach suggests that the trout S100 protein could be a new member of the S100 family. On Northern blots of ovarian tissue, the trout S100 cDNA hybridized with a 550bp transcript which appeared to progressively decrease throughout ovulation and was significantly down-regulated by 48h post-ovulation. A transcript was also detected in several other tissues such as the heart, skin, spleen, gills, intestine, stomach and testis. Using in situ hybridization the trout S100 mRNA was strongly detected in the granulosa cells of the ovulated follicle. These results suggest that a member of the S100 protein family may play a role in the trout ovary during the periovulatory period.

Conservation of steroidogenic acute regulatory (StAR) protein structure and expression in vertebrates.

Complementary DNAs for the open reading frames of the chicken, Xenopus and zebrafish StAR homologs were cloned along with a partial cDNA of the zebrafish homolog to MLN64, a StAR-related protein. A comparison of the amino acid sequences of piscine, amphibian, avian and mammalian StARs, indicates strong conservation of the protein across divergent vertebrate groups. On Northern blots probed with species specific StAR cDNAs, expression of StAR transcripts was observed in the ovary and adrenal of chicken, and the ovary, testis, kidney and head of zebrafish. The expression of StAR mRNA in various compartments of the hen ovary was consistent with the results of past studies on steroidogenesis; expression was first observed in follicles selected into the preovulatory hierarchy and was greatest in the largest preovulatory follicle. The expression of StAR mRNA was also consistent with aromatase expression in zebrafish ovaries. The conserved deduced protein sequence and expression pattern of StAR transcripts in chicken and zebrafish tissues, strongly suggest that StAR is also involved in the regulation of steroidogenesis in nonmammalian vertebrates.

Regulation of ovarian steroidogenesis in vitro by follicle-stimulating hormone and luteinizing hormone during sexual maturation in salmonid fish.

The regulation of ovarian steroidogenesis in vitro by coho salmon FSH and LH was investigated in intact coho salmon follicles and isolated follicular layers at various stages of oocyte maturation, from late vitellogenesis until the completion of germinal vesicle breakdown (GVBD). In granulosa layers from all stages, LH, but not FSH, stimulated 17alpha,20beta-dihydroxy-4-pregnen-3-one (17, 20beta-P) production. In theca-interstitial layers from all stages, FSH and LH stimulated steroid production, LH being more potent than FSH. The basal steroid output of intact follicles was significantly lower than that of isolated follicular layers, and their response to FSH and LH also differed. First, the intact follicles produced 17alpha-hydroxyprogesterone in response to FSH during the central germinal vesicle stage while theca-interstitial layers did not. Second, estradiol-17beta production was not inhibited by LH during final oocyte maturation in intact follicles, as observed for granulosa layers. Our results indicate that LH is the determining factor regulating the production of the maturation-inducing steroid, 17,20beta-P, and the induction of GVBD in the salmonid ovary. In summary, we have provided evidence for maturation-associated changes in the effects of FSH and LH in the salmonid ovary, which further supports the hypothesis that FSH and LH have distinct functions in the teleost ovary.

Cloning and characterization of prostaglandin endoperoxide synthase-1 and -2 from the brook trout ovary.

Using a combination of reverse transcription-PCR and library screening, the cDNAs for prostaglandin endoperoxide synthase-1 (PGS-1) and 2 (PGS-2) were isolated from the brook trout ovary. The brook trout PGS-1 cDNA encodes for a 598 amino acid protein that is 69% identical to mammalian PGS-1. PGS-1 transcripts were observed in the ovary, spleen, gills, head kidney, trunk kidney, intestine, stomach, skin and heart. To our knowledge, this is the first characterization of a non-mammalian PGS-1 cDNA. The brook trout PGS-2 encodes for a 607 amino acid protein that is 69% identical to mammalian PGS-2 and was observed in the skin, gills, stomach and heart. PGS-2 transcripts were highly upregulated in the ovaries by the phorbol ester, phorbol-12-myristate-13-acetate, in combination with the calcium ionophore, A23187. However, PGS-2 was not observed in the ovary of brook trout undergoing natural oocyte maturation and ovulation.