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.

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.

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.

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.

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.

The upregulation of messenger ribonucleic acids during 17alpha, 20beta-dihydroxy-4-pregnen-3-one-induced ovulation in the perch ovary.

While progestins appear to be involved in the local ovarian regulation of vertebrate ovulation, their specific role is unclear. In yellow perch (Perca flavescens) the progestin, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20-P), stimulates ovulation in vitro and this induction requires gene activation. Therefore, the perch model was used to isolate progestin-upregulated mRNAs. Perch ovaries were incubated for 32 h with or without 17,20-P (0.1 microg/ml). Messenger ribonucleic acids were isolated from the tissue and used for differential display PCR (DDPCR). From DDPCR, 5 bands were eventually obtained that were verified by Northern analysis to be consistently upregulated by 17,20-P at 32 h. Using these bands, full-length cDNAs were obtained by library screening and completely sequenced. Based on similarity to known sequences, four of the cDNAs presumably encode for perch forms of (1) neprilysin (PNEP-1; 63% identical); (2) a lysyl oxidase-type protein (PLO-2; 43.2% identical); (3) calmodulin (PCAL-1; 100% identical); and (4) a microtubule aggregate-like protein (PMAP-1; 29.6% identical). The fifth cDNA obtained from DDPCR most likely encodes for an egg protein and will be reported separately. Each of the cDNAs was used to probe Northern blots of ovarian mRNA taken at 0, 12, 24, 32 and 42 h of incubation with 17,20-P. This temporal Northern analysis verified that all four were upregulated by 32 h. In addition, PNEP and PMAP transcripts began to increase by 12 h, while PCAL and PLO transcripts remained elevated through 42 h. On Northern blots of RNA from other perch tissues, calmodulin was found in all tissues, PLO mRNA was ovarian specific, and PMAP mRNA was also present in the gills and liver. Multiple transcripts were observed for PNEP, but the ovarian form induced by 17,20-P was only found in high abundance in the heart. To our knowledge, this is the first report that specifically characterizes progestin upregulated mRNAs in the vertebrate ovary at ovulation.

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.

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.

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.

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.

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.

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.

Compartmentalization of prostaglandin synthesis within the fish ovary.

Numerous studies have shown that the follicle walls surrounding mature vertebrate oocytes are capable of producing primary prostaglandins. However, very few studies have looked at the prostaglandin synthetic potential of other ovarian tissue components. In brook trout (Salvelinus fontinalis) and goldfish (Carassius auratus), mature follicle walls can produce prostaglandins E and F (PGE and PGF, respectively); however, it is apparent that several other tissues within the fish ovary also produce specific primary PGs. Incubation of stroma or connective tissue from brook trout and goldfish ovaries with [14C]-arachidonic acid resulted in a very significant production of PGE2, whereas small immature oocytes of both species produced primarily PGF2 alpha. While the function of the primary prostaglandins produced by tissues external to the mature oocytes is unknown, it is important to recognize that multiple sites for prostaglandin synthesis are present within the fish ovary.

Involvement of protein kinase C in agonist-stimulated goldfish ovulation.

The effects of two protein kinase C (PKC) inhibitors, calphostin C and staurosporine, on the in vitro ovulation of goldfish (Carassius auratus) oocytes were investigated. Ovulation was stimulated by prostaglandin (PG) F2 alpha (PGF2 alpha, 2.0 micrograms/ml), by sodium orthovanadate (0.1 mM), by a combination of the phorbol ester phorbol 12-myristate-13-acetate (PMA, 0.1 micrograms/ml) and calcium ionophore A23187 (0.05 micrograms/ml), by thapsigargin (0.2 micrograms/ml), and by elevated pH (8.1). In addition, the effects of these inhibitors on the PKC activity of the goldfish follicle wall was determined by use of a specific peptide substrate phosphorylation assay. At 0.1 microM, staurosporine significantly blocked ovulation induced by all agents. However, at lower (0.01 microM) levels it blocked only PMA/A23187-induced ovulation. In contrast, calphostin significantly blocked only PMA/A23187-induced ovulation, although there was a decrease in pH-induced ovulation at lower calphostin concentrations. Both calphostin and staurosporine blocked follicular PKC activity at levels that were inhibitory to ovulation. In addition, staurosporine significantly blocked PKC activity at levels even lower than those needed to block ovulation. The combined results suggest that orthovanadate, PGF2 alpha, and thapsigargin do not require PKC activation for the induction of ovulation, whereas PMA/A23187 does.

Follicle and extrafollicular tissue interaction in 17alpha,20beta-dihydroxy-4-pregnen-3-one-stimulated ovulation and prostaglandin synthesis in the yellow perch (Perca flavescens) ovary.

Intact (attached to extrafollicular (EF) tissue) yellow perch follicles ovulate in vitro when stimulated with the steroid, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20-PG). However, follicles isolated from EF tissue will not ovulate under 17,20-PG stimulation. In the present study, the interaction of follicles and EF tissue in 17,20-PG-stimulated ovulation and prostaglandin synthesis was investigated by separating follicles from EF tissue at specific times during incubation. Following separation, the incubation of isolated follicles and EF tissue was continued. At 48 hr of incubation, isolated follicles were assayed for ovulation and the media of isolated follicle and EF tissue incubates were assayed by RIA for prostaglandin E (PGE) and prostaglandin F (PGF) levels. Steroid-stimulated follicles, isolated from EF tissue prior to 22 hr of incubation, ovulated less than 25%. However, there was a very large increase in the percentage ovulation in follicles separated from EF tissue from 24 to 32 hr. PGF levels increased significantly in 17,20-PG-stimulated follicles isolated from EF tissue after 22 hr. In contrast, mean PGE levels in steroid-stimulated follicle incubates appeared to be lower than control levels. Compared with controls, PGE and PGF levels were significantly lower in incubates of isolated EF tissue stimulated with 17,20-PG, and this did not appear to be related to the time of separation. The results demonstrate that follicles must interact with EF tissue for a specific length of time for ovulation and PGF synthesis to occur in isolated follicles. Further, the effects of 17,20-PG on prostaglandin synthesis appear to be specific for different tissue compartments.

The inhibitory effects of indomethacin, nordihydroguaiaretic acid, and pyrrolidinedithiocarbamate on ovulation and prostaglandin synthesis in yellow perch (Perca flavescens) follicle incubates.

This study specifically determined the correlation between ovulation and ovarian prostaglandin F (PGF) and prostaglandin E (PGE) levels in yellow perch follicles treated with several eicosanoid synthesis inhibitors. In incubates treated with indomethacin (IM; .00001 to 10.0 micrograms/ml) there was a significant dose-dependent correlation between ovulation and PGF/PGE levels, and between the two prostaglandins (PGs), further implicating PGs in the control of yellow perch ovulation. In incubates treated with nordihydroguaiaretic acid (NDGA), a traditional lipoxygenase inhibitor, there was a significant correlation between ovulation and PGF levels, yet there was no correlation between PGE levels and ovulation. A free-radical scavenger, pyrrolidinedithiocarbamate (PDTC) inhibited ovulation in a dose-dependent manner yet there was no correlation between either PGF or PGE levels and this inhibition, indicating that PDTC may block ovulation at other steps that involve free-radicals.

Cellular mechanisms for orthovanadate-, phorbol ester-, and calcium ionophore-stimulated prostaglandin production in brook trout (Salvelinus fontinalis) follicles.

Previous studies have shown that prostaglandin (PG) production in brook trout (Salvelinus fontinalis) follicles can be stimulated by sodium orthovanadate, phorbol-12 myristate 13-acetate ester (PMA), and the calcium ionophore, A23187. In the present study, the mechanisms by which these activators stimulate PG production in follicles were specifically investigated by examination of their effects on the release of 3H-labeled arachidonic acid (AA) from prelabeled follicles (AA release) and on the capacity to convert exogenous 3H-AA into prostanoids (AA conversion) by follicle walls. The release of AA from follicles was significantly stimulated by orthovanadate and A23187 after 4 h of incubation. Although AA release was not stimulated by PMA alone, levels of AA release in incubations with both PMA and A23187 were greater than in those with A23187 alone. In contrast, AA conversion in follicle walls was significantly increased by PMA and orthovanadate but not by A23187 alone. The results showed that 1) the calcium ionophore A23187 stimulated ovarian PG production mainly through its effects on the release of AA from follicles; 2) PMA enhanced follicular PG production through an increase in AA conversion in the follicle walls; and 3) orthovanadate mediated PG production by increasing both AA release and AA conversion. In addition, PMA-, A23187-, and orthovanadate-stimulated increases in AA release or conversion were significantly reduced by the translational inhibitor, cycloheximide, and the transcriptional inhibitor, actinomycin. These results indicate that PMA-, A23187-, and orthovanadate-stimulated AA release and AA conversion require protein synthesis and pretranslational activation in the follicles.(ABSTRACT TRUNCATED AT 250 WORDS)