An Emerging Cross-Species Marker for Organismal Health: Tryptophan-Kynurenine Pathway.

Tryptophan (TRP) is an essential dietary amino acid that, unless otherwise committed to protein synthesis, undergoes metabolism via the Tryptophan-Kynurenine (TRP-KYN) pathway in vertebrate organisms. TRP and its metabolites have key roles in diverse physiological processes including cell growth and maintenance, immunity, disease states and the coordination of adaptive responses to environmental and dietary cues. Changes in TRP metabolism can alter the availability of TRP for protein and serotonin biosynthesis as well as alter levels of the immune-active KYN pathway metabolites. There is now considerable evidence which has shown that the TRP-KYN pathway can be influenced by various stressors including glucocorticoids (marker of chronic stress), infection, inflammation and oxidative stress, and environmental toxicants. While there is little known regarding the role of TRP metabolism following exposure to environmental contaminants, there is evidence of linkages between chemically induced metabolic perturbations and altered TRP enzymes and KYN metabolites. Moreover, the TRP-KYN pathway is conserved across vertebrate species and can be influenced by exposure to xenobiotics, therefore, understanding how this pathway is regulated may have broader implications for environmental and wildlife toxicology. The goal of this narrative review is to (1) identify key pathways affecting Trp-Kyn metabolism in vertebrates and (2) highlight consequences of altered tryptophan metabolism in mammals, birds, amphibians, and fish. We discuss current literature available across species, highlight gaps in the current state of knowledge, and further postulate that the kynurenine to tryptophan ratio can be used as a novel biomarker for assessing organismal and, more broadly, ecosystem health.

Kynurenine to tryptophan ratio as a biomarker of acute stress in fish.

The aim of this study was to determine the kynurenine (KYN) to tryptophan (TRP) ratio (KTR) in fish tissue to assess its usefulness as a biomarker of acute stress. Laboratory held rainbow trout (Oncorhynchus mykiss) were subjected to an acute stressor and KYN, TRP and cortisol were measured in liver and brain tissues at 4- and 48-h post-stress. The analytical method used to determine our analytes was based on lyophilization, and liquid-solid extraction followed by isotope dilution high-performance liquid chromatography positive ion electrospray tandem mass spectrometry. The [KYN]/[TRP] ratio (KTR) was greater in fish liver and brain in the 48-h post-stress exposure group (n = 8) relative to controls (n = 8, p < 0.05); a similar increase was not observed in fish in the 4-h post-stress exposure group. Hepatic and brain cortisol levels were also elevated in fish from both stress-induced groups relative to their respective controls implying that cortisol responded more quickly to the stressful stimulus than KYN and TRP. Our results suggest that the KTR is a promising acute stress diagnostic biomarker in fish. Efforts are ongoing to assess whether the KTR can be used as a biomarker for chronic stress in fish exposed to aquatic contaminants and other environmental stressors and if similar assessments can be made on tissues collected via non-lethal approaches.

Absorption and elimination of per and poly-fluoroalkyl substances substitutes in salmonid species after pre-fertilization exposure.

Due to their relatively large production and few restrictions on uses, novel substitutes for historically used per and poly-fluoroalkyl substances (PFAS) are being used and accumulating in the environment. However, due to a lack of information on their toxicological properties their hazards and risks are hard to estimate. Before fertilization, oocytes of two salmonid species, Arctic Char (Salvelinus alpinus) and Rainbow Trout (Oncorhynchus mykiss), were exposed to three PFAS substances used as substitutes for traditional PFAS, PFBA, PFBS or GenX or two archetypical, historically used, longer-chain PFAS, PFOA and PFOS. Exposed oocytes were subsequently fertilized, incubated and were sampled during several developmental stages, until swim-up. All five PFAS were accumulated into egg yolks with similar absorption rates, and their concentrations in egg yolks were less than respective concentrations in/on egg chorions. Rapid elimination of the five PFAS was observed during the first 3 days after fertilization. Thereafter, amounts of PFOS and PFOA were stable until swim-up, while PFBA, PFBS and GenX were further eliminated during development from one month after the fertilization to swim-up. In these two salmonid species, PFBA, PFBS and GenX were eliminated faster than were PFOS or PFOA.

Remodeling of Arctic char (Salvelinus alpinus) lipidome under a stimulated scenario of Arctic warming.

Arctic warming associated with global climate change poses a significant threat to populations of wildlife in the Arctic. Since lipids play a vital role in adaptation of organisms to variations in temperature, high-resolution mass-spectrometry-based lipidomics can provide insights into adaptive responses of organisms to a warmer environment in the Arctic and help to illustrate potential novel roles of lipids in the process of thermal adaption. In this study, we studied an ecologically and economically important species-Arctic char (Salvelinus alpinus)-with a detailed multi-tissue analysis of the lipidome in response to chronic shifts in temperature using a validated lipidomics workflow. In addition, dynamic alterations in the hepatic lipidome during the time course of shifts in temperature were also characterized. Our results showed that early life stages of Arctic char were more susceptible to variations in temperature. One-year-old Arctic char responded to chronic increases in temperature with coordinated regulation of lipids, including headgroup-specific remodeling of acyl chains in glycerophospholipids (GP) and extensive alterations in composition of lipids in membranes, such as less lyso-GPs, and more ether-GPs and sphingomyelin. Glycerolipids (e.g., triacylglycerol, TG) also participated in adaptive responses of the lipidome of Arctic char. Eight-week-old Arctic char exhibited rapid adaptive alterations of the hepatic lipidome to stepwise decreases in temperature while showing blunted responses to gradual increases in temperature, implying an inability to adapt rapidly to warmer environments. Three common phosphatidylethanolamines (PEs) (PE 36:6|PE 16:1_20:5, PE 38:7|PE 16:1_22:6, and PE 40:7|PE 18:1_22:6) were finally identified as candidate lipid biomarkers for temperature shifts via machine learning approach. Overall, this work provides additional information to a better understanding of underlying regulatory mechanisms of the lipidome of Arctic organisms in the face of near-future warming.

Triiodothyronine reduces toxic effects of diazinon in Persian sturgeon (Acipenser persicus) embryos.

Thyroid hormones (THs) play an important role in early stages development of fish species. Manual elevation of THs in the embryos improves viability and hatching success. However, the impacts of endocrine disrupting chemicals on THs-treated embryos are unclear. This study investigated the effect of triiodothyronine (T3) to mitigate toxic effects of diazinon in the endangered Persian sturgeon (Acipenser persicus) eggs and embryos. Fertilized eggs were exposed to nominal concentrations of 0, 2, 4, 6, and 8 mg/L diazinon and the 96 h LC50 value was calculated at 3.5 mg/L. Eggs were then treated with exogenous T3 (1 ng/mL: LT3, and 10 ng/mL: HT3) and exposed to 3.5 mg/L diazinon (DLT3 and DHT3). Total THs concentrations, levels of cortisol, and expression of the igf-II gene were measured during embryogenesis. All the measured endpoints were significantly different between treatments or stages of incubation. Generally, despite insignificance in some cases, higher levels of T3 and Thyroxin (T4) were observed in T3-treated embryos regardless of the presence of diazinon. Cortisol was high in unfertilized eggs which reduced after fertilization. The igf-II gene up-regulated quickly after fertilization; was higher in T3-treated embryos. Exposure of eggs to diazinon reduced the levels of T3, T4, and igf-II gene expression, which corresponded to the lowest hatching. We concluded that exogenous T3 improves embryos development in A. persicus, which is a promising application for conservation strategies. Our study suggests that treating embryos with 10 ng/L T3 is a suitable way to overcome problems of incubation in diazinon-polluted water sources.

Effects of Chronic Dietary Selenomethionine Exposure on the Visual System of Adult and F1 Generation Zebrafish (Danio rerio).

The effects of chronic dietary selenomethionine (SeMet) exposure on the visual system of adult zebrafish and their progeny were investigated. Adult zebrafish were exposed to measured concentrations of 1.1 (control) and 10.3 µg Se/g dry mass as SeMet for 57 days, then encouraged to breed. Progeny were reared to swim-up and differences in mortality, eye size and visual behaviour were determined. Adult vision was also investigated using behavioural assays. Adults fed the SeMet-spiked diet exhibited significantly fewer positive reactions in the escape response assay when compared to controls. Larvae from adults fed elevated SeMet had smaller eyes and a lower proportion of positive responses in phototaxis, oculomotor and optokinetic response assays compared to controls. These results demonstrate that environmentally relevant elevated dietary SeMet exposure can affect the visual system of both exposed adult zebrafish and their progeny, which could affect fitness and survivability.

Interactive effects of chronic waterborne copper and cadmium exposure on tissue-specific metal accumulation and reproduction in fathead minnow (Pimephales promelas).

The present study was carried out to examine the interactive effects of chronic waterborne copper (Cu) and cadmium (Cd) on tissue-specific metal accumulation and reproduction in fathead minnow (Pimephales promelas). Trios (1 male: 2 female; n=5) of fish were exposed for 21days to: (i) control (no added Cu or Cd), (ii) waterborne Cu (75µg/L), (iii) waterborne Cd (5µg/L), and (iv) Cu and Cd mixture (75 and 5µg/L, respectively). Reproductive output (cumulative egg production) was significantly reduced by Cu but not by Cd. Interestingly however, no spawning occurred in fish exposed to the mixture of waterborne Cu and Cd. In general, both Cu and Cd accumulation in target tissues (gill, liver, gonad and carcass) increased significantly in fish exposed to Cu and Cd mixture, and no interaction between Cu and Cd accumulation was observed in any tissues, except in the liver where Cu accumulation was significantly reduced by Cd. The expression of female hepatic estrogen receptor genes (ER-α and ER-ß) was most significantly elevated in fish exposed to Cu and Cd mixture, whereas vitellogenin gene expression was reduced maximally in the same exposure. Similarly, the hepatic expression of the metallothionein gene was most significantly upregulated in fish exposed to Cu and Cd mixture. Moreover, the circulating estradiol level in females was significantly decreased only during the co-exposure of waterborne Cu and Cd. Overall, the present study indicates that the interaction of chronic waterborne Cu and Cd exposure may elicit greater than additive effect on reproductive output in fish.

Cross-species comparison of relative potencies and relative sensitivities of fishes to dibenzo-p-dioxins, dibenzofurans, and polychlorinated biphenyls in vitro.

Dioxin-like compounds of varying toxicities are found in complex mixtures. The toxic equivalency factor (TEF) approach was developed based on the potency of a dioxin-like compound relative to the potency of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) to streamline risk assessment. One limitation of the TEF approach is uncertainty regarding differences in the relative potency of dioxin-like compounds among different species. Relative potencies among fishes are limited, relative to relative potencies among birds and mammals, and TEFs for fishes are based entirely on the model species, rainbow trout (Oncorhynchus mykiss). An in vitro liver explant assay was used to characterize species-specific responses with regard to up-regulation of CYP1A transcript after exposure to 6 dioxin-like compounds in rainbow trout, white sturgeon (Acipenser transmontanus), lake sturgeon (Acipenser fulvescens), and northern pike (Esox lucius). Differences in sensitivities were observed among species after exposure to dioxin-like compounds. The relative potencies developed from liver explants of rainbow trout were comparable to relative potencies developed from embryo toxicity assays. Differences in relative potencies between species with the least and greatest relative potencies were up to 40-fold. To compare relative potencies among species, concentrations of dioxin-like compounds in fish eggs in the Fraser River and in Lake Ontario were used to calculate toxic equivalency quotients (TEQs) determined from TEFs or TCDD equivalents determined from relative potencies. The TEQs underestimated TCDD equivalents for white sturgeon, lake sturgeon, and northern pike, indicating uncertainty in application of TEFs to diverse fishes.

Mechanisms of toxicity of triphenyltin chloride (TPTC) determined by a live cell reporter array.

Triphenyltin chloride (TPTC), which has been extensively used in industry and agriculture, can occur at concentrations in the environment sufficient to be toxic. Here, potency of TPTC to modulate genes in a library containing 1,820 modified green fluorescent protein (GFP)-expressing promoter reporter vectors constructed from Escherichia coli K12 strains was determined. Exposure to TPTC resulted in 22 (fold change > 2) or 71 (fold change > 1.5) differentially expressed genes. The no observed transcriptional effect (NOTEC) and median transcriptional effect concentrations (TEC50) were determined to be 0.036 and 0.45 mg/L in E. coli. These responses were 1,230 and 97 times more sensitive than the acute median effect concentration (EC50) required to inhibit growth of cells, which demonstrated that this live cell array represents a sensitive method to assess toxic potency of chemicals. The 71 differentially expressed genes could be classified into seven functional groups. Of all the altered genes, three groups which encoded for catalytic enzymes, regulatory proteins, and structural proteins accounted for 28 %, 18 %, and 14 % of all altered genes, respectively. The pattern of differential expression observed during this study was used to elucidate the mechanism of toxicity of TPTC. To determine potential relationships among genes that were changed greater than 2.0-fold by exposure to TPTC, a correlation network analysis was constructed, and four genes were related to aroH, which is the primary target for metabolic regulation of aromatic biosynthesis by feedback inhibition in bacteria. The genes rnC, cld, and glgS were selected as potential biomarkers for TPTC, since their expression was more than 2.0-fold greater after exposure to TPTC.

Attenuation of the cortisol response to stress in female rainbow trout chronically exposed to dietary selenomethionine.

Selenomethionine (Se-Met) is the major dietary form of selenium (Se). While Se is a required nutrient, it can also influence the physiological stress response because it stimulates greater concentrations of cortisol in blood plasma of exposed fish. However, little is known about the effects of exposure to Se on the ability to cope with a secondary stressor. In the current study, female rainbow trout were exposed to an environmentally relevant dietary concentration (8.47 mg Se/kg dry mass (dm)) of Se-Met for 126 d, after which time fish were subjected to a 3-min handling stressor and sampled at 2h and 24h post-stressor exposure. Concentrations of cortisol, cortisone, glucose, and lactate in blood plasma and concentrations of glycogen and triglycerides in liver and muscle were determined. Abundances of transcripts of proteins involved in corticosteroidogenesis were determined using quantitative RT-PCR. Concentrations of cortisol were significantly greater in blood plasma of trout exposed to Se-Met, relative to control trout sampled prior to the handling stressor. A typical response of cortisol to the handling stressor was observed in the control trout. However, trout exposed to Se-Met were unable to mount a cortisol response to the handling stressor. Concentrations of cortisone, the inactive metabolite of cortisol, were significantly greater following the handling stressor in trout exposed to Se-Met. In trout exposed to Se-Met, transcript abundance of melanocortin 2 receptor (mc2r) and peripheral benzodiazepine receptor (pbr) were greater, which is consistent with the conclusion that synthesis of cortisol was greater. However, abundances of transcripts of cytochrome P450 side-chain cleavage (p450scc) and cytochrome P450 11B1 (cyp11b1) were not significantly different between controls and Se-Met exposed trout. Exposure to Se-Met affected accumulation and tissue partitioning of glycogen and triglycerides in liver and muscle as concentrations of these energy reserves were greater in muscle, but not liver. Concentrations of glycogen and triglycerides in muscle, but not in liver, were lesser following the handling stressor suggesting that the muscle energy reserves are an important source of energy required for recovery from the handling stressor. The results of the study demonstrate that chronic exposure to dietary Se-Met elicits a stress response, but prevents a cortisol response to a secondary handling stressor, most likely due to cortisol inactivation. Moreover, exposure to Se-Met has effects on concentrations of energy reserves that are important for providing the energy necessary to cope with a secondary stressor.

Chronic exposure to dietary selenomethionine increases gonadal steroidogenesis in female rainbow trout.

Selenomethionine (Se-Met) is the major dietary form of selenium (Se). Detrimental effects have been associated with exposure to elevated dietary selenium. Previous studies have demonstrated effects of Se on the endocrine system, in particular effects on cortisol and thyroid hormones. However, no information is available regarding effects of Se on sex steroid hormones. In the present study, effects of dietary exposure to an environmentally relevant concentration (4.54 mg/kg wet weight (ww)) of Se-Met for 126 days on concentrations of sex steroid hormones in blood plasma of female rainbow trout were determined. Furthermore, the molecular basis for effects of Se-Met on plasma sex steroid hormone concentrations was investigated. Concentrations of androstenedione (A), estrone (E1), and estradiol (E2) were 39.5-, 3.8-, and 12.7-fold greater in plasma of treated females than the untreated controls, respectively. Testosterone (T) was detected only in plasma of treated females. The greater E2 concentration stimulated greater transcript abundance of vitellogenin (vtg) and zona-radiata protein (zrp). Female rainbow trout exposed to Se-Met had greater transcript abundance of key steroidogenic proteins and enzymes, including peripheral benzodiazepine receptor (pbr), cytochrome P450 side-chain cleavage (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-hsd). Exposure to Se-Met did not affect transcript abundance of luteinizing hormone (lh) or follicle stimulating hormone (fsh). Similarly, there was no change in transcript abundance of luteinizing hormone receptor (lhr) or follicle stimulating hormone receptor (fshr). Long-term exposure to dietary Se-Met has the potential to stimulate vitellogenesis in female rainbow trout by directly stimulating ovarian tissue steroidogenesis. This is the first study to report effects of Se on sex steroid hormone production in fish.

In ovo thyroxine exposure alters later UVS cone loss in juvenile rainbow trout.

Thyroid hormones (THs) play a vital role in vertebrate neural development, and, together with the beta isoform of the thyroid hormone receptor (TRß), the development and differentiation of cone photoreceptors in the vertebrate retina. Rainbow trout undergo a natural process of cone cell degeneration during development and this change in photoreceptor distribution is regulated by thyroxine (T4; a thyroid hormone). In an effort to further understand the role of T4 in photoreceptor ontogeny and later developmental changes in photoreceptor subtype distribution, the influence of enhanced in ovo T4 content on the onset of opsin expression and cone development was examined. Juvenile trout reared from the initial in ovo-treated embryos were challenged with exogenous T4 at the parr stage of development to determine if altered embryonic exposure to yolk THs would affect later T4-induced short-wavelength-sensitive (SWS1) opsin transcript downregulation and ultraviolet-sensitive (UVS) cone loss. In ovo TH manipulation led to upregulation of both SWS1 and long-wavelength-sensitive (LWS) opsin transcripts in the pre-hatch rainbow trout retina and to changes in the relative expression of TRß. After 7 days of exposure to T4, juveniles that were also treated with T4 in ovo had greatly reduced SWS1 expression levels and premature loss of UVS cones relative to T4-treated juveniles raised from untreated eggs. These results suggest that changes in egg TH levels can have significant consequences much later in development, particularly in the retina.

Isolation and characterization of a mRNA encoding a novel insulin receptor (IR) subtype, IR2, from rainbow trout (Oncorhynchus mykiss) and patterns of expression of the four IR subtypes, IR1-IR4, in tissues and during embryonic development.

Insulin (INS) plays a critical role in the growth, development, and metabolism of vertebrates. In this study, a cDNA encoding a novel insulin receptor (IR) subtype was isolated, cloned, and sequenced from the liver of rainbow trout. A 1525-bp cDNA encoding a partial amino acid sequence of the ß-subunit including the transmembrane domain, the tyrosine kinase domain, and the 3' untranslated region (UTR) was obtained and designated IR2 based on comparison with known IR subtypes, including the three previously reported IR subtypes of trout. Trout IR2 shares 90.0%, 82.8%, and 84.3% nucleotide identity with previously characterized trout IR1, IR3 and IR4, respectively. Quantitative real-time PCR revealed that the four IR mRNAs were differentially expressed, both in terms of distribution among tissues as well as in terms of abundance within selected tissues of juvenile trout. IR1 mRNA was most abundant in spleen, liver, kidney, and muscle (white, red and cardiac), but least abundant in adipose. IR3 mRNA was most abundant in liver, spleen, kidney, and pancreas; in other tissues, levels of IR3 mRNA were uniformly abundant. By contrast, levels of IR2 and IR4 mRNA were uniformly abundant in most tissues, except in spleen where levels of IR4 were significantly lower. All IR subtypes were detected over the course of embryonic development. In head and tail regions, levels of IR2 and IR3 mRNA declined from pre-hatch (29 days post-fertilization, dpf) to post-hatch (68-90 dpf), whereas levels of IR1 and IR4 remained relatively unchanged. These findings contribute to our understanding of the evolution, distribution, and function of insulin receptors.

Differential expression of preprosomatostatin- and somatostatin receptor-encoding mRNAs in association with the growth hormone-insulin-like growth factor system during embryonic development of rainbow trout (Oncorhynchus mykiss).

Rainbow trout were used to evaluate the relationship between the somatostatin (SS) signaling and the growth hormone (GH)-insulin-like growth factor (IGF) systems during pre-hatch and post-hatch embryonic development. The expression of preprosomatostatins (PPSS), SS receptors (SSTR), GH receptors (GHR), IGF-1, IGF-2, and IGF type 1 receptors (IGFR1) was examined in various regions at the eyed-egg (29 days post-fertilization, dpf;), post-hatch (53dpf), swim-up (68dpf), and complete yolk-absorbed (90dpf) stages. In head, PPSSI mRNA abundance increased during development while that of PPSSII' decreased and that of PPSSII'' remained unchanged. In body and tail, mRNA abundance of all PPSSs remained unchanged except that of PPSSII'' which declined in the tail. SSTR expression increased as development progressed in all regions with the exception of SSTR1A mRNA which remained unchanged. mRNA levels of GHR1 declined in all regions of post-hatch embryos, whereas those of GHR2 remained unchanged. Expression of IGF-1 and IGF-2 in head and tail regions increased immediately after hatching, and then declined, whereas the expression of neither IGF changed during development in the body. The expression of IGFR1 mRNAs declined in all regions, reaching their lowest levels at 90dpf, with the exception of IGFR1A mRNA in the body which remained unchanged. The general decline in the expression of GH-IGF system components during development appears inversely related to a general increase in the expression of SS system elements, and suggests that these two systems interact to regulate the tissue expansion and tissue regression of embryogenesis.

Rainbow trout (Oncorhynchus mykiss) possess two insulin-encoding mRNAs that are differentially expressed.

Insulin (INS) plays a critical role in the growth, development, and metabolism of vertebrates. In this study, two unique cDNAs that encode preproinsulin were isolated, cloned and sequenced from the endocrine pancreas (Brockmann body) of rainbow trout. One 592-bp cDNA (INS 1) encodes a 105-amino acid protein and the other 587-bp cDNA (INS 2) encodes a 107-amino acid protein. The sequences share 93% nucleotide identity and 91.4% deduced amino acid identity. Quantitative real-time PCR revealed that the two INS-encoding mRNAs were differentially expressed, both in terms of distribution among tissues as well as in terms of abundance within selected tissues of juvenile trout. Both INS 1 and INS 2 mRNAs were detected in pancreas, adipose tissue, pyloric cecum, and brain; however, only INS 1 mRNA was detected in upper and lower intestine and pituitary. In all cases where INS 1 and INS 2 were co-expressed, INS 1 was more abundant. INS 1 and INS 2 also were differentially expressed in various body regions (head, body, and tail) during embryonic development. Both INS 1 and INS 2 mRNAs were detected early in development (29 days post-fertilization), but their expression declined as development proceeded (through 90 days post-fertilization); in most cases, unlike the situation in juveniles, INS 2 mRNA was more abundant than INS 1 mRNA in embryos. These findings contribute to our understanding of the evolution, distribution, and function of INS.

Expression profiles of growth-related genes during the very early development of rainbow trout embryos reared at two incubation temperatures.

Quantitative RT-PCR was used to determine the profiles of expression of 10 growth- or development-related genes in rainbow trout (Oncorhynchus mykiss) embryos prior to the formation of the somatotropic (ST) axis (pituitary somatotrops and liver); embryos were sampled immediately after fertilization and water-hardening (t(0)), 1-h post-fertilization, and 1-, 2-, 5-, 7-, 10- and 13-days post-fertilization (dpf); expression profiles were examined in embryos reared at two temperatures (6.0 and 8.5 degrees C), which had different developmental rates. Accumulation of mRNA encoding for GH1, GH2, IGF-1, IGF-2, two isoforms of GH receptors (GHR1, GHR2), two isoforms of IGF receptors (IGF-RIa, IGF-RIb) and two isoforms of thyroid receptor (TR), TRalpha and TRbeta, was measured. All of these genes were expressed in the t(0) samples, but the rates of expression of the different genes varied markedly. For most of the genes examined, the expression rates tended to fall within the first hour after fertilization, and remained at the lower level for between 2 and 7 days, after which there was a significant (P <0.05) and progressive increase in the number of accumulated copies of mRNA. This increase is probably associated with the commencement of embryonic genome transcription activity (EGTA), and it was generally, although not always, found later in embryos that were reared at 6.0 degrees C compared with the faster developing embryos reared at 8.5 degrees C. The study suggests that the EGTA begins between 2- and 5-dpf, with a staged increase in EGTA between 5- and 13-dpf.

Growth hormone and insulin-like growth factor gene expression prior to the development of the pituitary gland in rainbow trout (Oncorhynchus mykiss) embryos reared at two temperatures.

Real time RT-PCR was used to measure the changes in the rates of synthesis of mRNA encoding for growth hormone-1 (GH1) and -2 (GH2) and insulin-like growth factor-1 (IGF-1) and -2 (IGF-2), and whole embryo GH content was measured in early stage rainbow trout (Oncorhynchus mykiss) embryos reared at two incubation temperatures (8.5 and 6.0 degrees C). Particular attention was paid to the phase of embryo development that preceded the appearance of the pituitary gland. GH was present in zygotes, and there were no significant changes in whole embryo GH content of the two temperature treatment groups from fertilization (t0) until the time at which GH was detectable in the pituitary gland by immunostaining. The expression of the two GH genes decreased during the first 24 h post-fertilization, and then increased significantly by 17 dpf in embryos reared at both temperatures. There was a subsequent steep increase in the number of copies of GH1 and GH2 mRNA associated with the formation of the pituitary gland evident at 23 and 34 dpf in the 8.5 and 6.0 degrees C groups, respectively. The number of copies of mRNA encoding for IGF-1 and IGF-2 did not change during the first 24 h post-fertilization; however, there was a significant increase in the numbers of transcripts for both genes evident by 13 dpf in embryos reared at the two incubation temperatures. The differences in the timing of the increases in GH and IGF mRNA may suggest that IGF gene expression is not GH-dependent at that stage. Moreover, the increased expression of the GH genes prior to the formation of the pituitary gland suggests that tissues other than the pituitary are expressing these genes in early embryos. The pattern of changes in GH content was similar to the pattern of GH gene expression in embryos reared at the two incubation temperatures when the age of embryos was plotted using degree-days. There were no apparent compensatory responses in GH1, GH2, IGF-1 or IGF-2 gene expression related to altered growth rates. The number of copies of IGF-2 mRNA was higher than that of IGF-1 mRNA during the early developmental period; this is consistent with the hypothesis that IGF-2 predominates during embryonic development. A differential expression of GH2 and GH1 was also observed with the overall copy numbers of GH2 mRNA being consistently higher than those of GH1.

Trafficking of L-triiodothyronine between ovarian fluid and oocytes of rainbow trout (Oncorhynchus mykiss).

The study examines the dynamics of thyroid hormone (TH) trafficking between rainbow trout (Oncorhynchus mykiss) oocytes and ovarian fluid (OF) to explore the processes involved in the transfer of hormone to the oocytes. We also examined the effects of enhancing oocyte T(3) content and subsequent embryo survival. Oocytes incubated in OF alone had significant losses of THs within 12 h, whereas the T(3) content of oocytes retained in T(3)-enriched OF (10 and 100 microg ml(-1)) was significantly elevated in a dose-dependant manner within 3 h. When transferred to non-supplemented OF, the T(3) content of the 10 micro ml(-1) treatment group decreased significantly within 24 h with a concomitant significant increase in OF T(3) concentration. Although there was no significant change in the 100 microg ml(-1) treatment group the significant increase in the OF T(3) concentration was evidence for marked T(3) efflux during this period. These findings provide evidence for the independent trafficking of T(3) based on concentration gradients across the oocyte cell membrane, and suggest that it is not vitellogenin-dependent. Fertilization of in ovo T(3)-supplemented oocytes resulted in a small, albeit significant, increase in mortality rate, but there was no significant effect of treatment on embryo growth rates up to the hatching stage of development.

Suppressor of cytokine signaling 1 regulates an endogenous inhibitor of a mast cell protease.

Suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of c-Kit and interleukin-3 (IL-3) receptor signaling. We examined the role of SOCS1 in regulating IL-3-induced cell growth of primary bone marrow-derived mast cells (BMMCs) from SOCS1-/- mice. Instead of showing increased proliferation, SOCS1-deficient BMMCs responded poorly to IL-3 and stem cell factor. SOCS1-/- BMMCs showed increased apoptosis and defective cell cycle entry. We show that the growth retardation of SOCS1-/- BMMCs was due to a cell intrinsic defect. Protein tyrosine phosphorylation following IL-3 stimulation was markedly diminished in SOCS1-/- BMMCs. Intriguingly, JAK2 and STAT5 proteins were selectively diminished in SOCS1-/- BMMCs, which also showed lower molecular mass products of p85 and Vav suggesting proteolytic degradation. Incubation of the SOCS1-/- BMMC lysate with STAT5, p85, and Vav immunoprecipitated from SOCS1+/+ cells directly demonstrated the dysregulated proteolytic activity in SOCS1-/- BMMCs. The proteolytic activity in SOCS1-/- BMMCs was selectively inhibited by phenylmethylsulfonyl fluoride and soybean trypsin inhibitor, suggesting that the protease regulated by SOCS1 is a tryptase. The dysregulated tryptase in SOCS1-/- BMMCs is unlikely to be mMCP6 or mMCP7, because the enzyme activity was not inhibited by Polybrene but was inhibited by normal mouse plasma. SOCS1+/+ BMMC lysate inhibited the proteolytic activity present in SOCS1-/- BMMC lysate, indicating that SOCS1-/- BMMCs lack an endogenous protease inhibitor. These results show that SOCS1 is required for the expression and/or stability of an endogenous protease inhibitor, which protects mast cells from their own proteolytic enzymes.

A positive regulatory role for suppressor of cytokine signaling 1 in IFN-gamma-induced MHC class II expression in fibroblasts.

Suppressor of cytokine signaling 1 (SOCS1) is rapidly induced following stimulation by several cytokines. SOCS1 negatively regulates cytokine receptor signal transduction by inhibiting Janus family tyrosine kinases. Lack of such feedback regulation underlies the premature death of SOCS1(-/-) mice due to unbridled IFN-gamma signaling. We used mouse embryo fibroblasts derived from SOCS1(-/-) mice to investigate the role of SOCS1 in IFN-gamma signaling pathways. SOCS1(-/-) fibroblasts were exquisitely sensitive to the IFN-gamma-mediated growth arrest and showed sustained STAT1 phosphorylation. However, SOCS1(-/-) fibroblasts were inefficient in MHC class II surface expression following IFN-gamma stimulation, despite a marked induction of the MHC class II transactivator and MHC class II gene expression. Retroviral transduction of wild-type SOCS1 relieved the growth-inhibitory effects of IFN-gamma in SOCS1(-/-) fibroblasts by inhibiting STAT1 activation. SOCS1R105K, carrying a mutation within the phosphotyrosine-binding pocket of the Src homology 2 domain, did not inhibit STAT1 phosphorylation, yet considerably inhibited IFN-gamma-mediated growth arrest. Strikingly, expression of SOCS1R105K restored the IFN-gamma-induced MHC class II expression in SOCS1(-/-) cells, indicating that expression of SOCS1 facilitates MHC class II expression in fibroblasts. Our results show that SOCS1, in addition to its negative regulatory role of inhibiting Janus kinases, has an unanticipated positive regulatory function in retarding the degradation of IFN-gamma-induced MHC class II proteins in fibroblasts.