Feeding after spawning and energy balance at spawning are associated with repeat spawning interval in steelhead trout.

Consecutive and skip repeat spawning (1- or ≥2-year spawning interval) life histories commonly occur in seasonally breeding iteroparous fishes. Spawning interval variation is driven by energetic status and impacts fisheries management. In salmonids, energetic status (either absolute level of energy reserves or the rate of change of energy reserves, i.e., energy balance) is thought to determine reproductive trajectory during a critical period ∼1 year prior to initial spawning. However, information on repeat spawners is lacking. To examine the timing and the aspects of energetic status that regulate repeat spawning interval, female steelhead trout (Oncorhynchus mykiss) were fasted for 10 weeks after spawning and then fed ad libitum and compared to ad libitum fed controls. Plasma growth hormone (GH) and insulin-like growth factor-I (IGF-I) levels were measured to assess long-term energy balance. Plasma estradiol levels showed that some fish in both groups initiated a consecutive spawning cycle. In fasted fish, GH was lower at spawning in consecutive versus skip spawners. In consecutive spawners, GH was higher at spawning in fed versus fasted fish. These results suggest that fish with a less negative energy balance at spawning initiated reproductive development in the absence of feeding, but that feeding during the post-spawning period enabled initiation of reproduction in some fish with a more negative energy balance at spawning. Thus, both energy balance at spawning and feeding after spawning regulated reproductive schedules. These results show that the critical period model of salmonid maturation applies to regulation of repeat spawning, and that the reproductive decision window extends into the first 10 weeks after spawning.

Effects of post-spawning ration restriction on reproductive development and the growth hormone/insulin-like growth factor-1 axis in female rainbow trout (Oncorhynchus mykiss).

In iteroparous female salmonids, the growth and reproductive endocrine axes interact during the period after spawning. Energy depletion due to pre-spawn fasting, migration, and ovarian development must be restored, and the next reproductive cycle is initiated in consecutively maturing fish. In the natural environment, food availability is often limited during the post-spawn period. To investigate the growth and reproductive endocrinology of the post-spawn period, we sampled female rainbow trout over the 30 weeks following their first spawning. Fish were fasted for 2 months prior to spawning, then fed a standard or a restricted ration. Analysis was confined to reproductive fish. Plasma estradiol-17ß decreased during the 8 weeks following spawning and then began increasing in both ration groups and was lower in feed-restricted versus standard ration fish from 8 weeks onward. Plasma insulin-like growth factor-1 increased over the same period and then remained constant in both ration groups and was lower in feed-restricted versus standard ration fish from week 8 to week 30. Plasma growth hormone decreased following spawning in standard ration fish and became elevated in feed-restricted versus standard ration fish at 20- and 30-weeks post-spawn. Growth rates, condition factor, and muscle lipid levels were higher in standard ration versus feed-restricted fish within 2-4 weeks after spawning. These results suggest that two phases occurred during the post-spawn period: recovery from spawning and restoration of energy reserves over weeks 0 to 8, followed by adjustment of the growth and reproductive endocrine axes to ration level over weeks 8 to 30.

Establishment of time-resolved fluoroimmunoassays for detection of growth hormone and insulin-like growth factor I in rainbow trout plasma.

The GH/IGF-I axis influences many aspects of salmonid life history and is involved in a variety of physiological processes that are related to somatic growth (e.g., reproduction, smoltification, and the response to fasting and stress). As such, fisheries studies utilize GH/IGF-I axis components as indicators of growth and metabolic status. This study established time-resolved fluoroimmunoassays (TR-FIAs) for rainbow trout plasma GH and IGF-I using commercially available reagents. For the GH TR-FIA, the ED80 and ED20 were 0.6 and 28.1 ng/mL, the minimum detection limit was 0.2 ng/mL, and the intra- and inter-assay coefficients of variation (%CV) were 4.1% and 13.4%, respectively. Ethanol remaining from acid-ethanol cryoprecipitation (AEC) of plasma samples to remove IGF binding proteins reduced binding and increased variability in the IGF-I TR-FIA. Drying down and reconstituting extracted samples restored binding and reduced variability. The extraction efficiency of IGF-I standards through AEC, drying down, and reconstitution did not vary over the working range of the assay. For the IGF-I TR-FIA, the ED80 and ED20 were 0.2 and 6.5 ng/mL, the minimum detection limit was 0.03 ng/mL, and the intra- and inter-assay %CV were 3.0% and 6.5%, respectively. Biological validation was provided by GH injection and fasting studies in rainbow trout. Intraperitoneal injection with bovine GH increased plasma IGF-I levels. Four weeks of fasting decreased body weight, increased plasma GH levels, and decreased plasma IGF-I levels. The GH and IGF-I TR-FIAs established herein provide a cost-comparable, non-radioisotopic method for quantifying salmonid plasma GH and IGF-I using commercially available reagents.

A Computational Model of the Rainbow Trout Hypothalamus-Pituitary-Ovary-Liver Axis.

Reproduction in fishes and other vertebrates represents the timely coordination of many endocrine factors that culminate in the production of mature, viable gametes. In recent years there has been rapid growth in understanding fish reproductive biology, which has been motivated in part by recognition of the potential effects that climate change, habitat destruction and contaminant exposure can have on natural and cultured fish populations. New approaches to understanding the impacts of these stressors are being developed that require a systems biology approach with more biologically accurate and detailed mathematical models. We have developed a multi-scale mathematical model of the female rainbow trout hypothalamus-pituitary-ovary-liver axis to use as a tool to help understand the functioning of the system and for extrapolation of laboratory findings of stressor impacts on specific components of the axis. The model describes the essential endocrine components of the female rainbow trout reproductive axis. The model also describes the stage specific growth of maturing oocytes within the ovary and permits the presence of sub-populations of oocytes at different stages of development. Model formulation and parametrization was largely based on previously published in vivo and in vitro data in rainbow trout and new data on the synthesis of gonadotropins in the pituitary. Model predictions were validated against several previously published data sets for annual changes in gonadotropins and estradiol in rainbow trout. Estimates of select model parameters can be obtained from in vitro assays using either quantitative (direct estimation of rate constants) or qualitative (relative change from control values) approaches. This is an important aspect of mathematical models as in vitro, cell-based assays are expected to provide the bulk of experimental data for future risk assessments and will require quantitative physiological models to extrapolate across biological scales.

Acute hyperthermic responses of heat shock protein and estrogen receptor mRNAs in rainbow trout hepatocytes.

Heat shock proteins (HSPs) are induced upon elevated temperature in fishes. HSPs also function as molecular chaperones for cellular proteins, including steroid hormone receptors. Estrogen receptors (ERs) are critical for the hormone signaling necessary during the liver production of the yolk precursor protein vitellogenin in oviparous vertebrates. Considering the possible regulatory role of HSPs on the ER signaling pathway, the present study characterized the mRNA expression of all known isoforms of HSP70 (hsp70a, hsp70b), HSP90 (hsp90a1a, hsp90a1b, hsp90a2a, hsp90a2b, hsp90b1, hsp90b2), and ERs (erα1, erα2, erß1, erß2) in Rainbow Trout hepatocytes following an acute heat shock (1h at 25°C) compared to a control treatment (12°C). The results showed that the mRNA levels of hsp70a, hsp70b, hsp90a1b, hsp90a2a, and hsp90b2 were significantly increased after heat shock, while erα1 mRNA levels were significantly reduced by this treatment. hsp90a1a, hsp90a2b, hsp90b1, erα2, erß1 and erß2 were unaffected by this acute hyperthermic treatment. Comparatively, the responses of the two hsp70 isoforms were much greater than the hsp90 isoforms. Acute heat shock treatment of hepatocytes followed by a 24h exposure to 17ß-estradiol (E2) exposure also resulted in decreased expression of erα1 mRNA, but not vitellogenin (vtg) mRNA. This study showed that some hsp70 and hsp90 isoforms display a robust response to an acute hyperthermic treatment in Rainbow Trout hepatocytes. Among the transcripts measured here, the erα1 isoform uniquely showed significantly decreased mRNA levels upon acute heat treatment.

Metabolic endocrine factors involved in spawning recovery and rematuration of iteroparous female rainbow trout (Oncorhynchus mykiss).

To determine how energy balance affects metabolic hormones hypothesized to play a role in the onset of a new reproductive cycle in iteroparous salmonids, food availability after spawning was restricted in female rainbow trout. These fish were compared with a control group that was fed a standard brood stock ration. Bodyweight, length, and muscle lipid content were determined, and blood was collected from fish at regular intervals; a subset of fish from each group was sacrificed at each sampling time for the collection of liver and ovary tissue, and to calculate hepatosomatic index (HSI) and gonadosomatic index (GSI). Plasma hormone levels were quantified by radioimmunoassay, and tissue gene expression levels were analyzed using q-RT-PCR. The experiment was conducted twice, using two-year-old and three-year-old post-spawned fish. Food-restriction arrested ovarian growth and development within 15-20 weeks, as evidenced by lower GSI in restricted-ration fish. Food restriction also reduced Fulton's condition factor, muscle lipid content, and specific growth rate from one month onward, and reduced HSI after 3 months. In the liver, insulin-like growth factor (igf1 and igf2) gene expression was reduced in three-year-old food-restricted fish within 2 months; however, no effect of ration on igf1 or igf2 expression was detected in two-year-old fish. In both years, IGF binding protein-1 (igfbp1) gene expression decreased over time in both treatment groups. Liver leptin (slepA1) gene expression was lower in two-year-old food-restricted fish at 4 months. These results show that this feed restriction regime arrested reproductive development and affected factors associated with energy balance purported to play a role in initiating reproductive development within 2-4months after spawning.

Partial-year continuous light treatment reduces precocious maturation in age 1+ hatchery-reared male spring Chinook Salmon (Oncorhynchus tshawytscha).

Hatchery programs designed to conserve and increase the abundance of natural populations of spring Chinook Salmon Oncorhynchus tshawytscha have reported high proportions of males precociously maturing at age 2, called minijacks. High proportions of minijacks detract from hatchery supplementation, conservation and production goals. This study tested the effects of rearing juvenile Chinook Salmon under continuous light (LL) on minijack maturation in two trials. The controls were maintained on a simulated natural photoperiod for both trials. For trial 1, LL treatment began on the summer solstice 2019 or the autumn equinox 2019 and ended in late March 2020 (LL-Jun-Apr and LL-Sep-Apr, respectively). A significant reduction in the mean percent of minijacks (%MJ) was observed versus control (28.8%MJ) in both LL-Jun-Apr (5.4%MJ) and LL-Sep-Apr (9.3%MJ). Trial 2 was designed to evaluate whether stopping LL treatment sooner was still effective at reducing maturation proportions relative to controls. LL treatments began on the summer solstice 2020 and continued until the winter solstice (LL-Jun-Dec) or the final sampling in April 2021 (LL-June-Apr). LL-Jun-Dec tanks were returned to a simulated natural photoperiod after the winter solstice. Both photoperiod treatments showed a significant reduction in mean %MJ from the control (66%MJ): LL-Jun-Dec (11.6%MJ), LL-Jun-Apr (10.3%MJ). In both trials, minijacks had higher body weights, were longer and had increased condition factor when compared to females and immature males in all treatment groups at the final sampling. In both trials, there was little or no effect of LL treatment on fork length or body weight in immature males and females versus controls, but an increase in condition factor versus controls was observed. This study shows that continuous light treatment reduces minijack maturation in juvenile male spring Chinook Salmon and could provide an effective method for Spring Chinook Salmon hatcheries interested in reducing minijack production.

Estrogen receptor mRNA expression patterns in the liver and ovary of female rainbow trout over a complete reproductive cycle.

Estrogens are critical hormones involved in reproduction and need to bind to estrogen receptors in target organs for biological activity. Fishes have two distinct estrogen receptor subtypes, alpha (α) and beta (ß), with variable combinations of additional isoforms of each subtype dependent on the history of genome duplication within a taxon. The comparative expression patterns of estrogen receptor isoforms during the female reproductive cycle will provide important insights into the unique function and importance of each. The purpose of this study was to measure the mRNAs for the four estrogen receptor isoforms (erα1, erα2, erß1, erß2) in the liver and ovary of adult, female rainbow trout over the course of an annual reproductive cycle. The expression of estrogen receptor mRNA isoforms was measured by quantitative real-time RT-PCR. Several reproductive indices (gonadosomatic index, maximum oocyte diameter, plasma estradiol-17ß, plasma vitellogenin, and ovulation) were also quantified for comparison and used in a correlation analysis to examine any inter-relationships. Of the four isoforms, the expression of erα1 was highest in the liver, and had a significant positive correlation with liver erß1 expression. Liver expression of erα2 mRNA was the lowest, but showed a significant positive correlation with maximum oocyte diameter in the ovary. The pattern of the erß isoforms in liver was one of initially elevated mRNA expression followed by a gradual decrease as reproductive development proceeded. In the ovary the erß1 isoform had the highest mRNA expression of all estrogen receptor isoforms, at the beginning of the reproductive cycle, but then decreased afterward. Both ovarian erß isoforms had a significant positive correlation with one another. In contrast, erα2 mRNA expression showed a high maximum level in the ovary near the end of the cycle along with a significant positive correlation with plasma estradiol-17ß levels; the highest gonadosomatic indices, maximum oocyte diameter, and vitellogenin levels occurred then too.

Computational estimation of rainbow trout estrogen receptor binding affinities for environmental estrogens.

Environmental estrogens have been the subject of intense research due to their documented detrimental effects on the health of fish and wildlife and their potential to negatively impact humans. A complete understanding of how these compounds affect health is complicated because environmental estrogens are a structurally heterogeneous group of compounds. In this work, computational molecular dynamics simulations were utilized to predict the binding affinity of different compounds using rainbow trout (Oncorhynchus mykiss) estrogen receptors (ERs) as a model. Specifically, this study presents a comparison of the binding affinity of the natural ligand estradiol-17ß to the four rainbow trout ER isoforms with that of three known environmental estrogens 17α-ethinylestradiol, bisphenol A, and raloxifene. Two additional compounds, atrazine and testosterone, that are known to be very weak or non-binders to ERs were tested. The binding affinity of these compounds to the human ERα subtype is also included for comparison. The results of this study suggest that, when compared to estradiol-17ß, bisphenol A binds less strongly to all four receptors, 17α-ethinylestradiol binds more strongly, and raloxifene has a high affinity for the α subtype only. The results also show that atrazine and testosterone are weak or non-binders to the ERs. All of the results are in excellent qualitative agreement with the known in vivo estrogenicity of these compounds in the rainbow trout and other fishes. Computational estimation of binding affinities could be a valuable tool for predicting the impact of environmental estrogens in fish and other animals.

Non-sex specific genes associated with the secondary mitotic period of primordial germ cell proliferation in the gonads of embryonic rainbow trout (Oncorhynchus mykiss).

The purposes of this study were to quantify the secondary proliferation of primordial germ cells (PGCs) in both sexes of rainbow trout, determine if a sex difference in the timing of PGC proliferation and eventual pre-meiotic number exists, and use microarray data collected during this period to identify genes that are associated with PGC mitosis. The experiments used vasa-green fluorescent protein (vasa-GFP) transgenic rainbow trout of known genetic sex that allowed for the identification and collection of PGCs in vivo. An increase was observed in the number of PGCs counted in the gonads of both female and male embryonic vasa-GFP rainbow trout, from 300 to 700° days (water temperature in °C × days post-fertilization). For both sexes, a statistically significant (P < 0.05) increase in the PGC number was first noted at either 350 or 400° days of development. By 700° days, a 20-50-fold increase in germ cell number was apparent. No sex-specific differences in the timing of PGC proliferation or number were notable in any of the families until 700° days. In conjunction, a custom microarray based on cDNA libraries from embryonic rainbow trout gonads was used to identify genes involved in PGC mitosis. Five genes were discovered: guanine nucleotide binding protein, integral membrane protein 2B, transmembrane protein 47, C-src tyrosine-protein kinase, and the decorin precursor protein. All the genes identified have not been previously associated with germ cell mitosis, but are known to be involved with the cell plasma membrane and/or cell signaling pathways.

Aneuploid sperm formation in rainbow trout exposed to the environmental estrogen 17{alpha}-ethynylestradiol.

Environmental contaminants that mimic native estrogens (i.e., environmental estrogens) are known to significantly impact a wide range of vertebrate species and have been implicated as a source for increasing human male reproductive deficiencies and diseases. Despite the widespread occurrence of environmental estrogens and recognized detrimental effects on male vertebrate reproduction, no specific mechanism has been determined indicating how reduced fertility and/or fecundity is achieved. Previous studies show that male rainbow trout, Oncorhynchus mykiss, exposed to the environmental estrogen 17alpha-ethynylestradiol (EE2) before gamete formation and fertilization produce progeny with significantly reduced embryonic survival. To determine whether this observed decrease results from sperm chromosome alterations during spermatogenesis, male rainbow trout were exposed to 10 ng of EE2/l for 50 days. After exposure, semen was collected and sperm aneuploidy levels analyzed with two chromosome markers by fluorescent in situ hybridization. In vitro fertilizations were also conducted by using control and exposed sperm crossed to eggs from an unexposed female for offspring analysis. Evaluations for nucleolar organizer region number and karyotype were performed on developing embryos to determine whether sperm aneuploidy translated into embryonic aneuploidy. Results conclusively show increased aneuploid sperm formation due to EE2 exposure. Additionally, embryonic cells from propagated progeny of individuals possessing elevated sperm aneuploidy display high levels of embryonic aneuploidy. This study concludes that EE2 exposure in sexually developing male rainbow trout increases levels of aneuploid sperm, providing a mechanism for decreased embryonic survival and ultimately diminished reproductive success in EE2 exposed males.

Intracellular, not membrane, estrogen receptors control vitellogenin synthesis in the rainbow trout.

The synthesis of vitellogenin, via estrogens, by the liver of female oviparous vertebrates provides the precursor for yolk proteins in developing oocytes. There are two distinct estrogenic transduction pathways in vertebrates that could control vitellogenin synthesis. One provides direct genomic (i.e., nuclear) control in which estrogens bind to estrogen receptors (ERs) that function as transcription factors within the cell nucleus. The other involves a non-genomic pathway initiated by estrogens binding to membrane-bound ERs at the cell surface. The objective of this paper was to determine which ER transduction pathway regulates hepatic vitellogenin synthesis in rainbow trout. For this study an estrogenic molecule, 17alpha-ethynylestradiol (EE2), was conjugated to a peptide moiety (PEP) to make 17alpha-ethynylestradiol-peptide (EE2-PEP) to bind to membrane-bound ERs. This was compared with EE2 that is capable of crossing the cell membrane and binding to intracellular ERs. An in vivo experiment using male rainbow trout injected with either EE2-PEP or EE2 demonstrated that only EE2 stimulated a significant increase in plasma vitellogenin concentrations. This was further confirmed by treating male rainbow trout hepatocytes in primary culture for 24h with PEP, EE2-PEP or EE2. Only the EE2 treatment resulted in significantly higher vitellogenin expression in trout hepatocytes. These results demonstrate that estrogens must gain entry into hepatocytes to bind to intracellular ERs in order to stimulate vitellogenin synthesis. While this study cannot conclude that a membrane ER system is absent in the rainbow trout liver, it has established that the liver synthesis of vitellogenin in rainbow trout is regulated by intracellular ERs.

Regulation of hepatic estrogen receptor isoform mRNA expression in rainbow trout (Oncorhynchus mykiss).

The complete nuclear estrogen receptor family in rainbow trout consists of two subtypes (ERalpha and ERbeta) each of which consists of two isoforms (alpha1/alpha2 and beta1/beta2). Transcription rate and mRNA stability of ERalpha1 is affected by 17beta-estradiol (E2) but no information on estrogen regulation exists for the other isoforms. The objective of this study was to compare the mRNA expression patterns of the four ER isoforms in the liver of male trout and in immortalized trout hepatocyte lines (RTH-149 and SOB-15) treated with E2 or 17alpha-ethynylestradiol (EE2) using quantitative RT-PCR. To determine the in vivo dose-response, isogenic male trout were injected intra-peritoneally with 0, 1.5, 15 or 150 microg E2 or an equimolar amount of EE2 and the liver sampled 24 h later. Treatment with either E2 or EE2 significantly (p<0.05) increased the level of ERalpha1 mRNA at all doses tested compared to vehicle, while the response of mRNAs for the other three isoforms did not change. The in vitro dose-response was tested by treating both cell lines with 0, 0.1, 1.0 or 10.0 microM E2 for 48 h. In RTH-149 cells, ERalpha1, ERalpha2 and ERbeta2 mRNAs were significantly higher in cells incubated with 10 microM E2 as compared to cells treated with only vehicle (p<0.05). In SOB-15 cells, ERalpha2 and ERbeta1 mRNAs were significantly higher in cells incubated with 1.0 microM E2 as compared to cells incubated with only vehicle (p<0.05). These results support the conclusion that the mRNAs for the four ER isoforms respond differentially to estrogen regulation.

Elevated plasma triglycerides and growth rate are early indicators of reproductive status in post-spawning female steelhead trout (Oncorhynchus mykiss).

Many iteroparous fishes spawn after skipping one or more yearly cycles, which impacts recruitment estimates used for fisheries management and conservation. The physiological mechanisms underlying the development of consecutive and skip spawning life histories in fishes are not well understood. In salmonids, lipid energy reserves and/or growth are thought to regulate the initiation of reproductive maturation during a critical period ~1 year prior to spawning. The fasting spawning migration of summer-run steelhead trout (Oncorhynchus mykiss) results in significant depletion of energy reserves during the proposed critical period for repeat spawning. To determine whether and when lipid energy reserves and growth influence repeat spawning, measures of lipid energy reserves, growth rate and reproductive development were tracked in female steelhead trout from first to second spawning as a consecutive or skip spawner in captivity. Plasma triglyceride (TG) levels and growth rate were elevated by 10 weeks after spawning in reproductive (i.e. consecutive spawning) versus non-reproductive (i.e. skip spawning) individuals. Muscle lipid (ML) levels, condition factor and plasma estradiol levels increased at later time points. The early differences in plasma TG levels and increases in growth rate are attributable to differential rates of feeding and assimilation between the groups following spawning. A year after spawning, plasma TG levels, MLs and growth rate decreased in consecutive spawners, attributable to transfer of lipid reserves into the ovary. During the year prior to second spawning, energy reserves and plasma estradiol levels were higher in reproductive skip spawners versus consecutive spawners, reflecting the energy deficit after first spawning. These results suggest that the decision to initiate ovarian recrudescence occurs by 10 weeks after first spawning and are consistent with the differences in energy reserves acquired following spawning being a consequence of that decision. This information will increase the success of conservation projects reconditioning post-spawning summer-run steelhead trout.

Effect of parental exposure to trenbolone and the brominated flame retardant BDE-47 on fertility in rainbow trout (Oncorhynchus mykiss).

We exposed sexually maturing male rainbow trout (Oncorhynchus mykiss) to BDE-47 (a polybrominated diphenyl ether) and female rainbow trout to trenbolone (an anabolic steroid). Male trout were orally exposed for 17 days to 55 microg/kg/day BDE-47 and female trout continuously exposed for 60-77 days to a measured trenbolone water concentration of 35 ng/L. After the exposure, eggs and semen were collected and in vitro fertilization trials performed using a sperm:egg ratio of 300,000:1. In the BDE-47 study, eggs from control females were fertilized with semen from exposed males, while in the trenbolone study, eggs from exposed females were fertilized with semen from control males. All treatments were evaluated at two-three early developmental time-points representing first cleavage (0.5 day), embryonic keel (9 days), and eyed stages (19 days), respectively. The results indicated that BDE-47 exposure did not alter fertility as embryonic survival was similar between control and exposed groups. Trenbolone exposure also did not alter embryo survival. However, in the embryos fertilized with eggs from trenbolone exposed females, a noticeable delay in developmental progress was observed. On day 19 when eye development is normally complete, the majority of the embryos either lacked eyes or displayed under-developed eyes, in contrast to control embryos. This finding suggests steroidal androgen exposure in sexually maturing female rainbow trout can impact developmental timing of F1 offspring.

The complete nuclear estrogen receptor family in the rainbow trout: discovery of the novel ERalpha2 and both ERbeta isoforms.

Estrogen hormones interact with cellular ERs to exert their biological effects in vertebrate animals. Similar to other animals, fishes have two distinct ER subtypes, ERalpha (NR3A1) and ERbeta (NR3A2). The ERbeta subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of ERalpha have not been demonstrated in any fish species. In the rainbow trout (Oncorhynchus mykiss), the only ER described is an isoform of the ERalpha subtype (i.e. ERalpha1, NR3A1a). The purpose of this study was to determine whether the gene for the other ERalpha isoform, ERalpha2 (i.e., NR3A1b), exists in the rainbow trout. A RT-PCR and cloning strategy, followed by screening a rainbow trout BAC library yielded a unique DNA sequence coding for 558 amino acids. The deduced amino acid sequence had a 75.4% overall similarity to ERalpha1. Both the rainbow trout ERbeta subtypes, ERbeta1 [NR3A2a] and ERbeta2, [NR3A2b] which were previously unknown in this species, were also sequenced as part of this study, and the amino acid sequences were found to be very different from the ERalphas (approximately 40% similarity). ERbeta1 and ERbeta2 had 594 and 604 amino acids, respectively, and had 57.6% sequence similarity when compared to one another. This information provides what we expect to be the first complete nuclear ER gene family in a fish. A comprehensive phylogenetic analysis with all other known fish ER gene sequences was undertaken to understand the evolution of fish ERs. The results show a single ERalpha subtype clade, with the closest relative to rainbow trout ERalpha2 being rainbow trout ERalpha1, suggesting a recent, unique duplication event to create these two isoforms. For the ERbeta subtype there are two distinct subclades, one represented by the ERbeta1 isoform and the other by the ERbeta2 isoform. The rainbow trout ERbeta1 and ERbeta2 are not closely associated with each other, but instead fall into their respective ERbeta subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four ER isoforms (ERalpha1, ERalpha2, ERbeta1, and ERbeta2) in stomach, spleen, heart, brain, pituitary, muscle, anterior kidney, posterior kidney, liver, gill, testis and ovary samples from rainbow trout. The mRNAs for each of the four ERs were detected in every tissue examined. The liver tended to have the highest ER mRNA levels along with the testes, while the lowest levels were generally found in the stomach or heart. The nuclear ERs have a significant and ubiquitous distribution in the rainbow trout providing the potential for complex interactions that involve the functioning of many organ systems.

Dynamics of 17alpha-ethynylestradiol exposure in rainbow trout (Oncorhynchus mykiss): absorption, tissue distribution, and hepatic gene expression pattern.

17alpha-Ethynylestradiol (EE2) is a synthetic estrogen identified in sewage effluents. To understand better the absorption kinetics of EE2 and the induction of vitellogenin (VTG) and estrogen receptor alpha (ERalpha) mRNA, we subjected male rainbow trout (Onchorynchus mykiss) to continuous water exposures of 125 ng/L of EE2 for up to 61 d. Trout were either repetitively sampled for blood plasma or serially killed at selected time intervals. Vitellogenin, ERalpha mRNA, and EE2 were measured using enzyme-linked immunosorbent assay and using quantitative polymerase chain reaction and gas chromatography-mass spectrometry, respectively. In separate experiments, trout were exposed to EE2 for 7 d, and hepatic gene expression was assessed using a low- and high-density cDNA microarray. The EE2 was rapidly absorbed by the trout, with an apparent equilibrium at 16 h in plasma and liver. The ERalpha mRNA levels also increased rapidly, reaching near-peak levels by 48 h. In contrast, plasma levels of VTG continuously increased for 19 d. After 61 d, tissues with the highest levels of VTG were the liver, kidney, and testes. Microarray-based gene expression studies provided unexpected results. In some cases, known estrogen-responsive genes (e.g., ERalpha) were unresponsive, whereas many of the genes that have no apparent link to estrogen function or EE2 toxicity were significantly altered in expression. Of the two microarray approaches tested in the present study, the high-density array appeared to be superior because of the improved quality of the hybridization signal and the robustness of the response in terms of the number of genes identified as being EE2 responsive.

Determination of genetic sex in chinook salmon (Oncorhynchus tshawytscha) using the male-linked growth hormone pseudogene by real-time PCR.

This study used a real-time quantitative polymerase chain reaction (qPCR) method based on the growth hormone pseudogene (GHp) in chinook salmon (Oncorhynchus tshawytscha) to determine genetic sex. The GHp is present as a single copy in the genome of the male chinook salmon but is absent in the female, providing a means of using this real-time qPCR method to discriminate genetic sex. Comparisons between genomic DNA samples from 2 geographically distinct populations of chinook salmon (Columbia River, Washington, and Yukon River, Alaska) showed, within each population examined, that the males were clearly differentiated from the females. There were no interpopulation differences between males or females. The advantages of this real-time qPCR method are that it is rapid, is amenable to high sample throughput, and provides an accurate numerical value that allows comparisons between samples by statistical methods.

Status and opportunities for genomics research with rainbow trout.

The rainbow trout (Oncorhynchus mykiss) is one of the most widely studied of model fish species. Extensive basic biological information has been collected for this species, which because of their large size relative to other model fish species are particularly suitable for studies requiring ample quantities of specific cells and tissue types. Rainbow trout have been widely utilized for research in carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. They are distinctive in having evolved from a relatively recent tetraploid event, resulting in a high incidence of duplicated genes. Natural populations are available and have been well characterized for chromosomal, protein, molecular and quantitative genetic variation. Their ease of culture, and experimental and aquacultural significance has led to the development of clonal lines and the widespread application of transgenic technology to this species. Numerous microsatellites have been isolated and two relatively detailed genetic maps have been developed. Extensive sequencing of expressed sequence tags has begun and four BAC libraries have been developed. The development and analysis of additional genomic sequence data will provide distinctive opportunities to address problems in areas such as evolution of the immune system and duplicate genes.

Short-term exposure to 17 alpha-ethynylestradiol decreases the fertility of sexually maturing male rainbow trout (Oncorhynchus mykiss).

The synthetic estrogen 17 alpha-ethynylestradiol (EE2) is a commonly used oral contraceptive that has been increasingly detected in sewage effluents. This study determined whether EE2 exposure adversely affected reproduction in sexually maturing male rainbow trout (Oncorhynchus mykiss). We exposed male trout to graded water concentrations of EE2 (10, 100, and 1,000 ng/ L) for 62 d leading up to the time of spawning. Semen and blood plasma samples were removed from each fish. Semen was used to fertilize groups of eggs from one nonexposed female. As a measure of fertility, eggs were incubated for 28 d after fertilization to determine the proportion that attained the eyed stage of embryonic development. Additional endpoints also measured included sperm motility, spermatocrit, gonadosomatic and hepatosomatic indices, testis histology, and circulating plasma levels of the sex steroids 17 alpha, 20 beta-dihydroxyprogesterone (17,20-DHP) and 11-ketotestosterone (11-KT). Exposure to 1,000 ng/L of EE2 caused complete mortality of the treatment group by day 57. Exposure to lower EE2 water concentrations (10 and 100 ng/L) caused an increase in sperm density, while a significant reduction in testis mass was observed only in the 100-ng/L exposure group. Most significantly, semen harvested from fish exposed to 10 and 100 ng/L EE2 caused an approximately 50% reduction in the number of eggs attaining the eyed stage of embryonic development. Plasma levels of 17,20-DHP in exposed fish were roughly twice the level of the controls, while levels of 11-KT were significantly reduced in fish exposed to 100 ng/L EE2. These results suggest that sexually maturing male rainbow trout are susceptible to detrimental reproductive effects of short-term exposures to environmentally relevant levels of EE2.