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.

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.

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.

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.

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.

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.

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.

Plasma nesfatin-1 is not affected by long-term food restriction and does not predict rematuration among iteroparous female rainbow trout (Oncorhynchus mykiss).

The metabolic peptide hormone nesfatin-1 has been linked to the reproductive axis in fishes. The purpose of this study was to determine how energy availability after spawning affects plasma levels of nesfatin-1, the metabolic peptide hormone ghrelin, and sex steroid hormones in rematuring female rainbow trout (Oncorhynchus mykiss). To limit reproductive maturation, a group of female trout was food-restricted after spawning and compared with a control group that was fed a standard broodstock ration. The experiment was conducted twice, once using two-year-old trout (second-time spawners) and once using three-year-old trout (third-time spawners). During monthly sampling, blood was collected from all fish, and a subset of fish from each treatment was sacrificed for pituitaries. Pituitary follicle-stimulating hormone-beta (fsh-ß) mRNA expression was analyzed with q-RT-PCR; plasma hormone levels were quantified by radioimmunoassay (17ß-estradiol and ghrelin) and enzyme-linked immunosorbent assay (11-keto-testosterone and nesfatin-1). Although plasma nesfatin-1 levels increased significantly in the months immediately after spawning within both feeding treatments, plasma nesfatin-1 did not differ significantly between the two treatments at any point. Similarly, plasma ghrelin levels did not differ significantly between the two treatments at any point. Food restriction arrested ovarian development by 15-20 weeks after spawning, shown by significantly lower plasma E2 levels among restricted-ration fish. Pituitary fsh-ß mRNA levels were higher among control-ration fish than restricted-ration fish starting at 20 weeks, but did not differ significantly between treatment groups until 30 weeks after spawning. Within both treatment groups, plasma 11-KT was elevated immediately after spawning and rapidly decreased to and persisted at low levels; starting between 20 and 25 weeks after spawning, plasma 11-KT was higher among control-ration fish than restricted-ration fish. The results from these experiments do not provide support for plasma nesfatin-1 as a signal for the initiation of reproductive development in rematuring female rainbow trout.

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.

Toxicokinetic, toxicodynamic, and toxicoproteomic aspects of short-term exposure to trenbolone in female fish.

The toxicokinetics of trenbolone was characterized during 500 ng/l water exposures in female rainbow trout (Oncorhynchus mykiss) and fathead minnows (Pimephales promelas). Related experiments measured various toxicodynamic effects of exposure. In both species, trenbolone was rapidly absorbed from the water and reached peak plasma levels within 8h of exposure. Afterwards, trenbolone concentrations in trout (66-95 ng/ml) were 2-6 times higher compared with minnows (15-29 ng/ml), which was attributable to greater plasma binding in trout. During water exposures, circulating levels of estradiol (E2) rapidly decreased in both species to a concentration that was 25%-40% of control values by 8-24h of exposure and then remained relatively unchanged for the subsequent 6 days of exposure. In trout, changes in circulating levels of follicle-stimulating hormone were also significantly greater after trenbolone exposure, relative to controls. In both species, the pharmacokinetics of injected E2-d3 was altered by trenbolone exposure with an increase in total body clearance and a corresponding decrease in elimination half-life. The unbound percentage of E2 in trout plasma was 0.25%, which was similar in pre- or postvitellogenic female trout. Subsequent incubation with trenbolone caused the unbound percentage to significantly increase to 2.4% in the previtellogenic trout plasma. iTRAQ-based toxicoproteomic studies in minnows exposed to 5, 50, and 500 ng/l trenbolone identified a total of 148 proteins with 19 downregulated including vitellogenin and 18 upregulated. Other downregulated proteins were fibrinogens, α-2-macroglobulin, and transferrin. Upregulated proteins included amine oxidase, apolipoproteins, parvalbumin, complement system proteins, and several uncharacterized proteins. The results indicate trenbolone exposure is a highly dynamic process in female fish with uptake and tissue equilibrium quickly established, leading to both rapid and delayed toxicodynamic effects.

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.

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.

Relationships between the transcriptome and physiological indicators of reproduction in female rainbow trout over an annual cycle.

Normal transcriptomic patterns along the brain-pituitary-gonad-liver (BPGL) axis should be better characterized if endocrine-disrupting compound-induced changes in gene expression are to be understood. Female rainbow trout were studied over a complete year-long reproductive cycle. Tissue samples from pituitary, ovary, and liver were collected for microarray analysis using the 16K Genomic Research on Atlantic Salmon Project (GRASP) microarray and for quantitative polymerase chain reaction measures of estrogen receptor (ER) isoform messenger RNA (mRNA) levels. Plasma was collected to determine levels of circulating estradiol-17ß (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). As an a priori hypothesis, changes in gene expression were correlated to either circulating levels of E2, FSH, and LH, or ER mRNAs quantified by quantitative polymerase chain reaction. In the liver, most transcriptomic patterns correlated to levels of either E2, LH, or ERs. Fewer ovarian transcripts could be correlated to levels of E2, ERα, or FSH. No significant associations were obvious in the pituitary. As a post hoc hypothesis, changes in transcript abundance were compared with microarray features with known roles in gonadal maturation. Many altered transcripts in the ovary correlated to transcript levels of estradiol 17-beta-dehydrogenase 8 or 17 B HSD12, or to glycoprotein alpha chain 1 or 2. In the pituitary, genes involved with the growth axis (e.g., growth hormone, insulin-related growth factor binding protein) correlated with the most transcripts. These results suggest that transcriptional networks along the BPGL axis may be regulated by factors other than circulating steroid hormones.

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.

Variation among rainbow trout (Oncorhynchus mykiss) estrogen receptor isoform 3' untranslated regions and the effect of 17beta-estradiol on mRNA stability in hepatocyte culture.

Adenine and uridine (AU)-rich elements in the 3' untranslated region (3'UTR) have been implicated in the 17beta-estradiol (E2) stabilization of vertebrate estrogen receptor (ER) mRNAs. To date, fishes have the most complex arrangement of nuclear ERs with up to two isoforms of each of the two genes in some species (i.e., four different ERs). The objective of this study was to analyze the sequence variation of 3'UTRs among the four ER isoforms in the rainbow trout and determine to what degree it is responsible for the estrogen-induced increase of ER mRNAs in the liver of this fish. This was done by comparing the 3'UTR DNA sequence length and composition, and by measuring expression of ER isoform 3'UTR luciferase reporter constructs in primary cultures of trout hepatocytes treated with E2. There were large differences both in overall length and in sequence composition among the four ER isoform 3'UTRs. The ERalpha1 sequence was the longest and the only one of the four that contained multiple copies of the canonical AU-rich elements (AUUUA) as well as the stability sequence (GCUGAU). E2 treatment significantly increased the luciferase activity in cells transiently transfected with the ERalpha1 reporter construct, relative to cells transfected with reporter vectors containing the other three ER isoform 3'UTRs or the parental vector control. These results support the hypothesis that the E2-induced increase in hepatic ERalpha1 mRNA in rainbow trout is due in part to sequence variability among ER isoform 3'UTRs. We conclude that posttranscriptional stabilization of ER mRNA by E2 appears to be conserved among vertebrates.

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.

The ontogeny of nuclear estrogen receptor isoform expression and the effect of 17beta-estradiol in embryonic rainbow trout (Oncorhynchus mykiss).

Ligand bound nuclear estrogen receptor (ER) acts as a transcription factor regulating the expression of estrogen dependent genes. There are four nuclear ER isoforms in rainbow trout (Oncorhynchus mykiss). The objective of this study was to measure whole body mRNA levels of the two ERalpha isoforms (alpha1/alpha2) and the two ERbeta isoforms (beta1/beta2) in male and female embryos from 50 to 600 degree-days (DD; days post-fertilizationxwater temperature) and in embryos exposed to vehicle or 17beta-estradiol (E2) for 2h at 230, 240 and 250 DD. All four isoforms were detected at every time point in both sexes. Sexual dimorphism was rarely observed; at 50 DD the level of ERalpha2 mRNA was significantly greater in males than in females and at 100 DD the level of ERbeta1 mRNA was significantly greater in females than in males (p<0.05). Expression profiles of the two ERalpha isoforms were slightly different from one another, whereas the ERbeta isoforms exhibited similar expression patterns. The effect of E2 was not different between male and female embryos. The level of ERalpha1 mRNA increased significantly at 240 DD; a similar but not statistically significant trend was observed at 230 and 250 DD. Despite the critical role of estrogen during sex differentiation in rainbow trout, the receptivity to this hormone as measured by the response in mRNA levels of ER appears to be largely the same between males and females and ERalpha1 is the only E2 responsive isoform.

Lack of a heritable reproductive defect in the offspring of male rainbow trout exposed to the environmental estrogen 17alpha-ethynylestradiol.

Endocrine disruptors, including environmental estrogens, have been shown to induce heritable effects through both genetic and epigenetic mechanisms in mammals. Despite this information and the wealth of knowledge regarding the significant reproductive impacts endocrine disruptors impose on fishes, no studies have reported whether the observed effects are heritable. Without this information it is difficult to establish the long-term consequences for exposed populations. To determine potential consequences of long-term effects we must consider the possibility that induced reproductive defects in fishes may be heritable. Using rainbow trout (Oncorhynchus mykiss) as a model this study aims to determine whether a specific reproductive defect observed in 17alpha-ethynylestradiol exposed male parents, diminished progeny survival, is heritable in the unexposed surviving F1 males. Semen was collected from anesthetized males of the F1 generation upon sexual maturation at two time-points, one year old precocious males and two years old males. In vitro fertilization was used to produce an F2 generation. F2 embryos were then analyzed for survival at 19 days post-fertilization (eye pigmentation) and the different treatment groups statistically compared to the controls. Analysis indicated that F2 offspring survival from F1 males propagated from both exposed and unexposed parents survive normally and no heritable effect was observed in males from the F1 generation for this specific reproductive defect. These results provide scope for the recovery of fish populations exposed to environmental estrogens should the contaminant be removed.

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.

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.