James J. Nagler

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.

Estrogen Receptor-α Protein Localization in the Testis of the Rainbow Trout (Oncorhynchus mykiss) During Different Stages of the Reproductive Cycle

Abstract Estrogen receptor-alpha (ER-α) is important for male reproduction in mammals; however, no information is available on ER-α protein distribution in the testes of fishes. The cellular localization of the rainbow trout (Oncorhynchus mykiss) ER-α (rtER-α) protein, throughout the annual reproductive cycle was determined in this study. An antibody was designed based on a 15-amino acid sequence from the D-domain of the rtER-α, and its specificity was confirmed using Western blot analysis. Immunohistochemical analysis revealed rtER-α protein to be present only in the testicular interstitium, at every stage of the annual reproductive cycle. The localization of rtER-α protein in the interstitial fibroblasts, the Leydig cell precursor in the rainbow trout, suggests a role for estrogens in the differentiation of these precursor cells into mature Leydig cells. This is the first study to report the cellular localization of an estrogen receptor protein in the testis of any fish species.

Single pair mating indicates maternal effects on embryo survival in rainbow trout, Oncorhynchus mykiss

Abstract The maternal and paternal influences on early embryo survival in rainbow trout are not established. The purpose of this study was to determine whether variability in the survival of rainbow trout embryos could be attributed to either the female or male parent. Gametes from individual female and male rainbow trout were used in single pair matings to produce families whose survival was followed from fertilization to the time of swim-up (i.e., ∼7 weeks post-fertilization). Survival was assessed at 0.5, 9, 19, 33, and 48 days post-fertilization, corresponding to second cleavage, embryonic keel formation, retinal pigmentation, hatch, and swim-up, respectively. The variability of survival at all times was significantly ( P P >0.05). Therefore, in rainbow trout embryo survival can be equated with the quality of the egg. To predict survival at swim-up (i.e., after 48 days) it was found that embryonic keel formation, measured 9 days after fertilization, was the earliest time at which a highly significant positive correlation ( r =0.889, P

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.

Induction of gonadal intersex in genotypic male rainbow trout (Oncorhynchus mykiss) embryos following immersion in estradiol-17β.

This investigation was conducted to determine the initial period of gonadal sensitivity to estrogen in genetically male rainbow trout (Oncorhynchus mykiss). Fish were immersed in approximately 250 μg estradiol‐17β (E2)/l for two 2 hr periods during different stages of embryonic development beginning 30 days postfertilization (DPF) and continuing until 68 DPF. Histological analysis of gonad samples indicated a significant proportion of E2‐treated fish had intersex gonads; these gonads were primarily comprised of testicular tissue with one or more oocytes scattered throughout. The most sensitive period for altering normal testicular development was found to occur between 44 and 51 DPF (63% intersex), while the labile period in general was determined to span 24 days (from 30 to 54 DPF). Additionally, quantitative RT‐PCR was used to measure estrogen receptor (ER) mRNA expression in whole individual untreated embryos at six weekly time points throughout the period of E2‐exposure. Although the intersex condition was not observed throughout the entire E2‐exposure period, ER mRNA was detected at each time point assayed. Mol. Reprod. Dev. 56:495–501, 2000.

Effects of unilateral ovariectomy on recruitment and growth of follicles in the rainbow trout, Oncorhynchus mykiss

Virgin female rainbow trout, Oncorhynchus mykiss, were unilaterally ovariectomised at various stages of ovarian development to investigate the effect of the removal of one ovary on subsequent recruitment and growth of follicles in the remaining ovary. The right ovary was removed from groups of 12–15 fish, 12, 7 and 4 months before they were due to ovulate, and the gonadosomatic index and follicle number and size determined just prior to ovulation. There were no differences in fecundity or follicle size in fish unilaterally ovariectomised at 12 and 7 months prior to ovulation compared to the controls. However, in the females unilaterally ovariectomised 4 months prior to ovulation, the remaining ovary either had the normal number of follicles for a single ovary but of a significantly larger size than follicles in the controls, or alternatively had almost 70% more than the normal number of vitellogenic follicles but comprising two distinctly different size populations. Differences in plasma oestradiol-17β concentrations at the final sample were seen only in females unilaterally ovariectomised 4 months prior to ovulation, where the levels were significantly lower than both the sham operated and control fish (p < 0.05).These data show that in the rainbow trout, complete compensatory ovarian hypertrophy following unilateral ovariectomy can occur throughout a major part of ovarian development, but that follicle recruitment is limited to stages up to (and therefore fecundity is determined by) mid-vitellogenesis.

DYNAMICS OF 17α-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.

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 (β), 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, erβ1, erβ2) 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-17β, 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 erβ1 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 erβ isoforms in liver was one of initially elevated mRNA expression followed by a gradual decrease as reproductive development proceeded. In the ovary the erβ1 isoform had the highest mRNA expression of all estrogen receptor isoforms, at the beginning of the reproductive cycle, but then decreased afterward. Both ovarian erβ 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-17β levels; the highest gonadosomatic indices, maximum oocyte diameter, and vitellogenin levels occurred then too.

Reduced embryonic survival in rainbow trout resulting from paternal exposure to the environmental estrogen 17α-ethynylestradiol during late sexual maturation

Exposure of fishes to environmental estrogens is known to affect sexual development and spawning, but little information exists regarding effects on gametes. This study evaluated embryonic survival of offspring from male rainbow trout (Oncorhynchus mykiss) exposed to 17alpha-ethynylestradiol (EE(2)) using an in vitro fertilization protocol. Males were exposed at either 1800 or 6700 degree days ( degrees d) (i.e. 161 or 587 days post-fertilization (dpf)) to test for effects on testes linked to reproductive ontogeny. At 1800 degrees d, fish were beginning testicular differentiation and were exposed to 109 ng EE(2)/l for 21 days. At 6700 degrees d, fish have testes containing spermatocytes and spermatids and were exposed for 56 days to either 0.8, 8.3, or 65 ng EE(2)/l. Semen was collected at full sexual maturity in each group and used to fertilize eggs pooled from several non-exposed females. Significant decreases in embryonic survival were observed only with the 6700 degrees d exposure. In 0.8 and 8.3 ng EE(2)/l treatments, embryo survival was significantly reduced at 19 dpf when compared with the control. In contrast, an immediate decrease in embryonic survival at 0.5 dpf was observed in the 65 ng EE(2)/l treatment. Blood samples collected at spawning from 6700 degrees d exposed males revealed a significant decrease in 11-ketotestosterone and a significant increase in luteinizing hormone levels for the 65 ng EE(2)/l treatment when compared with the other treatment groups. Results indicate that sexually maturing male rainbow trout are susceptible to EE(2) exposure with these fish exhibiting two possible mechanisms of reduced embryonic survival through sperm varying dependant on EE(2) exposure concentrations experienced.

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.