Teleost fish are unique among vertebrates in that phenotypic sex or onset of sex inversion can be easily manipulated by hormonal treatments. In recent years, researchers have begun reporting concentrations of synthetic and natural hormones in the environment. Although concentrations are very low (in the parts per trillion to low parts per billion), they are still of concern because of the high potency of synthetic hormones and the enhanced susceptibility of teleost fishes, especially early life stages, to hormonal exposures. In this review, we will focus on sex differentiation in teleost fishes and how these processes in fish early life stages may be impacted by environmental hormones which are known to contaminate aquatic environments. We will start by reviewing information on sources and concentrations of hormones in the environment and continue by summarizing the state of knowledge of sex differentiation in teleost gonochoristic fishes, including information on genes involved (e.g. cyp19, dmrt1, sox9 and foxl2). We will end our review with a summary of studies that have examined the effects of androgens and estrogens on fish sex differentiation after exposure of fish embryos and larvae and with ideas for future research.
Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05–50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO) – an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors – flumioxazin – resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis-driven and mechanism-focused investigations within zebrafish and mammalian models.
Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is a high-production volume organophosphate-based plasticizer and flame retardant widely used within the United States. Using zebrafish as a model, the objectives of this study were to determine whether (1) TDCIPP inhibits DNA methyltransferase (DNMT) within embryonic nuclear extracts; (2) uptake of TDCIPP from 0.75 h postfertilization (hpf, 2-cell) to 2 hpf (64-cell) or 6 hpf (shield stage) leads to impacts on the early embryonic DNA methylome; and (3) TDCIPP-induced impacts on cytosine methylation are localized to CpG islands within intergenic regions. Within this study, 5-azacytidine (5-azaC, a DNMT inhibitor) was used as a positive control. Although 5-azaC significantly inhibited zebrafish DNMT, TDCIPP did not affect DNMT activity in vitro at concentrations as high as 500 μM. However, rapid embryonic uptake of 5-azaC and TDCIPP from 0.75 to 2 hpf resulted in chemical- and chromosome-specific alterations in cytosine methylation at 2 hpf. Moreover, TDCIPP exposure predominantly resulted in hypomethylation of positions outside of CpG islands and within intragenic (exon) regions of the zebrafish genome. Overall, these findings provide the foundation for monitoring DNA methylation dynamics within zebrafish as well as identifying potential associations among TDCIPP exposure, adverse health outcomes, and DNA methylation status within human populations.
Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a high-production volume organophosphate flame retardant widely used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) results in genome-wide alterations in methylation during cleavage (2 hpf) as well as epiboly delay or arrest (at higher concentrations) during late-blastula and early-gastrula (4–6 hpf). To determine whether these TDCIPP-induced effects were associated with impacts on the transcriptome, embryos were exposed to vehicle (0.1% DMSO) or 2 µM TDCIPP from 0.75 hpf to 6 hpf, and total RNA was extracted from triplicate embryo pools per treatment and hybridized onto duplicate Affymetrix Zebrafish Gene 1.0 ST Arrays per RNA sample. Based on transcriptome-wide profiling, TDCIPP resulted in a significant impact on biological processes involved in dorsoventral patterning and bone morphogenetic protein (BMP) signaling. Consistent with these responses, TDCIPP exposure also resulted in strongly dorsalized embryos by 24 hpf—a phenotype that mimicked the effects of dorsomorphin, a potent and selective BMP inhibitor. Moreover, the majority of dorsalized embryos were preceded by epiboly arrest at 6 hpf. Our microarray data also revealed that the expression of sizzled (szl)—a gene encoding a secreted Frizzled-related protein that limits BMP signaling—was significantly decreased by nearly 4-fold at 6 hpf. Therefore, we used a splice-blocking morpholino to test the hypothesis that knockdown of szl phenocopies TDCIPP-induced delays in epiboly progression. Interestingly, contrary to our hypothesis, injection of szl MOs did not affect epiboly progression but, similar to chordin (chd) morphants, resulted in mildly ventralized embryos by 24 hpf. Overall, our findings suggest that TDCIPP-induced epiboly delay may not be driven by decreased szl expression, and that TDCIPP-induced dorsalization may—similar to dorsomorphin—be due to interference with BMP signaling during early zebrafish development.
Concentrated animal feeding operation (CAFO) manure is a cost-effective fertilizer. In the Midwest, networks of subsurface tile-drains expedite transport of animal hormones and nutrients from land-applied CAFO manure to adjacent waterways. The objective of this study was to evaluate impacts of land-applied CAFO manure on fish populations and communities. Water chemistry including hormone, pesticide, and nutrient concentrations was characterized from study sites along with fish assemblage structure, growth, and endocrine disruption assessed in selected fish species. Although most CAFO water samples had hormone concentrations <1 ng/L, equivalent concentrations for 17β-E2 and 17α-TB peaked at >30 ng/L each during the period of spawning, hatching, and development for resident fishes. CAFO sites had lower fish species richness, and fishes exhibited faster somatic growth and lower reproductive condition compared to individuals from the reference site. Fathead minnows (Pimephales promelas) exposed to CAFO ditchwater during early developmental stages exhibited significantly skewed sex ratios toward males. Maximum observed hormone concentrations were well above the lowest observable effect concentrations for these hormones; however, complexities at the field scale make it difficult to directly relate hormone concentration and impacts on fish. Complicating factors include the consistent presence of pesticides and nutrients, and the difference in temperature and stream architecture of the CAFO-impacted ditches compared to the reference site (e.g., channelization, bottom substrate, shallow pools, and riparian cover).
Endocrine disrupting contaminants are of continuing concern for potentially contributing to reproductive dysfunction in largemouth and smallmouth bass in the Chesapeake Bay watershed (CBW) and elsewhere. Exposures to atrazine (ATR) have been hypothesized to have estrogenic effects on vertebrate endocrine systems. The incidence of intersex in male smallmouth bass from some regions of CBW has been correlated with ATR concentrations in water. Fish early life stages may be particularly vulnerable to ATR exposure in agricultural areas, as a spring influx of pesticides coincides with spawning and early development. Our objectives were to investigate the effects of early life stage exposure to ATR or the model estrogen 17α-ethinylestradiol (EE2) on sexual differentiation and gene expression in gonad tissue. We exposed newly hatched largemouth bass (LMB, Micropterus salmoides) from 7 to 80 days post-spawn to nominal concentrations of 1, 10, or 100 µg ATR/L or 1 or 10 ng EE2/L and monitored histological development and transcriptomic changes in gonad tissue. We observed a nearly 100% female sex ratio in LMB exposed to EE2 at 10 ng/L, presumably due to sex reversal of males. Many gonad genes were differentially expressed between sexes. Multidimensional scaling revealed clustering by gene expression of the 1 ng EE2/L and 100 µg ATR/L-treated male fish. Some pathways responsive to EE2 exposure were not sex-specific. We observed differential expression in male gonad in LMB exposed to EE2 at 1 ng/L of several genes involved in reproductive development and function, including star, cyp11a2, ddx4 (previously vasa), wnt5b, cyp1a and samhd1. Expression of star, cyp11a2 and cyp1a in males was also responsive to ATR exposure. Overall, our results confirm that early development is a sensitive window for estrogenic endocrine disruption in LMB and are consistent with the hypothesis that ATR exposure induces some estrogenic responses in the developing gonad. However, ATR-specific and EE2-specific responses were also observed.
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