Vitellogenin Gene Transcription Research Articles

Waterborne exposure to microcystin-leucine arginine induces endocrine disruption and gonadal dysplasia of Pelophylax nigromaculatus tadpoles via the hypothalamic-pituitary-gonadal-liver axis

The impact of microcystins on the gonad development and reproduction endocrine in the tadpole stage on amphibians remains unclear. In this study, the tadpoles (Pelophylax nigromaculatus) were exposed to 0, 1, and 10 μg/L of microcystin-leucine arginine (MC-LR) for 60 days to explore the impacts of environmental realistic concentration MC-LR on gonad development and reproduction endocrine, respectively. After MC-LR exposure, the germ cell structure has changed, especially in oocytes. The 10 μg/L MC-LR exposure group showed a significantly diminished gonad somatic index (GSI) in females. However, the sex ratio of tadpoles did not differ significantly. Moreover, gene transcription (figla and nobox) related to ovarian development and genes (sox9 and dmrt1) associated with testicular development were down-regulated after MC-LR exposure. After MC-LR exposure, the gene transcripts encoding gonadotropin-releasing hormone (gnrh1 and gnrh2) were down-regulated in the hypothalamus, while gonadotropins (FSH and LH) levels increased in serum. The transcripts of testosterone synthesis-related genes (star, cyp11a1, 3β-hsd, cyp17a1, and 17β-hsd) were up-regulated in the gonads, and the testosterone (T) concentration increased in serum. However, key gene transcript (cyp19a1) involved in estradiol synthesis was down-regulated and the estradiol (E2) concentration decreased in serum, resulting in the absence of a compensatory mechanism for positive feedback regulation of the hypothalamic-pituitary-gonadal (HPG) axis to maintain E2 levels. The vitellogenin gene (vtg1) transcription level was significantly down-regulated. The E2/T content ratio decreased in MC-LR concentration-dependent manner. Consequently, MC-LR exposure interfered with the hypothalamic-pituitary-gonadal-liver (HPGL) axis in tadpoles, which in turn affects gonadal development, especially the ovaries. Overall, this study provides the initial evidence that MC-LR exerts significant effects on reproductive endocrinology and gonadal development in amphibian tadpoles, highlighting the susceptibility of the tadpole reproductive system to the environmental risks of MC-LR.

ATPase subunits of the 26S proteasome are important for oocyte maturation in the brown planthopper.

The 26S proteasome is the major engine of protein degradation in all eukaryotic cells. Adenosine triphosphatase (ATPase) regulatory subunits (Rpts) are constituents of the proteasome that are involved in the unfolding and translocation of substrate proteins into the core particle. In this study, by using the brown planthopper Nilaparvata lugens as a model insect, we report the biological importance of Rpts in female reproduction. We identified six homologous Rpt genes (Rpt1-6) in N. lugens. These genes were detected at high transcript levels in eggs and ovaries of females but at low transcript levels in males. RNA interference-mediated knockdown of N. lugens Rpt genes significantly decreased the proteolytic activity of the proteasome and impeded the transcription of triacylglycerol lipase and vitellogenin genes in the fat bodies and ovaries of adult females and reduced the triglyceride content in the ovaries. The decrease in the proteolytic activity of the proteasome via knockdown of Rpts also downregulated the transcription of the CYP307A2 gene encoding an important rate-limiting enzyme in the 20-hydroxyecdysone biosynthetic pathway in the ovaries, reduced 20E production in adult females and impaired ovarian development and oocyte maturation, leading to the failure of egg production and egg-laying. These novel findings indicate that Rpts are required for the proteolytic activity of the proteasome, which is important for female reproductive success in N. lugens.

Lipin modulates lipid metabolism during reproduction in the cabbage beetle

Lipids are a critical source of stored energy in insects, and their metabolism is essential for growth, development, and reproduction. Adequate provisioning of lipids and yolk proteins in the oocytes is essential to ensure reproductive output. Therefore, it is particularly important to understand the molecular mechanisms linking lipid metabolism and reproduction. Lipin proteins are emerging as pivotal modulators of lipid metabolism. They exert a dual function as phosphatidate phosphatase enzymes involved in lipid synthesis and as transcriptional coactivators of genes related to lipid metabolism. However, the functional relationship between lipid metabolism and reproduction remains unclear. In this study, the role of lipin protein in the reproduction of female cabbage beetle Colaphellus bowringi was examined. It was found that Lipin was broadly expressed in the tissues of adult females, with relatively high transcript levels in the head, midgut, fat body, malpighian tubules, and epidermis. RNA interference experiments were conducted using double-stranded RNA against Lipin in C. bowringi females. Lipin silencing blocked ovarian development and strongly suppressed transcription of vitellogenin and vitellogenin receptor genes. In addition, the reduction in Lipin expression led to a rapid increase in lipid storage in the fat body and also promoted the expression of genes related to lipid synthesis and stress tolerance. Overall, these results suggest that a Lipin-mediated lipolytic system is essential for maintaining lipid homeostasis during reproduction in C. bowringi. The findings of this study provide a foundation for future studies on the relationship between lipid metabolism and reproduction in invertebrates.

Chronic exposure to di‐n‐butyl phthalate causes reproductive toxicity in zebrafish

Di-n-butyl phthalate (DBP) is known to have adverse effects on reproduction in mammals and is pervasive in the aquatic environment. The objective of the present study was to investigate whether long-term exposure to low concentrations of DBP can affect fish reproduction. In this study, zebrafish (Danio rerio) embryos (F0 ) were exposed to low concentrations (4.9, 13.6 and 43.8 μg/L) of DBP from 2 hours post-fertilization until sexual maturation. The results demonstrate that chronic exposure to DBP (43.8 μg/L) impaired the reproductive function of zebrafish, as verified by reduced egg production and modifications to gonadal histology of the treated fish. Plasma 17β-estradiol levels in female zebrafish decreased significantly in a concentration-dependent manner, while testosterone levels in males increased significantly when fish were exposed to 43.8 μg/L DBP. Real-time polymerase chain reaction was performed to examine selected genes in the hypothalamic-pituitary-gonadal (HPG) axis and liver. Hepatic vitellogenin gene transcription was downregulated in both males and females, suggesting that DBP possesses anti-estrogenic activity. The disturbed steroid hormones were accompanied by the significant alterations in gene expression along the HPG axis. Additionally, parental exposure to DBP caused reduced hatching and survival rate as well as decreased growth in the F1 generation. Taken together, these results demonstrate that long-term exposure to low concentrations of DBP in zebrafish could cause reproductive toxicity, implying that DBP could have significant adverse effects on fish populations, particularly in a highly DBP-contaminated aquatic environment.

Functional characterization of the cis-regulatory region for the vitellogenin gene in Plutella xylostella.

The vitellogenin gene promoter (VgP) is an essential cis-regulatory element that plays a significant role in transcription of the vitellogenin (Vg) gene, leading to the production of yolk protein in insects, including lepidopterans. However, the function of VgP is still not clear in Plutella xylostella. Here, we cloned a 5.1 kb DNA fragment of the cis-regulatory region adjacent to the 5' end of the Vg gene of P. xylostella (PxVg). We identified two promoter sites in that 5' upstream sequence of PxVg and performed in vitro analysis of two promoter sequences (PxVgP1, 4.9 kb, and PxVgP2, 2.9 kb) in the embryonic cell line of P. xylostella. PxVgP2 exhibited higher enhanced green fluorescent protein (EGFP) expression, so PxVgP2 was used for in vivo analysis. Strong EGFP fluorescence was observed in adult females and the fat body of females, with low expression in embryos. Our results suggest that PxVgP is an important stage-, tissue- and sex-specific endogenous cis-regulatory element in P. xylostella.

Influence of chronic exposure to thiamethoxam and chronic bee paralysis virus on winter honey bees.

Co-exposure to pesticides and viruses is likely to occur in honey bee colonies. Pesticides can be present in pollen, nectar, and persist in stored food (honey and bee bread), and viruses can be highly prevalent in honey bee colonies. Therefore, the present study describes the influence of chronic co-exposure to thiamethoxam and Chronic bee paralysis virus (CBPV) on bee survival, virus loads, expression level of immune and detoxication genes, and pesticide metabolism Experiments were performed on honey bees collected from a winter apiary with reduced viral contaminations. No synergistic effect of co-exposure was observed on bee survival, nor on the ability of bees to metabolise the pesticide into clothianidin. However, we found that co-exposure caused an increase in CBPV loads that reached the viral levels usually found in overt infections. The effect of co-exposure on CBPV replication was associated with down-regulation of vitellogenin and dorsal-1a gene transcription. Nevertheless, the observed effects might be different to those occurring in spring or summer bees, which are more likelyco-exposed to thiamethoxam and CBPV and exhibit a different physiology.

Molecular cloning of vitellogenin gene promoters and in vitro and in vivo transcription profiles following estradiol-17β administration in the cutthroat trout

Transcription of vitellogenin (vtg) genes are initiated when estradiol-17β (E2)-estrogen receptor (ER) complexes bind estrogen response elements (ERE) located in the gene promoter region. Transcriptional regulation of dual vtg subtypes (major salmonid A-type vtg: vtgAs; minor C-type vtg: vtgC) by E2 was investigated under co-expression of a potential major transcriptional factor, erα1, in cutthroat trout. Two forms of trout vtgAs promoters (1 and 2) and one vtgC promoter were sequenced. These promoters structurally differ based on the number of EREs present. The vtgAs promoter 1 exhibited the highest maximal transcriptional activity by in vitro gene reporter assays. The concentration of E2 that induces 50% of gene reporter activity (half-maximal effective concentrations, EC50) was similar among all vtg promoters and also to the EC50 of E2 administered to induce vtg transcription in vivo. This study revealed a difference in transcriptional properties of multiple vtg promoters for the first time in a salmonid species, providing the basis to understand mechanisms underlying regulation of vitellogenesis via dual vtg gene expression.

The white mullet (Mugil curema) as biological indicator to assess environmental stress in tropical coastal lagoons.

Several coastal lagoons and estuaries in Mexico receive untreated domestic and industrial discharges which contain complex mixtures of contaminants. In order to assess the effects of chemical contamination, we used the White mullet (Mugil curema) as biological indicator. We worked in two estuaries located in Northwest Mexico: Urias (highly polluted) and Teacapan (less polluted, therefore used as reference site). We measured several endpoints at different levels of biological organization: vitellogenin transcription in males as biomarker of estrogenic contamination, as well as reproductive, morphological (deformities), morphometric, and meristic parameters. Total RNA was isolated from the liver, and a partial sequence of the mullet vitellogenin gene was obtained; gene expression was analyzed by quantitative PCR. At the same time, gonad samples were analyzed by histologic techniques to determine sex ratios and the reproductive cycle stage. The reproductive season was detected from February to June in both sites, but the gonadosomatic index was consistently higher in Teacapan. Sex ratios were female-biased in both estuaries, and one intersex gonad and several malformations were found in fish from Urias. Vitellogenin gene transcription in males was detected in both sites, although gene expression was slightly higher in Urias. The results obtained in this study indicate that biological effects of contamination are evident in fish, environmental estrogens may be present in both estuaries, and the white mullet is useful as biological indicator to identify and characterize environmental stressors in tropical coastal ecosystems.

A 44 bp intestine-specific hermaphrodite-specific enhancer from the C. elegans vit-2 vitellogenin gene is directly regulated by ELT-2, MAB-3, FKH-9 and DAF-16 and indirectly regulated by the germline, by daf-2/insulin signaling and by the TGF-β/Sma/Mab pathway

The Caenorhabditis elegans vitellogenin genes are transcribed in the intestine of adult hermaphrodites but not of males. A 44-bp region from the vit-2 gene promoter is able largely to reconstitute this tissue-, stage- and sex-specific-expression. This “enhancer” contains a binding site for the DM-domain factor MAB-3, the male-specific repressor of vitellogenesis, as well as an activator site that we show is the direct target of the intestinal GATA factor ELT-2. We further show that the enhancer is directly activated by the winged-helix/forkhead-factor FKH-9, (whose gene has been shown by others to be a direct target of DAF-16), by an unknown activator binding to the MAB-3 site, and by the full C. elegans TGF-β/Sma/Mab pathway acting within the intestine. The vit-2 gene has been shown by others to be repressed by the daf-2/daf-16 insulin signaling pathway, which so strongly influences aging and longevity in C. elegans. We show that the activity of the 44bp vit-2 enhancer is abolished by loss of daf-2 but is restored by simultaneous loss of daf-16. DAF-2 acts from outside of the intestine but DAF-16 acts both from outside of the intestine and from within the intestine where it binds directly to the same non-canonical target site that interacts with FKH-9. Activity of the 44bp vit-2 enhancer is also inhibited by loss of the germline, in a manner that is only weakly influenced by DAF-16 but that is strongly influenced by KRI-1, a key downstream effector in the pathway by which germline loss increases C. elegans lifespan. The complex behavior of this enhancer presumably allows vitellogenin gene transcription to adjust to demands of body size, germline proliferation and nutritional state but we suggest that the apparent involvement of this enhancer in aging and longevity “pathways” could be incidental.

Toxic effects of bisphenol A on early life stages of Japanese medaka (Oryzias latipes).

The toxic effects of bisphenol A (BPA) in aquatic organisms have attracted global attention. However, few studies have investigated its effects at the gene transcription level. In this study, we measured the transcriptional response of a set of genes associated with the hypothalamic-pituitary-gonadal axis following BPA exposure during the early life stage of Japanese medaka. Transcription of vitellogenin genes was induced in both sexes, indicating estrogenic disruption. However, changes in transcription of the steroid hormone receptor gene and steroidogenesis-regulating genes suggest that BPA also acts as an androgen receptor antagonist. BPA exposure also decreased the hatchability of medaka embryos and increased the growth of female larvae. These pronounced gender-specific effects observed in this study demonstrate that it is important to identify the sex of fish in the early life stage.

Comparative responses to endocrine disrupting compounds in early life stages of Atlantic salmon, Salmo salar

Atlantic salmon (Salmo salar) are endangered anadromous fish that may be exposed to feminizing endocrine disrupting compounds (EDCs) during early development, potentially altering physiological capacities, survival and fitness. To assess differential life stage sensitivity to common EDCs, we carried out short-term (4 day) exposures using three doses each of 17 α-ethinylestradiol (EE2), 17 β-estradiol (E2), and nonylphenol (NP) on four early life stages; embryos, yolk-sac larvae, feeding fry and 1 year old smolts. Differential response was compared using vitellogenin (Vtg, a precursor egg protein) gene transcription. Smolts were also examined for impacts on plasma Vtg, cortisol, thyroid hormones (T4/T3) and hepatosomatic index (HSI). Compound-related mortality was not observed in any life stage, but Vtg mRNA was elevated in a dose-dependent manner in yolk-sac larvae, fry and smolts but not in embryos. The estrogens EE2 and E2 were consistently stronger inducers of Vtg than NP. Embryos responded significantly to the highest concentration of EE2 only, while older life stages responded to the highest doses of all three compounds, as well as intermediate doses of EE2 and E2. Maximal transcription was greater for fry among the three earliest life stages, suggesting fry may be the most responsive life stage in early development. Smolt plasma Vtg was also significantly increased, and this response was observed at lower doses of each compound than was detected by gene transcription suggesting plasma Vtg is a more sensitive indicator at this life stage. HSI was increased at the highest doses of EE2 and E2, and plasma T3 was decreased at the highest dose of EE2. Our results indicate that all life stages are potentially sensitive to endocrine disruption by estrogenic compounds and that physiological responses were altered over a short window of exposure, indicating the potential for these compounds to impact fish in the wild.

Bluegill Lepomis macrochirus vitellogenin gene transcription as a biomarker for xenoestrogenic contamination of water environments

Bluegill Lepomis macrochirus vitellogenin gene transcription as a biomarker for xenoestrogenic contamination of water environments

Assessing the effects of treated and untreated urban discharges to estuarine and coastal waters applying selected biomarkers on caged mussels

To assess effects of urban discharges, biomarkers were measured in caged mussels in northern Iberian Peninsula. Lysosomal membrane stability and histopathology of gonad and digestive gland were analysed as general effect biomarkers. Exposure to specific pollutants was evaluated by autometallographical detection of metals, peroxisomal acyl-CoA oxidase activity, micronucleus test and transcription levels of vitellogenin and MT20 genes. Health status of mussels was impaired after 3days of caging at the untreated outfall discharge and at the waste water treatment plant effluent discharge to the estuary. The most relevant finding was the significant up-regulation of vitellogenin gene transcription in male mussels exposed to the untreated outfall discharge. Metals and xenoestrogenic endocrine disruptors were bioavailable in some discharges and disturbed the health status of mussels. Biomarkers were effective in the assessment of effects of urban discharges and could be implemented in operative controls required to assess the risks associated to effluent discharges.

Endocrine disruption by di‐(2‐ethylhexyl)‐phthalate in Chinese rare minnow (Gobiocypris rarus)

Great concern has been raised over the potential impact of environmental contaminants on fish populations that inhabit the Three Gorge Reservoir. The present study investigated the endocrine-disrupting effects of di-(2-ethylhexyl)-phthalate (DEHP) on the Chinese rare minnow (Gobiocypris rarus), an endemic fish distributed in upstream waters in the Yangtze River. Adult rare minnow were exposed to environmentally relevant concentrations of DEHP (0 µg/L, 3.6 µg/L, 12.8 µg/L, 39.4 µg/L, and 117.6 µg/L) for a 21-d period. Then, concentrations of sex hormones in the plasma and relative transcription of various associated genes were measured in the hypothalamic-pituitary-gonadal (HPG) axis and liver of the fish. Exposure to DEHP resulted in greater circulating concentrations of testosterone (T) and lower concentrations of estradiol (E2), which were accompanied by upregulation of Cyp17 mRNA and downregulation of Cyp19a mRNA in the gonads of females. In males, increases of T and E2 levels were consistent with upregulation of Cyp17 and Cyp19a in the gonads. Furthermore, the T/E2 ratio was increased in females but reduced in males. A significant increase in the levels of hepatic vitellogenin (VTG) gene transcription was observed in both females and males. The present study showed that waterborne exposure to DEHP altered plasma sex hormone levels and modulated gene transcription profiles of associated genes in the HPG axis and liver, occurring mostly at higher concentrations (>39.4 µg/L), which suggests that environmental concentration of DEHP (5.4 µg/L) alone might not disturb the endocrine system of the rare minnow in the TGR.

Transcriptional responses in Japanese medaka ( Oryzias latipes) exposed to binary mixtures of an estrogen and anti-estrogens

Determining ecotoxicological risks of exposure to mixtures of endocrine disrupting chemicals (EDCs) remains a daunting challenge in environmental toxicology. Recently, some studies have illustrated that transcriptional profiling of genes offers the potential to identify the chemical causation of effects that are induced by exposure to complex mixtures. In the present study, the transcriptional responses of a set of genes involved in the hypothalamic–pituitary–gonadal (HPG, or HPG[L]-liver) axis of Japanese medaka ( Oryzias latipes) were systematically examined after treatment with a combination of an estrogen (17α-ethinylestradiol [EE2], 20 ng/L) and two model anti-estrogens, the aromatase inhibitor (AI) letrozole (LET) and the selective estrogen-receptor modulator (SERM) tamoxifen (TAM), at three concentrations (30, 100 and 300 μg/L) for 72 h. The data presented demonstrate that although gene transcription analyses increase our mechanistic understanding of the modes of action (MOAs) of EDCs, the characteristic of most genes altered by a certain single chemical exposure may not be useful for diagnostic chemical causation in a mixture exposure situation. For example, the induction of one vitellogenin gene (VTG1) transcription caused by EE2 in male fish was effectively blocked after exposure to a combination of EE2 and LET but not EE2 and TAM. Moreover, the responses in gene transcription to coexposure were elicited partially in a nonmonotonic concentration-dependent manner. Therefore, the application of transcriptional profiling of genes for screening complex environmental samples should be further evaluated until biomarker gene responses are robust and sensitive enough to properly assess the complex interactions.

A quantitative real‐time polymerase chain reaction method for the analysis of vitellogenin transcripts in model and nonmodel fish species

The measurement of vitellogenin (vtg) gene transcription has been shown to be a reliable indicator of exposure to estrogenic compounds. Unfortunately, the relatively poor molecular characterization of North American fish species has hindered its application to a larger number of ecologically important species. The current research aimed to demonstrate specific amplification of vtg gene transcripts in three model (zebrafish, rainbow trout, and medaka) and six nonmodel (emerald shiner, pearl dace, smallmouth bass, creek chub, white sucker, and golden redhorse) fish species. Quantitative polymerase chain reaction (QPCR) primers for model species were designed from publicly available vtg sequences. Successful amplification of vtg was demonstrated in fish exposed to 17alpha-ethinylestradiol (EE(2)) for all model species. Vitellogenin primers for selected nonmodel species were designed from published sequences of closely related species. Multiple primers were developed targeting different regions of the vtg gene. The successful amplification of vtg was confirmed through size and sequence analysis for all nonmodel species with the exception of the white sucker, in which amplifications failed. Furthermore, QPCR primers and conditions were quantitative over five orders of magnitude in at least one species (pearl dace) exposed to 5 ng/L of EE(2) for 24 h. The selected species are found in a wide array of ecological habitats that span the United States. Inclusion of vtg transcriptional analysis for wild, ecologically relevant fish in monitoring studies may aid in understanding the extent of estrogenic exposure in aquatic ecosystems across the United States.

Intersexuality and behavior in crayfish: The de-masculinization effects of androgenic gland ablation

In crustaceans, male differentiation and primary and secondary characteristics are regulated by the androgenic gland (AG). In gonochoristic crustaceans, the AG is also linked to intersexuality. Whereas the co-occurrence of various male and female characteristics has been demonstrated in intersex crustaceans, little is known regarding sexually dimorphic behavior patterns in such individuals. In the present study, we used an intersex crayfish model to investigate – for the first time in crustaceans – the agonistic and mating behavior of intersex individuals, and to explore the effects of AG ablation on behavior, morphology and physiology. As was the case for their morphological and physiological reproductive traits, intersex individuals – despite being genotypically females – generally resembled males in terms of behavior: they engaged in fighting with males and copulated with receptive females. However, fighting durations of intersex animals were intermediate between those of males and females, and the durations of the copulations were remarkably short. Adult intersex individuals that had been AG ablated at the juvenile stage were unlikely to engage in fighting with males (similar behavior to females) and did not exhibit any mating behavior with receptive females. AG ablation resulted in feminine morphological and physiological shifts in the treated intersex individuals and enabled vitellogenin gene transcription and the onset of secondary vitellogenesis. It thus appears that an as-yet-unknown AG-secreted factor(s) regulating maleness also seems to regulate the organization of male behavior in crustaceans.

Temporary impairment of reproduction in freshwater teleost exposed to nonylphenol

New born guppies, Poecilia reticulata, were exposed to a sublethal concentration of nonylphenol (NP) for 90 days, with the aim of evaluating the influence that this pollutant has on reproductive functions. At the end of treatment, the gender balance was significantly biased towards females ( sex ratio =0.3 males per female). Treated males displayed anomalous transcription of vitellogenin in correlation with a significant reduction of the gonadosomatic index (GSI); however, an increase of vitellogenin gene transcription, correlating directly with an increase in GSI, was observed in NP treated females. The hepatosomatic index (HSI) was significantly increased in exposed males and females. Females exposed to NP showed no alteration of sexual behavior or reproduction. These findings in guppies suggest that NP has estrogenic potency sufficient to disturb reproduction. Effects were transitory in nature because three months in uncontaminated water allowed males to recover normal sexual efficiency.

Quantity does matter. Juvenile hormone and the onset of vitellogenesis in the German cockroach

We aimed to elucidate why cockroaches do not produce vitellogenin in immature stages, by studying the appearance of vitellogenin mRNA in larvae of Blattella germanica. Treatment of female larvae in any of the last three instars with 1 μg of juvenile hormone (JH) III induces vitellogenin gene transcription, which indicates that the fat body is competent to transcribe vitellogenin at least from the antepenultimate instar larvae. In untreated females, vitellogenin production starts on day 1 after the imaginal molt, when corpora allata begin to synthesize JH III at rates doubling the maximal of larval stages. This coincidence suggests that the female reaches the threshold of JH production necessary to induce vitellogenin synthesis on day 1 of adult life. These data lead to postulate that larvae do not synthesize vitellogenin simply because they do not produce enough JH, not because their fat body is incompetent.

17α‐ethynylestradiol‐induced vitellogenin gene transcription quantified in livers of adult males, larvae, and gills of fathead minnows (Pimephales promelas)

We have applied a method for quantifying relative levels of messenger RNA (mRNA) transcription to assess chemically induced gene expression in fathead minnows (Pimephales promelas). Synthetic oligonucleotides designed for the fathead minnow vitellogenin gene transcription product were used in a reverse transcription polymerase chain reaction (RT-PCR) protocol. This sensitive and rapid strategy detected vitellogenin gene transcription in livers of male fathead minnows exposed to concentrations as low as 2 ng/L of the endocrine-disrupting compound 17alpha-ethynylestradiol for 24 h. Surprisingly, vitellogenin transcription products also were detected in gill tissue and in 48-h-old posthatch fathead minnow larvae. Relative levels of vitellogenin gene induction among individuals were quantified in a single-step reaction (PCR multiplex) with 18S rRNA universal primers and Competimers concurrently with fathead minnow vitellogenin oligonucleotides. This quantitative approach will markedly enhance detection of the first cellular event of estrogenic exposure to aquatic ecosystems in both field and laboratory systems. Use of the model provides sensitivity of detection at a concentration below those that cause mortality or visible signs of stress in fish or other aquatic organisms. The model may also provide an in vivo screening method for estrogenlike endocrine-disrupting compounds.