Induction Of Vitellogenin mRNA Research Articles

Bioavailability of pharmaceuticals in waters close to wastewater treatment plants: Use of fish bile for exposure assessment

Pharmaceuticals are ubiquitous in surface waters as a consequence of discharges from municipal wastewater treatment plants. However, few studies have assessed the bioavailability of pharmaceuticals to fish in natural waters. In the present study, passive samplers and rainbow trout were experimentally deployed next to three municipal wastewater treatment plants in Finland to evaluate the degree of animal exposure. Pharmaceuticals from several therapeutic classes (in total 15) were analyzed by liquid chromatography-tandem mass spectrometry in extracts of passive samplers and in bile and blood plasma of rainbow trout held at polluted sites for 10 d. Each approach indicated the highest exposure near wastewater treatment plant A and the lowest near that of plant C. Diclofenac, naproxen, and ibuprofen were found in rainbow trout, and their concentrations in bile were 10 to 400 times higher than in plasma. The phase I metabolite hydroxydiclofenac was also detected in bile. Hence, bile proved to be an excellent sample matrix for the exposure assessment of fish. Most of the monitored pharmaceuticals were found in passive samplers, implying that they may overestimate the actual exposure of fish in receiving waters. Two biomarkers, hepatic vitellogenin and cytochrome P4501A, did not reveal clear effects on fish, although a small induction of vitellogenin mRNA was observed in trout caged near wastewater treatment plants B and C.

Comparative responsiveness to natural and synthetic estrogens of fish species commonly used in the laboratory and field monitoring

Exposure to estrogenic chemicals discharged into the aquatic environment has been shown to induce feminization in wild freshwater fish and although fish species have been reported to differ in their susceptibility for these effects, empirical studies that directly address this hypothesis are lacking. In this study, in vitro ERα activation assays were applied in a range of fish species used widely in chemical testing (including, zebrafish, fathead minnow, medaka) and/or as environmental monitoring species (including, roach, stickleback, carp) to assess their comparative responsiveness to natural (estrone, estradiol, estriol) and synthetic (17α-ethinylestradiol (EE2), diethylstilbestrol (DES)) estrogens. In vivo exposures to EE2 via the water (nominal 2 and 10ng/L for 7 days) were also conducted for seven fish species to compare their responsiveness for hepatic vitellogenin (VTG) mRNA induction (an ER mediated response). Of the fish species tested, zebrafish ERα was found to be the most responsive and carp and stickleback ERα the least responsive to natural steroid estrogens. This was also the case for exposure to EE2 with an ERα-mediated response sensitivity order of zebrafish>medaka>roach>fathead minnow>carp>stickleback. For VTG mRNA induction in vivo, the order of species responsiveness was: rainbow trout (not tested in the ERα activation assays)>zebrafish>fathead minnow>medaka>roach>stickleback>carp. Overall, the responses to steroid estrogens in vitro via ERα compared well with those seen in vivo (VTG induction for exposure to EE2) showing in vitro screening of chemicals using fish ERα-mediated responses indicative of estrogenic responses (VTG induction) in vivo.

Effects of exposure water volume, depuration time, and feeding status on vitellogenin mRNA induction in male medaka ( Oryzias latipes) exposed to17 β-estradiol

Bioassays measuring the induction of vitellogenin gene expression in male fish are widely used for revealing estrogenic activity in water samples. Measuring induction of vitellogenin mRNA in males by means of RT-PCR analysis is a sensitive way to detect exposure to estrogenic chemicals. To date, little work has been done to examine variations in exposure conditions for assessing estrogenic activity in water samples using this model system. Here we report the results of experiments investigating the effects of volume of treatment water, time since removal from treatment water (depuration), and short-term food deprivation on vitellogenin mRNA induction in male Japanese medaka ( Oryzias latipes). Fish exposed to a single concentration of E 2 while volume was manipulated were found to have similar levels of vitellogenin mRNA, though more E 2 was present at larger volumes. Removal of fish from E 2-treated-water to clean water after exposures reduced vitellogenin levels in as little 24 h, however, the vitellogenin levels of the fish transferred to the clean water remained above those of the control fish for at least 72 h. Depriving fish of food for up to 72 h during exposure to E 2 did not significantly reduce vitellogenin induction. Together these results support the conclusion that real time RT-PCR measurement of vitellogenin in male fish can be used as a robust indicator of exposure to estrogenic contaminants in water.

Assessing the effects of exposure timing on biomarker expression using 17β-estradiol

Temporal and spatial variability in estrogenicity has been documented for many treated wastewater effluents with the consequences of this variability on the expression of biomarkers of endocrine disruption being largely unknown. Laboratory exposure studies usually utilize constant exposure concentrations which may produce biological effects that differ from those observed in organisms exposed in natural environments. In this study, we investigated the effects of differential timing of exposures with 17β-estradiol (E2) on a range of fathead minnow biomarkers to simulate diverse environmentally relevant exposure profiles. Two 21-day, replicate experiments were performed exposing mature male fathead minnows to E2 at time-weighted mean concentrations (similar average exposure to the contaminant during the 21-day exposure period; 17 ng E2/L experiment 1; 12 ng E2/L experiment 2) comparable to E2 equivalency values (EEQ) reported for several anthropogenically altered environments. A comparable time-weighted mean concentration of E2 was applied to five treatments which varied in the daily application schema: E2 was either applied at a steady rate (ST), in a gradual decreasing concentration (HI), a gradual increasing concentration (LO), applied intermittently (IN), or at a randomly varying concentration (VA). We assessed a range of widely used physiological (vitellogenin mRNA induction and plasma concentrations), anatomical (body and organ indices, secondary sex characteristics, and histopathology), and behavioral (nest holding) biomarkers reported to change following exposure to endocrine active compounds (EACs). All treatments responded with a rise in plasma vitellogenin concentration when compared with the ethanol carrier control. Predicatively, vitellogenin mRNA induction, which tracked closely with plasma vitellogenin concentrations in most treatments was not elevated in the HI treatment, presumably due to the lack of E2 exposure immediately prior to analysis. The ability of treatment male fish to hold nest sites in direct competition with control males was sensitive to E2 exposure and did yield statistically significant differences between treatments and carrier control. Other biological endpoints assessed in this study (organosomatic indices, secondary sex characteristics) varied little between treatments and controls. This study indicates that a broad suite of endpoints is necessary to fully assess the biological consequences of fish exposure to estrogens and that for at least field studies, a combination of vitellogenin mRNA and plasma vitellogenin analysis are most promising in deciphering exposure histories of wild-caught and caged fishes.

Induction of vitellogenin mRNA in juvenile chinese sturgeon (acipenser sinensis gray) treated with 17β‐estradiol and 4‐nonylphenol

It has been demonstrated that 4-nonylphenol (4-NP) exerts estrogenic effects in diverse fishes. The present study investigated the effects of 4-NP on Chinese sturgeon (Acipenser sinensis Gray) vitellogenin (VTG) gene expression. By reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers, a 462-base pair fragment of Chinese sturgeon VTG, corresponding to a 154-amino acid sequence, was amplified and sequenced. This sequence exhibited 152/154 identity to the amino acid sequence of white sturgeon (A. transmontano) VTG. Conventional RT-PCR and quantitative real-time RT-PCR were established and used to study the VTG and beta-actin gene expression in the liver of juvenile Chinese sturgeon injected three times with 17beta-estradiol (5 mg/kg body wt/week) or 4-NP (10 or 100 mg/kg/week) in the course of three weeks. Significant induction of VTG gene expression was detected in all treated groups, and no VTG mRNA was detected in the control group. The ratio of VTG to beta-actin analyzed from the results of quantitative real-time RT-PCR reached 0.041 +/- 0.024 (mean +/- SD) in the group receiving 10 mg/kg/week of 4-NP and 4.51 +/- 1.68 in the group receiving 100 mg/kg/week of 4-NP. Chemical analysis of 4-NP showed that the concentrations of 4-NP in the 10 mg/kg/week group and the 100 mg/kg/week group were 2.78 +/- 2.41 and 31.38 +/- 0.26 mcirog/ g wet weight, respectively. Compared with the 4-NP concentrations (0.8-1.92 microg/g wet wt) in fish from the Yangtze River, China, a potential hazard exists regarding 4-NP in Chinese sturgeon. These results represent the first indication of the risk of endocrine-disrupting chemicals for Chinese sturgeon.

Quantification of vitellogenin mRNA induction in mosquitofish (Gambusia affinis) by reverse transcription real-time polymerase chain reaction (RT-PCR)

A method to quantify induction of vitellogenin (Vtg) mRNA in adult male mosquitofish was developed. Male mosquitofish were exposed to 0, 1, 20 and 250 ng l−1 17β-oestradiol (E2) for 4 and 8 days in static exposures, and liver Vtg mRNA and 18S rRNA expression were quantified in duplex RT-PCR. Liver 18S rRNA expression was very consistent among individuals, and there was a highly significant increase in Vtg mRNA expression after exposure of mosquitofish for just 4 days at 250 ng l−1 E2. Lower doses did not induce Vtg mRNA expression even at 4 or 8 days. This method could be used as a rapid test to detect exposure of mosquitofish to oestrogenic chemicals. Further work is needed to determine if increased Vtg mRNA levels in male mosquitofish induce Vtg synthesis, and to determine the usefulness of the method in field sampling.

Use of vitellogenin mRNA as a biomarker for endocrine disruption in feral and cultured fish.

The presence of the female-specific yolk protein precursor vitellogenin in blood and liver from male fish is widely used as an indicator of endocrine disruption. We studied the induction of vitellogenin mRNA in liver from several species of fish, both maintained in fish tanks or captured in the wild. Our procedure requires minute amounts of liver samples (down to 50 mg), and can be applied to field samples if the appropriate RNA-stabilisation agent is used. We used reverse-transcriptase PCR and quantitative real-time PCR for detection and precise quantitation of vitellogenin mRNA levels. Male mummichog (Fundulus heteroclitus) exposed to 17 beta-estradiol contained levels of vitellogenin mRNA up to 30 times higher than in untreated females and treatment with nonylphenol resulted in a weak but consistent induction of this transcript. We also studied levels of vitellogenin mRNA in a population of common carp (Cyprinus carpio) from the Anoia river, a river known for its high levels of estrogenic alkylphenols. The results were consistent with recorded data for fish from this sampling site. Finally, we also detected vitellogenin mRNA in barbs (Barbus graellsi), a species for which no vitellogenin sequence was available. The use of mRNA quantitation techniques for analysis of feral and cultured fish of different species opens the possibility of more precise detection and further control of the noxious effects of contaminants on the local fauna exposed to them.

Preliminary investigations on vitellogenin m-RNA induction in some bioindicator Mediterranean fish species

This paper describes the development of a RT-PCR method for assaying Vtg gene expression in different marine fish as a potentially valuable and sensitive biomarker of exposure to estrogenic chemicals. The levels of Vtg mRNA have been analyzed using primers specifically designed for the various species and the procedures have been standardized relative to actine mRNA expression levels. Different species were analyzed including organisms with a great potential as bioindicators in the Mediterranean (i.e. the red mullet Mullus barbatus, the striped mullet Mugil cephalus, the European eel Anguilla anguilla) or exposed to biomagnification of halogenated hydrocarbons and with elevated commercial value (the bluefin tuna Thunnus thynnus). The analysis of vitellogenin mRNA levels has been standardized in feral fish providing suitable indications for a future development of this approach.

A Reverse Transcription–Polymerase Chain Reaction Bioassay for Avian Vitellogenin mRNA

A bioassay based on the measurement of vitellogenin (VTG) mRNA in avian embryo hepatocyte cultures by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) was developed. To allow sequence comparison and design of suitable PCR primers, a short region of VTG cDNA was cloned and sequenced for seven species of birds. Cell cultures were prepared from both chicken and herring gull embryos and treated with the estradiol analogue moxestrol or the organochlorine insecticide o,p′-DDT. Using primers based on an area of the VTG gene that was identical for herring gull and chicken, in vitro VTG mRNA induction was observed for both moxestrol- and o,p′-DDT-treated cultures. Herring gull embryo hepatocyte cultures responded with VTG mRNA induction at moxestrol concentrations of 1 nM compared with 10 nM in chicken embryo hepatocyte cultures. Both herring gull and chicken embryo hepatocyte cultures responded with substantial VTG mRNA induction when treated with 10,000 nM o,p′-DDT. These results suggest that the bioassay will be useful for comparing avian embryo hepatocyte culture concentration–response data in terms of intra- and interspecies sensitivities to pharmacological estrogens or environmental contaminants.

Effects of p-nonylphenol, methoxychlor, and endosulfan on vitellogenin induction and expression in sheepshead minnow (Cyprinodon variegatus).

Temporal and dose-response relationships of vitellogenin (VTG) mRNA induction and subsequent plasma VTG accumulation were established for sheepshead minnows (Cyprinodon variegatus) treated with p-nonylphenol (an alkylphenol) and the organochlorine pesticides methoxychlor and endosulfan. Thirty-two adult male fish per treatment were continuously exposed to measured concentrations of 0.64, 5.4, 11.8, 23.3, and 42.7 micrograms/L p-nonylphenol; 1.1, 2.5, 5.6, 12.1, and 18.4 micrograms/L methoxychlor; and in two separate tests, 15.9, 36.3, 68.8, 162, 277, 403, 590, and 788 ng/L endosulfan using an intermittent flow-through dosing apparatus. Separate triethylene glycol (50 microliters/L) and 17 beta-estradiol (65.1 ng/L) treatments served as the negative and positive controls, respectively. Four fish were randomly sampled from each test concentration on days 2, 5, 13, 21, 35, and 42 of exposure, and levels of hepatic VTG mRNA induction and serum VTG accumulation were determined for each individual. Overall, fish exposed to p-nonylphenol or methoxychlor demonstrated a rapid, dose-dependent synthesis of VTG mRNA up to day 5 of exposure, followed by a relatively constant dose-dependent expression through day 42. Both chemicals showed a dose-dependent increase in plasma VTG over the entire time course of exposure, with significantly elevated VTG levels by the fifth day of exposure to p-nonylphenol at concentrations of 5.4 micrograms/L or greater and to methoxychlor at concentrations of 2.5 micrograms/L or greater. Exposure to 0.64 microgram/L p-nonylphenol resulted in highly variable plasma VTG levels of less than 6 mg/ml. Exposures with endosulfan failed to induce measurable levels of either hepatic VTG mRNA or serum VTG at the chemical concentrations tested. Our results demonstrate that the sheepshead minnow bioassay is a suitable estuarine/marine teleost model for in vivo screening of potentially estrogenic substances.

Aluminium and Cadmium Inhibit Vitellogenin and Its mRNA Induction by Estradiol-17 β in the Primary Culture of Hepatocytes in the Rainbow Trout Oncorhynchus mykiss

Effects of Al and Cd on vitellogenin (VTG) and VTG mRNA induction by estradiol-17 β (E2) were examined in primary hepatocyte cultures of rainbow trout. Hepatocytes were precultured for 2 days and then E2 (2 × 10−6 M) and Al (10−6–10−4 M) or Cd (10−9–10−6 M) were simultaneously added to the incubation medium. Hepatocytes were cultured for 5 more days. Media and hepatocytes were then analyzed by SDS-PAGE and Northern blotting for VTG and VTG mRNA, respectively. These metals had no appreciable effect on the viability of hepatocytes in culture. However, Al and Cd interfered with VTG production and VTG mRNA expression. Al reduced VTG production in a concentration-dependent way and a significant reduction occurred at Al concentrations greater than 5 × 10−5 M. VTG mRNA expression also decreased with a negative correlation with Al concentrations (r = −0.98). The inhibition of VTG production by Cd was not concentration-dependent. This metal markedly inhibited VTG production and VTG mRNA expression at 10−6 M. The Al-induced inhibition of VTG production was restored 7 days after Al removal, but the Cd-induced inhibition was not restored. These results suggest that Al and Cd inhibit VTG production at the transcriptional level to reduce VTG mRNA expression by different mechanisms.

Analysis of vitellogenin mRNA by quantitive reverse transcription polymerase chain reaction (RT–PCR) in juvenile fish exposed for 12 months to nonylphenol

Nonylphenol (NP) is a degradation product of surfactants that enter aquatic systems mainly via sewage treatment plants. It has been shown that the estrogenic activity of NP results in the induction of vitellogenin (VG) in male fish after short-term exposure, but the effects after long-term exposure to environmentally relevant concentrations remain unclear. Vitellogenin is a precursor of egg yolk proteins, synthesized in the liver under the control of the female sex hormone estradiol and transferred to the ovaries via the blood. Induction of VG can therefore serve as a biomarker for estrogenicity. In this study, rainbow trout eggs were exposed after fertilization to NP concentrations of 1 and 10 μg/l. Exposure occurred throughout the embryonic, larval and juvenile period under controlled laboratory conditions. After 12 months, induction of VG mRNA was analyzed in the liver by quantitative RT–PCR, and VG protein using polyclonal antibodies in Western blots. The development of quantitative RT–PCR included primer design, competitive PCR using heterologous standards and titration. Both VG mRNA and protein were induced in NP-exposed rainbow trout in a dose-dependent manner. In male fish, increases in VG mRNA and protein were already observed at 1 μg/l NP. This study shows that chronic exposure of fish early life stages to environmentally realistic concentrations of NP leads to induction of vitellogenin.

Determination of a Screening System of Endocrine Disruptors by the Induction of Vitellogenin mRNA in C. elegans Larvae(PROCEEDINGS OF 24TH SYMPOSIUM ON TOXICOLOGY AND ENVIRONMENTAL HEALTH)

Determination of a Screening System of Endocrine Disruptors by the Induction of Vitellogenin mRNA in C. elegans Larvae(PROCEEDINGS OF 24TH SYMPOSIUM ON TOXICOLOGY AND ENVIRONMENTAL HEALTH)

Interaction of cadmium and oestradiol-17β on metallothionein and vitellogenin synthesis in rainbow trout (Oncorhynchus mykiss)

The induction of metallothionein and vitellogenin synthesis in rainbow trout liver was studied after injection of oestradiol-17 beta alone or in combination with cadmium or zinc. Intraperitoneal injection of oestradiol-17 beta increased the liver somatic index, with subsequent induction of vitellogenin synthesis. Oestradiol-17 beta did not induce metallothionein synthesis. Injection of cadmium induced the synthesis of metallothionein mRNA and metallothionein. Injection of oestradiol-17 beta in combination with cadmium resulted in inhibition of transcription and translation of both vitellogenin and metallothionein. Chromatography of liver cytosols revealed that cadmium, when co-injected with oestradiol-17 beta, did not bind to metallothionein but would initially bind to high-molecular-mass (HMr) cytosolic proteins. In fish injected with cadmium in combination with oestradiol-17 beta, cadmium was gradually redistributed from HMr proteins to metallothionein. This resulted in induction of metallothionein synthesis and in binding of most of the cadmium to metallothionein. Induction of vitellogenin mRNA was observed 15 days after injection, as cadmium was being redistributed to newly synthesized metallothionein. These findings indicate that cadmium inhibits the transcription of vitellogenin. The binding of cadmium to these non-metallothionein proteins represses the induction of metallothionein and results in increased toxicity of the metal. Preinduction of metallothionein by zinc injections resulted in decreased cadmium sensitivity of the fish and a decrease in the repression of vitellogenin mRNA. Furthermore, a role for metallothionein in the detoxification of cadmium is indicated by the induction of vitellogenin synthesis that occurs once metallothionein has begun sequestering cadmium.

Vitellogenin gene expression in male rainbow trout ( Salmo gairdneri

Estrogen has a marked effect on the expression of vitellogenin, the egg yolk precursor protein in the liver of egg-laying vertebrates. cDNA clones specific for trout vitellogenin mRNA have been used to study the expression of the vitellogenin genes in rainbow trout ( Salmo gairdneri. The steady-state levels of vitellogenin mRNA in the liver of male rainbow trout were measured during primary and secondary stimulation with estradiol. The kinetics of induction in trout appear to be very similar to those seen in Xenopusand chicken in that a lag of approximately 2 days is observed in the accumulation of serum vitellogenin during primary induction. This lag is not observed during the secondary stimulation. The primary induction of vitellogenin mRNA in trout liver, using a single injection of estradiol (3 mg/kg body wt) results in a short-lived rise, reaching a maximum level of 260 ppm total RNA on Day 2. Using silastic implants of estradiol to induce a primary response produces a large increase in the steady-state level of vitellogenin mRNA which reaches a maximum of 2750 ppm total RNA on Day 10. During secondary stimulation, using silastic tubing the maximum level reached was 1200 ppm of the total RNA on Day 7, approximately half the level seen in the primary induction using the silastic implants. The difference in these two levels is due to an increase in the steady-state levels of rRNA, which appear to increase between Days 10 and 21 after the primary stimulation. These results demonstrate that the induction of vitellogenesis in the trout by estradiol involves changes in the steady-state levels of a number of different mRNA and rRNA sequences and resembles that seen in Xenopus and chicken.

Persistent estrogen induction of hepatic Xenopus laevis serum retinol binding protein mRNA.

Administration of estradiol-17 beta to male Xenopus laevis induces the hepatic mRNA coding for the serum retinol binding protein (RBP) approximately 10-fold, both in vivo and in primary liver cultures. Estrogen induction of RBP mRNA is completely blocked by the anti-estrogen, hydroxytamoxifen. Testosterone administration reduces the elevated level of RBP mRNA observed in livers of female X. laevis to the constitutive level seen in livers of control male animals, and partially blocks the estrogen induction of RBP mRNA. Intracellular RBP mRNA levels therefore represent a balance between the opposing effects of estradiol-17 beta and testosterone. In marked contrast to the estrogen induction of vitellogenin mRNA, which requires the continuous presence of exogenous estrogen, induction of RBP mRNA persists for at least 4 months after a single injection of estrogen. Runoff transcription measurements demonstrate that persistent induction of RBP mRNA is due to an increased rate of RBP gene transcription. Administration of hydroxytamoxifen abolishes persistent induction of RBP mRNA, suggesting that residual hormone receptor complex plays a role in the persistent induction of RBP gene transcription. The persistent estrogen induction of RBP mRNA provides the first demonstration of long-term activation of the transcription of a hormone-responsive gene in response to a transient dose of a steroid hormone.

The preparation and characterization of vitellogenin messenger RNA from rainbow trout (Salmo gairdneri).

Agarose-gel electrophoresis of polyadenylated RNA from livers of oestrogen-treated male rainbow trout revealed a major high-Mr species (7200 nucleotides), which is absent from the polyadenylated RNA isolated from hormonally unstimulated male trout liver. Translation in vitro of the RNA from oestrogen-treated males in a mRNA-dependent rabbit reticulocyte lysate produced a protein (Mr 200 000) that could be immunoprecipitated with antibodies against trout serum vitellogenin, but no immunoprecipitable protein was synthesized with RNA from control animals. DNA complementary to the RNA from oestrogen-stimulated and control male trout liver was synthesized and back-hybridized, with R0t1/2 of 3.8 X 10(-2) and 1 X 10(-1) mol X litre-1 X s for RNA from hormone-treated and control animals respectively. The 9% increase in the abundant mRNA after oestrogen stimulation is due to the induction of vitellogenin mRNA.

Induction of estrogen receptor and reversal of the nuclear/cytoplasmic receptor ratio during vitellogenin synthesis and withdrawal in Xenopus laevis.

The levels of cytoplasmic and nuclear estrogen receptor have been determined in livers of male Xenopus laevis stimulated by estradiol-17 beta to synthesize vitellogenin mRNA. Estrogen receptor levels were also determined in unstimulated liver and following long term withdrawal of estrogen. In unstimulated liver cells, which do not contain detectable vitellogenin mRNA, more than 80% of the estrogen receptor is located in the nucleus (550 high affinity estrogen binding sites/nucleus), while the cytoplasm contains only 100 high affinity estrogen binding sites/cell. Administration of estradiol-17 beta, which induces massive synthesis and accumulation of vitellogenin mRNA, induces the estrogen receptor as well. The nuclear receptor level rises to approximately 2,000 estrogen binding sites/cell, while the cytosol receptor increases to only 150 sites/cel. Liver cells of male X. laevis which have been withdrawn from estrogen for 70 days exhibit a striking change in receptor levels. The nuclear receptor returns to the level prevailing in unstimulated cells (approximately 500 sites/cell) while the cytosol receptor level rises to more than 1,200 sites/cell (equivalent to 260 fmol/g of tissue). The existence of a pool of cytosol receptor, which is rapidly available for induction of vitellogenin mRNA, may in part explain the shorter lag period and more rapid induction of vitellogenin mRNA observed during secondary estrogen stimulation of withdrawn Xenopus liver cells.

Primary induction of vitellogenin mRNA in the rooster by 17beta-estradiol.

We have studied the kinetics of vitellogenin mRNA accumulation in rooster liver after a primary injection of 17beta-estradiol. The levels of vitellogenin mRNA have been determined both by hybridization of total cellular RNA to vitellogenin cDNA and by translation of vitellogenin mRNA in a wheat germ cell-free system. The results obtained by both methods of analysis are in good agreement and indicate that vitellogenin mRNA is present in the liver of normal roosters at a level of 0-5 molecules per liver cell and increases in amount during the 3 days following injection of estrogen, reaching a level of almost 6000 molecules per cell at the peak of the response. The level of vitellogenin mRNA declined exponentially during the next 14 days with a half-life of 29 hr, reaching a level of less than 10 molecules per cell at 17 days after injection of the hormone. The levels of vitellogenin mRNA after stimulation with estrogen have been correlated with the in vivo rate of synthesis of the vitellogenin polypeptide. The results indicate that the rate of vitellogenin synthesis is closely correlated with the level of vitellogenin mRNA. On the basis of these findings, we conclude that vitellogenin mRNA does not exist in the liver in an untranslated form after withdrawal from estrogen.