Biomedical subjects

Nancy D Denslow

Publications and source records attributed to Nancy D Denslow.

9 recordsLinked to original sources

PubMed2003

Relationship between reproductive success and male plasma vitellogenin concentrations in cunner, Tautogolabrus adspersus.

The gene for vitellogenin, an egg yolk protein precursor, is usually silent in male fish but can be induced by estrogen exposure. For this reason, vitellogenin production in male fish has become a widely used indicator of exposure to exogenous estrogens or estrogen mimics in the aquatic environment. The utility of this indicator to predict impacts on fish reproductive success is unclear because information on the relationship between male plasma vitellogenin and reproductive end points in male and female fish is limited. In the research reported in this article, we investigated whether the presence of male plasma vitellogenin is a reliable indicator of decreased reproductive success in mature fish. Adult and sexually mature male and female cunner (Tautogolabrus adspersus) were exposed to 17ss-estradiol, ethynylestradiol, or estrone, three steroidal estrogens that elicit the vitellogenic response. Data were gathered and pooled on egg production, egg viability, egg fertility, sperm motility, and male plasma vitellogenin concentrations. All males, including two with plasma vitellogenin levels exceeding 300 mg/mL, produced motile sperm. Neither percent fertile eggs nor percent viable eggs produced by reproductively active fish demonstrated a significant correlation with male plasma vitellogenin concentrations. Male gonadosomatic index and average daily egg production by females showed significant, but weak, negative correlation with male plasma vitellogenin concentrations. Results suggest that male plasma vitellogenin expression is not a reliable indicator of male reproductive dysfunction in adult cunner exposed to estrogens for 2-8 weeks during their reproductive season, at least in relation to capacity to produce motile sperm or fertilize eggs. Male plasma vitellogenin expression may serve as an indicator of reduced female reproductive function caused by estrogen exposure.

Animals↗

PubMed2003

Effects of pulp and paper mill effluents on reproductive success of largemouth bass.

This study evaluated the effects of bleached and unbleached kraft mill effluent on reproductive success of largemouth bass (Micropterus salmoides). Bass were exposed to effluent concentrations (0, 10, 20, 40, or 80%) for 28 and 56 d. Parameters measured included hepatosomatic index (HSI) and gonadosomatic index (GSI) and plasma concentrations of 17beta-estradiol (E2), 11-ketotestosterone (11-KT), and vitellogenin (VTG). At the end of the 56-d period, bass were moved to hatchery ponds to evaluate spawning success. Spawning mats with eggs either were brought indoors for evaluation of fecundities, hatchabilities, and egg and fry size (measured at age 3 d), or were left in ponds and fry number and size recorded (average age of 14 d). Effluent exposure was verified by measuring resin acids (isopimaric, abietic. and dehydroabietic acids) in bile. Compared to controls, exposed bass had greater concentrations of resin acids in bile. In general, exposed females had lower concentrations of E2 and VTG (> or = 20% effluent), whereas males had lower concentrations of 11-KT (> or = 20% effluent) and increased E2 (> or = 20% effluent). The HSI values increased in females (> or = 10% effluent), and GSI values decreased in both sexes (> or = 40% effluent). Fecundity, egg size, and hatchability did not differ across treatments, but an increase in the frequency of fry abnormalities and a decrease in fry weights was observed at effluent exposures of 40% and higher. However, results from the pond study, revealed a significant reduction in fry growth and survival (> or = 10%). This decline may have been caused by an increased frequency of deformities, in conjunction with alterations of growth. These changes could have resulted from alterations in egg quality because of failure of parental reproductive systems, from acute embryo toxicity after translocation of contaminants from the mother to the developing embryo, or from both.

Animals↗

PubMed2003

Expression profiling of estrogenic compounds using a sheepshead minnow cDNA macroarray.

A variety of anthropogenic compounds are capable of binding to the estrogen receptor (ER) of vertebrate species. Binding of these chemicals to the ER can interfere with homeostasis by altering normal gene expression patterns. The purpose of this study was to characterize the expression of 30 genes using a sheepshead minnow (Cyprinodon variegatus) cDNA macroarray. Many of the genes on the array were previously identified by differential display reverse transcriptase-polymerase chain reaction to be upregulated or downregulated in sheepshead minnows treated through aqueous exposure to known or suspected estrogenic chemicals. The results of this study show that 17 beta-estradiol (E2), 17 alpha-ethinyl estradiol (EE2), diethylstilbestrol (DES), and methoxychlor (MXC) have similar genetic signatures for the 30 genes examined. The genetic signature of fish treated with p-nonylphenol was identical in pattern to that in fish treated with E2, EE2, DES, and MXC except for the additional upregulation of a cDNA clone that shares similarity to ubiquitin-conjugating enzyme 9. Endosulfan produced results that resembled the gene expression patterns of untreated control fish with exception of the upregulation of estrogen receptor alpha and the downregulation of a cDNA clone that shares similarity to 3-hydroxy-3-methylglutaryl-coenzyme A reductase. We show that our estrogen-responsive cDNA macroarray can detect dose-dependent changes in gene expression patterns in fish treated with EE2.

Animals↗

PubMed2002

Estradiol-induced gene expression in largemouth bass (Micropterus salmoides).

Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E(2)). Single injections of E(2) ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ER alpha sequences. After acute E(2)-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36-42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E(2) in largemouth bass and that for Vtg a delayed primary response. The specific effect of E(2) on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen gamma was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E(2) involves a complex network of responses over time.

Amino Acid Sequence↗

PubMed2002

A comparison of the estrogenic potencies of estradiol, ethynylestradiol, diethylstilbestrol, nonylphenol and methoxychlor in vivo and in vitro.

Five natural, pharmaceutical, or xenobiotic chemicals [17beta-estradiol (E2), ethynylestradiol (EE2), diethystilbestrol (DES), methoxychlor (MXC), nonylphenol (NP)] were tested in two in vitro assays [yeast estrogen screen (YES), MCF-7 breast tumor cell proliferation (E-Screen)], and compared with previously reported results from two in vivo male sheepshead minnow vitellogenin (VTG) production studies. The purpose of this investigation was to determine how accurately the two in vitro assays predicted responses observed in live animals. EC50 values for all five chemicals were approximately one order of magnitude less sensitive in the YES assay than in the MCF-7 assay. Based on the EC50 values, DES was 1.1 (YES) to 2.5 (MCF-7) times more potent in these receptor binding assays than was E2, while EE2 was slightly less potent than E2 in the YES assay (0.7) and nearly twice as potent (1.9) as E2 in the MCF-7 assay. EE2 and DES were of approximately equal potency in the 13-day sheepshead minnow VTG production bioassay. Both MXC and NP were 10(7) times less potent than E2 in the YES assay, MXC was 10(5) times less estrogenic than E2 in the MCF-7 assay, while both were approximately 100 times less potent than E2 in the live animal bioassay. The in vitro tests were substantially less sensitive (at least 1000 times) than the sheepshead minnow VTG assay for estimating estrogenic potency of the two xenobiotic chemicals, which suggests that in vitro-based, large-scale screening programs could potentially result in many false negative evaluations.

Animals↗

PubMed2002

An evaluation of biomarkers of reproductive function and potential contaminant effects in Florida largemouth bass ( Micropterus salmoidesfloridanus) sampled from the St. Johns River.

The objective of this study was to describe and compare several reproductive parameters for Florida largemouth bass (Micropterus salmoides floridanus) inhabiting the St. Johns River and exposed to different types and/or degrees of contamination. Welaka was selected as the reference site in this study because of its low urban and agricultural development, Palatka is in close proximity to a paper mill plant, the Green Cove site is influenced by marine shipping activities and Julington Creek site receives discharges of domestic wastewater and storm water runoff from recreational boating marinas. For this study, bass were sampled both prior to (September 1996) and during the spawning season (February 1997). In order to characterize chemical exposure, bass livers were analyzed for up to 90 trace organics and 11 trace metal contaminants. Reproductive parameters measured included gonadosomatic index (GSI), histological evaluation of gonads and plasma concentrations of vitellogenin (VTG), 17beta-estradiol (E2) and 11-ketotestosterone (11-KT). In general, the sum of organic chemicals was highest in livers from Palatka bass and bass from Green Cove and Julington Creek had higher hepatic concentrations of low molecular polycyclic aromatic hydrocarbons and polychlorinated biphenyls when compared to fish from Welaka. Metals were more variable across sites, with highest mean concentrations found in bass from either Julington Creek (Ag, As, Cr, Cu, Zn) or Welaka (Cd, Hg, Pb, Se, Tn). Female bass from Palatka and Green Cove had lower concentrations of E2, VTG and lower GSI in relation to Welaka. Males from Palatka and Green Cove showed comparable declines in 11-KT in relation to males from Julington Creek and GSI were decreased only in Palatka males. These results indicate a geographical trend in reproductive effects, with changes being most pronounced at the site closest to the paper mill (Palatka) and decreasing as the St. Johns River flows downstream. Since reproductive alterations were most evident in bass sampled from the site closest to the paper mill discharge, it is possible that exposure to these effluents might explain at least some of the results reported here. However, the presence of reproductive alterations in fish sampled at a considerable distance from the mill discharge (Green Cove, 40 km) would suggest exposure to chemicals released from sources other than the paper mill plant. It is clear that additional studies are needed to evaluate the potential impact of these reproductive changes in populations of Florida largemouth bass inhabiting the St. Johns River.

Animals↗

PubMed2002

Vitellogenin mRNA regulation and plasma clearance in male sheepshead minnows, (Cyprinodon variegatus) after cessation of exposure to 17 beta-estradiol and p-nonylphenol.

Research was conducted to determine the kinetics of hepatic vitellogenin (VTG) mRNA regulation and plasma VTG accumulation and clearance in male sheepshead minnows (Cyprinodon variegatus) during and after cessation of exposure to either 17 beta-estradiol (E2) or para-nonylphenol (NP). Adult fish were continuously exposed to aqueous measured concentrations of 0.089 and 0.71 microg E2 per l, and 5.6 and 59.6 microg NP per l for 16 days using an intermittent flow-through dosing apparatus. Fish were sampled on days 8 and 16 of exposure followed by sampling at discrete intervals for up to 96 days post-exposure. At each interval five fish were randomly sampled from each concentration and hepatic VTG mRNA and serum VTG levels for individual fish determined by slot blot and direct enzyme-linked immunosorbent assay (ELISA), respectively. Exposure to E2 and NP resulted in a dose dependent increase in hepatic VTG mRNA and plasma VTG over the course of the 16-day exposure period. Mean plasma VTG levels at day 16 were >100 mg/ml for both high doses of E2 and NP, and >20 mg/ml for the low exposure treatments. Within 8 days post-exposure, hepatic VTG mRNA levels returned to baseline in both high and low E2 treatments but remained elevated 2-4 fold in the NP treatments. Due to a shortened sampling period, a clearance rate for plasma VTG in the 5.6 microg NP per l treatment could not determined. In the 0.089, 0.71 microg E2 per l, and 59.6 microg NP per l treatments, VTG levels began decreasing within 4 days after exposure cessation and exhibited an exponential rate of elimination from plasma. Clearance rates for 0.71 microg E2 per l and 59.6 microg NP per l were not significantly different (P=0.47), however, both demonstrated significantly higher rates of clearance (P<0.02) than observed in the 0.089 microg E2 per l treatment. Our results indicate that hepatic VTG mRNA rapidly diminishes after cessation of estrogenic exposure in sheepshead minnows, but plasma VTG clearance is concentration and time dependent and may be detected at measurable levels for months after initial exposure to an estrogenic compound.

Animals↗

PubMed2002

Vitellogenin-derived yolk proteins of white perch, Morone americana: purification, characterization, and vitellogenin-receptor binding.

The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a approximately 20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, beta'-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the beta'-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.

Amino Acid Sequence↗

PubMedDate not supplied

Array technology as a tool to monitor exposure of fish to xenoestrogens.

A variety of anthropogenic chemicals are capable of binding to the estrogen receptor of vertebrate species. Binding of these compounds can interfere with homeostasis by disrupting normal gene expression patterns. The purpose of this study was to investigate the feasibility of applying array technology as a monitoring tool for detecting the presence and distribution of estrogenic compounds in coastal habitats using sheepshead minnows as our model. cDNA clones that were isolated from differential display, including vitellogenin alpha and beta, vitelline envelope protein (ZP2), and transferrin, among others, were spotted on the macroarray. The results of these experiments demonstrate a characteristic expression pattern of estrogen responsive genes in sheepshead minnows exposed to 17 beta-estradiol (E2).

Animals↗