Vitelline Envelope Proteins Research Articles

Time course of hepatic gene expression and plasma vitellogenin protein concentrations in estrone-exposed juvenile rainbow trout (Oncorhynchus mykiss)

Estrone (E1), a natural estrogen hormone found in sewage effluents and surface waters, has known endocrine disrupting effects in fish, thus, it is a contaminant of emerging concern. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to an environmentally-relevant concentration of E1 (24ng/L E1 [0.1nM]) for 7d and then placed in clean water for a 9d recovery period. RNA sequencing showed transcripts from numerous affected biological processes (e.g. immune, metabolic, apoptosis, clotting, and endocrine) were altered by E1 after 4d of treatment. The time course of E1-inducible responses relating to vitellogenesis was examined daily during the two phases of exposure. Hepatic gene expression alterations evaluated by quantitative polymerase chain reaction (QPCR) were found during the treatment period for vitellogenin (VTG), vitelline envelope proteins (VEPs) α, β and γ, and estrogen receptor α1 (ERα1) transcripts. ERα1 was the only transcript induced each day during the treatment phase, thus it was a good indicator of E1 exposure. Gradual increases occurred in VEPβ and VEPγ transcripts, peaking at d7. VTG transcript was only elevated at d4, making it less sensitive than VEPs to this low-level E1 treatment. Inductions of ERα1, VEPα, VEPβ and VEPγ transcripts ceased 1d into the recovery phase. Plasma VTG protein concentrations were not immediately elevated but peaked 7d into the recovery phase. Thus, elevated vitellogenesis-related gene expression and protein production occurred slowly but steadily at this concentration of E1, confirming the sequence of events for transcripts and VTG protein responses to xenoestrogen exposure.

Characterizing the transcriptome of yellow-cheek carp (Elopichthys bambusa) enables evolutionary analyses within endemic East Asian Cyprinidae.

The identification of genes that may be responsible for the divergence of closely related species is one of the central goals of evolutionary biology. The species of endemic East Asian Cyprinidae diverged less than 8millionyears ago, and the morphological differences among these species are great. However, the genetic basis of their divergence remains unknown. In this report, we investigated the transcriptome of one endemic East Asian cyprinid - the yellow-cheek carp Elopichthys bambusa. A comparison with the publicly available transcriptomes of other endemic East Asian cyprinids, including the silver carp (Hypophthalmichthys molitrix) and blunt-nose black bream (Megalobrama amblycephala), revealed a number of candidate adaptive genes in each species, such as zona pellucida glycoprotein 2 in E. bambusa and zebrafish vitelline envelope protein in M. amblycephala. An enrichment test showed the enrichment of some specific gene ontology (GO) terms for these putatively adaptive genes. Taken together, our work is the first step toward elucidating the genes that may be related to the divergence of endemic East Asian Cyprinidae, and these genes identified as being probably under positive selection should be good candidates for subsequent evolutionary and functional studies.

Shotgun proteomics of rainbow trout ovarian fluid.

In the present study we used a shotgun proteomic approach to identify 54 proteins of rainbow trout ovarian fluid. The study has unravelled the identity of several proteins not previously reported in fish ovarian fluid. The proteome of trout ovarian fluid consists of diverse proteins participating in lipid binding and metabolism, carbohydrate and ion transport, innate immunity, maturation and ovulation processes. Most trout ovarian fluid proteins correspond to follicular fluid proteins of higher vertebrates, but 15% of the proteins were found to be different, such as those related to the immune system (precerebellin-like protein), proteolysis (myeloid cell lineage chitinase), carbohydrate and lipid binding and metabolism (vitellogenins), cell structure and shape (vitelline envelope protein gamma) and a protein with unknown functions (UPF0762 protein C6orf58 homologue). The present study could help in the decoding of the biological function of these proteins and in the discovery of potential biomarkers of oocyte quality.

Minimally invasive transcriptome profiling in salmon: Detection of biological response in rainbow trout caudal fin following exposure to environmental chemical contaminants

An increasing number of anthropogenic chemicals have demonstrated potential for disruption of biological processes critical to normal growth and development of wildlife species. Both anadromous and freshwater salmon species are at risk of exposure to environmental chemical contaminants that may affect migratory behavior, environmental fitness, and reproductive success. A sensitive metric in determination of the presence and impact of such environmental chemical contaminants is through detection of changes in the status of gene transcript levels using a targeted quantitative real-time polymerase chain reaction assay. Ideally, the wildlife assessment strategy would incorporate conservation-centered non-lethal practices. Herein, we describe the development of such an assay for rainbow trout, Oncorhynchus mykiss, following an acute 96h exposure to increasing concentrations of either 17α-ethinyl estradiol or cadmium. The estrogenic screen included measurement of mRNA encoding estrogen receptor α and β isoforms, vitellogenin, vitelline envelope protein γ, cytochrome p450 family 19 subfamily A, aryl hydrocarbon receptor, and the stress indicator, catalase. The metal exposure screen included evaluation of the latter two mRNA transcripts along with those encoding the metallothionein A and B isoforms. Exposure-dependent transcript abundance profiles were detected in both liver and caudal fin supporting the use of the caudal fin as a non-lethally obtained tissue source. The potential for both transcriptome profiling and genotypic sex determination from fin biopsy was extended, in principle, to field-captured Chinook salmon (Oncorhynchus tshawytscha).

Role of vitelline envelope during fertilization in the black tiger shrimp, Penaeus monodon

Animal eggs possess investments through which sperm must penetrate. The aim of the present study was to investigate the role of the egg coating, the vitelline envelope, during sperm–egg interactions in the black tiger shrimp, Penaeus monodon. The site(s) of primary binding between sperm and egg and the possible binding molecule(s) for sperm were identified. In vitro adsorption of the vitelline envelope protein onto the sperm surface showed that primary binding occurred between the sperm anterior spike of acrosome intact sperm and the vitelline envelope. Results from streptavidin blotting revealed that the component of the vitelline envelope that interacts with the sperm integral membrane protein is a 370kDa protein. In addition, it was shown that the vitelline envelope protein had no ability to induce acrosome reaction. These results suggest that the function of the vitelline envelope is as a primary binding site for sperm in shrimp, but not a sole trigger for the acrosome reaction.

Identification of estrogen‐responsive vitelline envelope protein fragments from rainbow trout (Oncorhynchus mykiss) plasma using mass spectrometry

Plasma peptides previously associated with exposure of juvenile male rainbow trout (Oncorhynchus mykiss) to the hormone 17β-estradiol (E2) were identified using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Specifically, plasma peptides of interest were fractionated and subsequently identified via spectra obtained by MALDI QqTOF MS/MS and LC-MALDI TOFTOF MS/MS analysis, de novo sequencing and database matching. The two peptide masses were identified as significant matches for fragments of the C-terminal propeptides from rainbow trout vitelline envelope protein (VEP)α and VEPγ isoforms. Our findings document the presence of the C-terminal propeptides from rainbow trout VEPα and VEPγ proteins in the bloodstream of juvenile male rainbow trout exposed to E2 via MALDI-TOF-MS detection. We provide three possible explanations for the presence of C-terminal propeptides in the bloodstream, as well as compare previously obtained hepatic transcriptomic results with the plasma proteomic results obtained in the present study.

Dicalcin Inhibits Fertilization through Its Binding to a Glycoprotein in the Egg Envelope in Xenopus laevis

Fertilization comprises oligosaccharide-mediated sperm-egg interactions, including sperm binding to an extracellular egg envelope, sperm penetration through the envelope, and fusion with an egg plasma membrane. We show that Xenopus dicalcin, an S100-like Ca(2+)-binding protein, present in the extracellular egg envelope (vitelline envelope (VE)), is a suppressive mediator of sperm-egg interaction. Preincubation with specific antibody greatly increased the efficiency of in vitro fertilization, whereas prior application of exogenous dicalcin substantially inhibited fertilization as well as sperm binding to an egg and in vitro sperm penetration through the VE protein layer. Dicalcin showed binding to protein cores of gp41 and gp37, constituents of VE, in a Ca(2+)-dependent manner and increased in vivo reactivity of VE with a lectin, Ricinus communis agglutinin I, which was accounted for by increased binding ability of gp41 to the lectin and greater exposure of gp41 to an external environment. Our findings strongly suggest that dicalcin regulates the distribution of oligosaccharides within the VE through its binding to the protein core of gp41, probably by modulating configuration of oligosaccharides on gp41 and the three-dimensional structure of VE framework, and thereby plays a pivotal role in sperm-egg interactions during fertilization.

ZP Domain Proteins in the Abalone Egg Coat Include a Paralog of VERL under Positive Selection That Binds Lysin and 18-kDa Sperm Proteins

Identifying fertilization molecules is key to our understanding of reproductive biology, yet only a few examples of interacting sperm and egg proteins are known. One of the best characterized comes from the invertebrate archeogastropod abalone (Haliotis spp.), where sperm lysin mediates passage through the protective egg vitelline envelope (VE) by binding to the VE protein vitelline envelope receptor for lysin (VERL). Rapid adaptive divergence of abalone lysin and VERL are an example of positive selection on interacting fertilization proteins contributing to reproductive isolation. Previously, we characterized a subset of the abalone VE proteins that share a structural feature, the zona pellucida (ZP) domain, which is common to VERL and the egg envelopes of vertebrates. Here, we use additional expressed sequence tag sequencing and shotgun proteomics to characterize this family of proteins in the abalone egg VE. We expand 3-fold the number of known ZP domain proteins present within the VE (now 30 in total) and identify a paralog of VERL (vitelline envelope zona pellucida domain protein [VEZP] 14) that contains a putative lysin-binding motif. We find that, like VERL, the divergence of VEZP14 among abalone species is driven by positive selection on the lysin-binding motif alone and that these paralogous egg VE proteins bind a similar set of sperm proteins including a rapidly evolving 18-kDa paralog of lysin, which may mediate sperm-egg fusion. This work identifies an egg coat paralog of VERL under positive selection and the candidate sperm proteins with which it may interact during abalone fertilization.

Cloning, characterization and expression of zvep, a novel vitelline envelope‐specific gene in the zebrafish ovary

The egg envelope is a specialized extracellular matrix that surrounds and protects the oocyte and plays significant roles in animal reproductive and developmental processes. Using the NCBI digital differential display program we identified an EST sequence (XM_001340234.1) acquired from zebrafish ovary cDNA libraries in GeneBank. The full-length cDNA of this transcript was obtained by 3'and 5' RACE and further confirmed by PCR and sequencing. The full-length cDNA of the novel gene is 2,720 bp and encodes a protein of 761 amino acids. RT-PCR and Western blot analysis showed its expression in ovary and brain but not in other tissues. In situ hybridization demonstrates that the mRNA is transcribed in ooplasm of stage I, II, and III oocytes. Interestingly, immunohistochemistry on zebrafish ovarian sections showed that protein expression in the vitelline envelope was located to two thin positive lines in the stage III oocytes. These ovarian expression patterns show that this is a new component of the vitelline envelope that is synthesized during early developing oocytes. This protein was named ZVEP (zebrafish vitelline envelope protein) and it did not have any homology with other known vitelline envelope genes. Thus, we found that zvep is a novel gene related to the vitelline envelope in zebrafish.

Purified trout egg vitelline envelope proteins VEβ and VEγ polymerize into homomeric fibrils from dimers in vitro

The rainbow trout egg vitelline envelope (VE) is composed of three proteins, called VEα (∼ 58–60kDa M r), VEβ (∼ 52kDa M r), and VEγ (∼ 47kDa M r). Each of these proteins is related to mouse egg zona pellucida (ZP) glycoproteins, called ZP1, ZP2, and ZP3, and possesses a ZP domain that has been implicated in the polymerization of the proteins into long, interconnected fibrils or filaments. Here, trout egg VEβ and VEγ were purified to homogeneity and analyzed under various experimental conditions (SDS-PAGE, Blue Native-(BN-)PAGE, size-exclusion chromatography, and transmission electron microscopy) to determine whether individual VE proteins would polymerize into fibrils in vitro. Such analyses revealed that in the presence of 6M urea each VE protein is present primarily as monomers and as small oligomers (dimers, tetramers, etc.). However, either a reduction in urea concentration or a complete removal of urea results in the polymerization of VEβ and VEγ dimers into very large oligomers. Mixtures of VEβ and VEγ also give rise to large oligomers . Under these conditions, VE proteins are visualized by transmission electron microscopy as aggregates of long fibrils, with each fibril composed of contiguous beads located periodically along the fibril. The relationship between the behavior of fish egg VE proteins and mouse ZP glycoproteins, as well as other ZP domain-containing proteins, is discussed.

Changes in hepatic gene expression related to innate immunity, growth and iron metabolism in GH-transgenic amago salmon ( Oncorhynchus masou) by cDNA subtraction and microarray analysis, and serum lysozyme activity

Growth hormone (GH) transgenic amago salmon ( Oncorhynchus masou) were generated with a construct containing the sockeye salmon GH1 gene fused to the metallothionein-B (MT-B) promoter from the same species. This transgene directed significant growth enhancement with transgenic fish reaching approximately four to five times greater weight than control salmon in F 2 and F 3 generations. This drastic growth enhancement by GH transgene is well known in fish species compared with mammals, however, such fish can show morphological abnormalities and physiological disorders like other GH transgenic animals. GH is known to have many acute effects, but currently there are no data describing the chronic effects of over-expression of GH on various hepatic genes in GH transgenic fish. Hepatic gene expression is anticipated to play very important roles in many physiological functions and growth performance of transgenic and control salmon. To examine these effects, we performed subtractive hybridization (using cDNA generated from liver RNA) in both directions to identify genes both increased and decreased in transgenic salmon relative to controls (576 clones were isolated and sequenced in total). Heme oxygenase, vitelline envelope protein, Acyl-coA binding protein, NADH dehydrogenase, mannose binding lectin-associated serine protease, hemopexin-like protein, leucyte-derived chemotaxin2 (LECT2), and many other genes were obtained in higher clone frequencies suggesting enhanced expression. In contrast, complement C3-1, lectin, rabin, alcohol dehydrogenase, Tc1-like transposase, Δ6-desaturase, and pentraxin genes were obtained in lower frequencies. Microarray analysis was also performed to obtain quantitative expression data for these subtracted cDNA clones. Analysis of fish across seasons was also conducted using both F 2 and F 3 salmon. Results of the microarray data essentially corresponded with those of the subtraction data when both F 2 and F 3 fish were completely immature, but the expression pattern was changed when fish approached maturation. Genes showing enhanced expression in GH transgenic fish in F 2 and F 3 by array analysis were vitelline envelope protein, hemopexin-like protein, heme-oxygenase, inter α-trypsin inhibitor, LECT2, GTP cyclohydrolase I feedback regulatory protein (GFRP), and bikunin. Reduced expression genes were lectin, Δ6-desaturase, apolipoprotein, and pentraxin. In particular, lectin was found to be highly suppressed in all F 2 and immature F 3 salmon. Further, serum lysozyme activity, one of innate immunity, was significantly ( p < 0.05) decreased in both F 2 and F 3 GH transgenic fish. These results indicate that the GH transgene fish had altered hepatic gene expression relating to iron-metabolism, innate immunity, reproduction, and growth.

Toxicogenomic responses in rainbow trout ( Oncorhynchus mykiss) hepatocytes exposed to model chemicals and a synthetic mixture

As more salmon gene expression data has become available, the cDNA microarray platform has emerged as an appealing alternative in ecotoxicological screening of single chemicals and environmental samples relevant to the aquatic environment. This study was performed to validate biomarker gene responses of in vitro cultured rainbow trout ( Oncorhynchus mykiss) hepatocytes exposed to model chemicals, and to investigate effects of mixture toxicity in a synthetic mixture. Chemicals used for 24 h single chemical- and mixture exposures were 10 nM 17α-ethinylestradiol (EE2), 0.75 nM 2,3,7,8-tetrachloro-di-benzodioxin (TCDD), 100 μM paraquat (PQ) and 0.75 μM 4-nitroquinoline-1-oxide (NQO). RNA was isolated from exposed cells, DNAse treated and quality controlled before cDNA synthesis, fluorescent labelling and hybridisation to a 16k salmonid microarray. The salmonid 16k cDNA array identified differential gene expression predictive of exposure, which could be verified by quantitative real time PCR. More precisely, the responses of biomarker genes such as cytochrome p4501A and UDP-glucuronosyl transferase to TCDD exposure, glutathione reductase and gammaglutamyl cysteine synthetase to paraquat exposure, as well as vitellogenin and vitelline envelope protein to EE2 exposure validated the use of microarray applied to RNA extracted from in vitro exposed hepatocytes. The mutagenic compound NQO did not result in any change in gene expression. Results from exposure to a synthetic mixture of the same four chemicals, using identical concentrations as for single chemical exposures, revealed combined effects that were not predicted by results for individual chemicals alone. In general, the response of exposure to this mixture led to an average loss of approximately 60% of the transcriptomic signature found for single chemical exposure. The present findings show that microarray analyses may contribute to our mechanistic understanding of single contaminant mode of action as well as mixture effects, but that its use in screening of complex environmental samples will need to be further evaluated.

Rapidly evolving zona pellucida domain proteins are a major component of the vitelline envelope of abalone eggs

Proteins harboring a zona pellucida (ZP) domain are prominent components of vertebrate egg coats. Although less well characterized, the egg coat of the non-vertebrate marine gastropod abalone (Haliotis spp.) is also known to contain a ZP domain protein, raising the possibility of a common molecular basis of metazoan egg coat structures. Egg coat proteins from vertebrate as well as non-vertebrate taxa have been shown to evolve under positive selection. Studied most extensively in the abalone system, coevolution between adaptively diverging egg coat and sperm proteins may contribute to the rapid development of reproductive isolation. Thus, identifying the pattern of evolution among egg coat proteins is important in understanding the role these genes may play in the speciation process. The purpose of the present study is to characterize the constituent proteins of the egg coat [vitelline envelope (VE)] of abalone eggs and to provide preliminary evidence regarding how selection has acted on VE proteins during abalone evolution. A proteomic approach is used to match tandem mass spectra of peptides from purified VE proteins with abalone ovary EST sequences, identifying 9 of 10 ZP domain proteins as components of the VE. Maximum likelihood models of codon evolution suggest positive selection has acted among a subset of amino acids for 6 of these genes. This work provides further evidence of the prominence of ZP proteins as constituents of the egg coat, as well as the prominent role of positive selection in diversification of these reproductive proteins.

Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm

Sperm–oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm–oocyte plasma membrane interactions.

Dose–response relationships in gene expression profiles in rainbow trout, Oncorhyncus mykiss, exposed to ethynylestradiol

Determining how gene expression profiles change with toxicant dose will improve the utility of arrays in identifying biomarkers and modes of toxic action. Isogenic rainbow trout, Oncorhyncus mykiss,were exposed to 10, 50 or 100 ng/L ethynylestradiol (a xeno-estrogen) for 7 days. Following exposure hepatic RNA was extracted. Fluorescently labeled cDNA were generated and hybridized against a commercially available Atlantic Salmon/Trout array (GRASP project, University of Victoria) spotted with 16,000 cDNAs. Transcript expression in treated vs control fish was analyzed via Genespring (Silicon Genetics) to identify genes with altered expression, as well as to determine gene clustering patterns that can be used as "expression signatures". Array results were confirmed via qRT PCR. Our analysis indicates that gene expression profiles varied somewhat with dose. Established biomarkers of exposure to estrogenic chemicals, such as vitellogenin, vitelline envelope proteins, and the estrogen receptor alpha, were induced at every dose. Other genes were dose specific, suggesting that different doses induce distinct physiological responses. These findings demonstrate that cDNA microarrays could be used to identify both toxicant class and relative dose.

Mass Spectrometric Evidence That Proteolytic Processing of Rainbow Trout Egg Vitelline Envelope Proteins Takes Place on the Egg

The rainbow trout egg vitelline envelope (VE) is constructed of three proteins, called VEalpha,VEbeta, and VEgamma, that are synthesized and secreted by the liver and transported in the bloodstream to the ovary, the site of VE assembly around eggs. All three proteins possess an N-terminal signal peptide, a zona pellucida domain, a consensus furin-like cleavage site (CFLCS) close to the C terminus, and a short propeptide downstream of the CFLCS. Proteolytic processing at the CFLCS results in loss of the short C-terminal propeptide from precursor proteins and enables incorporation of mature proteins into the VE. Here mass spectrometry (matrix-assisted laser desorption ionization time-of-flight-mass spectrometry and liquid chromatography-mass spectrometry with a micromass-quadrupole TOF hybrid mass and a QSTAR Pulsar i mass spectrometer) was employed with VE proteins isolated from rainbow trout eggs in a peptidomics-based approach to determine the following: 1) the C-terminal amino acid of mature, proteolytically processed VE proteins; 2) the cellular site of proteolytic processing at the CFLCS of VE precursor proteins; and 3) the relationship between proteolytic processing and limited covalent cross-linking of VE proteins. Peptides derived from the C-terminal region were found for all three VE proteins isolated from eggs, indicating that processing at the CFLCS occurs after the arrival of VE precursor proteins at the egg. Consistent with this conclusion, peptides containing an intact CFLCS were also found for all three VE proteins isolated from eggs. Furthermore, peptides derived from the C-terminal propeptides of VE protein heterodimers VEalpha-VEgamma and VEbeta-VEgamma were found, suggesting that a small amount of VE protein can be covalently cross-linked on eggs prior to proteolytic processing at the CFLCS. Collectively, these results provide important evidence about the process of VE formation in rainbow trout and other non-cyprinoid fish and allow comparisons to be made with the process of zona pellucida formation in mammals.

Structural characterization of fish egg vitelline envelope proteins by mass spectrometry.

The extracellular coat, or vitelline envelope (VE), of rainbow trout (Oncorhynchus mykiss) eggs consists of three proteins, called VEalpha (M(r) approximately 52 kDa), VEbeta (M(r) approximately 48 kDa), and VEgamma (M(r) approximately 44 kDa). Each of these proteins is related to mammalian egg zona pellucida (ZP) glycoproteins ZP1-3 and possesses an N-terminal signal sequence, a ZP domain, and a protease cleavage site near the C-terminus. VEalpha and VEbeta also have a trefoil domain. All three proteins possess a relatively large number of cysteine residues (VEalpha, 18; VEbeta, 18; VEgamma, 12), of which 8 are present in the ZP domain and 6 are present in the trefoil domain of VEalpha and VEbeta. Here, several types of mass spectrometry were employed, together with gel electrophoresis of chemical and enzymatic digests, to identify intramolecular disulfide linkages, as well as the N- and C-terminal amino acids of VEalpha, VEbeta, and VEgamma. Additionally, these methods were used to characterize two high molecular weight proteins (HMWPs; M(r) > 110 kDa) of rainbow trout VEs that are heterodimers of individual VE proteins. These analyses have permitted assignment of disulfide linkages and identification of N- and C-terminal amino acids for the VE proteins and determination of the protein composition of two forms of HMWPs. These experiments provide important structural information about fish egg VE proteins and filaments and about structural relationships between extracellular coat proteins of mammalian and nonmammalian eggs.

Quantification of serum levels of precursors to vitelline envelope proteins (choriogenins) and vitellogenin in estrogen treated masu salmon, Oncorhynchus masou

Previously two precursors to vitelline envelope proteins, choriogenin H (Chg H) and choriogenin L (Chg L), were identified in masu salmon, Oncorhynchus masou, and specific antisera against these two proteins were generated in rabbits. In this study, two methods of immunoassay have been developed using these specific antibodies: single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA). Non-competitive sandwich ELISAs for Chg H and Chg L were designed using digoxigenin-labeled antibodies and purified Chgs as assay components. The working range of the ELISAs was 1–128 and 2–256 ng/ml for Chg H and Chg L, respectively. Using these immunoassays and a chemiluminescent immunoassay for vitellogenin (Vg), the changes in these three estrogen-responsive proteins were measured in the serum of masu salmon after treatment with various doses of estradiol-17β (E 2). The changes in serum levels of Chgs and Vg in male fish differed according to the E 2 dose. When fish were given a 5 mg/kg body weight (BW) of E 2, Vg was induced to a greater extent than Chgs. By contrast, Chg levels were higher than that of Vg after a 10 μg/kg BW of E 2 injection. A similar trend was seen in the response time to exogenous E 2. Serum Chgs were induced from 8 h after E 2 injection and reached a peak of about 5 μg/ml at 24 h. Although Vg was not detected until 8 h after E 2 injection, its levels remained considerably low at around 0.03 μg/ml, even after 24 h. Chg H was more sensitive than was Chg L to 1 μg/kg BW of estrogen: the long-term exposure of fish to E 2 showed that Chg H could be induced from a lower dose of E 2 than could Chg L. Taken together, these results suggest that the serum levels of Chg H, Chg L, and Vg in masu salmon are regulated by circulating levels of E 2.

Dynamics of estrogen biomarker responses in rainbow trout exposed to 17β‐estradiol and 17α‐ethinylestradiol

We have investigated the response dynamics of the estrogen-dependent genes vitellogenin (VTG) and the vitelline envelope proteins (VEPs) as well as circulating VTG in immature female rainbow trout (Oncorhynchus mykiss) exposed to 17beta-estradiol (E2) and 17alpha-ethinylestradiol (EE2) for periods of 7 and 14 d. Gene responses were quantified by measurement of messenger RNA (mRNA) in liver extracts using a chemiluminescent hybridization protection assay. Circulating VTG was measured by a homologous enzyme-linked immunosorbent assay. Exposure to both E2 and EE2 induced concentration-dependent increases in all biomarkers. The data presented indicate that VEP genes may be more sensitive to estrogens than the VTG gene. The biomarker lowest-observed-effect concentrations (biomarkerLOEC) in the 14-d study with E2 were 14 ng/L (VTG protein, VTG mRNA, VEPbeta, and VEPgamma) or 4.8 ng/L (VEPalpha). The EE2 was 5- to 66-fold more potent depending on the biomarker studied. In the 7-d study, all biomarkers were elevated after 48-h exposure to E2, with biomarkerLOECs of 30 ng/L (VTG protein, VTG mRNA, and VEPgamma) or 9.7 ng/L (VEPalpha and VEPbeta). Vitellogenin mRNA was induced up to 1,000-fold above baseline, and this translated into an increase of approximately 50,000-fold in circulating VTG. In conclusion, all biomarkers responded to estrogen exposure at environmentally relevant concentrations.

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 α and β, 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 β-estradiol (E2).