Bluegill (Lepomis macrochirus) vitellogenin: purification and enzyme-linked immunosorbent assay for detection of endocrine disruption by papermill effluent

Vitellogenin (VTG) is a highly specific marker of exposure to environmental estrogens and has been used extensively in field and laboratory studies of estrogenic endocrine disruption in fishes. The purpose of this study was to develop and validate a sensitive, competitive, enzyme-linked immunosorbent assay (ELISA) specific for bluegill (Lepomis macrochirus) vitellogenin. Bluegill VTG was purified by anion exchange chromatography on DEAE-agarose. The polypeptide had an apparent mass of 170 kDa and was specifically recognized by the rabbit antiserum raised against bluegill female-specific plasma protein. Plasma samples from vitellogenic females diluted in parallel with the purified VTG standard curve in the ELISA. The detection limit of the assay was 29 ng/ml and the working range extended to 2700 ng/ml. Recovery of purified VTG was 85.8+/-9.5%, intra-assay variation was 6.4% and interassay variation was 12.3%. We used this ELISA to analyze the seasonal cycle of vitellogenesis in female bluegill and to evaluate potential disruption of this process by exposure to bleached kraft mill effluent (BKME). Captive female bluegill stocked in outdoor experimental streams in New Bern, NC had the lowest levels of VTG, estradiol-17beta (E2), and testosterone (T) and the smallest oocyte diameters in January, but these variables increased in March and remained elevated through August, suggesting an extended spawning season. Plasma VTG, E2, T and oocyte diameter were unaffected by exposure to BKME concentrations as high as 30%. Development of the VTG ELISA allowed rapid and convenient analysis of plasma samples to evaluate exposure to potential endocrine disrupting compounds.     

Purification of vitellogenin from smooth flounder (Pleuronectes putnami) and measurement in plasma by homologous ELISA

Male smooth flounder (Pleuronectes putnami) were induced to produce vitellogenin (VTG) by injection of 17beta-estradiol (E2). Anion exchange chromatography of precipitated plasma from E2-injected resulted in a single peak consisting of VTG. Smooth flounder VTG has an approximate molecular mass of approximately 520 kDa, determined by gel filtration with molecular weight standards. Purified VTG was used to develop a homologous enzyme-linked immunosorbent assay (ELISA). The flounder VTG ELISA is an indirect antigen competition assay with a detection limit of 15 ng.ml(-1) and a useful range of 30-950 ng.ml(-1) of diluted sample. Intra- and inter-assay precision (as %CV, n=7) ranged from 1.3% to 6.0% and 5.1%, respectively. The ELISA was evaluated using plasma samples collected from a smooth flounder population captured in the Saint Lawrence Estuary. The ELISA is sensitive enough to differentiate males and non-vitellogenic females from vitellogenic individuals during early vitellogenesis.     

Development and validation of a homologous zebrafish (Danio rerio Hamilton–Buchanan) vitellogenin enzyme-linked immunosorbent assay (ELISA) and its application for studies on estrogenic chemicals

Vitellogenin (VTG) was isolated by anion exchange chromatography from plasma of female zebrafish (Danio rerio) induced with 17α-ethinylestradiol (EE2). The purity of the VTG isolate was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE). Purified VTG was used to raise polyclonal antibodies in rabbits and the specificity of the antisera for VTG confirmed by Western blot analysis of plasma proteins separated by SDS-PAGE. The antibodies cross-reacted with two proteins in the plasma of female zebrafish, with molecular masses of approximately 142 and 171 kDa. No cross-reactivity was observed with any other plasma proteins. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using the polyclonal zebrafish VTG (z-VTG) antibodies and purified z-VTG as ligand and standard, respectively. The z-VTG ELISA was sensitive with a detection limit of between 2.0 and 3.0 ng purified VTG/ml, and a working range between 3 and 500 ng/ml (30–85% binding). The ELISA demonstrated precision, with inter- and intra-assay variations of 7.5±2.7 and 4.9±1.4%, respectively. Plasma from adult zebrafish and whole body homogenates from juvenile zebrafish diluted parallel with the z-VTG standard in the ELISA, validating the assay for quantifying z-VTG in both of these tissues. Exposure of adult male zebrafish to EE2 via water induced a concentration-dependent induction of VTG with a lowest observed effect concentration (LOEC) ≤1.67 ng EE2/l (for a 21-day exposure). The homologous z-VTG ELISA provides a valuable tool for the study of environmental estrogens in zebrafish.     

Paternal mouthbrooding in the black-chinned tilapia, Sarotherodon melanotheron (Pisces: cichlidae): changes in gonadal steroids and potential for vitellogenin transfer to larvae

The black-chinned tilapia (Sarotherodon melanotheron) is a paternal mouthbrooder. Pairs of adult black-chinned tilapia were raised in freshwater and the males were sampled during the mouthbrooding cycle. Sampling also occurred 10 days after release of the free-swimming fry for comparison. During the first week of incubation of the eggs, total androgens and estradiol were low (<5 and <0.3 ng/ml, respectively). During the second week of brooding, when the eggs have hatched and they are called newly hatched embryos, plasma levels of gonadal steroids increased (13-38 ng androgen/ml and >0.6 ng estradiol/ml). The plasma concentrations of vitellogenin (VTG) in male parents changed during mouthbrooding, with decreases occurring between egg pickup and hatching of the embryo (Day 6 of mouthbrooding). The pattern of change in concentrations of VTG in surface mucus of male parents differed from the pattern in plasma, with peak concentrations occurring at the time of hatching. The amount of VTG in mucus was similar to that measured in the female Oreochromis mossambicus during mouthbrooding of embryos. The appearance of peak VTG levels in the mucus at the time of hatching when plasma levels have declined and the availability of comparable amounts of mucus VTG in both maternal and paternal mouthbrooding tilapia, despite unequivalent plasma levels, support the possibility that parental provisioning of the young occurs during mouthbrooding in tilapia.     

Effects of estradiol-17beta treatment on in vitro and in vivo synthesis of two distinct vitellogenins in tilapia.

Two distinct vitellogenins (VTG) were purified from the blood of estradiol-17beta (E(2))-injected tilapia, Oreochromis mossambicus. Enzyme-linked immunosorbent assays (ELISA) of each VTG were developed to examine effects of E(2) treatment on induction of VTG synthesis in the primarily cultured tilapia hepatocytes. Two VTG molecules (VTG210 and VTG140) had apparent molecular masses of 370 and 220 kDa by gel filtration and 210 and 140 kDa by SDS-PAGE, respectively. Western blot analyses showed that antibodies raised against the purified VTG210 and VTG140 reacted only with each protein band. Furthermore, ELISA for each VTG was specific for target VTG. When E(2) was added into the media of primarily cultured tilapia hepatocytes, VTG210 and VTG140 were both detected from E(2) concentrations of 1x10(-7) M and 5x10(-7) M, respectively. Time course experiments showed that there was a difference in the detection time of VTG210 and VTG140 after the hormone treatment. Although the injection of different E(2) doses induced both VTGs in the plasma of male tilapia, the concentration of VTG210 was nearly five to eight times higher than that of VTG140. These results suggest that E(2) is a direct inducer of both VTGs in the tilapia hepatocytes in vitro and in vivo, and that there is difference in the hormone response in inducing the VTGs in the tilapia hepatocytes.     

Sandwich ELISAs for quantification of Xenopus laevis vitellogenin and albumin and their application to measurement of estradiol-17 beta effects on whole animals and primary-cultured hepatocytes

Sandwich enzyme-linked immunosorbent assay (ELISA) was developed for quantification of vitellogenin (VTG) and albumin (ALB) in Xenopus laevis. Working ranges of the ELISAs were 2-1000 ng/ml for VTG and 1-300 ng/ml for ALB. Recoveries of plasma VTG by ELISA were over 90% in dilutions of more than 200 times. The VTG-inducing activity of estradiol-17beta (E2) was measured in whole animals and primary cultured hepatocytes. Immersion of mature male animals in more than 1 nM E2 induced a detectable amount of plasma VTG. VTG induction in younger animals was less potent than in the mature animals but the youngest animals (1.5-3 g body mass) was applicable to the exposure test, irrespective of sex. In vitro exposure of hepatocytes to more than 0.1 nM E2 dose-dependently induced secretion of VTG into the culture medium, while ALB secretion was not significantly affected by E2 treatment. When the VTG-induction levels were normalized by use of a concentration ratio of VTG to ALB, the values obtained from three independent experiments were mutually comparable irrespective of differences in cell density and hepatocyte preparation. Thus, this ratio is thought to be useful for large-scale in vitro screening of estrogenic activities of chemical substances.     

Purification and establishment of ELISA for vitellogenin of Japanese sardine (Sardinops melanostictus)

Two female-specific serum proteins (FSSPs) were detected immunologically in estradiol-treated Japanese sardine Sardinops melanostictus. The major FSSP was demonstrated to be a high molecular estradiol-inducible glycolipophosphoprotein with an immunological relation to a major yolk protein, and was suggested to be vitellogenin (VTG). VTG was purified using negative immunoaffinity chromatography. The isolated VTG was used for raising the specific antiserum against VTG. A homologous enzyme-linked immunosorbent assay (ELISA) was developed using the antiserum and the isolated VTG. The sensitivity range of the ELISA was 44 ng/ml to 2670 ng/ml of VTG concentration for the conditions used in our investigation.     

One-step, non-denaturing purification method of carp (Cyprinus carpio) vitellogenin

A new single-step purification procedure was developed to purify carp (Cyprinus carpio) vitellogenin (VTG), from estradiol-treated carp plasma. This method was performed by high performance liquid weak anion-exchange chromatography, using a discontinuous elution gradient of NaCl (0-0.5 M, steps of 12.5 mM/4 min). SDS and native-PAGE analysis, of treated-fish plasma and purified solution, showed the appearance of a 370 kDa phospholipoprotein, composed of two 130 kDa monomers, with all VTG characteristics. The sequencing of a 130 kDa monomer confirmed that it was carp VTG. Consequently, this procedure is a rapid method, permitting high quantities of non-denatured carp VTG to be obtained.     

Size heterogeneity of epidermal growth factor in human body fluids

We measured the concentration of immunoreactive (IR) hEGF in various body fluids by radioimmunoassay (RIA) and evaluated its size heterogeneity by size exclusion high performance liquid chromatography combined with RIA or with time-resolved immunoflurometric assay (TR-IFMA). Mean concentration was 80 ng/ml in urine, 65 ng/ml in milk, 50 ng/ml in seminal plasma, 25 ng/ml in armpit sweat, 1 ng/ml in breast sweat, 0.3 ng/ml in third-trimester amniotic fluid, 3 ng/ml in saliva, 1.5 ng/ml in tears and 0.3 ng/ml in gastric juice.

All the fluids except armpit sweat and gastric juice contained two to five molecular sizes of IR-hEGF. As well as the 6200-dalton (6.2kDa) hEGF we found at least four other different molecular sizes with approximate weights of 300, 150, 70 and 20kDa. The authentic 6.2kDa form made up >90% of the total IR-hEGF in all except the amniotic fluid where its proportion was 71%, and the seminal plasma where the proportion could not be determined.     

Separation and detection of vitellogenin in fish plasma by capillary zone electrophoresis

A method for coupling capillary zone electrophoresis (CZE) with rapid membrane chromatography purification (RMCP) was established for the analysis of vitellogenin (VTG) in male fish plasma induced with 17ss-estrodiol. CZE analyses of purified VTG were performed in a buffer containing 25 mM sodium borate (pH 8.4). A 50 microm i.d. fused-silica capillary was used for separation and the detection was carried out by UV-diode array at 214 nm. Inter- and intra-assay variabilities of the proposed method were less than 10.06 and 1.95%, respectively. The method has good linear relationship over the scope of 15-2250 microg/ml with a correlation coefficient of R2 = 0.9965 and a detection limit of 7.0 microg/ml. The established CZE method was also applied to directly separate and identify VTG from fish plasma. The results indicated this method could minimize interferences from plasma proteins, allowing the detection of at least 62.5 microg/ml of VTG proteins in total proteins. This is a rapid and easy method to determine the quantity and purity of VTG compared to Bradford method and SDS-PAGE.     

Comparison of ELISAs for detecting vitellogenin in the fathead minnow (Pimephales promelas)

Measurement of vitellogenin (VTG) concentrations in the fathead minnow (Pimephales promelas) is currently being considered and evaluated for screening of endocrine active substances. One of the proposed methods, an enzyme-linked immunosorbent assay (ELISA) based on VTG from carp (Cyprinus carpio), was recently evaluated in an inter-laboratory ring test using whole body homogenates from juvenile fathead minnows. The objective of the current study was to compare the performance of three different ELISAs for measuring fathead minnow VTG: (1) a heterologous carp VTG (cVTG) ELISA used in the ring test, (2) a homologous fathead minnow VTG (fVTG) ELISA, and (3) a hybrid ELISA with the antibody developed for cVTG, but using fVTG for coating the plates and preparing standard curves. VTG was measured in whole body homogenates from juvenile fathead minnows exposed to 17alpha-ethynylestradiol (EE(2); 10 ng/l) and whole body homogenates and plasma from adult fathead minnows exposed to 17beta-estradiol (E(2); 5 mg/kg; i.p.). The cVTG assay showed lower specificity for fathead minnow VTG in whole body homogenates and plasma from treated fish, compared to the fVTG assay. VTG concentrations in juvenile fathead minnow homogenates from the EE(2)-exposed group were approximately 50-fold higher when measured using the fVTG method compared to the cVTG method. Use of the homologous fVTG in the hybrid cVTG assay yielded VTG concentrations in the range of the fVTG assay but the low specificity persisted. The homologous fVTG assay is recommended to achieve accurate quantification of VTG levels in fathead minnows.     

Sensitive liquid chromatography/tandem mass spectrometry method for the determination of the lipophilic antipsychotic drug chlorpromazine in rat plasma and brain tissue

A simple, sensitive and robust liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for quantification of chlorpromazine in rat plasma and brain tissue. Chlorpromazine was extracted from rat plasma and brain homogenate using liquid-liquid extraction. The compounds were separated on a Waters Atlantis dC-18 (30 mm x 2.1 mm i.d., 3 microm) column using a mobile phase of acetonitrile/20 mM ammonium formate (pH 4.25 adjusted with formic acid) with gradient elution. Chlorpromazine was detected in positive ion mode using multiple reaction monitoring (MRM). The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. The LLOQ was 0.2 ng/ml for plasma and 0.833 ng/g for brain tissue. The method was linear over the concentration range from 0.2 to 200.0 ng/ml for plasma and from 0.833 to 833.3 ng/g for brain tissue. The correlation coefficient (R(2)) values were more than 0.998 for both plasma and brain homogenate. The precision and accuracy for intra-day and inter-day were better than 7.54%. The relative and absolute recovery was above 84.9% and matrix effects were lower than 5.6%. This validated method has been successfully used to quantify the rat plasma and brain tissue concentration of chlorpromazine after chronic treatment.     

Vitellogenin dynamics during egg-laying: daily variation, repeatability and relationship with egg size

Daily variation in circulating levels of the avian yolk precursor, vitellogenin (VTG), throughout the laying cycle was investigated in female zebra finches Taeniopygia guttata and compared with predicted ovarian follicle demand (based on a model of follicular development for this species). In general, the pattern of variation in plasma VTG matched the predicted demand from the developing ovarian follicle hierarchy. Plasma VTG was non-detectable in non-breeders, but increased rapidly with onset of yolk development, remaining high (1.43–1.82 μg/ml, zinc) through to the 3-egg stage. Plasma levels then declined at the 5-egg stage (to 0.78±0.32 μg/ml) and were undetectable at clutch completion. This result is consistent with the hypothesis that yolk precursor production is costly and that selection has matched supply and demand. While inter-individual variation in plasma VTG was marked (e.g. 0.47–4.26 μg/ml at the 1-egg stage), it also exhibited high intra-individual repeatability (r=0.87–0.93). Finally, we examined the relationship between plasma VTG and primary reproductive effort. While individual variation in plasma VTG was independent of clutch size, laying interval and laying rate, there was a complex, diet-dependent relationship between VTG and egg size, with low plasma VTG levels being associated with both very small (<0.90 g) and very large (>1.15 g) egg sizes.     

Recruitment control and production of market-size Oreochromis niloticus with the predator Lates niloticus L. in the Sudan

Experiments were conducted in earth ponds (242–1260m²) to evaluate the Nile perch (Lates niloticus L.) as a predator for recruitment control and the production of marketable Oreochromis niloticus L. in the Sudan. Supply of the predatory fish was maintained by induced spawning of L. niloticus brooders by raising the water level in a 1–0 ha earth pond. Lates niloticus fingerlings (7–5 cm total length) were then stocked with same size O. niloticus at the ratios 1:5, 1:10 and 1:15, Lates:Oreochromis , respectively. Over a period of 7 months Lates niloticus reduced young 50g) Oreochromis population and enhanced the production of preferred-size ( 200 g) Oreochromis. The ratio 1:5 Lates:Oreochromis was established to be the most desirable for Lates -with a total production of 0.2428 kg/m² of O. niloticus with 55.7% (by weight) at the target size averaging 287.5g. The ratios 1:10 and 1:15 produced 0.2106 and 0.2153 kg m² of O. niloticus , with 49.4% and 16.9% averaging 235.0 and 210.0 g, respectively.     

A protein isolated from Escherichia coli,identified as GroEL,reacts with anti-beta spectrin antibodies

We found that a protein of molecular weight close to 65kDa, present in Escherichia coli cells, reacts with anti-beta spectrin antibodies. A method of purification of this protein was designed. The method consists of the following: nonionic detergent extraction, gel filtration chromatography, ion-exchange chromatography using DEAE-Servacell, and two FPLC ion-exchange chromatography runs: the first without urea, the second in its presence. This method allowed us to obtain a highly purified protein. The results of mass spectrometry analysis suggest that the investigated protein is GroEL (Hsp60 Class). Using computer programs, by sequence analysis of both proteins we tried to explain why GroEL isolated from E. coli reacts with anti-beta spectrin antibodies. Both proteins may share a single epitope for the antibodies on their surfaces. Additionally, such an assumption is supported by the results of experiments in which antibodies interacting with GroEL were obtained from anti-beta spectrin serum and were shown to react with both GroEL and beta spectrins.     

Identification of immuno-reactive lipocortin 1-like molecules in serum and plasma by an enzyme immunoassay for lipocortin 1.

A two-site enzyme immunoassay for lipocortin 1 (LC1) has been developed. The detection limit of LC1 was 0.2 ng/tube and the optimal assay range was 1 to 100 ng/tube. The assay system enabled us to identify immunoreactive lipocortin 1-like molecules (IR-LC1) in human serum and plasma. Normal human serum and plasma IR-LC1 concentrations were 44.6 ng/ml for males and 43.1 ng/ml for females with no significant difference between both sexes. The age-related analysis among nine age groups from newborn to 69 years old revealed that the serum or plasma level was high in infants (77.5 ng/ml for newborn and 75.6 ng/ml for 1 month-1 year group), and the 40-year-old (52.2 ng/ml) and 50-year-old (51.3 ng/ml) groups. The major population of plasma IR-LC1 was of 70 kDa in size corresponding to that of the LC1 homodimer. The present enzyme immunoassay system is sufficiently sensitive for the clinical study of LC1 in human body fluids, tissues and organs.     

Establishment of ELISA for murrel vitellogenin and choriogenin, as biomarkers of potential endocrine disruption

Vitellogenin (Vg) and choriogenin (Chg) are sensitive biomarkers for testing endocrine disruption in fish. Therefore, we have developed immunoassays for Vg and Chg in the Indian freshwater murrel, Channa punctatus. Vg is a known precursor of egg-yolk proteins, whereas Chg contributes to the formation of egg-envelope. Vg and Chg were induced in male murrel by administration of estradiol-17beta. Chg had an apparent native molecular mass of 180 kDa. It consisted of a single peptide with a molecular mass of 110 kDa, whereas native Vg protein (530 kDa) contained 175 kDa peptide. Highly specific polyclonal antibodies against purified plasma proteins, Vg and Chg, were employed for developing competitive enzyme linked immunosorbent assays (ELISAs). The sensitivity of Vg assay was 3.9 ng/mL (working range 15-500 ng/mL) and of Chg assay was 1.56 ng/mL (working range 6-200 ng/mL). The inter- and intra-assay variations were well within acceptable limits. The two antisera did not cross-react with male plasma proteins. Antiserum to Vg did not cross-react with Chg. Similarly, antiserum to Chg showed no correlation with Vg. Further, immunofluorescence and Western blotting confirmed the specificity of Vg and Chg antisera.     

Estimation of catecholamines in human plasma by ion-exchange chromatography coupled with fluorimetry

Estimation of catecholamines in human plasma was made by ion-exchange chromatography coupled with fluorimetry.Catecholamines in deproteinized plasma were adsorbed onto Amberlite CG-50 (pH 6.5, buffered with 0.4 M phosphate buffer) and selectively eluted by 0.66 M boric acid. The catecholamine fraction was separated further on a column of Amberlite IRC-50 which was coupled with a device for the automated performance of the trihydroxyindole method (epinephrine and norepinephrine) or the 4-aminobenzoic acid—oxidation method (dopamine). One sample could be analysed within 25 min with either method. The lower detection limits were 0.02 ng for epinephrine and dopamine, and 0.04 ng for norepinephrine.Plasma catecholamine contents of healthy adults at rest were epinephrine 0.07 ± 0.01 ng/ml (n = 19), norepinephrine 0.27 ± 0.03 ng/ml (n = 19) and dopamine 0.22 ± 0.03 ng/ml (n = 26).The procedure of adsorption and elution of the plasma catecholamines by ion-exchange resin was simple, the simplicity contributing to constant recovery. The catecholamine fraction could be analysed without evaporation of the eluate. The analytical column could be used for the analysis of more than 1000 samples before excessive back-pressure developed. Our method of continuous measurement of plasma catecholamine fulfils clinical requirements.     

Vitellogenin and its messenger RNA during ovarian development in the female blue crab, Callinectes sapidus: gene expression, synthesis, transport, and cleavage

Blue crab vitellogenin (VTG) cDNA encodes a precursor that, together with two other Brachyuran VTGs, forms a distinctive cluster within a phylogenetic tree of crustacean VTGs. Using quantitative RT-PCR, we found that VTG was primarily expressed in the hepatopancreas of a vitellogenic female, with minor expression in the ovary. VTG expression in the hepatopancreas correlated with ovarian growth, with a remarkable 8000-fold increase in expression from stage 3 to 4 of ovarian development. In contrast, the VTG levels in the hepatopancreas and hemolymph decreased in stage 4. Western blot analysis and N-terminal sequencing revealed that vitellin is composed of three subunits of approximately 78.5 kDa, 119.42 kDa, and 87.9 kDa. The processing pathway for VTG includes an initial hepatopancreatic cleavage of the primary precursor into approximately 78.5-kDa and 207.3-kDa subunits, both of which are found in the hemolymph. A second cleavage in the ovary splits the approximately 207.3-kDa subunit into approximately 119.4-kDa and approximately 87.9-kDa subunits. The hemolymph VTG profiles of mated and unmated females during ovarian development indicate that early vitellogenesis and ovarian development do not require mating, which may be essential for later stages, as VTG decreased to the basal level at stage 4 in the unmated group but remained high in the mated females. Our results encompass comprehensive overall temporal and spatial aspects of vitellogenesis, which may reflect the reproductive physiology of the female blue crab, e.g., single mating and anecdysis in adulthood.