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

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Abstract

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-17β (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.

Introduction

Vitellogenesis is a process by which oviparous female vertebrates produce and sequester nutrients into developing oocytes. In maturing females, this process is under hormonal regulation by estradiol-17β (E2), the major endogenous estrogen of vertebrates. Increasing titers of E2 stimulate the liver to produce vitellogenin (VTG), the major precursor to egg yolk proteins. This protein is modified in the liver, then secreted and transported in the bloodstream to the ovary, where it enters growing oocytes via specific receptor-mediated endocytosis (Wallace and Bergink, 1974, Specker and Sullivan, 1994). Once inside the oocyte, VTG is cleaved into the smaller yolk proteins, phosvitin, lipovitellin, and beta-component, which accumulate in yolk globules or granules (Mommsen and Walsh, 1988, Hiramatsu et al., 2002a, Hiramatsu et al., 2002b). Significant VTG production does not normally occur in male or immature female fish, but can be stimulated in either sex by exposure to exogenous E2 or estrogen mimics. Due to its specificity as a marker of estrogenic exposure, measurement of VTG has been used extensively in field (Harries et al., 1997, Jobling et al., 1998, Allen et al., 1999a, Allen et al., 1999b) and laboratory studies (Chen et al., 1986, Jobling and Sumpter, 1993, Flouriot et al., 1995, Jobling et al., 1995, Donohoe and Curtis, 1996, Monosson et al., 1996, Harries et al., 1997, Jobling et al., 1998, Allen et al., 1999a, Allen et al., 1999b, Gronen et al., 1999, Parks et al., 1999, Folmar et al., 2000, Cheek et al., 2001a, Cheek et al., 2001b) of estrogenic endocrine disruption in fishes.

Endocrine disruption is defined as the perturbation of endogenous hormone synthesis, transport, receptor interaction, intracellular signaling, and degradation by exogenous compounds, usually of anthropogenic origin (Guillette, 1996, McLachlan and Arnold, 1996, Crews et al., 2000). Many categories of chemicals and chemical mixtures appear to mimic estrogen activity in fish, including alkylphenols, pesticides, polychlorinated biphenyls (PCBs), plasticizers, and sewage effluents (Jobling and Sumpter, 1993, White et al., 1994, Sumpter and Jobling, 1995, Kime, 1998, Sumpter, 1998, Allen et al., 1999a, Allen et al., 1999b). These compounds can cause aberrant VTG production in immature and male fish. Bleached kraft mill effluent (BKME) clearly has endocrine disrupting effects, including reduced plasma steroid hormone concentrations, delayed sexual maturity, reduced gonad size, reduced secondary sexual characteristics, and VTG induction (Munkittrick et al., 1998, Tremblay and van der Kraak, 1999). Wild fish chronically exposed to BKME in Canada have shown reduced levels of reproductive hormones, reduced gonadal growth, and reduced expression of secondary sex characteristics (McMaster et al., 1996).

The purpose of this study was to develop and validate a sensitive, competitive, enzyme-linked immunosorbent assay (ELISA) specific for bluegill (L. macrochirus) VTG. Although VTG concentration can be estimated indirectly by measuring protein-bound phosphorus or calcium or directly by immunoblotting or radioimmunoassay (Specker and Sullivan, 1994), the ELISA approach provides a very specific, rapid, non-radioactive method for measuring VTG in large numbers of samples simultaneously. This capacity is important when considering the quantities of samples that are generated during extensive field studies of fish. Our goal was to use this assay to analyze the seasonality of vitellogenesis in female bluegill and to evaluate the potentially disruptive effects of BKME exposure on vitellogenesis. Based on previous studies showing suppressed GSI and hormone levels, we postulated that exposure to BKME would suppress steroid hormone levels and vitellogenesis in female bluegill. We chose to examine these questions in bluegill because wild populations are abundant throughout freshwaters of the US and because bluegill have been used extensively in toxicity testing (Spacie et al., 1983, Dimond et al., 1985, Oris and Giesy, 1985, Oris and Giesy, 1986, Oris et al., 1990).

Section snippets

VTG induction

Bluegill (L. macrochirus; 90–220 g) used for VTG induction and assay validation were captured by seine from an irrigation pond at the Central Crops Agricultural Research Station (North Carolina Agricultural Research Service, North Carolina State University (NCSU), Clayton, NC, USA). Six fish, identified as males by the expulsion of milt, were bled immediately after capture to provide plasma for antisera adsorption (discussed below). The remaining fish were transported to the NCSU Aquatic

VTG purification and antiserum specificity

Bluegill VTG was purified from estrogen-treated males by anion exchange chromatography on DEAE-agarose. As observed in other teleosts (Tao et al., 1993, Heppell et al., 1999, Parks et al., 1999), plasma proteins eluted in three peaks, with the third peak consisting of VTG (Fig. 1). The identity of VTG and specificity of the a-FSPP antiserum were verified by Western blotting. The antiserum recognized a single major polypeptide in the purified VTG sample, plasma from E2-injected males, and plasma

Discussion

The polyclonal antiserum generated against purified bluegill VTG appeared to be highly specific, interacting with a single major plasma protein of approximately 170 kDa molecular mass in plasma of vitellogenic females and E2-injected males. Previous studies using the same protocols reported here revealed VTG polypeptide monomers of similar mass and, as reported here, the VTG polypeptide was only detectable in vitellogenic females or E2-injected males (Tao et al., 1993, Heppell et al., 1999,

Acknowledgments

We thank N. Hiramatsu and G.M. Weber for assistance with vitellogenin purification, antibody production, and assay development and execution. The staff members of the Southeastern Aquatic Biology Facility are appreciated for their assistance with collecting, maintaining, and sampling fish for the BKME exposure studies. This work was funded under a cooperative agreement with the National Council of the Paper Industry for Air and Stream Improvement (CVS) and Louisiana Board of Regents grant

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