Development of an enzyme-linked immunosorbent assay for vitellogenin of Morelet's crocodile (Crocodylus moreletii)

Comparative Biochemistry and Physiology, Part C 143 (2006) 50 – 58 www.elsevier.com/locate/cbpc

Development of an enzyme-linked immunosorbent assay for vitellogenin of Morelet's crocodile (Crocodylus moreletii) Kyle W. Selcer a,⁎, Lisa M. Nespoli a , Thomas R. Rainwater b , Adam G. Finger b,1 , David A. Ray c,2 , Steven G. Platt d , Philip N. Smith b , Llewellyn D. Densmore c , Scott T. McMurry b a b

Department of Biological Sciences, Bayer School of Natural and Environmental Sciences, Duquesne University, Pittsburgh, PA 15282, USA The Institute of Environmental and Human Health, Department of Environmental Toxicology, Texas Tech University, Jefferson, TX 75657, USA c Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409, USA d Department of Math and Science, Oglala Lakota College, Kyle, South Dakota, 57752-0490, USA Received 5 August 2005; received in revised form 9 December 2005; accepted 10 December 2005 Available online 31 January 2006

Abstract The purpose of this study was to develop an immunoassay for vitellogenin in Morelet's crocodile (Crocodylus moreletii). Blood was collected from wild-caught crocodiles in Belize. Plasma samples from adult females taken during the breeding season were used for vitellogenin purification and samples from adult males were used for comparison. No differences were detected between males and females for plasma total protein concentration, as measured by Coomassie assay. However, denaturing polyacrylamide gel electrophoresis (SDS-PAGE) revealed that female plasma contained a 210-kDa protein, presumably the vitellogenin monomer, that was absent in adult male plasma. The identity of the putative vitellogenin was confirmed by its cross-reactivity in Western blots with a vitellogenin antiserum that was generated against a conserved vitellogenin peptide sequence. Crocodile vitellogenin was purified by two successive rounds of DEAE chromatography. The purified protein had an apparent molecular mass of 450 kDa, as determined by gel filtration chromatography, and 210 kDa on SDS-PAGE. An indirect enzyme-linked immunosorbent assay (ELISA) was then developed for C. moreletii vitellogenin. The detection limit of the assay was 20.0 ng/mL. The intra- and inter-assay coefficients of variation were 5.3% and 9.8%, respectively. The recovery of vitellogenin diluted into male plasma was 94.7%. The ELISA assay revealed that vitellogenin levels of adult female plasma during the breeding season ranged from 1.8 to 3.1 mg/mL with a mean of 2.5 ± 0.25 mg/mL. No vitellogenin was detected in adult male plasma. Induction of vitellogenin in Morelet's crocodile may be a useful model system for field studies of crocodile reproduction and for investigations of endocrine disruption in this species. © 2006 Elsevier Inc. All rights reserved. Keywords: Vitellogenin; ELISA; Crocodile; Crocodylus moreletii

1. Introduction Oviparous vertebrates rely on stores of yolk inside the egg as the source of metabolic energy for the developing embryo. The egg yolk is derived from vitellogenin, a phospholipoglycoprotein produced by the maternal liver during the reproductive season (Ho, 1987; Polzonetti-Magni et al., 2004). Vitellogenin ⁎ Corresponding author. Tel.: +1 412 396 5967; fax: +1 412 396 5907. E-mail address: [email protected] (K.W. Selcer). 1 Current address: V-tech Environmental Services, 1510 Buddy Holly Ave, Lubbock, TX 79401, USA. 2 Current address: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA. 1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2005.12.001

is secreted from the liver into blood, from which the developing oocytes take it up and cleave it into the egg-yolk proteins, lipovitellins and phosvitins (Bergink and Wallace, 1974). The synthesis, secretion and uptake of vitellogenin are regulated by the endocrine system (Wallace and Bergink, 1974; Tata and Smith, 1979; Ho, 1987; Polzonetti-Magni et al., 2004). Vitellogenin synthesis in vertebrates is largely under control of the steroid hormone estrogen (Follett and Redshaw, 1974; Tata and Smith, 1979; Ho, 1987), although other hormones may modulate the vitellogenic response (Wangh, 1982; Carnevali and Mosconi, 1992; Polzonetti-Magni et al., 2004). Vitellogenin levels are typically elevated in the blood of adult females during reproductive periods, when estrogen levels are elevated. Conversely, vitellogenin is usually absent from the blood of

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adult females during nonreproductive periods, and of males and immature females at all times of the year. However, vitellogenin can be induced in males and immature females by treatment with estrogens (Follett and Redshaw, 1968; Tata and Smith, 1979; Ho, 1987). The estrogen-dependence of vitellogenin synthesis makes this protein useful as a biomarker for environmental estrogens (Heppell et al., 1995; Sumpter and Jobling, 1995; Palmer and Selcer, 1996; Rotchell and Ostrander, 2003). Recently, it has become clear that a number of man-made chemicals have biological effects similar to those of the steroid hormone estrogen (Turner and Sharpe, 1997). These estrogenic compounds have the potential to interfere with physiological and developmental processes; consequently, there is substantial concern about the possible health effects of these agents on humans and wildlife (Colborn et al., 1993; Toppari et al., 1996; Danzo, 1998). The potential problems associated with environmental estrogens has created a need for assays that can screen chemicals for estrogenicity and that can detect the presence of estrogenic agents in the aquatic environment (McLachlan, 1993; Rotchell and Ostrander, 2003). A number of in vivo assays for estrogenicity have been developed that are based on vitellogenin induction. The vast majority of these use fish as model organisms (e.g., Sumpter and Jobling, 1995; Folmar et al., 1997; Fenske et al., 2001; Parks et al., 1999; Hemmer et al., 2001; Holbech et al., 2001; Roy et al., 2004; Cheek et al., 2004; Nakari, 2004; Kirby et al., 2004; Meucci and Arukwe, 2005). In contrast, reptiles are poorly represented in studies of endocrine disruption (Hopkins, 2000). There is a pressing need for more information on the effects of environmental contaminants on reptiles because they are experiencing a worldwide decline (Gibbons, 2000) and environmental toxicants are among the suspected causes for their reduction in numbers (Hopkins, 2000; Gibbons, 2000). Nevertheless, vitellogenin induction assays have been developed for only a few species of lizards and turtles (Brasfield et al., 2002; Irwin et al., 2001; Herbst et al., 2003; Tada et al., 2004; Rie et al., 2005). There are currently no established vitellogenin assays for evaluation of endocrine disruption in crocodilians. This is unfortunate because endocrine disruptors are suspected to have negatively impacted American alligators, Alligator mississippiensis (Guillette et al., 1996, 2000; Matter et al., 1998; Gunderson et al., 2004). Furthermore, environmental contaminants have been found in crocodilian tissues worldwide, raising the possibility of endocrine disruption in a number of other species (see Rainwater et al., 2002). Our laboratories have undertaken a long-term study of the effects of pollutants, including endocrine disruptors, on populations of Morelet's crocodile (Crocodylus moreletii) living in contaminated habitats of northern Belize and have demonstrated that these crocodiles are exposed to a number of contaminants (Wu et al., 2000a,b; Rainwater et al., 2002; Pepper et al., 2004.) In order to fully evaluate the possibility of endocrine disruption in this species, an assay capable of measuring blood vitellogenin concentrations is needed. In this

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paper, we describe the development of an assay for vitellogenin of Morelet's crocodile and use the assay to measure samples from wild-caught male and female crocodiles. 2. Materials and methods 2.1. Sample collection Morelet's crocodiles were captured and sampled from two sites in northern Belize, Gold Button Lagoon (17°55′N, 88°45′W) and the New River Watershed (17°42′N, 88°38′W), as part of a study on the ecotoxicology of this species (Rainwater, 2003). Crocodiles were captured at night by hand or breakaway snare during March–October, 1998–2001. Sex was determined by cloacal examination of the genitalia (Allsteadt and Lang, 1995), and total length (TL) and snoutvent length (SVL) were obtained for each animal. The sizes at which wild male and female Morelet's crocodiles reach reproductively maturity are unknown. Thus, for the purposes of this study the adult size class for males was based on that reported for alligators (≥ 180 cm; Ferguson, 1985), while the adult size class for females was based on the smallest known nesting female Morelet's crocodile in northern Belize (150 cm TL; Platt, 1996). Blood (ca. 1.0 to 10.0 mL, depending on animal size) was collected from the post-cranial sinus, transferred to an ethylenediaminetetraacetic acid (EDTA)-treated Vacutainer®, and centrifuged at 2000 rpm for 10 min. The plasma supernatant was then transferred to a collection tube and frozen at − 25 °C until shipment to Texas Tech University. Samples were then stored at − 80 °C until assayed for the presence of vitellogenin. Following sample collection, each crocodile was marked and released at its site of capture.” 2.2. Experimental design For this study, plasma from adult female Morelet's crocodiles collected during the breeding season were chosen as a source of vitellogenin for purification and to evaluate vitellogenin concentrations in wild individuals. Plasma samples from adult males from the same time period were used as negative controls and for comparative purposes. 2.3. Protein assay Protein concentrations of plasma were measured using a Coomassie protein assay (Bradford, 1976). Bovine serum albumin was used to generate the standard curve. Two μl of each plasma sample were added to 2 mL of water, and 2 mL Coomassie reagent (0.6% Coomassie G 250 in 3% perchloric acid) was added. The mixture was vortexed and then incubated for 10 min at room temperature, after which absorbance was read at 620 nm using a Genosys 20 (Themo Spectronic, Rochester, NY, USA) spectrophotometer. Bovine serum albumin was used for the standard curve.

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2.4. Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) Morelet's crocodile plasma samples were electrophoresed under denaturing conditions in polyacrylamide gels (SDSPAGE) using a BioRad (Hercules, CA, USA) minigel apparatus and BioRad 4–20% gradient gels. Samples were diluted 1 : 25 with water, mixed 1 : 1 volume : volume with Laemmli sample buffer (2% SDS, 62.5 mM Tris–HCl, pH 6.8, 0.01% bromophenol blue, 25% weight : volume glycerol combined with 10% weight : volume 2-mercaptoethanol), and heated 4 min in a boiling water bath. Running buffer was 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3. Plasma samples (10 μL per lane) were run at 25 mA until the dye-front reached the bottom of the gel. Gels were either used for immunoblotting (below) or were stained with Coomassie Blue stain (25% weight : volume trichloroacetic acid, 10% volume : volume methanol, 0.1% w/v Coomassie Blue R-250) overnight and then destained using 8% acetic acid. Molecular weight was assessed by comparison with unstained broad range molecular weight markers (Sigma-Aldrich Chemical Co, St. Louis, MO, USA). 2.5. Immunoblotting Proteins subjected to SDS-PAGE were transferred to PVDF membranes (BioRad, Hercules, CA, USA) for use in immunoblotting. Transfers were performed in a BioRad Trans-Blot apparatus packed in ice at 70 V for 3 h. Transfer buffer was 25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3. After transfer, the PVDF membrane was incubated in 5% nonfat dry milk for 1 h at room temperature. This solution was replaced with 5% nonfat dry milk containing rabbit anti-vitellogenin antiserum diluted 1 : 2000, and incubated for 16 h at room temperature with shaking. The PVDF membrane was then washed (10 min with shaking) once with 50 mM Tris–HCl (pH 7.5), 0.9% NaCl, 0.05% Tween-20 and twice with 50 mM Tris–HCl (pH 7.5), 0.9% NaCl. The PVDF membrane was then incubated in 5% nonfat dry milk containing peroxidasecoupled goat anti-rabbit IgG serum (BioRad, Hercules, CA, USA), diluted 1 : 3000, for 2 h at room temperature with shaking. The PVDF membrane was again washed (10 min with shaking) once with 50 mM Tris–HCl (pH 7.5), 0.9% NaCl, 0.05% Tween-20 and twice with 50 mM Tris–HCl (pH 7.5), 0.9% NaCl. Then, the PVDF membrane was developed for 1 min with peroxidase substrate (diaminobenzidine and ureahydrogen peroxide tablets, Sigma). The reaction was terminated by immersion in Tris–saline buffer for five minutes. Molecular weight was assessed by comparison with Kaleidoscope prestained molecular weight markers (BioRad, Hercules, CA, USA). 2.6. Purification of Morelet's crocodile vitellogenin Plasma from the five female and five adult male Morelet's crocodiles were pooled by sex and fractionated using a DEAE chromatography procedure that was previously effective for purification of Xenopus leavis vitellogenin (Palmer et al., 1998).

The procedure was run on a BioRad Econo Chromatography system (BioRad, Hercules, CA, USA) using a 1.0 × 15 cm column filled with DEAE-agarose (BioRad). The equilibration buffer was 25 mM Tris–HCl, 1 mM monothioglycerol, pH 7.5. The gradient buffer was 500 mM NaCl in equilibration buffer. The female and male plasma pools were diluted 1 : 4 volume : volume in equilibration buffer (400 μl total volume) and loaded onto the column. The column was washed with 30 mL equilibration buffer to remove proteins that did not adhere to the DEAE. A linear gradient from 0 to 500 mM NaCl was run through the column using a total volume of 90 mL. Protein was monitored by UV absorbance at 280 nm throughout the chromatography run. Fractions (3 mL) were collected throughout the procedure. Protein levels were determined separately for each fraction by Coomassie protein assay. Fractions found to contain significant amounts of protein were evaluated by denaturing polyacrylamide gel electrophoresis. DEAE fractions found to contain vitellogenin (210-kDa protein) were pooled and dialyzed against DEAE equilibration buffer overnight using Spectra/Por 1 dialysis membrane (Spectrum, Houston, TX, USA). After dialysis, the pooleddialyzed fractions were re-run on the DEAE column using the same conditions as for the plasma. The resulting vitellogenincontaining fractions were pooled, dialyzed against water, and lyophilized until use. The protein concentration of the purified vitellogenin after reconstitution in Tris–HCl (50 mM, pH 7.5) was determined by Coomassie protein assay, as described above. 2.7. Gel filtration The procedure was run on a BioRad Econo Chromatography system using a 1.5 × 150 cm column filled with Sepharose 6B (Sigma). The chromatography buffer was 25 mM Tris–HCl, 1 mM monothioglycerol, pH 7.5. Purified dialyzed vitellogenin-containing fractions from the second DEAE procedure were and loaded onto the column. The column was eluted with 300 mL buffer. Protein was monitored by UV absorbance at 280 nm throughout the chromatography run. The column was calibrated using gel filtration calibration standards (Sigma). 2.8. Vitellogenin antibody The vitellogenin antibody was generated against a peptide representing a highly conserved region of the vitellogenin protein (Selcer et al., 2001). The sequence for the peptide (EYRHIIPTTVGLPAELSLYQSAI) was selected based on multiple alignment of vitellogenin amino acid sequences from chicken (Gallus gallus), lamprey (Ichthyomyzon unicuspus), African clawed frog (X. laevis), sturgeon (Acipenser transmontano), and killifish (Fundulus heteroclitus). The antibody has been found to broadly cross-reactive against vertebrate vitellogenins (Selcer et al., 2001), including the frog X. laevis (African clawed frog), the turtles Trachemys scripta (red-eared slider) and Lepidochelys kempi (Atlantic ridley), the alligator A. mississippiensis (American alligator) and the fish Carassius auratus (goldfish).

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2.9. Vitellogenin enzyme-linked immunosorbent assay (ELISA)

3. Results

An indirect antibody-capture ELISA (Specker and Anderson, 1994) was developed for measuring Morelet's crocodile plasma vitellogenin levels. Initially, pooled male and pooled female plasma samples were compared to establish that the ELISA was able to detect difference between the two and to determine appropriate dilutions. Subsequently, results from individual male and female samples were compared with a standard curve generated using the purified Morelet's crocodile vitellogenin. Morelet's crocodile plasma samples were diluted (1 : 200 to 1 : 5000) with phosphate-buffered saline (PBS, 136 mM NaCl, 1.5 mM KH2PO4, 8.2 mM Na2HPO4, 2.7 mM KCl, and pH 7.2) and 100 μL of each was added to individual wells of an Immunosorb microtiter plate (Nunc, Denmark). PBS alone was added to wells designated as blanks. Antigen was allowed to bind to the plate overnight at 4 °C in a container filled with water and covered with plastic wrap to create a humid chamber. The plate was then washed three times with PBS. 100 μL of PBS-blotto (5 g nonfat dry milk in 100 mL PBS) was then used to block for one hour at room temperature, with shaking. The PBS-blotto was then replaced with 100 μL of PBS-blotto containing rabbit anti-vitellogenin antisera (1 : 2000 dilution). The plate was incubated for two h at room temperature, with shaking. The plate was then washed five times with PBS and incubated under the same conditions for an additional two h with goat anti-rabbit IgG conjugated to horseradish peroxidase (BioRad), diluted 1 : 1000 in PBS-blotto. The plate was again washed five times with PBS. Then, the plate was developed using TMB Peroxidase EIA Substrate Kit (BioRad) and incubated for ten minutes at room temperature. The reaction was stopped with 1 N H2SO4 and the optical density was read at 450 nm with background subtraction at 655 nm. For the standard curve, purified vitellogenin was diluted in PBS at various concentrations (1 to 500 ng/ mL) and the ELISA was performed as above. Blank wells contained PBS only as the antigen. Intra-assay variation was calculated from the values of the same female plasma sample plated in ten different wells. Inter-assay variation was calculated from the same female plasma sample plated in triplicate on six different occasions. Recovery was determined by adding known amounts of vitellogenin to a male plasma sample. Sensitivity was estimated by plating multiple wells of each of the lower vitellogenin concentrations of the standard curve and comparing absorbances from these with the values obtained for multiple wells of the zero concentration.

3.1. Plasma protein content and composition of male and female Morelet's crocodile

2.10. Statistical analysis Statistics were performed using the computer program Prism 4 (GraphPad Software, Inc, San Diego, CA, USA). Plasma protein concentration was compared between male and female crocodiles using Students t-test. The equations for the standard curves for the Coomassie protein assay and for the vitellogenin ELISA were generated by nonlinear regression. Probabilities of P b 0.05 were considered significant.

Plasma protein concentration from the adult female crocodiles averaged 101.6 ± 5.9 (mean ± SE) μg/μL compared to 97.5 ± 4.9 μg/μL for the adult male crocodiles. There was no significant difference in plasma protein concentration between the female and male samples (t = 0.611; 8 df; P N 0.05). Fig. 1 (panel A) shows a denaturing polyacrylamide gel (SDS-PAGE) of plasma from adult female and male Morelet's crocodiles. Plasma from females contained substantial amounts of a high molecular weight protein that was absent in male plasma. This presumptive vitellogenin was calculated to have a molecular mass of 210 kDa based on comparison of the rf value for this protein with a nonlinear regression of log molecular weight versus rf for the molecular weight standards (R = 0.98, 5 df, P b 0.001). 3.2. Crossreactivity of the anti-vitellogenin antibody with Morelet's crocodile plasma Western blotting was performed on PVDF membranes containing proteins transferred from SDS-PAGE gel electrophoresis of male and female Morelet's crocodile plasma, using MW (kDa) 205 119 66 55 45

A

36

216 132 78 46 32.5

B 1

2

3

4

5

6

7

M F M F S M F Fig. 1. Denaturing gel electrophoresis and vitellogenin Western blot of plasma from adult female and male Morelet’s crocodile (Crocodylus moreletii). Plasma was separated using a 4–20% denaturing polyacrylamide gel and stained with Coomassie (panel A). Western blot (panel B) shows immunoreactivity of polyclonal anti-vitellogenin rabbit serum against male and female plasma. Abbreviations : M = male plasma, F = female plasma, S = Sigma unstained molecular weight markers (for panel A) or BioRad Kaleidoscope prestained molecular weight markers (for panel B).

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the broadly crossreactive vitellogenin peptide antiserum as the source of antibody. As Fig. 1 (panel B) shows, the rabbit vitellogenin antiserum cross-reacted with several protein bands in the lanes containing female plasma but not with any bands in the lanes containing male plasma. The 210-kDa band presumed to be vitellogenin in the Coomassie-stained gels was one of the bands that cross-reacted with the anti-vitellogenin antiserum. Other bands in female plasma that cross-reacted with the vitellogenin antiserum had apparent molecular masses of 119, 78, and 60 kDa.

DEAE-chromatography was used to separate the proteins from female plasma in order to purify Morelet's crocodile vitellogenin. Male plasma was run as a control. The protein profile of the DEAE fractions from female plasma had five distinct peaks (Fig. 2). The first peak eluted during the wash (no salt) and represented proteins that did not adhere to the column. The second, third and fourth peaks eluted during the salt gradient at medium salt (≈ 175, 250, and 350 mM, respectively) and the fifth peak eluted at high salt (≈ 450 mM). In contrast, DEAE chromatography of plasma from male crocodiles resulted in only four major peaks, corresponding to the wash peak and the three medium-salt peaks. The high salt peak was lacking in males. SDS-PAGE analysis (not shown) of the fractions from the DEAE column revealed that the high-salt peak from the female plasma contained only the 210-kDa protein. The same fractions from male plasma contained no detectable protein. The DEAE procedure was repeated on fractions from the female plasma high salt peak, after dialysis against the chromatography buffer to remove the salt. SDS-PAGE and Western blotting of the purified vitellogenin revealed crossreactivity only to the 210-kDa band (Fig. 3). Gel filtration analysis of the purified vitellogenin, after dialysis, had an estimated molecular mass of 450 kDa compared with the migration of the calibration standards.

Protein

VTG

400

0.50 300 200 0.25 100

NaCl Concentration (mM)

(absorbance at 620 nm)

500

Male Female

0

0.00 0

10

20

30

40

132 kDa 78 kDa

45 kDa

A

B

1 2 3 V V K

3.3. Purification of Morelet's crocodile vitellogenin

0.75

216 kDa

50

4 M

5 F

1 2 3 V V K

4 M

5 F

Fig. 3. Denaturing gel electrophoresis (SDS-PAGE) and vitellogenin Western blot of purified vitellogenin from female Morelet’s crocodile (Crocodylus moreletii). Vitellogenin was run on a 4–20% denaturing polyacrylamide gel and stained with Coomassie (panel A). Western blot (panel B) shows immunoreactivity of polyclonal anti-vitellogenin rabbit serum against purified vitellogenin and male and female plasma. Abbreviations are as follows: V = purified vitellogenin, K = BioRad Kaleidoscope molecular weight markers, M = male plasma, F = female plasma.

3.4. Evaluation of the ELISA Cross-reactivity of the anti-Xenopus vitellogenin antibody against male and female Morelet's crocodile plasma was also tested using an indirect antibody-capture enzyme-linked immunosorbent assay (ELISA) procedure. In preliminary assays, there was substantially greater reactivity of the vitellogenin antiserum against pooled female crocodile plasma than pooled male plasma, with differences in optical density detected between female and male plasma dilutions up to 1 : 16,000. A standard curve was generated by diluting the purified vitellogenin at concentrations ranging from 1 to 500 ng/mL (Fig. 4). The detection limit of the assay was determined to be 20.0 ng/mL. The working range was from 20 to 500 ng/mL of diluted sample. The intra-assay coefficient of variation averaged 5.3% and the inter-assay coefficient of variation was 9.8%. The recovery of vitellogenin diluted into male plasma averaged 94.7%. Pooled plasma from females, diluted from 1 : 1000 to 1 : 20,000, ran parallel to the vitellogenin standard curve. Pooled 3

Absorbance (450 nm)

54

2

1

Fraction Number 0

Fig. 2. Comparison of DEAE-chromatography profiles of plasma from female and male Morelet’s crocodile (Crocodylus moreletii). Plasma proteins were separated on a column of DEAE BioGel A (BioRad) using a linear salt gradient (0–500 mM NaCl). Protein concentrations of each fraction were subsequently measured using a Coomassie protein assay. The position of the presumptive vitellogenin peak is labeled VTG.

1

10

100

1000

Vitellogenin (ng/ml) Fig. 4. Standard curve for the indirect, antibody-capture enzyme-linked immunosorbent assay (ELISA) for Morelet’s crocodile (Crocodylus moreletii) vitellogenin.

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Vitellogenin (mg/ml)

3

2

1

0 Females

Males

Fig. 5. Vitellogenin concentrations for plasma of wild-caught female and male Morelet’s crocodiles (Crocodylus moreletii) from northern Belize, as determined by ELISA. Bars represent the mean ± 1 standard error. Vitellogenin was undetectable in all male plasma samples. N = 5.

plasma from males had background optical density values at all dilutions tested. 3.5. Vitellogenin concentrations in plasma of wild-caught females and males Plasma samples from five adult male and five adult female Morelet's crocodiles collected in northern Belize during the breeding season were evaluated using the vitellogenin ELISA (Fig. 5). Vitellogenin concentrations of female plasma samples ranged from 1.8 to 3.1 mg/mL with an average of 2.54 ± 0.25 mg/mL. No vitellogenin was detected in any of the male samples. 4. Discussion In this study, we have provided new information on vitellogenin of Morelet's crocodile C. moreletii. We have purified the vitellogenin from this species and have developed an enzyme-linked immunosorbent assay for measurement of plasma vitellogenin. We have used this assay to measure levels of plasma vitellogenin from female Morelet's crocodile taken during the breeding season. Vertebrate vitellogenins are known to elute from DEAE chromatography under high-salt conditions (Wiley et al., 1979; Tyler and Sumpter, 1990; Carnevali and Belvedere, 1991; Kishida et al., 1992; Selcer and Palmer, 1995; Palmer et al., 1998). Therefore, DEAE chromatography is an effective means of confirming that a particular protein is vitellogenin and it is also a useful technique for purification purposes. DEAE chromatography of plasma from female Morelet's crocodiles resulted in a high-salt protein peak (450 mM NaCl) that was absent in the same fractions from the male plasma. Denaturing polyacrylamide gel electrophoresis revealed that this peak primarily contained a 210-kDa protein. Furthermore, the 210-kDa protein from the high-salt peak cross-reacted in Western blotting with the broadly crossreactive vitellogenin antiserum. This supports the contention that the 210-kDa protein in female Morelet's crocodile plasma is vitellogenin.

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The purified Morelet's crocodile vitellogenin had an apparent molecular mass of 450 kDa as determined by gel filtration, and 210 kDa as measured by SDS-PAGE. This suggests that the native protein is a dimer. Previous reports of vitellogenin in other species have also found that vitellogenin is a dimer in its native form (Callard and Ho, 1987; Mosconi et al., 1998). The 210-kDa size for the Morelet's crocodile vitellogenin monomer is similar to that reported for other reptiles (Callard and Ho, 1987; Selcer and Palmer, 1995; Heck et al., 1997). The molecular mass of the vitellogenin monomer in American alligator (A. mississippiensis) has been reported as 202–207 kDa by Gunderson et al. (2003) and as 220 kDa by Selcer et al. (2001). An anti-vitellogenin antibody was used for identification of vitellogenin and for the immunoassay. No vitellogenin antibody was available for Morelet's crocodile or any other crocodilian. Therefore, we used an antibody that was generated against a peptide representing a highly conserved region of the vitellogenin protein (Selcer et al., 2001). The sequence for the peptide (EYRHIIPTTVGLPAELSLYQSAI) was selected based on multiple alignment of vitellogenin amino acid sequences from chicken (G. gallus), lamprey (I. unicuspus), African clawed frog (X. laevis), sturgeon (A. transmontanus), and killifish (F. heteroclitus). The antibody has been found to broadly cross-reactive against vertebrate vitellogenins (Selcer et al., 2001), including the frog X. laevis (African clawed frog), the turtles T. scripta (red-eared slider) and L. kempi (Atlantic ridley), the alligator A. mississippiensis (American alligator) and the fish C. auratus (goldfish). In Western blotting of Morelet's crocodile plasma, the antivitellogenin peptide antibody strongly cross-reacted with bands from the female plasma but showed no cross-reactivity with bands from the male plasma, indicating that it recognized a female-specific protein. The reactivity was primarily against a protein in the 210-kDa region, in the same location as the putative vitellogenin band detected by Coomassie staining. Additional crossreactivity was present for several smaller bands in the female samples, but there was no apparent crossreactivity in the male samples. These smaller proteins may represent degradation products of the intact vitellogenin. Proteolytic fragments of vitellogenin have been observed in a number of other studies (Goodwin et al., 1992; Palmer et al., 1998; Tyler and Sumpter, 1990). Alternatively, the smaller proteins may represent other forms of vitellogenin. Multiple vitellogenin genes are known to exist in X. laevis (Wahli et al., 1979; Jaggi et al., 1980) and G. gallus (Wang and Williams, 1983). Furthermore, multiple vitellogenin proteins have been reported for several other species (Wiley and Wallace, 1978; Ding et al., 1989; Sawaguchi et al., 2005). An indirect, antibody-capture enzyme-linked immunosorbent assay (ELISA) was developed to measure Morelet's crocodile plasma vitellogenin levels. The first step was to demonstrate that the antiserum cross-reacted differently with plasma from male and female animals. Substantially greater reactivity was observed with female crocodile plasma compared to male crocodile plasma. Next, a purified Morelet's crocodile vitellogenin preparation was made by successive rounds of DEAE purification. The identity and purity of the final

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vitellogenin product was confirmed by SDS-PAGE and Western blotting. The purified Morelet's crocodile vitellogenin was then used to generate a standard curve for the vitellogenin ELISA assay. The standard curve developed revealed that the vitellogenin ELISA was dose-dependent and was reasonably sensitive. The sensitivity (20 ng/mL) and the working range (20–500 ng/mL of diluted plasma) of this assay should make it useful for studies of vitellogenin patterns in females during the reproductive season and for studies of endocrine disruption in this species. We used the ELISA to evaluate vitellogenin levels in wildcaught female and male Morelet's crocodiles taken during the breeding season from northern Belize. Females averaged 2.5 mg/mL vitellogenin in their plasma. Males did not contain any detectable vitellogenin. There are very few reports of vitellogenin levels from wild-caught reptiles for comparison. Tada et al. (2004) found that adult female pond turtles (Chinemys reevesii) taken from a Japanese river had vitellogenin levels ranging from 1 to 15 mg/mL. Herbst et al. (2003) reported a plasma vitellogenin level of 4.6 mg/mL for a single female nesting turtle. Thus, the vitellogenin concentrations found for female Morelet's crocodile during the breeding season are similar to those observed for other reptile species. One reason for the development of a vitellogenin assay for Morelet's crocodile was as a tool to study endocrine disruption in this species. Over the last decade, increasing evidence of contaminant-induced endocrine disruption in wildlife has highlighted the need for sensitive and reliable assays to screen populations for exposure to hormone-altering compounds (Colborn et al., 1993; McLachlan, 1993; Crain and Guillette, 1997). Vitellogenin induction has shown promise as a sensitive and non-destructive biomarker of wildlife exposure to xenobiotic estrogens, particularly in aquatic systems (Heppell et al., 1995; Sumpter and Jobling, 1995; Palmer and Selcer, 1996; Folmar et al., 1997; Tyler et al., 1999; Cheek et al., 2001). The majority of research examining contaminant-induced vitellogenesis in wildlife has involved laboratory and field studies on fish, and a considerable number of studies on wild fish have validated the use of this biomarker in the field (e.g., Folmar et al., 1997; Allen et al., 1999; Okoumassoun et al., 2002; Cheek et al., 2004; Kirby et al., 2004). Vitellogenin induction has also been observed in turtles and lizards exposed to estrogenic compounds in the laboratory (Palmer and Palmer, 1995; Brasfield et al., 2002; Herbst et al., 2003; Tada et al., 2004), suggesting the utility of this endpoint as a biomarker of environmental estrogen exposure in wild reptiles. However, despite evidence of population declines and widespread exposure to xenobiotic estrogens in these animals (Gibbons, 2000), few studies have examined vitellogenin in wild reptiles living in contaminated habitats (Irwin et al., 2001; Shelby and Mendonca, 2001; Gunderson et al., 2003; Rie et al., 2005). This is largely due to a lack of available antibodies specific for vitellogenin in these animals and the limited interspecies cross-reactivity of the antibodies that are available (Selcer et al., 2001). The availability of an assay capable of measuring plasma vitellogenin in Morelet's crocodile should facilitate studies of the effects of endocrine disruption in

populations of this species that are exposed to environmental contaminants. Acknowledgments We thank Robert Noonan (Gold Button Ranch) for providing access to his property and Mark and Monique Howells (Lamanai Outpost Lodge/Lamanai Field Research Center) for providing logistical support and accommodations in Belize during this study. Tanny Brown, Benjamin Cruz, Sean Richards, and Jim Stoker assisted in the field. This research was supported by Lamanai Field Research Center, Indian Church, Belize and U.S. EPA (Grant no. R826310 to STM). DAR and LDD were supported by National Geographic Society (Grant nos. 6529-99 and 7007-01 to LDD) and Texas Tech University. SGP was supported by Wildlife Conservation Society. Additional support for TRR was provided by the ARCS Foundation, Lubbock, Texas. References Allen, Y.A.P., Scott, A.P., Matthiessen, P., Haworth, S., Thain, J.E., Feis, S., 1999. Survey of estrogenic activity in United Kingdom estuarine and coastal waters and its effects on gonadal development in the flounder Platichthys flesus. Environ. Toxicol. Chem. 18, 1791–1800. Allsteadt, J., Lang, L.W., 1995. Sexual dimorphism in the genital morphology of young American alligators, Alligator mississippiensis. Herpetologica 51, 314–325. Bergink, E.W., Wallace, R.A., 1974. Precursor-product relationship between amphibian vitellogenin and the yolk proteins, lipovitellin and phosvitin. J. Biol. Chem. 249, 2897–2903. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brasfield, S.M., Weber, L.P., Talent, L.G., Janz, D.N., 2002. Dose–response and time course relationships for vitellogenin induction in male western fence lizards (Sceloporus occidentalis) exposed to ethinylestradiol. Environ. Toxicol. Chem. 21, 1410–1416. Callard, I.P., Ho, S.-M., 1987. Vitellogenesis and viviparity. In: Chester-Jones, I., Ingleson, P.M., Phillips, J.G. (Eds.), Fundamentals of Comparative Vertebrate Endocrinology. Plenum, New York, pp. 257–282. Carnevali, O., Belvedere, P., 1991. Comparative studies of fish, amphibian, and reptilian vitellogenins. J. Exp. Zool. 259, 18–25. Carnevali, O., Mosconi, G., 1992. In vitro induction of vitellogenin synthesis in Rana esculenta: role of the pituitary. Gen. Comp. Endocrinol. 86, 352–358. Cheek, A.O., Brouwer, T.H., Carroll, S., Manning, S., McLachlan, J.A., Brouwer, M., 2001. Experimental evaluation of vitellogenin as a predictive biomarker for reproductive disruption. Environ. Health Perspect. 109, 681–690. Cheek, A.O., King, V.W., Burse, J.R., Borton, D.L., Sullivan, C.V., 2004. Bluegill (Lepomis macrochirus) vitellogenin: purification and enzymelinked immunosorbent assay for detection of endocrine disruption by papermill effluent. Comp. Biochem. Physiol. C 137, 249–260. Colborn, T., vom Saal, F.S., Soto, A.M., 1993. Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ. Health Perspect. 101, 378–384. Crain, D.A., Guillette Jr., L.J., 1997. Endocrine-disrupting contaminants and reproduction in vertebrate wildlife. Rev. Toxicol. 1, 47–70. Danzo, B.J., 1998. The effects of environmental hormones on reproduction. Cell. Mol. Life Sci. 54, 1249–1264. Ding, J.L., Hee, P.L., Lam, T.J., 1989. Two forms of vitellogenin in the plasma and gonads of male Oreochromis aureus. Comp. Biochem. Physiol. B 93, 363–370.

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