Influence of tissue, age, and environmental quality on DNA methylation in Alligator mississippiensis

in Reproduction
Authors:
Benjamin B Parrott Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA
Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA

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John A Bowden Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA
Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA

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Satomi Kohno Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA
Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA

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Jessica A Cloy-McCoy Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA
Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA

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Matthew D Hale Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA
Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA

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Jacqueline T Bangma Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA
Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA

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Thomas R Rainwater Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA
Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA
Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA

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Phillip M Wilkinson Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA

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John R Kucklick Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA
Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA

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Louis J Guillette Jr Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA
Department of Obstetrics and Gynecology, Hollings Marine Laboratory, National Institute of Standards and Technology (NIST), United States Fish and Wildlife Service, Tom Yawkey Wildlife Center, Medical University of South Carolina, Charleston, South Carolina, USA

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Epigenetic modifications are key mediators of the interactions between the environment and an organism's genome. DNA methylation represents the best-studied epigenetic modification to date and is known to play key roles in regulating transcriptional activity and promoting chromosome stability. Our laboratory has previously demonstrated the utility of the American alligator (Alligator mississippiensis) as a sentinel species to investigate the persistent effects of environmental contaminant exposure on reproductive health. Here, we incorporate a liquid chromatography–tandem mass spectrometry method to directly measure the total (global) proportion of 5-methyl-2′-deoxycytidine (5mdC) in ovarian and whole blood DNA from alligators. Global DNA methylation in ovaries was significantly elevated in comparison with that of whole blood. However, DNA methylation appeared similar in juvenile alligators reared under controlled laboratory conditions but originating from three sites with dissimilar environmental qualities, indicating an absence of detectable site-of-origin effects on persistent levels of global 5mdC content. Analyses of tissues across individuals revealed a surprising lack of correlation between global methylation levels in blood and ovary. In addition, global DNA methylation in blood samples from juvenile alligators was elevated compared with those from adults, suggesting that age, as observed in mammals, may negatively influence global DNA methylation levels in alligators. To our knowledge, this is the first study examining global levels of DNA methylation in the American alligator and provides a reference point for future studies examining the interplay of epigenetics and environmental factors in a long-lived sentinel species.

Abstract

Epigenetic modifications are key mediators of the interactions between the environment and an organism's genome. DNA methylation represents the best-studied epigenetic modification to date and is known to play key roles in regulating transcriptional activity and promoting chromosome stability. Our laboratory has previously demonstrated the utility of the American alligator (Alligator mississippiensis) as a sentinel species to investigate the persistent effects of environmental contaminant exposure on reproductive health. Here, we incorporate a liquid chromatography–tandem mass spectrometry method to directly measure the total (global) proportion of 5-methyl-2′-deoxycytidine (5mdC) in ovarian and whole blood DNA from alligators. Global DNA methylation in ovaries was significantly elevated in comparison with that of whole blood. However, DNA methylation appeared similar in juvenile alligators reared under controlled laboratory conditions but originating from three sites with dissimilar environmental qualities, indicating an absence of detectable site-of-origin effects on persistent levels of global 5mdC content. Analyses of tissues across individuals revealed a surprising lack of correlation between global methylation levels in blood and ovary. In addition, global DNA methylation in blood samples from juvenile alligators was elevated compared with those from adults, suggesting that age, as observed in mammals, may negatively influence global DNA methylation levels in alligators. To our knowledge, this is the first study examining global levels of DNA methylation in the American alligator and provides a reference point for future studies examining the interplay of epigenetics and environmental factors in a long-lived sentinel species.

Introduction

Interactions between an organism's genome and the environment are thought to underlie the etiology of many complex disorders of the reproductive system. Epigenetic mechanisms have long been invoked as key mediators of these interactions, and more recent studies have begun to elucidate the molecular and biochemical nature of a broad array of epigenetic modifications (Packard 1901, Barker 1990). However, it remains unclear how combined exposures to ranges of environmental factors such as temperature, nutrition, and exposure to intricate mixtures of exogenous chemicals such as anthropogenic contaminants influence epigenetic programming. Here, we characterize tissue-specific global DNA methylation in a long-lived top predator, the American alligator (Alligator mississippiensis). Furthermore, we incorporate a model in which alligators originating from different environments are raised under controlled conditions to examine persistent effects of environmental quality and geographic location on global DNA methylation.

DNA methylation, consisting of the covalent addition of a methyl group to cytosine, represents the most fundamental and well-studied epigenetic modification to date. Cytosines directly preceding guanines (CpG dinucleotides) represent the predominant methylation sites in vertebrates. In mammals, the majority of DNA methylation patterning occurs during early development (Brandeis et al. 1993). However, the developmental dynamics of methylation patterning appear to be somewhat divergent across other vertebrates (Veenstra & Wolffe 2001, Stancheva et al. 2002, Potok et al. 2013). Once established, patterning is thought to retain a degree of temporal stability due to mitotic inheritance. Yet, studies investigating changes in DNA methylation patterning over short periods of time and in response to different stimuli have revealed a degree of context-dependent plasticity and variation (Woodfine et al. 2011, Barrès et al. 2012, Byun et al. 2012). For example, global hypomethylation of genomic DNA was observed in skeletal muscle in men and women shortly after exercise (Barrès et al. 2012). When taken together, these studies suggest that DNA methylation patterning remains stable over time but retains an underlying plasticity, allowing changes in response to specific cues.

Decreased levels of global genomic DNA methylation has been linked to aging, genomic instability, and malignant cancers (Gaudet et al. 2003, Rodriguez et al. 2006, Bollati et al. 2009, Hannum et al. 2013). In addition, environmental factors, such as exposure to air pollution and endocrine disrupting contaminants (EDCs), have also been shown to influence global levels of DNA methylation (Rusiecki et al. 2008, Baccarelli et al. 2009, Bromer et al. 2010). Yet, much of our current understanding of DNA methylation patterning in response to environmental cues is based on studies incorporating traditional laboratory models, cell culture, and associative epidemiological studies. Thus, a gap exists in our understanding of how long-term, chronic exposures to mixtures of contaminants affect DNA methylation patterning in real-world populations. The American alligator was chosen as a study model as this animal is long lived and has high site fidelity in the wild, and thus reflects ambient environmental conditions in its habitat. These characteristics provide an ideal model in which the aforementioned gap in our understanding can be addressed. Here, we report our findings utilizing the alligator as a sentinel to investigate persistent effects of environmental quality and geographic location on measures of genomic methylation levels.

Alligators living in Lake Apopka, Florida, undergo a combinatorial exposure to high levels of pesticides and agricultural contamination compared with those alligators living in a nearby, but relatively pristine lake, Lake Woodruff (Heinz et al. 1991, Guillette et al. 1999, Rauschenberger et al. 2007). A number of reproductive abnormalities, including altered gonadal development and decreased robustness of sexually dimorphic gene expression, have been reported in alligators originating from Lake Apopka (Milnes et al. 2005, 2008). Yet, understanding of the molecular mechanisms mediating the effects of poor environmental quality on reproductive health remain incomplete. Interestingly, our group has shown that many of these perturbations, including altered gonadal gene expression, persist in laboratory-reared juvenile alligators originating from field-collected eggs (Moore et al. 2010a, 2010b, 2011). Thus, the origins of these long-lasting impacts on reproductive health are likely to have roots in early development and may be mediated by epigenetic mechanisms. This study also incorporates animals derived from a different geographical location along the north central coast of South Carolina (Yawkey Wildlife Center). This population thrives near the northern boundary of the American alligator range and is exposed to more fluctuations in seasonal temperature and salinity compared with animals at Lake Woodruff or Lake Apopka.

A promising strategy to measure global DNA methylation is to measure the proportion of 5-methyl-2′-deoxycytidine (5mdC) in relation to total 2′-deoxycytidine (dC) or 2′-deoxyguanosine (dG) directly using liquid chromatography–tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) (Song et al. 2004, Kok et al. 2007, Ma et al. 2008, Quinlivan & Gregory 2008a, Liu et al. 2009, Le et al. 2011). Using direct measures that are independent of DNA sequence have become increasingly crucial to our understanding of global methylation as other assays, analyzing the methylation status of repetitive elements as a proxy for global methylation, have yielded inconsistent findings (Nelson et al. 2011). Here, we directly measure the relative genomic content of 5mdC employing dG as an internal calibrant. We examine global methylation levels in circulating blood cells and in ovarian tissue and find substantial differences between the two. Interestingly, we did not observe a correlation between global genomic methylation levels in blood and ovary across individuals, suggesting that methylation levels in the blood may not reflect those within the ovary. For female juvenile alligators originating from field-collected eggs but reared under identical conditions in the laboratory, no effect of site was observed. Finally, we compare levels of genomic methylation in juveniles with levels in wild-caught adults and find a dramatic decrease in global methylation in the latter, suggesting that age may be a crucial determinant of DNA methylation in alligators. This study provides the first data on the influence of tissue, site, and age on global DNA methylation in a crocodilian and provides a foundation to further explore epigenetic mechanisms in long-lived sentinel species.

Materials and methods

Field collections and animal husbandry

All experiments performed in this study conformed to the guidelines set forth by the Institutional Animal Care and Use Committee at the Medical University of South Carolina. All fieldwork and collections were performed under permits issued by the Florida Fish and Wildlife Commission, South Carolina Department of Natural Resources, and United States Fish and Wildlife Service. Adult alligators were captured at Lake Woodruff using a pole with a snare in April 2010 and a blood sample was immediately acquired and stored on ice in a heparinized vacutainer (BD Diagnostics, Franklin Lakes, NJ, USA) before centrifugation at 1500 g for 10 min at 4 °C. The pelleted cells were suspended in RNA later, placed on ice, and then stored at −20 °C.

For studies on juvenile alligators, eggs were collected shortly after oviposition from Lake Woodruff National Wildlife Refuge (FL), Lake Apopka (FL), and Yawkey Wildlife Center (SC) in June 2010 (as shown in Fig. 1). At least one egg from each clutch was necropsied shortly after collection and staged according to Ferguson (1985). For all clutches, embryos were collected before the stage at which temperature-dependent sex determination commences. Animals representing six clutches from Lake Apopka, ten clutches from Lake Woodruff, and ten clutches from Yawkey Wildlife Center were used in this study. Eggs were placed in damp sphagnum moss and incubated in commercial incubators (Thermo Scientific, Waltham, MA, USA, Forma Environmental Chamber, model #3920) at the female-producing temperature of 30 °C until hatching. After hatching, each alligator was marked with a number monel tag attached to the webbing of the right rear foot and individuals were co-housed in flow-thru tanks (1000 l) at the Hollings Marine Laboratory. Water temperature was maintained at 32 °C. Animal health was checked daily and animals were fed a commercial crocodilian diet (Mazuri, Arden Hills, MN, USA) ad libitum. When each animal reached 1 kg in body mass, a blood sample was acquired immediately before killing and treated as described above for adult samples. There was no significant variation in the age of animals at the time of killing across the three sites. Animals were then necropsied and ovaries were placed in RNA later on ice before storing at −80 °C.

Figure 1
Figure 1

Experimental design. (A) Three Southeastern (USA) sites were selected for alligator egg collection: Yawkey Wildlife Refuge (YK), Lake Woodruff (WO), and Lake Apopka (AP). (B) Alligator eggs were collected from each site and incubated under identical conditions at female-producing temperature. (C) Alligator hatchlings were allowed to reach 1 kg in weight. DNA was isolated from ovaries and blood. (D) 5mdC and dG, from the isolated and hydrolyzed DNA, were examined using LC-MS/MS. A resultant chromatogram is shown for the LC-MS/MS analysis of DNA extract from an alligator ovary. For quantitation of percentage of 5mdC, calibration curves were prepared in triplicate (with each curve in triplicate analyzed twice) and were constructed by varying the amount of 5mdC while holding dG constant.

Citation: REPRODUCTION 147, 4; 10.1530/REP-13-0498

Nucleic acid extraction and DNA hydrolysis

Both RNA and DNA were simultaneously extracted from each tissue. RNA was extracted using the one-step acid phenol–guanidium thiocyanate–chloroform method described by Chomczynski & Sacchi (1987) with minor modifications. RNA was then further purified using the SV Total RNA Isolation System (Promega). Genomic DNA was isolated from the organic phase resulting from the RNA extraction by addition of back extraction buffer (4 M guanidine thiocyanate, 50 mmol/l sodium citrate, and 1 M Tris base). Samples were briefly mixed and centrifuged at 12 000 g for 30 min. The genomic DNA, contained in the resulting aqueous phase, was further purified by two rounds of phenol–chloroform extraction, followed by precipitation with ethanol. DNA and RNA were both assayed for quality and quantity using standard gel electrophoresis and a Nanodrop spectrophotometer. For subsequent methylation analysis, 1 μg of DNA was hydrolyzed to nucleosides as described by Quinlivan & Gregory (2008b).

Confirming sex by morphology and gene expression

All juvenile alligators included in this study were incubated at the female-producing temperature (30 °C) until hatching. Sex was initially assessed at the time of necropsy by the presence of oviduct. To further confirm the sex of the juveniles used in this study, gonadal aromatase mRNA expression levels were analyzed by quantitative PCR. Juvenile alligators show clear sexual dimorphic patterns in the expression of aromatase, with females displaying levels at least threefold higher than males (Kohno et al. 2008, Milnes et al. 2008). Both assessments (gross morphology and aromatase expression) classified all juvenile alligators included in this study as female.

Preparation of standards and calibration curves for instrument validation

Neat standards of 5mdC, dC, and dG monohydrate were purchased from Sigma and Santa Cruz Biotechnology respectively. The materials were weighed to the nearest 0.00001 g into 10 ml Falcon tubes using a Mettler Toldeo XS205 balance. Stock solutions were prepared in deionized water (from a MilliQ Gradient A10, Millipore Corporation, Billerica, MA, USA) to final concentrations of 0.48917 mg/g (2.02 mmol/l) and 0.54870 mg/g (2.05 mmol/l) for 5mdC and dG respectively. A working solution for each 2′-deoxynucleoside was prepared to 20 μg/g (or ng/μl, at 82.8 and 74.8 μmol/l respectively) for the preparation of calibration curves. Two equimolar solutions of 5mdC and dG were prepared in hydrolysis buffer at concentrations of 5 and 50 μmol/l (each in triplicate) and were used to determine a relative response factor (RRF) to correct for variations in signal response resulting from differences in ionization efficiency. The equimolar solutions were used to tune the mass spectrometer.

Liquid chromatography–tandem mass spectrometry

2′-Deoxynucleoside standards and DNA extracts were analyzed in positive ion mode using an Applied Biosystems (AB) Sciex API 4000 triple quadrupole mass spectrometer with electrospray ionization using a TurboSpray Ion Source (Foster City, CA, USA). The instrument was operated in the MRM scan mode using ions that were the dominant protonated species, as well as the sodiated, dimer, and dimer-sodiated species ([M+H]+, [M+Na]+, [2M+H]+, and [2M+Na]+ respectively). The two dimer species were not detected for 5mdC. The following MRM transitions were monitored: 242.3→126.1 [5mdC+H]+, 268.1→152.0 [dG+H]+, 264.1→148.2 [5mdC+Na]+, 290.2→174.2 [dG+Na]+, 535.1→268.1 [2-dG+H]+, and 556.6→290.4 [2-dG+Na]+. Source (CAD 10, CUR 137 kPa, GS1 275 kPa, GS2 275 kPa, IS 5000 V, temperature 550 °C, and interface heater on) and compound-specific mass spectrometric parameters for each MRM transition were optimized. The compound-specific parameters (DP (V), EP (V), CE (eV), and CXP (V)) for each MRM transition were optimized to (25, 15, 20, and 10), (40, 10, 15, and 10), (25, 15, 20, and 10), (75, 15, 15, and 15), (75, 15, 15, and 15), and (75, 15, 15, and 15) for [5mdC+H]+, [dG+H]+, [5mdC+Na]+, [dG+Na]+, [2-dG+H]+, and [2-dG+Na]+ respectively. Nitrogen was used as the curtain gas. For each MRM transition, the dwell time was set to 200 ms.

The nucleosides were chromatographically separated using an Agilent 1100 LC (Palo Alto, CA, USA) equipped with a 2.1×150 mm Waters Atlantis dc18 column (5 μm i.d., Milford, MA, USA) and a gradient flow program consisting of solvent A (LC-grade water, BDH, West Chester, PA, USA) and solvent B (LC-grade methanol, Honeywell, Muskegon, MI, USA), with both solvents containing 0.1% formic acid (EMD, 98%, Darmstadt, Germany). For each injection, 5 μl was injected into a gradient flow rate set initially to 250 μl/min at 0% solvent A. The percentage of solvent A was increased to 20% from 0 to 10 min. At 10.01 min (to 17 min), the flow rate was changed to 220 μl/min and the percentage of solvent A was increased to 40%. At 17.01 min (to 22.0 min), the percentage of solvent A was decreased to 10%. At 22.01 min, the flow rate and percentage of solvent A were changed to 250 μl/min and 0% respectively, and these conditions were held until the end of the program (27 min). The column was heated to 35 °C using a column heater and the samples were kept at 8 °C in the autosampler tray until analysis. A sample chromatogram of 5mdC and dG separated in a DNA extract from ovary tissue is shown in Fig. 1.

Preparation of calibration curves for methylation measurements

For both the ovary and blood DNA samples, three separate calibration curves were included and analyzed in duplicate throughout a sample queue. Each calibration curve was prepared keeping dG constant at 10 μg/g (41.03 μmol/l). While dC was not used in our methods to determine methylation, it was necessary to include dC (stock solution at 10 μg/g or 37.43 mmol/l) for the purpose of preparing a calibration curve. At calibration levels from 82 nmol/l to 9.9 mmol/l (0.0, 0.1 (or 0.001 ng 5mdC/10 ng dG), 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, and 12% 5mdC), increasing amounts of 5mdC (mass) were added for each level while concomitantly decreasing amounts of dC (mass) were added, thus keeping the concentration of total dC=5mdC+dC constant across all calibration levels. The calibration levels were then designed to relate the percentage of 5mdC to the amount of dG. An overall calibration curve was then constructed from the sum of all the calibration curves analyzed, which was then used to determine the percent of 5mdC (to dG) present in the samples.

Handling of quality control and ovary/blood extracts

Post DNA hydrolysis, the samples (ovary and blood) were prepared in an autosampler vial and diluted to a total volume of 500 μl with hydrolysis buffer. The target DNA quantity per vial was 0.5 ng/μl, which resulted in ∼2.5 ng DNA/LC injection (employing 5 μl injections). A quality control (QC) DNA sample, extracted from visceral tissue from a juvenile alligator, was also included in this study to examine method performance and consistency. Since each matrix (for both ovary and blood samples) consisted of ∼300 sample injections per sample queue (which included ∼136 ovary and 104 blood DNA extract injections (n=1), along with 21 QCs, 80 calibration samples, and 50 blank injections), it was important to determine and evaluate method performance over the course of several days in regard to its feasibility as a population-based screening approach. For each sample type (ovary (O) and blood (B)), the QC material was included in triplicate (OQC1–3 and BQC1–3) and each sample in the triplicate was analyzed three times within a sample queue. The QC samples were processed in the same manner as the ovary and blood samples. The percentage of 5mdC (in relation to dG) for each individual replicate within the ovary and blood sample queue was 2.61±0.3% (or 0.0261±0.003 ng 5mdC/10 ng dG) for ovary OQC1–3 and 2.69±0.3% (or 0.0269±0.003 ng 5mdC/10 ng dG) for blood BQC1–3. The Relative Standard Deviation (RSD) for both sample queues combined for all the QC samples analyzed was 10%.

Sample and data processing prior to quantitation

Each ovary and blood DNA extract was examined (n=1) in a random order within a sample queue (two queues, one for each sample type). Each sample queue consisted of ∼300 injections, which included DNA extracts, QC samples (with replicates), calibration curve samples (in duplicate), and blanks. In post-sample analysis, the data were processed using Analyst v.1.5.2 (AB Sciex, Foster City, CA, USA).

During MS and MS/MS tuning of 5mdC and dG standards, it was evident that in addition to the detection of a protonated ion, [M+H]+, other forms of the 2′-deoxynucleosides were observed, including sodiated [M+Na]+, dimer [2M+H]+, and sodiated dimer [2M+Na]+ species. The presence of additional forms of 2′-deoxynucleosides has been previously reported (Song et al. 2004, Ma et al. 2008). It has been shown in previous work from our laboratory that several of these additional species (sodiated and dimer) for specific 2′-deoxynucleosides (e.g. dC) can be present at levels above 10% of the protonated species (data not shown). Further, it has been previously reported by others that additional species can constitute anywhere from 2/7 to 1/3 of the approximate signal of additional species vs total signal response for the sum of all species (Song et al. 2004, Ma et al. 2008). To compensate for these potential contributions, all nucleoside forms for 5mdC and dG were tuned by mass spectrometer, incorporated into the LC-MS/MS method, and quantitated using the sum of the nucleoside area for all species. It should be noted that for the detection of 5mdC, only the sodiated adduct was detected, while all the adduct forms were represented for dG (and dC). Unlike other 2′-deoxynucleosides, the percent presence (%) of each additional species for 5mdC and dG (additional species area over [M+H]+ species area) was negligible over the course of the ovary and blood sample batches and was recorded at 1.31±1.69 and 1.08±1.48 for [5mdC+Na]+, 2.40±0.13 and 2.61±0.18 for [dG+Na]+, 0.03±0.02 and 0.03±0.01 for [2dG+H]+, and 0.01±0.00 and 0.01±0.00 for [2-dG+Na]+ respectively.

Quantifying cytosine methylation

The peak areas for each 5mdC and dG species (including adducts and dimers) were examined and incorporated into a peak area ratio equation, Σ5mdCarea/(ΣdGarea×0.8971), where Σ is the sum of all the peak areas for the protonated, sodiated, and dimer species and 0.8971 is the experimentally determined RRF as described above. The RRF was determined by analyzing two equimolar solutions (5 and 50 μmol/l, each in triplicate) and comparing the relative peak areas of the sum of the 5mdC and dG peaks. The calibration curves were prepared in triplicate (with each curve in triplicate analyzed twice) and were constructed using the above peak area ratio calculation. Within the curve, only the amount (percentage, or mass fraction of ng 5mdc/10 ng dG) of 5mdC changed (0–12%, holding dG constant); thus, the percentage of 5mdC (in relation to dG) for each sample was calculated using the regression line derived from the sum of all the curves run for each sample matrix (ovary and blood). The calibration lines and R2 values for the ovary and blood calibration curves were y=0.0103x−0.0002 (1.00) and y=0.0127x+0.0003 (1.00) respectively.

Statistical analyses

GraphPad Prism, version 6.0, was used to perform all statistical analyses. For all analyses, statistical significance was set at P<0.05. All global genomic methylation levels were calculated as ratios and were arcsin transformed before statistical analyses. A two-way ANOVA was used to test for differences in measures of global DNA methylation among sites and tissues. A one-way ANOVA was used to test for clutch effects on global DNA methylation. Unpaired t-tests were employed to compare differences in global genomic methylation levels from alligators of different ages. A one-way ANOVA was used to test for differences among sites and clutch effects.

Results

Site and tissue analysis of DNA methylation

DNA methylation in mammalian genomes is tissue specific (Maegawa et al. 2010, Herzog et al. 2013). Studies have linked both increased and decreased DNA methylation levels to chemical exposures in traditional laboratory models and humans (Dolinoy et al. 2007, Baccarelli et al. 2009, Zhu et al. 2012). To test whether tissue-specific and persistent site differences in genomic methylation are present in juvenile alligators, we analyzed the genomic content of cytosine methylation using LC-MS/MS. In alligators originating from each site, genomic methylation (reported as the proportion of methylated cytosines relative to the number of total CpGs, 5mdC/CpGs) was higher in ovaries in comparison with blood (F2,234=136.71, P<0.001; Fig. 2A, Tables 1 and 2). However, both ovarian and blood methylation was similar among collection sites (F2,234=0.32, P=0.73). These data indicate genomic methylation is higher in ovaries than in blood and thus tissue-specific DNA methylation patterns are present in juvenile alligators. However, persistent site-of-origin differences in genomic methylation were not observed.

Figure 2
Figure 2

Comparison of global genomic methylation across tissues and sites. Different letters above groups indicate statistical significance for tissue comparisons. (A) Left y-axis indicates the proportion of methylated cytosines relative to CpGs dinucleotides in the genome, right y-axis indicates the proportion of methylated cytosines measured relative to guanosines. WO, Lake Woodruff; AP, Lake Apopka; and YK, Yawkey Wildlife Center. (B) Box-and-whisker plots of ovarian global genomic methylation across clutches. Left y-axis indicates the proportion of methylated cytosines relative to CpG dinucleotides in the genome, right y-axis indicates the proportion of methylated cytosines measured relative to guanosines. Whiskers indicate min. and max. No significant variation across clutches was observed.

Citation: REPRODUCTION 147, 4; 10.1530/REP-13-0498

Table 1

Descriptive Statistics comparing global methylation across sites. The uncertainty value is Standard Error from the Mean (SEM).

TissueCpG methylation mean % ±S.E.M. (n)
ApopkaWoodruffYawkey
Ovary57±1.5 (27)55±1.3 (45)57±1.1 (64)
Blood43±1.6 (28)44±1.2 (44)42±1.4 (32)
Table 2

Two-way ANOVA results comparing global methylation across tissues.

SourcedfFP
Site20.320.73
Tissue1136.71<0.001
Interaction21.930.15

DNA methylation among clutches

We next examined the potential of genetic contributions by examining variation among clutches. In late spring to early summer, female alligators lay a clutch of eggs that may be sired from one or multiple males (Lance et al. 2009). In an effort to determine whether a maternal effect on DNA methylation could be detected at the genome level, we compared variability of ovarian global genomic methylation among clutches (Fig. 2B). In all, DNA methylation was analyzed for individuals representing 26 clutches with an average of five eggs per clutch. A one-way ANOVA across all clutches revealed no significant variation among clutches (F25,106=0.82, P=0.71).

Relationship between DNA methylation in blood and ovary

The collection of peripheral blood is the most universal means of obtaining a consistent minimally invasive biological DNA sample across a large number of organisms within a given population. However, in many cases, an understanding of how transcriptional and epigenetic endpoints measured in blood relate to other tissues is either inferred through speculation or is completely lacking. In an effort to determine whether levels of DNA methylation in blood correlate with those measured in the ovary, we performed a correlation analysis across individuals (Fig. 3). When DNA methylation levels in blood and ovary from all individuals were examined, a correlation between the two tissues was not observed (n=101, P=0.94, r=0.008). We reasoned that perhaps correlations might exist in animals from certain sites and not others, and if so, comparing all animals together may obstruct detection. However, correlations between DNA methylation levels in blood and ovary were also absent when analyzing individuals originating from each site (Fig. 3). These data suggest that levels of global DNA methylation in the blood are not linked to those measured in the ovary.

Figure 3
Figure 3

Scatter plot of levels of global genomic methylation in ovary and blood. Axes indicate the proportion of methylated cytosines relative to CpG dinucleotides in the genome. Individuals from different sites are indicated by symbols. Results from a correlation analysis are reported and did not reveal a correlation regardless of site.

Citation: REPRODUCTION 147, 4; 10.1530/REP-13-0498

DNA methylation in blood and age

Decreased global DNA methylation levels are associated with aging and age-related cancers (Rodriguez et al. 2006, Bollati et al. 2009). Similar to humans, alligators are long-lived and undergo chronic exposure to a milieu of anthropogenic contaminants and thus represent an attractive model to study the effects of environmental quality on aging-related processes. To investigate whether the level of global DNA methylation within blood cells of alligators was decreased in adult alligators relative to juveniles, we measured DNA methylation from 16 adults captured on Lake Woodruff and compared the resulting levels to the juveniles reported in this study. Consistent with mammalian studies, juveniles originating from Lake Woodruff were observed to have elevated levels of global DNA methylation (44% CpG methylation, n=44) in comparison with their adult counterparts (Fig. 4; 36% CpG methylation, n=16, T=4.07, df=58, P=0.0001). Although alternative explanations are possible, taken at face value, these data suggest that age may influence global DNA methylation in these animals.

Figure 4
Figure 4

Comparison of global genomic methylation in blood samples from juvenile and adult animals from Lake Woodruff (FL). Left y-axis indicates the proportion of methylated cytosines relative to CpG dinucleotides in the genome; right y-axis indicates the proportion of methylated cytosines measured relative to guanosines. Measures of global genomic methylation are lower in adults (0.37±0.011 5mdC/CpGs) compared with juveniles (0.46±0.013 5mdC/CpGs). Statistical significance is indicated by different letters.

Citation: REPRODUCTION 147, 4; 10.1530/REP-13-0498

Discussion

The proposed strategy to characterize percent global methylation using the relative ratio of 5mdC:dG was designed to be simple, rapid, and inexpensive (no labeled standards), all being important components for providing a high-throughput platform for population-based screening. The most widely used approach for quantification of percent global methylation is to relate the peak area of 5mdC to the sum of the peak area for total dC (which is generally described as (5mdC)/(5mdC+dC)), either employing native or isotopically labeled 2′-deoxynucleosides for quantification by calibration curves (Friso et al. 2002, Kok et al. 2007, Ma et al. 2008, Quinlivan & Gregory 2008a, Liu et al. 2009, Le et al. 2011). However, recently, it was proposed to employ dG, instead of dC, to quantify percent methylation. This approach was based on the assumption that 5mdC+dC=dG (Parra et al. 2001, Song et al. 2004). While use of dC for quantitation has become the practiced approach, recent reports have indicated that dC can undergo multiple forms of modification, including 5mdC, 5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine, and 2′-deoxycytidine-5-carboxylic acid (Fu & He 2012). For monitoring subtle variations in percent global methylation, dG is more stable as an internal calibrant as it is less prone to modifications.

Currently, there are numerous methods for measuring global levels of genomic DNA methylation. In humans and traditional mammalian models, methylation levels of LINE-1 and Alu satellite repeats utilizing pyrosequencing of PCR-amplified bisulfite-treated DNA are most commonly utilized as a proxy for global levels of genomic methylation (Yang et al. 2004). LINE-1 and Alu repeats comprise a sizeable proportion of the genome and thus the methylation status of these repeats is likely to reflect that of the entire genome. However, some studies have failed to find a correlation between methylation levels of these elements and the methylation status of the genome in its entirety. Subsequently, calls for direct measures that are not biased in regard to sequence have recently been made for those studies investigating global methylation (Choi et al. 2009, Nelson et al. 2011). Another concern is that many epidemiological studies examining global genomic methylation have relied on DNA isolated from lymphocytes or buccal swabs as a surrogate for systemic genomic methylation. Yet, how global methylation in these tissues is correlated with other tissues is not well understood (Nelson et al. 2011).

By comparing our measures of global 5mdC content with the number of CpGs in the alligator genome, we were able to conclude that 55–57% of CpGs are methylated in ovarian tissues and 42–44% of CpGs are methylated in nucleated blood cells. These measures are lower than the 70–90% CpG methylation historically reported in human tissues (Ehrlich et al. 1982, Tucker 2001). Repetitive sequences are highly methylated in most vertebrates and are thought to comprise more than half of the human genome (Consortium 2001, de Koning et al. 2011). Interestingly, recent sequencing of the alligator genome revealed that repetitive elements only comprise an estimated 23% (St John et al. 2012), suggesting that the reduced global methylation in alligators relative to humans could be due to a decreased proportion of repetitive sequences. Further studies aimed at exploring methylation status at higher resolutions are needed to better understand these differences observed at the global level.

By examining global methylation in alligators raised under controlled conditions, but originating from sites with varying environmental qualities, we tested whether environmental quality during early development results in persistent alterations to global DNA methylation. Our inability to detect a site effect suggests that altered levels of global DNA methylation are not responsible for the persistent reproductive abnormalities previously described in juvenile alligators originating from Lake Apopka. However, whether alterations in methylation status are present at the locus or gene pathway level remain to be tested. Previous studies have documented rapid temporal fluctuations in levels of global DNA methylation in response to environmental cues (Baccarelli et al. 2009, Barrès et al. 2012). Thus, it is plausible that differences attributed to site might have been initially present but were subsequently lost while in captivity. Analyses examining the effects of site on wild-caught juvenile alligators are needed to determine whether site effects are present in wild populations.

Many human epidemiological studies to date have related global methylation, through both direct and indirect measures, to environmental exposures, the presence of cancer, and other diseases (Rusiecki et al. 2008, Choi et al. 2009, Terry et al. 2011). Most of these studies examine methylation levels in either white blood cells or cells obtained through buccal swabs, and it is unclear how these levels correlate with directly affected target tissues. In this study, we sought to examine tissue-specific differences in levels of global methylation in an environmental sentinel species and to investigate how these measures are related. We did not observe a correlation between global methylation levels in blood and ovary across individuals. Our findings suggest that measures of global methylation in the blood may not reflect relative methylation levels in other tissues. Studies examining global methylation across tissues in humans have reported similar findings, with correlations between tissues being rare (van Bemmel et al. 2012). These findings along with those presented here raise further questions regarding the influence or lack thereof of metabolic factors, such as one-carbon metabolism, on systemic global methylation levels across individuals. Collectively, these studies point toward the use of global methylation levels in circulating blood cells as a potential biomarker or as a means to understanding exposure histories, but at this point, conclusions regarding the methylation status of other tissues are premature.

We found that average global methylation levels were substantially higher in juvenile alligators originating from Lake Woodruff compared with adults from the same lake. However, there did appear to be some juvenile alligators with levels of DNA methylation that were similar to adults. It is well established that aging is associated with decreased levels of global methylation in peripheral blood cells in mammals (Fuke et al. 2004, Bollati et al. 2009). Yet, to our knowledge, this association has not been demonstrated in non-mammalian vertebrates. While other influences, such as captivity, cannot be ruled out, given our current knowledge of the relationship between aging and global methylation, it is probable that the differences observed between juvenile and adult alligators in this study are due to age. Further studies focused on measuring global methylation levels in wild alligator populations across a range of ages are needed to better understand the relationship between DNA methylation and aging. Furthermore, studies comparing the relationships between DNA methylation and aging across sites with differing environmental factors, such as varying degrees of contamination, could potentially reveal the influence of environmental factors on the aging process.

DNA methylation plays a critical role in mediating interactions between the genome and the environment. Numerous studies in humans and traditional laboratory models have demonstrated a sensitivity of global methylation levels to environmental factors. Measures of global methylation remain a cost-effective strategy for assessing epigenetic status and potential environmental impacts across a population. Further studies aimed at determining environmental impacts in wild populations of alligators and other sentinel species should aid in our understanding of how DNA methylation in blood cells, and also in other tissues, is affected by long-term combinatorial exposures.

Discussion from the meeting

Laura Vandenberg (Medford, USA): You have seen environmental effects on animals at every age you have studied, and the same individuals continue to have these defects on repeated sampling. Is that because the animals continue to live in the same contaminated areas, or is it an organizational effect due to an early hit which persists during life?

L J Guillette (Charleston, USA): Neonatal exposure and continued exposure are both important, and it is difficult to separate the two components. Alligators have problems when they hatch. We have kept some in captivity with a clean environment, and the problems persist. However, same age animals in the wild with continued contaminated exposure for 1–3 years have greater severity of the same problems. Therefore, birth defects are augmented by continued environmental exposure, possibly due to exactly the same mechanisms. Early studies on endocrinology, before the concept of endocrine disrupting chemicals (EDCs) had been considered, showed that developing embryos exposed to abnormal hormones had more dramatic effects when stimulated again with hormones in later life, whether steroid or non-steroid hormone. The embryonic hit is pump priming for future exposure.

Åke Bergman (Stockholm, Sweden): The environment around the NASA space center must have other chemical pollutants in addition to flame retardants and metals, particularly fluorinated compounds such as PFOS and polyfluorinated hexosulphonates. These are present around airports in Sweden. Have you examined them?

L J Guillette: Rocket fuel contains perchlorate, 2½ tons of which are projected from the nozzles of the rocket at 9000 °C at every launch. This produces a cloud of HCl and water with a pH of 0.5. There are thyroid defects around NASA and we were asked to investigate perchlorate levels: however, none was present. All the perchlorate is complete vaporized, and the highly acid cloud at extreme temperatures oxidizes and vaporizes metal compounds in the vicinity. The biggest environmental problem around rocket sites is the presence of many weird metals and metal compounds. We are also looking at flame retardants and fluorinated compounds.

Sander van den Driesche (Edinburgh, UK): You demonstrated DNA methylation in the SOX9 promoter gene and indicated a difference in expression between males and females. Given that gender determination is temperature dependent, is there a difference in methylation of SOX9 at different temperatures?

L J Guillette: We incubated embryos at a temperature which would induce male differentiation by sex reversal, and added an estrogen. This resulted in a male pattern of methylation although the confounding factor of an added steroid hormone has to be considered in case the estrogen was having an effect. There was no difference in the methylation enzymes between males and females which is to be expected because other genes are being methylated. This indicates that SOX9 methylation is not controlled by temperature but is regulated by other upstream events. The trigger for environmental sex determination is not clear but it is upstream of SOX9. Aromatase is not the trigger for ovarian differentiation but the determining factor can be overridden by an estrogen signal.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology; nor does it imply that the materials or equipment identified are necessarily the best for the purpose.

Funding

This article is based on work presented at the 7th Copenhagen Workshop on Endocrine Disrupters, which was supported by the Danish Ministry of the Environment – Environmental Protection Agency. Publication of this special issue was supported by the Society for Reproduction and Fertility. This work was funded in part by the South Carolina Center of Economic Exellence Center for Marine Genomics and an exploratory research grant to L J Guillette from NIST.

Acknowledgements

The authors would like to acknowledge employees of the Florida Fish and Wildlife Conservation Commission for logistical support. This study would not be possible without the continued support of Allan Woodward, Arnold Brunell, and Patrick Delaney in regard to fieldwork and permitting. They would also like to thank Lance Koch, the refuge manager at Lake Woodruff, for his assistance in this project. In addition, they would also like to acknowledge Jamie Dozier of the South Carolina Department of Natural Resources for providing access and logistical support at the Yawkey Wildlife Center. Finally, they would like to acknowledge an anonymous reviewer and Lori Cruze of the Medical University of South Carolina for their guidance and suggestions regarding statistical analyses.

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This paper forms part of a special issue of Reproduction on Endocrine Disrupters. This article was presented at the 7th Copenhagen Workshop on Endocrine Disrupters, 28–31 May 2013. The meeting was supported by the Danish Ministry of the Environment – Environmental Protection Agency as an activity under the Danish Centre on Endocrine Disrupters. Publication of this special issue has been supported by the Society for Reproduction and Fertility. The opinions or views expressed in this special issue are those of the authors, and do not necessarily reflect the opinions or recommendations of the Danish Ministry of the Environment – Environmental Protection Agency or the Society for Reproduction and Fertility. The Guest Editors for this special issue were Anna-Maria Andersson, Hanne Frederiksen, Niels Erik Skakkebæk, Rigshospitalet, Denmark, Kenneth M Grigor, Western General Hospital, Edinburgh, UK and Jorma Toppari, University of Turku, Finland.

*

(B B Parrott and J A Bowden are co-first authors)

 

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  • Experimental design. (A) Three Southeastern (USA) sites were selected for alligator egg collection: Yawkey Wildlife Refuge (YK), Lake Woodruff (WO), and Lake Apopka (AP). (B) Alligator eggs were collected from each site and incubated under identical conditions at female-producing temperature. (C) Alligator hatchlings were allowed to reach 1 kg in weight. DNA was isolated from ovaries and blood. (D) 5mdC and dG, from the isolated and hydrolyzed DNA, were examined using LC-MS/MS. A resultant chromatogram is shown for the LC-MS/MS analysis of DNA extract from an alligator ovary. For quantitation of percentage of 5mdC, calibration curves were prepared in triplicate (with each curve in triplicate analyzed twice) and were constructed by varying the amount of 5mdC while holding dG constant.

  • Comparison of global genomic methylation across tissues and sites. Different letters above groups indicate statistical significance for tissue comparisons. (A) Left y-axis indicates the proportion of methylated cytosines relative to CpGs dinucleotides in the genome, right y-axis indicates the proportion of methylated cytosines measured relative to guanosines. WO, Lake Woodruff; AP, Lake Apopka; and YK, Yawkey Wildlife Center. (B) Box-and-whisker plots of ovarian global genomic methylation across clutches. Left y-axis indicates the proportion of methylated cytosines relative to CpG dinucleotides in the genome, right y-axis indicates the proportion of methylated cytosines measured relative to guanosines. Whiskers indicate min. and max. No significant variation across clutches was observed.

  • Scatter plot of levels of global genomic methylation in ovary and blood. Axes indicate the proportion of methylated cytosines relative to CpG dinucleotides in the genome. Individuals from different sites are indicated by symbols. Results from a correlation analysis are reported and did not reveal a correlation regardless of site.

  • Comparison of global genomic methylation in blood samples from juvenile and adult animals from Lake Woodruff (FL). Left y-axis indicates the proportion of methylated cytosines relative to CpG dinucleotides in the genome; right y-axis indicates the proportion of methylated cytosines measured relative to guanosines. Measures of global genomic methylation are lower in adults (0.37±0.011 5mdC/CpGs) compared with juveniles (0.46±0.013 5mdC/CpGs). Statistical significance is indicated by different letters.

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