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BOR - Papers in Press, published online ahead of print January 2, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.064915
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BIOLOGY OF REPRODUCTION 78, 932–938 (2008)
DOI: 10.1095/biolreprod.107.064915
© 2008 by the Society for the Study of Reproduction, Inc.

Toxicology; 

Increased Posthatching Mortality and Loss of Sexually Dimorphic Gene Expression in Alligators (Alligator mississippiensis) from a Contaminated Environment1

Matthew R. Milnes 2 3, Teresa A. Bryan 3, Yoshinao Katsu 4, Satomi Kohno 3, Brandon C. Moore 3, Taisen Iguchi 4, and Louis J. Guillette, Jr. 3

Department of Zoology,3 University of Florida, Gainesville, Florida 32611 Okazaki Institute for Integrative Bioscience,4 National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki 444-8787, Japan

ABSTRACT

A previous study from our laboratory examining development in neonatal alligators from polluted Lake Apopka, Florida, found numerous differences relative to neonates from a reference site, Lake Woodruff National Wildlife Refuge. We postulated that the differences were the result of organizational changes derived from embryonic exposure to environmental contaminants and are related to the poor reproductive success reported in alligators from Lake Apopka. In this study we examine differences in alligators collected as eggs from these two populations and raised under similar conditions for 1 yr. Egg hatch rates did not differ between lake populations; however, posthatching mortality was much higher among Lake Apopka hatchlings. Snout-vent length and body mass were greater in Lake Apopka hatchlings, but no differences were detected between lake populations in thyroid, liver, and spleen mass corrected for body size or in plasma concentrations of testosterone and estradiol. Males from Lake Woodruff exhibited greater relative expression of gonadal mRNA for steroidogenic factor 1 (Nr5a1) and steroidogenic acute regulatory protein (Star) than males from Lake Apopka. Alligators from Lake Woodruff also expressed all genes examined in a sexually dimorphic pattern. In contrast, mRNA expression did not differ between males and females from Lake Apopka for Nr5a1, Star, cytochrome P450 11A1 (Cyp11a1), and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1). Our results document persistent differences in development, survivorship, and gene expression in alligators from a contaminated environment. Because these animals were raised under similar laboratory conditions, the differences are most likely of embryonic origin and organizational in nature.

estradiol, gene regulation, steroid hormones, testosterone, toxicology

INTRODUCTION

Low reproductive success and alterations in endocrine function are commonly reported in wildlife associated with aquatic environments where exposure to endocrine-disrupting contaminants occurs [1]. In the Great Lakes region, organochlorine pesticides, polychlorinated biphenyls, and polychlorinated dibenzodioxins have been implicated in population declines and/or reproductive impairment of Forster terns (Sterna forsteri), double-crested cormorants (Phalacrocorax auritus), herring gulls (Larus argentatus), and bald eagles (Haliaeetus leucocephalus) [for review see 2]. Herring gulls of Lake Ontario were significantly affected in the 1960s and 1970s when use of organochlorine pesticides and polychlorinated biphenyl contamination peaked. A high incidence of embryonic and chick mortality, abnormal gonadal development, female-female pairings, and supernormal clutches, in which multiple females lay eggs in a single nest, was reported [3]. In the United Kingdom, wild roach (Rutilus rutilus) collected from rivers contaminated with treated sewage effluent exhibited the loss of sexually dimorphic steroid concentrations, intersex gonads, reduced sperm production, and poor fertilization success [4, 5]. Studies from our laboratory and others have shown that American alligators (Alligator mississippiensis) living in lakes contaminated with organochlorine pesticides, municipal runoff, and nutrients exhibit low egg hatch rates, altered plasma hormone concentrations, and reduced phallus size relative to those of alligators from less contaminated lakes [for review see 6].

Possibly related to effects on sex steroid signaling, certain environmental contaminants have been shown capable of altering primary sex determination—especially in species with temperature-dependent sex determination. The egg incubation temperature during a particularly thermosensitive period of development is the primary factor influencing the sex of reptiles with temperature-dependent sex determination. However, numerous studies have shown that natural and synthetic steroids are capable of overriding the effects of temperature that typically produce either sexes or all males [7–9]. Two well-characterized model species for investigating the effects of endocrine-disrupting contaminants on sex determination are the freshwater turtle, Trachemys scripta, and the American alligator. In both species, a number of organochlorine pesticide contaminants that have been detected in Lake Apopka alligator tissues have been shown to induce male-to-female sex reversal. These include metabolites of dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (p,p'-DDE) and dichlorodiphenyldichloroethane (p,p'-DDD), trans-nonachlor, and chlordane [10–12]. Furthermore, a previous study comparing neonatal alligators incubated at a temperature that produces both sexes resulted in 76% females from Lake Apopka compared to 60% females from Lake Woodruff National Wildlife Refuge (NWR) [13].

The numerous cases documenting alterations in circulating steroid concentrations has led researchers to examine steroidogenesis at the enzymatic level in contaminant-exposed vertebrates. In the steroidogenic pathway, cholesterol is initially converted to the 21-carbon (C21) steroid pregnenolone by the cytochrome P450 (CYP) enzyme designated CYP11A1, formerly side chain cleavage. Pregnenolone is then converted by a series of hydroxysteroid dehydrogenases and CYP enzymes to mineralocorticoids and glucocorticoids in the adrenal glands or sex steroids such as C19 androgens and C18 estrogens in the gonads [14, 15]. The majority of research on steroidogenesis has focused on enzymes downstream from the conversion of cholesterol to pregnenolone such as 3 beta- and steroid delta-isomerase 1 (HSD3B1) and various CYP enzymes such as 17 alpha-hydroxylase (CYP17A1) and aromatase (CYP19A1). For instance, the enzyme responsible for the conversion of androgens to estrogens, CYP19A1, is usually expressed in a sexually dimorphic manner in various tissues including the gonads, liver, and brain. The herbicide atrazine was shown to increase gonadal aromatase activity in male alligators to levels similar to those of control females following embryonic exposure [16].

Noticeably absent from studies relating contaminant exposure to altered steroid concentrations in nonmammalian systems are studies of the transcription factors and proteins involved in regulating steroidogenesis prior to the conversion of cholesterol to pregnenolone. Nuclear receptor 5A1 (NR5A1, formerly steroidogenic factor 1) is directly involved in regulating transcription of 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS1), which is necessary for the synthesis of cholesterol [17]. Furthermore, it has been shown to bind to the promoter region of CYP11A1 in association with hormonally regulated steroidogenesis [18]. Although CYP11A1 activity is the first chronically regulated step in the steroidogenic pathway, its activity is limited by the availability of intramitochondrial cholesterol. The steroidogenic acute regulatory protein (STAR) is responsible for mediating the transfer of cholesterol from the outer to the inner mitochondria membrane [19]. In mammalian systems, environmental contaminants have been shown to alter expression of STAR. Posttranscriptional disruption of STAR was observed following treatment of MA-10 (mouse Leydig tumor) cells with the antifungal drugs econazole and miconazole [20], whereas the insecticides lindane (organochlorine) and dimethoate (organophosphate) inhibit steroidogenesis by disrupting transcription of Star mRNA [21, 22].

A previous study from our laboratory examining development and endocrine function in neonatal alligators (age < 1 mo) from Lake Apopka, Florida, historically contaminated with pollutants from agricultural activity, storm water runoff, and sewage, found numerous differences relative to neonates from Lake Woodruff NWR [13]. We postulated that the differences were the result of organizational changes derived from the embryonic environment. A review of similar endpoints compared between juveniles captured from both sites suggests that organizational alterations might be subject to further modification by environmental exposure to contaminants or ontogenetic changes in endocrine function. Although a host of studies provide evidence that environmental contaminants are associated with alterations of the endocrine system in wildlife, few have looked at the persistence of contaminant-induced effects of embryonic origin. In particular, we know little about how embryonic exposure to environmental contaminants manifests itself over time in long-lived species. In this study, alligator eggs were collected early in development, and hatchlings were raised under identical conditions, thus eliminating the effect of posthatching environmental differences. We examined endpoints relevant to embryonic exposure to endocrine-disrupting contaminants that have also been compared in previous studies between neonates from these two study sites, including embryonic mortality, somatic indexes, sex determination, plasma steroid concentrations, and posthatching mortality. In addition, we compared the expression of mRNA in the gonads associated with steroidogenesis, as previous studies suggest this pathway is highly susceptible to alterations related to chemical exposure.

MATERIALS AND METHODS

Egg Incubation and Animal Care

All work involving alligators was performed under the guidelines specified by the Institutional Animal Care and Use Committee at the University of Florida. All field work was conducted under permits from the Florida Fish and Wildlife Conservation Commission and the U.S. Fish and Wildlife Service. Six clutches of alligator eggs were collected within the first 2 wk following oviposition from Lake Apopka and Lake Woodruff NWR. One egg from each clutch was opened to determine embryonic stage based on criteria described by Ferguson [23]. At embryonic stage 19, the stage just prior to the thermosensitive period of sex determination, eggs were candled to determine fertility and viability by the presence of a vascularized, opaque band indicating development of extraembryonic membranes associated with the embryo. Ten viable eggs from each of six clutches from both study sites were selected and incubated as previously described [24] at 32°C, a temperature known to produce a relatively even ratio of males and females. Upon hatching, neonates were web-tagged on both hind legs with unique identification numbers and maintained in greenhouse enclosures under natural light conditions for 13 mo at the University of Florida. Animals were fed commercial alligator chow (Burris Mill and Feed, Franklinton, LA) ad libitum, and water changes were performed every other day. Animals were checked each day for general health, and dead animals were immediately removed and placed in Bouin fixative for future determination of sex by examination of the gonads and reproductive tract as described later.

Tissue Collection and Radioimmunoassays

Thirteen months after the eggs hatched, body mass (BM) and snout-vent length (SVL) were determined to the nearest 1.0 g and 1.0 mm, respectively. A blood sample was obtained, juveniles were killed with a lethal dose (0.5 mg/g BM) of sodium pentobarbital (Sigma, St. Louis, MO), and sex was determined under 10x magnification. Females were identified based upon the presence of an oviduct and the comparatively larger, textured, and light pink ovary, whereas the testis of males has a smooth, dark red appearance. Gonads were removed, weighed to the nearest 1.0 mg, flash frozen in liquid nitrogen, and stored at –70°C. The thyroid, liver, and spleen were also removed and weighed to the nearest 1.0 mg as somatic indexes of development.

Blood samples were centrifuged in heparinized Vacutainers (Becton, Dickinson, and Company, Franklin Lakes, NJ) at 1500 x g for 20 min; after which plasma was drawn off and stored at –70°C until assayed. Plasma samples from males and females were assayed for testosterone (T) and estradiol-17β (E2), respectively. These radioimmunoassays have been previously described and validated for alligator plasma [24–26]. All samples for each hormone were analyzed in a single assay. Intraassay variance for T and E2 was 1.6% and 2.4%, respectively.

RNA Isolation and Primer Design

The right gonad of each animal was homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA) using 1 ml TRIzol for every 100 mg of tissue, and total RNA was isolated according to the manufacturer's protocol. All samples were then purified using the RNeasy kit (Qiagen, Valencia, CA). Total RNA concentration was determined with a spectrophotometer, and the quality of each sample was verified on a formaldehyde agarose gel. First strand cDNA synthesis was carried out on 1.2 µg total RNA in the presence of SuperScript II RNase H– Reverse Transcriptase and Oligo (dT) 12–18 Primer (Invitrogen).

Nucleotide sequences for alligator Nr5a1 (accession no. AF180296) and Cyp19a1 (accession no. AY029233) have been reported elsewhere [27, 28]. A partial sequence for Star (accession no. AB186355) was directly submitted to GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) by S. Kohno. Partial sequences of three additional genes relevant to the steroidogenic pathway were obtained for this study. Using degenerative oligonucleotide primers derived from conserved regions, we amplified fragments of the alligator Cyp11a1, Hsd3b, and Cyp17a1 genes (Table 1). Amplified PCR products of the expected size were sequenced using the ABI Prism 3100 and BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA), and checked for nucleotide and amino acid homology using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/).


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  		TABLE 1. Forward and reverse degenerative primers.

Quantitative Real-Time PCR

Real-time PCR primers were designed using Primer Express (Applied Biosystems) and are reported in Table 2. Quantitative real-time PCR (Q-PCR) was performed using SYBR Green PCR Master Mix in the ABI Prism 5700 (Applied Biosystems) following manufacturer's protocol in a reaction volume of 15 µl as previously described [29]. Conditions for Q-PCR were 2 min at 50°C, 10 min at 95°C, and 40 cycles of 95°C for 15 sec, 60°C for 1 min. The relative expression of mRNA in each sample was calculated from a standard curve obtained from a serially diluted, pooled sample. Each sample was run in triplicate and normalized for the expression of ribosomal protein L8 (Rpl8) [29].


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  		TABLE 2. PCR primers used for quantitative real-time PCR.

Statistical Analysis

The SAS System for Windows version 9.0 (SAS Institute, Cary, NC) was used for all analyses. Data are presented as means ±SEM where appropriate. Hatching success, posthatching mortality, and sex ratios were compared among clutches and between lake populations using chi-square tests. Measurements of body size, organ weights, and plasma steroid concentrations were log transformed to reduce heterogeneity of variance [30]. An initial one-way ANOVA was used to determine if sexual dimorphism was present in any of the somatic indexes within each lake population prior to comparing means between lake populations. No sexual dimorphism was detected for BM, SVL, liver, thyroid, or spleen mass (P > 0.05 for all), so values from males and females were combined for further statistical analyses of those variables. Both SVL and BM varied between lake populations, therefore thyroid, liver, and spleen mass were compared between lake populations using BM as a covariate. Relative expression of each steroidogenic gene, expressed as a ratio with ribosomal protein L8, was arcsine transformed prior to two-way ANOVA (lake population x sex). When overall variation was significant (P < 0.05), least square means were analyzed using Tukey-Kramer post hoc comparisons.

RESULTS

Mortality and Sex Determination

Embryonic and posthatching mortality are reported by clutch in Figure 1. Embryonic mortality did not vary by clutch (P = 0.060) or lake population (P = 0.125). Overall, 80% of the eggs from Lake Apopka hatched, whereas 90% of the eggs from Lake Woodruff NWR produced hatchlings. Following 13 mo under standardized conditions, the percentage of posthatching mortality was higher among Lake Apopka animals (44%) compared to those from Lake Woodruff NWR (4%; P < 0.0001). Sex ratios were calculated from all individuals that hatched (n = 48 for Apopka; n = 54 for Woodruff), including hatchings that had died prior to 13 mo of age. Sex ratios did not vary among clutches (P = 0.109), but a difference was detected between study sites (P = 0.049). A female biased sex ratio from Lake Woodruff NWR (33:21 female:male) and a male biased sex ratio from Lake Apopka at hatching (20:28, female:male) was exacerbated by female biased posthatching deaths among animals from Lake Apopka. As a result, there were only 8 females and 19 males from Lake Apopka 13 mo after hatching, whereas Lake Woodruff NWR was represented by 33 females and 19 males.


Figure 01
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  		FIG. 1. Survivorship at hatching and 13 mo posthatching by clutch in alligators from Lake Apopka (AP) and Lake Woodruff NWR (WO). Asterisk denotes difference in posthatching mortality between lakes (P < 0.05).

Body Size and Somatic Indexes

Snout-vent length and BM (Fig. 2) were greater in Lake Apopka juveniles compared to Lake Woodruff NWR juveniles (P = 0.0003 for SVL and BM). No differences were detectable in thyroid (P = 0.716), liver (P = 0.593), and spleen (P = 0.947) mass.


Figure 02
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  		FIG. 2. Mean (±SEM) snout-vent length (SVL) and body mass (BM) in 13-mo-old alligators. Asterisk denotes difference in SVL and BM between lakes (P < 0.05).

Plasma Steroids and Steroidogenic Gene Expression

Plasma T averaged 186.8 + 10.8 and 189.3 + 9.0 pg/ml for Lake Apopka and Lake Woodruff NWR males, respectively. Plasma E2 averaged 19.2 + 3.7 and 24.3 + 11.0 pg/ml for Lake Apopka and Lake Woodruff NWR females, respectively. No difference in plasma T (P = 0.73) or E2 (P = 0.35) was detected between lake populations. The relative expression of mRNA associated with steroidogenesis is reported in Figure 3. Lake Woodruff NWR males exhibited higher levels of gene expression for Nr5a1 (P = 0.039) and Star (P = 0.027) than did Lake Apopka males, whereas no differences were detected between females. Juveniles from Lake Woodruff NWR exhibited sexual dimorphic expression of all genes examined. In contrast, no differences could be detected in expression of Cyp11a1 and Hsd3b1 in females from Lake Apopka when compared to males from either lake. Only Cyp17a1 and Cyp19a1 were expressed in a sexually dimorphic pattern in juveniles from Lake Apopka. Expression of Nr5a1 and Star in Lake Apopka males appeared slightly feminized, in that male expression was not different from female expression from either lake population. Lake Woodruff NWR males typically displayed 0.5–3.0 times higher expression levels of all genes with the exception of Cyp19a1, which was nearly 40-fold greater in females than males from both lakes.


Figure 03
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  		FIG. 3. Mean (±SEM) expression of Nr5a1 (A), Star (B), Cyp11a1 (C), Hsd3b1 (D), Cyp17a1 (E), and Cyp19a1(F) in 13-mo-old alligators. Bars with different letters are significantly different at P < 0.05.

DISCUSSION

The goal of this study was to examine the persistence of alterations in development and endocrine function derived from embryonic exposure to environmental contaminants, with an emphasis on the expression of genes involved in steroidogenesis. Other proposed mechanisms to explain the low reproductive success and developmental differences reported in Lake Apopka alligators include nutritional and genetic differences. Nutritional differences resulting from several decades of nutrient and contaminant loading impacting prey species composition is a plausible explanation that has not been studied to date. Previous research has shown dietary differences and altered egg yolk lipid composition to be associated with reduced egg viability in captive alligators [31]. If the prey species composition has been drastically altered on Lake Apopka, it is possible that essential nutrients are lacking in the diet, thus leading to deficiencies in specific fatty acids or nutrients incorporated into the egg yolk during vitellogenesis. Genetic differences seem an unlikely cause based on the lack of genetic diversity found among alligator populations in the Southeastern United States. No genetic marker could be found to distinguish populations (including Lake Apopka and Lake Woodruff NWR) across Florida using microsatellite DNA [32].

Although there was no statistical difference in embryonic mortality between lake populations in this study, the percentage of eggs that gave rise to viable hatchlings from each study site was remarkably similar to that of previous studies [13]. When data are combined from both studies (n = 60 eggs distributed across six clutches per lake per yr), the percentage of viable hatchlings from Lake Woodruff NWR (90.8%) is greater than the percentage from Lake Apopka (81.7%; P = 0.011). This is a highly conservative measure of egg viability, as previous reports of low hatching success on Lake Apopka indicate relatively high rates of infertility or early embryonic mortality [33, 34]. These data represent eggs that appeared viable at the end of the first third of development (stage 19, about Day 22), as indicated by a highly vascularized, opaque band where the extraembryonic membranes have attached to the inner eggshell membrane. The slightly higher incidence of embryonic mortality in Lake Apopka alligators combined with a greater incidence of posthatching mortality resulted in the loss of 55% of the individuals from Lake Apopka compared to 13% of alligators from Lake Woodruff NWR at 13 mo of age. There was no obvious cause or commonly observed symptom related to posthatching deaths, which occurred sporadically throughout the 13 mo. Although results were skewed toward females, there was no statistical difference in the percentage of each sex that died from Lake Apopka.

Based on experimental studies with contaminants found in Lake Apopka eggs [11] and the sex ratios previously reported from these sites [13], we expected a greater percentage of females from Lake Apopka when compared to Lake Woodruff NWR. Lake Woodruff NWR clutches resulted in approximately 60% females in both studies. In contrast, Lake Apopka clutches produced 41% females in the current study compared to 76% females reported in the 2005 study. These ratios include individuals that died after hatching but prior to 13 mo of age but not individuals that died prior to hatching. The possibility exists that eggs highest in contaminant concentrations, hence more likely to result in female hatchlings, are also most likely to fail to produce a viable hatchling. If the data from both studies are combined, there is no detectable difference in sex ratios between lake populations (P = 0.813), with Lake Apopka and Lake Woodruff NWR clutches averaging 62.1% and 60.6% females, respectively, at 32°C. Conely et al. [35] described significant annual and interclutch variation in alligator egg yolk steroid concentrations from Louisiana, with a significant decrease in steroid concentrations coinciding with the thermosensitive period of sex determination. An examination of egg yolk steroids in association with annual and interclutch variation in sex ratios is warranted for future studies.

That SVL and BM were greater in Lake Apopka hatchlings compared to Lake Woodruff NWR hatchlings was not expected, as the animals were raised under identical conditions, and previous work has shown Lake Apopka neonates were the same size as or smaller than those from Lake Woodruff NWR [36]. One possible explanation is that only the most robust individuals from Lake Apopka were represented at 13 mo of age, thus biasing the estimate of mean size. Another possibility is that endocrine function related to metabolism and growth such as thyroid function or growth hormones are differentially affected in animals from Lake Apopka. Alterations in the relationship between size and thyroxine (T4) concentrations have been shown in juveniles from Lake Apopka compared to juveniles from Lake Woodruff NWR [37], and variation in T4 concentration was reported in juvenile alligators from several sites in central Florida [38] and the Everglades drainage basin [39], varying in pesticide and nutrient contamination. Thyroid morphology and relevant gene expression in these animals is currently being examined for publication elsewhere.

Expression of NR5A1 is typically positively correlated with steroidogenic activity [40], and STAR and CYP11A1 are regarded as the acute and chronically regulated rate-limiting steps in steroidogenesis, respectively [41]. We observed lower relative expression of mRNA for Nr5a1 and Star in male alligators from Lake Apopka relative to males from Lake Woodruff NWR. Lower expression of Nr5a1 could lead to reduced synthesis of cholesterol or decrease the abundance of steroidogenic enzymes such as CYP11A1, and lower expression of Star could reduce the availability of cholesterol to steroidogenic enzymes. We did not observe a difference in plasma T in this study corresponding to differences in steroidogenic mRNA expression in males. This could be due to posttranscriptional regulation of the genes examined or compensation via hepatic metabolism, and it warrants further investigation. We feel, however, that the mRNA data are significant in that they provide a potential mechanism for several previous studies that found lower plasma T concentrations in hatchling and juvenile males from Lake Apopka compared to those of Lake Woodruff NWR [26, 37, 42, 43].

Sexually dimorphic expression of Cyp11a1 and Hsd3b1 in Lake Woodruff NWR alligators and the lack of sexual dimorphism in juveniles from Lake Apopka is consistent with previously reported circulating concentrations of sex steroids in juvenile alligators from these study sites [42]. Because different hormones were assayed in males (T) and females (E2) in this study, we cannot determine how the sexual dimorphic expression of steroidogenic genes corresponds to sexual dimorphism in circulating steroid concentrations in these animals. Our results regarding expression of Cyp19a1 are consistent with expected differences between sexes but are contrary to previous studies showing elevated plasma E2 in males and females from Lake Apopka relative to those from Lake Woodruff NWR [42, 44]. Studies examining in vitro E2 production or aromatase activity in alligators from these two sites also show varying results. No differences in aromatase activity were reported in neonates from Lake Apopka and Lake Woodruff NWR [13], whereas Guillette et al. [25] found lower E2 production in 6-mo-old female alligators from Lake Apopka relative to females from Lake Woodruff NWR. Similarly, Crain et al. [16] observed depressed aromatase activity in 9-mo-old alligators from Lake Apopka compared to those from Lake Woodruff NWR. The apparent lack of reconciliation between circulating E2, aromatase activity, and Cyp19a1 mRNA expression might indicate that the major regulatory sites of steroidogenesis occur upstream in the steroidogenic pathway. In other words, the availability of substrate to the aromatase enzyme, which is modulated by multiple genes, could be the limiting factor as opposed to transcription or translation of Cyp19a1.

It is important to emphasize that these results do not represent an acute, toxicological response to chemical exposure but rather differences observed 13 mo following hatching and rearing under identical conditions. Furthermore, the causes of embryonic and posthatching mortality and their connection, if any, to the differences in gene expression are not known. We offer two explanations that are not mutually exclusive for the changes in mRNA expression of steroidogenic genes. First, differences in mRNA expression patterns could be explained by morphological alterations in steroidogenic tissues. For instance, a reduction in the density of Leydig, theca, or granulosa cells would be perceived as a decrease in mRNA expression derived from whole gonad homogenates. Structural differences in the gonads were noted between juvenile alligators from these two study sites. These abnormalities included the presence of polynuclear oocytes and polyovular follicles in Lake Apopka females and poorly organized seminiferous tubules with unidentified bar-shaped structures in the testes of males [26]. Second, chemically induced epigenetic effects such as DNA methylation have been associated with permanent and transgenerational changes in gene expression [45–47]. Genetic imprinting at any point along the hypothalamus-pituitary-gonad axis could lead to changes in steroidogenic gene expression. The lack of a concomitant change in plasma steroids could be accounted for by compensatory mechanisms along the axis, including hepatic metabolism.

The results of this study document persistent differences in development, survivorship, and gene expression between juvenile alligators from contaminant-exposed and reference populations. This is the first study to show alterations in mRNA expression patterns for steroidogenic genes in alligators associated with a contaminated environment. While quantification of mRNA is highly sensitive to and generally predictive of physiological changes, posttranscriptional and translational regulatory processes further modulate gene expression. Because the differences in mRNA expression did not correlate with plasma steroid concentrations, caution must be observed in the interpretation of the significance of these data. The fact that these animals were raised under identical conditions supports the hypothesis that embryonic exposure to environmental contaminants results in permanent organizational alterations that can vary in phenotypic expression with ontogenetic development.

ACKNOWLEDGMENTS

We thank Allan Woodward and Dwayne Carbonneau of the Florida Fish and Wildlife Conservation Commission for their field assistance and Harry Dutton for issuing collection permits. Dr. Shinichi Miyagawa provided valuable technical support. We are indebted to Elizabeth Swiman and Maite Cintrón for their assistance with animal care and dissections.

FOOTNOTES

1Supported in part by grants to L.J.G. from the UF Opportunity Fund, grants to T.I. from the Japanese Ministry of Education, Culture, Sports, Science, and Technology and from the Japanese Ministry of Environment, and a grant to M.R.M. from the National Science Foundation and Japanese Society for the Promotion of Science (OISE-0413602). Back

Correspondence: 2Correspondence and current address: Matthew R. Milnes, Conservation and Research for Endangered Species, Zoological Society of San Diego, 15600 San Pasqual Valley Road, Escondido, CA 92027-7000. FAX: 760 291 5428; e-mail: mmilnes{at}sandiegozoo.org

Received: 8 August 2007.

First decision: 24 October 2007.

Accepted: 2 January 2008.

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