YGCEN 12086 No.

of Pages 9, Model 5G
8 April 2015

General and Comparative Endocrinology xxx (2015) xxx–xxx

1

Contents lists available at ScienceDirect

General and Comparative Endocrinology

journal homepage: www.elsevier.com/locate/ygcen

5
6

3 Developmental exposure to bisphenol A (BPA) alters sexual

4 differentiation in painted turtles (Chrysemys picta)
7 Caitlin M. Jandegian a,b,c, Sharon L. Deem a,d, Ramji K. Bhandari b,e, Casey M. Holliday f, Diane Nicks b,
8 Cheryl S. Rosenfeld c,g,h, Kyle W. Selcer i, Donald E. Tillitt b,e, Frederick S. vom Saal e, Vanessa Vélez-Rivera b,
9 Ying Yang c, Dawn K. Holliday f,j,⇑
10 a
Saint Louis Zoo Institute for Conservation Medicine, One Government Drive, St. Louis, MO 63110, United States
11 b
USGS Columbia Environmental Research Center, 4200 New Haven Rd, Columbia, MO 65201, United States
12 c
Bond Life Sciences Center, 1201 E. Rollins St., University of Missouri, Columbia, MO 65201, United States
13 d
Veterinary Medicine and Surgery, 1600 E. Rollins St., University of Missouri, Columbia, MO 65201, United States
14 e
Biological Sciences, University of Missouri, Columbia, MO 65201, United States
15 f
Pathology and Anatomical Sciences, School of Medicine, One Hospital Drive, University of Missouri, Columbia, MO 65212, United States
16 g
Biomedical Sciences, 1600 E. Rollins St., University of Missouri, Columbia, MO 65201, United States
17 h
Genetics Area Program, University of Missouri, Columbia, MO 65201, United States
18 i
Biological Sciences, Duquesne University, 600 Forbes Ave., Pittsburgh, PA 15282, United States
19 j
Biology and Environmental Science, Westminster College, 501 Westminster Ave, Fulton, MO 65251, United States

20
21
a r t i c l e i n f o a b s t r a c t
2 7
3 3
24 Article history: Environmental chemicals can disrupt endocrine signaling and adversely impact sexual differentiation in 38
25 Received 20 November 2014 wildlife. Bisphenol A (BPA) is an estrogenic chemical commonly found in a variety of habitats. In this 39
26 Revised 1 April 2015 study, we used painted turtles (Chrysemys picta), which have temperature-dependent sex determination 40
27 Accepted 3 April 2015
(TSD), as an animal model for ontogenetic endocrine disruption by BPA. We hypothesized that BPA would 41
28 Available online xxxx
override TSD and disrupt sexual development. We incubated farm-raised turtle eggs at the male-produc- 42
ing temperature (26 °C), randomly assigned individuals to treatment groups: control, vehicle control, 43
29 Keywords:
17b-estradiol (E2, 20 ng/g-egg) or 0.01, 1.0, 100 lg BPA/g-egg and harvested tissues at hatch. Typical 44
30 Endocrine disrupting chemical
31 Feminization
female gonads were present in 89% of the E2-treated ‘‘males’’, but in none of the control males 45
32 SOX9 (n = 35). Gonads of BPA-exposed turtles had varying amounts of ovarian-like cortical (OLC) tissue and 46
33 b-Catenin disorganized testicular tubules in the medulla. Although the percentage of males with OLCs increased 47
34 Vitellogenin with BPA dose (BPA-low = 30%, BPA-medium = 33%, BPA-high = 39%), this difference was not significant 48
35 Aquatic reptile (p = 0.85). In all three BPA treatments, SOX9 patterns revealed disorganized medullary testicular tubules 49
36
and b-catenin expression in a thickened cortex. Liver vitellogenin, a female-specific liver protein com- 50
monly used as an exposure biomarker, was not induced by any of the treatments. Notably, these results 51
suggest that developmental exposure to BPA disrupts sexual differentiation in painted turtles. Further 52
examination is necessary to determine the underlying mechanisms of sex reversal in reptiles and how 53
these translate to EDC exposure in wild populations. 54
Ó 2015 Published by Elsevier Inc. 55
57
56
58
59

Abbreviations: BPA, bisphenol A; E2, 17b-estradiol; EDCs, endocrine disrupting

compounds; HIER, heat-induced epitope retrieval; IHC, immunohistochemistry;
1. Introduction 60

PBS, phosphate buffered saline; PCBs, polychlorinated biphenyls; SOX9, sex-

determining region box 9, protein; Sox9, gene transcript; TBS, Tris-buffered saline; Many anthropogenic environmental chemicals are endocrine 61
TBST, Tris-buffered saline and Tween 20; TSD, temperature-dependent sex deter- disrupting chemicals (EDCs) and can disrupt normal physiological 62
mination; TSP, temperature sensitive period; VTG, vitellogenin.
⇑ Corresponding author at: Biology and Environmental Science, Westminster and developmental signaling. Some EDCs act as xenoestrogens and 63

College, 501 Westminster Ave, Fulton, MO 65251, United States. may impact wildlife species through feminization or demasculin- 64

E-mail addresses: cmjkwd@missouri.edu (C.M. Jandegian), deem@stlzoo.org ization, especially if exposure occurs during the developmental 65
(S.L. Deem), Bhandarir@missouri.edu (R.K. Bhandari), Hollidayca@missouri.edu period. Bisphenol A (BPA) is one such synthetic estrogen mimic. 66
(C.M. Holliday), dnicks@usgs.gov (D. Nicks), RosenfeldC@missouri.edu At least 8, but up to as much as 15 billion pounds of BPA are 67
(C.S. Rosenfeld), selcer@duq.edu (K.W. Selcer), dtillitt@usgs.gov (D.E. Tillitt),
produced annually (GrandViewResearch, 2014) in a variety of 68
vomsaalf@missouri.edu (F.S. vom Saal), vvelez@usgs.gov (V. Vélez-Rivera),
yangyin@missouri.edu (Y. Yang), dawn.holliday@westminster-mo.edu everyday plastics and epoxy resins making it one of the most ubi- 69
(D.K. Holliday). quitous EDCs (Vandenberg et al., 2010, 2013). In the environment, 70

http://dx.doi.org/10.1016/j.ygcen.2015.04.003
0016-6480/Ó 2015 Published by Elsevier Inc.

Please cite this article in press as: Jandegian, C.M., et al. Developmental exposure to bisphenol A (BPA) alters sexual differentiation in painted turtles
(Chrysemys picta). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.04.003
 YGCEN 12086 No. of Pages 9, Model 5G
8 April 2015

2 C.M. Jandegian et al. / General and Comparative Endocrinology xxx (2015) xxx–xxx

71 anaerobic conditions decrease biodegradation allowing BPA to 2009) to sewage ponds (Lindeman, 1992). C. picta is small in size, 137
72 accumulate in aquatic sediments (Crain et al., 2007; Environment has a long life span, is oviparous with TSD, and thus meets the 138
73 Canada, 2008). As such, BPA is a persistent environmental chemical criteria as an excellent sentinel of EDCs and other toxicants 139
74 in wildlife habitats and has been detected in rivers across the U.S. (Irwin and Irwin, 2006). We hypothesized that in ovo exposure to 140
75 (Kolpin et al., 2002). BPA will override TSD and disrupt sexual development at the 141
76 Studies on aquatic species offer a way to monitor and better male-inducing temperature as evidenced by partial to full gonad 142
77 understand the potential impacts of contaminants like BPA on sex reversal. Specifically, our prediction is that BPA will produce 143
78 human and animal health. Turtles are important indicators of similar effects to estradiol in generating an ovotestis, a gonad with 144
79 environmental health because of their extended juvenile period, a proliferated ovarian cortical region, while still maintaining some 145
80 high site fidelity, low metabolic rate, use of large fat deposits for medullary structure containing Sertoli cells within the testicular 146
81 egg development (Selcer, 2006), and maternal transfer of lipid-sol- tubules (Barske and Capel, 2010; Bergeron et al., 1999; Crews 147
82 uble xenoestrogens during egg formation (Basile et al., 2011). et al., 1991; Pieau et al., 1999). 148
83 Additionally, aquatic turtles reside in the water column and
84 sediments of aquatic environments, which enhance the potential
85 for exposure to BPA and other EDCs (Bhandari et al., 2014; 2. Materials and methods 149
86 Kassotis et al., 2013). Furthermore, female turtles nest in adjacent
87 terrestrial sites potentially exposing their eggs to contaminants in 2.1. Turtle egg incubation, dosing, and tissue collection 150
88 the soils. As a first step in determining potential impacts of BPA on
89 free-living aquatic turtle populations, it is imperative that we Freshly laid (<24 h post oviposition) painted turtle eggs were 151
90 determine sensitivity under laboratory conditions. purchased from Louisiana Cypress Turtle Farms (Pierre Part, LA). 152
91 One life trait of most turtle species is their temperature-depen- Eggs were transported to the USGS Columbia Environmental 153
92 dent sex determination (TSD). Three TSD patterns are found in tur- Research Center (Columbia, MO) under the appropriate state and 154
93 tle species, with painted turtles (Chrysemys picta) demonstrating federal permits. This research was conducted under an approved 155
94 pattern 1a (Bull, 1980; Ernst and Lovich, 2009). In this pattern of Animal Welfare Plan (AWP) at the Columbia Environmental 156
95 TSD, the temperature at which the eggs develop during incubation Research Center (CERC). All personnel involved in the animal care 157
96 determines the sex of the hatchlings such that cool temperatures and use were required to adhere to the AWP which followed the 158
97 produce males and warmer temperatures produce females (Bull, spirit and intent of the policies and regulations that assure humane 159
98 1980; Ewert et al., 2004). Previous studies have shown that tem- and ethical treatment of research animals. The AWP was reviewed 160
99 perature determination can be overridden when reptile eggs are by the CERC Animal Use Committee for compliance with the 161
100 exposed to EDCs including: several legacy organochlorine contami- Animal Welfare Act and associated amendments, principles and 162
101 nants in red-eared slider turtles (Trachemys scripta elegans; guidelines. Additionally, we followed the NIH guidelines for the 163
102 Bergeron et al., 1994; Matsumoto et al., 2014; Willingham and care and use of laboratory animals. 164
103 Crews, 1999); BPA and 17b-estradiol in Caiman latirostris (Stoker At the laboratory, we candled eggs to determine viability and 165
104 et al., 2003); and TCDD, o0 p0 -DDE, p0 p0 -DDE, indole-3-carbinol, developmental stage according to Mahmoud et al. (1973), weighed, 166
105 and 17a-ethinylestradiol (EE2) in Alligator mississippiensis (Matter and to minimize clutch effects and treatment bias randomly 167
106 et al., 1998). Finally, Sheehan et al. (1999) determined 17b-estra- assigned the eggs to a treatment and a location within BPA-free 168
107 diol, an endogenous estrogen and positive control for many EDC incubation boxes. Eggs were placed in moistened vermiculite 169
108 studies, induced sex reversal in turtles at even very low doses (1:1 v/v autoclaved vermiculite: water) and incubated at male- 170
109 and with evidence for the absence of a threshold dose. The authors producing (26 ± 1 °C) temperatures. Three times per week, the 171
110 suggest that other EDCs mechanistically similar to 17b-estradiol incubation boxes were weighed, eggs rehydrated accordingly, 172
111 may also show no threshold dose for sex reversal. and the boxes were rotated within the incubator. We monitored 173
112 Most turtle sexual development begins with a bipotential embryo development by candling and staged the eggs throughout 174
113 gonad, which under the influence of temperature or chemicals, will incubation. At approximately stage 17 (the beginning of the tem- 175
114 develop into an ovary or testis. Initially Sox9 (sex determining perature sensitive period), we topically applied dosing solutions 176
115 region box 9) is expressed in the bipotential gonad (Morrish and using a micropipette. Stage 17 was selected as this represents a 177
116 Sinclair, 2002). However, with the development of Sertoli cells in very sensitive time period for exogenous influence on sexual devel- 178
117 the male testis, Sox9 expression is up-regulated (Spotila et al., opment and is thus commonly chosen in studies examining toxi- 179
118 1998). If the gonad is to develop into an ovary, the existing cant effects on sexual differentiation (e.g., Matsumoto et al., 180
119 Sertoli cells become lacunae (Mork and Capel, 2013), medullary 2014). Dosing solutions of 0.01, 1.0 and 100 lg BPA/lL (low, 181
120 Sox9 expression is down-regulated (Barske and Capel, 2010) and medium and high, respectively) and 20 ng 17b-estradiol/lL (the 182
121 cortical b-catenin expression is up-regulated. estrogen positive control for chemically-induced sex reversal) 183
122 Vitellogenin (VTG), a lipoprotein produced in the liver of mature were delivered topically to each egg in 5 lL of 100% medical grade 184
123 female turtles, has been shown to be a potential biomarker of EtOH. Control eggs received either no treatment (control) or 5 lL of 185
124 exposure to environmental estrogens (Ho et al., 1981). EtOH (vehicle control). Mean egg mass at dosing was 4.97 g 186
125 Production of this female-specific protein has been induced by lab- (±0.69 g) which resulted in the following nominal concentrations: 187
126 oratory exposures to estrogen in pre-reproductive sea turtles (Heck 20 ng E2/g-egg (20 the ED50 presented in Sheehan et al., 1999) 188
127 et al., 1997), juvenile pond turtles (Tada et al., 2008) and adult and 0.01, 1, and 100 lg BPA/g-egg. Concentrations of BPA in terres- 189
128 male red-eared sliders (Palmer and Palmer, 1995). In the present trial soils range from 0.6–10.6 ng/g in control soils and up to 190
129 study, any production of VTG in pre-reproductive male hatchlings 167.9 ng/g in sewage-fertilized agricultural fields (Zhang et al., 191
130 provides a signal of BPA-induced systemic changes. 2015). Therefore, our low dose represents an acute exposure possi- 192
131 The painted turtle (C. picta) is the most widespread native turtle ble in fertilized agricultural fields in which painted turtles may 193
132 species in North America (Ernst and Lovich, 2009) and is dis- nest. The two higher doses are more representative of concentra- 194
133 tributed across aquatic and terrestrial environments throughout tions found in aquatic habitats near contaminated sites, such as 195
134 much of the northern and eastern United States (Gervais et al., landfills (Bhandari et al., 2014; Masoner et al., 2014). The eggs 196
135 2009). This species can live in a variety of aquatic habitats from were returned to the incubators and allowed to continue develop- 197
136 relatively undisturbed slow-moving creeks (Ernst and Lovich, ing at the male-producing temperature. Additionally, we incubated 198

Please cite this article in press as: Jandegian, C.M., et al. Developmental exposure to bisphenol A (BPA) alters sexual differentiation in painted turtles
(Chrysemys picta). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.04.003
 YGCEN 12086 No. of Pages 9, Model 5G
8 April 2015

C.M. Jandegian et al. / General and Comparative Endocrinology xxx (2015) xxx–xxx 3

199 forty eggs at female-producing temperatures (30 ± 1 °C) under had a thicker epithelial cortex with some medullary testicular 225
200 otherwise identical conditions as non-treated controls or vehicle tubules with varying amounts of disorganization were considered 226
201 controls for comparisons with sex-reversed males. to be disrupted and categorized as ovarian-like cortex (OLC) sensu 227
202 At hatch, we measured straight-line carapace and plastron (Pieau et al., 1998). The term ovotestis or intersex was not used 228
203 lengths and carapace width using vernier calipers and we deter- because it was unclear if the gonad at maturation would be capable 229
204 mined mass using a portable Ohaus Trooper balance. Hatchlings of producing both eggs and sperm (Crews et al., 1991). We did not 230
205 were then euthanized and liver and urogenital tissues were har- collect data on the presence or absence of Müllerian ducts. 231
206 vested. The right kidney and gonad were fixed in Bouin’s solution
207 for 12 h, washed in 70% EtOH over 3 days and then stored in 70%
2.3. Gonad Immunohistochemistry 232
208 EtOH until histological analysis. The left gonad was collected for
209 future gene expression studies.
Immunohistochemistry with gonadal markers was performed 233
following methods similar to Mork and Capel (2013) on a subset 234
210 2.2. Gonad histology of individuals (n = 7 per treatment). This subset of individuals 235
represented multiple clutches to minimize any clutch effects and 236
211 We serially dehydrated and embedded the urogenital tissues in had complete histological sections through the entire gonad. 237
212 paraffin. Sections (5–7 lm) were stained using Richard-Allan™ Tissues were deparaffinized, rehydrated, and antigens retrieved 238
213 Hematoxylin 7211 and Richard-Allan™ Eosin-Y, Saturated by Sodium Citrate Heat-Induced Epitope Retrieval (Larsson, 239
214 (Thermo Scientific, Waltham, MA, USA). Histology was performed 1988). The slides were then submerged in blocking buffer (3.0% 240
215 by Idexx Radil Bioresearch Laboratory (Columbia, MO) or by one Bovine Serum Albumin and 3.0% Donkey Serum in PBS) and incu- 241
216 of the authors (CMJ). Sex was determined by visual inspection of bated for 1 h. Primary antibodies, 1:500 dilution of SOX9 (EMD 242
217 the histological slides independently and blindly by three of the Millipore, Billerica, MA, USA) and 1:200 dilution of b-catenin 243
218 authors (CMJ, SLD and DKH) and assessments made by comparison (Sigma–Aldrich, St. Louis, MO, USA), diluted in blocking buffer 244
219 to images in Pieau and Dorizzi (2004), Wyneken et al. (2007). The were applied, and slides incubated overnight at 4 °C. We then incu- 245
220 following definitions were agreed upon by all observers. Testes bated the slides at 24 °C for one hour with Cy-3 Conjugated Donkey 246
221 were defined as a simple single-layered epithelial cortex and a anti-mouse diluted 1:100 in PBS (Jackson Immunoresearch, West 247
222 well-formed medulla with testicular tubules; whereas ovaries Grove, PA, USA) and Alexa Fluor 488 Donkey anti-rabbit IgG diluted 248
223 exhibited a well-developed multi-layer cortex and a disorganized 1:300 in PBS (Abcam, Cambridge, MA, USA). Finally, Vectashield 249
224 medulla occasionally with large lacunae (Fig 1). Gonads which (Vector Laboratories, Burlingame, California, USA) and Prolong 250

Fig. 1. The effect of in ovo exposure of BPA and E2 during the temperature sensitive period on Chrysemys picta gonad structure. Photomicrographs of H&E images of full
gonads at 10 (top row) and 20 (bottom row) showing details. Labeled are the single-layered epithelium (E) and testicular tubules (TT) characteristic of males and a thicker
cortex (C) and lacunae (L) characteristic of females and those with an ovairian-like cortex (OLC). Scale bar = 50 lm.

Please cite this article in press as: Jandegian, C.M., et al. Developmental exposure to bisphenol A (BPA) alters sexual differentiation in painted turtles
(Chrysemys picta). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.04.003
 YGCEN 12086 No. of Pages 9, Model 5G
8 April 2015

4 C.M. Jandegian et al. / General and Comparative Endocrinology xxx (2015) xxx–xxx

251 Antifade Reagent (Life Technologies, Grand Island, New York, USA) morphological measurements were analyzed using ANOVA for 300
252 were added with the cover slip. Negative controls included sec- males and a t-test for females. The percentage of OLC or female 301
253 tions incubated with the secondary antibodies alone. Imaging was compared across treatment groups, and then between the 302
254 was performed at the USGS Columbia Environmental Research three BPA treatments using Chi square Goodness of Fit tests. 303
255 Center using a Nikon 90i eclipse Widefield Microscope with FITC Because the vitellogenin data were not normally distributed, data 304
256 and Texas Red filters. We used Nikon’s NIS-Elements software for were log transformed prior to an ANOVA. Figures were constructed 305
257 image analysis. using SigmaPlot Version 13.0 (Systat Software, San Jose, CA) and 306
CorelDraw Version X3 (Ottawa, Canada). 307
258 2.4. Vitellogenin ELISA

259 We quantified vitellogenin protein using an enzyme-linked 3. Results 308

260 immunosorbent assay (ELISA) following methods similar to that
261 of Irwin et al. (2001) on the same individuals used for IHC (n = 7 3.1. Hatchling success and size 309
262 per treatment). Approximately one-third of each liver was weighed
263 and placed in a 1.5 ml cryotube. Lysis beads, the same weight as At male- and female-producing temperatures, overall hatching 310
264 the liver, were added to the cryotubes and 1 TBS added at twice success was 82% and was independent of treatment (Fisher’s 311
265 the original tissue mass. Cryotubes were placed in the Bullet Exact Test = 11.7, p = 0.07; Table 1). At hatch, there were no 312
266 Blender and mixed for <1 min. Homogenized livers were then differences in size among male treatment groups (carapace length 313
267 centrifuged for 10 min at 3600 rpm at 4 °C. The supernatant was F = 0.29, p = 0.92; carapace width F = 1.09, p = 0.36; plastron length 314
268 collected and stored at 80 °C until analysis. Standards were F = 0.57, p = 0.72; mass F = 0.79, p = 0.55; Fig. 2) or female control 315
269 diluted in TBS (25 mM Tris, 0.3 M NaCl, pH 7.4) and loaded into a groups (data not shown; carapace length F = 0.31, p = 0.58; cara- 316
270 96 well microtiter Nunc Maxisorb plates (50 lL per well). In addi- pace width F = 0.07, p = 0.79; plastron length F = 0.38, p = 0.54; 317
271 tion to the samples, each plate contained standards, adult female mass F = 0.53, p = 0.47). Similarly, all gross abnormalities (e.g., a 318
272 box turtle plasma as a positive control (provided by SLD), estro- deformity other than additional carapace bony elements) were 319
273 gen-induced T. scripta purified vitellogenin protein as a positive not associated with any specific treatment or sex. 320
274 control (provided by KWS), and non-specific binding wells. All
275 samples including positive controls were diluted 1:2500 and run
276 in duplicate. The plate was incubated overnight at 4 °C. After
3.2. Histological analysis 321
277 incubation, plates were warmed to room temperature for 30 min
278 and then washed three times with 200 lL of TBST (TBS + 0.2%
Histological examination of the gonads showed a clear effect of 322
279 Tween 20) and once more with TBS followed by blocking with
treatment. All control males and those exposed to the EtOH vehicle 323
280 150 lL Blotto Tween (5% BSA, 0.2% Tween 20) for two hours at
were classified as male (Fig. 3) and presented with well-formed 324
281 room temperature. The plates where then washed three additional
medullary testicular tubules and a uniformly single layered cortex 325
282 times with 200 lL of TBST and once with TBS. We added 50 lL of
(Fig. 1). Temperature-determined (control and vehicle control) 326
283 primary antibody (provided by KWS) diluted 1:1000 in Blotto
individuals classified as female had a thickened cortex with lacu- 327
284 Tween and incubated the plates at room temperature for two
nae present (Fig. 1). There was a significant difference in feminiza- 328
285 hours. Plates were again washed three times with TBST and once
tion between the treatment groups (v2 = 45.2, p < 0.001; Fig. 3). 329
286 with TBS. Secondary Antibody, Alexa FluorÒ Goat anti-rabbit IgG
Treatment of eggs with estradiol (E2; n = 19) resulted in only two 330
287 (H + L) conjugated to alkaline phosphatase (Molecular ProbesÒ,
males. Of the 17 remaining individuals, twelve were clearly 331
288 Life Technologies, Grand Island, NY, USA), diluted 1:1000 in
sex-reversed females with a hyper-developed cortex and a disorga- 332
289 Blotto Tween was added and incubated for two hours. Plates were
nized medulla with multiple lacunae and five were categorized as 333
290 then washed four times with TBST and four times with TBS. After
OLC with a thinner cortex and a disorganized medulla (Fig. 1). Of 334
291 the last wash, plates were developed using 0.1% (w/v) p-nitro-phe-
those exposed to BPA, we observed a significant number of 335
292 nyl phosphate (PNPP) in developing buffer (0.1 M NaCl, 0.1 M Tris,
individuals with disruption of sexual development at all concen- 336
293 5 mM MgCl2, pH 9.5) and incubated at room temperature for
trations, but none were fully sex-reversed. Exposure to high 337
294 15 min. Optical density was read with a Synergy4 BioTek
concentrations of BPA (100 lg/g-egg) produced seven with OLCs 338
295 Microplate Reader at 405 nm.
and eleven non-sex reversed males (39%; n = 18). At medium con- 339
centrations of BPA (1 lg/g-egg), six individuals had OLCs and 340
296 2.5. Statistics twelve were males (33%; n = 18). Even at the lowest dose of 341
0.01 lg BPA/g-egg, we observed a significant amount of sexual dis- 342
297 Data were statistically analyzed using NCSS Version 8.0 ruption with 7 OLC individuals and sixteen males (30%; n = 23). The 343
298 (Kaysville, UT) with an alpha = 0.05. We used a Fisher’s Exact percentage of OLCs increased with BPA dose, but not significantly 344
299 Test to compare hatching success among treatments and (v2 = 0.3, p = 0.85). 345

Table 1
The percent of painted turtle eggs that hatched was independent of treatment (p = 0.07).

Treatment Eggs incubated Eggs hatched Percent hatched (%) 95% CIs
Male control 46 35 76 61.2–87.4
Male control (EtOH) 47 42 89.4 76.9–96.5
Female control 20 17 85 62–97
Female control (EtOH) 20 18 90 68–99
E2 20 ng/g-egg 45 33 73.3 58.1–85.4
0.01 lg BPA/g-egg 44 42 95 84.5–99.4
1 lg BPA/g-egg 48 40 83.3 69.8–92.5
100 lg BPA/g-egg 48 39 81.3 67.4–91.1

Please cite this article in press as: Jandegian, C.M., et al. Developmental exposure to bisphenol A (BPA) alters sexual differentiation in painted turtles
(Chrysemys picta). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.04.003
 YGCEN 12086 No. of Pages 9, Model 5G
8 April 2015

C.M. Jandegian et al. / General and Comparative Endocrinology xxx (2015) xxx–xxx 5

increasing BPA concentration (Fig. 4). The expression of b-catenin 361

was consistent with the thickness of the developing cortex. 362
Those individuals with more cortical development (Fig. 4, 1 363
lg BPA/g-egg) appeared to have greater expression of cortical 364
b-catenin; whereas those with a thinner cortex had less expression 365
(Fig. 4, 0.01 lg and 100 lg BPA/g-egg), but still followed the pat- 366
tern observed in both temperature-induced and E2-produced 367
females. 368

3.4. Vitellogenin induction 369

Even though gonads from E2 and BPA-treated individuals 370

exhibited evidence of full to partial sex-reversal, these histological 371
and functional changes did not result in the predicted induction of 372
hepatic VTG (F = 1.24, p = 0.31). Pre-reproductive female hepatic 373
VTG concentrations were not significantly different from male 374
hepatic concentrations. All values measured were an order of mag- 375
nitude lower than the positive controls (not shown). Interestingly, 376
Fig. 2. Carapace length, carapace width, and plastron length of Chrysemys picta there was more variation in the female and BPA high samples than 377
hatchlings at hatch with respect to treatment. At hatch, there were no differences in the other treatments and control males; however, this could be a 378
size among male treatment groups (ANOVA carapace length p = 0.92; carapace function of our small sample size (Fig. 5; n = 7 per treatment). 379
width p = 0.36; plastron length p = 0.72; mass p = 0.55). Bars represent standard
error.
4. Discussion 380

346 3.3. Immunohistochemical expression of SOX9 and b-catenin

Here we demonstrated that BPA exposures topically applied to 381
painted turtle eggs incubated at male-producing temperatures 382
347 Immunohistochemistry supported our histological findings
resulted in the disruption of sexual development. As expected, 383
348 above. Control male gonads expressed a distinct pattern of SOX9
exposure to E2 overrode the male-producing temperature-induced 384
349 associated with the Sertoli cells (Sc) of the testicular tubules (tt)
testicular differentiation and produced predominantly females. In 385
350 and b-catenin expression was present in the testicular tubules
ovo BPA exposure resulted in sexual disruption, as evidenced by 386
351 (Fig. 4). Temperature-determined female gonads did not have
disruption of the testicular tubules and formation and thickening 387
352 SOX9 expression and b-catenin expression was predominantly
of the cortex producing OLCs. Exposure to 100 lg/g-egg resulted 388
353 localized to the cortex. Unlike the temperature-induced females,
in sexual disruption in 39% of our turtles, whereas a similar expo- 389
354 the sex-reversed E2-treated individuals continued to express
sure of 138 lg/g-egg caused 100% sex reversal in 10 days and 390
355 SOX9 along the perimeters of the testicular tubules, but a thick-
6 month-old Caiman latirostris (Stoker et al., 2003). However, 391
356 ened cortical region with strong b-catenin expression was also
exposure to a lower dose of BPA resulted in very similar degrees 392
357 clearly visible (Fig. 4). Exposure to each of the BPA concentrations
of sexual disruption in both studies (40% in caiman at 1.3 lg/g- 393
358 produced varying degrees of disorganization in the testicular
egg, 33% in our painted turtles at 1.0 lg/g-egg). The only paper 394
359 tubules as evidenced by the disruption of medullary SOX9 signal-
examining the effects of BPA on turtles (Clairardin et al., 2013) 395
360 ing. The degree of disorganization seemed to increase with
determined that in T. scripta BPA interfered with estradiol metabo- 396
lism. Trachemys eggs treated with 40 lg/egg BPA early in develop- 397
ment prior to the TSP had greater concentrations of yolk estradiol 398
and estrone and lesser concentrations of estrone sulfate than 399
untreated control eggs. This suggests that developmental exposure 400
to BPA alters metabolism of maternal estrogens and possibly 401
makes them bioavailable during later times in development, 402
including the temperature sensitive period (TSP). 403
We applied the BPA and E2 topically to the eggs during the 404
known TSP to ensure a controlled exposure to all embryos across 405
the treatment groups. Although, this method does not exactly 406
mimic natural exposure via maternal transfer and/or from the 407
nesting substrate for the duration of incubation, it does target 408
the most sensitive period for disruption of sex determination. 409
The method we used in this study is similar to a number of other 410
EDC studies in reptilian species (Crews et al., 1991; Matsumoto 411
et al., 2014; Stoker et al., 2003). Although we did not explore 412
mechanisms in the current study, our results of BPA-induced sex- 413
reversal may be associated with altered hormone metabolism 414
(Clairardin et al., 2013), in addition to binding to estrogen or other 415
receptors (Reif et al., 2010), and thus both mechanisms and their 416
Fig. 3. The number of males, females, and gonads with ovarian-like cortices (OLC) relative importance warrant further study. 417
across all treatments identified during histological examinations of Chrysemys picta Because we collected samples at hatch, we were unable to 418
hatchlings. There was a significant difference between all treatment groups
(p > 0.001) but no difference between the three BPA treatment groups (p = 0.85).
determine if the observed effects persist into adulthood. 419
The percent OLCs in BPA treatments were 39% for high exposure (n = 18), 33% for However, our findings of BPA overriding TSD, even in this young 420
medium exposure (n = 18) and 30% for low exposure (n = 23). cohort, suggest low doses of BPA can disrupt turtle sex 421

Please cite this article in press as: Jandegian, C.M., et al. Developmental exposure to bisphenol A (BPA) alters sexual differentiation in painted turtles
(Chrysemys picta). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.04.003
 YGCEN 12086 No. of Pages 9, Model 5G
8 April 2015

6 C.M. Jandegian et al. / General and Comparative Endocrinology xxx (2015) xxx–xxx

Fig. 4. Immunohistochemistry of Chrysemys picta gonads showing expression on of SOX9 (green), b-catenin (red) and both overlaid at 100 and 200 (merged). Abbreviations
are as follows: Sc = Sertoli cells, co = cortex, tt = testicular tubules. Scale bar = 100 lm at 100 and 50 lm at 200. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Jandegian, C.M., et al. Developmental exposure to bisphenol A (BPA) alters sexual differentiation in painted turtles
(Chrysemys picta). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.04.003
 YGCEN 12086 No. of Pages 9, Model 5G
8 April 2015

C.M. Jandegian et al. / General and Comparative Endocrinology xxx (2015) xxx–xxx 7

possible that with age further evidence of feminization of OLC 465

gonads would be observed and thus our results may underestimate 466
the true extent of changes from exposure to BPA. We are currently 467
testing this hypothesis in a follow-up study. 468
The present results demonstrated that in ovo exposure to BPA 469
causes sexual disruption in painted turtle embryos. We observed 470
variation within treatments in the thickness of the cortex and 471
the degree of testicular tubule disruption. Although turtle eggs 472
are quite porous, the degree to which BPA can penetrate eggshells 473
has not been studied. Another possible explanation for the degree 474
of variation is that there may be individual differences in embry- 475
onic metabolism of these chemicals or possibly a distribution of 476
sensitivities to BPA. Future studies should therefore examine 477
uptake and pharmacokinetics of BPA in developing turtle embryos. 478
Recently, Mork et al. (2014) demonstrated that although sexual 479
differentiation of the gonad happens during the temperature sensi- 480
tive period, there may be an earlier genetic or environmental sex- 481
determining signal. Possibly, exposure at an earlier developmental 482
stage would have resulted in a greater percentage of OLCs. 483
Fig. 5. Log transformed vitellogenin protein concentration measured by ELISA in
Unfortunately, such a comprehensive temporal study would 484
liver tissue did not differ across treatments (p = 0.31). Bars represent standard error.
require a very large sample size and be cost-prohibitive. 485
One other endpoint tested in the current study was the induc- 486

422 determination and future studies of hatchlings in older age classes tion of hepatic VTG. We chose this biomarker because it has been 487

423 is warranted to better understand possible effects at the pop- shown to be a powerful biomarker in fish (Marin and Matozzo, 488

424 ulation level. In this young age class, we observed that with 2004) and has been demonstrated in turtles when assessing the 489

425 increased concentrations of in ovo BPA, the percentage of OLCs impacts of EDCs on reproduction (Heck et al., 1997; Irwin et al., 490

426 tended to increase. Interestingly, a low dose of BPA (0.01 lg/g- 2001; Palmer and Palmer, 1995). The lack of positive VTG induc- 491

427 egg) seemed to produce the same amount of disorganization tion in our study may be a result of the young age of the hatchlings 492

428 within the gonad as the highest dose (100 lg/g-egg), suggesting tested or the low single dose of E2 as positive control (Irwin et al., 493

429 that although BPA has previously been characterized as a weak 2001). Other studies with reptiles have failed to show VTG induc- 494

430 estrogen with regard to its binding potency to estrogen receptors tion in the presence of a wide array of environmental contaminants 495

431 (Ben-Jonathan and Steinmetz, 1998; Vandenberg et al., 2009), it (Irwin et al., 2001; Matter et al., 1998; Rie et al., 2005; Valverde 496

432 can produce gonadal disorganization and potential sex reversal et al., 2008). These collective findings may be an indication that 497

433 across a wide range of exposures. Other research has demonstrated VTG is not a good biomarker of estrogenized turtles and other rep- 498

434 significant sex reversal in turtles (e.g., red-eared sliders) after topi- tiles and perhaps the use of other biomarkers should be further 499

435 cal exposure of eggs to a variety of EDCs (Bergeron et al., 1994; evaluated in these species. 500

436 Crews et al., 1991; Matsumoto et al., 2014; Willingham and Potential biomarkers may include biomolecular or epigenetic 501

437 Crews, 1999). Although none of these prior studies examined changes (such as altered DNA methylation status for select genes 502

438 BPA, all showed some degree of sex reversal, with up to 100% or altered expression of non-coding RNAs). The sequencing and 503

439 reversal in some instances. A number of these studies found a annotation of the painted turtle genome (Shaffer et al., 2013) will 504

440 dose-dependent sex reversal (Bergeron et al., 1994; Crews et al., assist in identifying these additional biomarkers that may be 505

441 1991; Willingham and Crews, 1999). Factors such as species, stage induced in BPA/E2 treated turtles and hold across other species 506

442 of development, length of exposure and dose seem to contribute to (such as mammals) where currently no such reliable biomarkers 507

443 the variations in the degree of sex reversal observed in turtles. have been discovered. The sequencing of this genome also allows 508

444 Along with the histological examination of transforming for examination of potential molecular mechanisms driving the 509

445 gonads, it is essential to examine differentiation of gonadal cell BPA/E2 induced sexual disruption in painted turtles. 510

446 types at the molecular level as these events can precede histologi- Reptiles, including turtles, are one of the most threatened verte- 511

447 cal transformations. The expression of SOX9 and b-catenin brate taxa with habitat degradation, climate change, disease, and 512

448 together provide definitive information on the fate of a differ- unsustainable harvest for food and the pet trade cited as the pri- 513

449 entiating and developing gonad (Mork et al., 2014). Temperature- mary threats to their long-term survival (Gibbons et al., 2000). In 514

450 determined ovaries and E2-treated sex-reversed ovaries showed addition to these commonly cited conservation challenges, it is 515

451 cortical enhancement of b-catenin expression and a reduction or imperative to explore other anthropogenic changes that may be 516

452 absence of medullary SOX9 expression (Fig. 4). Recently, driving shifts in population structure. Reliant on TSD, most turtles 517

453 (Matsumoto et al., 2014) reported a similar reduction in Sox9 fol- are increasingly threatened by climate change and environmental 518

454 lowing exposure of otherwise male turtle embryos to EE2 or 4-hy- EDCs which both may lead to changes in the sex ratio of pop- 519

455 droxy-20 ,40 ,60 -trichlorobiphenyl. As predicted, testes continued to ulations even prior to hatching (Bergeron et al., 1994; Janzen, 520

456 express b-catenin in the medulla (in association with the testicular 1994; Matsumoto et al., 2014; Sheehan et al., 1999; Willingham 521

457 tubules) and in the surface epithelium and SOX9 expression was and Crews, 1999). Our findings of disruption during sexual differ- 522

458 clearly associated with the Sertoli cells. The gonads exposed to entiation following acute exposure to low doses of BPA may have 523

459 BPA produced altered expression patterns for these markers. far reaching consequences on sex ratios of wild populations and 524

460 Some gonads were clearly still male and histologically classified potentially exacerbate population decline. As sentinels for aquatic 525

461 as such. Others were undergoing medullary reorganization with habitats, these findings have implications not only for turtle con- 526

462 dissolution of testicular cords and varying amounts of cortical servation and health, but for other aquatic organisms, and ulti- 527

463 expansion. Typically during normal development, lacunae forma- mately humans who also are reliant on a safe water supply 528

464 tion precedes cortical development (Wibbels et al., 1991) so it is (Bhandari et al., 2014). 529

Please cite this article in press as: Jandegian, C.M., et al. Developmental exposure to bisphenol A (BPA) alters sexual differentiation in painted turtles
(Chrysemys picta). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.04.003
 YGCEN 12086 No. of Pages 9, Model 5G
8 April 2015

8 C.M. Jandegian et al. / General and Comparative Endocrinology xxx (2015) xxx–xxx

530 5. Conclusions Heck, J., MacKenzie, D.S., Rostal, D., Medler, K., Owens, D., 1997. Estrogen induction 602
of plasma vitellogenin in the Kemp’s ridley sea turtle Lepidochelys kempii. Gen. 603
Comp. Endocrinol. 107, 280–288. 604
531 We demonstrated that developmental exposure to relevant Ho, S.-M., Danko, D., Callard, I.P., 1981. Effect of exogenous estradiol-17b on plasma 605
532 concentrations of BPA or E2 caused a disruption in sexual differ- vitellogenin levels in male and female Chrysemys and its modulation by 606
testosterone and progesterone. Gen. Comp. Endocrinol. 43, 413–421. 607
533 entiation in painted turtle hatchlings. Histological analyses and 608
Irwin, L., Irwin, K., 2006. Global threats affecting the status of reptile populations.
534 immunohistochemistry revealed disorganization of SOX9-express- In: Gardner, S.C., Oberdörster, E. (Eds.), New Perspectives: Toxicology of 609
535 ing testicular tubules and the development of a b-catenin express- Reptiles. Taylor & Francis, Boca Raton, FL, pp. 10–34. 610
Irwin, L.K., Gray, S., Oberdörster, E., 2001. Vitellogenin induction in painted turtle, 611
536 ing ovarian-like cortex in the gonads of the BPA treated embryos. 612
Chrysemys picta, as a biomarker of exposure to environmental levels of estradiol.
537 Further studies are warranted to identify reliable biomarkers and Aquat. Toxicol. 55, 49–60. 613
538 elucidate the underlying mechanisms responsible for these func- Janzen, F.J., 1994. Climate change and temperature-dependent sex determination in 614
reptiles. Proc. Natl. Acad. Sci. U.S.A. 91, 7487–7490. 615
539 tional and morphological changes.
Kassotis, C.D., Tillitt, D.E., Davis, J.W., Hormann, A.M., Nagel, S.C., 2013. Estrogen and 616
androgen receptor activities of hydraulic fracturing chemicals and surface and 617
540 Acknowledgments ground water in a drilling-dense region. Endocrinology 155, 897–907. 618
Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, S.D., Barber, L.B., 619
Buxton, H.T., 2002. Pharmaceuticals, hormones, and other organic wastewater 620
541 This project was supported by funds provided by the University contaminants in US streams, 1999–2000: a national reconnaissance. Environ. 621
542 of Missouri Mizzou Advantage Program, Bond Life Sciences Center, Sci. Technol. 36, 1202–1211. 622
Larsson, L.-I., 1988. Immunocytochemistry: Theory and Practice. CRC Press, Boca 623
543 Office of Research, and Biology Department. Additional support Raton, FL, p. 283. 624
544 was provided by the USGS Contaminant Biology Program, Lindeman, P.V., 1992. Nest-site fixity among painted turtles (Chrysemys picta) in 625
545 Environmental Health Mission Area. We also thank the northern Idaho. Northwest. Natural. 73, 27–30. 626
Mahmoud, I.Y., Hess, C.L., Klicka, J., 1973. Normal embryonic stages of the western 627
546 University of Missouri Cytology Core for use of their equipment 628
painted turtle, Chrysemys picta bellii. J. Morph. 141, 269–279.
547 during early troubleshooting and imaging. Technical support was Marin, M.G., Matozzo, V., 2004. Vitellogenin induction as a biomarker of exposure to 629
548 provided by Jamie Palmer from the Saint Louis Zoo Institute for estrogenic compounds in aquatic environments. Mar. Pollut. Bull. 48, 835–839. 630
Masoner, J.R., Kolpin, D.W., Furlong, E.T., Cozzarelli, I.M., Gray, J.L., Schwab, E.A., 631
549 Conservation Medicine and Doug Hardesty from the USGS. This 632
2014. Contaminants of emerging concern in fresh leachate from landfills in the
550 manuscript was improved from helpful comments provided by conterminous United States. Environ. Sci. Proc. Impacts 16, 2335–2354. 633
551 Bethany Kunz, USGS internal reviewer, and an anonymous Matsumoto, Y., Hannigan, B., Crews, D., 2014. Embryonic PCB exposure alters 634
phenotypic, genetic, and epigenetic profiles in turtle sex determination, a 635
552 reviewer.
biomarker of environmental contamination. Endocrinology 155, 4168–4177. 636
Matter, J.M., McMurry, C.S., Anthony, A.B., Dickerson, R.L., 1998. Development and 637
553 References implementation of endocrine biomarkers of exposure and effects in American 638
alligators (Alligator mississippiensis). Chemosphere 37, 1905–1914. 639
Mork, L., Capel, B., 2013. Conserved action of b-catenin during female fate 640
554 Barske, L.A., Capel, B., 2010. Estrogen represses SOX9 during sex determination in
determination in the red-eared slider turtle. Evol. Dev. 15, 96–106. 641
555 the red-eared slider turtle Trachemys scripta. Dev. Biol. 341, 305–314.
Mork, L., Czerwinski, M., Capel, B., 2014. Predetermination of sexual fate in a turtle 642
556 Basile, E.R., Avery, H.W., Bien, W.F., Keller, J.M., 2011. Diamondback terrapins as
with temperature-dependent sex determination. Dev. Biol. 386, 264–271. 643
557 indicator species of persistent organic pollutants: using Barnegat Bay, New
Morrish, B., Sinclair, A., 2002. Vertebrate sex determination: many means to an end. 644
558 Jersey as a case study. Chemosphere 82, 137–144.
Reproduction 124, 447–457. 645
559 Ben-Jonathan, N., Steinmetz, R., 1998. Xenoestrogens: the emerging story of
Palmer, B.D., Palmer, S.K., 1995. Vitellogenin induction by xenobiotic estrogens in 646
560 bisphenol A. Trends Endocrinol. Metab. 9, 124–128.
the red-eared turtle and African clawed frog. Environ. Health Perspect. 103 647
561 Bergeron, J.M., Crews, D., McLachlan, J.A., 1994. PCBs as environmental estrogens:
(Suppl. 4), 19–25. 648
562 turtle sex determination as a biomarker of environmental contamination.
Pieau, C., Dorizzi, M., 2004. Oestrogens and temperature-dependent sex 649
563 Environ. Health Perspect. 102, 780–781.
determination in reptiles: all is in the gonads. J. Endocrinol. 181, 367–377. 650
564 Bergeron, J.M., Willingham, E., Osborn 3rd, C., Rhen, T., Crews, D., 1999.
Pieau, C., Dorizzi, M., Richard-Mercier, N., Desvages, G., 1998. Sexual differentiation 651
565 Developmental synergism of steroidal estrogens in sex determination.
of gonads as a function of temperature in the turtle Emys orbicularis: endocrine 652
566 Environ. Health Perspect. 107, 93.
function, intersexuality and growth. J. Exp. Zool. 281, 400–408. 653
567 Bhandari, R.K., Deem, S.L., Holliday, D.K., Jandegian, C.M., Kassotis, C.D., Nagel, S.C.,
Pieau, C., Dorizzi, M., Richard-Mercier, N., 1999. Temperature-dependent sex 654
568 Tillitt, D.E., vom Saal, F.S., Rosenfeld, C.S., 2014. Effects of the environmental
determination and gonadal differentiation in reptiles. Cell. Mol. Life Sci. CMLS 655
569 estrogenic contaminants bisphenol A and 17b-ethinyl estradiol on sexual
55, 887–900. 656
570 development and adult behaviors in aquatic wildlife species. Gen. Comp.
Reif, D.M., Martin, M.T., Tan, S.W., Houck, K.A., Judson, R.S., Richard, A.M., Knudsen, 657
571 Endocrinol., accepted.
T.B., Dix, D.J., Kavlock, R.J., 2010. Endocrine profiling and prioritization of 658
572 Bull, J.J., 1980. Sex determination in reptiles. Q. Rev. Biol. 55, 3–21.
environmental chemicals using ToxCast data. Environ. Health Perspect. 118, 659
573 Clairardin, S.G., Paitz, R.T., Bowden, R.M., 2013. In ovo inhibition of steroid
1714–1720. 660
574 metabolism by bisphenol-A as a potential mechanism of endocrine
Rie, M.T., Kitana, N., Lendas, K.A., Won, S.J., Callard, I.P., 2005. Reproductive 661
575 disruption. Proc. Biol. Sci. 280, 20131773.
endocrine disruption in a sentinel species (Chrysemys picta) on Cape Cod, 662
576 Crain, D.A., Eriksen, M., Iguchi, T., Jobling, S., Laufer, H., LeBlanc, G.A., Guillette Jr.,
Massachusetts. Arch. Environ. Contam. Toxicol. 48, 217–224. 663
577 L.J., 2007. An ecological assessment of bisphenol-A: evidence from comparative
Selcer, K.W., 2006. Reptile ecotoxicology: studying the effects of contaminants on 664
578 biology. Reprod. Toxicol. 24, 225–239.
populations. In: Gardner, S., Oberdoerster, E. (Eds.), Toxicology of Reptiles. CRC 665
579 Crews, D., Bull, J.J., Wibbels, T., 1991. Estrogen and sex reversal in turtles: a dose-
Taylor and Francis, Boca Raton, FL, pp. 267–298. 666
580 dependent phenomenon. Gen. Comp. Endocrinol. 81, 357–364.
Shaffer, H.B., Minx, P., Warren, D.E., Shedlock, A.M., Thomson, R.C., Valenzuela, N., 667
581 Environment Canada, 2008. Screening assessment for the challenge phenol, 4,40 -(1-
Abramyan, J., Amemiya, C.T., Badenhorst, D., Biggar, K.K., 2013. The western 668
582 methylethylidene)bis-(bisphenol A). Chemical Abstracts Service Registry
painted turtle genome, a model for the evolution of extreme physiological 669
583 Number 80-05-7. Environment Canada and Health Canada, Ottawa, https://
adaptations in a slowly evolving lineage. Genome Biol. 14, R28. 670
584 www.ec.gc.ca/ese-ees/default.asp?lang=En&n=3C756383-1 (accessed
Sheehan, D.M., Willingham, E., Gaylor, D., Bergeron, J.M., Crews, D., 1999. No 671
585 25.03.15).
threshold dose for estradiol-induced sex reversal of turtle embryos: how little is 672
586 Ernst, C.H., Lovich, J.E., 2009. Chrysemys Picta Painted Turtle. Turtles of the United
too much? Environ. Health Perspect. 107, 155. 673
587 States and Canada, 2nd ed., Johns Hopkins University Press, Baltimore, pp. 184–
Spotila, L.D., Spotila, J.R., Hall, S.E., 1998. Sequence and expression analysis of WT1 674
588 211.
and Sox9 in the red-eared slider turtle, Trachemys scripta. J. Exp. Zool. 281, 417– 675
589 Ewert, M.A., Etchberger, C.R., Nelson, C.E., 2004. Turtle Sex-determining Modes and
427. 676
590 TSD Patterns, and Some TSD Pattern Correlates. Temperature-dependent Sex
Stoker, C., Rey, F., Rodriguez, H., Ramos, J.G., Sirosky, P., Larriera, A., Luque, E.H., 677
591 Determination in Vertebrates. Smithsonian Books, Washington, DC, pp. 21–32.
Munoz-de-Toro, M., 2003. Sex reversal effects on Caiman latirostris exposed to 678
592 Gervais, J., Rosenberg, D., Barnes, S., Puchy, C., Stewart, E., 2009. Conservation
environmentally relevant doses of the xenoestrogen bisphenol A. Gen. Comp. 679
593 assessment for the western painted turtle in Oregon: (Chrysemys picta bellii)
Endocrinol. 133, 287–296. 680
594 version 1.1 (technical report). U.S.D.A. Forest Service, pp. 4–61.
Tada, N., Nakao, A., Hoshi, H., Saka, M., Kamata, Y., 2008. Vitellogenin, a biomarker 681
595 Gibbons, J.W., Scott, D.E., Ryan, T.J., Buhlmann, K.A., Tuberville, T.D., Metts, B.S.,
for environmental estrogenic pollution, of Reeves’ pond turtles: analysis of 682
596 Greene, J.L., Mills, T., Leiden, Y., Poppy, S., 2000. The global decline of reptiles,
similarity for its amino acid sequence and cognate mRNA expression after 683
597 Déjà Vu amphibians. BioScience 50, 653–666.
exposure to estrogen. J. Vet. Med. Sci. 70, 227–234. 684
598 GrandViewResearch, 2014. Global bisphenol A (BPA) market by application
Valverde, R.A., Selcer, K.W., Lara, L.R., Sibaja-Cordero, J.A., 2008. Lack of 685
599 (appliances, automotive, consumer, construction, electrical & electronics)
xenoestrogen-induced vitellogenin in male olive ridley sea turtles 686
600 expected to reach USD 20.03 billion by 2020, http://www.digitaljournal.com/
601 pr/2009287 (accessed 18.03.15).

Please cite this article in press as: Jandegian, C.M., et al. Developmental exposure to bisphenol A (BPA) alters sexual differentiation in painted turtles
(Chrysemys picta). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.04.003
 YGCEN 12086 No. of Pages 9, Model 5G
8 April 2015

C.M. Jandegian et al. / General and Comparative Endocrinology xxx (2015) xxx–xxx 9

687 (Lepidochelys olivacea) from the Pacific coast of Costa Rica. Rev. Biol. Trop. 56, Wibbels, T., Bull, J.J., Crews, D., 1991. Chronology and morphology of temperature- 700
688 49–57. dependent sex determination. J. Exp. Zool. 260, 371–381. 701
689 Vandenberg, L.N., Maffini, M.V., Sonnenschein, C., Rubin, B.S., Soto, A.M., 2009. Willingham, E., Crews, D., 1999. Sex reversal effects of environmentally relevant 702
690 Bisphenol-A and the great divide: a review of controversies in the field of xenobiotic concentrations on the red-eared slider turtle, a species with 703
691 endocrine disruption. Endocr. Rev. 30, 75–95. temperature-dependent sex determination. Gen. Comp. Endocrinol. 113, 429– 704
692 Vandenberg, L.N., Chahoud, I., Heindel, J.J., Padmanabhan, V., Paumgartten, F.J., 435. 705
693 Schoenfelder, G., 2010. Urinary, circulating, and tissue biomonitoring studies Wyneken, J., Epperly, S.P., Crowder, L.B., Vaughan, J., Blair Esper, K., 2007. 706
694 indicate widespread exposure to bisphenol A. Environ. Health Perspect. 118, Determining sex in posthatchling loggerhead sea turtles using multiple 707
695 1055–1070. gonadal and accessory duct characteristics. Herpetologica 63, 19–30. 708
696 Vandenberg, L.N., Ehrlich, S., Belcher, S.M., Ben-Jonathan, N., Dolinoy, D.C., Hugo, Zhang, Z., LeVelly, M., Rhind, S.M., Kyle, C.E., Hough, R.L., Duff, E.I., Mckenzie, C., 709
697 E.S., Hunt, P.A., Newbold, R.R., Rubin, B.S., Saili, K.S., Soto, A.M., Wang, H.-S., vom 2015. A study on temporal trends and estimates of fate of bisphenol A in 710
698 Saal, F.S., 2013. Low dose effects of bisphenol A: an integrated review of in vitro, agricultural soils after sewage sludge amendment. Sci. Total Environ. 515–516, 711
699 laboratory animal and epidemiology studies. Endocr. Disrupt. 1, E1–E20. 1–11. 712
713

Please cite this article in press as: Jandegian, C.M., et al. Developmental exposure to bisphenol A (BPA) alters sexual differentiation in painted turtles
(Chrysemys picta). Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.04.003