Effects of developmental exposure to bisphenol A and ethinyl estradiol on spatial navigational learning and memory in painted turtles (Chrysemys picta)
Introduction
Endocrine disrupting chemicals (EDCs) are widely distributed throughout aquatic and terrestrial environments (Reviewed in (Bhandari et al., 2015)). Two abundant EDCs are bisphenol A (BPA) and ethinyl estradiol (EE2). As a plasticizer, BPA is present in a wide range of commonly used household items, including plastic storage containers and cardboard products. Current estimates indicate global production of BPA at 15 billion pounds annually (GrandViewResearch, 2014). Bisphenol A has been identified in almost all aquatic habitats tested to date (Reviewed in (Bhandari et al., 2015)), thus, causing concern for widespread and continued exposure of humans and wildlife. Ethinyl estradiol is the estrogen present in birth control pills. The unmetabolized form can be excreted in the urine, and thus, can also collect in aquatic ecosystems (Laurenson et al., 2014).
Many xenoestrogens can induce gonadal sex reversal in reptilian species that exhibit temperature sex determination (TSD) (Bergeron et al., 1999, Bull et al., 1988, Clairardin et al., 2013, Crews et al., 1991, Crews et al., 1995, Crews et al., 1996, Freedberg et al., 2006, Gutzke and Chymiy, 1988, Jandegian et al., 2015, Kohno et al., 2015, Sheehan et al., 1999, Willingham and Crews, 1999). Recently, we showed that in ovo exposure of eastern painted turtles (Chrysemys picta) to BPA and 17β-estradiol results in partial to full gonadal sex reversal in animals incubated at the male-inducing temperature, 26⁰C (Jandegian et al., 2015). In Olive Ridley sea turtles (Lepidochelys olivacea), estradiol treatment leads to hypoplastic ovaries but delayed remodeling of the medullary cords and suppression of testicular factor Sox9 (Diaz-Hernandez et al., 2015). While it is generally thought that in ovo exposure to exogenous estrogens fully replicate the effects of ovarian-promoting temperature incubation, this study showed that estradiol but not the female-permissive temperature causes an up-regulation of FoxL2 prior to the expression of aromatase.
In mammalian species, brain sexual differentiation is driven by the organizational and activational effects of gonadal hormones (Arnold and Breedlove, 1985, Morris et al., 2004, Phoenix et al., 1959). Organizational and activational effects have also been reported in male leopard geckos (Eublpharis macularius) (Rhen and Crews, 2000). An initial prenatal surge of testosterone, which may be converted to estrogen in certain brain regions, guides the initial programming. Full elaboration of sex-dependent behaviors requires a later adult increase in testosterone (Arnold and Breedlove, 1985, Morris et al., 2004). As sexual differentiation of brain is guided by sex hormones, it is likely vulnerable to disruption by EDCs found in the environment.
Past rodent and other animal studies show that early exposure to BPA or EE2 can disrupt in a sex-dependent manner various behavioral domains, such as cognition, anxiety, and socio-sexual (Reviewed in (Rosenfeld and Trainor, 2014)). For instance, male deer mice (Peromyscus maniculatus bairdii) exposed to BPA or EE2, during the perinatal period through the maternal diet, demonstrate compromised spatial navigational learning and memory (Jasarevic et al., 2011); whereas, performance of exposed females was comparable to control females. In contrast, Sprague-Dawley female but not male rats developmentally exposed to BPA exhibited impairment in this behavior (Johnson et al., 2015). Exploratory behavior is reduced in BPA-exposed female California mice (Peromyscus californicus) (Williams et al., 2013), who rely on this behavior to locate food sources for their young.
To verify that endogenous or exogenous hormones alone can impact brain sexual differentiation, the behavioral effects of these chemicals should be tested in a species lacking sex chromosomes (e.g. painted turtles). While many turtles exhibit TSD instead of sex-chromosomal dependent gonadal sexual differentiation, there are similarities in the brain organization, such as the hippocampus, between the turtle/archosaur lineage and mammals (Striedter, 2015). During the temperature sensitive period (TSP) of embryogenesis, activity of brain aromatase, the enzyme that converts testosterone to estrogen, rises in potential female turtles relative to presumptive males and then declines prior to hatching (Willingham et al., 2000). This pattern of expression suggests that elevated brain estrogen concentrations during this critical window of time may orchestrate neurobehavioral programming.
The objective of the current study was to determine whether painted turtles incubated at the male-inducing temperature and exposed to BPA or EE2 would demonstrate changes in learning and memory compared to non-exposed, control males. To test for such effects, treated and control individuals were tested as juveniles with a spatial behavioral test designed for turtles (López et al., 2001). This semi-aquatic species has previously been shown to possess excellent spatial memory abilities (Bowne and White, 2004, Bowne, 2008, Krochmal et al., 2015, Petrillo et al., 1994, Roth and Krochmal, 2015, Roth and Krochmal, 2016).
Section snippets
Incubation of Turtle Eggs and Distribution of Treatments
Recently laid painted turtle eggs (203) were purchased from Louisiana Cypress Turtle Farms (Pierre Part, LA). The eggs were maintained and transported in clutches as laid. Beforehand, an import permit was obtained from the Missouri Department of Conservation (MDC), and in June 2014, eggs were transported by automobile from Louisiana to Columbia, Missouri. The experiments were performed in accordance with the NIH guidelines for the care and use of laboratory animals and under the approved MU
Total Distance Traveled and Velocity
For distance traveled, there was a significant interaction between treatment and testing stages (p = 0.009). On the late trial days, BPA High animals traveled less distance than Control and EE2-exposed turtles (p ≤ 0.05, Fig. 2A). For velocity, there were no main or interaction effects for treatment and testing stages (treatment*testing stages, p = 0.7, Fig. 2B).
Number of Entries into the Correct Target Zone and Latency
There was a significant two-way interaction (treatment*testing stages) for the number of times spent in the correct target zone (p = 0.02). On
Discussion
The primary goal of this study was to determine if in ovo exposure of painted turtles to BPA or EE2 would exhibit changes in spatial learning and memory relative to unexposed controls. We and others have previously shown that turtle eggs incubated at the male-inducing temperature that are developmentally exposed to EDCs are partially to fully sex-reversed to females (Bergeron et al., 1999, Bull et al., 1988, Clairardin et al., 2013, Crews et al., 1991, Crews et al., 1995, Crews et al., 1996,
Funding
This project was funded by the Mizzou Advantage Program, the Bond Life Sciences Center, and the University of Missouri Office of Research. One of the coauthors (SJB) was supported by an endowment from IDEXX-BioResearch.
Acknowledgements
The authors are grateful to Roger Meissen (MU Bond Life Sciences Center) for the photographic images and supplementary video. The authors also express gratitude to Wayne Shoemaker, Leon Toebben, and Danny Patterson who constructed the maze and associated apparatuses.
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