* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download GnRH Protein Levels in Atrazine-Treated Axolotls
Survey
Document related concepts
Causes of transsexuality wikipedia , lookup
Biochemistry of Alzheimer's disease wikipedia , lookup
Aging brain wikipedia , lookup
Central pattern generator wikipedia , lookup
Trans-species psychology wikipedia , lookup
Environmental enrichment wikipedia , lookup
Nervous system network models wikipedia , lookup
Premovement neuronal activity wikipedia , lookup
Synaptic gating wikipedia , lookup
Feature detection (nervous system) wikipedia , lookup
Pre-Bötzinger complex wikipedia , lookup
Clinical neurochemistry wikipedia , lookup
Neuropsychopharmacology wikipedia , lookup
Hypothalamus wikipedia , lookup
Neuroanatomy wikipedia , lookup
Optogenetics wikipedia , lookup
Transcript
Page 1 of 8 Impulse: The Premier Journal for Undergraduate Publications in the Neurosciences 2008 GnRH Protein Levels in Atrazine-Treated Axolotls (Ambystoma mexicanum) Sara Nienaber and Sarah Leupen Department of Zoology, Ohio Wesleyan University, Delaware, OH, 43015 Atrazine is the most widely used agricultural herbicide in the United States and a known endocrine disruptor. In amphibians, it has been shown to cause gonadal malformations, feminization of males, behavioral changes, and immune suppression; however, its mechanism of action is unknown. We hypothesized that atrazine reduces the production of gonadotropin releasing hormone (GnRH) in the hypothalamus. Axolotls (Ambystoma mexicanum) were exposed to atrazine, 10-8 M ß-estradiol-3-benzoate, or no treatment and were sacrificed at 6, 8, and 10 months of age. GnRH neurons were labeled using immunocytochemistry, and labeled neurons were then counted using confocal microscopy. Although no significant difference was found in the total number of GnRH neurons, ectopic GnRH expression was seen in some brains. A significant negative correlation was found between presence of ectopic GnRH and number of normal GnRH neurons. Atrazine-treated animals were more likely than control or estrogentreated animals to have ectopic GnRH expression. The data implicate a central site of action of atrazine. Keywords: amphibians, herbicides, endocrine disruption, hypothalamic-pituitary-gonadal axis, aromatase, estradiol Introduction Atrazine is the most widely used agricultural herbicide in the world, with more than 60 million lbs used annually in the United States alone (Hayes et al., 2002). Atrazine was long considered safe because it is not as directly toxic as many other agricultural chemicals. More recently, however, atrazine has been characterized as an endocrine disrupting compound (EDC): a chemical that mimics the action of a hormone, often at very low doses (Hayes et al., 2002). Many studies have focused on the effects of atrazine on amphibians. Due to their partially aquatic lifestyle and environmental sensitivity, these animals are especially vulnerable to contaminants in their habitat (Rohr and Palmer, 2005). Many amphibian breeding seasons occur in the spring, when agricultural chemical application is at its peak, making young, developing amphibians even more susceptible to the effects of endocrine disruptors (Freeman et al., 2005). The Environmental Protection Agency sets the maximum amount of atrazine allowed in drinking water at 3 ppb (Brodkin et al., 2007). However, malformations and abnormalities in amphibians have been seen at as low as 0.10 ppb (Hayes et al., 2002). Abnormalities in amphibians following exposure to atrazine include hermaphroditism and other gonadal abnormalities (Coady et al., 2005; Hayes et al., 2002; 2006), changes in testosterone levels (Hecker et al., 2005), poorly developed larynxes in male frogs (Coady et al., 2005; Hayes et al., 2006), hyperactivity and other behavioral changes (Rohr and Palmer, 2005), and immune system suppression (Brodkin et al., 2007; Houck and Sessions, 2006). Some of these malformations appeared a year or more after exposure had ceased (Rohr et al., 2006). Because of atrazine’s effects, it, along with other EDCs, has been named a factor in the global decline of amphibian populations (Rohr and Palmer, 2005). Atrazine’s mechanism of endocrine disruption is unknown. Because it raises levels of circulating estrogens in all animals studied so far, it has been hypothesized that activation of the enzyme aromatase, which converts testosterone into estrogen, may be its Page 2 of 8 GnRH Protein Levels in Atrazine Treated Axolotls (Ambystoma mexicanum) 2008 mechanism of action (Hayes et al., 2006). However, several studies have shown no effect of atrazine on aromatase (Hecker et al., 2005; Murphy et al., 2006). Though possible mechanisms of endocrine disruption, such as aromatase activation, have been previously investigated, few studies have focused on the central effects of atrazine and other endocrine-disrupting compounds. Gonadotropin Releasing Hormone (GnRH) is the master hormone of the reproductive axis in all vertebrate animals. Its pattern and level of release from the hypothalamus dictate the activity of the entire system. As neuroendocrine controllers, these GnRH neurons receive not only direct feedback from gonadal steroids as well as pituitary gonadotropins, but also an array of sensoryderived neural information communicating the season (Doer-Schott and Dubois, 1978; Ralph, 1978), the metabolic state (Schneider, 2004; Wade and Schneider, 1992) and immune status of the animal (e.g. Bouret et al., 2004; Igaz et al., 2006), physiological stress (e.g. Moore and Zoeller, 1985), presence of potential mates (Boehm et al., 2005; Propper and Moore, 1991), and other factors. We hypothesized that atrazine exposure may reduce the production of GnRH in the hypothalamus of developing amphibians, resulting in disrupted steroidogenesis and sexual development. We chose the axolotl, Ambystoma mexicanum, as our model organism because its long history as an experimental amphibian provides many well-established laboratory protocols, and because it is large, hardy, and aquatic throughout its lifecycle. Materials and Methods Animal Care The following protocols were approved by Ohio Wesleyan University's Institutional Animal Care and Use Committee. At four months of age, axolotls (Ambystoma mexicanum) were divided into three groups of 20 animals each: control, atrazine-treated, and estradiol-treated. These animals are neotenic salamanders, which means that they remain aquatic through their entire lifecycle, and never metamorphose into terrestrial adults. Four months of age was chosen as the starting point for this study because we wanted to begin atrazine exposure during development, while GnRH-producing neurons were being formed. The control group was raised in 40% Holtfreter’s solution, a slightly saline solution that is the standard bowl water for axolotl care. A second group of animals were raised in 3 ppb atrazine (Supelco, Bellefonte, PA) in 40% Holtfreter’s solution, which corresponds to the maximum amount of atrazine allowed in drinking water by the EPA. Because atrazine has been shown to elevate estrogen levels, possibly by inducing aromatase, a third group was raised in 10-8 M ß-estradiol-3-benzoate (Sigma Aldrich, St. Louis, MO) in 40% Holtfreter’s solution. If atrazine’s effects are mediated in full by increased estrogen levels, similar effects should be seen in atrazine- and estradiol-treated animals. By contrast, if atrazine has effects independent of estrogen activation, these effects ought to be revealed by direct comparison of atrazine- and estradiol-treated groups. Animals were housed in 2-L plastic containers and fed salmon pellets ad libitum. Treatment water was changed as needed. Axolotls were initially housed three to a bowl, then slowly transitioned to living in pairs, and then alone. This shift was necessary as they grew and in some cases in response to cannibalistic behavior, which occurs frequently in juvenile animals of this species. By eight months of age, all animals were living alone in 2-L containers. Arbitrarily chosen subsets of animals, representing small, medium, and large specimens from each group, were sacrificed by decapitation at 6, 8, and 10 months of age, respectively. Previous, unpublished observations from our lab indicate that GnRH neurons are formed in most juvenile axolotls between 6 and 10 months of age. Thus, this range of ages was chosen as the ideal range to reveal potential differences in GnRH development among the groups. The heads were preserved in 4oC 4% paraformaldehyde, as were the weighed gonads of animals 8 and 10 months of age; gonads of animals sacrificed at six months of age were not Page 3 of 8 Impulse: The Premier Journal for Undergraduate Publications in the Neurosciences 2008 sufficiently differentiated from associated renal tissue to be collected. fraction of body weight) could be calculated for each animal. Brain Preparation After at least 24 hours of fixation in paraformaldehyde at 4oC, brains were removed from the heads and set in small blocks of 5% agarose (Fisher Chemical, Fair Lawn, NJ). Brains were sliced with a vibrating microtome (Vibratome Co., St. Louis, MO) into 80 µm slices. The slices were then collected into sixwell plates in phosphate buffered saline (PBS), pH 7.2. Immunocytochemistry was then performed as follows to identify the GnRH neurons. Slices were blocked in 1% goat serum in PBS for 30 minutes, and GP6 primary antibody (generously donated by Lothar Jennes) was added at a concentration of 1:500 and incubated for 72 hours at 4oC. Samples were then rinsed three times with PBS plus 0.3% triton (PBS-T), and Biotin SP-conjugated goat anti-guinea pig secondary antibody (Jackson ImmunoResearch, West Grove, PA) was added for two hours at a 1:500 concentration. After three further rinses in PBS-T, samples were incubated in fluorescein DTAF Streptavidin (Jackson ImmunoResearch) at 1:500 for one hour. After rinsing samples three times with PBS-T, brain slices were mounted onto slides and confocal microscopy was used to count the number of GnRH neurons in each brain. Slides were coded with neutral names, so that the person counting the neurons was blind to experimental group until the completion of the study. The number of normal GnRH neurons in each section was counted, and any abnormalities in GnRH expression were also recorded. Minitab and Excel were used for calculations of t-tests and analysis of variance (ANOVA) when appropriate. Results The GnRH signal was more complex than expected. In addition to normal GnRH neurons (Fig 1, top), a subset of brains displayed additional specific fluorescent labeling (“ectopic” GnRH expression; (Fig 1, bottom). Normal GnRH neuron Ectopic GnRH Figure 1: Digital images of GnRH fluorescence in axolotl brains. Top, a normal GnRHproducing neuron; bottom, ectopic GnRH expression. Other measurements Monthly weights were recorded for each animal. At the eight and 10 month sacrifice dates, gonads were also removed from and weighed for each animal sacrificed so that the gonadosomatic index (GSI; gonad weight as a Figure 2: Proportion of animals per group with ectopic GnRH expression. Ectopic GnRH was seen in animals of all treatment groups, but with greatest frequency in the group of axolotls treated with atrazine (p=.005 vs. control, p=0.03 vs. estradiol). No previous literature mentioned the presence of ectopic GnRH which was surprising considering the frequency of this occurrence among our experimental animals. Though it may have been useful to somehow quantify the areas of ectopic GnRH, there was no satisfying Page 4 of 8 GnRH Protein Levels in Atrazine Treated Axolotls (Ambystoma mexicanum) 2008 Differences in proportion of animals displaying ectopic GnRH were calculated using a z-test for proportions. As indicated in Figure 2, ectopic GnRH expression was predominantly seen in atrazine-treated animals (11 of 14), significantly more so than in control (2 of 12, p=0.003) or estradiol-treated (3 of 9, p=0.04) animals. There was no significant difference between estradiol-treated and control animals. 100 25 Normal GnRH Neurons (mean number/individual) or objective way to do so, because ectopic GnRH usually occurred in branching streaks or overlapping blocks, rather than being separated into distinct elements. 20 15 10 5 0 Control Estradiol Atrazine Figure 3: Mean number of normal GnRH neurons per axolotl brain for control, estradiol-treated, and atrazine-treated groups. There were no significant differences among the groups (p=0.10). 80 70 60 50 40 30 20 10 0 Control Estradiol Atrazine Figure 2: Proportion of animals per group with ectopic GnRH expression. Ectopic GnRH was seen in animals of all treatment groups, but with greatest frequency in the group of axolotls treated with atrazine (p=.005 vs. control, p=0.03 vs. estradiol). A one-way ANOVA was performed to compare the number of normal GnRH neurons across treatment groups. There was no significant difference in mean number of normal GnRH neurons per axolotl among treatment group(s) (Figure 3, Fdfb,dfw=32, p=0.10); although a trend is apparent, it is not statistically significant, due to the large variation in number of neurons observed within groups. A one-way ANOVA was performed to compare the number of normal GnRH neurons in brains with and without ectopic GnRH. As is indicated in Figure 4, there were significantly fewer GnRH neurons in those brains with ectopic GnRH than those without, regardless of treatment group (Fdfb,dfw=33, p=0.02). 20 18 16 Normal GnRH Neurons (total number) % Animals with Ectopic GnRH 90 14 12 10 8 6 4 2 0 Brains with Ectopic GnRH Brains without Ectopic GnRH Figure 4: Brains expressing ectopic GnRH had fewer normal GnRH neurons (p=0.02) than those without ectopic GnRH. Repeating this ANOVA analysis using only atrazine-treated animals, the same pattern is revealed, though the groups are not significantly different (Fdfb,dfw=12, p=0.10). Page 5 of 8 Impulse: The Premier Journal for Undergraduate Publications in the Neurosciences 2008 Normal GnRH Neurons (total number) 20 18 16 14 12 10 8 6 4 2 0 Brains with and without ectopic GnRH Figure 5: Brains expressing ectopic GnRH had fewer, though not significantly (p=0.10), normal GnRH neurons than those without ectopic GnRH. Due to the great variation in size of axolotls of the same age which normally occurs, no difference was seen in the weight of animals in each treatment group over the course of this study (in all monthly comparisons p=NS). Because gonadal abnormalities have been seen regularly in male amphibians exposed to atrazine (ex. Hayes et al., 2002), gonadosomatic index was calculated for each animal sacrificed at eight and ten months of age. Prior to this age, most gonadal tissue was too poorly differentiated from renal tissue to be measured accurately. No differences were seen among the gonadosomatic indices of the treatment groups. Full characterization of gonadal morphology will require histological work with the isolated gonadal tissue. Discussion An underappreciated danger of EDCs is that they defy the basic principles of toxicology; larger doses do not necessarily produce greater harm (Hayes et al., 2002). Rather, minute amounts of a compound can have dire consequences for an organism. Many of these compounds, like atrazine, produce few if any outward malformations, so a chemical may be on the market for decades before it is connected to a problem such as global amphibian population declines. With so many chemicals on the market and many more being introduced annually, it is impossible to monitor the safety of each compound, especially if compounds are assumed to be safe until proven otherwise. It is important to understand the mechanisms by which chemicals such as atrazine act as endocrine disruptors, so that newly developed chemicals may be designed to minimize endocrine disruption. GnRH is an important target to examine as a possible site of endocrine disruption, since endocrine disruptors (including atrazine) have been shown to cause sexual malformations, immune disruption, and behavioral changes, all of which could conceivably be downstream of changes in GnRH release, and more directly, because gross abnormalities in reproductive function are likely to be attributable to a disruption “high” within the reproductive axis. The most surprising result of this study was the occurrence of ectopic GnRH, which was previously undocumented. Our results indicate that the presence of ectopic GnRH occurs rarely in axolotls under normal conditions. However, exposure to chemicals such as atrazine may increase the likelihood of this occurring. The presence of ectopic GnRH was associated with a reduced number of normal GnRH neurons, which may lead to changes in gonadotropin synthesis and release, endogenous sex steroid production, gametogenesis, and sexual behavior. Since the presence of ectopic GnRH was a surprise finding in this study and has not previously been documented, it may be beneficial to design further studies to investigate what this ectopic GnRH actually is, how it forms, and how to quantify its production. A challenge of this study was that the amount of ectopic GnRH could not be easily measured, because it occurs in streaks rather than welldefined cell bodies. In the future, other methods of measuring GnRH levels could be used to quantify GnRH expression or release. One such method is an ex vivo assay, which has previously been used to measure GnRH release in amphibians (Tsai, Moenter, and Cavolina, 2003). This protocol involves hypothalamic perfusion and frequent sampling in order to assay the GnRH release of the tissue. An advantage of this method is that it measures GnRH release directly, rather than protein levels, which may or may not be directly Page 6 of 8 GnRH Protein Levels in Atrazine Treated Axolotls (Ambystoma mexicanum) 2008 correlated with release. However, this method does not allow for observations of the anatomical pattern of GnRH expression, the presence of ectopic GnRH, or the existence of other abnormalities at the cellular level. Though this experiment began with 20 individuals in each group, cannibalism of bowlmates reduced the number of individuals in each group. Despite the fact that it may be more labor intensive, in the future it may be useful to house animals individually from the beginning, to prevent cannibalism. It is possible that atrazine affects the GnRH expression of males differently than females. Indeed, sexual malformations in response to atrazine have thus far only been found in male amphibians. We would ideally like to examine our data separately by sex. However, many of our animals were too young or too early in sexual development to accurately determine sex, and genetic or chromosomal sex determination methods for axolotls do not exist. In future studies, it may be beneficial to raise all specimens into adulthood, and divide the animals up by sex to process the data. Of course, further perturbations of the hypothalamic-pituitary-gonadal axis may also occur in older animals, or conversely, earlier perturbations may diminish. This study did not establish atrazine’s mechanism of action as an EDC, but the data are consistent with an aromatase-inducing or other estrogen-stimulating action. Estradiol treatment was expected to inhibit GnRH expression and therefore lower the number of countable GnRH neurons, through physiological negative feedback in the hypothalamo-pituitary-gonadal axis. Although it did not statistically significantly do so, the similar number of mean GnRH neurons in the estradiol- and atrazinetreated groups is consistent with this interpretation. Importantly, independent of the “aromatase question”, which predicts that atrazine exposure up regulates aromatase, and therefore conversion of testosterone to estradiol, this is the first study to demonstrate that atrazine may effect the morphology of the central nervous system. The significance of ectopic GnRH expression is unclear. The morphology suggests induced GnRH production by endothelial cells in cerebral blood vessels. This may represent a generalized stress or immune response, which could be induced or exacerbated by exposure to a stressor such as a chemical pollutant. However, it is also possible that the induced peptide is not GnRH, but rather another peptide which is cross-reacting with the GnRH antibody. No previous studies have reported cross-reacting of the antibodies used in this experiment, so it is assumed that the likelihood of this occurring is low. It is important to delineate the developmental window of harm by exposure to atrazine in developing amphibians. What dose, for what duration, at what time in development, maximizes (or minimizes) central and peripheral reproductive disruption, and by what mechanism? Future work will prioritize these questions. Acknowledgements We would like to thank the Support of Mentors and their Students in the Neurosciences (SOMAS) for their funding of this work, as well as the Ohio Wesleyan University Summer Science Research Program (SSRP) for their support of this project. We are very grateful to Lothar Jennes for the generous donation of the GP6 primary antibody, and to Katie Ayers and Tov Nordbo for their assistance in animal care over the course of this project. Corresponding Author Sara Ann-Marie Nienaber Ohio Wesleyan University Email: [email protected] Sara Nienaber HWCC Box 1931 Delaware, OH 43015 Page 7 of 8 Impulse: The Premier Journal for Undergraduate Publications in the Neurosciences 2008 References Boehm U, Zou Z, Buck LB. (2005). Feedback Loops Link Odor and Pheromone Signaling with Reproduction. Cell. 123, 683-695. Bouret S, De Seranno S, Beauvillain JC, Prevot V. (2004). Transforming growth factor beta1 may directly influence gonadotropin-releasing hormone gene expression in the rat hypothalamus. Endocrinology. 145, 1794-1801. Brodkin MA, Madhoun H, Rameswaran M, Vatnick I. (2007). Atrazine is an immune disruptor in adult northern leopard frogs (Rana pipiens). Environ Toxicol Chem. 26, 80-84. Coady KK, Murphy MB, Villeneuve DL, Hecker M, Jones PD, Carr JA, Solomon KR, Smith EE, Van Der Kraak G, Kendall RJ, Giesy JP. (2005). Effects of atrazine on metamorphosis, growth, laryngeal and gonadal development, aromatase activity, and sex steroid concentrations in Xenopus laevis. Ecotoxicol Environ Saf. 62, 160-173. Doerr-Schott J and Dubois MP. (1978). Immunohistochemical Localisation of Different Peptidergic Substances in the Brain of Amphibians and Reptiles. In ‘Comparative Endocrinology’ (Eds. PJ Gaillard and HH Boer) p 367-370. Elsevier/North Holland Biomedical Press, Amsterdam. Freeman JL, Beccue N, and Rayburn AL. (2005). Differential metamorphosis alters the endocrine response in anuran larvae exposed to T3 and atrazine. Aquat Toxicol. 75, 262-276. Hayes TB, Collins A, Lee M, Mendoza M, Noregia N, Stuart AA, Vonk A. (2002). Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proc Natl Acad Sci. 99, 5476-5480. Hayes TB, Stuart AA, Mendoza M, Collins A, Noriega N, Vonk A, Johnston G, Liu R, Kpodzo D. (2006). Characterization of atrazine-induced gonadal malformations in African clawed frogs (Xenopus laevis) and comparisons with effects of an androgen antagonist (cyproterone acetate) and exogenous estrogen (17_estradiol): Support for the demasculinization/feminization hypothesis. Environ Health Perspect. 114, 134-141. Hecker M, Park J, Murphy MB, Jones PD, Solomon KR, Van Der Kraak G, Carr JA, Smith EE, du Preez L, Kendall RJ, Giesy JP. (2005). Effects of atrazine on CYP19 gene expression and aromatase activity in testes and on plasma sex steroid concentrations of male African clawed frogs (Xenopus laevis). Toxicol Sci. 86, 273-280. Houck A and Sessions SK. (2006). Could atrazine effect the immune system of the frog, Rana pipiens?. Bios. 77, 107-112. Igaz P, Salvi R, Rey JP, Glauser M, Pralong FP, Gaillard RC. (2006). Effects of cytokines on gonadotropin-releasing hormone (GnRH) gene expression in primary hypothalamic neurons and in GnRH neurons immortalized conditionally. Endocrinology. 147, 1037-1043. Moore FL and Zoeller RT. (1985). StressInduced Inhibition of Reproduction: Evidence of Suppressed Secretion of LHRH in an Amphibian. Gen Comp Endocrinol. 60, 252-258. Murphy MB, Hecker M, Coady KK, Tompsett AR, Higley EB, Jones PD, du Preez LH, Solomon KR, Carr JA, Smith EE, Kendall RJ, Van Der Kraak G, Giesy JP. (2006). Plasma steroid hormone concentrations, aromatase activities and GSI in ranid frogs collected from agricultural and non-agricultural sites in Michigan. Aquat Toxicol. 77, 153-166. Propper CR and Moore FL. (1991). Effects of courtship on brain gonadotropinreleasing hormone and plasma steroid concentrations in a female amphibian ( Taricha granulosa) Gen Comp Endocrinol. 81, 304-312. Ralph CL. (1978). Pineal control of reproduction: nonmammalian vertebrates. Progress in Reproductive Biology. 4, 30-50. Page 8 of 8 GnRH Protein Levels in Atrazine Treated Axolotls (Ambystoma mexicanum) 2008 Rohr JR and Palmer BD. (2005). Aquatic herbicide exposure increases salamander desiccation risk eight months later in a terrestrial environment. Environ Toxicol Chem. 24, 1253-1258. Rohr JR, Sager T, Sesterhenn TM, Palmer BD. (2006). Exposure, postexposure, and density-mediated effects of atrazine on amphibians: breaking down net effects into their parts. Environ Health Perspect. 114, 46-50. Schneider JE. (2004). Energy balance and reproduction. Physiol Behav. 81, 289317. Tsai PS, Moenter SM, and Cavolina JM. (2003). Development of a novel enzyme immunoassay for the measurement of in vitro GnRH release from rat and bullfrog hypothalamic explants. Comp Biochem Physiol A Mol Integr Physiol. 136, 693-700. Wade GN and Schneider JE. (1992). Metabolic Fuels and Reproduction in Female Mammals. Neurosci Biobehav Rev. 16, 235-272.