Phytoestrogens are endocrine disruptors produced by plants that are analogous to estrogens produced by animals. Given this close analogy, phytoestrogens can mimic estrogen action in the bodies and brains of humans or non-human animals. This project will systematically characterize the effects of three representative phytoestrogens on normal behavior patterns of adult male fighting fish Betta splendens, an ideal animal model because their behavior and physiology are relatively well understood and because their neuroendocrinology is similar to that of other vertebrates, including mammals. The project will determine whether observed behavioral changes occur because phytoestrogens bind directly to estrogens receptors localized in the testes and determine whether such phytoestrogen exposure reduces the production of testosterone and 11-ketotestosterone, decreases sperm production, and causes testicular atrophy. Subsequently, the project will determine whether the observed behavioral effects are the direct result of phytoestrogen binding to estrogen receptors in the brain, which could alter brain neurochemistry and affect the production and metabolism of neurotransmitters such as serotonin, dopamine and norepinephrine. This research will contribute to a better understanding of how phytoestrogens affect animal behavior and physiology and indicate how reproduction persists in the face of environmental estrogen exposure. Funding from the NSF will also provide numerous research and training opportunities for undergraduate students at Amherst College.
Intellectual merit Phytoestrogens are plant compounds that can interfere with the normal functioning of the endocrine system in vertebrate animals. Even though they are "natural" compounds, phytoestrogens can occur at unnaturally high levels due to human activity; examples include many aquatic habitats near wood pulp and paper mills, biofuel manufacturing plants, sewage-treatment plants, and agricultural fields. There are many studies of the endocrine-disrupting effects of phytoestrogens in rodents, but relatively few on aquatic vertebrates. Furthermore, few of these studies measure endpoints that are pertinent to reproductive success and the long-term stability of wild animal populations. In our research, we used the fighting fish Betta splendens as a study species. We chose this fish because it is easy to maintain and breed in captivity, its behavior is stereotyped and easy to quantify, and because it suffers from phytoestrogen exposure in the wild. We focused our research on two phytoestrogens, the isoflavone genistein and the phytosterol beta-sitosterol. These compounds were chosen because they are found at elevated levels in nature and because they are some of the most potent and biologically active phytoestrogens. In our first set of studies, we found evidence that phytoestrogens can interfere with the expression of male aggressive behavior (for which this species is particularly well-known) and that this interference may be the result of phytoestrogen action in the brain. More specifically, phytoestrogens were shown to affect the action of the neurotransmitters serotonin, dopamine, and norepinephrine. Separate studies by our research group had confirmed the role of these neurotransmitters in the expression of aggressive behavior in this species. We then focused our attention on the reproductive consequences of phytoestrogen exposure. These experiments used both high (pharmacological) and low (environmentally relevant) doses of the compounds; adult fish were exposed to the test chemical for several weeks and then several reproductive endpoints were measured. These included courtship behavior, gonad size, gonad development, gamete quality, and levels of sex steroid hormones circulating in the bloodstream. For these studies, we extended our work to include adult females as well as adult males, as the effects of phytoestrogens on females have been poorly studied thus far. On the whole, we found that sexually mature adult fish suffered little from phytoestrogen exposure in terms of the parameters listed above. There was some minor evidence of endocrine disruption, but primarily at the pharmacological doses. This does not imply that phytoestrogens do not pose risks for fish populations, but rather than sexually mature fish – whose reproductive system is already fully developed – are relatively immune from such risks. Immature, developing fish are likely to be much more sensitive to endocrine disruption by phytoestrogens. Many of our studies used positive controls, or compounds known or suspected to have measurable effects. We used the synthetic estrogen ethinylestradiol as a positive control, and found that it did indeed have significant, measurable effects on male reproductive performance. In particular, male fighting fish exposed to ethinylestradiol had slower sperm and that the reduced sperm velocity was due to a reduction of adenosine triphosphate (ATP) in the sperm. Given the prevalence of ethinylestradiol in the environment, this finding is significant. Broader impacts Amherst College is an institution that emphasizes teaching and research excellence at the undergraduate level. Therefore, the primary broader impacts are in the training of future scientists. This NSF award provided salary and supplies money for 13 undergraduate researchers, of whom 11 were women and 5 were from groups under-represented in the sciences. Six of these students have already been admitted into top medical or graduate programs, and several others will be applying this year. Their research experience, made possible by this grant, has been a prominent factor in their success thus far. Furthermore, this awarded allowed for the training of one postdoctoral fellow and one graduate student; both were women and one was from a group under-represented in the sciences. In terms of broader impacts within the scientific community, this award has resulted in six scientific papers published or currently in review, as well as several oral and poster papers at scientific meetings.
