An Examination of the Effect of Estrogen on Neural and Behavioral Development in African Clawed Frogs (Xenopus laevis)

AbstractAn Examination of the effects Estrogen on Neural and Behavioral Development in African Clawed Frogs (Xenopus laevis)/nAlthough there is strong evidence that estrogens (E) play an important role in sexual differentiation of behavior in mammals, birds, and amphibians, where and how they act in the brain is still unclear. Estradiol (E2) may masculinize the brain during development, but in adulthood, exposure to E2 in males, de-masculinizes adult copulatory behavior. Most researchers who examined exogenous compounds and amphibian sexual and reproductive behaviors have focused on the impacts that these environmental compounds have on the animal’s physiology, morphology, and adult sexual behaviors. Few scientists have examined the molecular and neural mechanisms that underlie the hormonal changes that induce adult morphology and sexual behavior in amphibians. To address these gaps in the literature, I used exogenous compounds known to affect E2 synthesis and activity to understand how changes in E2 during development induce morphological changes in the brain that may influence adult sexual behaviors in amphibians. Chapter 1 provides a general review of how same-sex behavior is observed and analyzed in literature and provides an overview of previous research conducted on the effects of E2 during development to affect adult sexual behavior. In Chapter 2, I examined the effects of atrazine (ATZ), a chemical that induces aromatase --the enzyme that converts testosterone (T) into E2-- on sexual behavior and reproductive function in male African clawed frogs (Xenopus laevis). Tadpoles generated from breeding lab-bred X. laevis from two populations (San Diego [SD] and Golden Gate [GG]) were exposed throughout development to ATZ Sex ratios, E2 and T concentrations, and reproductive behavior were examined. Four percent of males exposed ATZ were sex-reversed. Males in the ATZ-treated group that were not sex-reversed displayed same-sex behavior in which males allowed other males to clasp them. Males that allowed other males to copulate with them (males on bottom) had elevated plasma E2 concentration and a higher E2 to T concentration relative to the males on top. In addition, males that displayed female typical behavior (bottom) were larger in size compared to males on top. Same-sex behavior in ATZ-exposed males is likely due to increased plasma E2 during development. Together, these results suggest that changes in E2 concentration during development can induce same-sex behavior in exposed animals. In chapter 3, I examined sexually dimorphic areas in the brain of adult X. laevis. I used immunohistochemistry (IHC) to analyze oxytocin (OT) and vasotocin (AVT) cells in the brains of control males, control females, and ATZ-exposed males. I sought to determine whether changes in E2 concentration due to ATZ-exposure during development could induce sexually dimorphic changes in OT/AVT cell number and/or distribution in the brain. Control males had twice the number of AVT cells throughout the brain compared to control females. ATZ-exposed males did not significantly differ in AVT cell number compared to control males. IHC for OT in the brain was unsuccessful. In addition to examining OT/AVT cells in the brain, I also analyzed the vocalization, contractile function of the laryngeal muscles, and fictive calls in females, control males, and ATZ-treated males. Although click rates and laryngeal fused tension differed between females and males (female laryngeal muscle fused tension at approximately 20 Hz whereas males laryngeal muscle fused tension at 70 Hz), click rates and laryngeal fused tension (both at 70 Hz) did not significantly differ between control males and ATZ-treated males. These results suggest that ATZ does not affect the vocal nuclei in the brain or laryngeal muscles in ATZ-exposed males. In chapter 4, I used compounds that affect E2-synthesis and activity in adult female X. laevis as tools to understand how changes in E2 during develop can affect adult sexual behavior and reproduction. In this chapter I exposed ovariectomized and sham-operated female X. laevis to tamoxifen (TAM), a compound that can act as an E2 antagonist or agonist depending on the tissues, miconazole (MIC), an aromatase inhibitor, ATZ (which induces aromatase), and ethinylestradiol (EE2), a synthetic estrogen. Ovariectomy significantly reduced plasma E2 and vitellogenin (VTG) expression. Treatment with MIC (3 ppb and 300 ppb) did not affect plasma E2 and VTG expression compared to control in females after four weeks of exposure. ATZ-exposed females had increased E2 and VTG compared to control females. EE2-treated (20 ppb) females had decreased plasma E2 and VTG expression compared to control females. TAM significantly increased plasma E2 concentration but significantly decreased VTG expression in exposed females. TAM did not induce significant changes in plasma E2 and VTG expression in OVX TAM-treated females. TAM, ATZ, EE2, nor MIC affected oviductal morphology. Lastly, in chapter 5, I provide a summary of the results and potential future studies for the experiment conducted. Chapter 5 also discusses the potential impact of my results on the field regarding how we examine sexual differentiation of the brain and sexual behaviors. Finally, this chapter concludes with implications and final thoughts on how society should rethink the view that sex is a binary construct, and explore without bias the diversity of sexuality.

Advisor: Hayes, Tyrone B