Hormonal Regulation of Prostaglandin F2α Receptor Expression: Identifying Mechanisms of Female Reproductive Behavior in a Cichlid Fish Anusha Kumar May 2014 HORMONAL REGULATION OF PROSTAGLANDIN F2α RECEPTOR EXPRESSION: IDENTIFYING MECHANISMS OF FEMALE REPRODUCTIVE BEHAVIOR IN A CICHLID FISH An Honors Thesis Submitted to the Department of Biology in partial fulfillment of the Honors Program STANFORD UNIVERSITY by Anusha Kumar May 2014 ! 2 Acknowledgements I would like to thank my postdoctoral mentor Scott Juntti for guiding me through the experimental design process, for overseeing my use of molecular biology techniques, and for providing invaluable suggestions as I analyzed my data. I also thank postdoctoral fellow Mariana Jimenez for helping me develop the ovariectomy procedure I make extensive use of in this study. Special thanks to my PI Russell Fernald, for providing me continuing support and a friendly lab environment to conduct my research. I will treasure the memories from these four, stellar years, Fernald Lab. Also, many thanks to all my readers, Scott Juntti and Professors Russ Fernald and Craig H. Heller, who helped me edit and revise this document. Finally, this project would not have been possible with out the generous funding provided by the UAR Major Grant, for the 2013-2014 year. ! 4 Table of Contents ABSTRACT............................................................................................................7! INTRODUCTION..................................................................................................8! METHODS .......................................................................................................... 11! RESULTS............................................................................................................. 16! DISCUSSION ...................................................................................................... 18! REFERENCES..................................................................................................... 23! FIGURES ............................................................................................................. 26! ! ! ! ! 5 List of Figures Figure 1. Ovarian hormone cycle in the A. burtoni female Figure 2. Timeline for hormonal and vehicle injections of OVX A. burtoni Figure 3. Identification and confirmation of DHP receptor (PR) sequence in A. burtoni genome Figure 4. Ptgfr expression in the POA of OVX+DHP treated female A. burtoni is significantly higher than in OVX females Figure 5. Ptgfr expression in the POA of OVX A. burtoni females Figure 6. PR expression in the female A. burtoni brain is localized in two areas, the POA and NLT Figure 7. Overlapping Ptgfr and PR expression in the POA of female A. burtoni ! 6 Abstract Several hormones work in concert to mediate female reproduction, a behavior that drives evolution. Previous studies in mammals have identified the ovarian steroids estradiol and progesterone as hormones that act together to induce female mating. In other organisms, the identity of and relationship between reproductive hormones can vary, and this divergence is informative from an evolutionary and molecular standpoint. For example, though ovarian steroids are involved in the reproduction of teleost fish, the post-ovulatory hormone prostaglandin F2α (PGF2α) is sufficient to induce spawning (mating) behavior in females of many teleost species. PGF2α thus activates a distinct, important pathway for reproduction. However, the regulation of this pathway has yet to be defined. Here, we sought to address whether and how ovarian steroids regulate PGF2α -mediated female reproductive behavior. To do so, we used the African cichlid fish Astatotilapia burtoni, a model organism for the study of social behaviors and neural plasticity. In female A. burtoni, the PGF2α receptor (Ptgfr) is expressed in a behaviorally relevant brain region, the preoptic hypothalamus (POA), and increases expression during spawning. This spatio-temporal expression pattern implicates the POA as a locus of PGF2α signaling during reproductive behaviors. We investigated the mechanism of PGF2α-mediated spawning by searching for the regulators of POA Ptgfr expression in female A. burtoni. We hypothesized that ovarian steroids account for the observed variation in Ptgfr mRNA expression. Using in situ hybridization, we demonstrated upregulated POA Ptgfr expression in ovariectomized females treated with teleost progesterone 17α,20β- dihydroxy-4-pregnen-3-one (DHP). We further demonstrated that DHP receptors are expressed in the POA, suggesting DHP directly regulates Ptgfr expression in that region. These results are consistent with a model where DHP-regulated PGF2α signaling mediates reproductive behavior. This model can help identify conserved PGF2α pathways in other vertebrates or track the diversification of PGF2α’s reproductive role over evolutionary time. Key Words: Biology, prostaglandin F2α (PGF2α), receptor, social behavior, reproductive regulation, teleost, 17α,20β-dihydroxy-4-pregnen-3-one (DHP) ! 7 Introduction Reproductive behaviors comprise an important subset of social behaviors (1). These behaviors are key substrates upon which selection pressures act to maximize reproductive success (28); they are a vehicle for adaptation and drive evolution. Reproductive behaviors arise from signaling through multiple pathways, which integrate environmental and internal cues to activate hardwired neural substrates (1). Many of the molecules that constitute these reproductive pathways are conserved across species. Even when reproductive molecules and signaling pathways differ between organisms, this divergence is informative from an evolutionary standpoint, helping us understand the mechanisms by which molecules and neural circuits accommodate adaptive behaviors. Some progress has been made in elucidating the control of reproductive behaviors, since some of the neural circuitry involved in reproduction feeds into a conserved hypothalamic-pituitary-gonad (HPG) axis. The HPG axis produces several steroid hormones (e.g. estrogen, testosterone, etc.) that have been established as mediators of gonadal and gametic maturation (2-4). Through considerable research in mammals, many of these same hormones have since been identified as molecular effectors of reproductive behavior. In particular, estrogen and progesterone elicit lordosis (female sexual behavior) in mice, thereby implicating these hormones as the key translators of reproductive state to sexual and receptive behaviors (2-4). Nonetheless, the precise mechanisms by which these steroid hormones signal in the brain and influence reproductive behaviors have yet to be determined. These hormones could act through several pathways, in conjunction with many other hormones, to mediate the different stages of sexual behavior. Extending reproductive research to other vertebrates, such as teleost fish, will broaden the understanding of these various hormonal mechanisms and better define evolutionarily conserved or divergent pathways controlling reproductive behaviors. To this end, we have chosen to use a social behavioral model system, the African cichlid Astatotilapia burtoni, to investigate the mechanisms of reproductive behavior. This teleost species lends itself to such investigations for a variety of reasons. A. burtoni requires only simple maintenance and has a relatively short generation time. Moreover, unlike other traditional model organisms that suffer from a stunted repertoire of ! 8 reproductive behaviors in laboratory housing, A. burtoni continues to display a rich suite of well-defined social behaviors (6). In addition, molecular investigations in this species have been aided in recent years by the sequencing of the A. burtoni genome. Thus, behavioral, neural, and molecular changes that proceed or follow changes in reproductive state can be readily assayed using A. burtoni. The post-ovulatory hormone prostaglandin F2α (PGF2α) plays a critical role in regulating reproductive behaviors in teleosts including A. burtoni. In female fish, ovulation triggers synthesis of PGF2α, which in turn activates spawning behavior (7,8). Spawning in A. burtoni comprises several stereotyped mating behaviors, including male courtship, female following into the male’s territory, egg laying, and female mouth- brooding after fertilization (9). Thus, in teleosts, PGF2α appears to be a key translator of internal state (the occurrence of ovulation) into outward reproductive behavior, a role associated with steroid hormones in mammals. Functional tests confirm that this postovulatory PGF2α acts as a spawn-inducing hormone in several teleosts (5, 10). In both goldfish and A. burtoni, treating non- reproductive females with PGF2α is sufficient to induce spawning behaviors (11, 12, 5). The necessity of PGF2α for such behaviors was further confirmed in goldfish by treating mature, gravid (post-ovulation) females with a prostaglandin synthesis inhibitor, which prevented the females from displaying spawning behaviors (12). However, the neural and molecular mechanisms by which PGF2α stimulates reproductive behaviors has yet to be demonstrated. As a step towards bridging this gap, recent work identified the PGF2α receptor (Ptgfr) in A. burtoni (5). Ptgfr expression is localized to four brain areas: the ventral telencephalon (Vd-c), the preoptic area (POA), the lateral tuberal nucleus (NLT), and the vagal lobe (VL) (5). Of these areas, the POA has been identified as a crucial node in the mating neural circuit. Evidence