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MIAMI UNIVERSITY The Graduate School CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation Of Jill Marie Russell Candidate for the Degree: Doctor of Philosophy Director Phyllis Callahan, PhD Reader James Janik, PhD Reader Paul James, PhD Graduate School Representative Emily Murphree, PhD ABSTRACT THE EFFECT OF STEROIDS ON NEUROENDOCRINE FUNCTION IN IMMATURE RATS By Jill Marie Russell The hypothesis tested was that a developmental difference in the effects of estrogen and progesterone on nitric oxide and dopamine influences the steroid-induced luteinizing hormone and prolactin surges. I investigated the stimulatory effects of steroid hormones (estrogen and progesterone) on luteinizing hormone and prolactin secretion in pre- pubertal (4 weeks), peripubertal (6 weeks) and sexually mature (12 & 16 weeks) female rats. Additionally, the effects of L-arginine, on the steroid induced luteinizing hormone and prolactin secretions were examined in peripubertal female rats. I also investigated the regulatory role of steroid hormones on nitric oxide synthase and tyrosine hydroxylase levels in the hypothalamus of pre-pubertal (4 weeks) and sexually mature (12 weeks) female rats. For each age, intact, ovariectomized and ovariectomized plus steroid replaced females were examined. These studies advanced our understanding of the neural regulation of puberty and cyclicity by examining the regulatory role of ovarian steroids on nitric oxide and dopamine and its regulatory role on puberty and the development of cyclicity in females. Importantly, these studies investigated the changes that occur during pubertal development. THE EFFECT OF STEROIDS ON NEUROENDOCRINE FUNCTION IN IMMATURE RATS A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Zoology by Jill Marie Russell Miami University Oxford, OH 2004 Dissertation Director: Phyllis Callahan, PhD Table of Contents Certificate for Approving the Dissertation Abstract Title page………………………………………………………………………………..i Table of Contents………………………………………………………………………..ii List of tables……………………………………………………………………………..iii List of figures……………………………………………………………………………iv Dedication………………………………………………………………………………..v Acknowledgements………………………………………………………………………vi Chapter 1…………………………………………………………………………………1 Chapter 2…………………………………………………………………………………15 Chapter 3…………………………………………………………………………………39 Chapter 4…………………………………………………………………………………70 ii List of Tables Table 2.1..………………………………………………………………………………..28 iii List of Figures Figure 1.1…………………………………………………………………………………10 Figure 2.1..………………………………………………………………………………..31 Figure 2.2…………………………………………………………………………………32 Figure 2.3…………………………………………………………………………………33 Figure 2.4…………………………………………………………………………………34 Figure 2.5…………………………………………………………………………………35 Figure 2.6…………………………………………………………………………………36 Figure 2.7…………………………………………………………………………………37 Figure 2.8…………………………………………………………………………………38 Figure 3.1.a……………………………………………………………………………….61 Figure 3.1.b……………………………………………………………………………….62 Figure 3.2.a……………………………………………………………………………….63 Figure 3.2.b……………………………………………………………………………….64 Figure 3.3…………………………………………………………………………………65 Figure 3.4.a……………………………………………………………………………….66 Figure 3.4.b……………………………………………………………………………….67 Figure 3.5.a……………………………………………………………………………….68 Figure 3.5.b……………………………………………………………………………….69 iv Dedication This work is dedicated to my family. To Mom & Dad; who persevered and always saw the potential in me. To Vickie, Sam, Tim, Suzie & Gardiner; who took care of me. To Dave; who would not let me quit. To Leslie, Amanda & Paul; for accepting me and enriching my life. To Katlyn and Dylan; who gave my life purpose and motivated me to better myself. You are my inspiration. I could not have accomplished this work without your unending love and support. Thank you. v Acknowledgments To my co-advisors, Phyllis Callahan & Jim Janik, a big thank you. I would not have stayed in graduate school, if you had not taken me into your lab. The level of scientific research in your lab is superior. I learned valuable experimental techniques from you. You have been great mentors and friends. Thank you for keeping me on track and dealing with my disabilities. I know it has not been easy for you, and I appreciate your patience and tutelage. To Paul James, for teaching me how to run Westerns and for keeping me in your lab even after I broke your equipment. To Paul Harding, for your regular words of encouragement and sense of humor. To Emily Murphree, for surviving the brain storming sessions with Phyllis and me. Thanks for running all the stats all these years, and for having patience with us. vi CHAPTER 1 General Introduction Puberty Puberty is a dynamic stage in the mammalian female life cycle, which is typically associated with increases in body size and the development of reproductive function. In the female rat, puberty is characterized by several events including vaginal opening, onset of receptive behavior patterns (soliciting and lordosis), initiation of cyclic pituitary gland hormone secretion, and ovulation (for review see Gore 2001, Ojeda et al. 1994). The hypothalamus is the site of vital neurochemical activity during both the onset of puberty and in the regulation of the adult pattern of cyclic pituitary secretory patterns which stimulate the release of eggs from the ovaries on a consistent basis (Clough et al. 1983, Grumbach 2002). These cyclical hormone patterns are undetected in the immature female rat until the onset of puberty (for review see Ojeda et al. 1983b, Ojeda et al. 1994). The anatomical and physiological changes that occur within the female mammal during puberty are dependent upon neural changes during embryonic and prepubertal development that lead to the activation of gonadotropin releasing hormone (GnRH) neurons in the hypothalamus (Gore 2001). The ovaries and pituitary gland of immature rats function normally when transplanted into mature animals, indicating that these organs are functionally mature, even prior to puberty (Debeljuk et al. 1972). GnRH stimulates the release of the reproductive hormones, follicle stimulating hormone (FSH) and luteinizing hormone (LH), from the anterior pituitary gland. FSH stimulates the initial development and maturation of ovarian follicles, as well as the secretion of estrogen (E2) by the follicles. In response to positive feedback effects of E2, an LH surge occurs and the increased LH induces the first ovulation, marking the onset of sexual maturity (Gore 2001, Grumbach 2002). In the rat, a concomitant prolactin (PRL) surge occurs, and serves a luteotropic function (Freeman et al. 2000). This cyclic pattern of hormonal secretion continues during the reproductive lifespan of the adult female. Postnatal prepubertal development of the female rat can be divided into four phases: (1) a neonatal period initiated at birth and ending on postnatal day 7; (2) an infantile period that extends from days 7 – 21; (3) a juvenile period that ends around days 30-32 and; (4) a 1 peripubertal period of variable duration that culminates with the occurrence of the first ovulation (around day 38 for most laboratory stocks) (for review see (Ojeda et al. 1994). During the juvenile period of development there are sporadic LH spikes. The magnitude of these spikes becomes attenuated during the juvenile period due to E2 negative feedback, which keeps circulating gonadotropin levels low. In order to induce an LH surge during the early juvenile period, E2 doses twice as high as those on proestrus must be administered (for review see Ojeda et al. 1994). As juvenile development continues, E2 levels stimulate norepinephrine (NE) and glutamate production in the hypothalamus, as well as increases in neuronal growth and synaptic connections, and increases in uterine growth and intrauterine fluid. Norepinephrine and dopamine (DA) turnover increase and pituitary DA receptor sensitivity decreases (Hohn et al. 1979, Lamberts et al. 1981, Raum et al. 1980). Blockade of catecholamine synthesis in late juvenile (30 day old) rats reduces LH levels, suggesting that catecholamines have a stimulatory effect on GnRH during puberty (Moguilevsky et al. 1995). While gonadotropin levels are low during juvenile development, PRL levels increase. PRL stimulates follicular growth, and increases steroid sensitivity to LH and FSH via interaction with LH receptors in the ovary (Advis et al. 1979). These levels of PRL also activate dopaminergic neurons, increasing DA release, which desensitizes pituitary DA receptors. PRL begins to be secreted in a variable, yet adult type pattern, and the ovary grows. This pattern of release is characterized by discharges occurring approximately every 3 hours. The most prominent PRL secretions occur at midafternoon and during the early morning hours. As E2 levels continue to rise and after E2 and progesterone (P4) levels have remained high for an extended period, a GnRH surge is induced, via steroid positive feedback. Whether this is a direct effect, or one mediated by intermediate factors is still unclear. This shift from negative feedback to positive feedback sets in motion the events of puberty and the beginning of cyclical hormone secretion. The nocturnal increases in PRL levels disappear, but the afternoon surge of both PRL and LH become even more pronounced (Ojeda et al. 1983a, Ojeda et al. 1994). It is believed that the shift in LH pulses is due to the final maturation of the GnRH circuitry, because GnRH is the primary stimulator of LH secretion.