Rhabdomyosarcoma (RMS) is a malignant soft-tissue sarcoma in children, accounting for about 40% of pediatric soft-tissue tumours. Five-year survival for metastatic RMS is only about 25%. Furthermore, there has been no significant improvement in RMS survival since 1975, pointing to a need for improved therapy. Previous work in our lab has shown that 4-hydroxytamoxifen (4OHT) leads to increased apoptosis and decreased viability in RMS cells. Expanding on this work, the current project aims to elucidate the mechanisms behind 4OHT-induced apoptosis in RMS cells, focusing on the roles of estrogen receptors (ER) and MAP kinases (MAPK). We found that: 1) 4OHT-induced apoptotic signaling was associated with increased MAPK phosphorylation, 2) Inhibition of MAPK protected cells against 4OHT, 3) Inhibition of ER also protected against 4OHT, and 4) ER inhibition blocked 4OHT-associated MAPK phosphorylation.

Male rat mating is a suite of individual behaviors mediated by the actions of two metabolites of testosterone (T), dihydrotestosterone (DHT) and estradiol (E2), on the brain. Individually, neither metabolite fully maintains or restores mating in castrated males, but both combined are as effective as T. Two hormone-responsive areas of the brain, the medial preoptic area (MPO) and the medial amygdala (MEA), are crucial for mating. These studies ask: by what mechanism(s) does E2 act in the MPO and MEA? We blocked the conversion of T to E2 in the MEA of intact male rats and sexual behavior was not maintained. We then infused antisense oligodeoxynucleotides (ODNs) to estrogen receptor-alpha (ER-á) mRNA bilaterally to the MPO or the MEA of intact male rats to block ER-á expression. ODN infusion of the MPO attenuated mating but infusion of the MEA had no effect. These results suggest that ER-á is the behaviorally relevant estrogen receptor (ER) in the MPO but not in the MEA. ER was originally described in the cytoplasm and nucleus of cells. Recently plasma membrane associated ERs (mER) have been reported. We conjugated E2 to Bovine Serum Albumin (BSA-E2), a large protein that will not penetrate the plasma membrane, thus restricting the action of E2 to mER, and chronically delivered it to the MPO and MEA. BSA-E2 maintained mating if put in the MPO, but not in the MEA, suggesting a surface action of E2 is sufficient in the MPO. The MPO and MEA are reciprocally connected and probably constitute elements of a larger, steroid-responsive neural network that mediates male mating behavior. To begin to describe this purported circuit, we injected Pseudorabies virus (PRV) into the prostate gland and dually labeled PRV-immunoreactive cells for ER or androgen receptors. We found dual labeling in a forebrain diencephalic circuit that includes the MPO, the medial preoptic nucleus, bed nucleus of stria terminalis, the zona incerta, the periaqueductal gray and other areas that presumably mediate both autonomic and motor aspects of male mating. Together, the results of these studies begin to elucidate locations and mechanisms of E2 mediation of male sexual behavior.

The Australian zebra finch (Taeniopygia guttata) serves as an excellent model organism for studying the mechanisms that influence brain sexual differentiation. The brain and behavior of the zebra finch are sexually dimorphic. The regions of the brain that control the learning and production of song (song control nuclei) are significantly larger in the male brain than in the female brain and only males sing courtship songs, thus the majority of past research has focused on the development of these sex differences. In the majority of mammals, brain sexual differentiation occurs because hormones secreted from the gonads act to initiate male or female brain development. In zebra finches, estradiol is sufficient to masculinize the male brain, however manipulations of developmental hormone exposure fail to fully reverse the sex differences in song nuclei size. Furthermore, genetic females induced to develop functional testicular tissue do not develop a completely masculinized song system and castration has no effect on development of the song system in males. The source of the increased estrogenic signal in male zebra finch brain has yet to be identified, but data suggest that other neuronal factors play a role in development of the song control nuclei. Coregulators, such as coactivators and corepressors, are proteins and RNA activators that work by enhancing or depressing transcriptional activity of the nuclear steroid receptor with which they associate. Coregulators also modulate the development of sex-specific brain morphology and behavior in rodents and birds and may help to explain the difficulties observed in altering song nuclei development via castration and gonadal hormone replacement. As an estrogen receptor-α coactivator, ribosomal protein L7 (RPL7) is able to make the brain more sensitive to estradiol by enhancing the effects of steroid receptor action. Therefore, this dissertation addressed the following questions regarding RPL7: (1) is RPL7 expression sexually dimorphic in the song nuclei of the zebra finch brain?; (2) is RPL7 protein expression regulated by steroid hormones?; and (3) does decreasing RPL7 protein expression with antisense oligonucleotides alter neuronal survival in vivo and song nuclei size and neuron number in vitro? Collectively, these studies will provide valuable information about the role of steroid receptor coactivators in development of the zebra finch song system and on the role of coactivators on sexual differentiation of the brain.

Male rat copulation is mediated by estrogen-sensitive neurons in the medial preoptic area (MPO) and medial amygdala (MEA); however, the mechanisms through which estradiol (E2) acts are not fully understood. We hypothesized that E2 acts through estrogen receptor α (ERα) in the MPO and MEA to promote male mating behavior. Antisense oligodeoxyneucleotides (AS-ODN) complementary to ERα mRNA were bilaterally infused via minipumps into either brain area to block the synthesis of ERα, which we predicted would reduce mating. Western blot analysis and immunocytochemistry revealed a knockdown of ERα in each brain region; however, compared to saline controls, males receiving AS-ODN to the MPO showed significant reductions in all components of mating, whereas males receiving AS-ODN to the MEA continued to mate normally. These results suggest that E2 acts differently in these brain regions to express sexual behavior and that ERα in the MPO, but not in the MEA, promotes mating.

Male rat copulatory behavior is dependent on Testosterone (T) and its metabolites, estradiol (E2) and dihydrotestosterone (DHT). The estrogen receptor (ER) isoforms, ERα and ERβ, exist in the medial Amygdala (MEA) and either receptor might mediate mating behavior. Therefore, the effects of selective estrogenic MEA implants: propyl pyrazole triol (PPT, ERα agonist), diarylpropionitrile (DPN, ERβ agonist), and 1-methyl-4-phenyl pyridinium (MPP, ERα antagonist) were compared to E2 in maintaining sexual behavior. Four groups of male rats were castrated and administered DHT s.c. and bilateral MEA implants containing either cholesterol, E2, PPT or DPN. An additional group of gonadally intact male rats received bilateral MPP-MEA implants. The post-surgical trials showed a significant decrease in the mating behavior of groups that received cholesterol, PPT, or DPN-MEA implants. However, sexual behavior was maintained in male rats that received the E2 or MPP-MEA implants. These results suggest a differential response of the MEA to E2.

Moderate energy restriction in young, exercising women attenuates the positive effects of exercise on bone density. Studies have shown that in the absence of adequate levels of circulating estrogen, there may not be enough functional estrogen receptor-a (ER-a) to respond adequately to loading. The experiment described in this document is significant because this model has not been explored under conditions of energy restriction (EnR) which are known to reduce circulating estrogen levels; it has been tested only in ovariectomized animals. The central hypothesis of this research is that reductions in estrogen due to EnR limit the ability of bone to respond to mechanical loading (LOAD) through a down-regulation of ER-a. Study one determined which nutrient’s (calcium or energy) restriction (-40 percent) had the greatest negative effects on the skeletal integrity of exercising female rats and whether exercise (EX) could mitigate these deleterious changes. EnR caused detrimental effects in many of the structural properties of bone; however EX attenuated losses in cancellous bone. Study two ascertained whether EX maintained cancellous bone mass in female rats subjected to graded EnR (-20 or -40 percent) and whether changes in endocrine factors were related. EX preserved cancellous bone volume and osteoblast activity under both levels of EnR, in addition to total body lean mass and bone mineral content. A similar maintenance of serum insulin-like growth factor and estradiol occurred in the EX EnR(40 percent) group suggesting that these changes may be related to the protective effects of EX. Study three determined the effects of 40 percent EnR on bone formation rate to LOAD in young adult female rats and tracked alterations in ovarian function (estradiol). Although higher than non-loaded animals, the response of bone to LOAD in EnR animals was dampened in comparison to energy-replete animals. The experiments described in this document are significant because these are the first experiments to explore the relationship between EnR and estrogen levels on cancellous bone response to LOAD. This is particularly important for physically active, energy restricted women because cancellous bone in these women will not experience the same effects of loading which can increase their risk for developing osteoporosis.

Neurotransmitters are widely distributed in the central nervous system of tilapia during development. In the present study, the effects of gonadal steroids, temperature, and neurotransmitters on the development of central neurotransmitter systems in tilapia, Oreochromis mossambicus, were investigated. Exogenous E2 before 10 days posthatching resulted in an inhibitory effect on the activity of central 5-HT system via decreasing TPH activity and increasing MAO activity, a decrease of the gene expression of brain aromatase and ERa, which is involved in the feminization. The masculinizing actions of 17a-methyltestosterone (MT) are most potent later at up to day 20 of age, and may depend on MT-induction of aromatase activity, aromatase mRNA gene expression and estrogen receptor-b mRNA gene expression. The development of central neurotransmitter systems is influenced by aquatic temperature during its respective restricted period. The influence of both lower and elevated temperatures on the neurotransmitter activity, either increasing or suppressing, is dependent on its developing stage. Neurotransmitters have an effect to influence the development of central neurotransmitter and this effect may mediated by the neural proliferation.

Thesis (Ph. D.)--Case Western Reserve University, 2010. / [School of Medicine] Department of Pharmacology. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 93-113).

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