In the United States, there are currently more than 40 million postmenopausal women. These women are faced with a variety of physiological changes including ovarian steroid withdrawal and alterations in hypothalamic neurons. Within the hypothalamic infundibular nucleus of postmenopausal women, there is neuronal hypertrophy and an increase in neurokinin B gene expression. Recent studies identified the kisspeptins and dynorphins as major regulators of reproduction. In our first experiment, we examined the location and alterations of KiSS-1 mRNA-expressing neurons in the hypothalami of pre and postmenopausal women. KiSS-1 neurons were largely confined to the infundibular nucleus, and in postmenopausal women, exhibited neuronal hypertrophy and increased gene expression. To determine if these changes could result from alterations in ovarian steroids, we investigated KiSS-1 gene expression in the hypothalamic infundibular nucleus of non-human primates. Similar to the findings in postmenopausal women, ovariectomy of monkeys resulted in neuronal hypertrophy and increased KiSS-1 gene expression within the infundibular nucleus. Further, estrogen treatment of ovariectomized monkeys yielded a dramatic decrease in KiSS-1 gene expression. Together, these findings suggest that the postmenopausal alterations in KiSS-1 neurons are secondary to ovarian failure.In a second study, we examined alterations in dynorphin gene expression in the hypothalami of pre and postmenopausal women. Dynorphin mRNA-expressing neurons were identified in multiple nuclei. Numbers of dynorphin neurons were decreased within the mPOA and infundibular nucleus of postmenopausal women. In the infundibular nucleus of postmenopausal women, dynorphin neurons were hypertrophied. To determine the contribution of ovarian steroids on dynorphin gene expression, we examined dynorphin mRNA in a monkey model of menopause. Young ovariectomized monkeys exhibited hypertrophy of dynorphin neurons, with no changes in dynorphin gene expression. Estrogen replacement yielded a decrease in neuronal size and an increase in dynorphin neuron number.In future studies, we will use Quantum Dot FISH to determine if NKB, KiSS-1, and dynorphin are colocalized in the hypertrophied neurons. These neuropeptides are involved in the regulation of GnRH and changes in their gene expression likely contribute to postmenopausal alterations in reproductive hormones. Our findings provide greater understanding of the postmenopausal condition and offer opportunities for pharmaceutical investigation and treatment.

Introdução: Novos medicamentos trouxeram um considerável aumento na chance de gravidez no momento em que a estimulação ovariana controlada permitiu aumentar o número de oócitos a serem recuperados e, consequentemente, um maior número de embriões para serem transferidos. Os análogos de hormônio liberador de gonadotrofina, em associação com gonadotrofinas, trouxeram a solução para um dos problemas da estimulação ovariana controlada: o pico precoce de hormônio luteinizante, que prejudicava a coleta de oócitos em torno de 20% dos casos. Rotineiramente, usamos o protocolo com agonista, mas a possibilidade do uso de antagonistas resulta em maior facilidade de manuseio pelo usuário, envolve um único menstrual ciclo e dispensa orientação para uso de drogas, no final do ciclo anterior. Assim, buscamos avaliar o uso dos dois protocolos e analisar seus resultados. Objetivos: Análise e comparação de dados entre dois protocolos de indução (longo com agonista e flexível com antagonista) em pacientes submetidas a técnicas de reprodução assistida na SEGIR – Serviço de Ecografica, Genética e Reprodução Assistida – Porto Alegre. Métodos: Estudo transversal comparando os resultados intermediários com o uso de dois diferentes protocolos de estimulação ovariana com de agonista versus antagonista do hormônio liberador de gonadotrofina para técnicas de reprodução assistida. A análise estatística dos dados recuperados (idade, índice de massa corpórea, número de oócitos recuperados, número de oócitos fertilizados, número de oócitos clivados, dose total de FSH utilizada e ocorrência de síndrome do hiperestímulo ovariano) foi realizada através de teste t de Student para dados paramétricos e análise de covariância para as variáveis dependentes, calculados com o programa SPSS 16.0. Resultados: Um total de 50 pacientes preencheram os critérios para inclusão no estudo entre janeiro e março no ano de 2010, sendo 25 em cada grupo. Houve diferença estatística apenas na idade média entre os grupos (p=0,031). Não houve diferença estatística para os demais dados analizados (índice de massa corpórea, número de oócitos recuperados, número de oócitos fertilizados, número de oócitos clivados e dose de FSH utilizada) entre os grupos. Não houve casos de síndrome do hiperestímulo ovariano. Conclusão: Ambos protocolos os são iguais em termos de resultados. O agonista tem vantagens sobre o agendamento do procedimento, mas leva muito tempo para começar a estimulação e tem a possibilidade de iniciar a medicação em uma paciente grávida. Somado a isso, temos a possibilidade de ter síndrome do hiperestímulo ovariano como complicação. No grupo antagonista, está claro a maior facilidade de uso da medicação e o início mais rápido da estimulação ovariana. / Background: New medications have brought a considerable increase in the chance of pregnancy at the time that controlled ovarian stimulation allowed an increase in the number of oocytes to be recruited and, consequently, a greater number of embryos to be transferred. The gonadotropin-releasing hormone analogues, in association with gonadotropins, brought the solution to one of the controlled ovarian stimulation's problems: the early peak of luteinizing hormone, which harmed the oocytes collection around 20% of the cases. We, routinely, used the agonist protocol, but the possibility of antagonists usage results in greater ease of handling by the user, involves a single menstrual cycle and dispensation guidance for drug use, at the end of the previous cycle. Thus we seek to evaluate the use of the two protocols and analyze their results. Objective: Analysis and compare data between two induction protocols (long agonist and flexible antagonist) in patients submitted an assisted reproduction technique in SEGIR – Serviço de Ecografia Genética e Reprodução assistida – Porto Alegre. Methods: Cross-sectional study comparing the intermediate results with the use of two different ovarian stimulation protocols with gonadotropin-releasing hormone agonist versus antagonist to assisted reproductive techniques. The statistical analysis of the retrieved data (age, body mass index, number of oocytes recovered, number of fertilized oocytes, number of oocytes cleaved, total dose of FSH used and occurrence of ovarian hyperstimulation syndrome) was performed by Student t test for parametric data and analysis of covariance for the dependent variables, calculated with the program SPSS 16.0. Results: A total of 50 patients, 25 in each group, met the criteria for inclusion in the study between January and March in the year 2010. There was statistically significant difference only in the middle ages between the groups (p = 0 .031). There was no statistical difference for the remaining data analyzed (body mass index, number of oocytes recovered, number of fertilized oocytes, number of oocytes cleaved and dose of FSH utilized) between the groups. There were no cases of ovarian hyperstimulation syndrome. Conclusion: Both protocols are equal in terms of results. The agonist has advantages about scheduling of the procedure, but it takes too long to start the stimulation and have possibility to start medication in a pregnant patient. Added to this, we have the possibility of getting the ovarian hyperstimulation syndrome as complication. In the antagonist group, is clear the ease-of-use of the medication and the fastest start of the ovarian stimulation.

Introdução: Novos medicamentos trouxeram um considerável aumento na chance de gravidez no momento em que a estimulação ovariana controlada permitiu aumentar o número de oócitos a serem recuperados e, consequentemente, um maior número de embriões para serem transferidos. Os análogos de hormônio liberador de gonadotrofina, em associação com gonadotrofinas, trouxeram a solução para um dos problemas da estimulação ovariana controlada: o pico precoce de hormônio luteinizante, que prejudicava a coleta de oócitos em torno de 20% dos casos. Rotineiramente, usamos o protocolo com agonista, mas a possibilidade do uso de antagonistas resulta em maior facilidade de manuseio pelo usuário, envolve um único menstrual ciclo e dispensa orientação para uso de drogas, no final do ciclo anterior. Assim, buscamos avaliar o uso dos dois protocolos e analisar seus resultados. Objetivos: Análise e comparação de dados entre dois protocolos de indução (longo com agonista e flexível com antagonista) em pacientes submetidas a técnicas de reprodução assistida na SEGIR – Serviço de Ecografica, Genética e Reprodução Assistida – Porto Alegre. Métodos: Estudo transversal comparando os resultados intermediários com o uso de dois diferentes protocolos de estimulação ovariana com de agonista versus antagonista do hormônio liberador de gonadotrofina para técnicas de reprodução assistida. A análise estatística dos dados recuperados (idade, índice de massa corpórea, número de oócitos recuperados, número de oócitos fertilizados, número de oócitos clivados, dose total de FSH utilizada e ocorrência de síndrome do hiperestímulo ovariano) foi realizada através de teste t de Student para dados paramétricos e análise de covariância para as variáveis dependentes, calculados com o programa SPSS 16.0. Resultados: Um total de 50 pacientes preencheram os critérios para inclusão no estudo entre janeiro e março no ano de 2010, sendo 25 em cada grupo. Houve diferença estatística apenas na idade média entre os grupos (p=0,031). Não houve diferença estatística para os demais dados analizados (índice de massa corpórea, número de oócitos recuperados, número de oócitos fertilizados, número de oócitos clivados e dose de FSH utilizada) entre os grupos. Não houve casos de síndrome do hiperestímulo ovariano. Conclusão: Ambos protocolos os são iguais em termos de resultados. O agonista tem vantagens sobre o agendamento do procedimento, mas leva muito tempo para começar a estimulação e tem a possibilidade de iniciar a medicação em uma paciente grávida. Somado a isso, temos a possibilidade de ter síndrome do hiperestímulo ovariano como complicação. No grupo antagonista, está claro a maior facilidade de uso da medicação e o início mais rápido da estimulação ovariana. / Background: New medications have brought a considerable increase in the chance of pregnancy at the time that controlled ovarian stimulation allowed an increase in the number of oocytes to be recruited and, consequently, a greater number of embryos to be transferred. The gonadotropin-releasing hormone analogues, in association with gonadotropins, brought the solution to one of the controlled ovarian stimulation's problems: the early peak of luteinizing hormone, which harmed the oocytes collection around 20% of the cases. We, routinely, used the agonist protocol, but the possibility of antagonists usage results in greater ease of handling by the user, involves a single menstrual cycle and dispensation guidance for drug use, at the end of the previous cycle. Thus we seek to evaluate the use of the two protocols and analyze their results. Objective: Analysis and compare data between two induction protocols (long agonist and flexible antagonist) in patients submitted an assisted reproduction technique in SEGIR – Serviço de Ecografia Genética e Reprodução assistida – Porto Alegre. Methods: Cross-sectional study comparing the intermediate results with the use of two different ovarian stimulation protocols with gonadotropin-releasing hormone agonist versus antagonist to assisted reproductive techniques. The statistical analysis of the retrieved data (age, body mass index, number of oocytes recovered, number of fertilized oocytes, number of oocytes cleaved, total dose of FSH used and occurrence of ovarian hyperstimulation syndrome) was performed by Student t test for parametric data and analysis of covariance for the dependent variables, calculated with the program SPSS 16.0. Results: A total of 50 patients, 25 in each group, met the criteria for inclusion in the study between January and March in the year 2010. There was statistically significant difference only in the middle ages between the groups (p = 0 .031). There was no statistical difference for the remaining data analyzed (body mass index, number of oocytes recovered, number of fertilized oocytes, number of oocytes cleaved and dose of FSH utilized) between the groups. There were no cases of ovarian hyperstimulation syndrome. Conclusion: Both protocols are equal in terms of results. The agonist has advantages about scheduling of the procedure, but it takes too long to start the stimulation and have possibility to start medication in a pregnant patient. Added to this, we have the possibility of getting the ovarian hyperstimulation syndrome as complication. In the antagonist group, is clear the ease-of-use of the medication and the fastest start of the ovarian stimulation.

Introdução: Novos medicamentos trouxeram um considerável aumento na chance de gravidez no momento em que a estimulação ovariana controlada permitiu aumentar o número de oócitos a serem recuperados e, consequentemente, um maior número de embriões para serem transferidos. Os análogos de hormônio liberador de gonadotrofina, em associação com gonadotrofinas, trouxeram a solução para um dos problemas da estimulação ovariana controlada: o pico precoce de hormônio luteinizante, que prejudicava a coleta de oócitos em torno de 20% dos casos. Rotineiramente, usamos o protocolo com agonista, mas a possibilidade do uso de antagonistas resulta em maior facilidade de manuseio pelo usuário, envolve um único menstrual ciclo e dispensa orientação para uso de drogas, no final do ciclo anterior. Assim, buscamos avaliar o uso dos dois protocolos e analisar seus resultados. Objetivos: Análise e comparação de dados entre dois protocolos de indução (longo com agonista e flexível com antagonista) em pacientes submetidas a técnicas de reprodução assistida na SEGIR – Serviço de Ecografica, Genética e Reprodução Assistida – Porto Alegre. Métodos: Estudo transversal comparando os resultados intermediários com o uso de dois diferentes protocolos de estimulação ovariana com de agonista versus antagonista do hormônio liberador de gonadotrofina para técnicas de reprodução assistida. A análise estatística dos dados recuperados (idade, índice de massa corpórea, número de oócitos recuperados, número de oócitos fertilizados, número de oócitos clivados, dose total de FSH utilizada e ocorrência de síndrome do hiperestímulo ovariano) foi realizada através de teste t de Student para dados paramétricos e análise de covariância para as variáveis dependentes, calculados com o programa SPSS 16.0. Resultados: Um total de 50 pacientes preencheram os critérios para inclusão no estudo entre janeiro e março no ano de 2010, sendo 25 em cada grupo. Houve diferença estatística apenas na idade média entre os grupos (p=0,031). Não houve diferença estatística para os demais dados analizados (índice de massa corpórea, número de oócitos recuperados, número de oócitos fertilizados, número de oócitos clivados e dose de FSH utilizada) entre os grupos. Não houve casos de síndrome do hiperestímulo ovariano. Conclusão: Ambos protocolos os são iguais em termos de resultados. O agonista tem vantagens sobre o agendamento do procedimento, mas leva muito tempo para começar a estimulação e tem a possibilidade de iniciar a medicação em uma paciente grávida. Somado a isso, temos a possibilidade de ter síndrome do hiperestímulo ovariano como complicação. No grupo antagonista, está claro a maior facilidade de uso da medicação e o início mais rápido da estimulação ovariana. / Background: New medications have brought a considerable increase in the chance of pregnancy at the time that controlled ovarian stimulation allowed an increase in the number of oocytes to be recruited and, consequently, a greater number of embryos to be transferred. The gonadotropin-releasing hormone analogues, in association with gonadotropins, brought the solution to one of the controlled ovarian stimulation's problems: the early peak of luteinizing hormone, which harmed the oocytes collection around 20% of the cases. We, routinely, used the agonist protocol, but the possibility of antagonists usage results in greater ease of handling by the user, involves a single menstrual cycle and dispensation guidance for drug use, at the end of the previous cycle. Thus we seek to evaluate the use of the two protocols and analyze their results. Objective: Analysis and compare data between two induction protocols (long agonist and flexible antagonist) in patients submitted an assisted reproduction technique in SEGIR – Serviço de Ecografia Genética e Reprodução assistida – Porto Alegre. Methods: Cross-sectional study comparing the intermediate results with the use of two different ovarian stimulation protocols with gonadotropin-releasing hormone agonist versus antagonist to assisted reproductive techniques. The statistical analysis of the retrieved data (age, body mass index, number of oocytes recovered, number of fertilized oocytes, number of oocytes cleaved, total dose of FSH used and occurrence of ovarian hyperstimulation syndrome) was performed by Student t test for parametric data and analysis of covariance for the dependent variables, calculated with the program SPSS 16.0. Results: A total of 50 patients, 25 in each group, met the criteria for inclusion in the study between January and March in the year 2010. There was statistically significant difference only in the middle ages between the groups (p = 0 .031). There was no statistical difference for the remaining data analyzed (body mass index, number of oocytes recovered, number of fertilized oocytes, number of oocytes cleaved and dose of FSH utilized) between the groups. There were no cases of ovarian hyperstimulation syndrome. Conclusion: Both protocols are equal in terms of results. The agonist has advantages about scheduling of the procedure, but it takes too long to start the stimulation and have possibility to start medication in a pregnant patient. Added to this, we have the possibility of getting the ovarian hyperstimulation syndrome as complication. In the antagonist group, is clear the ease-of-use of the medication and the fastest start of the ovarian stimulation.

Introduction : La mucoviscidose est une maladie génétique autosomale récessive causée par des mutations dans le gène CFTR (cystic fibrosis transmembrane conductance regulator). Ce dernier code un canal chlorure AMPc-dépendant localisé dans la membrane apicale des cellules épithéliales, dont l’activité est régulée par de nombreuses interactions protéine-protéine. Dans le cadre de la recherche de nouveaux partenaires du CFTR, une interaction directe entre le canal (sauvage et muté F508del) etl’annexine A5 (AnxA5) a été mise en évidence dans notre laboratoire. Des stratégies de sur et de sousexpression nous ont également permis d’établir un lien fonctionnel entre les deux protéines. En effet, nos travaux montrent que les sécrétions ioniques dépendantes du CFTR sont corrélées au niveau d’expression intracellulaire de l’AnxA5. Par ailleurs, une élévation des courants médiés par le CFTR ainsi qu’une augmentation de la quantité de canaux dans la membrane plasmique sont observées suite à la surexpression de l’AnxA5 dans des cellules exprimant le CFTR muté F508del.But de l’étude : Au vu de ces observations, l’AnxA5 apparaît comme une cible potentielle pour la correction de certains défauts engendrés par la mutation F508del. Une piste thérapeutique pourrait être l’identification de composés capables d’augmenter son expression dans des cellules épithéliales exprimant le mutant F508del de la protéine CFTR. Considérant les informations fournies par la littérature, notre choix s’est porté sur la GnRH (gonadotropin-releasing hormone), molécule utilisée en thérapeutique humaine depuis plus de 25 ans. Ainsi, nous avons évalué l’effet de la GnRH sur la modulation de l’expression de l’AnxA5 et sur le transport ionique dépendant du CFTR dans nos différents modèles d’étude Résultats : Outre la présence du récepteur de la GnRH dans nos modèles cellulaires, nous montrons également que l’expression de l’AnxA5 y est augmentée dès 60 minutes de traitement avec l’hormone (1 nM). De plus, comparativement à des cellules non stimulées, des cellules prétraitées avec la GnRH présentent une hausse significative des sorties actives d’iodure, corrélant avec une augmentation de la quantité de CFTR à la surface cellulaire. Ces observations ont été faites dans les modèles exprimant le CFTR muté F508del ainsi que dans ceux exprimant le CFTR sauvage. Conclusion : Dans nos modèles et selon nos conditions de stimulation, un traitement avec la GnRH augmente l’expression intracellulaire de l’AnxA5 et conduit à une élévation des sécrétions ioniques médiées par le canal CFTR. Néanmoins, au vu de la multitude de voies de signalisation susceptibles d’être activées et de gènes pouvant être régulés suite à la liaison de la GnRH sur son récepteur, l’effet observé sur l’AnxA5 ne représente probablement pas le seul évènement cellulaire à l’origine de l’impact positif enregistré sur l’activité du canal CFTR. / Background: Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-stimulated chloride channel. CFTR is primarily located at the apical surface of epithelial cells, where its activity is regulated by some protein-protein interactions. As part of new CFTR’s partners research, we previously showed that annexin A5 (AnxA5) binds directly to both normal and F508del-CFTR. Moreover, under and overexpression strategies led us to establish a functionnal link between these two proteins. In fact, CFTR-dependent ion secretions are correlated to the intracellular level of AnxA5. Otherwise, in transfected epithelial cells, AnxA5 overexpression increases CFTR’s level in plasma membranes and raises CFTR-mediated currents in F508del-CFTR expressing cells. Aim of the study: In the light of these findings, AnxA5 appears as a potential target in order to correct some defects caused by the F508del mutation. A therapeutic approach would be to find some compounds capable of increasing AnxA5 expression in F508del-CFTR expressing epithelial cells. Reviewing the literature, our choice fell on GnRH (gonadotropin-releasing hormone), a commonly used molecule for diverse clinical applications for 25 years. So, the effects of GnRH on the modulation of AnxA5 expression and on CFTR-dependent ion transport were assessed in our different cellular models. Results: Beside the GnRH receptor expression, we show that AnxA5 expression is augmented in all cell lines after one hour incubation with the hormone (1 nM). Moreover, compared to untreated cells, a significant iodide efflux peak is measured in GnRH pretreated, which is correlated with an increased cell surface expression of CFTR. It is of interest to note that these observations have been made in CF and non-CF cells. Conclusion: In our models and according to our stimulation conditions, GnRH treatment enhances AnxA5 intracellular expression and leads to a rise of CFTR-dependent ion secretions. Nevertheless, given the multitude of activated signaling pathways and regulated genes in response to GnRH binding to its receptor, the positive impact on CFTR activity is probably not solely explained by the effect on AnxA5 expression.

Master of Science / Department of Animal Sciences and Industry / David M. Grieger / Mares in estrus can exhibit erratic and fractious behavior that may adversely affect their ease of handling. Current treatments for suppression of estrus in the mare include an oral progestin, Regu-mate® (Hodgson et al., 2005), uterine insertion of a glass ball (Nie et al., 2003), fetal crushing through rectal palpation (Lefranc, 2004) and surgical sterilization (McCue, 2003). Although effective, all of these procedures are problematic for different reasons. Immunization against an endogenous hormone critical to fertility is an attractive alternative in suppressing estrus; especially if it is less intrusive and reversible. This strategy has been demonstrated by targeting gonadotropin-releasing hormone (GnRH) in gilts (Esbenshade and Britt, 1985), ewes (Clarke et al., 1978), mares (Garza et al., 1986), and heifers (Johnson et al., 1988). The antigen developed for this study was produced using the sequence of the bacterial protein, glutathione-S-transferase (GST) linked to three in-tandem repeats of the mammalian GnRH gene coding sequence (GST-GnRH3). Six reproductively sound mares, between the ages of 3 and 8 years, were used in this study. The anti-GnRH group (n=4) received 1 mg of GST-GnRH3 in 2ml of incomplete Freund’s adjuvant (IFA) as the primary injection. Four weeks later, mares received a single booster injection of 0.5mg of GST-GnRH3 in 1ml of IFA. The control mares (n=2) received similar doses of GST protein only, in identical injection volumes of IFA as the anti-GnRH group. The entire duration of the study ran for 22 weeks from early May through September. Ovaries were monitored three times weekly to track follicular growth and ovulation via transrectal ultrasonography. In addition, all mares were exposed to a stallion twice weekly and observed for estrous behavior. Weekly blood samples were collected to evaluate progesterone levels and serum binding of GnRH. The GST-GnRH3 antigen suppressed follicular activity in all treatment mares within 45 days following the second injection. Estrous behavior was suppressed in all but one mare in the anti-GnRH group. When exposed to the stallion, this mare displayed strong estrous behavior for seven weeks despite her lack of ovarian cyclicity. Follicular activity and estrous behavior remained normal in one of the control mares (avg. cycle length = 20 days). For the final 10 weeks of the study, however, the other control mare developed large follicles but failed to ovulate according to the ultrasound data. This mare did not display estrous behavior during this period, and her progesterone levels remained greater than 2 ng/ml for most of the final 10 weeks of the study. Approximately 2 weeks after the booster injection all anti-GnRH mares had progesterone levels of <1ng/ml. GnRH antibody binding peaked two weeks following booster immunization in all treated mares and remained undetectable in control mares throughout the study. The GST-GnRH3 treatment induced GnRH binding, suppressed follicular activity and reduced progesterone concentrations in all four mares. Although estrous behavior was abolished in 3 of the 4 treated mares, one did continue to demonstrate estrous behavior in the presence of a stallion. This dissociation of ovarian activity and estrous behavior was evident in our study with a limited number of animals, but the vaccine does show promise in reducing unwanted estrous behavior.

No description available.

Pheromones are chemosensory cues released by an individual to cause a behavioral or physiological change in a conspecific. These changes can range from increasing a female's receptivity to altering hormone secretions in the body. The red-legged salamander (Plethodon shermani) is an emerging non-mammalian model for understanding the evolution of chemical communication due to their well-characterized pheromones. Plethodontid salamanders secrete pheromones from their submandibular gland, called mental gland pheromones. Previous studies showed that mental gland pheromones increased receptivity in females during courtship and also increased corticosterone (CORT) concentrations in males. CORT is a metabolic hormone that mediates vertebrate stress responses. To further understand the neuroendocrine mechanisms involved in behavioral and physiological responses to pheromones, I conducted several experiments. First, I investigated whether pheromones altered levels of two neuromodulators in the brain, gonadotropin-releasing hormone (GnRH) and arginine vasotocin (AVT) in P. shermani. GnRH is both a neuromodulator and hormone, and has strong effects on reproductive behavior. Additionally, AVT has broad behavioral effects in vertebrates, particularly in newts. I found that application of mental gland pheromones altered the number of GnRH-immunoreactive neurons, but had no effect on the number of AVT-immunoreactive neurons. Second, I examined the effects of AVT and AVP antagonist on courtship and mating in dusky salamanders (Desmognathus spp.). There was no effect of AVT or antagonist on reproductive behaviors in dusky salamanders. Finally, I investigated the role of increased CORT concentrations in male P. shermani. I first validated a non-invasive method to transdermally deliver CORT through a dermal patch. I then used dermal patches to exogenously elevate plasma CORT and examine the effects of CORT on behavior (activity and chemoinvestigation) and metabolism. CORT had no effect on behavior, but increased metabolic rates in male red-legged salamanders. Together these studies provide insight into the mechanisms by which pheromones work to alter behaviors and physiological functions in vertebrates. / Bayer School of Natural and Environmental Sciences; / Biological Sciences / PhD; / Dissertation;

The physiological mechanisms through which nutrition mediates its effects in controlling reproduction are not well characterized. Both neural and endocrine components have been implicated in the communication of nutritional status to the central nervous system. Leptin, a hormone synthesized and secreted mainly by adipocytes, is heavily involved in this communication network. The objectives of studies reported herein were 1) to determine the effects of short-term restriction of nutrients on circulating leptin, leptin gene expression in adipose tissue, and leptin receptor (LR) gene expression in the adenohypophysis of ovariectomized cows; and 2) to investigate the responsiveness of the hypothalamic-adenohypophyseal (AP) axis of fasted and non-fasted cattle to leptin. Studies demonstrated that circulating concentrations of leptin and leptin gene expression in subcutaneous adipose tissue are decreased by fasting. Although 2 to 3 days of fasting did not affect patterns of release of luteinizing hormone (LH), cerebroventricular infusions of leptin increased mean circulating concentrations of LH in fasted, but not normal-fed cows, without affecting frequency or amplitude of pulses of LH. In vitro studies were conducted to determine whether the in vivo effects of leptin could be accounted for at the hypothalamic and/or AP levels. Leptin did not affect the release of gonadotropin-releasing hormone (GnRH) from hypothalamic-infundibular explants from either normal-fed or fasted cattle. Moreover, leptin did not affect the basal release of LH from bovine AP cells or AP explants from normal-fed cows. However, leptin induced a higher basal release of LH from AP explants of fasted cows and increased GnRH-stimulated release of LH from AP explants of normal-fed cows. Results demonstrate that leptin acts directly at the AP level to modulate the secretion of LH, and its effects are dependent upon nutritional status. Cellular mechanisms associated with the increased responsiveness of gonadotropes to leptin in fasted cows were investigated. Expression of LR and suppressor of cytokine signaling-3 (SOCS-3) in the adenohypophysis did not account for the increased responsiveness of fasted cows to leptin. Therefore, although leptin clearly stimulates the hypothalamic-gonadotropic axis in nutrient-restricted cattle, it is unclear why cattle maintained under neutral or positive energy balance are resistant to leptin.

Four experiments were conducted to test the hypothesis that RF-amide related peptide 3 (RFRP3) negatively regulate the secretion of LH in mares. In Exp. 1, mares received native gonadotropin-releasing hormone (GnRH) continuously at a rate of 20 microgram/h, delivered subcutaneously using Alzet osmotic pumps during the luteal phase of the estrous cycle. Mares were treated with i.v. bolus injections of 0, 500 and 1,000 microgram eRFRP3 on days 4, 6 and 8 of cycle. Mean concentrations of LH in the peripheral circulation averaged 1.2 +/- 0.2 ng/mL and did not differ among groups before or following RFRP3 treatment. In Exp. 2, pituitary venous effluent was sampled for characterization of episodic release of LH. Mares received either saline or eRFRP3 (250 microgram) i.v. every 10 min for 6 h beginning 2 h after onset of sampling. At hour 6, each mare was challenged with 1 mg GnRH. Neither mean ICS concentrations of LH (1.3 +/- 0.2 ng/ml), nor frequency (3.6 +/- 0.55 episodes/h), amplitude (0.2 +/- 0.03 ng/ml), or duration (36.3 +/- 3.5 min) of individual secretory episodes, differed between groups before or after eRFRP3 treatment. Area under the GnRH-induced LH curve (arbitrary units) also did not differ between control and RFRP3 treated mares (175.9 +/- 11.4 vs. 192.6 +/- 10.6). In Exp. 3, winter anovulatory mares (n=6) were treated continuously for 7 d with GnRH (100 microgram/h) to stimulate synthesis of LH and increase circulating concentrations of LH to values similar to the breeding season. The ICS was catheterized for blood sampling and mares were treated with saline or RFRP3 (5 mg) in a replicated Latin square design. Treatment with RFRP3 failed to alter ICS mean concentration of LH (0.95 +/- .03 ng/ml). Finally in Exp. 4, mares in the follicular phase of the estrous cycle were assigned randomly to receive either saline (n=3) or 10 microgram/kg BW of oRFRP3 (n=3) in a single injection. No effect on mean concentration of LH was observed. In contrast to observations in birds and other mammals, results of the current experiments fail to provide evidence for functional activity of eRFRP3 or oRFRP3 in regulating LH release in the mare.