O corpo lúteo (CL) canino é responsável pela síntese de E2 durante o diestro. O E2 atua de forma autócrina e/ou parácrina sobre essa glândula. O mecanismo de atuação do E2 depende da razão entre receptor alfa (ERα) e receptor beta (ERβ). A ligação ao ERα tem efeito proliferativo e ao ERβ antiproliferativo. O objetivo deste trabalho foi entender a sinalização mediada pelo ERα e ERβ no processo de formação e regressão do CL. CLs foram obtidos de cadelas não prenhes (n=30) nos dias 10, 20, 30, 40, 50 e 60 (n=5/grupo) após a ovulação (po). No dia da ovariossalpingohisterectomia foi colhido sangue para mensuração das concentrações de P4 e E2. Dezoito CLs (n=3/grupo) foram submetidos ao sequenciamento de RNA (RNAseq). Os genes diferencialmente expressos (DE) identificados pelo RNAseq foram submetidos ao software oPOSSUM3 para identificação das regiões de ligação mais representadas nos fatores de transcrição relacionados aos genes do ERα (ESR1) e ERβ (ESR2). A análise de expressão temporal revelou a presença de 5.116 diferencialmente expressos (DE) em pelo menos uma comparação e 1.106 não foram anotados ainda no genoma canino, destes genes DE 295 apresentavam regiões de ligação de fatores de transcrição em comum com ESR1 e ESR2. Dez genes DE foram selecionados para validação dos resultados de RNAseq através do qPCR; usando o GAPDH como gene de referência. Desses genes, quatro apresentaram regioes em comum com ESR2 (LEF-1; PAPPA; NDGR2 ATP1A1) e um com ESR1 (CAV1) e os demais controlam proliferação celular (CTNNB1; CCND1; IGFBP 3, 4 e 5). A expressão proteíca de PAPPA e IGFBP 3, 4 e 5 (componentes do sistema IGF) foi avaliada também por imunohistoquímica. Durante a primeira metade do diestro, nossos resultados indicam que a sinalização mediada pelo E2 ocorre via interação do ERα com CAV-1 (sinalização não genômica), ativando as vias de sinalização IGF e WNT/βcatenina, regulando o processo de proliferação celular. Enquanto na segunda metade, o ERβ regularia a expressão gênica de NDGR2 e ATP1A1, contribuindo para a regressão do CL. Assim nos resultados sugerem que o E2 atue tanto como um fator luteotrófico e quanto regulador da regressão do CL canino. / The canine corpus luteum (CL) is responsible for E2 synthesis during diestrus, which acts in an autocrine and / or paracrine manner in this gland. The E2 mechanism of action depends on the ratio between alpha (ERα) and beta (ERβ) receptor The binding to ERα has a proliferative effect whereas to ERβ an antiproliferative. The objective of this study was to understand the signaling mediated by ERα and ERβ in the formation and regression of the CL. CLs were obtained from non-pregnant bitches (n = 30) on days 10, 20, 30, 40, 50 and 60 (n = 5 / group) post-ovulation (po). On the day of ovariohysterectomy blood was collected for measurement of P4 and E2 concentrations. Eighteen CLs (n = 3 / group) were subjected to RNA sequencing (RNA-Seq). Genes differentially expressed (DE) identified by RNA-Seq were submitted to oPOSSUM3 software for detection of over-represented transcription factors binding sites (TFBS) related to the ERα (ESR1) and ERβ (ESR2) genes. The temporal expression analysis revealed the presence of 5116 differentially expressed (DE) genes in at least one comparison, and 1106 genes, which have not been recorded to the canine genome yet From these, 295 genes showed TFBS in common with ESR1 and ESR2. Ten DE genes were selected to validate the results of RNA-Seq by qPCR; using GAPDH as reference gene. Of these genes, four had TFBS in common with ESR2 (LEF-1; PAPPA; NDGR2; ATP1A1) and one with ESR1 (CAV1) and others genes were chosen because they control cell proliferation (CTNNB1; CCND1, IGFBP 3, 4 and 5). Protein expression of the IGF related genes (PAPPA and IGFBP 3, 4 and 5) was also evaluated by immunohistochemistry. During the first half of diestrus, it appears that E2 mediated signaling occurs via interaction of ERα with CAV-1 (non-genomic signaling), activating signaling pathways of IGF and WNT / βcatenin, then regulating the cell proliferation process. Whereas in the second half, ERβ appears to regulate NDGR2 and ATP1A1 gene expression, contributing to the regression of the CL. Thus our results suggest that E2 may act as a luteotrophic and also a luteolytic factor in the canine CL.

Exposure to exogenous estrogens such as synthetic 17α-ethinyl estradiol (EE) occurs via multiple sources (i.e. hormonal contraceptives, environmental contamination, hormone replacement therapy). The natural estrogen, 17β-estradiol (E2), is a well-studied immunomodulatory hormone at both environmental and pharmacologic levels. Conversely, little data exist regarding the immune effects of EE at either environmentally-relevant exposure levels or at pharmacological levels. Further, EE is delivered to patients in a clinical setting via different routes of exposure (e.g. subcutaneous or oral). Many key questions in relation to potential immunological effects of EE are unanswered. Important variables in estrogen-modulation of the immune system include: (i) dose, (ii) age, (iii) gender, and (iv) route of exposure. Thus, pertinent questions emerge. Does exposure to EE at low concentrations for a subacute duration affect the immune or reproductive systems? Are the effects similar in both hormones and between sexes? Are these effects similar in juvenile and aged mice? How do the effects compare across two common routes of exposure (subcutaneous versus oral)? To address these questions, three separate studies were performed. In the first study, we investigated whether very low, but environmentally relevant, doses of EE, E2 (10 ng/kg body weight), or vehicle orally administered every other day for 21 days to young (6 week-old) and aged (>15 month-old) C57BL/6 mice had immunomodulatory effects. As expected, significant gender and age-related differences were noted with regard to thymus weight, thymocyte recovery, spleen weight, and splenocyte recovery. However, low dose treatment of either E2 or EE had no marked effects on the thymus or spleen organ to body weight ratios, cell numbers, or lymphocyte subsets. Low dose oral estrogens did not alter the ability of activated splenocytes to induce interferon-γ or nitric oxide. No effects on male reproductive organ to BW ratios of young or aged mice were found. Similarly, with the exception of E2-stimulating effects on the female reproductive tract of young mice, there were no pronounced effects in females. In separate studies, intact juvenile female and male C57BL/6 mice were given daily subcutaneous (second study) or oral (third study) doses of either EE or E2 (0.04, 0.4, or 4.0 μg per 25 g BW) for 21 days. In the subcutaneous exposure study, both EE and E2 morphologically altered uterine and seminal vesicle weights. However, EE had a more pronounced effect compared to E2, especially in males, even at the lowest dose administered. Additionally, like E2, EE induced thymic atrophy in both sexes. In female mice, thymic atrophy and thymic cellularity were significantly decreased by subcutaneous EE and E2 at doses of 0.4 and 4.0 μg/25 g body weights. EE elicited significantly more pronounced thymic atrophy-inducing effects compared to E2 at the 4.0 μg/25g dose. In males, thymocyte cellularity was decreased by both subcutaneous EE and E2 only at the highest dose tested (4.0 μg/25 g body weight), whereas only 4.0 EE significantly decreased thymus to body weight ratios. Neither splenic weights, splenic cellularity, nor splenic cell phenotype were affected by either estrogenic compound regardless of route of exposure. Oral exposure of EE or E2 did not induce marked immunological effects. Collectively, these data demonstrate that select thymic and reproductive endpoints are significantly altered following a 21-day subcutaneous exposure to either EE or E2 and that the thymus is a more sensitive target than the spleen with regard to subacute exposure to EE. In addition, EE at a comparable dose was more potent than E2 at exerting thymic and reproductive organ morphological alterations. Furthermore, route of administration is critical, as subcutaneous exposure induced far more dramatic thymic and reproductive morphological alterations than did oral administration. Future studies need to address the precise mechanism through which EE induces thymic atrophy and diminished thymus cellularity. Are these effects mediated directly through the thymus, perhaps through estrogen-induced increased thymocyte apoptosis or alterations to thymic epithelial cells? Or could EE be mediating alterations via bone marrow stem cells targeted for distribution to the thymus? Our novel findings regarding EE-induced effects on the thymus are of health significance and set the stage for future work. / Master of Science

A perimenopausa representa a transição da vida reprodutiva para não reprodutiva. É geralmente caracterizada por alterações neuroendócrinas, metabólicas e comportamentais, um possível resultado da depleção folicular ovariana e consequente redução do número de folículos ovarianos. É o período em que as mulheres podem apresentar maior susceptibilidade a manifestar transtornos afetivos e de ansiedade. A exposição de roedores ao resíduo químico 4-diepóxido de vinilciclohexeno (VCD) é um modelo bem estabelecido para estudos sobre perimenopausa, pois o VCD acelera o processo natural de atresia folicular. Embora as concentrações plasmáticas de estradiol estejam normais ou elevadas durante a perimenopausa, a terapia com estradiol pode ser benéfica para mulheres sintomáticas na perimenopausa. Portanto, o objetivo do presente trabalho foi investigar se a depleção folicular gradativa acelerada pelo VCD resulta em alterações na neuroquímica de ratas fêmeas em núcleos cerebrais que controlam o humor, além de avaliar se o estradiol seria capaz de reverter as possíveis alterações. Ratas da linhagem Wistar (28 dias pós-natal) receberam diariamente, durante 15 dias consecutivos, injeções subcutâneas de VCD (160 mg / kg) ou óleo de milho (O). Aproximadamente 55 dias após a primeira injeção, cápsulas de silastic contendo 17?-estradiol (E) ou O foram inseridas subcutaneamente (Grupos O+O; VCD+O; VCD+E). Cerca de 21 dias após o implante das cápsulas, as ratas dos grupos O+O e VCD+O foram decapitadas na manhã do diestro, enquanto que as do grupo VCD+E foram decapitadas exatamente 21 dias após o implante das cápsulas contendo estradiol, entre 0900 h e 1100 h. O sangue foi colhido para avaliação das concentrações plasmáticas de estradiol e progesterona por radioimunoensaio (RIE). Os cérebros foram removidos para microdissecção do hipocampo, amígdala, Locus coeruleus (LC) e Núcleo Dorsal da Rafe (NDR), para posterior análise dos níveis de RNAm para os receptores de progesterona (PR) e estradiol do tipo beta (ER?) por meio de RT/PCR. Este experimento foi replicado para remoção do hipocampo e amígdala para dosagem dos conteúdos de noradrenalina (NA) e serotonina (5-HT) por meio de cromatografia líquida de alta performance, seguida de detecção eletroquímica (HPLC/ED). Outro conjunto de ratas submetidas às mesmas condições10 experimentais foi perfundido para imunohistoquímica para TPH no NDR e TH no LC. Como esperado, na periestropausa (grupo VCD+O) as concentrações plasmáticas de estradiol não foram diferentes daquelas das ratas controles (O+O). As concentrações plasmáticas de progesterona na periestropausa foram menores que as do grupo controle, o que foi revertido pelo estradiol. No LC, a expressão de PR na periestropausa foi igual à das ratas controles, enquanto a expressão do ER? foi menor; a terapia com estradiol não modificou a expressão de nenhum destes receptores. A densidade de neurônios noradrenérgicos (TH+) no LC não foi alterada nem pela depleção folicular nem pela terapia estrogênica. Na periestropausa, o conteúdo de NA foi menor na amígdala, mas não no hipocampo, e o estradiol não alterou este conteúdo em nenhuma das áreas. No NDR, a expressão de PR e de ER? nas ratas na periestropausa foi menor que nas ratas controles; o estradiol preveniu o declínio da expressão de ER?, mas não de PR. O NDR foi analisado separadamente por toda a extensão rostro-caudal em 3 níveis anatômicos: rostral, médio e caudal, cada um dividido em 3 sub-regiões: lateral, dorsal e ventral. O número de neurônios serotonérgicos (TPH+) no NDR foi menor na periestropausa, e o estradiol foi capaz de reverter esse efeito, atuando principalmente na região caudal. A expressão gênica de PR não foi alterada nem pela depleção folicular nem pela terapia estrogênica tanto na amígdala como no hipocampo. A expressão de ER? também não foi diferente na periestropausa, quando comparada ao grupo controle, mas o estradiol aumentou esta expressão no hipocampo. Tanto na amígdala como no hipocampo houve redução no conteúdo de 5-HT na periestropausa e estradiol foi capaz de reestabelecer os níveis deste neurotransmissor aos valores controles apenas no hipocampo. Estes dados elucidam, pelo menos em parte os mecanismos do efeito positivo da terapia estrogênica nos sintomas de mulheres normoestrogênicas na perimenopausa. Estes efeitos parecem não envolver de forma importante o sistema noradrenérgico central, mas resultar do aumento da biossíntese de progesterona periférica em associação com a regulação positiva de ER? no NDR e hipocampo, que parece potencializar a via serotonérgica NDR/HPC. Portanto, o desenvolvimento de novas terapias que ativem os ER? pode ser uma alternativa para obter os efeitos positivos da ação do estradiol, eliminando os efeitos colaterais das terapias de estradiol que normalmente resultam da ativação do ER?. / Perimenopause represents the transition from reproductive to non-reproductive life. It is usually characterized by neuroendocrine, metabolic and behavioural changes, which result from a follicular depletion and reduced number of ovarian follicles. During this period, women are more likely to express mood disorders and anxiety. The exposure of animals to diepoxide 4-vinylcyclohexene (VCD) is a well-established experimental model for perimenopause studies, as VCD induces loss of ovarian small follicles (primary and primordial) in mice and rats by accelerating the natural process of atresia. Although estrogens levels are normal or even high during perimenopause, estrogen therapy can be beneficial for symptomatic perimenopausal women. The aim of this study was to investigate whether gradual follicular depletion induced by VCD results in changes in the neurochemistry of female rats in brain nuclei that control mood and the role of estradiol on these changes. Female rats (28 days) were daily injected with VCD or corn oil (O) for 15 days. Around 55 days after the first injection, pellets of 17?-estradiol (E) or O were inserted s.c (Groups O+O; VCD+O; VCD+E). Around 21 days after, rats O+O and VCD+O were decapitated between 0900 h and 1100 of diestrus while rats VCD+E were decapitated exactly 21 days after the onset of E therapy. Another set of rats followed the same experimental design and were perfused for TH and TPH immunohistochemistry in Locus coeruleus (LC) and Dorsal Raphe Nuclei (DRN), respectively. Blood was collected for estradiol and progesterone measurement by radioimmunoassay (RIA). The brains were removed from decapitated rats to punch out LC, DRN, hippocampus and amygdala to analyse the expression of mRNA for ER? and PR by RT/PCR. This experiment was replicated to punch out the hippocampus and amygdala for the determination of noradrenaline (NE) and serotonin (5-HT) contents by High Performance Liquid Chromatography, followed by Electrochemical Detection (HPLC/ED). As expected, plasma concentrations of estradiol were not different from those of control rats (O + O). Plasma concentrations of progesterone in the periestropause were lower than those in the control group, which was reversed by estradiol. In the LC, the PR expression in the periestropause was similar to that of the control rats, whereas the ER? expression was lower; estradiol therapy did not modify the expression of any of these receptors. The12 density of noradrenergic (TH +) neurons in LC was not altered by either follicular depletion or estrogen therapy. In periestropause, NA content was lower in the amygdala, but not in the hippocampus, and estradiol did not alter this content in any of the areas. In NDR, the expression of PR and ER? in periestropausal rats was lower than in controls; estradiol prevented the decrease of ER? expression, but not PR. The NDR was analyzed separately for the entire rostrocaudal axis in three anatomical levels: rostral, middle and caudal, each divided into three sub-regions: lateral, dorsal and ventral. The number of serotonergic neurons (TPH +) in NDR was lower in the periestropause, and estradiol was able to reverse this effect, acting mainly in the caudal region. PR gene expression was not altered by either follicular depletion or estrogen therapy in either the amygdala or the hippocampus. ER? expression was also no different in periestropause compared to the control group, but estradiol increased this expression in the hippocampus. Both in the amygdala and in the hippocampus there was a reduction in 5-HT content in the periestropause, and estradiol was able to reestablish the levels of this neurotransmitter at the control values only in the hippocampus. These data elucidate, at least in part, the mechanisms of the positive effect of estrogen therapy on the symptoms of normoestrogenic women in perimenopause. These effects do not appear to significantly involve the central noradrenergic system but result from increased peripheral progesterone biosynthesis in association with positive regulation of ER? in the NDR and hippocampus, which appears to potentiate the serotonergic NDR/HPC pathway. Therefore, the development of new therapies that activate ER? may be an alternative to obtain the positive effects of the estradiol action, eliminating the side effects of the estradiol therapies that normally result from the activation of ER?.

A perimenopausa representa a transição da vida reprodutiva para não reprodutiva. É geralmente caracterizada por alterações neuroendócrinas, metabólicas e comportamentais, um possível resultado da depleção folicular ovariana e consequente redução do número de folículos ovarianos. É o período em que as mulheres podem apresentar maior susceptibilidade a manifestar transtornos afetivos e de ansiedade. A exposição de roedores ao resíduo químico 4-diepóxido de vinilciclohexeno (VCD) é um modelo bem estabelecido para estudos sobre perimenopausa, pois o VCD acelera o processo natural de atresia folicular. Embora as concentrações plasmáticas de estradiol estejam normais ou elevadas durante a perimenopausa, a terapia com estradiol pode ser benéfica para mulheres sintomáticas na perimenopausa. Portanto, o objetivo do presente trabalho foi investigar se a depleção folicular gradativa acelerada pelo VCD resulta em alterações na neuroquímica de ratas fêmeas em núcleos cerebrais que controlam o humor, além de avaliar se o estradiol seria capaz de reverter as possíveis alterações. Ratas da linhagem Wistar (28 dias pós-natal) receberam diariamente, durante 15 dias consecutivos, injeções subcutâneas de VCD (160 mg / kg) ou óleo de milho (O). Aproximadamente 55 dias após a primeira injeção, cápsulas de silastic contendo 17?-estradiol (E) ou O foram inseridas subcutaneamente (Grupos O+O; VCD+O; VCD+E). Cerca de 21 dias após o implante das cápsulas, as ratas dos grupos O+O e VCD+O foram decapitadas na manhã do diestro, enquanto que as do grupo VCD+E foram decapitadas exatamente 21 dias após o implante das cápsulas contendo estradiol, entre 0900 h e 1100 h. O sangue foi colhido para avaliação das concentrações plasmáticas de estradiol e progesterona por radioimunoensaio (RIE). Os cérebros foram removidos para microdissecção do hipocampo, amígdala, Locus coeruleus (LC) e Núcleo Dorsal da Rafe (NDR), para posterior análise dos níveis de RNAm para os receptores de progesterona (PR) e estradiol do tipo beta (ER?) por meio de RT/PCR. Este experimento foi replicado para remoção do hipocampo e amígdala para dosagem dos conteúdos de noradrenalina (NA) e serotonina (5-HT) por meio de cromatografia líquida de alta performance, seguida de detecção eletroquímica (HPLC/ED). Outro conjunto de ratas submetidas às mesmas condições10 experimentais foi perfundido para imunohistoquímica para TPH no NDR e TH no LC. Como esperado, na periestropausa (grupo VCD+O) as concentrações plasmáticas de estradiol não foram diferentes daquelas das ratas controles (O+O). As concentrações plasmáticas de progesterona na periestropausa foram menores que as do grupo controle, o que foi revertido pelo estradiol. No LC, a expressão de PR na periestropausa foi igual à das ratas controles, enquanto a expressão do ER? foi menor; a terapia com estradiol não modificou a expressão de nenhum destes receptores. A densidade de neurônios noradrenérgicos (TH+) no LC não foi alterada nem pela depleção folicular nem pela terapia estrogênica. Na periestropausa, o conteúdo de NA foi menor na amígdala, mas não no hipocampo, e o estradiol não alterou este conteúdo em nenhuma das áreas. No NDR, a expressão de PR e de ER? nas ratas na periestropausa foi menor que nas ratas controles; o estradiol preveniu o declínio da expressão de ER?, mas não de PR. O NDR foi analisado separadamente por toda a extensão rostro-caudal em 3 níveis anatômicos: rostral, médio e caudal, cada um dividido em 3 sub-regiões: lateral, dorsal e ventral. O número de neurônios serotonérgicos (TPH+) no NDR foi menor na periestropausa, e o estradiol foi capaz de reverter esse efeito, atuando principalmente na região caudal. A expressão gênica de PR não foi alterada nem pela depleção folicular nem pela terapia estrogênica tanto na amígdala como no hipocampo. A expressão de ER? também não foi diferente na periestropausa, quando comparada ao grupo controle, mas o estradiol aumentou esta expressão no hipocampo. Tanto na amígdala como no hipocampo houve redução no conteúdo de 5-HT na periestropausa e estradiol foi capaz de reestabelecer os níveis deste neurotransmissor aos valores controles apenas no hipocampo. Estes dados elucidam, pelo menos em parte os mecanismos do efeito positivo da terapia estrogênica nos sintomas de mulheres normoestrogênicas na perimenopausa. Estes efeitos parecem não envolver de forma importante o sistema noradrenérgico central, mas resultar do aumento da biossíntese de progesterona periférica em associação com a regulação positiva de ER? no NDR e hipocampo, que parece potencializar a via serotonérgica NDR/HPC. Portanto, o desenvolvimento de novas terapias que ativem os ER? pode ser uma alternativa para obter os efeitos positivos da ação do estradiol, eliminando os efeitos colaterais das terapias de estradiol que normalmente resultam da ativação do ER?. / Perimenopause represents the transition from reproductive to non-reproductive life. It is usually characterized by neuroendocrine, metabolic and behavioural changes, which result from a follicular depletion and reduced number of ovarian follicles. During this period, women are more likely to express mood disorders and anxiety. The exposure of animals to diepoxide 4-vinylcyclohexene (VCD) is a well-established experimental model for perimenopause studies, as VCD induces loss of ovarian small follicles (primary and primordial) in mice and rats by accelerating the natural process of atresia. Although estrogens levels are normal or even high during perimenopause, estrogen therapy can be beneficial for symptomatic perimenopausal women. The aim of this study was to investigate whether gradual follicular depletion induced by VCD results in changes in the neurochemistry of female rats in brain nuclei that control mood and the role of estradiol on these changes. Female rats (28 days) were daily injected with VCD or corn oil (O) for 15 days. Around 55 days after the first injection, pellets of 17?-estradiol (E) or O were inserted s.c (Groups O+O; VCD+O; VCD+E). Around 21 days after, rats O+O and VCD+O were decapitated between 0900 h and 1100 of diestrus while rats VCD+E were decapitated exactly 21 days after the onset of E therapy. Another set of rats followed the same experimental design and were perfused for TH and TPH immunohistochemistry in Locus coeruleus (LC) and Dorsal Raphe Nuclei (DRN), respectively. Blood was collected for estradiol and progesterone measurement by radioimmunoassay (RIA). The brains were removed from decapitated rats to punch out LC, DRN, hippocampus and amygdala to analyse the expression of mRNA for ER? and PR by RT/PCR. This experiment was replicated to punch out the hippocampus and amygdala for the determination of noradrenaline (NE) and serotonin (5-HT) contents by High Performance Liquid Chromatography, followed by Electrochemical Detection (HPLC/ED). As expected, plasma concentrations of estradiol were not different from those of control rats (O + O). Plasma concentrations of progesterone in the periestropause were lower than those in the control group, which was reversed by estradiol. In the LC, the PR expression in the periestropause was similar to that of the control rats, whereas the ER? expression was lower; estradiol therapy did not modify the expression of any of these receptors. The12 density of noradrenergic (TH +) neurons in LC was not altered by either follicular depletion or estrogen therapy. In periestropause, NA content was lower in the amygdala, but not in the hippocampus, and estradiol did not alter this content in any of the areas. In NDR, the expression of PR and ER? in periestropausal rats was lower than in controls; estradiol prevented the decrease of ER? expression, but not PR. The NDR was analyzed separately for the entire rostrocaudal axis in three anatomical levels: rostral, middle and caudal, each divided into three sub-regions: lateral, dorsal and ventral. The number of serotonergic neurons (TPH +) in NDR was lower in the periestropause, and estradiol was able to reverse this effect, acting mainly in the caudal region. PR gene expression was not altered by either follicular depletion or estrogen therapy in either the amygdala or the hippocampus. ER? expression was also no different in periestropause compared to the control group, but estradiol increased this expression in the hippocampus. Both in the amygdala and in the hippocampus there was a reduction in 5-HT content in the periestropause, and estradiol was able to reestablish the levels of this neurotransmitter at the control values only in the hippocampus. These data elucidate, at least in part, the mechanisms of the positive effect of estrogen therapy on the symptoms of normoestrogenic women in perimenopause. These effects do not appear to significantly involve the central noradrenergic system but result from increased peripheral progesterone biosynthesis in association with positive regulation of ER? in the NDR and hippocampus, which appears to potentiate the serotonergic NDR/HPC pathway. Therefore, the development of new therapies that activate ER? may be an alternative to obtain the positive effects of the estradiol action, eliminating the side effects of the estradiol therapies that normally result from the activation of ER?.

Doctor of Philosophy / Department of Animal Sciences and Industry / Jeffrey S. Stevenson / Two experiments assessed pregnancy outcomes (pregnancy per AI; P/AI) after 5-d Ovsynch-56 Resynch (RES; GnRH injection 5 d before [GnRH-1; d 0] and 56 h (GnRH-2) after the last PGF2α [PGF] injection on d 6 given 24 h after first PGF injection on d 5, and TAI on d 8) with and without a 5-d progesterone insert. In Exp. 1, only 76% of 1,023 nonpregnant cows enrolled on d 34 post-AI had high (≥1 ng/mL) progesterone. The RES-CIDR cows with low progesterone at treatment initiation had greater P/AI than RES-CON (37.7 vs. 29.4%), whereas RES-CIDR cows with high progesterone had lesser P/AI than RES-CON (27.4 vs. 34.3%) suggesting that supplemental progesterone is progesterone-dependent. In Exp. 2, 381 cows were enrolled in similar treatments on d 31 with RES on d 41post-AI plus a third treatment including PG-3-G (Pre-PGF on d 31, Pre-GnRH on d 34, and RES on d 41. The P/AI was similar among treatments but was greater in cows starting RES on d 41 when progesterone was low (44%) than high (33%).Experiment 3 determined LH and ovulatory responses in cows enrolled in two treatments before AI: 1) Pre10 (n = 37): PGF-1 and PGF-2 given 14 d apart (Presynch); or PG3G (n = 33): PGF given concurrent with the PGF-2, 3 d before GnRH-1 followed in 7 d by Ovsynch [injection of GnRH (GnRH-2) 7 d before PGF (PGF-3) and GnRH-3 at either 56 or 72 h after PGF-3] that was initiated 10 d after PGF-2 for Pre10 or 7 d after GnRH-1 of PG3G. The GnRH- 1 increased incidences of LH surges and ovulation in PG3G compared with Pre10. The LH in serum of Pre10 was greater than that of cows receiving PG3G after GnRH-2. Following GnRH- 3, cows receiving GnRH at 72 h had increased incidence of spontaneous LH surges before GnRH-3. The P/AI for PG3G vs. Pre10 and for 56 vs. 72 h was similar, but the Pre10-72 h treatment combination was less than all other treatment combinations. Release of LH is protocol dependent and flexibility of GnRH timing is an advantage for PG3G before first-service TAI.

published_or_final_version / abstract / Pharmacology / Doctoral / Doctor of Philosophy

Estradiol (Compudose®, COM) implants are extensively used in beef cattle production systems to alter body composition and feed efficiency. Little information exists about the physiological mechanisms affected by COM treatment in growing, mature, and senescent female cattle. Moreover, no reports describe the level of blood estradiol resulting from COM treatment. The effect of COM on levels of plasma estradiol and blood metabolites and proteins, and relative content of glutamine synthetase (GS) and other amino acid nitrogen-metabolizing enzymes in liver tissue, was studied using three experimental models relevant to cow-calf production regimens: senescent cows (Trial 1), young mature (young) versus senescent (old) cows (Trial 2), and growing heifers (Trial 3). In Trial 1, plasma estradiol concentrations were 222 % more after 14 and 28 d in COM-implanted than sham implanted (Control) cows. COM treatment did not affect measured blood metabolites and enzymes, but increased hepatic GS protein expression by 350% after 14 d and 200% after 28 d of implantation. In contrast, protein expression of alanine transaminase, aspartate transaminase, glutamate dehydrogenase, and two glutamate transporters was not affected by COM. In Trial 2, plasma estradiol concentrations of COM implanted young and old cows were 48% higher than Control groups, whereas blood metabolites were not affected. COM implantation did not affect GS protein expression in young cows, but tended to increase GS expression in the old cows by 283% after 14 d and 41% after 28 d. GS mRNA content was increased about 38% in both young and old COM-treated cows. Hepatic content of beta-catenin and G protein-coupled receptor 30 (GPR30) content was not affected by COM treatment, indicating that estradiol-mediated GS expression was not regulated by beta-catenin- or GPR30-controlled pathways. In Trial 3, plasma estradiol levels in COM-treated heifers were 70% higher in COM heifers, concomitant with increased levels of total bilirubin and creatine kinase, and decreased creatinine. Correlation analysis of plasma estradiol levels and blood constituents only identified a positive correlation between plasma estradiol and potassium. Collectively, these data describe positive estradiol-mediated effects on hepatic metabolism and blood parameters in female cattle.

Demands are placed on cattle producers to provide a steady supply of beef at a competitive price. Producers must maximize beef output while minimizing input expenses without compromising product quality. The use of growth implants has become a common practice among cattle producers. The objective of this study was to evaluate the effects of two implant strategies on the average daily gain of suckling calves rotationally grazed on Kentucky – 31 endophyte-infected tall fescue and Kentucky – 31 non-endophyte-infected tall fescue. Eighteen cows with spring calves (N = 18) were used in this study. Calves were grouped by birth date into four blocks. Within each block, calves were stratified by sex and 45d of age body weight into three implant treatment groups for a total of six calves per treatment (n = 6, control 90.3 ± 9.7 kg, zeranol 102.9 ± 10.9 kg, and progesterone (100 mg) and estradiol benzoate (10 mg) 92.4 ± 10.3 kg). Calves were weighed and re-implanted at 129 d of age (84 d after initial implant). Zeranol treated calves were re-implanted using the same implant as the initial implant. Progesterone and estradiol benzoate treated calves were re-implanted after reaching a minimum body weight of 181 kg with either 200 mg progesterone and 20 mg estradiol benzoate or 200 mg testosterone propionate and 20 mg estradiol benzoate depending on sex. Data were analyzed using the REPEATED function in the MIXED procedure of SAS. No interactions were found among sex and treatments for 84d weight gains and 140d weights. There were also no main effects found for 84d weight gains and 140d weight gains. Forage analysis suggested that low crude protein and energy content may have contributed to the low ADG. Low endophyte concentrations may also have played a role.

El efecto del estradiol (E2) y la progesterona (P4), aplicadas alrededor del momento de reconocimiento maternal de la preñez (RMP), 8-10 días gestación, sobre la sobrevivencia embrionaria (SE), fue evaluada en llamas. Para éste propósito, hembras adultas con descanso post parto ³ 15 días, fueron ecografiadas para determinar presencia de un folículo preovulatorio (³7mm) y luego fueron sometidas a empadre con macho fértil por un tiempo de cópula ³15 minutos. Se seleccionaron 80 llamas y fueron distribuidas al azar en 4 grupos: G0 (placebo/animal/día, días 8 y 9 post cópula); G1 (0.2 mg/animal/día estradiol, días 8 y 9 post cópula); G2 (15mg/animal/día proligestona, días 8 y 9 post cópula) y G3 (0.2 mg/animal/día estradiol y 15 mg/animal/día proligestona, días 8 y 9 días post cópula). El día del empadre fue considerado el día 0. Posteriormente se hicieron evaluaciones ecográficas: día 2 para determinar ovulación por desaparición del folículo dominante; el día 9 para determinar tamaño y presencia del cuerpo lúteo; y los días 20, 25, 30 y 35 para observar vesícula embrionaria y determinar presencia del embrión. La conducta sexual fue evaluada para determinar receptividad frente al macho el día 0 y para diagnosticar gestación temprana el día 15. Del total de hembras empadradas, sólo dos no ovularon las que fueron separadas del análisis de sobrevivencia. La tasa de sobrevivencia embrionaria (TSE) a partir del día 20 fue mayor en el G1 (75 %), observándose una elevada tendencia hacia un mayor efecto en comparación con el G0 (57.89%), G2 (63.16%) y G3 (55%); permaneciendo esta tendencia alta en G1 (75%) hasta el día 35 en comparación con el G0 (57.89%), G2 (52.63%) y G3 (55%), pero sin encontrarse diferencia estadística entre grupos (p<0.05). Por otro lado al evaluar el tamaño del cuerpo lúteo, medido el día 9, y al relacionar con la SE al día 35, se encontró que el diámetro promedio en preñadas (12.79mm) fue mayor que en no preñadas (10.77mm). En conclusión, estos resultados indican que con la aplicación de E2 entre los días 8 y 9 post servicio se presentaría una mejor respuesta sobre la tasa de sobrevivencia embrionaria. Palabras clave: Estradiol, progesterona, sobrevivencia embrionaria, reconocimiento maternal de la preñez, llamas.

Memoria para optar al título profesional de Médico Veterinario / En este estudio se realizó un programa de manejo del ciclo estral en yeguas fina sangre de carrera (F.S.C.), mediante la utilización de un dispositivo intravaginal con 1,55 g de progesterona y 10 mg de benzoato de estradiol (PRID), durante el inicio de la temporada reproductiva de 2000, entre los meses de agosto y diciembre. Se utilizaron 28 yeguas provenientes de 3 haras de la Región Metropolitana, las cuales fueron distribuídas al azar en dos grupos, el grupo control (n = 15) y el grupo tratado (n = 13) con el PRID durante 12 días desde el inicio de la temporada reproductiva en el hemisferio Sur. En ambos grupos se midió por medio del ultrasonido: el tamaño ovárico, el número de folículos ováricos a 5 mm de diámetro y el diámetro del folículo dominante; y por medio del radioinmunoanálisis (RIA) se analizaron las concentraciones séricas de progesterona. Las muestras se tomaron el primer día, sexto día, duodécimo día y al tercer día de celo durante el ensayo. El primer día del ensayo se determinó como el inicio de la temporada reproductiva. Todas las yeguas fueron cubiertas cerca del momento de la ovulación. El diagnóstico de gestación se realizó a los 15 días post-monta por medio de ultrasonografía. En ambos grupos de yeguas la duración del primer y segundo ciclo estral al inicio de la temporada reproductiva fueron similares (p > 0,05). El tiempo transcurrido entre el inicio de la temporada reproductiva y el inicio del primer celo no difirieron (p > 0,05) entre las yeguas tratadas y las yeguas controles. En las yeguas tratadas se necesitó un menor número de celos (p < 0,05) durante la temporada reproductiva para quedar preñadas respecto a las yeguas controles. El primer día de muestreo las yeguas tratadas mostraron un mayor número de folículos en ambos ovarios (p < 0,05) respecto a las yeguas controles, pero en los siguientes días de muestreo no se observaron diferencias significativas (p > 0,05). En cada uno de los cuatro tiempos de muestreo, el diámetro del folículo preovulatorio no fue diferente (p > 0,05) entre yeguas tratadas y controles. El análisis de regresión de la variable días desde el inicio de la temporada reproductiva y diámetro del folículo preovulatorio indicó que existe una asociación (p < 0,05), para ambos grupos de yeguas. Las yeguas tratadas tuvieron su primera ovulación antes que las yeguas controles (p < 0,05; 18 ± 3,76 y 36,5 ± 28,9 días, respectivamente) desde el inicio del tratamiento. El lapso de tiempo entre el inicio del primer celo de la temporada reproductiva y la primera ovulación de la estación fue menor para las yeguas tratadas (4,15 ± 1,51 días; p < 0,05) que para las yeguas controles (6,9 ± 3,66 días; p < 0,05). Las concentraciones séricas de progesterona fueron similares para ambos grupos de yeguas en los distintos tiempos de muestreo (p > 0,05). El análisis de regresión mostró que no existe una asociación (p > 0,05) entre las concentraciones plasmáticas de progesterona y el tiempo desde el inicio de la temporada reproductiva durante el ensayo, para ambos grupos de yeguas. El análisis de regresión mostró que no existe una asociación (p > 0,05) entre el diámetro del folículo dominante y las concentraciones séricas de progesterona durante el ensayo, para ambos grupos de yeguas. En el grupo control se realizó un mayor número de montas durante la temporada reproductiva en comparación con las yeguas tratadas (p < 0,05). El 61,53% de las yeguas tratadas y el 26,66% de las yeguas controles quedaron preñadas durante el primer celo de la temporada reproductiva (p > 0,05); el 30,76% de las yeguas tratadas y el 6,66% de las yeguas controles quedaron preñadas durante el segundo celo de la temporada reproductiva (p < 0,05). Las yeguas tratadas quedaron preñadas antes que las yeguas controles a partir del inicio de la temporada reproductiva (p < 0,05; 27,75 ± 17,06 y 51,5 ± 33,42 días, respectivamente). El número de yeguas que presentó mellizos no difiere (p > 0,05) entre los grupos. Del presente estudio se concluye que el uso de un dispositivo intravaginal liberador de progesterona y estradiol al inicio de la temporada reproductiva, muestra un efecto positivo sobre la fertilidad equina, siendo una herramienta útil en la sincronización del celo y de la ovulación en yeguas que se encuentran en la etapa de transición, lo cual ayuda a predecir en forma más exacta el momento de la monta.