Abstract The luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) that has a large N-terminal ligand binding ectodomain. The LHR ectodomain splice variant, expressed concomitantly with the full-length LHR in tissues, has an unknown biological function. GPCRs are a major pharmacological target, however, very little is known about the intracellular regulation of these receptors. In the present work, expression and maturation of the rat LHR and its variant were elucidated using both tissues and heterologous expression systems. A special effort was made to identify the role of developmental stage and tissue type on the LHR maturation and to find out about the molecular role of the ectodomain splice variant. We found two sites of localization for the receptor, namely the sensory system and urogenital tissues. This was demonstrated at mRNA and protein level and by rat LHR promoter-driven β-galactosidase (β-Gal) expression in the mice. In neurons, the β-Gal co-localized with the cytochrome P450 side chain cleavage enzyme, which may indicate a novel role in the neurosteroid synthesis. The neuronal LHR was expressed in the mature and immature protein forms in both developing and adult tissues, being able to bind hormone with similar high-affinity as gonadal receptors. In contrast, only immature receptors were detected in the fetal rat urogenital structures. A significant novel finding was substantial upregulation of the LHR in pregnant female rat adrenal glands and kidneys at a time that coincides with the differentiation of the fetal urogenital tissues. The mice overexpressing the ectodomain splice variant showed interference in pituitary-gonadal functions and morphological changes in the urogenital tissues. The studies showed that the variant was an endoplasmic reticulum (ER)-retained soluble protein. It accumulated in juxtanuclear regions of the ER together with ER folding chaperones and was a substrate for ER associated degradation (ERAD). The co-expression of the variant with the full-length receptor decreased the amount of receptors and misrouted them to the juxtanuclear ER subcompartment. Taken together, we suggest that the maturation of the LHR protein is developmentally and physiologically regulated at the post-translational level in tissues. The LHR ectodomain splice variant possibly modulates post-translationally the number of full-length receptors through physiological signals. Our observation of the chaperone and protein accumulation into a specific ER subcompartment may represent a protein quality control holding compartment for inefficiently/misfolded ERAD substrates.

The role of endogenous dopamine, utilizing a dopamine antagonist (fluphenazine; FLU), in modulation of gonadotropin, growth hormone (GH) and prolactin (PRL) secretion during the early postpartum period and various stages of the estrous cycle was investigated in Holstein cows. Experiment 1 was conducted in anovulatory early postpartum cows. Fluphenazine caused a decrease (P < .05) in mean serum LH concentration and LH pulse frequency. Likewise, FLU caused a (P < .05) decrease in mean GH concentration. These results suggest that endogenous dopamine, at least in part, is responsible for regulation of LH and GH secretion in anovulatory Holstein cows. Experiment 2 was conducted in cyclic lactating Holstein cows during the mid-luteal phase of the estrous cycle. Mean serum LH and FSH concentrations, pulse frequencies, and peak amplitudes did not change in response to FLU. FLU did not affect mean serum GH concentration. These results suggest that a dopamine-mediated mechanism for modulation of gonadotropin and GH secretion is absent or perhaps overridden by high progesterone concentration during the luteal phase of the estrous cycle in lactating dairy cows. Experiment 3 was conducted during the early follicular phase of the estrous cycle in Holstein cows. During the follicular phase, FLU caused a decrease (P < .05) in mean serum LH concentration and LH pulse frequency. However, FLU had no effect on mean serum FSH concentration or pulse frequency. Further, FLU increased (P < .05) GH concentrations during the follicular phase. Experiment 4 was conducted during the early metestrus phase of the estrous cycle. During the metestrus phase, FLU tended to decrease (P < .1) mean LH concentration and suppressed (P < .05) LH pulse frequency but had no effect on FSH secretion. Fluphenazine caused a transient increase (P < .05) in mean serum GH concentration. The results of the third and fourth experiments suggest that, during the early follicular and metestrus phases of the estrous cycle, when progesterone concentration is low, modulation of LH and GH secretion, at least in part, is modulated by endogenous dopamine. However, a dopamine mediated mechanism for FSH secretion is absent during both phases of the estrous cycle in lactating Holstein cows. In all experiments FLU increased (P < .01) PRL secretion indicating that endogenous dopamine suppresses PRL secretion in cattle regardless of ovarian status. It is concluded that: 1) endogenous dopamine plays a stimulatory role in LH secretion during the anovulatory postpartum period and during the estrous cycle only when serum progesterone is low. 2) FLU decreased GH secretion in anovulatory postpartum Holstein cows but it increased GH secretion during the follicular and metestrus phases of the estrous cycle. However FLU had no effect on GH secretion during the luteal phase of the estrous cycle. Thus it appears that, modulation of GH secretion is dependent upon reproductive status and ovarian hormones secretion. / Ph. D.

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No description available.

Inseminação artificial em tempo fixo (IATF) associada à inseminação artificial pós-cervical (IAPC) permite uma maior utilização de machos geneticamente superiores e uma redução expressiva de mão de obra na produção de suínos. O objetivo deste estudo foi avaliar a eficiência da IATF de acordo com diferentes protocolos de (IA), usando pLH - hormônio luteinizante suíno - como indutor da ovulação. Um total de 597 matrizes desmamadas com detecção de estro realizada uma vez ao dia (08:00) foram alocados em três tratamentos: Controle (n = 199) - primeira inseminação realizada no início do estro (0 h) e repetida a cada 24 h, durante o estro; IATF1 (n = 199) - fêmeas receberam 5 mg (4 ml) i.m. de pLH no início do estro, e foram inseminadas 24 horas depois, e IATF2 (n = 199) – fêmeas receberam 5 mg de pLH mas foram inseminadas no início do estro (0 h) e 24 horas depois. Foram realizadas IAPC com doses homospérmicas (1,5 x 109 de espermatozoides totais/50 ml) em todos os tratamentos. O tratamento hormonal não afetou o intervalo do início do estro à ovulação (P> 0,05). O número de inseminações foi de 2,9, 1,0 e 2,0 para Controle, FTAI1 e FTAI2 respectivamente. Não houve diferença entre os tratamentos para taxa de parto e leitões nascidos totais (P> 0,05). Leitões nascidos totais por dose inseminante foi diferente (P <0,0001) entre os tratamentos (4,5, 12,5 e 6,2 para Controle, FTAI1 e FTAI2 respectivamente). O uso de pLH no início do estro associado à uma única inseminação em tempo fixo IAPC 24 horas após, não comprometeu o desempenho reprodutivo de porcas multíparas. / Fixed-time artificial insemination (FTAI) associated to post cervical artificial insemination (PCAI) allows a wider use of high indexed boars and an expressive reduction on labor requirements in swine production. The aim of this study was to evaluate FTAI efficiency according to different AI protocols, using pLH – porcine luteinizing hormone - as ovulation inductor. A total of 597 weaned sows whose estrus detection was performed once daily (08:00 am) were allocated into three treatments: Control (n= 199) – the first insemination was performed at estrus onset (0 h) and repeated every 24 h thereafter, during estrus; FTAI1 (n= 199) - sows received a 5 mg (4 ml) i.m. injection of pLH at estrus onset, and were inseminated 24 h after, and FTAI2 (n= 199) - sows received 5 mg of pLH but were inseminated at estrus onset (0 h) and 24 h after. PCAI with homospermic doses (1.5 x 109 total sperm cells/50 ml) were performed in all treatments. Hormonal treatment did not affect the interval onset of estrus to ovulation (P>0.05). The number of inseminations was 2.9, 1.0 and 2.0 for Control, FTAI1 and FTAI2 respectively. Treatments did not affect farrowing rate and total born (P>0.05). Total piglets born per insemination dose was different (P<0.0001) among treatments (4.5, 12.5 and 6.2 for Control, FTAI1 and FTAI2 respectively). The use of pLH at estrus onset associated to a single fixed-time PCAI 24 h after does not compromise the reproductive performance of multiparous sows.

Ovarian cancer is thought to be derived from the ovarian surface epithelium (OSE), but it is often diagnosed during the late stages and therefore the events that contribute to the initiation and progression of ovarian cancer are poorly defined. Epidemiological studies have indicated an association between the female reproductive hormones and ovarian cancer etiology, but the direct effects of 17β-estradiol (E2), progesterone (P4), luteinizing hormone (LH) and follicle stimulating hormone (FSH) on disease pathophysiology are not well understood. A novel transgenic mouse model of ovarian cancer was generated that utilized the Cre/loxP system to inducibly express the oncogene SV40 large and small T-Antigen in the OSE. The tgCAG-LS-TAg mice developed poorly differentiated ovarian tumours with metastasis and ascites throughout the peritoneal space. Although P4 had no effect; E2 significantly accelerated disease progression in tgCAG-LS-TAg mice. The early onset of ovarian cancer was likely mediated by E2’s ability to increase the areas of putative preneoplastic lesions in the OSE. E2 also significantly decreased survival time in ovarian cancer cell xenografts. Microarray analysis of the tumours revealed that E2 mainly affects genes involved in angiogenesis and cellular differentiation, proliferation, and migration. These results suggest that E2 acts on the tumour microenvironment in addition to its direct effects on OSE and ovarian cancer cells. In order to examine the role of the gonadotropins in ovarian cancer progression, the tgCAG-LS-TAg mice were treated with 4-vinylcyclohexene-diepoxide (VCD) to induce menopause. Menopause slowed the progression of ovarian cancer due to a change in the histological subtype from poorly differentiated tumours to Sertoli tumours. Using a transgenic mouse model, it was shown that E2 accelerated ovarian cancer progression, while P4 had little effect on the disease. Menopause (elevated levels of LH and FSH) altered the histological subtype of the ovarian tumours in the tgCAG-LS-TAg mouse model. These results emphasize the importance of generating animal models to accurately recapitulate human disease and utilizing these models to develop novel prevention and treatment strategies for women with ovarian cancer.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 6, 2010). Thesis advisor: M. Cathleen Kuehl-Kovarik. Includes bibliographical references.

Ovarian cancer is thought to be derived from the ovarian surface epithelium (OSE), but it is often diagnosed during the late stages and therefore the events that contribute to the initiation and progression of ovarian cancer are poorly defined. Epidemiological studies have indicated an association between the female reproductive hormones and ovarian cancer etiology, but the direct effects of 17β-estradiol (E2), progesterone (P4), luteinizing hormone (LH) and follicle stimulating hormone (FSH) on disease pathophysiology are not well understood. A novel transgenic mouse model of ovarian cancer was generated that utilized the Cre/loxP system to inducibly express the oncogene SV40 large and small T-Antigen in the OSE. The tgCAG-LS-TAg mice developed poorly differentiated ovarian tumours with metastasis and ascites throughout the peritoneal space. Although P4 had no effect; E2 significantly accelerated disease progression in tgCAG-LS-TAg mice. The early onset of ovarian cancer was likely mediated by E2’s ability to increase the areas of putative preneoplastic lesions in the OSE. E2 also significantly decreased survival time in ovarian cancer cell xenografts. Microarray analysis of the tumours revealed that E2 mainly affects genes involved in angiogenesis and cellular differentiation, proliferation, and migration. These results suggest that E2 acts on the tumour microenvironment in addition to its direct effects on OSE and ovarian cancer cells. In order to examine the role of the gonadotropins in ovarian cancer progression, the tgCAG-LS-TAg mice were treated with 4-vinylcyclohexene-diepoxide (VCD) to induce menopause. Menopause slowed the progression of ovarian cancer due to a change in the histological subtype from poorly differentiated tumours to Sertoli tumours. Using a transgenic mouse model, it was shown that E2 accelerated ovarian cancer progression, while P4 had little effect on the disease. Menopause (elevated levels of LH and FSH) altered the histological subtype of the ovarian tumours in the tgCAG-LS-TAg mouse model. These results emphasize the importance of generating animal models to accurately recapitulate human disease and utilizing these models to develop novel prevention and treatment strategies for women with ovarian cancer.

O objetivo deste trabalho foi verificar a presença e a localização de receptores para hormônios esteróides e gonadotróficos, através da técnica de imunohistoquímica, pelo método de peroxidase-antiperoxidase (PAP), nos diferentes tecidos que compõe o trato genital da égua e a variação de reatividade destes receptores durante o ciclo estral e no anestro fisiológico. Também se objetivou verificar se há diferença de reatividade em éguas com e sem endometrose. Foram coletadas amostras de útero, cérvice e oviduto, de 41 éguas sem raça definida e com histórico reprodutivo desconhecido, em um abatedouro. Quinze éguas se encontravam em estro, dezoito em diestro e oito éguas em anestro. Concluiu-se que a intensidade e a distribuição da coloração para os receptores de estrógeno (RE), progesterona (RP) e hormônio luteinizante (RLH) variaram de acordo com o tipo de célula e o estágio do ciclo estral. Nas amostras de endométrio observou-se imunorreatividade alta no epitélio luminal para RE e RP tanto no estro quanto no diestro; o epitélio glandular, estroma e miométrio mostraram reatividade moderada para os dois receptores durante as duas fases. Durante o anestro os resultados foram semelhantes aos encontrados durante a fase cíclica. Na avaliação da reatividade para RLH, durante o estro e diestro, o epitélio luminal mostrou reatividade de fraca a moderada, mas no diestro houve maior reatividade média. O epitélio glandular apresentou menor reatividade do que o luminal. No miométrio a coloração foi fraca durante todo o ciclo. Durante o anestro a reatividade foi fraca no epitélio luminal, ausente em quase todas as amostras no epitélio glandular e de fraca a ausente no miométrio. Neste experimento, não foi observada diferença significativa de reatividade entre os endométrios com e sem endometrose, mas as áreas afetadas mostraram coloração assíncrona para RE, RP e RLH. Na cérvice, foi observada imunorreatividade moderada a alta para RE e RP no epitélio luminal, no estroma e no músculo. A intensidade de coloração das células epiteliais e musculares variou pouco entre o estro e o diestro, mas durante o anestro houve maior reatividade no tecido muscular e no estroma. Foi observada reatividade para RLH no epitélio e camada muscular, sem variação significativa nas fases do ciclo. A intensidade de coloração foi de fraca a moderada no epitélio e fraca na camada muscular. No oviduto, observou-se imunorreatividade para RE e RP nos três tecidos, durante a fase cíclica e o anestro. No epitélio, os valores encontrados foram de moderados a altos, sem variação significativa nas três fases. A coloração das células epiteliais do oviduto foi nitidamente irregular, com o núcleo muito corado no que parecem ser células secretoras e pouco corado ou sem coloração nas células ciliadas, refletindo provavelmente as diferentes funções das células epiteliais neste órgão. No estroma a reatividade foi moderada durante a fase luteal, mostrando reatividade mais alta no estro e no anestro. A camada muscular apresentou reatividade máxima para RE no estro e no diestro. A reatividade para RLH no epitélio luminal foi de fraca a moderada durante todo o ciclo. No músculo também foi observada reatividade, porém bem mais fraca do que no epitélio. Durante o anestro somente três das oito amostras apresentaram reatividade no tecido muscular. No diestro foi observada maior reatividade do que no estro. Os resultados do presente estudo evidenciam, pela primeira vez, a presença de receptores para LH nos diferentes tecidos do trato reprodutor extragonadal da égua. Embora existam relatos da expressão e localização de RE e RP no endométrio equino, esta é a primeira vez que se utiliza a técnica de imunohistoquímica para localizar estes receptores na cérvice e no oviduto desta espécie. Foi observada variação individual bastante acentuada entre as amostras, em uma mesma fase cíclica. Provavelmente estes resultados sejam o reflexo da variação entre o dia do ciclo em que os animais se encontravam, bem como da complexidade dos mecanismos envolvidos na presença desses receptores. Os achados deste estudo indicam que tanto os hormônios gonadais quanto o LH atuam por meio de seus receptores nos diferentes tecidos do trato reprodutivo da égua, podendo servir para a elaboração de novas estratégias para melhorar a eficiência reprodutiva nesta espécie. / The aim of this study was to demonstrate the presence and localization of gonadotropic and steroid hormone receptors, in different tissues of the mare genital tract and the different reactivity to these receptors during the endometrial cycle and physiologic anestrus. Another objective was to compare the reactivity to theses receptors in mares with and without endometrosis. Immunohistochemistry was performed using the peroxidase anti-peroxidase technique (PAP). Uterus, cervix and oviduct of 41 criollo mares were collected in an abattoir. There was variation in the intensity of the staining and distribution for estrogen receptors (PR), progesterone receptors (PR) and luteinizing hormone receptors (LHR) with the endometrial cycle and different tissues. The endometrial surface epithelial cells were stained strongly for ER and PR in the estrous and dioestrus; glandular epithelial cells, stromal cells and smooth muscle cells of the myometrium had moderate staining for ER and PR during these two phases and in anestrus too. The immunoreactive score for LHR in the surface epithelial cells during endometrial cycle was weak to moderate but, in general, strong staining was observed in dioestrus. More weak staining intensity was observed in the glandular epithelial cells than luminal epithelial cells. Smooth muscle cells of the myometrium showed weak staining for LHR throughout the endometrial cycle. During the anestrus, the immunoreactivity score was weak in the surface epithelial cells. In general, the glandular epithelium was not stained. Myometrium cells were weak to not staining for LHR, in this phase. In this study there was no significant difference in immunoreactive score for ER, PR and LHR in endometrium with or without endometrosis but fibrotic glands showed different expression patterns of ER, PR and LHR, could evidence for functionally glandular maldifferentiation in endometrosis. The cervical epithelial surface, stromal cells and smooth muscle cells were moderate to strongly staining for ER and PR, with little variation throughout the endometrial cycle but the immunorectivity was strongest during the anestrus in muscular and stromal cells. Surface epithelial cells of cervix were weak to moderate stained for LHR; smooth muscle cells showed weak staining for these receptors. There was no variation during cycle. In the oviduct, epithelial, stromal and muscle cells showed reactivity for RE and RP, during cycle and anestrus. Epithelial cells were moderate to strongly staining for these receptors, with evident irregularity in different types of cells. Apparently ciliated epithelial cells were stained but the intensity was much less than that observed in nonciliated epithelial cells, probably reflecting different functions of these cells. Stromal cells showed moderate staining during dioestrus and strongest reactivity in estrous and anestrus; muscle cells showed strong reactivity for ER throughout the cycle. The reactivity for LHR was weak to moderate throughout the cycle in the epithelial cells and weak in the muscle cells. During anestrus only three strains of muscle cells showed reactivity for LHR. In dioestrus the intensity was strongest. These findings evidence for the firs time the presence for LHR in extra-gonadal reproductive organs of mare. Though there were reports of ER and PR expression in equine endometrium, this is the first report of localization of these receptors in cervix and oviduct of mare using immunohistochemistry. It was found marked individual variation among the strains. These results probably were caused by the variation among the day of cycle and the complexity of mechanisms involved in the presence of these receptors. The findings of the present study allow us to infer that the ovarian steroid hormones nad LH function through their receptors in different tissues of mare reproductive tract, can help us to elaborate new strategies to improve the reproductive efficiency in this specie.