Glycodelin, a homodimeric glycoprotein belonging to the lipocalin superfamily, is synthesised predominantly by the cells of the reproductive system of certain primates including humans. Of the four different known glycoforms of the molecule, glycodelin A (GdA), secreted by the glandular epithelial cells of the endometrium in response to progesterone, is involved in the immunosuppression of the maternal immune response to the semi-allograft fetus. GdA secretion onsets few days after ovulation. In the absence of fertilization, GdA levels drop, but subsequent to a successful fertilization, the concentrations peak till the 12th week of pregnancy and fall steadily to low levels. The importance of GdA has been implicated in implantation, endometrial receptivity, trophoblast invasion and differentiation, and modulating the functions of almost all immune cells. GdA has profound influence on the activity of T cells. It inhibits the proliferation of T cells, induces apoptosis in activated T cells, inhibits the IL-2 production and leads to skewing of the Th-1/Th-2 balance towards Th-2 type of immune response. Cytotoxic T lymphocytes are more resistant to the induction of apoptosis by GdA, but, it suppresses their cytolytic activity Additionally, GdA induces apoptosis in monocytes and natural killer (NK) cells, inhibits the proliferation of B cells and induces tolerogenic phenotype in dendritic cells. Clinical studies showing that women undergoing recurring spontaneous abortions have low levels of GdA supports its role in prevention of fetus rejection. The immunomodulatory activity of Gd resides in the protein backbone, however, apart from GdA and GdF which have similar oligosaccharide chains, other glycoforms do not possess this activity. Glycosylation seems to dictate the stability, folding and activity of Gd. In absence of glycosylation, the expression of the recombinant Gd is compromised and the protein is improperly folded while over-mannosylation of Gd impairs its immunomodulatory function. Additionally, sialylation seen on the glycan chain regulates the activity. Therefore, in order to obtain adequate amounts of active recombinant Gd (rGd), expression of the protein was attempted in three different systems, insect, yeast and bacteria (Chapter 1). In all of the described systems, the rGd protein was found apoptotically active. The protein expressed in the Sf21 insect cells was demonstrated to be differentially glycosylated compromising the activity. Hence, a genetically modified yeast strain, Pichia pastoris SuperMAN5 was explored for expression. Though presence of a single glycosylated protein species was observed in small-scale cultures, similar to the case of Sf21 cell expression, differentially glycosylated proteins were detected in large-scale fermentation and even the yield was low. Eventually, mutant Gd, modified to increase the stability and aid in proper protein folding, was expressed in E.coli and demonstrated to be able to induce apoptosis in Jurkat cells (T cell leukemia cell line). This active rGd was used for further studies. The immunomodulatory function of GdA during pregnancy protects the semi-allograft fetus from rejection by the maternal immune system. In the process, GdA tweaks the T cell immune response from pro-inflammatory to anti-inflammatory in a specific and localized manner. Allograft rejection seen during mis-match transplantations is basically a pro-inflammatory condition which is mediated by the activation of cellular immune response, NK cell cytotoxicity and antibody-dependent immune response, the same processes that are suppressed for a successful pregnancy. Chapter 2 discusses whether it is feasible to use Gd to prevent allograft rejection. Killing of target graft cells by the cytotoxic T lymphocytes (CTLs) predominantly presides acute graft rejection. GdA treatment has been shown to suppress the cytotoxicity of in vitro generated CTLs. On this basis, the earlier study was translated to in vivo conditions by establishing an allograft nude mouse model. The tumor rejection mediated by the action of in vitro generated cytotoxic alloactivated PBMCs in the nude mouse imitated the allograft rejection. A heterogenous population of immune cells with the predominance of CTLs was chosen to accommodate a more interactive immune response in the tumor microenvironment and enabled the study of other cells which may contribute to the rejection. Reactivation and proliferation of CD4+ and CD8+ T cells following their infiltration in the tumor validated our hypothesis. On treatment with rGd, the cytotoxicity of the alloactivated PBMCs was suppressed, thereby inhibiting the tumor rejection in the nude mouse. Real time PCR analysis showed that rGd treatment was able to affect the functions of the immune cells in vivo. It decreased the T cell population most probably by inducing apoptosis. As expected, the reduction was more prominent in case of CD4+ T cells than CD8+ T cells. The their expression of key molecules responsible for the cytotoxicity such as IL-2, granzyme B and EOMES, was observed to be downregulated by rGd. Concomitantly, decreased levels of pro-inflammatory cytokines, TNFα and IL-6 were also seen. Expression of Foxp3, marker for regulatory T cells, was upregulated in the tumor infiltrating immune cells suggesting an expansion of the concerned population upon rGd treatment. Overall, rGd seems to suppress the cellular immune response to the tumor by modulating the T cell population and their functions. Since, T cell-dependent immune response is central to allograft rejection, the ability of rGd to regulate it could be of therapeutic use in the management of allograft rejection. NK cells are essential for the maintenance of pregnancy, evident from their abundance (70% of total leukocytes) at the first trimester decidua. The third chapter focuses on how Gd regulates the NK cell function. The cytokine production from CD56bright subset of NK cells and their interaction with the HLA antigens expressed by the trophoblast cells helps in creating a favourable environment for the growth of the fetus. It is important to note that the NK cell population present in the decidua exclusively express Gd, implicating a role of Gd in their differentiation from the peripheral CD56bright cells. However, an increased number of CD56dimCD16+ cells in the peripheral blood dictates a negative outcome for the pregnancy. The study, presented in Chapter 3, demonstrated that rGd treatment induces caspase-dependent apoptosis in the activated CD56dimCD16+ cells and reduces their cytotoxicity by downregulating granzyme B and IFNγ production. Similar effect of rGd is also seen on the NKT cells characterised as CD3+CD56dimCD16-. Furthermore, in YT-Indy cells, an activated NK cell line, it was shown that the induction of apoptosis by rGd involves Ca2+ signalling which could explain why Gd affects activated immune cells only. This study therefore reinforces the role of Gd in modulating the NK cell activity during pregnancy. Cytotoxicity of NK and NKT cells also plays an important role during allograft rejection. Decrease in the mRNA levels of CD56 upon rGd treatment in the allograft mouse model indicates that the effect of Gd on NK cells observed in cell culture system can be translated to in vivo conditions. In conclusion, suppression of the cellular immune response and NK cell mediated cytotoxicity by rGd could potentiate its’ probable use in the management of allograft rejection. .

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.

Este trabalho teve por objetivo avaliar a dinâmica folicular de vacas de corte tratadas com protocolos para a sincronização da ovulação onde se utilizou implantes vaginais impregnados com progesterona ou com acetato de medroxi-progesterona e um implante auricular impregnado com norgestomet, além da utilização de ésteres de estradiol em diferentes doses. Foram utilizadas nove vacas Braford, multíparas, não-lactantes, cíclicas e com CC>=3. Os animais foram mantidos confinados e alimentados com feno e ração. Os animais foram divididos em três tratamentos, sendo que todos os animais passaram pelos três tratamentos em quatro repetições. Em cada repetição os animais foram divididos entre os três grupos de forma homogênea. No grupo 1 (n = 14), os animais receberam no dia 0, uma esponja vaginal impregnada com 250 mg de acetato de medroxi-progesterona (MAP) e uma aplicação intramuscular (i.m.) de 2 mg de benzoato de estradiol (BE); no dia 8, a esponja vaginal foi retirada e aplicado i.m. 0,5 mg de Cloprostenol e 24 horas após foi aplicado i.m. 1 mg de BE. No grupo 2 (n = 7), os animais receberam no dia 0, um implante vaginal de silicone impregnado com 1 g de progesterona e uma aplicação i.m. de 2 mg de BE; no dia 8, o implante foi retirado e aplicado i.m. 0,5 mg de Cloprostenol e 24 horas após foi aplicado i.m. 1 mg de BE. No grupo 3 (n = 8), os animais receberam no dia 0, um implante auricular de silicone impregnado com 3 mg de Norgestomet e uma aplicação i.m. de 3 mg de Norgestomet e 5 mg de Valerato de Estradiol (VE); no dia 9 o implante auricular foi retirado. Os animais foram examinados diariamente por via trans-retal com um ultra-som equipado com um transdutor linear de 8 MHz para monitorar a dinâmica folicular e luteal nos ovários. Após a retirada do implante auricular no grupo 3 e após a aplicação de 1 mg de BE nos grupos 1 e 2, os animais foram avaliados por ultra-sonografia duas vezes por dia até que a ovulação fosse detectada. No grupo 1, sete animais perderam a esponja vaginal impregnada com MAP e os dados foram retirados das análises. Os três tratamentos foram eficientes em promover uma nova onda de crescimento folicular sincronizada em 3,7 ± 1,1, 3,7 ± 0,7 e 4,9 ± 1,1 dias (P = 0,059) nos grupos 1, 2 e 3, respectivamente. Além de promover a ovulação de um folículo dominante com poucos dias de dominância e com diâmetro satisfatório em 66 ± 12, 66 ± 0 e 70,5 ± 12,7 horas após a retirada do implante (P = 0,62) nos grupos 1, 2 e 3, respectivamente. Portanto, os três protocolos foram eficientes em promover a sincronização da ovulação e são indicados para utilização em programas de inseminação artificial a tempo fixo em vacas de corte. / This experiment was done to evaluate the ovarian follicular dynamics in beef cows treated with protocols of synchronization of ovulation where were used vaginal implants impregnated with progesterone or with medroxi-progesterone acetate and an ear implant impregnated with norgestomet, than the use of estradiol esters in different doses. Nine Braford cows, multiparous non-lactating, cyclic and BCS>=3 where used in these trial. The animals were kept confined and fed with hay and concentrated. The animals were divided into three treatments, which all animals went for three treatments in four replicates. In each replicate the animals were divided among groups homogeneously. Group 1 (n = 14), animals received on day 0, a vaginal sponge impregnated with 250 mg medroxi-progesterone acetate (MPA) and an intramuscular (i.m.) application of 2 mg of estradiol benzoate (EB); on day 8, the vaginal sponge was removed and applied i.m. 0.5 mg cloprostenol and 24 hours latter was applied i.m. 1 mg of EB. Group 2 (n = 7), the animals received on day 0, a silicone vaginal implant with 1 g of progesterone and an application i.m. of 2 mg of EB; on day 8, the implant was removed and applied i.m. 0.5 mg cloprostenol and 24 hours latter was applied i.m. 1 mg of EB. Group 3 (n = 8), the animals received on day 0, a silicone ear implant impregnated with 3 mg norgestomet and an application i.m. of 3 mg norgestomet and 5 mg of estradiol valerate; on the 9th day the implant was removed. From day 0, animals were examined daily by trans-rectal ultrasound with an 8 MHz linear transducer to monitor follicular and luteal dynamics. After removal of the implant in group 3 and after application of 1 mg EB in the other groups, the animals were evaluated by ultrasound two times per day until ovulation was detected. In group 1, seven animals lost a vaginal sponge impregnated with MPA and the data were removed from analysis. The three treatments groups were effective in promoting a new follicular wave emergence synchronously in 3,7 ± 1,1, 3,7 ± 0,7 and 4,9 ± 1,1 days (P = 0,059) in the groups 1, 2 and 3, respectively. Besides promoting ovulation a dominant follicle with few days of dominance and with satisfactory diameter in 66 ± 12, 66 ± 0 and 70,5 ± 12,7 hours after implant removal (P = 0,62) in the groups 1, 2 and 3, respectively. Therefore, the three protocols were effective in promoting the synchronization of ovulation and are indicated for use in fixed-time artificial insemination programs in beef cows.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-05-04T12:31:58Z No. of bitstreams: 1 Guilherme Aniceto Veras.pdf: 755618 bytes, checksum: abf9279c842f5f903daa9429771d78c0 (MD5) / Made available in DSpace on 2017-05-04T12:31:58Z (GMT). No. of bitstreams: 1 Guilherme Aniceto Veras.pdf: 755618 bytes, checksum: abf9279c842f5f903daa9429771d78c0 (MD5) Previous issue date: 2013-08-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The aim of this study was to evaluate the effect of different progesterone concentrations (P4) on follicular dynamics in Holstein heifers (Bos taurus) subjected to synchronization of estrus and ovulation. The experiment was conducted at the Experimental Station of São Bento do Una/PE Agronomic Institute of Pernambuco / IPA, between September and December 2012. There were used 19 pubertal Holstein heifers presenting BSC between 3.0 and 3.5 and kept in intensive system. The heifers were divided into two groups, receiving an intravaginal devices at D0 containing 0.75 g of P4 in G1 (n = 10) and 1 g of P4, in G2 (n = 9) along with 2 mg BE im. At D7, there were applied 530μg of cloprostenol and 300 IU of eCG im, at D8 intravaginal devices were removed. At D9, heifers in both groups received 1mg of EB im and at D10 TAI were preceded. The ovarian activity was monitored at D0, D4, D8, D10 and at each 6 hours after TAI until ovulation. Pregnancy diagnosis were performed 30 days after TAI. The data were subjected to analysis of variance (ANOVA) and means were compared by the test of Turkey at 5% by the statistical program SPSS 16 for Windows. Binomial variables were analyzed by chi-square. No significant effect was observed between wave synchronization (100 vs. 100%), ovulation rate (70 vs. 89%), time of ovulation (60.5 ± 2.65 vs. 64.5 ± 0.33 h) and conception rate (40 vs. 44.4%) between G1 and G2, respectively. The dominant follicle average diameter at D9 between heifers that ovulated from G1 was 14,8±0,6 mm and in G2 11,2±1,6mm presenting no statistical difference (P=0.07) the same way was not also observed statistical difference between heifers that did not ovulate from G1 (9.6±0.7mm and in G2 (7.7±0.03)(P=0.42). .) Already when comparing the average diameters of dominant follicles in D9 between the heifers that ovulated (14.8 ± 0.6 mm) and those that did not ovulate (9.6 ± 0.7 mm) from G1, it was observed significant difference (P ˂ 0.05), as well as from G2 (11,2 ± 1.6 vs. 7.7 ± 0.03 mm). The ovulation rate increased significantly in the follicles larger than 11 mm, measured on D9 However, conception rates were higher when the follicles were larger than 8 mm on D9. In the present study, no significant effect was observed in DFØ, OFØ, time of ovulation, ovulation and conception rates in pubertal Holstein heifers, but animals receiving device with lower concentrations of P4 in D0 showed higher daily rate growth follicular. / O objetivo deste estudo foi avaliar o efeito de diferentes concentrações de progesterona (P4) sobre a dinâmica folicular em novilhas Holandesas (Bos taurus) submetidas à sincronização de estro e ovulação. O experimento foi realizado na Estação Experimental de São Bento do Una/PE do Instituto Agronômico de Pernambuco/IPA, entre setembro e dezembro de 2012. Foram utilizadas 19 novilhas púberes Holandesas, com ECC entre 3,0 e 3,5 e mantidas em sistema intensivo. Foram distribuídas em dois grupos, onde no D0, receberam implantes intravaginal contendo 0,75g de P4 no G1 (n=10) e 1g de P4 no G2 (n=9), associado a 2 mg de BE i.m. No D7, foram aplicados 530μg de cloprostenol sódico e 300 UI de eCG i.m. e no D8 os implantes intravaginais foram removidos. No D9, foi aplicado 1mg de BE i.m. em ambos os grupos e no D10 a IATF. A atividade ovariana foi acompanhada no D0, D4, D8, D10 e após a IA de 6/6 horas até a ovulação. O diagnóstico de gestação, realizado 30 dias após a IATF. Os dados foram analisados pelo SPSS 16.0, empregando-se a análise de variância (ANOVA). Sendo evidenciada diferença estatística, aplicou-se o teste de Turkey. As variáveis binominais foram analisadas pelo Qui-quadrado. Não foi observado efeito significativo entre a sincronização da onda (100 vs. 100%), taxa de ovulação (70 vs. 89%), momento da ovulação (60,5±2,65 vs. 64,5±0,33 h) e taxa de concepção (40 vs. 44,4%) entre G1 e G2, respectivamente. O diâmetro do folículo dominante no D9 (ØFD) (13,5±0,1 vs. 10,8±1,7 mm) não diferiu entre os grupos, assim como o Ø máx. FD (14,3±1,5 vs. 11,9±0,86 mm) e do Ø máx. FO (14,7±0,74 vs. 12,3±1,37 mm), porém a taxa de crescimento diário dos folículos foi significativamente maior no G1 que no G2 (1,6±0,12 vs. 1,0±0,18 mm/dia). O diâmetro médio do folículo dominante no D9 entre as novilhas que ovularam do grupo G1 foi 14,8±0,6 mm e no G2 11,2±1,6 mm não se observando diferença estatística entre os tratamentos (P=0,07), da mesma forma que também não se observou diferença estatística entre novilhas que não ovularam do G1 (9,6±0,7) e do G2 (7,7±0,03)(P=0,42). Já ao se comparar os diâmetros médios dos folículos dominantes no D9 entre as novilhas que ovularam (14,8±0,6 mm) e as que não ovularam (9,6±0,7 mm) do G1, observou-se diferença significativa (P˂0,05), assim como no G2 (11,2±1,6 vs. 7,7±0,03 mm. A taxa de ovulação aumentou significativamente nos folículos maiores de 11 mm, mensurados no D9, no entanto, as taxas de concepção foram maiores quando os folículos se encontravam maiores que 8 mm, no D9. No presente estudo não foi observado efeito significativo da concentração de P4 no ØFD, ØFO, momento da ovulação, taxas de ovulação e concepção em novilhas púberes Holandesas, porém os animais que receberam dispositivo com menor concentração de P4 no D0 apresentaram maior taxa de crescimento folicular diário.

A presente tese foi dividida em 5 Experimentos. Os objetivos do Experimento 1 foram avaliar a utilização da gonadotrofina coriônica equina (eCG) e/ou do cipionato de estradiol (ECP) na dinâmica folicular e taxa de concepção de vacas holandesas submetidas a inseminação em tempo fixo (IATF). No D0, todos os animais (n = 782) receberam 2 mg de benzoato de estradiol (BE) e um dispositivo intravaginal de progesterona (CIDR). Oito dias depois, o CIDR foi retirado e todos os animais receberam PGF2?. Simultaneamente, os animais foram divididos em 4 grupos: G1) eCG + ECP no Dia 8; G2) eCG no Dia 8 + GnRH após 48h; G3) ECP no Dia 8; G4) GnRH após 48h. Amostras de sangue e exames utlra-sonográficos foram realizados frequentemente em um subgrupo de animais (n = 96). As análises estatísticas de todos os experimentos foram efetuadas com o proc GLIMMIX e proc MIXED do SAS. O uso de eCG e o escore de condição corporal (ECC) dos animais afetaram as concentrações circulantes de progesterona no diestro. Os animais do G2 apresentaram maior taxa de concepção que os do G4 (33,8% vs. 28,9%). Além disso, para animais de menor ECC, ficou evidente o benefício da aplicação de eCG (G2 = 44,4% vs. G4 = 6,1%). No Experimento 2 (n = 26), o objetivo foi comparar o efeito da administração da eCG no dia da remoção do CIDR em animais de menor (2,0-2,5) ou maior (3,0-3,5) ECC. Foram avaliadas algumas características do corpo lúteo (CL) como o volume e histologia, assim como as concentrações plasmáticas de progesterona no diestro. Independentemente da condição corporal dos animais, a eCG aumentou o volume do CL e a concentração plasmática de progesterona no diestro. O ECC afetou negativamente o volume e concentração de progesterona sérica no diestro. Não foi encontrado diferença na proporção de células grandes/pequenas, assim como no fluxo sanguíneo no CL entre os grupos experimentais. No Experimento 3, foi comparado a taxa de concepção em vacas de leite de alta produção (n = 388) após o uso do protocolo G2 do Experimento 1 (Capítulo I), com ou sem adição de ECP no momento da retirada do CIDR (novo ou usado). Não foi verificado efeito da adição do tratamento com ECP e nem do tipo do dispostivo na taxa de concepção. No Experimento 4 (n = 199), apesar no aumento verificado no diâmetro folicular no grupo tratado com GnRH 56h (17,8 mm) comparado com GnRH 48h (16,5 mm); e do atraso no momento da ovulação após a retirada do CIDR (GnRH 56h = 75,3h; GnRH 48h = 79,8h) não foi constatado qualquer diferença na concepção ao se atrasar a aplicação do GnRH de 48h para 56h em vacas inseminadas 16h depois do GnRH. No Experimento 5 (n = 185), a taxa de concepção não diferiu em animais que receberam o GnRH 48h ou 56h (momento da IATF) após a retirada do CIDR, indicando a possibilidade do emprego de um protocolo com apenas 3 manejos em vacas de leite de alta produção. / The current thesys has been divided in 5 Experiments. Objectives of Experiment 1 were to evaluate the effects of equine chorionic gonadotropin (eCG) and/or estradiol cypionate (ECP) on follicular dymanics and conception rate in Holstein cows receiving fixed timed artificial insemination (TAI). On D0, all cows (n = 782) received 2 mg of estradiol benzoate (EB) and one intravaginal progesterone device (CIDR). Eight days later, CIDR was removed and all animals were treated with PGF2?. Simultaneously, animals were divided in 4 groups: G1) eCG + ECP on Day 8; G2) eCG on Day 8 + GnRH 48h later; G3) ECP on Day 8; G4) GnRH 48h later. Blood samples and ultrasound exams were frequently performed in a subset of the animals (n = 96). All the statistical analyses for all experiments were performed with proc GLIMMIX and proc MIXED of SAS. Equine chorionic gonadotropin (eCG) treatment and body condition score (BCS) affected circulating progesterone in the diestrus. Cows in G2 had greater conception rates than cows in G4 (33,8% vs. 28,9%). In addition, in cows with lower BCS, eCG seems to be even more affective (G2 = 44,4% vs. G4 = 6,1%). In Experiment 2 (n = 26), the objective was to compare the effect of eCG the day of CIDR removal in animals with lower (2,0-2,5) or higher (3,0-3,5) BCS. Some variables such as corpus luteum (CL) volume, histology and circulating progesterone concentration in the diestrus were evaluated. Regardless of the body condition of the animals, eCG increased CL volume and circulating progesterone concentration in the diestrus. BCS negatively affected CL volume and circulating progesterone. There were no differences in large/small CL cell ratio, as well as CL blood flow between experimental groups. In Experiment 3, it was compared conception rate in dairy cows (n = 388) after using the same protocol G2 from Experiment 1 (Chapter I), with or without an ECP treatment at the time of CIDR (new or used) removal. Both ECP treatment and type of CIDR did not significantly affected conception rates. In Experiment 4 (n = 199), despite the fact that follicular diameter was increased in group GnRH 56h (17,8 mm) compared with GnRH 48h (16,5 mm); and of the delayed time of ovulation after CIDR removal (GnRH 56h = 75,3h; GnRH 48h = 79,8h), there were no differences in conception rates after delaying the GnRH treatment from 48h to 56h in cows inseminated 16h after GnRH. In Experiment 5 (n = 185), conception rate did not differ in animals that received GnRH 48h or 56h (at the time of TAI) after CIDR removal, indicating the possibility of using a protocol with only 3 handlings in high producing dairy cows.

A aplicação de drogas durante o diestro para melhorar a taxa de prenhez na égua inseminada e na receptora de embrião tem sido o foco de alguns grupos de pesquisa. Estudos com hCG encontraram resultados promissores nas taxas de prenhez e características uterinas de receptoras de embrião no dia da transferência. Com o objetivo de avaliar o efeito da aplicação da hCG em diferentes momentos do ciclo estral sobre as características do trato reprodutivo, vascularização e concentração sérica de progesterona, os animais foram submetidos à aplicação da hCG para induzir ovulação, no dia da ovulação e no 5o dia pós ovulação, além do grupo controle. O presente trabalho foi dividido em estudo preliminar com 4 animais em cada grupo e um estudo principal, com 12 animais por grupo sendo que todos os animais foram submetidos a todos os tratamentos. As características examinadas ao longo dos 15 dias pós ovulação foram tônus, morfoecogenicidade e vascularização do útero; tônus da cérvix; o diâmetro e área do corpo lúteo e do pedículo ovariano e seu RI; RI e vascularização mesometrial. Além disso, amostras de sangue para mensuração sérica de progesterona foram coletadas. Não foi encontrado efeito do tratamento nas características avaliadas com hCG em nenhum dos grupos. As características somente apresentaram variação ao longo do tempo, conforme descrito na literatura. Novos estudos avaliando o efeito da hCG em éguas devem ser realizados para averiguar outras variáveis e possíveis efeitos. / The use of drugs during diestrus in order to improve conception rates in inseminated and recipient mares has been the focus of several research groups. Studies using hCG found promising results regarding pregnancy rates and recipient uterine characteristics on the Day of the transfer. The objective of the study was to evaluate the effect of hCG administration in different moments of the oestrous cycle on reproductive tract characteristics, vascularization and serum progesterone concentration. Therefore, groups consisted of control, hCG to induce ovulation; hCG on day 0 and hCG on day 5 postovulation. The current study was performed as a preliminar study with 4 animals per group, one cycle each animal and a main study with 12 animals per group, each animal receiving all the treatments during consecutive cycles. The evaluations were performed daily from day 0 until day 15 postovulation. Characteristics examined were uterine tone, morphoecogenicity and vascularization; cervical tone; area, diamaterand vascularization of the corpus luteum, ovarian pedicle vascularization and RI; mesometrial vascularization and RI. Besides, blood samples were collected for serum progesterone concentration. No diferences were detected comparing treated and control groups. The characteristics only varied through the days, as described in the literature. Therefore, it can be concluded that hCG administered to induce ovulation, on the day of ovulation and on day 5 postovulation did not alter the characteristics evaluated in the current study.

Reef-building coral populations throughout the world are being threatened by numerous stressors and continue to decline. As potent endocrine-disrupting compounds, exogenous sex steroid contamination has been a largely overlooked stressor to corals. Previous research indicates these compounds are prevalent in marine environments, fluctuate annually along with reproductive cycles, can bioaccumulate, and have had variable effects on growth and reproduction in several cnidarian species. This project had three primary objectives: (1) establish environmental estradiol and progesterone concentrations in Broward County and lower Florida Keys reef environments, (2) conduct 17 β-estradiol and progesterone larval assays on P. astreoides larvae to determine the effects of these compounds on settlement and viability, and (3) conduct 17 β-estradiol and progesterone dosing experiments on adult Acropora cervicornis and Porites astreoides fragments to determine the effects on growth, zooxanthellae, reproduction, and overall tissue health. Estradiol was detected in surface and at-depth water samples from Broward County and lower Keys reef sites at effect level concentrations for marine organisms. Broward County larvae treated with low progesterone (5 ng/L) had decreased survival, while lower Keys larvae in low estradiol treatments (1 ng/L) had increased on-disc settlement. No other treatment effects were observed, however, lower Keys larvae had greater overall survival in comparison to Broward County larvae. There were no significant differences between estradiol and progesterone treatments in the adult-dosing experiment for growth, zooxanthellae density, reproduction, and overall tissue health. This is the first study to detect estradiol at Broward County reefs sites and our results, while inconclusive, indicate these compounds may have the potential to affect coral reef ecosystems.

After the findings were reported for the Women’s Health Initiative (WHI) study in the past decade, there has been a significant decline in the overall use of hormone replacement therapy (HRT) among women. However, there are still millions of middle-aged, menopausal women in the U.S. who are currently undergoing hormone therapy. Their reasons for continuing treatment include relief of severe menopausal symptoms, aid in the management of osteoporosis and reduction in the risk of colon cancer (Ness et al., 2005). The purpose of the following investigation was to evaluate the impact of HRT on the central and peripheral auditory systems both during and after treatment. Over the course of the study, hormone treatments were administered to female aging CBA/CaJ mice to observe what effects estrogen (E) and progestin (P) have on the peripheral and central auditory systems. Female CBA/CaJ middle age mice were ovariectomized and placed into 4 HRT groups (E, P, E+P and Placebo [Pb]). Hormone treatment lasted 6 months followed by a recovery/washout period of 1 month. During this time, electrophysiology tests such as auditory brainstem responses (ABR) and ABR gap in noise (GIN) were used to measure neural activity for the auditory nerve and brainstem. Distortion product otoacoustic emission (DPOAE) testing was also implemented to assess the functional status of the outer hair cells (OHC) and their ability to amplify sound in the cochlea. After 6 months of treatment, animals treated with E exhibited the least amount of changes in ABR thresholds and ABR GIN amplitudes than any other subject groups. Interestingly, P animals exhibited an abrupt increase in ABR thresholds only 3 months after treatment; however, for ABR GIN amplitude levels a progressive reduction observed throughout the study. E+P and Pb animals showed signs of accelerated age-related hearing loss (ARHL) with significantly elevated ABR thresholds and dwindling ABR GIN amplitude levels. No significant signs of recovery were observed for any of the hormone groups. Therefore, in the present murine investigation, the effects of HRT were long lasting. To further expand on the results obtained for the electrophysiology tests, molecular biology experiments were performed to evaluate the expression of IGF-1R and FoxO3 in the cochlea during hormone therapy, from both in vitro and in vivo perspectives. Both genes play significant roles in the PI3K/AKT pathway and were specifically chosen because of their role in anti-apoptotic responses and cell survival. It was hypothesized that E attenuates the effects of ARHL via the PI3K/AKT pathway by up-regulating IGF-1R and FoxO3 to counteract the effects of oxidative stress in the aging mammalian cochlea. qPCR experiments were performed with stria vascularis (SV) lateral wall cells extracted from the cochlea of each animal in the hormone groups post-treatment (in vivo) and in SVK-1 cells treated with HRT over various lengths of time (in vitro) to evaluate the expression levels of IGF-1R and FoxO3. In-vivo experiments showed that the E-treated animals had significantly higher IG-1R levels compared to the other subject groups after treatment was discontinued. Similarly, IGF-1R levels steadily increased for E-treated SVK-1 cells over the course of hormone therapy, compared to P and E+P cells. FoxO3 expression, on the other hand, declined for all of the hormone-treated cells groups, relative to control SVK-1 cells (in vitro), and no statistical differences were detected for FoxO3 levels among the post-treatment animals (in vivo). These findings indicate that there is cross talk between E and IGF-1R involving the PI3K/AKT pathway, which contributes to the delayed onset of ARHL observed during HRT with E. Meanwhile, FoxO3 may not play a role in neuro-protective properties in the cochlea during HRT, as initially hypothesized.

Homocysteine (Hcy) is a sulfur containing amino acid and is a potent neurotoxin. It has been shown that elevated levels of Hcy, termed hyperhomocysteinemia, plays a role in the pathologies of Alzheimer’s disease (AD) and age-related cognitive decline. Hcy is a glutamate agonist, which causes in increase in Ca[superscript (2+)] influx via the activation of NMDA class of excitatory amino acid receptors, which results in neuronal cell death and apoptosis. Estrogen and progesterone are female hormones that are responsible for reproduction and maternal behaviour. However, in the last decade, it is evident that both female hormones have neuroprotective properties in many animal models of neurodegeneration. Collectively, both estrogen and progesterone reduce the consequences of the oxidative stress by enhancing the antioxidant defence mechanisms, reducing excitotoxicity by altering glutamate receptor activity and reducing the damage caused by lipid peroxidation. However, the mechanisms by which estrogen and progesterone provide such neuroprotection probably depend on the type and concentration of hormone present. Moreover, numerous studies have shown that hormone replacement therapy (HRT, estrogen and progestins) or estrogen-only replacement therapy (ERT) may prevent or delay the onset of AD and improve cognition for women with AD. Clinical trials have also shown that women taking HRT may modify the effects of Hcy levels on cognitive functioning. Oxidative stress increases in the aging brain and thus has a powerful effect on enhanced susceptibility to neurodegenerative disease. The detection and measurement of lipid peroxidation and superoxide anion radicals in the brain tissue supports the involvement of free radical reactions in neurotoxicity and in neurodegenerative disorders. The hippocampus is an important region of the brain responsible for the formation of memory. However, agents that induce stress in this area have harmful effects and could lead to dementia. This study aims to investigate and clarify the neuroprotective effects of estrogen and progesterone, using Hcy-induced neurodegenerative models. The initial studies demonstrate that estrogen and progesterone have the ability to scavenge potent free radicals. Histological studies undertaken reveal that both estrogen and progesterone protect against Hcy-induced neuronal cell death. In addition, immunohistochemical investigations show that Hcy-induced apoptosis in the hippocampus can be inhibited by both estrogen and progesterone. However, estrogen also acts at the NMDA receptor as an agonist, while progesterone blocks at the NMDA receptor. These mechanisms reduce the ability of Hcy to cause damage to neurons, since Hcy-induced neurotoxicity is dependent on the overstimulation of the NMDA receptor. SOD and GPx are important enzymatic antioxidants which can react with ROS and neutralize them before these inflict damage in the brain. Hcy can increase oxidative stress by inhibiting expression and function of these antioxidants. However, it has been shown that the antioxidant abilities of both estrogen and progesterone can up-regulate the activities of SOD and GPx. These results provide further evidence that estrogen and progesterone act as antioxidants and are free radical scavengers. The discovery of neuroprotective agents is becoming important as accumulating evidence indicates the protective role of both estrogen and progesterone in Hcy-induced neurodegeneration. Thus further work in clinical trials is needed to examine whether reducing Hcy levels with HRT can become the treatment of neurodegenerative disorders, such as Alzheimer’s disease.

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.