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Orientador: Fernanda da Cruz Landim e Alvarenga / Coorientador: Paula Ripamonte / Banca: Frederico Ozanan Papa / Banca: Cezinande Meira / Banca: Márcio Teoro do Carmo / Banca: Claudia Barbosa Fernandes / Resumo: As alterações observadas nos folículos pré-ovulatórios dos mamíferos a partir da onda pré-ovulatória de LH são mediadas por fatores de crescimento como AR e ER, que induzem a expansão das células do cumulus e maturação oocitária. O LH também estimula a deposição de ácido hialurônico mediada pela enzima HAS-2. Em eqüinos, já foi caracterizada a expressão de HAS-2, AR e ER em resposta à administração de hCG em folículos pré-ovulatórios, mas sua ação em folículos imaturos não está esclarecida, embora tenha sido demonstrada a expansão precoce do cumulus após sua administração em folículos menores de 30mm. O objetivo deste estudo foi caracterizar o padrão de expressão de ER, AR e HAS-2 em folículos de eqüinos em diferentes estágios de maturação, submetidos ou não ao tratamento com EPE e hCG. Para isso, recuperamos células foliculares por OPU de folículos de diversos tamanhos, em 3 ou 24 horas após estímulo gonadotrófico exógeno, e avaliamos a expressão gênica de ER, AR e HAS-2 por PCR em tempo real. O tratamento com hCG induziu a expressão de ER em folículos imaturos e pré-ovulatórios, demonstrando sua capacidade de indução da maturação folicular precoce, e houve tendência do mesmo efeito ser obtido com a expressão de HAS-2, embora não tenha sido estatisticamente comprovado. O EPE apresentou efeito menor que a hCG na indução da expressão de ER, o que pode ser justificado pelo mecanismo de down regulation que o LH exerce sobre a ativação dos fatores de crescimento ligados ao EGF, como a ER e a AR / Abstract: The modifications observed on mammalia n follicles after the pré-ovulatory surge of LH are mediated by growth factors like epiregulin (ER), which is responsible for the cumulus cells expansion and interfere with the oocyte maturation . The LH also stimulates the deposition of hyaluronic acid mediated by the hialuronan synthase -2 (HAS-2). In horses , the expression of HAS-2 and ER was already characterized in pre-ovulatory follicles , after the administration of hCG. In the present experiment we were able to demonstrate that the hCG is also able to induce the expression of ER in equine follicles smaller than 30mm in diameter , confirming an ability of immature follicles in responding to hormonal stimuli. Although no statistic differences were observed among groups a tendency to the same effect was also observed for the HAS-2. Similarly, the use of EPE stimulated the expression of ER, but when EPE and hCG were combined a decrease in expression was observed. This observation indicates a mechanism of down regulation exerted by the LH on the ER and HAS expression / Doutor

Made available in DSpace on 2014-06-11T19:35:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-12Bitstream added on 2014-06-13T19:05:26Z : No. of bitstreams: 1 blanco_idp_dr_botfmvz.pdf: 464375 bytes, checksum: 98e141ce5fa2b921f8f4ddf393292d45 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As alterações observadas nos folículos pré-ovulatórios dos mamíferos a partir da onda pré-ovulatória de LH são mediadas por fatores de crescimento como AR e ER, que induzem a expansão das células do cumulus e maturação oocitária. O LH também estimula a deposição de ácido hialurônico mediada pela enzima HAS-2. Em eqüinos, já foi caracterizada a expressão de HAS-2, AR e ER em resposta à administração de hCG em folículos pré-ovulatórios, mas sua ação em folículos imaturos não está esclarecida, embora tenha sido demonstrada a expansão precoce do cumulus após sua administração em folículos menores de 30mm. O objetivo deste estudo foi caracterizar o padrão de expressão de ER, AR e HAS-2 em folículos de eqüinos em diferentes estágios de maturação, submetidos ou não ao tratamento com EPE e hCG. Para isso, recuperamos células foliculares por OPU de folículos de diversos tamanhos, em 3 ou 24 horas após estímulo gonadotrófico exógeno, e avaliamos a expressão gênica de ER, AR e HAS-2 por PCR em tempo real. O tratamento com hCG induziu a expressão de ER em folículos imaturos e pré-ovulatórios, demonstrando sua capacidade de indução da maturação folicular precoce, e houve tendência do mesmo efeito ser obtido com a expressão de HAS-2, embora não tenha sido estatisticamente comprovado. O EPE apresentou efeito menor que a hCG na indução da expressão de ER, o que pode ser justificado pelo mecanismo de down regulation que o LH exerce sobre a ativação dos fatores de crescimento ligados ao EGF, como a ER e a AR / The modifications observed on mammalia n follicles after the pré-ovulatory surge of LH are mediated by growth factors like epiregulin (ER), which is responsible for the cumulus cells expansion and interfere with the oocyte maturation . The LH also stimulates the deposition of hyaluronic acid mediated by the hialuronan synthase -2 (HAS-2). In horses , the expression of HAS-2 and ER was already characterized in pre-ovulatory follicles , after the administration of hCG. In the present experiment we were able to demonstrate that the hCG is also able to induce the expression of ER in equine follicles smaller than 30mm in diameter , confirming an ability of immature follicles in responding to hormonal stimuli. Although no statistic differences were observed among groups a tendency to the same effect was also observed for the HAS-2. Similarly, the use of EPE stimulated the expression of ER, but when EPE and hCG were combined a decrease in expression was observed. This observation indicates a mechanism of down regulation exerted by the LH on the ER and HAS expression

The Bureau of Land Management (BLM) maintains a growing number of feral horses on public rangelands. With population growth rates as high as 22% annually, herds are exceeding their carrying capacity and millions of dollars are spent maintaining captured horses in holding facilities awaiting adoption. To manage the feral horse population, the BLM is seeking a contraceptive that is safe, can be remotely delivered, requires only a single administration and is effective for several years. Contraceptive strategies have been developed for feral horses that include hormone implants, chemical intrauterine devices, and immunocontraception. Porcine zona pellucida (pZP) immunocontraceptive vaccines have shown great potential for providing safe, long-term contraception in feral horses. ImmunoVaccine Technologies (Halifax, Nova Scotia, Canada) has developed a liposome encapsulated pZP formulation known as SpayVac™ (SpayVac), which after a single-dose provides multi-year contraceptive efficacy. In a continued effort to optimize the acceptability and efficacy of SpayVac, ImmunoVaccine Technologies developed alternative adjuvant preparations using either killed Mycobacterium butyricum (Modified Freund's Adjuvant; MFA) or a proprietary non-Mycobacterium based adjuvant (IVT) that are proposed to have less of the undesirable side-effects associated with Mycobacterium tuberculosis. Therefore, the objective of this research was to evaluate SpayVac in different adjuvant formulations for efficacy of contraception as measured by pZP titers and estrous cyclicity in treated mares. Domestic mares (n=28) were randomly assigned to four treatments (7 mares per treatment): adjuvant alone or saline (Control) or SpayVac vaccines in one of three adjuvant preparations: IVT or MFA in either an aqueous (MFA aq) or non-aqueous (MFA non-aq) suspension. Pre-immune blood samples were collected from each mare and mares were injected in the neck with a single injection of the Control or SpayVac. Subsequent blood samples were collected at weekly intervals for 26 weeks. Sera were analyzed for pZP titers and progesterone using ELISA. At the conclusion of the study, ovaries were recovered by ovariectomy (16 mares) or at necropsy (12 mares) for histologic analysis and collection of morphometric data and oocytes. Titers for pZP were greater (P<0.05) in IVT and MFA mares compared to Control mares and for MFA compared to IVT mares. Mares vaccinated with MFA aq had greater (P<0.05) pZP titers at 2 weeks post-injection compared to mares injected with IVT or MFA non-aq and at 3 weeks post-injection compared to mares injected with IVT. MFA non-aq mares had greater (P<0.05) pZP titers at 6 weeks post-injection compared to IVT mares and, although not significantly different, titers in MFA non-aq mares remained greater during weeks 8, 10, 14, 18 and 22 compared to IVT and MFA aq mares. Mean serum progesterone concentrations were greater (P<0.05) in Control compared to MFA non-aq mares. Mean ovarian weights, oocyte diameters, zona pellucida thicknesses and the number of horse sperm bound to oocytes recovered from vaccinated mares were greater (P<0.05) in Control mares compared to IVT and MFA mares. As judged by pZP titers and serum progesterone, these results suggest that SpayVac suspended in the MFA non-aqueous formulation exerted the greatest contraceptive effects in treated mares. This preparation of SpayVac may meet the criteria cited by the BLM for their most desirable immunocontraceptive. / Graduation date: 2012