published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

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Multiplex polymerase chain reaction (PCR) assays were designed that incorporated primer pairs for the sex determining region on the Y chromosome, Sry, and one or two control sequences. A triplex and a duplex assay were created involving Sry and Dax, a single copy X chromosome gene which is involved in the female sex determination pathway. The third sequence in the triplex assay was a repetitive Y chromosome sequence, YR. A minimum of 2.5 x 10-3 to 2.5 x 10 -4 m g/ m L of male DNA and 2.5 x 10-5 m g/ m L of female DNA was required if a single multiplex PCR was performed. To demonstrate that sex determination of preimplantation porcine embryos is possible, morulae were collected 5 d post insemination from pregnant mare serum gonadotrophin (PMSG)/human chorionic gonadotrophin (hCG)-treated 60--70 kg prepubertal gilts. Embryos were biopsied using a micromanipulator and cells were placed into individual microcentrifuge tubes for further analysis. Embryos were then cultured to the blastocyst stage. A total of 315 embryos were sexed via the PCR assay with a resultant female/male (%) ratio of 73/27. When 94 embryos from heavier gilts (90--100 kg) were sexed in a similar assay, the resultant female/male ratio was 60/40. Attempts were made to correlate these results with karyotypes. Five transfers of sexed embryos into synchronized recipients were attempted. None of these resulted in pregnancies; although return to estrus was delayed by two to eight days, in four out of the five recipients. Our findings suggest that PCR amplification of the Sry gene can be a reliable method for sexing porcine embryos. It does appear that embryo quality is critical for both the PCR assay and subsequent successful culture. (Abstract shortened by UMI.)

Currently more than 300 million adults are obese and 1 billion are overweight throughout the world. Obesity is frequently accompanied by an array of health conditions such as hyperglycemia, hyperlipidemia, hypertension, cardiovascular diseases, and diabetes which are all considered to be part of what is now known as the metabolic syndrome. The role of adipose tissue as an endocrine organ has been emphasized by the characterization of its hormones: leptin, adiponectin and resistin. All three proteins regulate energy utilization. Over the past decade, leptin and resistin have also been shown to affect the reproductive system. This suggests that other adipocytokines, such as adiponectin, may also affect reproduction. This relationship was investigated using a porcine in vitro maturation system. When porcine cumulus oocyte complexes were matured in the presence of 30mug/mL of recombinant adiponectin an improvement in the meiotic maturation was observed. Moreover, maturation of denuded oocytes revealed that adiponectin acts through the cumulus cells to improve meiotic maturation of porcine oocytes. Finally, maturation of cumulus-oocyte complexes in the presence of MAPK pathway inhibitors suggested that adiponectin acts at or downstream of MEK1/2 and 38MAPK. This study shows, for the first time, an effect of adiponectin on porcine oogenesis. Further investigation will determine whether adiponectin also affects embryo development.

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Regulation of tubulin gene expression in embryos of the sea urchin Lytechinus pictus has been experimentally investigated by use of cloned recombinant tubulin DNA and anti-tubulin antiserum. Tubulin synthesis appears to be autogenously regulated at the level of tubulin mRNA stability by the level of unpolymerized tubulin; i.e., the more unpolymerized tubulin, the less stable the tubulin mRNA. Destabilization of tubulin mRNA requires continued protein synthesis. Most of tubulin stored in eggs is unpolymerized; during embryogenesis the mass of tubulin per embryo changes little, but unpolymerized tubulin is increasingly polymerized into microtubules. There is a transcriptional stimulation of tubulin genes at the time of ciliogenesis but thereafter autoregulation by the ontogenetic decrease of the level of unpolymerized tubulin plays a predominant role for an increasing accumulation of tubulin mRNA. Deciliation results in a transient enhancement of transcription of tubulin genes, which is independent of the level of unpolymerized tubulin and does not require protein synthesis.

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Orientadora: Prof. Dra. Marcella Pecora Milazzotto. / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2015. / A bioenergia destaca-se como um substituto relevante para os combustíveis fósseis e os A capacidade de selecionar o embrião com maior viabilidade para transferência às receptoras é um componente crucial para o sucesso das técnicas de reprodução assistida. Atualmente, avaliações morfológicas são utilizadas para selecionar os embriões com maior potencial de implantação, e apesar de se tratar de um método não invasivo e relativamente bem sucedido, ainda apresenta uma série de limitações. Nos últimos anos, o desenvolvimento de novas tecnologias tem possibilitado análises quantitativas e qualitativas para determinação da viabilidade dos embriões para melhoria da seleção embrionária. Em especial as tecnologias espectroscópicas e espectrométricas permitiram o desenvolvimento de novos métodos para a caracterização embrionária e predição do potencial de estabelecimento da prenhez. Assim, o objetivo deste trabalho foi a caracterização não invasiva de embriões bovinos pela análise dos perfis espectroscópicos e espectrométricos dos meios de cultivo de embriões com diferentes cinéticas de desenvolvimento visando a obtenção de padrões específicos baseados no fenótipo embrionário. Para isso, embriões bovinos foram produzidos in vitro por protocolos convencionais. Os zigotos foram transferidos para meios de cultivo individualmente e classificados em dois grupos: rápido (4 células-40hpi) e lento (2 ou 3 células-40hpi). Os meios de cultura foram coletados às 40, 96 e 168 horas pós-inseminação (hpi), tranferidos para criotubos e congelados a -80ºC até o momento das análises. A análise do metaboloma embrionário foi realizada por espectroscopia Raman. Para tal, gotas de meios de cultura foram cobertas com óleo mineral, sendo escaneadas por um sistema Raman triplo. Os dados foram normalizados e analisados por Análises de Componentes Principais (PCA), de Agrupamentos (Clusters) e por Loading plot. A análise do secretoma embrionário foi feita por MALDI-MS-TOF, através da extração dos metabólitos, sendo os espectros adquiridos pelo espectrômetro em modo de ionização positiva. Foram feitas analises estatísticas univariadas e multivariadas pelo software online Metaboanalyst. Os resultados obtidos pela espectroscopia Raman e espectrometria de massas demonstram que embriões com diferentes cinéticas de desenvolvimento possuem diferentes perfis espectroscópicos e espectrométricos ao longo do desenvolvimento embrionário, indicando que estes embriões consomem e/ou produzem diferencialmente metabólitos nos meios de cultura. Com base em nossos resultados, propomos com este estudo que a análise dos meios de cultura por espectroscopia Raman pode ser utilizada para fins de diagnóstico embrionário, pois permite uma caracterização rápida e global dos perfis embrionários relacionados a cinética, enquanto a espectrometria de massas pode ser utilizada para caracterização qualitativa dos embriões, permitindo a identificação do secretoma de embriões durante o cultivo in vitro. / The ability to select embryo with greater viability to transfer to the receptors is a crucial component to the success of assisted reproduction techniques. Currently, morphological assessments are used to select embryos with the highest implantation potential, although it¿ s a noninvasive and relatively successful, has still a number of limitations. In recent years, the development of new technologies has enabled quantitative and qualitative analyzes for the determination of viability of embryos to improve embryo selection. In particular spectroscopic and spectrometric technologies have permitted the development of new methods for embryo characterization and prediction of potential establishment of pregnancy. The objective of this study was non-invasive characterization of bovine embryos by analysis of spectroscopic and spectrometric profiling of embryo culture media with different kinetics of development in order to obtain specific patterns based on embryonic phenotype. For this, bovine embryos were produced in vitro by standard protocols. The zygotes were transferred to individual culture medium and divided into two groups: Fast (4 cells-40hpi) and slow (2 or 3 cells-40hpi). The culture media were collected at 40, 96 and 168 hours post-insemination (hpi) tranfered to cryotubes and frozen at -80 until the time of analysis. The analysis of the metabolome embryo was made by Raman spectroscopy. For this, droplets of culture media were overlaid with mineral oil being scanned by a triple Raman system. The data were normalized and analyzed by Principal Component Analysis (PCA), clusters and Loading plot. Analysis of embryonic secretome was made by MALDI-TOF-MS, through the extraction of the metabolites, and the spectra acquired by the spectrometer in positive ionization mode. Analyzes were performed univariate and multivariate statistics by the online software Metaboanalyst. The results obtained by Raman spectroscopy and mass spectrometry demonstrated that the development of embryos with different kinetics have different spectroscopic and spectrometric profiles during embryonic development, indicating that these embryos consume and /or produce differentially metabolites in the culture media. Based on our results, we propose that the analysis of Raman spectroscopy culture media may be used for diagnostic purposes embryo, since it allows a fast and comprehensive characterization of embryonic profiles related to kinetics, while mass spectrometry can be used for qualitative characterization of the embryo, allowing the identification of secretome embryo during in vitro culture.

Made available in DSpace on 2014-06-11T19:35:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2006Bitstream added on 2014-06-13T18:46:35Z : No. of bitstreams: 1 pyles_escs_dr_botfmvz.pdf: 345793 bytes, checksum: f8cd21a8887d03897e049aa3fce53820 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Neste estudo vitrificou-se em OPS ovócitos (Experimentos 1, 2 e 3) e embriões bovinos (Experimentos 4, 5 e 6) em três soluções de vitrificação distintas: Experimentos 1 e 4) 20%EG + 20%DMSO + 0,5M sacarose; Experimentos 2 e 5) 20%DF + 20%EG + 0,5M sacarose; Experimentos 3 e 6) 20%DF + 20%DMSO + 0,5M sacarose. Os embriões foram vitrificados na presença ou não de citocalasina B. Os ovócitos foram vitrificados ou somente expostos aos crioprotetores imaturos (G0h e G0hexp), ou após 6 horas (G6h e G6hexp), 12 horas (G12h e G12hexp) ou 22 horas de MIV (G22h e G22hexp). Após a exposição ou aquecimento, parte dos ovócitos completou as 22 horas de MIV e foi destinada à PIV, para avaliação da capacidade de se desenvolverem até blastocisto. No restante avaliou-se o estágio de maturação nuclear e a distribuição dos grânulos corticais e das mitocôndrias. Observou-se que a exposição dos ovócitos imaturos (0 ou 6 horas de MIV) aos crioprotetores nos experimentos 1, 2 e 3 resultou em perda substancial de viabilidade, avaliada pelas taxas de clivagem (GC=79,4%; 1G0hexp=37,1%; 1G6hexp=51,1%; 2G0hexp=33%; 2G6hexp=52,5%; 3G0hexp=36,2%; 3G6hexp=49,8%), o que foi menos notável nos ovócitos expostos após 12 ou 22 horas de MIV (1G12hexp=59,1%; 1G22hexp=71,1%; 2G12hexp=61,5%; 2G22hexp=62,5%; 3G12hexp=58,8%; 3G22hexp=68,6%). A realização da MIV antes da vitrificação foi benéfica. Apesar de nenhuma das soluções de crioprotetores ter sido capaz de promover proteção adequada aos ovócitos submetidos à vitrificação em qualquer momento da MIV, no Experimento 1 foi observada clivagem (1G12h=7,3%; 1G22h=4,2%) indicando que a mistura de crioprotetores EG+DMSO foi a mais eficiente nas condições utilizadas. Entretanto, não houve produção de blastocistos em nenhum experimento. Nos Experimentos 4, 5 e 6 notou-se que em todos os grupos... / In this study bovine oocytes (Experiments 1, 2 and 3) and embryos (Experiments 4, 5 and 6) were vitrified in OPS using 3 different cryoprotectant solutions: Experiments 1 and 4) 20%EG + 20%DMSO + 0,5M sucrose; Experiments 2 and 5) 20%DF + 20%EG + 0.5M sucrose; Experiments 3 and 6) 20%DF + 20%DMSO + 0.5M sucrose. Embryos were vitrified in the presence or not of cytocalasin B. Oocytes were vitrified or just exposed to the cryoprotectant solutions after 0h (G0h and G0hexp), 6h of IVM (G6h and G6hexp), 12h of IVM (G12h and G12hexp) and 22h of of in vitro maturation (IVM) (G22h and G22hexp). Oocytes from groups 0 and 6h were considered immature and from 12 and 22h groups were considered mature. After exposure to cryoprotectants or warming, part of the oocytes that completed 22h of IVM were submitted to IVF to evaluate their developmental ability. Another part of the oocytes were used to evaluate nuclear maturation in the distribution of mitochondria and cortical granules. The results showed that the exposure of immature oocytes (0 and 6 hours of MIV) to cryoprotectants reduce their viability, since cleavage rates of all groups were significantly lower compared to control group (GC=79.4%; 1G0hexp=37.1%; 1G6hexp51.1%; 2G0hexp=33%; 2G6hexp=52.5%; 3G0hexp=36.2%; 3G6hexp=49.8%). This effect was less evident in the groups of mature oocytes submitted to IVM for 12 or 22 hours (1G12hexp=59.1%; 1G22hexp=71.1%; 2G12hexp=61.5%; 2G22hexp=62.5%; 3G12hexp=58.8%; 3G22hexp=68.6%). The previous IVM is beneficial to vitrification. Although none of the cryoprotectant solutions utilized were totally efficient in protecting the oocytes form cryodamage, in Experiment 1 cleavage was observed (1G12h=7.3%; 1G22h=4.2%) indicating that the combination of EG+DMSO was more effective than the other two. However in none of the groups blastocyst was formed. When embryos... (Complete abstract click electronic access below)