While the central role of spermatozoa in sexual reproduction and fertilization is well understood, many functional attributes of sperm have yet to be elucidated at the molecular level. One key to ultimately understand the molecular basis of sperm function is to comprehensively characterize its biochemical composition. This crucial information has been lacking, as molecular characterization of the sperm cell cannot be assessed by classic gene expression assays since mature spermatozoa are transcriptionaly inert. Whole-cell shotgun proteomic approaches have revolutionized the molecular analysis of sperm form and function. We have utilized improved methodologies to re-analyze the D. melanogaster sperm proteome and characterize five additional Drosophila species sperm proteomes. This methodology, which included a 1D SDS-PAGE prefractionation step, resulted in good reproducibility between biological replicates and high quality sperm proteomes. An interspecific analysis of the sperm proteomes revealed that despite variation in protein composition, Drosophila sperm proteomes have a consistent functional profile and 519 proteins were identified a being conserved across the melanogaster subgroup within a phylogenetic framework. Evolution of the sperm proteome was explored in Mus musculus through the utilization of targeted proteomic datasets completed, which provided subcellular localizations for sperm components. This study resulted in several novel findings, including evidence for accelerated evolution as well as an enrichment of positive selection on genes found in the cell membrane and acrosome. This may be a result of the selective pressures encountered by these membrane proteins during sperm development, maturation and transit through the female reproductive tract where the sperm cell membrane, and eventually the acrosome, are exposed to the extracellular milieu and are available for direct cell-cell interactions. These findings not only reveal the varying evolutionary pressures acting on a single cell type but also highlights the utility of the proteomics technique in clarifying protein interaction and evolutionary history.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O presente estudo foi conduzido com o objetivo de avaliar os efeitos da inclusÃo de 13% de farelo de castanha de caju na dieta de carneiros da raÃa Morada Nova sobre o ganho de peso, consumo de matÃria seca, rendimento de carcaÃa, pesos dos ÃrgÃos sexuais, qualidade seminal e proteÃnas seminais e membranares do espermatozoide. Para tanto, vinte carneiros foram divididos em dois grupos alimentados com dietas contendo 13% (GCA, n=10) ou 0% (GCO, n=10) de farelo de castanha de caju. As dietas foram isoproteicas e isoenergeticas, com uma relaÃÃo de 50/50 de concentrado/volumoso (feno de Tifton 85) e Ãgua e sal mineral à vontade. Durante noventa dias, os animais foram mantidos em baias individuais e avaliados quanto ao consumo de matÃria seca e ganho de peso. As coletas seminais por eletroejaculaÃÃo ocorreram semanalmente com a determinaÃÃo do volume ejaculado e, concentraÃÃo, motilidade e morfologia espermÃtica. ApÃs os noventa dias, as proteÃnas seminais e espermÃticas foram analisadas por meio de eletroforese bidimensional em gel de poliacrilamida. Os mapas proteÃcos foram avaliados por meio do aplicativo PDQuest, version 8.0; Bio Rad, USA. Ao final do experimento, os carneiros foram abatidos e, o peso vivo, rendimento de carcaÃa e peso dos ÃrgÃos sexuais foram mensurados. O consumo de matÃria seca total permaneceu sem alteraÃÃes nos dois grupos experimentais durante os primeiros 60 dias do estudo. PorÃm, apÃs 60 dias, houve e reduÃÃo no consumo de matÃria seca no grupo alimentado com castanha (P < 0,05). NÃo houve efeito da inclusÃo do farelo de castanha sobre o ganho de peso, rendimento de carcaÃa, desenvolvimento dos ÃrgÃos sexuais e parÃmetros seminais dos animais. No entanto, a inclusÃo do farelo de castanha de caju esteve associada à expressÃo de proteÃnas seminais e espermÃticas. Sete spots proteicos presentes nos mapas de plasma seminal foram expressos diferencialmente entre os grupos castanha e controle (P < 0,05) e um spot proteico (21,8 kDa; pI: 5) correlacionou-se negativamente com o vigor espermÃtico (r = -0,49; P<0,05). Adicionalmente, sete spots proteicos dos gÃis com proteinas da membrana dos espermatozoides tambÃm diferiram entre os carneiros alimentados com e sem farelo de castanha de caju (P < 0,05). Conclui-se, portanto, que a inclusÃo de 13% de farelo de castanha de caju na dieta altera a expressÃo de proteÃnas seminais e espermÃticas em ovinos. / The cashew nut meal is alternative food for ruminant nutrition which is rich in lipids, proteins and minerals. However, its nutrition influences on male development and reproduction are still unknown. In this context, seminal and sperm protein analysis can show metabolic consequences from cashew nut meal on sheep reproductive efficiency. Therefore, the current dissertation aims evaluate the effects of 13% of cashew nut meal in the ration on the weight gain, food intake, carcass yield, weight of sexual organs, sperm quality and, seminal and sperm membrane proteins of Morada Nova Rams. Twenty rams were divided in two groups: cashew nut and control, which received 13% or 0% of cashew nut meal in the diet, respectively. During ninety days, the animals were kept in individual boxes and evaluated for ration consumption and weight gain. The sperm collections by eletroejaculation were made by weekly and, the volume, sperm concentration and motility were analyzed. After ninety days, the seminal and sperm membrane proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. The gels were digitalized, and their images were evaluated by computer software. At the end of the experiment, the rams were slaughtered and, the weight gain, carcass yield and weight of sexual organs were measured. There was not effect of cashew nut meal addition in the diet on the weight gain, carcass yield and weight of sexual organs. But, after sixty days, there was a reduction on food intake in the cashew nut group (P < 0,05). The sperm quality was not influenced by the diet. We observed an effect of cashew nut diet on the expression of seminal and sperm proteins. Seven spots from seminal plasma were expressed differently between cashew nut and control groups (P < 0,05). The spot 2206 (21,82 kDa; pI: 5,04) was negatively correlated with the individual motility score (r = -0,49). Additionally, seven spots from sperm membrane protein also differ between rams from cashew nut and control groups (P < 0,05). In conclusion, the addition of 13% of cashew nut meal in the diet alters the expression of sheep seminal plasma and sperm membrane proteins.

Reproduction is an essential feature of all animals and a fundamental step to produce new generations. Study of sperm proteins is crucial for understanding of the sperm-egg recognition. We searched out sperm surface proteins involving in the zona pellucida (ZP) binding and studied whether these proteins are preserved throughout mammalian species. Indirect immunofluorescent technique was used to test a panel of monoclonal antibodies prepared against boar sperm surface proteins on spermatozoa of bull and mice. We found a cross-reactivity of some antibodies against boar sperm with bull ejaculated and mouse epididymal spermatozoa. Further, we isolated sperm proteins from different mammalian species, such as pig, bull, dog, cat, mouse and human. Proteins were separated by SDS- electrophoresis and protein/glycoprotein profiles from epididymal, ejaculated and in vitro capacitated sperm were compared. The interaction of sperm with ZP was studied on electrophoretically-separated sperm surface proteins from pig and bull with biotin-labeled ZP glycoproteins. Antibodies, which reacted with boar sperm surface proteins with ZP- binding activity, therefore could be potential egg-binding receptors, were used for monitoring of the sperm protein origin in reproductive fluids and tissues. (In Czech) Keywords: sperm...

Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-08-03T13:15:24Z No. of bitstreams: 1 2013_dis_lsfonseca.pdf: 641605 bytes, checksum: 04f084807466b026c5bf42f0ceccacd9 (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-08-03T13:15:46Z (GMT) No. of bitstreams: 1 2013_dis_lsfonseca.pdf: 641605 bytes, checksum: 04f084807466b026c5bf42f0ceccacd9 (MD5) / Made available in DSpace on 2016-08-03T13:15:46Z (GMT). No. of bitstreams: 1 2013_dis_lsfonseca.pdf: 641605 bytes, checksum: 04f084807466b026c5bf42f0ceccacd9 (MD5) Previous issue date: 2013 / FONSECA, Lucas dos Santos. Desempenho, consumo de matéria seca, parâmetros seminais e proteômica do plasma seminal e da membrana espermática de carneiros Morada Nova alimentados com dieta à base de castanha de caju. 2013. 65 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Zootecnia, Fortaleza-CE, 2013 / The cashew nut meal is alternative food for ruminant nutrition which is rich in lipids, proteins and minerals. However, its nutrition influences on male development and reproduction are still unknown. In this context, seminal and sperm protein analysis can show metabolic consequences from cashew nut meal on sheep reproductive efficiency. Therefore, the current dissertation aims evaluate the effects of 13% of cashew nut meal in the ration on the weight gain, food intake, carcass yield, weight of sexual organs, sperm quality and, seminal and sperm membrane proteins of Morada Nova Rams. Twenty rams were divided in two groups: cashew nut and control, which received 13% or 0% of cashew nut meal in the diet, respectively. During ninety days, the animals were kept in individual boxes and evaluated for ration consumption and weight gain. The sperm collections by eletroejaculation were made by weekly and, the volume, sperm concentration and motility were analyzed. After ninety days, the seminal and sperm membrane proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. The gels were digitalized, and their images were evaluated by computer software. At the end of the experiment, the rams were slaughtered and, the weight gain, carcass yield and weight of sexual organs were measured. There was not effect of cashew nut meal addition in the diet on the weight gain, carcass yield and weight of sexual organs. But, after sixty days, there was a reduction on food intake in the cashew nut group (P < 0,05). The sperm quality was not influenced by the diet. We observed an effect of cashew nut diet on the expression of seminal and sperm proteins. Seven spots from seminal plasma were expressed differently between cashew nut and control groups (P < 0,05). The spot 2206 (21,82 kDa; pI: 5,04) was negatively correlated with the individual motility score (r = -0,49). Additionally, seven spots from sperm membrane protein also differ between rams from cashew nut and control groups (P < 0,05). In conclusion, the addition of 13% of cashew nut meal in the diet alters the expression of sheep seminal plasma and sperm membrane proteins. / O presente estudo foi conduzido com o objetivo de avaliar os efeitos da inclusão de 13% de farelo de castanha de caju na dieta de carneiros da raça Morada Nova sobre o ganho de peso, consumo de matéria seca, rendimento de carcaça, pesos dos órgãos sexuais, qualidade seminal e proteínas seminais e membranares do espermatozoide. Para tanto, vinte carneiros foram divididos em dois grupos alimentados com dietas contendo 13% (GCA, n=10) ou 0% (GCO, n=10) de farelo de castanha de caju. As dietas foram isoproteicas e isoenergeticas, com uma relação de 50/50 de concentrado/volumoso (feno de Tifton 85) e água e sal mineral à vontade. Durante noventa dias, os animais foram mantidos em baias individuais e avaliados quanto ao consumo de matéria seca e ganho de peso. As coletas seminais por eletroejaculação ocorreram semanalmente com a determinação do volume ejaculado e, concentração, motilidade e morfologia espermática. Após os noventa dias, as proteínas seminais e espermáticas foram analisadas por meio de eletroforese bidimensional em gel de poliacrilamida. Os mapas proteícos foram avaliados por meio do aplicativo PDQuest, version 8.0; Bio Rad, USA. Ao final do experimento, os carneiros foram abatidos e, o peso vivo, rendimento de carcaça e peso dos órgãos sexuais foram mensurados. O consumo de matéria seca total permaneceu sem alterações nos dois grupos experimentais durante os primeiros 60 dias do estudo. Porém, após 60 dias, houve e redução no consumo de matéria seca no grupo alimentado com castanha (P < 0,05). Não houve efeito da inclusão do farelo de castanha sobre o ganho de peso, rendimento de carcaça, desenvolvimento dos órgãos sexuais e parâmetros seminais dos animais. No entanto, a inclusão do farelo de castanha de caju esteve associada à expressão de proteínas seminais e espermáticas. Sete spots proteicos presentes nos mapas de plasma seminal foram expressos diferencialmente entre os grupos castanha e controle (P < 0,05) e um spot proteico (21,8 kDa; pI: 5) correlacionou-se negativamente com o vigor espermático (r = -0,49; P<0,05). Adicionalmente, sete spots proteicos dos géis com proteinas da membrana dos espermatozoides também diferiram entre os carneiros alimentados com e sem farelo de castanha de caju (P < 0,05). Conclui-se, portanto, que a inclusão de 13% de farelo de castanha de caju na dieta altera a expressão de proteínas seminais e espermáticas em ovinos.

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