Orientador: Maria Tercília Vilela de Azeredo Oliveira / Resumo: A subordem Heteroptera possui uma enorme diversidade de insetos distribuída em sete infraordens, dentre as quais utilizamos Gerromorpha, Nepomorpha, Cimicomorpha e Pentatomomorpha. As espécies que foram estudadas da primeira infraordem, representando os insetos semi-aquáticos, são Limnogonus aduncus e Mesovelia mulsanti, da segunda infraordem, representando os aquáticos, são Belostoma anurum, Martarega sp. e Buenoa unguis, da terceira infraordem Zelus sp. e Teleonemia sp. e da última infraordem, representando juntas os terrestres, são Largus sp., Stenocoris sp., Zicca pulchra e Dysdercus sp. para descrever as ultraestruturas de suas espermátides e posterior comparação. Durante o processo de espermiogênese em geral, ocorre uma série de modificações nas espermátides antes dessas serem transformadas em espermatozoide, inclusive nos insetos. Neste trabalho verificamos por meio da Microscopia Eletrônica de Transmissão, as modificações ocorridas nas espermátides das infraordens anteriormente mencionadas. Na maioria dos insetos com hábitat aquático foi identificada uma maior variação morfológica com relação aos derivados mitocondriais, apresentando-se tanto com simetria reniforme, com simetria e padrão diferente, quanto com assimetria. Com relação aos terrestres, foi identificado predominantemente um padrão morfológico reniforme com uma área de cada derivado mitocondrial menor que a do axonema. Somente a espécie B. unguis, de hábitat aquático, evidenciou um padrão atípico, assimétri... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The suborder Heteroptera has an enormous diversity of insects distributed in seven infraorders, among which Nepomorpha, Gerromorpha, Pentatomomorpha e Cimicomorpha, The species that have been studied from the first infraorder, representing semi-aquatic insects, are Limnogonus aduncus and Mesovelia mulsanti, from the second order, representing the aquatic ones, Belostoma anurum, Martarega sp. and Buenoa unguis, from the third infraorder Zelus sp. and Teleonemia sp. and the last infraorder, representing terrestrials together, are Largus sp., Stenocoris sp., Zicca pulchra and Dysdercus sp. to describe the ultrastructures of their spermatides and further comparison. During the spermiogenesis process in general, a series of changes in spermatids occur before they are transformed into sperm, including in insects In this work we verified through Transmission Electron Microscopy, the changes occurred in the spermatids of the abovementioned infraorders. In the majority of insects with aquatic habitat, a greater morphological variation was identified in relation to mitochondrial derivatives, presenting both with reniform symmetry, with different symmetry and pattern, and with asymmetry. With respect to terrestrials, a predominantly reniform morphological pattern was identified with an area of each mitochondrial derivative smaller than the axoneme. Only the species B. unguis, of aquatic habitat, showed an atypical, asymmetric pattern of mitochondrial derivatives, in relation to that com... (Complete abstract click electronic access below) / Doutor

The aim of my thesis was to identify proteins involved in chemical communication and especially those that are involved in sexual signalling. Volatile chemical signals are transported with lipocalins in their beta-barrel structure to present their ligands to receptors or out of the body. Thus, I focused on the identification of these proteins in saliva and vaginal secretion of the house mouse using proteomic and transcriptomic approaches. Due to a cyclic manner of reproduction and its hormonal control, I have also focused on the role of estradiol on sperm phenotype in the laboratory mouse. We have identified an elevated sexual dimorphism in several lipocalins (i.e. 10 out of 20) in the saliva proteome where they may play a role in sexual signalling (i.e. similar to their described roles in the mouse urine). Interestingly, vaginal secretion also contains lipocalins and they rise from proestrus to estrus and remain steady during metestrus. Such variation provides evidence that they serve sexual signalling, however, due to their elevated levels during metestrus it is most likely that their ligands function as signals and not the proteins themselves. On the level of sperm phenotype, we have provided evidence, that experimental concentrations of estradiol have differential effects on sperm. This is due...

There are an increasing amount of compounds in the environment that can have a negative effect on reproductive parameters in both male and female organism. There has been a worldwide decline of sperm quality during past decades and this fact lead to an increase of unnatural ways of conception through assisted reproduction techniques in the specialised centres. Natural estrogens are one of these compounds and they get into waste water after being excluded from the body by the urine. They get back into the human body from drinking water or from the food, and they can interfere with function of endogenous hormones in very low concentrations. For these reasons it is up to date to deal with the influence of these compounds on mammalian sperm. For many years, estrogens have been considered typically female sex hormones. It is now certain that they are also very important in the regulation of male reproduction. Endogenous estrogens in mammalian males are an important part of the endocrine system. Estrogens play an important role in the development of germ cells, spermatogenesis and processes leading to successful egg fertilization such as a capacitation or acrosomal reaction. Tyrosine phosphorylation is one of the essential steps for the properly ongoing process of capacitation in sperm followed by a...

O presente estudo visou investigar o efeito da refrigeração do sêmen da jaguatirica sobre o Índice de Motilidade Espermática [IME=(%M+MPx5)/2; %M = proporção de espermatozóides móveis; MP = motilidade progressiva], integridade acrossomal (IA) e capacitação espermática; assim como avaliar a eficácia da técnica FITC-PNA/IP na avaliação simultânea da viabilidade espermática (VE) e IA. Sete jaguatiricas foram eletroejaculadas, sendo utilizados apenas ejaculados (n=16) apresentando %M>=60% e MP>=3. Avaliou-se a IA por meio da Coloração Simples. Os ejaculados foram diluídos 1:1 na Variante do Diluente de PLatz e submetidos aos Protocolos de Transporte: Temperatura Ambiente e Refrigeração, - 0,23ºC/min, (Experimento 1); ou apenas Temperatura Ambiente (Experimentos 2 e 3). Após 2h, as alíquotas foram reaquecidas, reavaliando-se os parâmetros observados antes do transporte. Os espermatozóides foram lavados por centrifugação em meio F10 de Ham, ressuspensos nesse meio e processados conforme o experimento: (1) após pré-incubação (38ºC; 5%CO2) durante 0, 1, 2 e 4 horas, foram retiradas alíquotas a cada intervalo para serem incubadas (30 min) na ausência e na presença do cálcio ionóforo A23187 (Ca2+Ion) (1mM), avaliando-se IA e IME; (2) após pré-incubação por 0, 1 e 2h, foram incubadas alíquotas na ausência e presença de 1 e 2mM de Ca2+Ion, avaliado-se IA e IME; (3) pré-incubados por 9h, sendo retiradas alíquotas a cada hora, para as avaliações da IA e VE, (a) separadamente através da Coloração Simples e do IME, ou (b) simultaneamente através da técnica FITC-PNA/IP. A refrigeração causou declínio (p<0,02) da IA (71,0%) e IME (67,1), em comparação aos valores observados antes do transporte (88,5%; 85,4), enquanto a manutenção das amostras à temperatura ambiente não afetou (p>0,1) essas variáveis (84,8%; 76,4). Dentre as amostras refrigeradas, aquelas expostas ao Ca2+Ion sofreram redução (p<0,01) na IA (52,4%) frente ao controle (55,56%). Já nas amostras transportadas à temperatura ambiente, não foi observada diferença (p>0,1) entre os grupos com e sem ionóforo (64,41% vs. 63,87%). Quando analisados os tempos separadamente, o único tratamento em que houve efeito (p<0,05) do Ca2+Ion sobre a IA foi aquele refrigerado e pré-incubado por 2h. Foi verificada redução (p<0,05) nos valores de IME e IA devida à simples incubação, mesmo na ausência do Ca2+Ion. A concentração de 2µM dessa substância foi mais efetiva na indução da reação acrossômica que 1µM. Apesar dos fluorocromos FITC-PNA/IP terem se ligado aos espermatozóides, nas regiões esperadas, a proporção de células marcadas variou aleatoriamente durante pré-incubação, sem correlação (p>0,1) com IME. A IA avaliada pela Coloração Simples apresentou correlação positiva (r=0,77; p<0,0001) com IME, decrescendo (p<0,0001) durante pré-incubação. A refrigeração mostrou-se desvantajosa frente à manutenção do sêmen à temperatura ambiente, pois foi deletéria à função e às membranas dos espermatozóides. A refrigeração tornou-os capazes de responder ao estímulo do Ca2+Ion, característica observada nos espermatozóides capacitados. O ensaio de reação acrossômica induzida pelo Ca2+Ion deve ser aperfeiçoado para permitir avaliação acurada da capacitação espermática na jaguatirica. A Coloração Simples associada à avaliação do IME foi mais eficiente e menos laboriosa, frente á técnica FITC-PNA/IP, na avaliação da IA e VE. / This study aimed to investigate the effect of ocelot semen refrigeration on Sperm Motility Index [SMI=(%M+PMx5)/2; %M = proportion of motile spermatozoa ; PM = Progressive Motility], acrossomal integrity (AI) and sperm capacitation. Another objective was to evaluate the FITC-PNA/IP technique efficacy on evaluating simultaneously sperm viability (SV) and AI. Five ocelots, were electroejaculated, the semen was evaluated and only ejaculates (n=16) presenting %M>=60% and PM>=3 were used. Sperm AI was evaluated using Fast Green / Rose Bengal staining (FGRB). The ejaculates were diluted 1:1 in Platz Diluent Variant and subjected to the transportation protocols: Room Temperature and Cooling, -0.23ºC/min, (experiment 1); or only Room Temperature (experiments 2 and 3). After 2 hours, the aliquots were rewarmed and samples were taken to re-evaluate the parameters observed before the transport. The spermatozoa were washed in Ham’s F10 medium, ressuspended in fresh medium and processed differently, according the experiment: (1) after pre-incubation (38ºC; 5%CO2) during 0, 1, 2 and 4 hours, samples were taken at each time point to be incubated in the absence and presence of 1mM calcium ionophore A23187 (Ca2+Ion), SMI and AI were evaluated; (2) after pre-incubation during 0, 1 and 2h, aliquots were incubated in the absence and presence of 1 and 2 mM Ca2+Ion; SMI and AI were evaluated; (3) after pre-incubation during 9h, aliquots were taken every hour to compare the evaluation of SV and AI (a) separately by the FGRB staining and SMI or (b) simultaneously by the FITC-PNA / IP technique. Cooling caused decline (p<0.02) on AI (71.0%) and SMI (67.1), when compared to values observed before transportation (88.5%; 85.4). Maintenance at room temperature didn’t affect (p>0.1) these variables (84.8%; 76.4). Among cooled samples, spermatozoa exposed to Ca2+Ion showed smaller (P<0.01) AI value (52.4%) compared to the group incubated without that substance (55.56%). For samples transported at room temperature, it wasn’t observed difference (P>0.05) between the groups with and without ionophore (64.41% vs. 63.87%). When time intervals were analysed separately, the only treatment in which there was effect (p<0,05) of Ca2+Ion on AI was the group refrigerated and pre-incubated for 2h. There was a reduction (p<0,05) on SMI and AI due simply to incubation, even in the absence of Ca2+Ion. The 2µM concentration of this substance was more effective to induce acrosome reaction than 1µM. FITC-PNA and IP fluorocromes bound spermatozoa at the expected sites. However, proportion of marked cells varied randomly during pre-incubation, and didn’t correlate (p>0,1) with SMI. IA evaluated by FGRB staining showed positive correlation (r=0,77; p<0,0001) with SMI, decreasing (p<0,0001) during incubation. Cooling was disadvantageous compared to maintaining semen at room temperature, since it was deleterious to spermatozoa membranes and function, and made those cells capable to answer the Ca2+Ion challenge, a characteristic observed in capacitated spermatozoa. Ca2+Ion induced acrosome reaction assay must be improved to allow accurate evaluation of sperm capacitation on ocelots. FGRB staining associated to SMI evaluation was more efficient and easier to perform, than FITC-PNA/IP technique, for AI and SV investigation.

Orientadora: Marion Burkhardt de Koivisto / Banca: Gisele Zoccal Mingoti / Banca: Sony Dimas Bicudo / Resumo: O objetivo deste trabalho foi avaliar os efeitos da gota citoplasmática proximal (GCP) no sêmen de bovinos quanto à integridade do DNA, das membranas citoplasmática, acrossomal, no potencial mitocondrial e verificar a taxa de produção de embriões in vitro. Três amostras descongeladas de cinco (Controle: espermiograma normal), e oito touros Bos indicus (Gota: GCP ≥15%) foram avaliadas. Foram realizados os seguintes testes: motilidade e vigor pós-descongelação, concentração, morfologia espermática, teste de termo-resistência lento (TTL), integridade da membrana acrossomal, plasmática e potencial mitocondrial utilizando sondas fluorescentes (PI, FITC-PSA e JC-1) e integridade da cromatina pelo método de coloração com laranja de acridina. Dois touros com índices elevados de GCP e três animais controle foram selecionados para fertilização in vitro (FIV). As análises estatísticas foram efetuadas empregando-se o programa Statistical Analysis System. O nível de significância foi de 5%. Os resultados obtidos demonstraram que altos índices de GCP não afetaram a motilidade e o vigor, antes e após o TTL, assim como não interferiram na porcentagem de acrossomas intactos. Os resultados destas avaliações mostraram que a alta incidência de GCP afetou a integridade da membrana acrossomal e plasmática bem como a presença de potencial mitocondrial. No entanto, a alta incidência de GCP não promoveu aumento na porcentagem de injúrias à cromatina após descongelação, mas os resultados sugerem que podem ser mais sensíveis à desnaturação quando incubados por três horas. No experimento II, os índices de produção de embriões in vitro podem ter sido afetados pela interação da alteração morfológica e o efeito individual do touro. / Abstract: The objective of this study was to evaluate the effects of the proximal cytoplasmic droplets (PCD) in bovine semen, on the integrity of DNA, cytoplasmic membrane, acrossome, mitochondrial function and the rate of in vitro embryo production. Three batches of five (control group G1: normal sperm parameters) and eight Bos indicus bulls (G2: PCD ≥15%) were analysed. The following tests were carried out: post thaw motility and, vigor, concentration, sperm morphology, slow thermo-resistance (TRT), membrane integrity, acrossome status, mitochondrial function through fluorescent probes (FITC-PSA, PI and JC-1) and integrity of chromatin was accessed by acridine orange stain. Two bulls with high rates of PCD and three animals (control group) were selected for in vitro fertilization (IVF). Statistical analyses were performed using the Statistical Analysis System. The significance level was 5%. The results showed that high rates of PCD did not affect motility and vigor, before and after the TRT, and did not affect the percentage of intact acrossome. The results showed that the high incidence of PCD affected membrane integrity, acrossome status and mitochondrial function when compared to the G1 group due. However, the high incidence of PCD did not affect the percentage of chromatin injury after thawing, but results suggest that spermatozoa may be more susceptible to damage when incubated for three hours. In experiment II the embryo production rate may have been affected by the interaction of the morphology traits and the bull effect. / Mestre

O objetivo deste estudo foi avaliar as alterações morfo-funcionais do sêmen bovino submetido à sexagem através da técnica de citometria de fluxo e comparar com o sêmen convencional e a diferença entre as subespécies. O sêmen foi obtido de seis touros, sendo três taurinos (Holandês preto e branco) e três zebuínos (Gir leiteiro), com quatro ejaculados de cada animal (n = 24). Cinco tratamentos foram realizados: sêmen convencional com diluidor L (Lagoa); sêmen convencional com diluidor S (Sexing) e sêmen sexado (nos tempos de zero, três e seis horas após a ejaculação para avaliar se existem alterações de membranas ao longo do tempo). Foram avaliados pela citometria de fluxo quanto à integridade de membrana plasmática e reação acrossomal (PI/FITC-PSA); peroxidação lipídica (C11-BODIPY581/591) e capacitação espermática através de análises da fosforilação da tirosina e aumento da fluidez e desorganização da membrana plasmática (Merocianina 540 e Yo-Pro1). Os efeitos de tratamentos foram avaliados por análises de variância (ANOVA), empregando-se o programa estatístico Stat-View&reg; (SAS Institute, Inc. 1998, Cary, NC, USA). Foram considerados significativos os valores com P<0,05 e com tendência a significância (P<0,10). O sêmen proveniente da subespécie taurina teve um efeito negativo mais significante do que a zebuína na qualidade do sêmen. O sêmen sexado apresentou maior peroxidação lipídica nos espermatozóides com membrana íntegra e a presença de proteínas fosforiladas na superfície da membrana plasmática, fatores que são indicativos de capacitação espermática. Porém, ao contrário do esperado, este aumento não foi acompanhado pela quantidade de células classificadas como capacitadas pela análise de associação de sondas Yo-Pro-1 e Merocianina 540, pois o sêmen convencional apresentou maior população espermática com membrana plasmática íntegra desorganizada (P<0,05). Não houve piora na qualidade do sêmen sexado devido ao tempo de espera do ejaculado (P<0,05). / The objective of this study was to evaluate the morpho-functions alterations in bovine semen submitted on sorting through flow cytometry technology and compare with conventional semen and the difference between the subspecies. Semen was obtained from six bulls, that three were taurine (Holstein Black and White) and three were zebuine (Dairy Gir) with four ejaculates from each animal (n = 24). Five treatments were performed: conventional semen with L (Lagoa) media; conventional semen with S (Sexing) media and sexed semen (zero, three and six hours after ejaculation to evaluate if there were membrane alterations over the time). The treatments were analyzed with flow cytometry as for plasmatic membrane integrity and acrosome reaction (PI/FITC-PSA); lipid peroxidation (C11-BODIPY581/591) and sperm capacitation through protein tyrosine phosphorylation and the increase in plasma membrane fluidity and disorganization (Merocyanine 540 and Yo-Pro-1). The treatments effects were evaluated by analysis of variance (ANOVA) (Stat-View&reg; SAS Institute, Inc. 1998, Cary, NC, USA). Effects were considered significant if the values were P<0.05 and with significant tendency (P<0.10). Semen from the taurine subspecie had a more significant negative effect than zebuine on semen quality. Sexed semen showed more lipid peroxidation in sperm with membrane integrity and the presence of phosphorylated proteins in plasma membrane surface that seemed to be sperm capacitation. However, in the contrary of expected, this increase did not accompany with quantity of cells classified as capacitated by the association probes Yo-Pro-1 e Merocyanine 540 analyse because the conventional semen showed more sperm population with plasma membrane integrity disorganized (P<0.05). There was not worsening in sexed semen quality over the time ejaculate waiting (P<0.05).

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 5 uppsatser.

La dégradation des fonctions de reproduction masculine dans les dernières décennies, et par suite, la baisse du taux de fertilité chez l'homme a renforcé les démarches de recherche des causes d'infertilité masculine, qu'elles soient génétiques, ou environnementales, mettant en œuvre les perturbateurs endocriniens ou d'autres produits reprotoxiques. Par ailleurs, les traitements palliatifs tels que l'aide médicale à la procréation ne permettent qu'à 1 couple sur deux d'obtenir une grossesse. La recherche vise donc également à définir de nouvelles pistes d'amélioration de ces techniques. Les phospholipases A2 (PLA2) sont des enzymes abondamment exprimées dans les organes reproducteurs mâles, le sperme éjaculé et dans le tractus génital femelle. Elles jouent des rôles importants dans la capacitation, la réaction acrosomique (RA) et la fécondation. Nous avons montré au laboratoire que la PLA2 murine secrétée de groupe X (mGX) est présente dans l'acrosome des spermatozoïdes de souris. Elle est libérée au cours de la RA et s'avère un inducteur puissant de la RA. Les mécanismes permettant ces effets sont encore mal connus. Par l'utilisation d'inducteurs et d'inhibiteurs des PLA2, sur des modèles murins wild type ou KO pour certaines PLA2, ce travail montre que durant la capacitation, l'activation des iPLA2 β permet de déclencher une RA spontanée dans une sous-population spécifique de spermatozoïdes. Au cours de la RA induite par la progestérone (P4), On a pu aussi valoriser le rôle des iPLA2 β qui initie la cascade de la RA et permet que les sPLA2 soient activées pour amplifier le déroulement de la réaction acrosomique. Par ailleurs, ce travail montre que l'effet de mGX sur le taux de fécondation et de développement embryonnaire ne dépend pas du taux de la RA. Cet effet obtenu grâce à mGX n'est pas observé avec d'autres sPLA2 (murine ou humaine), ni avec la P4 ce qui lui confère une propriété espèce dépendante. / For the last ten years, the impairment of the male reproductive functions has highly increased, while the use of assisted reproductive techniques has also increased. Despite this evolution, the pregnancy rates obtained in in vitro fertilization (IVF) remain low, as only one in two couples will obtain a pregnancy after 4 attempts. Research has to discover new ways to gain in reproductive impact. Hence, many studies have recently been developed to test molecules that could improve the IVF results. Phospholipases A2 (PLA2s) are part of these molecules. They play an important role, because of their abundant expression in: male reproductive organs, in ejaculated sperm and in the female tract. Several studies have suggested a role for members of the secreted phospholipase A2 family in capacitation, acrosome reaction (AR), and fertilization. We demonstrated previously that sperm from mGX knock-out mice had a severely impaired fertilization potential in vitro, but the molecular nature of these enzymes and their specific functions have remained elusive. Our aims were to study the mechanism of the acrosome reaction by focusing on different kinds of PLA2 using inhibitors and knockout mice for each type of PLA2. We demonstrate the importance of iPLA2β in spontaneous AR occurring during capacitation. We also show that iPLA2β and sPLA2 of group X are both involved in progesterone (P4)-induced AR in mouse sperm. In addition we show that in the mouse neither P4 nor any of the other sPLA2s tested are able to mimic the IVF improvement obtained with mGX-treatment. We also demonstrate that this improvement obtained with phospholipase A2 murine group X is not dependent on the rate of AR. These results demonstrate that sPLA2s are not commutable in the context of mouse sperm fertility, indicating that group X sPLA2 is unique to improve fertility outcome.

Made available in DSpace on 2015-12-06T23:46:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2014 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / funcionais nos espermatozoides e o nivel seminal de peroxidacao lipidica. Metodo: Um estudo transversal foi realizado incluindo 156 pacientes normozoospermicos. Apos a coleta do semen por masturbacao, uma aliquota foi utilizada para a analise seminal e outra para a avaliacao da atividade mitocondrial, da integridade do acrossoma e da integridade do DNA dos espermatozoides. O volume remanescente de semen foi centrifugado e o plasma seminal sobrenadante foi utilizado para a avaliacao do nivel seminal de peroxidacao lipidica e para a analise proteomica. Posteriormente, os pacientes foram divididos em percentis (15%) para formacao dos grupos experimentais de cada estudo: Estudo 1 - alta (grupo controle) e baixa (grupo alterado) atividade mitocondrial dos espermatozoides, Estudo 2 - alta (grupo controle) e baixa (grupo alterado) integridade do acrossoma dos espermatozoides, Estudo 3 - baixa (grupo controle) e alta (grupo alterado) fragmentacao do DNA dos espermatozoides e Estudo 4 - baixo (grupo controle) e alto (grupo alterado) niveis seminais de peroxidacao lipidica. A analise proteomica foi realizada utilizando LCMS/MS. Os grupos foram comparados por meio de analise univariada (teste t de Student) e analise multivariada (PLS-DA e analise discriminante). As proteinas significantes foram posteriormente submetidas a analise de enriquecimento funcional. Resultados: Nos estudos 1, 2, 3 e 4 foram observadas 506, 493, 474 e 629 proteinas, respectivamente. As funcoes enriquecidas no estudo 1 foram detoxificacao de EROs e ligacao a NADP (controle) e atividade de oxidoredutase intramolecular, catabolismo de aminoglicanos, inibicao de endopeptidases, lisossomos e resposta imune de fase aguda (alterado). As principais funcoes enriquecidas no estudo 2 foram resposta imune (controle) e inibicao de fosfolipase, metabolismo do acido araquidonico, exocitose, resposta inflamatoria aguda, resposta ao peroxido de hidrogenio e transporte lisossomal (alterado). As principais funcoes enriquecidas no estudo 3 foram metabolismo de carboidratos, regulacao de lipoproteinas, regulacao negativa da apoptose, metabolismo de hormonios, atividade de metalopeptidases, ligacao ao NAD e lisossomos (controle) e biossintese de prostaglandinas e ligacao a acidos graxos (alterado). As principais funcoes enriquecidas no estudo 4 foram biossintese de acidos graxos insaturados, atividade de oxidantes e antioxidantes e resposta celular ao estresse termico (alterados). Nos estudos 1, 2, 3 e 4 foram sugeridos 8, 6, 8 e 7 biomarcadores seminais de atividade mitocondrial, integridade acrossoma, fragmentacao de DNA e peroxidacao lipidica, respectivamente. Conclusoes: O perfil proteomico do plasma seminal reflete alteracoes funcionais dos espermatozoides e o nivelseminal de peroxidacao lipidica e diversas funcoes pos-genomicas estao relacionadas as alteracoes estudadas. Proteinas relacionadas as alteracoes funcionais dos espermatozoides e ao nivel seminal de peroxidacao lipidica constituem potenciais biomarcadores seminais para cada alteracao / Objective: To verify if the seminal plasma proteomic profile reflects sperm functional alteration and semen lipid peroxidation levels. Method: A cross-sectional study was performed including 156 normozoospermic patients. After semen retrieval by masturbation, an aliquot was utilized for semen analysis and another for evaluation of sperm mitochondrial activity, acrosome integrity and DNA fragmentation. The remaining semen volume was centrifuged and the supernatant seminal plasma was utilized for semen lipid peroxidation levels evaluation, and proteomic analysis. Patients were divided into percentiles (15%) to form the experimental groups: Study 1 – high (control group) and low (altered group) sperm mitochondrial activity; Study 2 – high (control group) and low (altered group) sperm acrosome integrity; Study 3 – low (control group) and high (altered group) sperm DNA fragmentation; Study 4 – low (control group) and high (altered group) semen lipid peroxidation levels. Proteomic analysis was performed by a LC-MS/MS approach. Groups were compared using univariate (Student’s t test) and multivariate (PLS-DA and discrimant analysis) analyses. Differentially expressed proteins were then utilized for functional enrichment analysis. Results: 506, 493, 474, and 629 proteins were observed in studies 1, 2, 3, and 4, respectively. Enriched functions in study 1 were reactive oxygens species detoxification, and NADP binding (control group), and intramolecular oxidoreductase activity, aminoglycans catabolism, endopeptidases inhibition, lysosomes, and acute-phase response (altered group). In study 2, main enriched functions were acute-phase response (control group), and phospholipase inhibition, arachidonic acid metabolism, exocytosis, regulation of acute inflammatory response, response to hydrogen peroxide and lysosomal transport (altered group). In study 3, main enriched functions were carbohydrates metabolism, lipoprotein regulation, negative regulation of apoptosis, hormone metabolism, metalopeptidases activity, NAD binding, and lysosomes (control group), and prostaglandin biosynthesis, and fatty acid binding (altered group). In study 4, enriched functions were unsaturated fatty acid biosynthesis, oxidants and antioxidants activity, and cellular response to heat stress (altered group). In total, 8, 6, 8, and 7 seminal biomarkers were proposed for studies 1 (mitochondrial activity), 2 (acrosome integrity), 3 (DNA fragmentation), and 4 (lipid per oxidation), respectively. Conclusions: The seminal plasma proteomic profile reflects sperm functional alterations and semen lipid perodixation levels, and several post-genomic functions are related to the studied alterations. Proteins related to sperm functional alterations, and semen lipid peroxidation levels constitute potential seminal biomarkers for each alteration. / BV UNIFESP: Teses e dissertações

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No. of bitstreams: 0 Previous issue date: 2009-02-27 / Objetivos:Avaliar o efeito da varicocele na integridade do DNA nuclear,na atividade mitocondrial,na peroxidação lipídica e na integridade acrossômica dos espermatozóides.Métodos:as amostras foram obtidas e analisadas de acordo com os parâmetros da OMS(1999) e morfologia segundo critério estrito de Kruger.O grupo de estudo incluiu 30 homens com varicocele grauII ou III e o grupo controle incluiu 32 homens sem varicocele.A integridade do DNA nuclear dos espermatozóides foi avaliada pelo ensaio Cometa alcalino, e as células foram classificadas de acordo com a intensidade de dano no DNA:grau I(alta integridade do DNA),grau II(DNA ainda íntegro ou em início de fragmentação),grau III(DNA moderadamente fragmentado)e grau IV(DNA altamente fragmentado).A atividade mitocondrial foi avaliada pelo método colorimétrico proposto por Hrudka(1987),e as células foram classificadas em:classe I(mitocôndrias todas ativas),classe II (mais de 50 por cento de mitocôndrias ativas),classe III(menos de 50 por cento de mitocôndrias ativas)e classe IV(mitocôndrias todas inativas).A peroxidação lipídica foi determinada usando-se o método descrito por Ohkawa(1979),que se baseia na determinação de MDA devido à sua reação com o TBA, e o nível de peroxidação lipídica foi descrito em nanogramas de TBARS/mL de sêmen.A integridade do acrossoma foi avaliada através da sonda fluorescente PNA(Peannut Agglutinin)-FITC (Fluorescein isothiocyanite)conjugada, e o resultado foi expresso em porcentagem de espermatozóides com acrossoma íntegro.Resultados: quanto à integridade do DNA,o grupo de homens com varicocele apresentou uma menor porcentagem de espermatozóides com DNA nuclear íntegro(grau II, p=0,040).Não houve diferença na porcentagem de células grau I,III e IV.Quanto à atividade mitocondrial, o grupo de homens com varicocele apresentou uma porcentagem maior de espermatozóides com mitocôndrias inativas(classe III, p=0,020)e uma porcentagem menor de espermatozóides com mitocôndrias ativas(classe I, p=0,005).Não houve diferença na porcentagem de células classe II e IV.Quanto à integridade acrossômica, o grupo de homens com varicocele apresentou uma menor porcentagem de espermatozóides com acrossoma íntegro(p=0,0002).Por fim, com relação ao nível de peroxidação lipídica,não foi encontrada nenhuma diferença estatisticamente significante entre os grupos com e sem varicocele.Conclusões: Neste estudo, homens com varicocele apresentaram um aumento nas taxas de fragmentação de DNA e uma redução na atividade mitocondrial e na integridade acrossômica dos espermatozóides.Todavia, nenhuma diferença entre os níveis seminais de MDA foi encontrada, o que sugere que talvez as alterações funcionais encontradas não estejam associadas diretamente com o estresse oxidativo, ou que o estresse oxidativo leve a alterações no DNA, mitocôndrias e acrossoma durante a espermatogênese, e não após a ejaculação / Objectives: to assess the effect of varicocele on sperm nuclear DNA integrity, mitochondrial activity, lipid peroxidation and acrosome integrity. Methods: semen samples were obtained and analyzed according to the World Health Organization guidelines (1999) and sperm morphology was evaluated by Kruger’s strict criteria (1986). The study group included 30 men with varicocele grades II or III and the control group included 32 men without varicocele. Sperm nuclear DNA integrity was assessed by the alkaline Comet assay, and cells were graded according to the intensity of DNA damage: class I (high DNA integrity), class II (DNA still intact or initiating fragmentation), class III (DNA fairly fragmented) and class IV (DNA extremely fragmented). Mitochondrial activity was evaluated by the colorimetric method proposed by Hrudka (1987). Cells were classified according to the proportion of active mitochondria: class I (100% of active mitochondria), class II (more than 50% of active mitochondria), class III (less than 50% of active mitochondria) and class IV (100% of inactive mitochondria). Lipid peroxidation was determinated by Ohkawa’s method, which is based on the measurement of malondialdehyde (MDA) due to its reaction with thiobarbituric acid (TBA), and the levels of lipid peroxidation were described as nanograms of TBARS/mL. Acrosome integrity was assessed by use of the conjugated fluorescent probe PNA-FITC and the results were expressed in percentages of intact acrosomes (fluorescence was observed over the entire acrosomal region of the sperm head). Results: Concerning DNA integrity, the varicocele group showed less spermatozoa with intact nuclear DNA (grade II, p=0,040). There was no significant difference in classes I, III and IV between the two groups. Regarding mitochondrial activity the varicocele group showed more cells with inactive mitochondria (class III, p=0,001) and less cells with active mitochondria (class I, p=0,005). There was no difference in classes II and IV. Also, the varicocele group showed less spermatozoa with intact acrosomes (p=0,0002), when compared to the controls. Finally, no significant differences were observed in lipid peroxidation levels. Conclusions: This study was able to demonstrate that varicocele in adults is associated with increased DNA fragmentation, reduced mitochondrial activity and decreased acrosome integrity even when semen quality does not differ from men without varicocele. However, levels of seminal products of lipid degradation (MDA) are not increased in these patients, suggesting that perhaps the functional changes found are not directly associated with oxidative stress, or that oxidative stress leads to changes in DNA, acrosomes and mitochondria during spermatogenesis, and not after ejaculation. / TEDE / BV UNIFESP: Teses e dissertações