In mammals, freshly ejaculated spermatozoa do not possess the ability to fertilize a mature oocyte. They acquire fertilization competence upon residing for a period of time in the female reproductive tract. The physiological changes that bring about these time-dependent changes in motility pattern and acquisition of fertilizing ability of spermatozoa are collectively referred to as capacitation, culminating in sperm hyperactivation. Capacitation-associated increase in sperm protein tyrosine phosphorylation (PYP), exhibited by mammalian sperm, is one of the major downstream events, regulating hyperactivated motility. However, it is still unclear which are the tyrosine kinases and phosphatases involved in modulating the capacitation-associated increase in global PYP. In order to determine this, our laboratory earlier showed the role of PYP in hamster sperm capacitation using a specific EGFR protein tyrosine kinase (PTK) inhibitor, tyrphostin A47 (TP-47). Interestingly, inhibition of capacitation by 0.5 mM TP-47 was associated with induction of a slow circular motility pattern, accompanied by inhibition of PYP of certain proteins (Mr. 45,000-52,000), localized to the principle piece of the sperm flagellum. Two such proteins, hypo-tyrosine phosphorylated, were found to be tektin-2 and ODF-2, using 2D-PAGE followed by MS/MS analysis. Interestingly, a global phosphoproteome analysis of human spermatozoa showed that PYP changes are associated with capacitation and asthenospermic condition in infertile men is attributed to the failure of capacitation-associated increase in PYP. Such individuals exhibited impaired sperm motility. There is a need to understand the exact mechanism of phosphorylation of sperm flagellar proteins, which is necessary to assess sperm’s ability to fertilize the mature oocyte. Therefore, the focus of the present work was to elucidate the role of receptor tyrosine kinases (RTKs) and the non-receptor tyrosine kinases (NRTKs) in mammalian (hamster) sperm capacitation. Recent studies have shown that apart from EGFR other RTKs like IGF1R, FGFR, VEGFR, MuSK, TrkA are expressed in mammalian spermatozoa and actively involved in sperm capacitation. However, there is very little information available in the context of sperm capacitation and associated PYP. Therefore, attempts were made to understand the role of various RTKs (IGF1R, FGFR and VEGFR) in hamster sperm capacitation and associated PYP. Initially, the role of IGF1R tyrosine kinase during sperm capacitation was studied. Immunolocalization of IGF1R in spermatozoa showed a strong signal in the sperm acrosome and the principal piece of the sperm flagellum. Inhibition of IGF1R kinase with an IGF1R-specific inhibitor TP-1-O-Me-AG538 (TP-538) showed inhibitory effect on sperm capacitation and the associated hyperactivation. But, inhibitors of FGFR and VEGFR tyrosine kinases did not show such an effect. Interestingly, inhibition of IGF1R by TP538 was associated with inhibition of PYP of certain proteins (Mr. 45,000-120,000), localized to head, mid piece and principle piece regions of the sperm flagellum. Phosphoproteomic analysis using 2D-PAGE-western blot with anti-phosphotyrosine antibodies identified 17 differentially phosphorylated protein spots. Out of the 17 spots, 12 were identified by MALDI-MS/MS analysis. The proteins identified to be differentially phosphorylated, upon inhibition of IGF1R, were PDHE1, ODF-2, Tubulin β 2C chain, PDHE2 and ATP synthase β subunit. The RTKs being present in the membrane level may not be directly involved in the phopshorylation of downstream target proteins associated with the mitochondrial membrane, sperm axonemal structures and outer dense fibers. Therefore, the RTKs may interact directly or indirectly with the downstream NRTKs, which may be involved in the phosphorylation of target sperm proteins. Till date, six different families of NRTKs are shown to be expressed in mammalian spermatozoa. The major family of NRTKs involved in sperm function is the Src family of kinases. However, there is very little information available in the context of sperm capacitation and the associated PYP. Therefore, studies were carried out to understand the role of Src family of NRTKs in sperm capacitation and associated PYP. Presence of active Src signaling was observed by the immunolocalization of activated Src (pY416) in the acrosome, mid piece and the principal piece regions of the sperm flagellum. Inhibition of Src family of kinase with a specific Src family kinase inhibitor PP2, showed inhibition of sperm capacitation and the associated hyperactivation. Inhibition of Src family of kinases with PP2 was associated with decrease in PYP of several proteins (Mr. 45,000-120,000), localized mainly to the mid piece region, followed by the principle piece region of the sperm flagellum. Phosphoproteomic analysis using 2D-PAGE-western blot with anti-phosphotyrosine antibodies identified 38 differentially phosphorylated protein spots. Out of the 38 spots, 16 were identified by MALDIMS/MS analysis and these corresponded to seven proteins which included PDHE1, ODF-2, Tubulin β 2C chain, Tektin-2, GAPDS, PDHE2 and ATP synthase β subunit. Additionally, the biochemical and molecular characteristics of the identified proteins were also studied. Bioinformatic analysis predicted the presence of phosphorylation motifs for several kinases and interestingly, all the proteins identified had a Src kinase motif. Comparing the current observations and the previous work in the laboratory, two proteins ODF-2 and Tektin-2 were found to be regulated by EGFR, IGF1R and Src family of kinases. Therefore, characterization of the capacitation-associated tyrosine phoposphorylated proteins ODF-2 and Tektin-2 was performed. By employing PCR and Northern blotting techniques, the presence of the transcripts of both the proteins was shown. Additionally, the ontogeny of expression of ODF2 and Tektin-2 in hamster testis development was studied and the results indicated that the expression of both the proteins started from week 3 onwards till week 8. To confirm the meiotic stage-associated expression of ODF-2 and Tektin-2, germ cells were sorted based on their DNA content. ODF-2 and Tektin-2 transcripts were first expressed in the meiotic germ cells (pachytene spermatocytes) and their expression was upregulated in the post-meiotic germ cells (round spermatids). Sequential extraction of sperm proteins showed that, Tektin-2 was majorly extracted out in the Triton X-100 and DTT fraction, whereas, ODF-2 was maximally extracted in the presence of urea and DTT. In conclusion, these observations indicate that IGF1R and Src family of tyrosine kinases are critical for mammalian sperm capacitation and associated global PYP. Inhibition of sperm capacitation was associated with hypo-tyrosine-phospohorylation of certain proteins associated with mitochondrial membrane, axonemal structures and outer dense fibers of the sperm flagellum. Future work can be directed towards understanding the role of other RTKs and NRTKs involved in sperm capacitation and the molecular characterization of hypophosphorylated proteins critical for sperm function and its fertilization competence.

INTRODUÇÃO: Este trabalho avalia a capacitação dos multiplicadores do Projeto \"Nossas Crianças: Janelas de Oportunidades\", a partir da percepção dos sujeitos participantes. Tal Projeto tem como tema o desenvolvimento infantil, na perspectiva da Promoção da Saúde, por meio da atuação das equipes do PSF, junto às famílias com gestantes e crianças na faixa etária de 0 a 6 anos. O estudo considera a consonância entre a Metodologia Pedagógica empregada na capacitação e os referenciais teóricos que embasaram o Projeto, tendo em vista o empowerment dos profissionais. A estratégia escolhida para a capacitação foi a Oficina Problematizadora. OBJETIVO: Avaliar o processo de capacitação dos multiplicadores do Projeto Janelas a partir da percepção dos mesmos, por meio do grau de apreensão dos conteúdos apresentados, e da adequação da estratégia pedagógica empregada à finalidade de reproduzirem-na junto às equipes. MATERIAL E MÉTODO: A pesquisa realizada foi de natureza qualitativa na modalidade estudo de caso, com a utilização de entrevistas semi-estruturadas e posterior análise temática. A amostra foi intencional e o critério de inclusão dos participantes foi o maior grau de manutenção de suas atividades, relacionadas à implementação do Projeto em suas unidades de origem. Foram entrevistados oito participantes, número suficiente para contemplar o critério de saturação dos dados. RESULTADOS: Foram entrevistados sete mulheres e um homem, nas faixas etárias entre 25 e 50 anos, sendo cinco enfermeiras, uma técnica em enfermagem, uma assistente social e um médico, dentre eles, dois bacharéis, duas especialistas em Saúde da Família, uma sanitarista, um Mestre e uma Doutora. A grande maioria permanece de 3 a 4 anos na estratégia. A análise das falas demonstrou uma avaliação fortemente positiva pelos entrevistados quanto à sistematização e apreensão de conteúdos como: cuidados, resiliência, competências familiares, rede social e família, que os instrumentalizou para o trabalho. A Metodologia Problematizadora possibilitou um resgate de suas realidades, da experiência de grupo, da participação e da vivificação do trabalho. CONCLUSÃO: A avaliação demonstra que houve apreensão dos conteúdos pelos participantes, bem como adequação entre a metodologia e os referenciais teóricos do Projeto, possibilitando aos mesmos transmitirem seus conteúdos junto às suas equipes / INTRODUCTION: This work evaluates the qualification of the multipliers of the Project Our Children: Windows of Chances, from the perception of the participant citizens. This Project has as subject the infantile development, in the perspective of the Health Promotion, by means of the performance of the PSF teams, together to the families with pregnant women and children aged from 0 to 6 years. The study considers the accord between the employed Pedagogical Methodology in the theoretical qualification and referenciais that had based the Project, considering the empowerment of the professionals. The strategy chosen for the qualification was the Problemized Workshop. OBJECTIVE: To evaluate the process of qualification of the multipliers of the Project Windows from the perception of the same ones, by means of the degree of apprehension of the presented contents, and the adequacy of the pedagogical strategy used to the purpose to reproduce it together to the teams. MATERIAL AND METHOD: The research was of qualitative nature in the modality of case study, with the use of half-structuralized interviews and posterior thematic analysis. The sample was intentional and the criterion of inclusion of the participants was the biggest degree of maintenance of its activities, related to the implementation of the Project in its units of origin. Eight participants had been interviewed, number enough to contemplate the criterion of saturation of the data. RESULTS: Seven women had been interviewed and a man, in the age from 25 to 50 years, being five nurses, one technique in nursing, a social assistant and a doctor, amongst them, two graduated people, two specialists in Family Health, a sanitary agent, a Master and a Doctor. The great majority remains from 3 to 4 years in the strategy. The analysis of the conversations demonstrated a positive evaluation for the interviewed ones related to the systematization and apprehension of contents as: familiar cares, resilience, abilities, social net and family, who instrumentalized them for the work. The Problematized Methodology made possible a rescue of its realities, of the experience of group, the participation and the experience of the work. CONCLUSION: The evaluation demonstrates that the apprehension of the contents happened between the participants, as well as adequacy between the methodology and the theoretical referenciais of the Project, making possible the same ones to transmit its contents together to its teams

Analisar a trajetória profissional e pedagógica do enfermeiro docente para o desenvolvimento da simulação clínica no ensino de enfermagem, considerando suas interações sociais e culturais ao longo da vida e repercussões no ensino de graduação em enfermagem. Metodologia: trata-se de estudo de abordagem qualitativa, realizado em Instituição de Ensino Superior Pública do estado de São Paulo, a qual mantém dois cursos de graduação em enfermagem e realiza práticas de ensino simulado sistematicamente atreladas às disciplinas de graduação dos respectivos cursos. Para identificar as docentes que aplicam a simulação no ensino de enfermagem, foram analisados os cronogramas de todas as disciplinas de graduação ofertadas para os dois cursos. Desse modo, de um universo de 23 docentes, dez foram convidadas para participar da pesquisa. Para coleta de dados, foi realizada entrevista gravada, mediada por 17 questões norteadoras. As respostas foram transcritas e analisadas segundo análise temática e discutidas considerando o referencial da abordagem históricocultural. O período de coleta de dados foi de junho a agosto de 2017. Resultados: entre as dez professoras convidadas para participar da pesquisa, oito delas aceitaram. A partir da análise dos dados, foi possível eleger três temas para discussão que trataram de questões relacionadas: às trajetórias profissionais e pedagógicas para formação de professores no que se referem às convivências das docentes ao longo da vida; aos encontros com a simulação clínica em enfermagem enquanto agentes potencializadores ou desafiadores da aprendizagem dos estudantes e da prática pedagógica dos professores e; às repercussões na formação em enfermagem mediadas pela motivação, intenção e percepções de professores. Considerações finais: a atuação do professor na simulação clínica no ensino de graduação está diretamente relacionada ao modo como o professor vem interagindo com o mundo ao seu redor e, aparentemente, depende principalmente das suas concepções pessoais de mundo e de ensino / Goal: Analyze the professional and pedagogical background of the nursing professor for the development of the clinical simulation in the nursing teaching, taking into account their social and cultural interactions throughout life, as well as the repercussions in the nursing undergraduation teaching. Methodology: a qualitative approach study conducted in a public university institution in the state of São Paulo, which holds two nursing undergraduation courses and runs teaching practices systematically simulated coupled to the undergraduation subjects of the courses. In order to identify the professors who apply the simulation in the nursing teaching, we analyzed the chronograms of all the undergraduation subjects offered for both courses. Thus, out of 23 professors, 10 were invited to take part in the research. For the data collection, a recorded interview mediated by 17 guiding questions was used. The answers were transcripted, submitted to the thematic analysis and discussed in light of the referential of the historical-cultural approach. The data collection period was from June to August 2017. Results: Among the 10 professors invited to take part in the research, 8 accepted to do so. The data analysis enabled the selection of three subjects for the discussion, which dealt with questions related to: professional and pedagogical development for the formation of the professors regarding their interactions throughout life; the experiences with the nursing clinical simulation as boosters or challengers of students´ learning and of the professors´ teaching practice; the repercussions in the formation of the nurse mediated by the professors´ motivation, intention and perception. Final considerations: The role of the professor in the clinical simulation in the undergraduation teaching is closely related to how professors have interacted with the world around them. Apparently, it depends mainly on their personal conceptions of both the world and the teaching

A capacitação é um pré-requisito fisiológico importante para que a célula espermática fertilize o oócito. O óxido nítrico (NO) é sintetizado in vivo durante a conversão da L-arginina em L-citrulina por reações oxidativas catalisadas pela enzima óxido nítrico sintase (NOS) desempenhando um papel importante na regulação da motilidade e na capacitação dos espermatozoides. Estudos indicam que o NO é capaz de regular a concentração da AMP cíclico e, por conseguinte, através da atividade da adenil ciclase, estimular a capacitação espermática em várias espécies. O objetivo deste estudo foi avaliar a função do NO na capacitação de espermatozoides equinos criopreservados. Três ejaculados foram colhidos de três garanhões (n=9). O sêmen foi diluído em meio Botu-Crio&reg; na concentração final de 200×106 células/mL, envasado em palhetas de 0,5 mL e criopreservado usando um sistema automatizado. Para cada análise, foram descongeladas quatro palhetas da mesma partida e do mesmo garanhão em banho-maria a 37oC/30 s. e em seguida, o sêmen foi submetido à centrifugação em meio FIV. Posteriormente, o sêmen foi incubado neste mesmo meio na presença de L-arginina, com ou sem inibidor da enzima óxido nítrico sintase o (L-NAME), e com ou sem o removedor de NO (azul de metileno) nos tratamentos: 1) C= (FIV); 2) A= L-arginina (10 mM); 3) L = L-NAME (1 mM); 4) M = azul de metileno (100 mM); 5) AL = L-arginina (10 mM) + L-NAME (1 mM); 6) AM = L-arginina (10 mM) + azul de metileno (100 mM). As amostras foram incubadas a 38oC e 5 % de CO2. Após a incubação realizou-se a análise computadorizada da motilidade do espermatozoide e as análises por citometria de fluxo. Para a análise computadorizada da motilidade espermática foram avaliados os tempos de incubação de 0, 60, 120 e 300 min. e para as análises por citometria de fluxo os tempos de 60, 120 e 300 min. Para avaliar a integridade das membranas plasmática e acrossomal usou-se a associação FITC-PSA e IP. Para a detecção da fosforilação do aminoácido tirosina, usou-se o anticorpo antifosfotirosina conjugado a uma fluoresceína (DAF-2). A fim de dosar a quantidade de NO produzido pelo espermatozoide equino criopreservado foi utilizada a sonda DAF e para avaliar a peroxidação lipídica da membrana espermática utilizou a sonda C11-BODIPY. A sonda H33342 foi usada com a finalidade de evitar que partículas do mesmo tamanho e granulosidade da célula espermática fossem incluídas na contagem das análises por citometria de fluxo. Os dados foram analisados por meio da ANOVA e a comparação das médias, dentro de cada tempo, pelo teste de Tukey, com o nível de significância de 5 %, usando o software SAS. A remoção do NO do meio de cultura inibiu a motilidade das células espermáticas em todos os tempos de incubação. A motilidade total e motilidade progressiva foram reduzidas nos grupos M e AM. Os espermatozoides incubados com o removedor do NO apresentaram maior porcentagem de células com membrana plasmática e acrossomal íntegras nos 60 e 120 minutos de incubação (p<0,05). A reação acrossomal foi induzida nos tratamentos que receberam L-arginina (A; AL). Dentro de cada tratamento, a quantidade de NO produzido pelo espermatozoide, a fosforilação do aminoácido tirosina e a peroxidação lipídica não apresentaram diferenças entre os tempos (p>0,05). Foi verificada uma redução destas variáveis nos grupos M e AM (p<0,05). Contudo, a dose de 1 mM de L-NAME, não foi suficiente para inibir a NOS em espermatozoides criopreservados de equinos. A remoção do NO mantém a integridade das membranas plasmática e acrossomal, entretanto inibe totalmente a motilidade espermática, sugerindo um papel benéfico do NO endógeno na manutenção da motilidade dos espermatozoides equinos criopreservados. / Capacitation is an essential physiological prerequisite in order to sperm cell fertilize the oocyte. Nitric oxide (NO) is synthesized in vivo during the conversion of L-arginine in L-citruline by oxidative reactions catalyzed by nitric oxide synthase enzyme (NOS) and plays an important role in regulation of motility and in sperm capacitation. Studies indicated that NO is capable of regulating cAMP concentration and, therefore, by adenylyl cyclase, stimulate sperm capacitation in several species. The aim of this study was to evaluate the function of nitric oxide in cryopreserved equine sperm capacitation.Three ejaculates from three stallions were collected (n=9). Semen samples were diluted with Botu-Crio&reg; extender to a final concentration of 200×106 sperms/mL, and then packaged in 0.5mL straws and cryopreserved using an automated freezing system. For each analysis, four straws from the same batch and the same stallion were thawed in a water bath at 37oC/30 s. washed by centrifugation in FIV medium. Thereafter, samples were incubated in FIV medium in the presence of L-arginine, with or without the inhibitor of nitric oxide sinthase (L-NAME), and with or without the scavenger of NO (Methylene blue) in the following treatments: 1) C = Control (FIV); 2) A = L-arginine 10 mM; 3) L = L-NAME 1mM; 4) M = Methylene blue 100 mM; 5) AL = L-arginine (10 mM) + L-NAME (1 mM); 6) AM = L-argine (10 mM) + Methylene blue (100 mM). The treatments were incubated at 38oC and CO2 at 5 %. After incubation, the computer-assisted sperm motility (CASA) and flow cytometry analyses were performed. For CASA analysis, the incubation times of 0, 60, 120 e 300 min. were evaluated and for flow cytometry analyses times 60, 120 e 300 min. were evaluated. Plasma and acrosomal membranes integrity were evaluated by FITC-PSA and PI association. In order to detect amino acid tyrosine phosphorylation, we used the anti-phosphotyrosine antibody conjugated to a fluorescein (DAF-2). In order to quantify the amount of nitric oxide produced by cryopreserved equine sperm, the fluorescent probe DAF was used, and to evaluate the lipid peroxidation of sperm membrane we used the probe BODIPY-C11. The probe H33342 was used in order to prevent that particles of the same size and granularity of sperm cell were included in the counting of flow cytometry analyses. Data were analyzed by ANOVA and comparison of means within each time by the Tukey test, at a significance level of 5%, using SAS software. Removing NO from the culture medium inhibited the motility of sperm cells at all incubation times. Total and progressive motilities were reduced in both groups, M and AM. Sperms incubated with the scavenger of NO had the highest percentage of cells with intact plasma and acrosomal membranes at 60 and 120 minutes of incubation (p <0.05). Acrosomal reaction was induced in treatments with L-arginine (A, AL). Within each treatment, the amount of NO produced by sperms, the level of amino acid tyrosine phosphorylation and lipid peroxidation had no differences between the times used (p> 0.05). A reduction of these variables in groups M and AM (p <0.05) was observed. However, a dose of 1 mM L-NAME was not sufficient to inhibit NOS in cryopreserved equine sperm. Removal of NO maintains plasma and acrosomal membranes integrity, however completely inhibits sperm motility, suggesting a beneficial role of endogenous NO in the maintenance of motility of cryopreserved equine spermatozoa.

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.

Made available in DSpace on 2016-04-27T14:32:15Z (GMT). No. of bitstreams: 1 Fabiola Gonzalez dos Santos.pdf: 1428417 bytes, checksum: 8c4e91521962843fc329b4f4b562dc9f (MD5) Previous issue date: 2008-06-12 / Secretaria da Educação do Estado de São Paulo / This study aims to understand the formation process of the people responsible for the execution of the School Family Program in state net of education. For this, first it is presented a brief historic social and political context in which the program has its origin, showing the pretensions of the program according to the official discourse of State Secretary of Education, the people involved in its execution, its organizational structure and pointing out some concepts involved in the design of this program and its possible consequences. We aim to comprehend the formation of the people responsible for the execution of the Program in school. Our criterion was to understand the existence of an effective contribution of these people in the constitution of the school as a democratic and social transformation space and not only the offer of actions of compensation of the social inequalities. So, our concern is to identify how the formation of those responsible and involved in the implementation and operation process of the School Family Program happened, in the sense of understanding the process of formation occurred and seek the learning of new repertoires of action with the objective of expanding the resources and the possibilities of formation of the School Family Program educators / A presente pesquisa tem o objetivo de compreender o processo de formação dos sujeitos responsáveis pela execução do Programa Escola da Família na rede estadual de ensino. Para tal, primeiramente, apresenta-se um breve contexto histórico-social e político no qual o Programa tem sua origem, mostrando suas pretensões segundo o discurso oficial da Secretaria de Estado da Educação, os sujeitos envolvidos em sua execução, sua estrutura organizacional e apontando alguns conceitos envolvidos na concepção deste programa e suas possíveis conseqüências. Objetivamos a compreensão da formação dos sujeitos responsáveis pela execução do Programa na escola. Nosso critério foi compreender a existência de uma efetiva contribuição desses sujeitos na constituição da escola em um espaço democrático e de transformação social e não apenas a oferta de ações de compensação das desigualdades sociais. Assim, nossa preocupação é identificar como ocorreu a formação dos responsáveis envolvidos no processo de implantação e funcionamento do Programa Escola da Família, no sentido de compreender o processo de formação ocorrido e buscar o aprendizado de novos repertórios de ação com o objetivo de ampliar os recursos e as possibilidades de formação dos educadores do Programa Escola da Família

A capacitação é um pré-requisito fisiológico importante para que a célula espermática fertilize o oócito. O óxido nítrico (NO) é sintetizado in vivo durante a conversão da L-arginina em L-citrulina por reações oxidativas catalisadas pela enzima óxido nítrico sintase (NOS) desempenhando um papel importante na regulação da motilidade e na capacitação dos espermatozoides. Estudos indicam que o NO é capaz de regular a concentração da AMP cíclico e, por conseguinte, através da atividade da adenil ciclase, estimular a capacitação espermática em várias espécies. O objetivo deste estudo foi avaliar a função do NO na capacitação de espermatozoides equinos criopreservados. Três ejaculados foram colhidos de três garanhões (n=9). O sêmen foi diluído em meio Botu-Crio&reg; na concentração final de 200×106 células/mL, envasado em palhetas de 0,5 mL e criopreservado usando um sistema automatizado. Para cada análise, foram descongeladas quatro palhetas da mesma partida e do mesmo garanhão em banho-maria a 37oC/30 s. e em seguida, o sêmen foi submetido à centrifugação em meio FIV. Posteriormente, o sêmen foi incubado neste mesmo meio na presença de L-arginina, com ou sem inibidor da enzima óxido nítrico sintase o (L-NAME), e com ou sem o removedor de NO (azul de metileno) nos tratamentos: 1) C= (FIV); 2) A= L-arginina (10 mM); 3) L = L-NAME (1 mM); 4) M = azul de metileno (100 mM); 5) AL = L-arginina (10 mM) + L-NAME (1 mM); 6) AM = L-arginina (10 mM) + azul de metileno (100 mM). As amostras foram incubadas a 38oC e 5 % de CO2. Após a incubação realizou-se a análise computadorizada da motilidade do espermatozoide e as análises por citometria de fluxo. Para a análise computadorizada da motilidade espermática foram avaliados os tempos de incubação de 0, 60, 120 e 300 min. e para as análises por citometria de fluxo os tempos de 60, 120 e 300 min. Para avaliar a integridade das membranas plasmática e acrossomal usou-se a associação FITC-PSA e IP. Para a detecção da fosforilação do aminoácido tirosina, usou-se o anticorpo antifosfotirosina conjugado a uma fluoresceína (DAF-2). A fim de dosar a quantidade de NO produzido pelo espermatozoide equino criopreservado foi utilizada a sonda DAF e para avaliar a peroxidação lipídica da membrana espermática utilizou a sonda C11-BODIPY. A sonda H33342 foi usada com a finalidade de evitar que partículas do mesmo tamanho e granulosidade da célula espermática fossem incluídas na contagem das análises por citometria de fluxo. Os dados foram analisados por meio da ANOVA e a comparação das médias, dentro de cada tempo, pelo teste de Tukey, com o nível de significância de 5 %, usando o software SAS. A remoção do NO do meio de cultura inibiu a motilidade das células espermáticas em todos os tempos de incubação. A motilidade total e motilidade progressiva foram reduzidas nos grupos M e AM. Os espermatozoides incubados com o removedor do NO apresentaram maior porcentagem de células com membrana plasmática e acrossomal íntegras nos 60 e 120 minutos de incubação (p<0,05). A reação acrossomal foi induzida nos tratamentos que receberam L-arginina (A; AL). Dentro de cada tratamento, a quantidade de NO produzido pelo espermatozoide, a fosforilação do aminoácido tirosina e a peroxidação lipídica não apresentaram diferenças entre os tempos (p>0,05). Foi verificada uma redução destas variáveis nos grupos M e AM (p<0,05). Contudo, a dose de 1 mM de L-NAME, não foi suficiente para inibir a NOS em espermatozoides criopreservados de equinos. A remoção do NO mantém a integridade das membranas plasmática e acrossomal, entretanto inibe totalmente a motilidade espermática, sugerindo um papel benéfico do NO endógeno na manutenção da motilidade dos espermatozoides equinos criopreservados. / Capacitation is an essential physiological prerequisite in order to sperm cell fertilize the oocyte. Nitric oxide (NO) is synthesized in vivo during the conversion of L-arginine in L-citruline by oxidative reactions catalyzed by nitric oxide synthase enzyme (NOS) and plays an important role in regulation of motility and in sperm capacitation. Studies indicated that NO is capable of regulating cAMP concentration and, therefore, by adenylyl cyclase, stimulate sperm capacitation in several species. The aim of this study was to evaluate the function of nitric oxide in cryopreserved equine sperm capacitation.Three ejaculates from three stallions were collected (n=9). Semen samples were diluted with Botu-Crio&reg; extender to a final concentration of 200×106 sperms/mL, and then packaged in 0.5mL straws and cryopreserved using an automated freezing system. For each analysis, four straws from the same batch and the same stallion were thawed in a water bath at 37oC/30 s. washed by centrifugation in FIV medium. Thereafter, samples were incubated in FIV medium in the presence of L-arginine, with or without the inhibitor of nitric oxide sinthase (L-NAME), and with or without the scavenger of NO (Methylene blue) in the following treatments: 1) C = Control (FIV); 2) A = L-arginine 10 mM; 3) L = L-NAME 1mM; 4) M = Methylene blue 100 mM; 5) AL = L-arginine (10 mM) + L-NAME (1 mM); 6) AM = L-argine (10 mM) + Methylene blue (100 mM). The treatments were incubated at 38oC and CO2 at 5 %. After incubation, the computer-assisted sperm motility (CASA) and flow cytometry analyses were performed. For CASA analysis, the incubation times of 0, 60, 120 e 300 min. were evaluated and for flow cytometry analyses times 60, 120 e 300 min. were evaluated. Plasma and acrosomal membranes integrity were evaluated by FITC-PSA and PI association. In order to detect amino acid tyrosine phosphorylation, we used the anti-phosphotyrosine antibody conjugated to a fluorescein (DAF-2). In order to quantify the amount of nitric oxide produced by cryopreserved equine sperm, the fluorescent probe DAF was used, and to evaluate the lipid peroxidation of sperm membrane we used the probe BODIPY-C11. The probe H33342 was used in order to prevent that particles of the same size and granularity of sperm cell were included in the counting of flow cytometry analyses. Data were analyzed by ANOVA and comparison of means within each time by the Tukey test, at a significance level of 5%, using SAS software. Removing NO from the culture medium inhibited the motility of sperm cells at all incubation times. Total and progressive motilities were reduced in both groups, M and AM. Sperms incubated with the scavenger of NO had the highest percentage of cells with intact plasma and acrosomal membranes at 60 and 120 minutes of incubation (p <0.05). Acrosomal reaction was induced in treatments with L-arginine (A, AL). Within each treatment, the amount of NO produced by sperms, the level of amino acid tyrosine phosphorylation and lipid peroxidation had no differences between the times used (p> 0.05). A reduction of these variables in groups M and AM (p <0.05) was observed. However, a dose of 1 mM L-NAME was not sufficient to inhibit NOS in cryopreserved equine sperm. Removal of NO maintains plasma and acrosomal membranes integrity, however completely inhibits sperm motility, suggesting a beneficial role of endogenous NO in the maintenance of motility of cryopreserved equine spermatozoa.

La baisse de la fertilité masculine mondiale ces dernières décennies est devenue préoccupante. Ainsi, un homme sur quinze est subfertile. Si les causes de la baisse de fertilité semblent être essentiellement liés à l'environnement et au mode de vie, il est essentiel de comprendre tous les mécanismes qui régulent le pouvoir fécondant du spermatozoïde afin d'optimiser les techniques de procréation médicalement assistée. Le développement de nouveaux axes de recherche, expérimentaux et théoriques, pour identifier les mécanismes sous-jacents de l'infertilité masculine est indispensable. C'est dans ce contexte, de développement de nouvelles stratégies expérimentales que se situe ce travail de thèse. La stratégie que j'ai employée ici est la recherche de nouveaux outils pharmacologiques à partir de venin d'animaux par des tests d'activités biologiques. Cette recherche nous a permis de mettre en évidence une nouvelle voie de régulation de la physiologie spermatique : la voie des phospholipases A2 secrétées. Ainsi, nous avons montré pour la première fois que les phospholipases A2 secrétées, régulent 4 étapes clé de la fécondation : la capacitation, la mobilité spermatique, la réaction acrosomique et l'interaction gamétique.

La capacitación espermática puede ser mimetizada in vitro eliminando el plasma seminal por distintos sistemas de lavado. Entre los tratamientos espermáticos empleados habitualmente en los laboratorios para capacitar a los espermatozoides se encuentran los lavados que se realizan con medios enriquecidos con albúmina o a través de gradientes de Percoll. El objetivo de este trabajo ha sido determinar los cambios que acontecen en los espermatozoides (procedentes de epidídimo y eyaculados) sometidos a tres sistemas de capacitación in vitro evaluados mediante una batería de técnicas que determinan distintos estadios de la capacitación espermática.De los resultados obtenidos se desprende que tanto la procedencia de los espermatozoides (epidídimo o eyaculado) como el tratamiento de capacitación al que se les somete afecta en gran medida a los resultados de la penetración in vitro y por lo tanto, la capacitación se produce de manera diferente entre estos grupos. / Sperm capacitation may be defined as a set of molecular modifications that occurs in the spermatozoa, after maturation in the epididymis, which enables them to fertilize the oocyte. In vitro, this process can be imitated in vitro by separation of the seminal plasma by different systems of washing. Between the sperm treatments routinely used in the laboratories, semen samples are washed of albumin or centrifugations through a Percoll. The aim of this work was examine and characterize the changes that happen in the sperms (from epididymis and ejaculated) submitted to three systems of in vitro capacitation, evaluated by means of a battery of tests to determine different levels of the sperm capacitation. Sperm capacitation was dependent on sperm treatment, whetherepididymal or ejaculated and to whichever parameter measured.Nevertheless, all these parameters, in spite of the fact that they have beendescribed as tools to evaluate the sperm capacitation, really are not capable of discriminating or indicating the level of capacitation.

Fertilization is a process that generates the first cell of a new organism. In mammals, fertilization occurs in the female reproductive tract. The male gametes (spermatozoa) are rendered fertilization-competent only after they undergo capacitation and acrosome reaction (AR). The set of physiological changes, characterised by the acquisition of hyperactivated motility, that render the spermatozoa fertilization competent is known as capacitation. Using in vitro models, the complex intracellular signaling events mediating this process are still being understood. This thesis explores the role of protein tyrosine phosphorylation during capacitation using the golden hamster (Mesocricetus auratus) spermatozoa. The knowledge about the molecular components involved in capacitation, apart from enriching our understanding about a basic cellular process could also provide leads in the management of male (in)fertility. A comprehensive review on the perspectives of male reproduction, spermatogenesis, the structural features of a spermatozoon and sperm maturation, relevant to the content of the thesis is provided in Chapter-1 (General Introduction). Molecular mediators that initiate capacitation include cAMP, Ca2+and HCO3- ions. These signalling molecules regulate activities of protein kinases and phosphatases, which control the level of protein phosphorylation in spermatozoa. Capacitation-associated increase in protein phosphorylation, specifically protein tyrosine phosphorylation (PYP) has been demonstrated in a few species such as mouse, rat and human. The unique nature of PYP signaling during sperm capacitation has been exemplified by discoveries of several male germ cell-specific signalling molecules like soluble adenylate cyclase. However,molecular identities of tyrosine-phosphorylated proteins and their functional role during sperm capacitation are yet to be investigated in detail. In this context, the effect of modulating intracellular levels of signaling molecules upstream of protein phosphorylation was sought using pentoxifylline (PF), a cAMP phosphodiesterase inhibitor. Interestingly, PF-induced capacitation was associated with an early induction of tyrosine phosphorylation of proteins (45-80 kDa) localized to the mid piece of the sperm tail. Interestingly, the ultrastructural localization of tyrosine-phosphorylated proteins in the sperm tail by immunoelectron microscopy (IEM) revealed most intense immunolabelling in the fibrous sheath, followed by outer dense fibers (ODFs)and the axoneme. Data pertaining to the effect of PF on sperm capacitation and the associated protein-phosphorylation is presented in Chapter-2. Since PYP was determined to be extremely critical for hyperactivation in spermatozoa, the involvement of protein tyrosine kinases (PTKs) in this process was assessed using a specific PTK inhibitor, tyrphostin A47 (TP-47: EGFR-TK specific). The third chapter deals with the effect of tyrphostins on sperm capacitation and PYP. A dose-dependent inhibition by TP-47 of capacitation and principal piece associated-PYP of ~45-60 kDa proteins was observed. Interestingly, TP-47 treated-spermatozoa exhibited a circular motility pattern; when assessed for kinematic parameters, by computer aided sperm analysis, sperm showed lower values for key kinematic parameters as compared to the controls. While sperm viability in TP-47- treated samples was not affected, the ATP content reduced towards latter (4-5 h) part of culture as compared to the controls. When spermatozoa were treated with two other PTK inhibitors, tyrphostin AG1478 (EGFR-TK specific) and tyrphostin AG1296 (PDGFR-TK specific), they did not show any changes in kinematic parameters or PYP, indicating that the TP-47-effect was compound-specific. The fourth chapter of this thesis involves the molecular analysis of proteins hypo-tyrosine phosphorylated in the presence of TP-47, which started with the enrichment of sperm flagellar proteins that are tyrosine phosphorylated during capacitation, using various detergents. Detergent extractions established that most tyrosine-phosphorylated proteins were non-membranous in nature, which complemented the IEM data. Therefore, phosphoproteome analysis of the untreated and TP-47-treated sperm samples was performed. For this, protein extracts were subjected to 2D-PAGE-phosphotyrosine immunoblots. A 51 kDa spot and two 45 kDa spots, corresponding to the hypo-tyrosine phosphorylated spots, were analyzed by MS/MS. While peptides from the 51 kDa protein matched with tektin-2 (a microtubular protein), those of the 45 kDa spots matched with ODF-2 protein of the sperm flagellum. Validation of the presence of tektin-2 and ODF-2 protein and their tyrosine-phosphorylated forms on sperm capacitation in the hamster spermatozoa has also been performed. In addition to detailing the role of PYP in hamster sperm capacitation, this study revealed the identities of a few of these proteins, whose tyrosine phosphorylated status could be critical for optimal sperm flagellar bending, required for sperm hyperactivation. By understanding causes that lead to altered sperm function, for example, as observed with hamster spermatozoa, new insights could be achieved into molecular regulatory mechanisms that govern sperm function in clinical cases of non-obstructive male infertility in the human.