Navegando por Palavras-chave "Spermatozoa"

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    Análise morfofuncional de órgãos reprodutivos masculinos e investigação do comportamento sexual em modelo animal de mucopolissacaridose do Tipo I (MPS I)
    (Universidade Federal de São Paulo (UNIFESP), 2018-03-29) Nascimento, Cinthia Castro do [UNIFESP]; D'Almeida, Vania [UNIFESP]; http://lattes.cnpq.br/7220411418339421; Universidade Federal de São Paulo (UNIFESP)
    A Mucopolissacaridose do Tipo I (MPS I) é uma doença de depósito lisossômico causada pela mutação do gene IDUA, codificador da enzima α-L-iduronidase. Trata-se de uma hidrolase lisossômica que degrada heparan e dermatan sulfatos, dois tipos de glicosaminoglicanos (GAGs). GAGs são moléculas estruturais e sinalizadoras, abundantes na matriz extracelular e importantes para a manutenção fisiológica de células e tecidos. Devido à deficiência enzimática, os GAGs são continuamente acumulados em inúmeros tipos celulares, o que caracteriza a doença como multissistêmica e progressiva. Com o aumento da expectativa de vida dos pacientes em decorrência das possibilidades terapêuticas, julgou-se relevante avaliar o efeito da doença sobre parâmetros reprodutivos masculinos e sobre o comportamento sexual do modelo animal de MPS I. Utilizamos camundongos machos C57BL, de 6 meses de idade, com genótipo normal (Idua+/+) e afetado (Idua-/-) para avaliar a morfologia de testículos, epidídimos, vesículas seminais e próstatas. O comportamento sexual foi observado aos 3 e 6 meses de idade. Alguns acasalamentos foram monitorados para que a ocorrência de prenhez fosse registrada. As quantificações hormonais e de GAGs também foram obtidas. Na análise morfológica, encontramos células intersticiais vacuolizadas em todos os órgãos avaliados referentes ao grupo Idua-/-. Danos epiteliais epididimários foram detectados, embora a morfologia e motilidade espermáticas estivessem mantidas. As vesículas seminais apresentaram menores pesos absolutos e relativos (p=0,013 e p=0,009; respectivamente) e sinais de atrofia, enquanto as próstatas foram bastante comprometidas, contendo alguns ácinos com sinais de necrose. Sob análise ultraestrutural, detectamos alterações nas células mioides, de Leydig e de Sertoli. As últimas com maior quantidade de vesículas contendo material em digestão semelhantes a autofagossomos (p=0,0005) e menor número de vesículas semelhantes a lisossomos (p=0,01). Porém, sob análise molecular, não comprovamos aumento de proteínas autofágicas oriundas do homogenato testicular. Quanto ao comportamento sexual, detectamos maiores latências de montas no grupo Idua-/- independentemente da idade (p=0,05) e também de intromissões no grupo Idua-/- de 3 meses (p=0,02), provavelmente devido à limitação motora comprovada pelo teste do campo aberto, que indicou menor atividade horizontal e vertical independentemente da idade dos animais Idua-/- (p=0,01 e p=0,04 respectivamente). As concentrações de hormônios foram similares entre os grupos e houve acúmulo de dermatan sulfato nos testículos (p<0,0001), epidídimos (p=0,007) e próstatas (p=0,0004). Concluímos que a deficiência da enzima IDUA afeta a morfologia de importantes órgãos reprodutivos, porém, os gametas são morfologicamente normais e móveis. Embora apresentem limitações motoras, machos Idua-/- são aptos à copula e muitos foram capazes de emprenhar fêmeas e gerar filhotes.
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    Avaliação do efeito precoce da orquiectomia radical unilateral no perfil de proteínas do plasma seminal de homens portadores de tumor de células germinativas de testículo
    (Universidade Federal de São Paulo (UNIFESP), 2019-11-06) Andrade, Maria Beatriz Ribeiro De [UNIFESP]; Spaine, Deborah Montagnini [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Objective: To evaluate the effect of orchiectomy on the seminal plasma proteomic profile of men with testicular germ cell tumors. Methods: Seventeen men with Testicular Germ Cell Tumors provided one semen sample before (Pre-orchiectomy) and another 30 days after orchiectomy (Post-orchiectomy). Following liquefaction, an aliquot was used for semen analysis and other one was centrifuged for collection of seminal plasma. The remaining volume was criopreservated. The seminal plasma was used to proteomic analysis .For semen analysis a Student’s t-test for paired samples was used and to proteomic analysis a one sample Student’s t-test was performed. For both analysis was adopted p˂0,05. Effect size was assessed using Cohen’s d coefficient. Results: No significant difference was observed in semen analysis. Two hundred and seven proteins were identified and quantified with high fidelity, of which five were increase in the pre-orchiectomy period and eight proteins were increase in post-orchiectomy period. Conclusion: Removal of the affect testis alters the seminal plasma molecular environment.
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    Bovine papillomavirus DNA in milk, blood, urine, semen, and spermatozoa of bovine papillomavirus-infected animals
    (Funpec-editora, 2009-01-01) Lindsey, Charles Julian [UNIFESP]; Almeida, Marcos Eduardo de [UNIFESP]; Vicari, Camilo Alfredo Faigle [UNIFESP]; Carvalho, Claudemir de; Yaguiu, Andrea; Freitas, Antonio Carlos de; Beçak, Willy; Stocco, Rita de Cassia [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Inst Butantan; Universidade Federal de Pernambuco (UFPE)
    Papillomavirus infection in bovines is associated with cutaneous papillomatosis on the hide, udders and other epithelial tissues, as well as in oral respiratory, alimentary and urinary tract mucosa. Bovine papillomavirus (BPV) is also considered the etiological agent of esophageal tumors and the malignant bladder tumors that characterize the clinical condition associated with chronic enzootic hematuria. After infective viral DNA was found in cattle blood and BPV1, 2 and 4 DNA in cattle reproductive and embryonic tissues, we looked for and found BPV DNA in blood, milk, urine, seminal fluid, and spermatozoa of BPV-infected animals. Peripheral blood lymphocyte cultures from BPV-infected animals had high rates of chromosome aberrations, including radial rearrangements that signal oncogenic potential and viral interaction with telomeric regions. The finding of BPV DNA in body fluids and tissues other than the epithelium demonstrates co-infection of other tissues or cell types by papillomavirus and shows the potential role of lymphocytes, seminal fluid and spermatozoa in BPV transmission. Our findings reinforce a peremptory need for prophylactic and therapeutic instruments to curtail this disease in bovine livestock.
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    Efeito da amifostina(WR-2721)sobre a fertilidade de ratos pré-puberes tratados com doxorrubicina, com ênfase na integridade do DNA dos espermatozoides e na qualidade dos embriões concebidos
    (Universidade Federal de São Paulo (UNIFESP), 2009) Vilela, Vanessa Vendramini [UNIFESP]; Miraglia, Sandra Maria [UNIFESP]
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    Efeito da varicocele na função dos espermatozóides
    (Universidade Federal de São Paulo (UNIFESP), 2009-02-27) Blumer, Camile Garcia [UNIFESP]; Cedenho, Agnaldo Pereira [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    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.
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    O efeito do processamento seminal e a integridade do DNA nuclear do espermatozoide humano
    (Universidade Federal de São Paulo (UNIFESP), 2006) Stevanato, Juliana [UNIFESP]; Cedenho, Agnaldo Pereira [UNIFESP]
    Objetivo: Avaliar o efeito da centrifugacao em gradiente de densidade descontinuo na taxa de fragmentacao em fita dupla do DNA dos espermatozoides. Metodos: Foi realizado um estudo prospectivo e pareado, constituido de 35 amostras seminais antes e depois do processamento seminal em gradiente de densidade descontinuo, provindas de pacientes encaminhados ao Ambulatorio de Reproducao Humana para tratamento de infertilidade conjugal atraves das tecnicas de Reproducao Assistida. As amostras foram colhidas por masturbacao apos um periodo de 2 a 7 dias de abstinencia ejaculatoria e em seguida analisadas de acordo com os criterios da Organizacao Mundial da Saúde (OMS). As amostras foram divididas em duas aliquotas (antes do processamento seminal e depois do processamento seminal) para subsequente avaliacao da motilidade e integridade do DNA espermatico atraves da tecnica do TUNEL, que avalia fragmentacao apoptotica do DNA dos espermatozoides. A integridade do DNA foi avaliada por microscopia optica e expressa em porcentagem de espermatozoides com fragmentacao apoptotica no DNA por amostra. Resultados: A motilidade pos-processamento seminal foi maior do que a motilidade pre¬-processamento (86,6; 17,7 por cento e 60; 14,4 por cento, respectivamente, p <0,0001), no entanto nao foi observada diferenca na fragmentacao apoptotica do DNA dos espermatozoides apos o processamento seminal nas amostras analisadas (antes do processamento seminal: 5; 4,3 por cento e apos 5; 4,5 por cento, p=0,958). Ja as amostras seminais com alteracoes nas variaveis seminais apresentaram maiores porcentagens de celulas com fragmentacao apoptotica do DNA, em relacao as amostras que nao apresentaram alteracoes seminais (Pre-processamento - 8; 4 por cento e 4,5; 3,3 por cento respectivamente, p= 0,011 e pos-processamento - 7,7; 5,1 por cento e 4,4; 3,1 por cento respectivamente, p= 0,027). Mas nao houve melhora na porcentagem de fragmentacao apoptotica do DNA das amostras com e sem variacao seminal apos o processamento (p= 0,929). Conclusao: Nas condicoes deste estudo, o processamento seminal pela centrifugacao em gradiente de densidade descontinuo de duas camadas nao produz efeito na fragmentacao apoptotica do DNA dos espermatozoides, e apesar de pacientes com alteracao seminal apresentarem maior taxa de fragmentacao apoptotica no DNA dos espermatozoides, o processamento seminal nao melhorou os resultados em nenhum dos dois grupos. Portanto atencao especial deve ser dada aos pacientes que apresentam altas taxas de...(au)
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    O efeito do tratamento com carnosina durante o processamento seminal de amotras humanas por gradiente de densidade descontínuo
    (Universidade Federal de São Paulo (UNIFESP), 2018-02-27) Adami, Luana Nayara Gallego [UNIFESP]; Nichi, Marcilio; http://lattes.cnpq.br/7054360633543023; Universidade Federal de São Paulo (UNIFESP)
    Introdução: A infertilidade masculina pode ser causada pelo estresse oxidativo seminal que leva prejuízos aos espermatozoides. Entretanto, o plasma seminal possui mecanismos antioxidantes para tentar neutralizar esses danos celulares. Por outro lado, as biotécnicas utilizadas durante o processamento seminal tendem a diminuir essa proteção antioxidante aumentando os danos oxidativos ao gameta. Na tentativa de minimizar os danos, antioxidantes exógenos são suplementados nos sistemas in vitro. A carnosina, reportada por sua potente função antioxidante, tem capacidade de reagir com produtos responsáveis por provocar danos celulares. Dessa forma suplementar carnosina nos meios de processamento seminal levaria a uma melhora na qualidade funcional dos espermatozoides. Objetivo: Avaliar o efeito de diferentes concentrações da carnosina no processamento das amostras seminais. Método: As amostras seminais (n=34) foram divididas em 3 grupos e seguiram para o processamento seminal: grupo controle sem suplementação com a carnosina (0) recebeu as camadas de gradiente de densidade descontínua – Percoll (80% e 40%) e a amostra; o grupo 20 mM, por sua vez, teve as camadas de Percoll suplementadas com 20 mM de carnosina; finalmente, o grupo 50mM teve as camadas de Percoll suplementadas com 50 mM de carnosina. As amostras passaram pelo processo de gradiente de densidade descontínuo por 20 minutos a 600xG e foram lavadas com meio de cultura fluido tubário humano (HTF) por 10 minutos a 600xG. Após o processamento, os espermatozoides foram avaliados quanto ao potencial de membrana mitocondrial, à produção de ânion superóxido intracelular, à fragmentação de DNA espermático, à integridade acrossômica, à atividade mitocondrial, à integridade de membrana plasmática e à motilidade espermática. Para analisar a normalidade e esfericidade dos dados, utilizamos os testes de Kolmogorov-Smirnov e de Mauchly respectivamente, e então o General Linear Model (GLM) com teste post-hoc de Sidak. Para as variáveis não normais foi utilizado o teste não paramétrico de Friedman com post-hoc de Games-Howell, considerando α = 5% e utilizando o programa SPSS21. Resultados: A suplementação com a carnosina na concentração de 50 mM levou à melhora da atividade mitocondrial dos espermatozoides quando comparado ao grupo controle. Para variáveis como % de espermatozoides móveis e % de espermatozoides progressivos, velocidade média de percurso, retilínea e curvilínea, e linearidade dos espermatozoides houve uma melhora nos índices após o gradiente de densidade descontínuo, entretanto as concentrações de carnosina não foram efetivas. Já para a frequência de batimento dos flagelos a presença de carnosina em ambas concentrações elevou as frequências quando comparadas à amostra antes do gradiente de densidade descontínuo. Conclusão: A suplementação da carnosina nas amostras seminais humanas pode apresentar efeito benéfico para a atividade mitocondrial espermática e a frequência de batimento cruzado, amenizando os possíveis danos provocados pelo processamento seminal.
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    Efeitos de cádmio na barreira hematotesticualr e do estrogeno em espermatozóide de rato.
    (Universidade Federal de São Paulo (UNIFESP), 2009) Siu, Erica Rosanna [UNIFESP]; Porto, Catarina Segreti [UNIFESP]
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    Effect of the exposure to fine inhalable particulate matter (pm2.5) on sperm functional quality of mice
    (American Society for Reproductive Medicine, 2020-09-01) Intasqui, Paula; Tamashiro, Letícia Kaory; Yariwake, Victor Yuji; Souza, Rosana Xavier; Kanashiro, Camila Matie; Veras, Mariana Matera; http://lattes.cnpq.br/5188240479960852
    Objective: To evaluate the effect of exposure to pollution (fine inhalable particulate matter - PM2.5) from the city of São Paulo on sperm functional quality. Design: Male isogenic BALB/c mice were used, distributed in two groups, control (n=6) and polluted air (n=8). For the polluted air group, after weaning (21 days), animals were daily exposed to 600 μg/m3 of PM2,5 for 96 days in an Ambient Particle Concentrator (APC). Control group was simultaneously exposed to filtered air in the APC. On postnatal day 118, animals were sacrificed (isoflurane overdose), body was weighted and the epididymis were collected. Materials and Methods: Sperm obtained from the cauda epididymis were used for the evaluation of motility, mitochondrial activity (DAB staining), acrosome integrity (PNA staining), DNA fragmentation (alkaline comet assay), oxidative stress (DHE staining) and cell viability (PI staining). Groups were compared using an unpaired Student’s t test (p<0.05). Results: Groups did not differ regarding body weight, and sperm motility. Furthermore, air pollution did not alter sperm functional quality (Table 1). Conclusions: Exposure to high concentrations of PM2.5 does not affect sperm motility and functional parameters.
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    Estudo das vias proteômicas do plasma seminal e dos espermatozoides associadas à infertilidade masculina
    (Universidade Federal de São Paulo (UNIFESP), 2018-05-08) Lopes, Paula Intasqui [UNIFESP]; Bertolla, Ricardo Pimenta [UNIFESP]; http://lattes.cnpq.br/8479803539567479; Universidade Federal de São Paulo (UNIFESP)
    Objetivo: A composição proteica do plasma seminal e dos espermatozoides é essencial para a sua função correta e, consequentemente, para a fertilidade. Assim, estudos proteômicos vêm sendo realizados a fim de entender os mecanismos pós-genômicos subjacentes e de identificar as proteínas alteradas na infertilidade masculina. Portanto, o objetivo deste estudo foi avaliar proteínas de plasma seminal e de espermatozoides e sua associação com alterações funcionais dos espermatozoides e com a infertilidade masculina primária ou secundária. Métodos: Este trabalho foi subdividido em dois estudos. No estudo 1, amostras seminais de 197 homens normozoospérmicos foram utilizadas. Após a coleta e liquefação do sêmen, uma alíquota foi utilizada para a análise seminal, outra para a avaliação da integridade funcional dos espermatozoides e o volume remanescente foi centrifugado para a separação do plasma seminal. Este foi então utilizado para a avaliação dos níveis seminais dos biomarcadores sugeridos: (i) de alterações na atividade mitocondrial dos espermatozoides – GSTM3, (ii) de defeitos no acrossoma dos espermatozoides – PLTP e (iii) de integridade do DNA dos espermatozoides – CRISPLD1, CRISPLD2 e RARRES1. No estudo 2, sete homens com fertilidade comprovada (n=7), nove homens com infertilidade primária (n=9) e sete homens com infertilidade secundária (n=7) foram incluídos. Após a coleta e liquefação do sêmen, uma alíquota foi utilizada para a análise seminal e o volume remanescente foi congelado a –80 °C. No momento da análise proteômica, a amostra foi descongelada e centrifugada para separação dos espermatozoides. Após a extração das proteínas dos espermatozoides, essas foram utilizadas para a análise proteômica shotgun por 1D-LC-MS/MS. As proteínas diferencialmente expressas foram utilizadas para análise de redes de interação proteína-proteína. As proteínas centrais nessas redes, envolvidas com funções biológicas alteradas em ambos os grupos de homens inférteis (BAG6, HSPA2 e SPA17), assim como as proteínas específicas de testículos (HIST1H2BA e SPA17) foram validadas nas amostras de espermatozoides. Nos dois estudos, os biomarcadores foram avaliados por Western blotting. No estudo 2, as proteínas foram também analisadas por imunocitoquímica utilizando microscopia confocal. Resultados: No estudo 1, o nível seminal da proteína GSTM3 estava aumentado em amostras com baixa atividade mitocondrial dos espermatozoides. Por outro lado, o nível das proteínas CRISPLD2 e RARRES1 estava diminuído no plasma seminal de homens com alta fragmentação de DNA dos espermatozoides. As proteínas PLTP e CRISPLD1 não variaram entre os grupos estudados. No estudo 2, um total de 1.305 proteínas de espermatozoides foram identificadas, sendo 102 diminuídas e 15 aumentadas em ambos os grupos de infertilidade. As proteínas diminuídas estavam relacionadas, principalmente, a modificações pós-traducionais e dobramento de proteínas. A proteína BAG6 demonstrou-se significativamente diminuída em homens inférteis, enquanto a proteína HIST1H2BA estava aumentada nesses pacientes. Os níveis das proteínas HSPA2 e SPA17 não diferiram entre os grupos. Na análise por imunocitoquímica, nenhuma diferença quanto à localização dessas proteínas nos espermatozoides foi observada. Conclusão: As proteínas do plasma seminal e dos espermatozoides refletem alterações funcionais dos espermatozoides e a infertilidade masculina.
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    Investigação celular e molecular sobre as condições de agravo na fertilidade do homem
    (Universidade Federal de São Paulo (UNIFESP), 2019-12-11) Belardin, Larissa Berloffa [UNIFESP]; Bertolla, Ricardo Pimenta [UNIFESP]; http://lattes.cnpq.br/8479803539567479; http://lattes.cnpq.br/4341099347238414; Universidade Federal de São Paulo (UNIFESP)
    Objective: To evaluate whether the extracellular environment and its interaction with sperm are related to the two main described causes of alteration in male fertile potential, namely varicocele and sperm DNA fragmentation. Methods: This thesis was divided into 2 chapters, where the first one aims to identify molecular changes in the extracellular environment of men with varicocele. This chapter has been divided into 2 articles. In article I it was verified: (i) the effect of varicocele; and (ii) effect of varicocelectomy on the levels of cysteine rich secretory protein 3 (CRISP-3) in seminal plasma by western blotting. In article II, the expression of microRNAs present in seminal plasma extracellular vesicles in men with varicocele, and before and after varicocelectomy was evaluated using microarray. The second chapter sought to better understand whether the extracellular environment and its interaction with sperm reflect the sperm DNA fragmentation status. The first article evaluated, using a Multiplex assay, the expression of proteins from the Matrix metalloproteinases (MMPs) family and their inhibitors named Tissue inhibitors of metalloproteinases (TIMPs) in seminal plasma of men with high and low sperm DNA fragmentation. The second article verified the presence of Epididymal sperm-binding protein 1 (ELSPBP1) in seminal plasma samples and sperm with high and low DNA fragmentation by western blotting and identified its location in the sperm by immunocytochemistry and verified if this protein is capable of separating sperm with high levels of DNA fragmentation from the healthy population, using immunomagnetic cell separation. Results: Regarding the study of varicocele (chapter I), in article I it was found that men with varicocele have higher CRISP-3 seminal levels when compared to controls. CRISP-3 levels decreased after varicocelectomy. In Article II, it was observed that there are microRNAs present in seminal plasma extracellular vesicles that reflect the pathogenic process of varicocele before varicocelectomy. While other microRNAs are involved in fertility protective processes. In Chapter II, which studied the pathology of sperm DNA fragmentation, in article I, lower levels of seminal plasma MMP-2, MMP-7, TIMP-1, TIMP-2 and TIMP-4 were observed in high when compared to the low sperm DNA fragmentation group. In logistic regression analysis, proteins MMP-2, MMP-7 and TIMP-4 classified the samples as low and high DNA fragmentation. With respect to article II, the protein ELSPBP1 is more expressed in sperm from samples with higher levels of sperm DNA fragmentation when compared to samples with lower levels of DNA fragmentation. This protein, in human sperm is preferentially located in the region of the head and was efficient to separate sperm with higher levels of DNA fragmentation from the health population. Conclusion: The extracellular environment and its interaction with sperm are related to varicocele and sperm DNA fragmentation.
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    Investigação da expressão de microRNAs nos espermatozoides de ratos wistar modelo de transtorno do espectro do autismo induzido por ácido valproico
    (Universidade Federal de São Paulo (UNIFESP), 2019-05-30) Roloff, Helena Bertazolli [UNIFESP]; Teixeira, Taiza Stumpp [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Epigenetics deals with changes in the genome that do not alters the sequence of base pairs of DNA, but affect the modulation of gene expression. These modifications can occur in different ways, including DNA methylation and post-transcriptional regulation by microRNAs (miRNAs), which modulate messenger RNA (mRNA) activity. The miRNAs have been identified as potential biomarkers for the diagnosis and evaluation of response of psychiatric disorders. Among psychiatric syndromes, ASD (Autism Spectrum Disorder) is a neurodevelopmental disorder that involves a broad spectrum of clinical phenotypes and a complex genetic contribution. Many miRNAs differentially expressed in this disorder are involved in neuronal differentiation, maturation and proliferation, so that their dysregulation may be associated with the neurodevelopmental deficits observed in ASD. The environmental factors involved in the ASD etiology may be pre- or postnatal, highlighting, in the present study, in utero exposure to the teratogenic drug valproic acid (VPA), a broad spectrum anticonvulsant drug. VPA acts through epigenetic mechanisms and studies have been using prenatal exposure to VPA in animals to produce a model of autism that presents a constructive and face validity. Two important events of epigenetic reprogramming occur during embryonic development: the first immediately after fertilization and the second during the development of primordial germ cells (PGCs), which are the precursors of gametes. During this reprogramming period, PGCs become more susceptible to epigenetic changes, which can remain in the gametes and can be passed on to the next generation and generate an epigenetic inheritance mechanism. It is important to mention that during the epigenetic reprogramming of PCG, alternative silencing mechanisms of retrotransposons, whose expression is controlled by methylation, need to be activated. Thus, impairments during this process may lead to inappropriate mobilization of retrotransposons, which may result in mutations and other important changes in the genome. Copy number variations of retrotransposon Line-1 (long interspersed element 1), for example, are considered a possible cause of neurobehavioral disorders, such as ASD. Aim: Given these data, it was decided to use the ASD model based on the treatment with valproic acid to investigate the expression of miRNAs related to this disorder, as well as to investigate possible XIII changes in the number of Line-1 copies in the spermatozoa of these animals. Methods: For this, Wistar rats were treated with 800mg / kg VPA at 12.5 days post-conception (dpc). Male descendants were submitted to basic behavioral tests to verify the induction of behaviors that could confirm the model. The spermatozoa, pre-frontal cortex and cerebellum of the male offspring from these rats were collected and subjected to miRNA or DNA extraction. The testicles were also collected and fixed in Bouin's liquid for morphological analysis. The expression of miR-132-5p, miR-106b-5p, miR-182, miR-18a-5p, miR-34a-5p, miR134-5p, miR219a-5p, miR3596 e miR-195-5p was evaluated. Expression changes those miRNAs has been described in patients with ASD or other psychiatric disorders. For this analysis, TaqMan assays were used. The extracted DNA was used for the analysis of the number of copies variation (CNV) of Line-1 by qPCR with Sybr assays. Results: The results show that the male offspring of VPA treated rats present behavioral changes, although it was not possible to carry out more conclusive tests because they caused stress, which could add more bias to the experiment. These animals presented significant alterations in sperm morphology when compared to controls, although they did not show relevant testicular morphological alterations. The miRNAs miR-132-5p, miR-106b-5p, miR-182, miR-34a-5p, miR134-5p, miR219a-5p, miR3596 and miR-195-5p had reduced expression in cerebellar samples from VPA animals compared to CT animals. There was no change in the expression of any of the nine miRNAs comparing pre-frontal cortex samples from VPA and CT animals. Expression of miRNAs miR-132-5p, miR-34a-5p and miR-219a-5p was shown to be altered in the spermatozoa of the offspring of VPA-treated females in contrast to CT animals. Unfortunately, no results could be obtained from the investigation of the number of Line-1 copies in brain and sperm samples. Conclusion: With this study, it was possible to establish strategies and obtain basic data to study mechanisms of epigenetic inheritance of ASD in animal models.
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    MALDI-TOF Fingerprinting of Seminal Plasma Lipids in the Study of Human Male Infertility
    (Springer, 2014-09-01) Camargo, Mariana [UNIFESP]; Intasqui, Paula [UNIFESP]; Lima, Camila Bruna de [UNIFESP]; Montani, Daniela Antunes [UNIFESP]; Nichi, Marcilio; Pilau, Eduardo Jorge; Gozzo, Fabio Cesar; Lo Turco, Edson Guimaraes [UNIFESP]; Bertolla, Ricardo Pimenta [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Universidade Estadual de Campinas (UNICAMP); Natl Inst Sci & Technol Bioanalyt; Universidade Estadual de Maringá (UEM)
    This study proposed lipid fingerprinting of human seminal plasma by mass spectrometry as an analytical method to differentiate biological conditions. for this purpose, we chose infertile men as a model to study specific conditions, namely: high and low seminal plasma lipid peroxidation levels (sub-study 1.1), high and low sperm nuclear DNA fragmentation (sub-study 1.2), and intervention status: before and after subinguinal microsurgical varicocelectomy (study 2). Study 1 included 133 patients, of which 113 were utilized for sub-study 1.1 and 89 for sub-study 1.2. Study 2 included 17 adult men submitted to subinguinal varicocelectomy, before and 90 days after varicocelectomy. Lipids were extracted from seminal plasma and submitted to Matrix-Assisted Laser Desorption Ionization Quadrupole-Time-of-Flight Mass Spectrometry in the positive ionization mode. Spectra were processed using Waters(A (R)) MassLynx, and MetaboAnalyst online software was used for statistical analyses. for sub-studies 1.1 and 1.2, and study 2, univariate analysis revealed 8, 87 and 34 significant ions, respectively. Multivariate analysis was performed through PCA and PLS-DA. PCA generated 56, 32 and 34 components respectively for each study and these were submitted to logistic regression. A ROC curve was plotted and the area under the curve was equal to 97.4, 92.5 and 96.5 %. PLS-DA generated a list of 19, 24 and 23 VIP ions for sub-studies 1.1 and 1.2, and study 2, respectively. Therefore, this study established the lipid profile and comparison of patterns altered in response to specific biological conditions.
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    Molecular Characterization, Electrophysiological and Contraceptive Effect of Chilean Latrodectus Venom
    (Soc Chilena Anatomia, 2011-01-01) Gomez, Patricia Navarrete [UNIFESP]; Ormeno, David; Miranda, Antonio [UNIFESP]; Gutierrez, Raul Sanchez; Romero Mejia, Fernando Gonzalo [UNIFESP]; Rivera, Jorge Parodi; Univ La Frontera; Universidade Federal de São Paulo (UNIFESP)
    Since the 1970s, There have been studies of the venom of Latrodectus sp. spiders, in particular the latrotoxin (LTX) of Latrodectus mactans. Many of the studies were aimed at understanding the action of the venom on the muscular system. Now accepted that LTX is able to generate a calcium-permeable membrane pore and modulate the release of synaptic vesicles that activate a receptor and induce cellular changes. Interestingly, when work began with venom obtained from the Latrodectus sp present in Chile, it generated clinical indications similar to the bite of this spider in another country, with some differences in intensity. The purpose of the first studies was to understand the systemic mechanisms of this venom, and other active compounds were studied for biological interest. It was found that these molecules are capable of causing systemic effects such as changes in muscle contraction; of generating vascular relaxation and synaptic and cellular modulation; and of altering potassium conductance channels. Based on this evidence, we suggested biotechnological applications to characterize low molecular-weight compounds obtained from the Chilean Latrodectus venom and exploring the effects on the electrophysiology in oocytes and neurons, and the contraceptive effect on spermatozoa.
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    Sperm characteristics in a sample of healthy adolescents in São Paulo, Brazil
    (Escola Nacional de Saúde Pública Sergio Arouca, Fundação Oswaldo Cruz, 2002-04-01) Mori, Marcos Mitsuyoshi [UNIFESP]; Cedenho, Agnaldo Pereira [UNIFESP]; Koifman, Sergio; Srougi, Miguel [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Fundação Oswaldo Cruz Escola Nacional de Saúde Pública Departamento de Epidemiologia e Métodos Quantitativos em Saúde
    The article presents preliminary data from a prospective investigation in a sample of healthy 14-17-year-old students from a technical school in São Paulo, Brazil. Ninety-six Tanner stage 5 and thirty-one Tanner stage 4 adolescents were evaluated: testicular volume was measured using a Prader orchidometer, and semen analysis was performed according to standard procedures. Median testicular volume was 20.0ml among Tanner 5 students and 15.0ml in Tanner 4 students in both the right and left testes. No significant differences were found in sperm volume or motility. Median concentration was 66.0 million/ml for Tanner 5 and 47.0 million/ml for Tanner 4 subjects. Morphological patterns showed abnormal forms in 81.9% of Tanner 5 and 93.6% of Tanner 4 adolescents. Oligospermia (sperm concentration < 5 million/ml) was observed in 7.3% of Tanner stage 5 and 12.9% of Tanner stage 4 individuals. Azoospermia was observed in 3 students (1.8%), with counts less than 1.0 in 8 students (4.8%). The authors discuss the observed results, analyzing the potential implications arising from biological development and potential environmental exposures.