Efeito da varicocele na capacitação espermática.
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Data
2018-01-30
Autores
Andretta, Rhayza Roberta [UNIFESP]
Orientadores
Bertolla, Ricardo Pimenta [UNIFESP]
Tipo
Tese de doutorado
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Introdução: A varicocele é definida como uma dilatação anormal das veias espermáticas internas do plexo pampiniforme com fluxo sanguíneo retrógrado nas veias testiculares internas. É uma das principais doenças associadas à infertilidade masculina e é responsável por alterações na qualidade do sêmen, levando a falhas nos processos associados à fertilização, como por exemplo, a capacitação espermática. Na capacitação espermática, diversas mudanças fisiológicas ocorrem no espermatozoide, como: ativação da proteína quinase A, estímulo na produção de espécies reativas de oxigênio, aumento dos níveis de fosforilação, mudanças no padrão da motilidade e reação acrossômica. Entretanto, estudos associados a varicocele e capacitação espermática são escassos na literatura. Objetivos: Avaliar os efeitos da varicocele na capacitação espermática de homens adultos, em espermatozoides viáveis. Métodos: Amostras seminais dos homens do grupo controle (n=19) e varicocele (n=37) foram coletadas. Uma alíquota do plasma seminal foi separada a fim de verificar os níveis de expressão das proteínas fosforiladas pela PKA. O volume excedente foi submetido ao gradiente de densidade descontínuo. Posteriormente, o pellet obtido foi lavado, com meio de cultura HTF enriquecido com soro bovino de albumina (1%) (p:v). Uma alíquota foi submetida à análise da motilidade computadorizada (CASA). O excedente foi induzido à capacitação, por período de 2 horas, com meio de cultura HTF, com BSA (1%) (p:v), 37 ºC, 5% CO2. Após esse procedimento, foram avaliados: motilidade computadorizada (CASA), capacitação espermática, estado acrossomal (não íntegro/reagido) (com e sem progesterona), integridade da membrana plasmática, atividade intracelular dos ânions superóxidos e atividade mitocondrial. Os grupos foram comparados utilizando o teste T de Student para amostras independentes. A motilidade espermática computadorizada (CASA) e o estado acrossomal, foram avaliados pelo teste General Linear Models (GLM) para medidas repetidas. Os resultados foram considerados estatisticamente significantes, quando p < 0,05. Resultados: Homens com varicocele apresentaram menor atividade mitocondrial e menor capacitação espermática. A integridade da membrana plasmática, o estado acrossomal, a atividade intracelular dos ânions superóxidos e as proteínas fosforiladas pela PKA não foram alteradas nos pacientes com varicocele. A capacitação espermática teve efeito sobre a motilidade, na velocidade média do trajeto e na retilinearidade, mas não houve efeito da varicocele sobre essa alteração. Conclusão: A varicocele está associada a menor atividade mitocondrial e menor capacitação dos espermatozoides, mesmo após a seleção espermática pelo método de gradiente de densidade descontínuo. A capacitação aumentou a velocidade média do trajeto e diminuiu a retilinearidade dos espermatozoides. Não houve interação dos efeitos da capacitação nos homens com varicocele.
Introduction: Varicocele is defined as an abnormal dilation of the testicular veins in the pampiniform plexus with retrograde blood flow in the internal spermatic veins. It is considered one of the main causes of male infertility, and is responsible for alterations to semen quality, leading to failures in the processes associated with fertilization, such as sperm capacitation. Many changes occur during sperm capacitation, such as activation of protein kinase A, stimulation of reactive species of oxygen production, increase protein phosphorylation levels, changes in motility pattern and acrosome reaction. However, studies associated to the varicocele and sperm capacitation are scarce in the literature. Objectives: To evaluate the effects of varicocele on sperm capacitation in viable sperm of adult men. Methods: Semen samples from controls (n = 19) and men with varicocele (n= 37) were collected. An aliquot of seminal plasma was separated in order to verify the expression levels of PKA – phosphorylated proteins. The remaining volume was submitted to a discontinuous density gradient. The pellet was then washed in culture media HTF suplememnted with BSA (1%) (w:v). An aliquot was used to evaluate motility by computer-assisted sperm analysis (CASA). Capacitation was then induced using culture media HTF supplemented with BSA 1% (w:v) for 2 hours at 37 oC, 5 % CO2. Following capacitation induction, the following measures were assessed: computer - assisted sperm analysis (CASA), capacitation assay, acrosome reaction (spontaneous and with progesterone inductor), membrane integrity, intracellular superoxide anion activity and mitochondrial activity. Groups were compared using a Student's T test for independent samples. CASA and acrossomal status analysis were assessed using a General Linear Model (GLM) for repeated measures test. For acrossomal status analysis, which were assessed with and without progesterone, after sperm capacitation, GLM test was applied. Results were considered statistically significant when p < 0.05. Results: Men with varicocele presented lower mitochondrial activity and lower capacitation. Membrane integrity, acrosome reaction, intracellular activity of superoxide anions, and PKA phosphorylated proteins were not altered in patients with varicocele. The sperm capacitation had effect on motility, in path velocity and straightness, but there was no effect of varicocele on this alteration. Conclusion: Varicocele is associated with lower mitochondrial activity and lower sperm capacitation even after sperm selection by discontinuous density gradient method. Induction of capacitation altered motility, by increasing path velocity and decreasing straightness. There was no interaction of capacitation effects in men with varicocele.
Introduction: Varicocele is defined as an abnormal dilation of the testicular veins in the pampiniform plexus with retrograde blood flow in the internal spermatic veins. It is considered one of the main causes of male infertility, and is responsible for alterations to semen quality, leading to failures in the processes associated with fertilization, such as sperm capacitation. Many changes occur during sperm capacitation, such as activation of protein kinase A, stimulation of reactive species of oxygen production, increase protein phosphorylation levels, changes in motility pattern and acrosome reaction. However, studies associated to the varicocele and sperm capacitation are scarce in the literature. Objectives: To evaluate the effects of varicocele on sperm capacitation in viable sperm of adult men. Methods: Semen samples from controls (n = 19) and men with varicocele (n= 37) were collected. An aliquot of seminal plasma was separated in order to verify the expression levels of PKA – phosphorylated proteins. The remaining volume was submitted to a discontinuous density gradient. The pellet was then washed in culture media HTF suplememnted with BSA (1%) (w:v). An aliquot was used to evaluate motility by computer-assisted sperm analysis (CASA). Capacitation was then induced using culture media HTF supplemented with BSA 1% (w:v) for 2 hours at 37 oC, 5 % CO2. Following capacitation induction, the following measures were assessed: computer - assisted sperm analysis (CASA), capacitation assay, acrosome reaction (spontaneous and with progesterone inductor), membrane integrity, intracellular superoxide anion activity and mitochondrial activity. Groups were compared using a Student's T test for independent samples. CASA and acrossomal status analysis were assessed using a General Linear Model (GLM) for repeated measures test. For acrossomal status analysis, which were assessed with and without progesterone, after sperm capacitation, GLM test was applied. Results were considered statistically significant when p < 0.05. Results: Men with varicocele presented lower mitochondrial activity and lower capacitation. Membrane integrity, acrosome reaction, intracellular activity of superoxide anions, and PKA phosphorylated proteins were not altered in patients with varicocele. The sperm capacitation had effect on motility, in path velocity and straightness, but there was no effect of varicocele on this alteration. Conclusion: Varicocele is associated with lower mitochondrial activity and lower sperm capacitation even after sperm selection by discontinuous density gradient method. Induction of capacitation altered motility, by increasing path velocity and decreasing straightness. There was no interaction of capacitation effects in men with varicocele.
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ANDRETTA, Rhayza Roberta. Efeito da varicocele na capacitação espermática. 2018. Tese (Doutorado em Medicina: urologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2018.