Empleo de la citrometría de flujo como herramienta para el estudio de las características espermáticas de ovino manchego durante procesos de fecundación in vitrorelación con fertilidad in vivo

Resumo

Resumen de la tesis doctoral “Empleo de la citometría de flujo como herramienta para el estudio de las caracteristicas espermaticas de ovino manchego durante procesos de fecundación in vitro: relación con la fertilidad in vivo” Defendida por el Dr. Enrique del Olmo de Medina el 21 de enero del 2016 En los centros de Inseminación Artificial se vienen evaluando de forma rutinaria diferentes pruebas de evaluación seminal con el objetivo de discriminar aquellas muestras espermaticas que no tengan una calidad mínima. No determinar de forma precoz la fertilidad supone un coste económico para los centros de sementales puesto que es posible que se mantengan animales con bajas tasas de fertilidad. Por lo tanto, determinar la fertilidad de un macho de forma temprana a partir de evaluaciones seminales eficientes mejoría el rendimiento económico puesto que aquellos machos con problemas de fertilidad podrían retirarse y no ser utilizados para inseminación artificial. El objetivo general de la tesis se orientó a la búsqueda de nuevos parámetros espermáticos, en muestras descongeladas de ovino, que evaluados in vitro pudieran correlacionarse con fertilidad tanto in vivo como in vitro. En primer lugar, decidimos utilizar la citometría de flujo y la evaluación de la motilidad mediante sistemas automáticos de análisis de imagen como herramientas para el análisis espermático debido al alto número de espermatozoides que estas técnicas son capaces de analizar en un breve espacio de tiempo y debido a su objetividad, y buscamos relaciones con fertilidad in vivo. En el primer experimento encontramos una relación directa entre dos parámetros de velocidad espermática, VCL y VAP, y la fertilidad in vivo, tras 2h de incubación a 37 °C. Sin embargo, no obtuvimos el mismo éxito en nuestro intento de encontrar relaciones entre parámetros espermáticos evaluados mediante citometría de flujo [viabilidad y apoptosis (YO-PRO-1/PI), evaluación del potencial de membrana mitocondrial (JC-1), integridad del acrosoma (FITC-PNA) y determinación de la concentración de calcio intracelular (Fluo-3)] y fertilidad. Los resultados obtenidos nos llevaron a diseñar un nuevo experimento, con el fin de buscar nuevas técnicas de citometría de flujo que pudieran presentar una relación directa con la fertilidad. Así, decidimos incluir el estudio de la producción de especies reactivas del oxígeno (ROS) por parte del espermatozoide y el posible daño que estos ROS podrían causar a la fisiología espermática y a su ADN. Para ello, utilizamos nuevas sondas como la fluoresceína (CM-H2DCFDA) para la detección de la producción de especies reactivas del oxígeno (ROS) y diferentes test para la evaluación de la integridad del ADN (SCSA®, TUNEL y Cromomicina A3). Tras la evaluación de los resultados, observamos que existía una relación directa entre la producción de ROS y los resultados de fertilidad in vivo, de tal modo, que cuanto más fértil es un macho, menor es su producción de ROS in vitro, en condiciones que simulan el tracto reproductor femenino (medio SOF, 38 °C, 5% CO2). En una segunda fase, basándonos en resultados previos de nuestro grupo de investigación que apuntaban a que la FIV era una de las técnicas evaluadas in vitro que mayor correlación presentaba con la fertilidad in vivo, obtenida por inseminación artificial intrauterina con semen descongelado de ovino, decidimos estudiar en profundidad el proceso de capacitación in vitro y ver la relación que tenía este proceso con los resultados de fertilidad in vivo. En el tercer experimento, simulamos in vitro las condiciones de capacitación (medio SOF, 38 °C, 5% CO2, 10% de suero de oveja en celo (SOC)). Nos preguntamos si verdaderamente durante este proceso de “capacitación in vitro” los espermatozoides se capacitaban o de si su respuesta estaba más relacionada con procesos de apoptosis. En esta ocasión nuestros análisis se centraron en la expresión por parte de los espermatozoides de actividad caspasa. Siendo las caspasas unas proteínas de expresión constitutiva en su forma de zimógeno en la mayoría de las células optamos por añadir un tratamiento inhibidor con el fin de poder comparar su efecto con el del suero de oveja en celo. Los resultados de esta experiencia mostraron la capacidad inhibitoria del SOC sobre la actividad caspasa. El suero de oveja en celo, durante la inducción in vitro de la capacitación de los espermatozoides ovino descongelado, parece regular a la baja las vías de apoptosis. Este mecanismo podría asegurar que los espermatozoides mantienen su competencia fisiológica en este proceso crítico previo a la fecundación. Además, tras la evaluación de diferentes parámetros espermáticos, se constató que el suero de oveja en celo no solo favorece la capacitación sino que además previene el deterioro de los espermatozoides. Nuestros resultados mostraron, además, una relación directa entre la actividad caspasa y la fertilidad in vivo, siendo los machos más fértiles los que presentaban una menor actividad caspasa durante la capacitación in vitro. Siguiendo con nuestro estudio en profundidad de la capacitación espermática, en un cuarto experimento, decidimos evaluar si, como otros autores habían indicado, el proceso de capacitación estaba directamente relacionado con la apoptosis a través de la producción de ROS, en el sentido de que ciertos ROS son necesarios para la capacitación pero si se sobrepasan ciertos límites, entonces lo que se provoca es la apoptosis y por tanto la muerte espermática. Nuestros resultados mostraron, que en condiciones capacitantes (medio SOF, 38 °C, 5% CO2, 10% de suero de oveja en celo), el SOC mantiene niveles suficientes de ROS para que se produzca la capacitación y previene de un exceso de los mismos que pudiera desencadenar la apoptosis. 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