Publication: Función del sistema cannabinoide en maduración de ovocitos, fecundación y desarrollo embrionario en mamíferos
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2017-08-02
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Universidad Complutense de Madrid
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Abstract
La señalización cannabinoide ha sido asociada con diferentes eventos reproductivos como la gametogénesis, la fecundación, la implantación, la placentación, la preñez y el parto (Battista, et al. 2008, Maccarrone 2009). Existen pruebas convincentes de que los receptores cannabinoides CB1 y CB2 se expresan en ovarios, ovocitos y espermatozoides de diferentes especies como humanos, rata y ratón (Agirregoitia, et al. 2015a, Agirregoitia, et al. 2016). Sin embargo, se desconoce su función y si tiene algún efecto en la maduración o la fecundación del ovocito. El objetivo de esta tesis doctoral es analizar en primer lugar, los efectos de los cannabinoides en la maduración del ovocito y sus consecuencias en la fecundación y el desarrollo embrionario; en segundo lugar, identificar algunas de las posibles vías de señalización por las que pueden estar actuando; y en tercer lugar, analizar el efecto de los cannabinoides en el espermatozoide. En el capítulo 1 hemos utilizado el modelo murino en un abordaje genético y farmacológico, para examinar si los cannabinoides actúan a través de los receptores CB1 y/o CB2 modulando la maduración de ovocitos. En primer lugar evaluamos cual era la dinámica de expresión de ambos receptores y su localización celular durante el reinicio de la meiosis. Nuestros resultados confirman la expresión y localización de ambos receptores en los ovocitos inmaduros en estadio de vesícula germinal (GV), lo que convierte al ovocito en una posible diana para cannabinoides durante el proceso de maduración. De los dos receptores analizados, sólo CB1 tiene un cambio en su dinámica de expresión durante la maduración del ovocitos, disminuye en el paso de GV a metafase I (MI) y desaparece en el ovocito madurado en estadio de metafase II (MII). En cuanto a la localización celular, solo el receptor CB1 tiene cambios, pasando del núcleo en el estadio GV, hacia una ubicación periférica a medida que se dan los eventos característicos del reinicio de la meiosis como la fase de ruptura de la vesícula germinal (GVBD), el paso a MI y finalmente el detenimiento en MII donde se observa ubicado claramente en la membrana del ovocito, sitio en el que el receptor puede activar la maquinaria de las proteínas G, al contrario CB2 permanecio ubicado en el citoplasma y no se detecto ningún cambio en la localización durante las diferentes etapas. El receptor CB1 continúa localizado en la membrana durante todo el desarrollo embrionario, mientras que para el receptor CB2 no se observan cambios en su localización, lo cual indica que los posibles efectos de los cannabinoides deben ser mediados por la unión al receptor CB1...
Cannabinoid signaling is associated with different reproductive events like gametogenesis, fertilization, implantation, placentation, pregnancy and parturition (Battista, et al. 2008, Maccarrone 2009). There are convincing evidences indicating that cannabinoid receptors CB1 and CB2 are expressed in the ovaries, oocytes and spermatozoa of different species like human, rats and mice (Agirregoitia, et al. 2015a, Agirregoitia, et al. 2016). However, its function and effects on oocyte maturation and fecundation remain unknown. The objectives of this thesis are: first, to analyse the effects of cannabinoids on oocyte maturation and their consequences on fecundation and embryo development; second, to identify the signalling pathways by which they can act; and third, to analyse the effects of cannabinoids on spermatozoa. In the first chapter we used a mouse model from a genetic and pharmacological approach, to examine whether cannabinoids act through the CB1 and/or CB2 receptors to modulate oocyte maturation. First, we evaluated the expression dynamics of both receptors and their cellular localization during meiosis resumption. Our results confirmed the expression and localization of both receptors in immature oocytes in germinal vesicle (GV) stage, which points to the oocyte as a possible target for cannabinoids during the maturation process. Among both receptors, only CB1 showed dynamic expression changes during oocytes maturation, decreasing from GV to metaphase I (MI), and disappearing in the mature oocyte in metaphase II (MII) stage. Regarding cellular localization, only CB1 receptor experienced changes, moving from the nucleus in GV stage to a peripheral location along meiosis resumption, specifically during the germinal vesicle breakdown (GVBD), metaphase I (MI) and finally the arrest in MII, where it is clearly localized in the oocyte membrane, being able to activate the G proteins signaling. The CB1 receptor remain localized in membrane during the whole embryo development. On the contrary, CB2 remained localized in the cytoplasm during oocyte maturation and whole embryo development. This may indicate that the effects of cannabinoids may be mediated by the binding to CB1 receptor...
Cannabinoid signaling is associated with different reproductive events like gametogenesis, fertilization, implantation, placentation, pregnancy and parturition (Battista, et al. 2008, Maccarrone 2009). There are convincing evidences indicating that cannabinoid receptors CB1 and CB2 are expressed in the ovaries, oocytes and spermatozoa of different species like human, rats and mice (Agirregoitia, et al. 2015a, Agirregoitia, et al. 2016). However, its function and effects on oocyte maturation and fecundation remain unknown. The objectives of this thesis are: first, to analyse the effects of cannabinoids on oocyte maturation and their consequences on fecundation and embryo development; second, to identify the signalling pathways by which they can act; and third, to analyse the effects of cannabinoids on spermatozoa. In the first chapter we used a mouse model from a genetic and pharmacological approach, to examine whether cannabinoids act through the CB1 and/or CB2 receptors to modulate oocyte maturation. First, we evaluated the expression dynamics of both receptors and their cellular localization during meiosis resumption. Our results confirmed the expression and localization of both receptors in immature oocytes in germinal vesicle (GV) stage, which points to the oocyte as a possible target for cannabinoids during the maturation process. Among both receptors, only CB1 showed dynamic expression changes during oocytes maturation, decreasing from GV to metaphase I (MI), and disappearing in the mature oocyte in metaphase II (MII) stage. Regarding cellular localization, only CB1 receptor experienced changes, moving from the nucleus in GV stage to a peripheral location along meiosis resumption, specifically during the germinal vesicle breakdown (GVBD), metaphase I (MI) and finally the arrest in MII, where it is clearly localized in the oocyte membrane, being able to activate the G proteins signaling. The CB1 receptor remain localized in membrane during the whole embryo development. On the contrary, CB2 remained localized in the cytoplasm during oocyte maturation and whole embryo development. This may indicate that the effects of cannabinoids may be mediated by the binding to CB1 receptor...
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Tesis inédita de la Universidad Complutense de Madrid, Facultad de Veterinaria, Departamento de Fisiología Animal, leída el 23-09-2016