CEFYBO   02669
CENTRO DE ESTUDIOS FARMACOLOGICOS Y BOTANICOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Sperm release from the Oviductal Epithelium depends on Ca2+ influx through TRPV1 and the sequential activation of CB1 and TRPV1 by AEA in bovines
Autor/es:
MARÍA G. GERVASI, CLAUDIA OSYCKA-SALUT, CARMEN LLADOS, MANUEL VILLALÓN, SILVINA PEREZ MARTINEZ
Lugar:
Montreal
Reunión:
Congreso; Society for Study of Reproduction SSR 46th Annual meeting; 2013
Resumen:
Sperm release from the Oviductal Epithelium depends on Ca2+ influx through TRPV1 and the sequential activation of CB1 and TRPV1 by AEA in bovines.María G. Gervasi1, Claudia Osycka-Salut1, Carmen Llados2, Manuel Villalón2, Silvina Perez-Martinez1. (1)Centro de Estudios Farmacológicos y Botánicos (CEFYBO)-CONICET, Buenos Aires, Argentina. (2)Pontificia Universidad Católica de Chile, Santiago de Chile, Chile.   Sperm transit in the female tract is a critical event for the success of fertilization. Anandamide (AEA) is an endocannabinoid that binds to cannabinoid (CB1 and CB2) and vanilloid (TRPV1) receptors and activates several different pathways. Previously, we studied the participation of AEA in the regulation of sperm-oviduct interaction in bovines and we found that AEA promotes sperm release from oviductal epithelium probably by inducing sperm capacitation through the activation of CB1 and TRPV1. Taking into account that sperm capacitation is one of the causes of sperm release from oviductal reservoir, and that CB1 and TRPV1 are activated during this event, the aims of the present work were 1) to study the possible crosstalk between these receptors and 2) to investigate the participation of TRPV1-mediated gating of Ca2+, in the regulation of sperm release from oviductal cells induced by AEA in bovines. The crosstalk between CB1 and TRPV1 depends on the activation of a phospholipase C (PLC). Thus we performed experiments of sperm-oviduct interaction incubating the co-cultures with a combination of different CB1 and TRPV1 agonists, antagonists or a PLC inhibitor. Our results showed that either capsaicin (a TRPV1 agonist) or ACEA (a CB1 agonist) decreased the number of spermatozoa bound to the epithelial cells (p<0.05). Moreover, the incubation with capsaicin plus a CB1 antagonist (SR141716A) reversed the effect of capsaicin alone suggesting that the activation of both receptors is required to promote sperm release. Interestingly, the PLC inhibitor prevented sperm release due to capsaicin, ACEA or capsaicin plus ACEA, indicating that a PLC is involved in the activation of CB1 and TRPV1 pathways. As TRPV1 is also a Ca2+ channel, we investigated whether Ca2+ ions might be involved in the AEA pathway activation in spermatozoa bound to oviductal cells. Spermatozoa preloaded with BAPTA2-AM (an intracellular Ca2+ scavenger) were incubated with bovine oviductal epithelial cells during 60 min. After that, co-cultures were treated with AEA or capsaicin to promote sperm release. Our results indicate that the sperm release promoted by cannabinoid agonists was abolished in co-cultures with spermatozoa preloaded with BAPTA2-AM (p<0.05 vs spermatozoa without BAPTA), suggesting that Ca2+ is involved in AEA pathway in that physiological process. Then we evaluated Ca2+ content in spermatozoa bound to oviductal epithelial cells. Spermatozoa were preloaded with FURA2-AM, then co-cultured with oviductal epithelial cells and incubated with AEA or capsaicin. Data analysis indicates that approximately a 30% of bound spermatozoa, including control treatment, increased Ca2+ flux. However, this increase was higher in spermatozoa treated with AEA or capsaicin than in control cells. Moreover, CB1 or TRPV1 antagonists completely inhibited Ca2+ elevation observed with AEA (p<0.05). Overall these results suggest that the activation of PLC is required for the activation of CB1 and TRPV1 crosstalk pathway to promote sperm detachment from the oviduct epithelial cells. In addition, CB1 and TRPV1 activation leads to an increase in Ca2+ content in spermatozoa, suggesting that this ion is also involved in sperm release by AEA. The present study indicates that the intracellular pathways modulated by the activation of CB1 and TRPV1 are related with the molecular events involved in activation and release of sperm cells thus emphasizing the role of the endocanabinoid system in sperm physiology.