INVOLVEMENT OF THE NOVEL CANNABINOID RECEPTOR GPR55 IN SPERM FUNCTION IN BOVINES

LOTTERO LECONTE RAQUEL; PEREZ MARTINEZ S; VERNAZ Z; ALONSO CAI

Jornada; XX JORNADAS ANUALES DE LA SOCIEDAD ARGENTINA DE BIOLOGÍA (SAB) XVII JORNADAS DE LA SOCIEDAD URUGUAYA DE BIOCIENCIAS (SUB); 2018

Mammals oviduct acts as a functional reservoir of spermatozoa, providing a proper environment for their maintenance and for competence to oocyte fertilization. Several molecules from the oviductal fluid are involved in sperm- oviduct interaction. Previous results from our laboratory have shown the involvement of endocannabinoid system in sperm capacitation and in sperm-release from oviductal epithelial cells (OECs) in bovines. We previously reported that anandamide (AEA), a known endocannabinoid, fluctuated in the oviductal fluid during the estral cycle in bovines. In addition, AEA induces sperm capacitation and sperm release from OECs through CB1 and TRPV1 cannabinoid receptors activation. In these molecular pathways are involved a calcium increase and cAMP/PKA activation. On the other hand, G-protein-coupled receptor 55 (GPR55) was proposed as a novel component of the endocannabinoid system, which molecular pathway includes a Protein Kinase C (PKC) activation. The aims of this work were to characterize GPR55 receptor in bovine spermatozoa and to study its participation in sperm function. Our studies were performed with bovine cryopreserved spermatozoa (without incubation (T=0), capacitated, and released from OECs) and co-cultures of sperm-OEC. First, we analyzed the presence (by Western Blot) and localization (by immunocytochemistry) of GPR55 in bovine spermatozoa. GPR55 was presented as a band of an apparent molecular of 37 kDa, as expected for this protein. At T=0, it was localized in the acrosomal region of bovine spermatozoa. However, when sperm were released from the oviductal epithelium, the receptor was mainly found in the equatorial segment and flagellum; a similar localization was observed in capacitated sperm. Subsequently, we evaluated the involvement of GPR55 in sperm capacitation induced by AEA by analyzing of increase of tyrosine phosphorylation (pY) levels and substrates of PKA (pPKA), essential events associated with this process. Results indicated that the presence of CID16020046 (GPR55 antagonist) produced a decrease of 15% in pY and ∼25% of pPKA levels, suggesting a participation of GPPR55 in sperm capacitation. In addition, we evaluated, the involvement of PKC in GPR55 activation during sperm capacitation induced by AEA. The presence of a PKC inhibitor diminished (30%) serine/threonine phosphorylated proteins levels. Considering that released and capacitated spermatozoa presented GPR55 mainly in the flagellum, we studied its participation in motility. Spermatozoa were incubated in capacitating conditions with different concentrations of GPR55 agonist AM251 (10-5-10-9 M). Results showed that AM251 (10-7M) induced a significant increase in sperm motility (vigor), similar than that observed in the presence of AEA, which was attenuated with GPR55 antagonist. Overall, results indicate that sperm bovine present a functional GPR55 that may play a role in sperm capacitation and motility, suggesting that its activation would contribute to the acquisition of sperm fertilizing ability in bovine.