103 Characterization of the receptors of the endocannabinoid system in equine sperm: Possible role of anandamide in sperm function

Abstract

Conventional IVF in horses remains challenging. In particular, stallion sperm fails to penetrate the zona pellucida, possibly due to incomplete in vitro sperm capacitation. Therefore, there is a need to elucidate, in horses, molecules with a proven role during capacitation in other mammals. Our laboratory has described the relevance of the endocannabinoid system in capacitation of bovine and murine sperm. We reported that anandamide (AEA), an endocannabinoid present in follicular and oviducal fluids, induced capacitation-associated events. The aims of this work were to characterise the localization of cannabinoid receptors in equine sperm and to evaluate the effects of AEA on levels of tyrosine-phosphorylated proteins (pY) and substrates phosphorylated by protein kinase A (pPKA). Both cannabinoid receptors (CB1, CB2, TRPV1) and pPKA and pY were localised in sperm by indirect immunofluorescence. Sperm (15 × 10⁶ mL⁻¹) were incubated, at 38.5°C in air, in modified Tyrode’s-albumin-lactate-pyruvate (TALP) with 25 mM NaHCO₃, 5 mM dextrose and 1 mg mL⁻¹ polyvinyl alcohol (PVA; TALP-Bic-PVA) or TALP-Bic-PVA supplemented with AEA (0.1, 1, 10, 100 nM, and 1 µM) for 4 h. After incubation, Western blot was used to determine levels of pY and pPKA in 4.5 × 10⁶ sperm. Cryopreserved sperm samples from three stallions were evaluated. The normality of data distributions and homoscedasticity were verified with the Shapiro-Wilk and Levene tests, respectively. Data were analysed by one-way ANOVA and Bonferroni post hoc test, with P < 0.05 considered significant. Based on immunofluorescence, CB1 was mainly localised in the post-acrosomal region and flagellum (93.4% ± 5.5, mean ± s.d.), CB2 in the post-acrosomal region and middle piece (89.9% ± 28.3), and TRPV1 in the post-acrosomal region and flagellum (89.3% ± 9). Sperm positive for pPKA had fluorescence in the middle piece and principal piece of the flagellum. Incubation with 1 nM AEA for 4 h induced a 61% increase in pPKA levels compared with TALP-Bic-PVA medium alone, with no induction of pY levels in any treatment. In conclusion, cannabinoid receptors were present in equine sperm, and incubation with AEA induced an increase in PKA activity, an essential event associated with sperm capacitation. To our knowledge, this was the first report describing the presence of receptors of the endocannabinoid system in equine sperm and the potential role of AEA in the acquisition of sperm fertilizing ability.