FIG. 3. Characterization of CNR1 and TRPV1activity in caput and cauda spermatozoa. Dose-response curves of AEA on spermatozoa collected from caput (A) and cauda (B) epididymis (in vitro treatment);1 iM AEA was also combined with the CNR1 antagonist AM281. Evaluation of CNR1 and MAPK1 (housekeeping gene) expression in caput and cauda spermatozoa by Western blot analysis (C). Data are means 6 SEM. SPZ, spermatozoa.
FIG. 4. Evidence for the presence of a 2-AG gradient along the epididymis. Qualitative and quantitative analysis of endocannabinoids (AEA and 2-AG) in WT caput and cauda spermatozoa (A) or epididymal tissue (B). Spermatozoa and epididymal tissues were from the same animals. The concentrations of endocannabinoids (AEA, 2-AG) were expressed as pmol per gram (g) of spermatozoa and expressed as mean 6 SEM. SPZ, spermatozoa.
This decrease perfectly mirrored the result obtained in the cauda of WT mice treated with SR in vivo (Fig. 1C), in which, however, the compound did elevate motility in the caput as observed with AM281 but suggested an additional effect of SR as TRPV1 agonist on cauda spermatozoa. With this in mind, we attempted to counteract SR effect on cauda spermatozoa using the IRTX, the most potent TRPV1 antagonist tested so far. The combined treatment restored the percentage of motile spermatozoa collected from cauda to control values, thus confirming the dual effect (CNR1 antagonist-TRPV1 agonist) of SR in our system (Fig. 2B).
Furthermore, incubation of spermatozoa collected from caput and cauda in the presence or absence of the TRPV1 agonist CPS, alone or in combination with another TRPV 1 antagonist, CPZ, confirmed the existence of a TRPV1-mediated inhibitory effect on motility only in cauda spermatozoa (P < 0.01) (Fig. 2C). In agreement with the relative potency of these two compounds as TRPV1 agonists, SR was less efficacious as compared to CPS, being the percentage of motile spermatozoa lower when CPS rather than SR was used (40.4 6 1.54% vs. 58.3 6 1.39%; P < 0.01). itat on
Characterization of CNR1 and TRPV1 Activity in Caput and Cauda Spermatozoa
Spermatozoa separately collected from caput and cauda of WT animals were washed to get rid of endocannabinoids and stimulated with increasing AEA doses, the true ligand for both CNR1 and TRPV1 receptors. The first observation to emerge was that washed spermatozoa acquired (once again) higher capability to move although preserving their own properties. Indeed, the percentage of both caput (Fig. 3A) and cauda (Fig. 3B) motile spermatozoa was higher as compared with unwashed sperm analyzed in in vivo experiments (P < 0.01). Worthy of note, we observed that AEA decreased the percentage of motile spermatozoa collected from the caput (Fig. 3A; P < 0.01) or cauda (Fig. 3B; P < 0.01) with the same IC50 (0.5 iM). This effect was counteracted by the selective CNR1 antagonist AM281. Interestingly, high AEA concentration (10 iM) further decreased the percentage of motile spermatozoa collected from cauda, suggesting an additional TRPV1-mediated AEA effect only in cauda epididymis (Fig. 3B). Furthermore, Western blot analysis revealed that CNR1 levels in caput and cauda spermatozoa did not change (Fig. 3C).