FIG. 2. Characterization of SR effect on cauda spermatozoa. Sperm analysis after in vitro treatment with SR alone (A) or in combination with TRPV1 antagonist, IRTX (B). Effect of the TRPV1 agonist CPS, with or without the TRPV1 antagonist CPZ, on spermatozoa motility (C).
Evaluation of CNR1 Role in Epididymal Sperm Cell ”Start-Up”
We determined the percentage of motile spermatozoa from epididymis, caput, and cauda separately of control WT and Cnr1KO mice (Fig. 1A). In control animals, the percentage of motile spermatozoa was significantly higher (P < 0.01) in cauda than in caput. In Cnr1KO animals, the percentage of motile spermatozoa from caput was significantly higher (P < 0.01) as compared with values of spermatozoa collected from the caput of WT mice. Furthermore, no differences were observed in the percentage values of spermatozoa collected from the cauda of WT or caput and cauda of CnrlKO animals. Interestingly, caput spermatozoa of Cnr1KO animals were characterized by vigorous motility.
The percentage of motile spermatozoa was also counted in the epididymis of WT animals pharmacologically treated with selective CNR1 antagonists AM281 or SR. The administration of AM281 (Fig. 1B) mirrored the Cnrl’KO phenotype by increasing vigorously motile spermatozoa number in the caput (P < 0.01) reaching cauda control values. Cauda values of AM281 treated animals did not change as compared with those of cauda control WT mice. more
Such results did not perfectly match those obtained from WT mice injected with SR (Fig. 1C). Indeed, while SR increased the number of vigorously motile spermatozoa in the caput, it decreased their percentage in cauda, suggesting an additional effect of SR per se in our system. Interestingly, although SR is reported to be a specific CNR1 antagonist, it has also been reported to act as partial TRPV1 agonist at concentrations >1 iM, and TRPV1 is expressed in spermatozoa. Starting from the previously mentioned observations, we carried out a series of in vitro experiments to characterize 1) the SR effect on cauda spermatozoa and 2) the receptor(s) regulating spermatozoa motility and their activity in caput and cauda spermatozoa.
Characterization of SR Effect on Caudal Spermatozoa Motility
Spermatozoa separately collected from caput and cauda of WT animals were washed to get rid of endocannabinoids and then directly stimulated with different drugs (SR 6 IRTX; CPS 6 CPZ). Interestingly, washed spermatozoa acquired higher capability to move. Indeed, the percentage of both caput and cauda motile spermatozoa was higher (Fig. 2A, control values) as compared with unwashed (Fig. 1A, control values) spermatozoa analyzed in in vivo experiments (P < 0.01). SR alone did not affect the percentage of motile spermatozoa collected from caput (as expected due to the absence of inhibitory stimuli to be counteracted in vitro), but it inhibited that in those collected from the cauda (P < 0.01) (Fig. 2A).