FIG. 5. EMT inhibitors disrupt the 2-AG gradient. Disruption of 2-AG gradient by treatment of rats with the inhibitors of endocannabinoid cellular release and reuptake, AM404 (A) or OMDM-1 (B). The treatment lasted 7 days since this is the interval necessary for caput spermatozoa to reach the cauda. For sperm collection and analysis, see Material and Methods. C) Quantitative analysis of 2-AG in caput and cauda spermatozoa of WT mice in vivo treated with OMDM-1. Spermatozoa were from mice different from those used for the experiment in Figure 3. Data are means 6 SEM. SPZ, spermatozoa.
Accordingly, in vitro experiments demonstrated that SR (10 iM), as well as the prototypical TRPV1 agonist CPS (1 iM), decreased percentage of motile spermatozoa in the cauda but not in the caput being SR less efficacious as compared with CPS. Both the effects were counteracted by TRPV1 antagonists, thus strongly suggesting 1) the dual effect (CNR1 antagonist-TRPV1 agonist) of SR in our system, 2) the activation of TRPV1 in spermatozoa, and 3) the existence of a TRPV1-mediated inhibitory effect on motility only in cauda spermatozoa (P < 0.01). Since spermatozoa are translationally inactive cells and protein modifications (i.e., changes in glycosylation and phosphorylation rates) are usually described during the journey through the epididymis, we suggest that the observed TRPV1-mediated response is due to a protein modification specifically occurring in cauda spermatozoa. Accordingly, it is well established that phosphorylation modulates TRPV1 activity. fully
The unexpected TRPV1 effect hints that both CNR1 and TRPV1, or a shifted activity of the former versus the latter, might induce spermatozoa to acquire potential progressive motility in cauda. In order to verify the true role of both receptors, we directly stimulated caput or cauda spermatozoa with increasing AEA doses. We observed that AEA, via CNR1, decreased the percentage of motile spermatozoa collected from the caput and cauda with the same IC50 of 0.5 iM.
Furthermore, high AEA concentration (>1 iM) further decreased the percentage of motile spermatozoa collected from cauda, suggesting, as expected, a TRPV1-mediated additional AEA effect only in cauda epididymis. These results suggest that CNR1 activation inhibits either caput or cauda spermatozoa motility, while high AEA concentrations are requested to further inhibit motility of cauda spermatozoa via TRPV1. Therefore, differences in percentage of motile spermatozoa between caput and cauda are not likely to derive either from the different function of CNR1 or TRPV1 or from the combined activity of both receptors and not even from the different expression of CNR1. Indeed, Western blot analysis demonstrated that CNR1 expression levels were not different in spermatozoa from caput or cauda.
The previously described data suggest that, although either CNR1 or TRPV1 activation reduced the percentage of motile spermatozoa, changes in the activity triggered by the former rather than the latter (functionally active only in cauda spermatozoa at high AEA concentrations) might be responsible for spermatozoa start-up when passing from caput to cauda of the epididymis.