We reasoned that, since CNR1 functional activity, assessed using submicromolar and canna-binoid receptor-specific concentrations of the same agonist (AEA), was identical in spermatozoa from these two parts of the epididymis, the acquisition of their potential to become motile, if due to changes in endocannabinoid tone (as suggested by experiments with Cnr1KO mice and AM281), might be caused by a decrease of endocannabinoid levels. With this in mind, and in order to determine which ligand was actually involved, we measured the levels of AEA and 2-AG in isolated caput and cauda spermatozoa. The levels of 2-AG but not AEA significantly (P < 0.01) and dramatically (~30-fold) decreased from caput to cauda spermatozoa. Link
2-AG but not AEA levels also decreased in the controlateral cauda epididymal tissue used to prepare spermatozoa for endocannabinoid measurement but to a much lower extent as compared with levels detected in spermatozoa (~1-9-fold). Since we used whole epididymal caput and cauda tissue containing spermatozoa (technically it is very hard to achieve spermatozoa-free tissues) to measure AEA and 2-AG, it is possible that the change in endocannabinoid levels observed in the cauda epididymis was a mere reflection of the much stronger effect found in isolated spermatozoa. Therefore, our analytical findings support the hypothesis that the increased percentage of motile spermatozoa collected from the cauda is caused by decreased levels of 2-AG, resulting, in turn, in a decreased activity of CNR1. It is hard to reconcile this observation with the known protective effect exerted by endocannabinods against oxidative stress. In this respect, additional protective substances may be used for cauda spermatozoa despite low 2-AG levels.
Recently, Sun et al. 2009 reported the compromised motility and fertilizing capacity of epididymal Faah null (Faah-/-) spermatozoa. Authors concluded that this phenotype was due to high AEA/ CNR1 activity, as the normal phenotype was recovered in double-knockout spermatozoa (Faah-/-/Cnr1-/-). Although our results confirmed that motility of epididymal spermatozoa is responsive to AEA via both CNR1 and TRPV1, however, they indicate that 2-AG/CNR1 activity is the physiological signal regulating spermatozoa start-up in cauda epididymis. Further studies are necessary to reveal activity of all elements of the endocannabinoid system in the epididymis, possibly focusing on specific mechanisms operating in caput and cauda Faah-/- or FaahTh/Cnr1~h epididymis.
Finally, we wanted to investigate the possible mechanism through which the gradient of 2-AG levels in spermatozoa is generated when passing from the caput to the cauda of the epididymis. Apart from the expression/activity of its major biosynthesizing (DAGLA) and degrading (MGLL) enzymes, the levels of 2-AG acting on CNR1 are controlled by cellular release and reuptake via a still putative, albeit seemingly specific, transport mechanism that has been pharmacologically characterized in boar spermatozoa.
However, DAGLA and MGLL have not yet been identified in mammalian spermatozoa or in the epididymis. Here, we provided evidence for the occurrence of these enzymes in 1) spermatozoa, in which, being that these cells not translationally active, we used specific enzyme assays, and 2) the epididymis, in which, being that this tissue is strongly contaminated with transcriptionally/translationally inactive spermatozoa, we used quantitative real-time PCR (Table 2). We showed evidence that the gradient of 2-AG levels in spermatozoa cannot be caused directly by changes in the activity/expression of DAGLA or MGLL since the enzymatic activity of DAGLA significantly increased in cauda spermatozoa, whereas MGLL activity decreased.