By contrast, the expression of these enzymes in the epididymal tissue appeared to be regulated in the opposite way, with an increase of Mgll mRNA and a decrease of Dagla mRNA in the cauda. This was a puzzling result, and the only way to reconcile these findings with the observed decreased 2-AG levels in cauda spermatozoa was to speculate that this latter phenomenon is the result of a progressive gradient-driven transport of 2-AG from sperm cells to epididymal epithelial cells during the travel of spermatozoa from the caput to the cauda. This process would also explain why the levels of 2-AG in caput epididymis and spermatozoa were comparable, whereas those in cauda epididymis were significantly higher than those in cauda spermatozoa despite the fact that changes in metabolic enzymes expression/activity should have caused the opposite effect. comments
We postulated that the ‘‘stripping’’ of 2-AG from spermatozoa by the epididymis would be 1) driven by the observed increased MGLL expression in the cauda of this tissue and by decreased 2-AG degradation and increased 2-AG biosynthesis in cauda spermatozoa and 2) facilitated by the putative specific transport mechanism in both spermatozoa and epididymal epithelial cells, a process that is known to be driven by the gradient of endocannabinoid concentrations across plasma membranes. In agreement with this hypothesis, we found that two inhibitors of endocannabinoid cellular uptake and release, AM404 and OMDM-1, disrupted the increase in the number of motile spermatozoa found in the cauda. Furthermore, the effect of OMDM-1 was accompanied by a significant enhancement of the levels of 2-AG in cauda spermatozoa despite the fact that, in this set of experiments, the extent of the gradient was lower than in the first set of experiments but still very marked (3.5-fold higher levels of 2-AG in caput).
In conclusion, along the epididymis, the decrease of 2-AG levels from caput to cauda promotes sperm start-up by releasing spermatozoa from the inhibition exerted by CNR1 activation. These changes correspond to a functional 2-AG gradient that, by keeping the inhibitory activity of CNR1 high in caput and low in cauda spermatozoa, controls their potential to become motile during the travel in the epididymis. It is intriguing to observe how the endocannabinoid gradient seems to regulate both male and female reproduction since the gradient of AEA levels underlies both oviductal transport of embryos and their uterine implantation in the mouse.
Interestingly, the 2-AG gradient in mouse spermatozoa is not due to the ‘‘dilution mechanism’’ (evolutionarily selected by the external ‘‘aquatic’’ fertilization) postulated for amphibian spermatozoa but seems the result of 2-AG sequestration from the epididymis driven by changes in 2-AG metabolic enzymes in this tissue that are opposite to those observed in spermatozoa. Clearly, the conclusive confirmation of this mechanism, which was suggested here by the results of biochemical, pharmacological, and analytical experiments, will require the molecular characterization of the endocannabinoid transporter and its detection in mouse epididymis.
However, both mechanisms ensure the right number of motile spermatozoa by decreasing endocannabinoid activity although using, of course, different strategies. Disruption of this mechanism, if translated to humans, might explain in part those cases of male infertility that are due to the impairment of sperm motility. As a consequence, our findings might open new avenues for the pharmacological treatment of this ever-increasing medical and social problem.