AEA and 2-AG Measurement along the Epididymis
The levels of 2-AG, but not AEA, significantly (P < 0.01) and dramatically (~ 30-fold) decreased from caput to cauda spermatozoa (Fig. 4A). 2-AG but not AEA levels also decreased (P < 0.05) in the controlateral cauda epididymal tissue used to prepare spermatozoa for endocannabinoid measurement, but to a much lower extent as compared with levels measured in spermatozoa (~ 1.9-fold) (Fig. 4B). Expression/Activity of Synthesizing and Degrading Enzymes for 2-AG along the Epididymis We analyzed, through quantitative real-time PCR, the expression/activity of 2-AG major biosynthesizing (DAGLA) and degrading (MGLL) enzymes. We showed that the enzymatic activity of DAGLA significantly (P < 0.01) increased in cauda spermatozoa, whereas MGLL activity decreased (Table 2). By contrast, the expression of these enzymes in the epididymal tissue appeared to be regulated in the opposite way, with an increase (P < 0.01) of Mgll mRNA and a decrease (P < 0.01) of Dagla mRNA in the cauda (Table 2). in detail
Effects of AM404 and OMDM-1 on Sperm Motility and 2-AG Levels
In control animals (Fig. 5, A and B), the percentage of motile spermatozoa significantly (P < 0.01) increased in the cauda. When administered i.p., both AM404 and OMDM-1 (two different EMT inhibitors), while being ineffective on caput values, significantly (P < 0.01) decreased the percentage of vigorously motile spermatozoa in the cauda (Fig. 5, A and B). Measurement of 2-AG, after OMDM-1 treatment (Fig. 5C), revealed that its levels significantly (P < 0.01) increased in cauda reaching caput values. However, both caput and cauda 2-AG levels after OMDM-1 treatment were significantly lower (P < 0.05) as compared with caput control values.
The percentages of motile spermatozoa collected from the caput and cauda epididymis of WT and Cnr1KO mice were assessed in order to investigate the involvement of CNR1 in the regulation of the potential of sperm for progressive motility. The percentage of motile spermatozoa collected from caput was higher in Cnrl KO than in WT mice. Conversely, the percentage of motile spermatozoa in the cauda of both WT and KO animals was similar. Interestingly, vigorous motility was observed in the caput of KO mice.
We concluded that the absence of functional CNR1 causes a precocious acquisition of motility in spermatozoa present in the caput, which becomes comparable to that observed in the cauda. Accordingly, we found that either SR or AM281 administration, two different selective CNR1 antagonists, mimicked the Cnr1KO phenotype by increasing number of vigorously motile spermatozoa in the caput. Unexpectedly, SR decreased the percentage of motile spermatozoa in the cauda, suggesting an additional effect of SR per se in our system. We ascribed this effect to the capability of SR to activate TRPV1 at concentrations >1 iM. Indeed, the presence of TRPV1 in mouse spermatozoa has recently been demonstrated by Grimaldi et al..