Cell Start-Up: Conclusion 2

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…

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Cell Start-Up: Conclusion

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…

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Cell Start-Up: DISCUSSION 2

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…

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Cell Start-Up: DISCUSSION

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...

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Cell Start-Up: RESULTS 2

FIG. 3. Characterization of CNR1 and TRPV1activity in caput and cauda spermatozoa. Dose-response curves of AEA on spermatozoa collected from caput (A) and cauda (B) epididymis (in vitro treatment);1 iM AEA was also combined with the CNR1 antagonist AM281. Evaluation of CNR1 and MAPK1 (housekeeping gene) expression in caput and cauda spermatozoa by Western blot…

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Cell Start-Up: RESULTS

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…

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Cell Start-Up: Quantitative Real-Time PCR Analysis

Caput or cauda of the epididymis was homogenized in 1 ml of Trizol (Invitrogen). Total RNA was extracted according to the manufacturer’s recommendations, dissolved in RNA storage solution (Ambion), UV quantified by a Bio-Photometer (Eppendorf), and stored to —80°C. RNA aliquots (6 ig) were digested by RNAse-free DNAse I (Ambion deoxyribonucleic acid [DNA]-free kit) in…

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