Effect of azorellanone, a diterpene from Azorella yareta hauman, on human sperm physiology

Previous studies have shown that cyclic terpenes extracted from plants decrease sperm motility and concentration in rats. In this work, we studied the effect 13-α-hydroxy-7-oxoazorellano (azorellanone), a cyclic diterpene extracted from Azorella yareta Hauman, on in vitro human sperm physiology. Sperm aliquots, capacitated for 4.5 or 20 hours, were incubated for 15 minutes with different concentrations of azorellanone. Then, the effects of azorellanone on sperm motility, viability, binding to the human zona pellucida, progesterone-induced acrosome reactions and increase in intracellular Ca ^2- concentration, and trypsin- and chymotrypsin-like protease activities were evaluated. Sperm motility was evaluated according to World Health Organization (WHO) guidelines; sperm viability with the supravital dye Hoescht 33258; sperm-zona binding with the hemizona assay; progesterone-induced acrosome reaction with fluorescent lectin, intracellular Ca²⁺ level with fura 2; and protease activity with the synthetic substrates N-t-Boc-Gln-Ala-Arg-Amido-4-methylcoumaryn and Succinyl-Leu-Leu-Val-Tyr-Amido-4- methylcoumaryn. The results obtained indicate that azorellanone inhibited sperm motility in a concentration-dependent manner at 0.15, 1.5, and 3 mM, while sperm viability was only inhibited at 3 mM. Treatment with azorellanone significantly inhibited sperm-zona binding, progesterone-induced acrosome reactions, and intracellular Ca^2- concentration Treatment of free-swimming sperm with azorellanone did not affect protease activity; however, the incubation of sperm extracts with azorellanone significantly inhibited both trypsin-like and chymotrypsin-like protease activities. In conclusion, azorellanone has a significant effect on the different parameters that characterize human sperm physiology.