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Journal of Andrology, Vol. 24, No. 3, May/June 2003
Copyright © American Society of Andrology

Effect of Azorellanone, a Diterpene From Azorella yareta Hauman, on Human Sperm Physiology

PATRICIO MORALES*, MILENE KONG*, EDUARDO PIZARRO*, CONSUELO PASTEN*, GLAUCO MORALES{dagger}, JORGE BORQUEZ{dagger} AND LUIS A. LOYOLA{dagger}

From the * Unit of Reproductive Biology, Faculty of Health Sciences, and the {dagger} Chemistry Department, Faculty of Basic Sciences, University of Antofagasta, Antofagasta, Chile.

Correspondence to: Dr Patricio Morales, Unit of Reproductive Biology, Faculty of Health Sciences, University of Antofagasta, PO Box 170, Antofagasta, Chile (e-mail: pmorales{at}uantof.cl).


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-{alpha}-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 Ca2+ 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 Ca2+ 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.

     Key words: Sperm–zona binding, cyclic terpenes, intracellular Ca2+, protease activity






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Copyright © 2003 by The American Society of Andrology.