The objective of this study was to examine the influence of reactive oxygen species (ROS) on equine sperm capacitation. Motile equine spermatozoa were separated on a discontinuous Percoll gradient, resuspended at 10 x 10 6 ml in Tyrode's medium supplemented with BSA (0.5%) and polyvinyl alcohol (0.5%) and incubated at 39°C for 2 h with or without the xanthine (X; 0.1 mM)-xanthine oxidase (XO; 0.01 U/ml) system or NADPH (0.25 mM). The importance of hydrogen peroxide or superoxide for capacitation was determined by the addition of catalase (CAT; 150 U/ml) or superoxide dismutase (SOD; 150 U/ml), respectively. Following incubation, acrosomal exocytosis was induced by a 5 min incubation at 39°C with progesterone (3.18 μM), and sperm viability and acrosomal integrity were then determined by staining with Hoechst 33258 and fluoroisothiocyanate-conjugated Pisum sativum agglutin. To examine tyrosine phosphorylation, treatments were subjected to sodium dodecyl sulfate-polyacrylaminde gel electrophoresis (SDS-PAGE) followed by Western blot analysis with the anti-phosphotyrosine antibody (α-PY; clone 4G10). Capacitation with the X-XO system or NADPH led to a significant (P < 0.0001) increase in live acrosome-reacted spermatozoa compared to controls. The addition of CAT or SOD prevented the increase in live acrosome-reacted spermatozoa associated with X-XO treatment. Incubation with the X-XO system was also associated with a significant (P < 0.005) increase in tyrosine phosphorylation when compared to controls, which could be prevented by the addition of CAT but not SOD. This study indicates that ROS can promote equine sperm capacitation and tyrosine phosphorylation, suggesting a physiological role for ROS generation by equine spermatozoa.
|Number of pages||9|
|State||Published - Oct 15 2003|
Bibliographical noteFunding Information:
This research was supported by the John P. Hughes Endowment and by the Center for Equine Health with funds provided by the Oak Tree Racing Association, the State of California pari-mutual fund, and contributions by private donors. The authors would like to thank Dr. Jan Roser and the Department of Animal Science for use of some of the stallions in this study; Barbara Stewart, Dr. Deirdre Carver and Dr. Myrthe Wessel of the School of Veterinary Medicine for assistance with semen collections and the laboratory of Dr. Stuart Meyers, Department of Physiology and Cell Biology, for assistance with the detection of tyrosine phosphorylation.
- Acrosome reaction
- Reactive oxygen species
ASJC Scopus subject areas
- Small Animals
- Food Animals
- Animal Science and Zoology