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Hum. Reprod. Advance Access published online on January 20, 2006
Human Reproduction, doi:10.1093/humrep/dei435
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© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received September 7, 2005
Revised November 13, 2005
Accepted November 16, 2005

Article

Tyrosine phosphorylation on capacitated human sperm tail detected by immunofluorescence correlates strongly with sperm-zona pellucida (ZP) binding but not with the ZP-induced acrosome reaction

D.Y. Liu 1 *, G.N. Clarke 2, and H.W.G. Baker 3

1 Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, Australia
2 Department of Andrology Laboratory, University of Melbourne, Melbourne, Australia
3 Department of Obstetrics and Gynaecology, University of Melbourne, Australia; Reproductive Services, Royal Women’s Hospital; Melbourne IVF, Melbourne, Australia

* To whom correspondence should be addressed.
D.Y. Liu, E-mail: dyl{at}unimelb.edu.au


  Abstract

BACKGROUND: Protein tyrosine phosphorylation (TP) of human sperm is related to sperm capacitation and zona pellucida (ZP) binding. The aim of this study was to determine whether the TP of capacitated sperm is a useful marker for the ability of sperm to bind to the ZP and undergo the ZP-induced acrosome reaction (AR). METHODS: Semen samples were obtained from 115 subfertile men with sperm count ≥20 x 106/ml, motility ≥25% and variable morphology. Motile sperm (2 x 106/ml) selected by swim-up were incubated with four oocytes for 2 h, and the number of sperm bound to the ZP and the ZP-induced AR was examined. TP of sperm tail was assessed by immunofluorescence (IF) with anti-phosphotyrosine monoclonal antibody. The time course and effects of dibutyryl cyclic adenosine mono-phosphate (dbcAMP) and phorbol myristate acetate (PMA) on TP were also studied. RESULTS: TP was stimulated more by dbcAMP (P < 0.001) and less by PMA (P < 0.05). TP increased significantly with time of incubation of sperm. TP was not detectable on the surface of unfixed live sperm by either Dynabeads or IF. Sperm TP at 2, 4 and 20 h incubation was all significantly correlated with sperm-ZP binding but not with the ZP-induced AR. CONCLUSION: Sperm TP detected by IF correlates strongly with sperm-ZP binding capacity but not with the ZP-induced AR. This simple IF assay of TP may be a clinically useful test of sperm function that is predictive of normal sperm ZP-binding capacity.

Keywords: immunofluorescence/sperm-zona binding/tyrosine phosphorylation/zona-induced acrosome reaction.
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