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.
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 > or =20 x 10(6)/ml, motility > or =25% and variable morphology. Motile sperm (2 x 10(6)/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 monophosphate (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.


CONCLUSIONS
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.
Liu
D Y
DY
Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, Australia. dyl@unimelb.edu.au
Clarke
G N
GN
Baker
H W G
HW
D020101:Acrosome Reaction / Q000502:physiology; D015415:Biomarkers; D003994:Bucladesine / Q000494:pharmacology; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007248:Infertility, Male / Q000175:diagnosis; D008297:Male; D010766:Phosphorylation / Q000187:drug effects; D013075:Sperm Capacitation / Q000502:physiology*; D013082:Sperm Tail / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D013084:Sperm-Ovum Interactions / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*; D015044:Zona Pellucida / Q000502:physiology*
D015415:Biomarkers; D014443:Tyrosine; D003994:Bucladesine; D013755:Tetradecanoylphorbol Acetate