D Y Liu1, G N Clarke, H W G Baker. 1. Department of Obstetrics and Gynecology, University of Melbourne, Reproductive Services and Andrology Laboratory, Royal Women's Hospital and Melbourne IVF, Melbourne, Australia. dyl@unimelb.edu.au
Abstract
BACKGROUND: The aim of this study was to determine the effect of culture medium osmolality, in the range known to occur in the male and female reproductive tracts, on human sperm tyrosine phosphorylation and sperm-zona pellucida (ZP) interaction in vitro. METHODS: Motile sperm (2x10(6)), selected by swim-up from semen of normozoospermic men with normal sperm-ZP binding, were incubated with or without four oocytes in 1 ml human tubal fluid (HTF) medium with different osmolalities (150, 200, 280, 350, 400 mOsm/kg) adjusted by variation of the NaCl concentration. After 2 h incubation, the number of sperm bound to the four ZP was examined, sperm motility and velocities were assessed by Hamilton-Thorn Motility Analyzer (IVOS 10) and sperm tyrosine phosphorylation was assessed by both western immunoblotting and immunofluorescence with an anti-phosphotyrosine monoclonal antibody (PY20). The effect of hyper-osmolality (400 mOsm/kg) on the ZP-induced acrosome reaction (AR) was also determined. RESULTS: Incubation of human sperm in hyper-osmotic medium significantly increased tyrosine phosphorylation and the number of sperm bound to the ZP. In contrast, hypo-osmotic medium significantly decreased both tyrosine phosphorylation and sperm-ZP binding. Medium with high osmolality (400 mOsm/kg) significantly reduced the ZP-induced AR. Both hypo- and hyper-osmotic media significantly decreased average sperm percentage progressive motility and velocities. CONCLUSION: Incubation of human sperm in hyper-osmotic media was associated with significantly increased tyrosine phosphorylation and ZP-binding ability but severely reduced the ZP-induced AR.
BACKGROUND: The aim of this study was to determine the effect of culture medium osmolality, in the range known to occur in the male and female reproductive tracts, on human sperm tyrosine phosphorylation and sperm-zona pellucida (ZP) interaction in vitro. METHODS: Motile sperm (2x10(6)), selected by swim-up from semen of normozoospermic men with normal sperm-ZP binding, were incubated with or without four oocytes in 1 ml human tubal fluid (HTF) medium with different osmolalities (150, 200, 280, 350, 400 mOsm/kg) adjusted by variation of the NaCl concentration. After 2 h incubation, the number of sperm bound to the four ZP was examined, sperm motility and velocities were assessed by Hamilton-Thorn Motility Analyzer (IVOS 10) and sperm tyrosine phosphorylation was assessed by both western immunoblotting and immunofluorescence with an anti-phosphotyrosine monoclonal antibody (PY20). The effect of hyper-osmolality (400 mOsm/kg) on the ZP-induced acrosome reaction (AR) was also determined. RESULTS: Incubation of human sperm in hyper-osmotic medium significantly increased tyrosine phosphorylation and the number of sperm bound to the ZP. In contrast, hypo-osmotic medium significantly decreased both tyrosine phosphorylation and sperm-ZP binding. Medium with high osmolality (400 mOsm/kg) significantly reduced the ZP-induced AR. Both hypo- and hyper-osmotic media significantly decreased average sperm percentage progressive motility and velocities. CONCLUSION: Incubation of human sperm in hyper-osmotic media was associated with significantly increased tyrosine phosphorylation and ZP-binding ability but severely reduced the ZP-induced AR.