Visit our Expo - Redox and Inflammation signaling 2012

Session XVII : Cell signaling in health and disease Poster XVII, 21
Selection of non-apoptotic human spermatozoa provides superior tyrosine
phosphorylation
during capacitation and improved acrosome reaction
Sonja Grunewald, Thomas Baumann, Uwe Paasch, Hans-Juergen Glander
EAA Center University of Leipzig, Philipp-Rosenthal-Str. 23-25, 04103 Leipzig,
Germany
sonja.grunewald@medizin.uni-leipzig.de
Introduction: Capacitation and acrosome reaction (AR) of human spermatozoa are
prerequisites for fertilization. Major changes during capacitation result from tyrosine
phosphorylations, a process regulated by a cAMP-dependent pathway involving protein
kinase A. Annexin-V-MACS is able to separate apoptotic from non-apoptotic sperm based on
their externalization of phosphatidylserine (EPS). The non-apoptotic (EPS-) fraction is
characterized by lowest amounts of caspase activation, disrupted mitochondrial potential and
DNA fragmentation. The aim of our study was to investigate the separation effect of
Annexin-V-MACS on capacitation-related tyrosine phosphorylations and acrosome reaction.
Methods: Semen specimens from 10 healthy donors were separated into 2 samples each, one
was left untreated (control) and the second was subjected to Annexin-V-MACS. Two aliquots
of both, the control as well as the EPS negative fraction after Annexin-V-MACS were
incubated in BWW at 37°C, 5% CO2 for 3h either with 3% BSA (capacitation) or without
additives. Capacitation (CAP) was monitored by tyrosine phosphorylation (TyrP, western
blot). AR was determined by labeling with mab CD46-FITC before and after stimulation with
calcium ionophore A23187, followed by flow cytometric evaluation of the percentage of
CD46+ sperm.
Results: Densitometric analyses of the 105kDa and 80 kDa bands of the TyrP western blots
demonstrated significantly higher TyrP in the capacitated aliquots compared to the noncapacitated
aliquots. Furthermore, EPS- samples presented with significant more TyrP
compared to the non-separated semen samples (TyrP: Control 100%, Control-CAP 136±30%,
EPS- 115±13%. EPS--CAP 165±34%). There was no difference in spontaneous AR in all
groups (CD46+ sperm: Control 3.5±0.7%, Control-CAP 4.8±1.2%, EPS- 3.4±1.6%, EPS--
CAP 4.9±1.9%). In contrast, after induction of AR the capacitated as well as EPS- aliquots
showed significantly increased amounts of CD46 positive sperm. AR was best inducible in
EPS- sperm after capacitation (CD46+ sperm: control 25.9±11.4%, control-CAP 44.3±9.3%,
EPS- 37.4±8.0%, EPS--CAP 55.7±15.1%).
Conclusions: Non-apoptotic human spermatozoa are characterized by superior ability to
capacitate and consequently by maximum potential to perform acrosome reaction after
stimulation. Selection of EPS negative sperm by Annexin-V-MACS may be of advantage for
assistant reproduction in order to prepare the sperm subpopulation with the highest fertilizing
potential.
- 628 -