Untersuchungen zur Kapazitation und Hyperaktivierung equiner ...

Ergebnisse
2.0 mM CaCl2, 0.4 mM MgCl2, 0.3 mM NaH2PO4, 25 mM NaHCO3, 21.6 mM Na-
lactate, 1.0 mM Na-pyruvate, 10 mM HEPES, pH 7.5, 3 g L -1 BSA, and 50 µg mL -1
gentamycin. Tyrode B control medium (for non-capacitating conditions) lacked CaCl2,
NaHCO3 and BSA, and was supplemented with 1 mM Na2EGTA. Whittens A medium
is composed of 100 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 5.5 mM glucose, 22 mM
HEPES, 4.8 mM Ca-lactate and 1.0 mM pyruvic acid Na-pyruvate, 25 mM NaHCO3
and 7 mg mL -1 BSA. Whittens B control medium (for non-capacitating conditions)
lacked NaHCO3 and BSA.
Incubations in Tyrode or Whittens A and B medium were done for 2 h, in
medium equilibrated at 37 ˚C in the presence and ab sence of 5% CO2, respectively.
Sperm were diluted in Tyrode or Whittens medium at 1 x 10 6 cells mL -1 . This was
done by diluting 5 µL 100 x 10 6 cells mL -1 in skim milk extender in a final volume of
500 µL Tyrode or Whittens medium. Aliquots were prepared for evaluation of
capacitation characteristics at subsequent time points. Fluorescent dyes were added
to these aliquots 10 min prior to flow cytometric analysis.
For determination of percentages of sperm that have damaged plasma and
acrosomal membranes, a double staining with propidium iodide (PI; Sigma-Aldrich,
St. Louis, MO, USA) and fluorescein labeled peanut agglutinin (FITC-PNA; Vector
Laboratories, Burlingame, CA, USA) was performed (Rathi et al., 2001). For PI/FITC-
PNA staining, 5 µL 100 x 10 6 cells mL -1 in extender was diluted in 490 µL Tyrode or
Whittens medium, and 2 µL 0.75 mM PI and 3 µL 75 µM FITC-PNA were added;
resulting in 1 x 10 6 cells mL -1 , 3 µM PI and 0.45 µM FITC-PNA.
A flow cytometer (Cell Lab Quanta SC MPL; Beckman-Coulter, Fullerton, CA,
USA) was used that is equipped with a 488 nm argon ion laser of 22 mw for
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