Capacitation status was determined by the dual staining method described by Pérez et al. [19] (link) with some modifications. Briefly, 135 µl of treated spermatozoa were added to 15 µl of H33258 solution (10 µg H33258/ml DPBS) and incubated for 2 min at RT. Excess dye was removed by layering the mixture over 250 µl of 2% (w/v) polyvinylpyrrolidone in DPBS. After centrifuging at 100× g for 2.5 min, the supernatant was discarded and the pellet resuspended in 100 µl of DPBS; 100 µl of a freshly prepared chlortetracycline fluorescence (CTC) solution (750 mM CTC in 5 µl buffer: 20 mM Tris, 130 mM NaCl, and 5 mM cysteine, pH 7.4). Samples were observed with a Microphot-FXA microscope (Nikon) under epifluorescence illumination using ultraviolet BP 340–380/LP 425 and BP 450–490/LP 515 excitation/emission filters for H33258 and CTC, respectively. The spermatozoa were classified as live non-capacitated (F type, bright green fluorescence distributed uniformly over entire sperm head, with or without stronger fluorescent line at equatorial segment), live capacitated (B type, green fluorescence over acrosomal region and a dark post acrosome), or live acrosome reacted (AR type, sperm showing a mottled green fluorescence over head, green fluorescence only in post acrosomal region or no fluorescence over the head) [20] (link). All spermatozoa had bright green fluorescent mid-pieces. Two slides per sample were evaluated with at least 400 spermatozoa per slide.
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