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Files in this Data Supplement:
Fig. S1. Kinetics of antibody uptake in acrosome-reacted human spermatozoa. The lag time between exposure of binding sites for anti-CD46 and binding of the fluorescent anti-CD46 antibody (FITC-anti-CD46) was measured. AR was pre-induced using 10 µM A23187 (1 hour) to enable maximum exposure of inner acrosomal membrane before addition of fluorescent anti-CD46. During image capture on the microscope, fluorescent anti-CD46 was added at a recorded timepoint, with images captured every 10 seconds. (A) Image series showing uptake of fluorescent-anti-CD46 in a single sperm (green fluorescence). Scale bar: 5 µm. (B) Timing of uptake of fluorescent-anti-CD46 in five acrosome-reacted (black squares) and five acrosome-intact (white squares) sperm (i; relative fluorescence units are shown) and the average response of 70 acrosome-reacted sperm from one experiment (ii; normalised to maximum for each cell). Six experiments were undertaken (1346 sperm). Bars indicate standard deviation. Numbers on the image series indicate time in seconds.
Fig. S2. Histograms showing latency of AR and the time of death following AR. (A) Latency of AR was measured as the time between addition of stimulus and initiation of AR (measured as initial uptake of fluorescent anti-CD46 antibody). (B) Time between AR initiation (measured as initial uptake of fluorescent anti-CD46 antibody) and cell death (measured as initial uptake of propidium iodide). Measurements were taken in cells undergoing AR following stimulation with 10 µM A23187 (119 sperm, four experiments) or 3 µM progesterone (108 sperm, seven experiments).
Movie 1. Real-time imaging of living human sperm showing the stages of acrosome reaction. Sperm are imaged in the presence of Alexa-488-SBTI (green fluorescence) and fluorescent anti-CD46 (red fluorescence). Exposure of acrosomal content is detected by uptake of green fluorescence and exposure of the inner acrosomal membrane is detected by uptake of red fluorescence. AR is stimulated using 10 µM A23187. Video focuses on two sperm using a fluar 40× (1.3 NA) oil-immersion objective. Video shows 12 minutes of image capture.
Movie 2. Real-time AR in living human sperm induced by 10 µM A23187. Sperm are imaged in the presence of fluorescent anti-CD46 (green fluorescence) and propidium iodide (red fluorescence). AR is visualised by uptake of green fluorescence into the acrosome, and cell death is visualised by uptake of red fluorescence. Video shows a subsection of an experimental field using a fluar 40× (1.3 NA) oil-immersion objective. Video shows 20 minutes of image capture.
Movie 3. Real-time AR in living human sperm induced by 3 µM progesterone. Sperm are imaged in the presence of fluorescent anti-CD46 (green fluorescence) and propidium iodide (red fluorescence). AR is visualised by uptake of green fluorescence into the acrosome, and cell death is visualised by uptake of red fluorescence. Video shows a subsection of an experimental field using a fluar 40× (1.3 NA) oil-immersion objective. Video shows 40 minutes of image capture.
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