Temporal characteristics of the biphasic nature of the AR in human sperm. Temporal characteristics of the biphasic nature of the AR in human sperm. (A)

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Temporal characteristics of the biphasic nature of the AR in human sperm. Temporal characteristics of the biphasic nature of the AR in human sperm. (A) Detection of the AR was measured using a two-probe technique. (i) Fluorescent anti-CD46 monoclonal antibody (red circles) and Alexa-488–SBTI (green circles) are present in the medium surrounding live acrosome-intact human sperm. (ii) Onset of the AR involves fenestration of the plasma membrane with the outer acrosomal membrane, allowing binding of Alexa-488–SBTI to acrosomal content [accumulation of green fluorescence in the acrosome (iii)]. (iv) Acrosomal content is then shed, enabling fluorescent anti-CD46 to accumulate and bind to previously concealed CD46 sites located on the inner acrosomal membrane, visualised by an accumulation of red fluorescence over the inner acrosomal membrane, as portrayed in the acrosome-reacted sperm (v). (B) Image series showing two sperm undergoing A23187-induced AR (10 μM). Exposure and dispersal of acrosomal content is detected using Alexa-488–SBTI (green) and exposure of the inner acrosomal membrane is detected using fluorescent anti-CD46 (red). Numbers represent time in minutes. Scale bar: 5 μm. (C) Kinetics of accumulation and loss of Alexa-488–SBTI (green), and uptake of fluorescent anti-CD46 (red). Mean ± s.e.m. of 72 acrosome-reacted cells from five experiments is shown. (Di) Initial rates of acrosomal content dispersal and fluorescent anti-CD46 uptake [expressed as change in normalised (% maximum) fluorescence minute–1] measured over 2 minutes following initial Alexa-488–SBTI loss. Most points are above the dashed line (plotting equivalent rates). (Dii,iii) Single-sperm plots of Alexa-488–SBTI (green) and fluorescent anti-CD46 (red) showing heterogeneous responses. Traces are presented as % fluorescence normalised to maximum for each probe separately. (Diii) Horizontal red line (y=0) represents no binding of fluorescent anti-CD46 (red). (E) Histogram showing time to 90% uptake of fluorescent anti-CD46 that occurred spontaneously (white bar; 41 sperm, four experiments) or was induced by 10 μM A23187 (black bar; 119 sperm, four experiments) or by 3 μM progesterone (grey bar; 108 sperm, seven experiments). Claire V. Harper et al. J Cell Sci 2008;121:2130-2135 © The Company of Biologists Limited 2008