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First published online 3 June 2008
doi: 10.1242/jcs.030379

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Dynamic resolution of acrosomal exocytosis in human sperm

¹ Centre for Cell Imaging, School of Biological Sciences, Biosciences Building, Crown Street, University of Liverpool, L69 7ZB, UK
² Division of Immunology, School of Infection and Host Defence, Duncan Building, Daulby Street, University of Liverpool, L69 3GA, UK
³ School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK

View larger version (43K): [in this window] [in a new window]   Fig. 1. 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).

View larger version (90K): [in this window] [in a new window]   Fig. 2. Spatial characteristics of the AR in human sperm. (A) The acrosome of each acrosome-reacting sperm was divided into three regions: (1) anterior, (2) mid-section and (3) posterior. (B) Histogram showing the initial site of uptake of Alexa-488–SBTI (green) and fluorescent anti-CD46 (red) in 72 acrosome-reacted sperm from five experiments. Bars show s.e.m. (C) Image series showing uptake of Alexa-488–SBTI (green) in three different sperm (i-iii); numbers represent time in seconds. (D) Image series showing uptake of fluorescent anti-CD46 (red) in three different sperm (i-iii); numbers represent time in minutes. Scale bars: 2 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 3. Kinetics of induced and non-induced AR, and its relationship to cell death and motility. (Ai-iv) Single-sperm response graphs showing the timing of A23187 (10 µM)-induced AR (uptake of fluorescent anti-CD46; green lines) and cell death (uptake of PI; red lines). Each graph represents a different cell. (B) Image series of sperm stimulated with 10 µM A23187. (Ci-iv) Single-sperm response graphs show the timings of AR and cell death occurring spontaneously; (D) corresponding image series showing a single sperm undergoing spontaneous AR. (E) Single-sperm response graphs showing the timings of progesterone (3 µM)-induced AR and cell death; (F) corresponding image series showing a single sperm undergoing progesterone (3 µM)-induced AR. On all graphs, the x-axis is time relative to initiation of AR for that individual cell. Traces are normalised to 100% increase in fluorescence for each probe. Arrows on the single-cell graphs show the time at which that cell became immotile. In all image series, uptake of green fluorescence represents sperm undergoing AR (uptake of fluorescent anti-CD46) and uptake of red fluorescence represents sperm undergoing cell death (uptake of PI). Numbers on the image series indicate time in minutes. Scale bars: 5 µm.

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