Fig. 4.
Plasma membrane GM1 dynamics in sperm. We saturated all surface-accessible GM1 using 250 μg/ml FITC-conjugated CTB, and then added 5 μg/ml AlexaFluor 555-conjugated CTB to see if there was exposure of additional GM1 during CTB-induced redistribution to the PAPM. (A) Live sperm were incubated with FITC-CTB, fixed using 0.004% PF to induce redistribution, and then AlexaFluor 555-CTB was added. All sperm had an intense FITC-CTB fluorescence and no detectable AlexaFluor 555-CTB fluorescence, showing that FITC-CTB completely saturated GM1 in murine sperm. (B) Sperm were incubated with FITC-CTB, then simultaneously treated with AlexaFluor 555-CTB and fixed using 0.004% PF to induce redistribution. Some sperm had intense FITC-CTB fluorescence and no detectable AlexaFluor 555-CTB fluorescence [similar to (A); not shown] and others showed AlexaFluor 555-CTB labeling over the APM and PAPM (shown). This finding suggested that additional molecules of GM1 appeared in the plasma membrane during redistribution to the PAPM. The percentages of cells showing the two patterns corresponded with the percentage of dead and live cells, respectively. (C) Fluorescence intensity measurement over the region of the APM with and without saponin permeabilization in fixed cells. Box-whisker plots show mean pixel intensities measured in the region of the APM (inset; region of quantification outlined) in saponin permeabilized and control, unpermeabilized cells. The lower and upper ends of the box mark the 25th and 75th quantiles; the median is represented as a horizontal line within the box, and the mean as a horizontal line through the box. Vertical whiskers extend from the ends of the box to the 10th and 90th quantiles. A student’s t test showed significant differences between the permeabilized and control cells (P<0.0001). This finding provides evidence for an acrosomal pool of GM1 accessible to CTB after saponin permeabilization.