(Fertilization. 15) (mean SEM). The numbers of females examined are in parentheses. Based on the foregoing evidence, we observed the subcellular localization of CD9-EGFP in living Tg+CD9?/? eggs (Fig. 2are enlarged images of the boxed areas. (= 10), CD9?/? (= 9), and Tg+CD9?/? eggs (= 10) (mean SEM). The average values of the wild-type eggs were arranged to 100%. We next examined the manifestation of ganglioside GM3, identified as a CD9-connected molecule (12) and a component of exosomes (10), in CD9+/+, CD9?/?, and Tg+CD9?/? eggs. First, we confirmed the manifestation of GM3 synthase (ST3GalV/SAT-1) (13) in these eggs by RT-PCR (Fig. 2and Fig. 2and Fig. 2and Fig. 2and are enlarged. Level pub: 20 m. (= 204; 4.0 m, = 66) (mean SEM). CD9+/+ (= 120) and CD9?/? (= 112) served as positive and negative settings, respectively. (and and and CD9?/? Eucalyptol eggs cultured in the medium containing CD9 collected from wild-type eggs. (= 51), CD9?/? eggs cultured in the medium containing CD9 (= 112), and CD9?/? eggs cultured in the medium depleted of CD9 by antibody-conjugated beads (= 74). The total numbers of eggs examined are in parentheses. (= 34; 3 h: = 55), CD9+/+ (1 h: = 71; 3 Eucalyptol h: = 79), and CD9?/? eggs (1 h: = 100; 3 h: = 115) were separately incubated with sperm. Total number of coincubated eggs examined: CD9+/? eggs (= 54) coincubated with CD9?/? eggs (= 60), and CD9+/+ eggs (= 65) coincubated with CD9?/? eggs (= 74) at 1 h; CD9+/? eggs (= 51) coincubated with CD9?/? eggs (= 33), and CD9+/+ eggs (= 98) coincubated with CD9?/? eggs (= 90) at 3 h. (Fertilization. To observe the fusion with the sperm, zona-intact and zona-free eggs were incubated with DAPI (10 g/ml) in the medium for 20 min, then washed before the sperm were added. This procedure allowed the staining of only fused sperm nuclei by dye-transfer into sperm after membrane fusion. At 1 h or 3 h after incubation inside a 30-l drop of medium, the eggs were fixed with a mixture of paraformaldehyde and glutaraldehyde for 20 min at 4C. Monitoring the Association of CD9-Comprising Vesicles with Sperm. Eggs collected from Tg+CD9?/? females were set in a 30-l drop of TYM medium. The sperm were added to the eggs at a final concentration of 1 1.5 105/ml after incubation in the medium for 2 h. Articles of latex beads were deposited round the eggs. A glass coverslip was cautiously pressed down onto the articles until the egg were fixed. The medium comprising eggs and sperm was cooled to 10C before Rabbit Polyclonal to ADCK1 observation. Cooling reduced the sperm motility. This procedure allowed us to measure the CD9-EGFP fluorescence within the sperm head using a confocal microscope. Images of the sperm were captured at 1 framework/s. The average value of the fluorescent intensities of CD9-EGFP at 0 s was arranged to 100%, and the final concentration of antibodies was modified to 50 g/ml. The data are measurements of serial images from 15 wild-type sperm in triplicate dishes. Collection of CD9-Comprising Vesicles. The medium comprising the vesicles was collected from denuded wild-type eggs. The eggs were cultured inside a 60-l drop of medium for 2 h after the zona pellucida was removed from the eggs. Eucalyptol Collecting the medium comprising the vesicles required an incubation time of 2 h. The collected medium was utilized for analysis of vesicle parts and evaluation of sperm-fusing ability. CD9-depleted medium was used.