Table 1.
RT-PCR primers and conditions.
Figure 1.
PS exposure in in vitro fertilized eggs.
Phase contrast, Hoechst staining and FITC-ANX5 labeling of mouse eggs. ZP-free eggs were incubated with (a–c) or without (d–f) sperm in the presence of FITC-ANX5. After 1 h, eggs were stained with Hoechst 33342 for DNA visualization (b and e), and observed. Note the fluorescent labeling on a fertilized egg (c, right egg) and its absence in a non-fertilized egg present in the same fertilization drop (c, left egg) and in a non-inseminated egg (f). Also note the presence of bound sperm on both fertilized and non-fertilized eggs (a). ZP-intact eggs were inseminated and gametes co-incubated during 3 h (g–l). FITC-ANX5 was added during the last hour of gamete co-incubation. Eggs were then stained with Hoechst 33342 (h and k), and observed. Note the green fluorescent labeling on a fertilized egg (l), absent in a non-fertilized egg (i). In both cases, the first polar body (1 pb) is brightly fluorescent. Arrowheads: metaphase II chromosomes. Arrows: penetrating sperm head. Results are representative of 3 independent experiments in each of which 6–15 eggs/group were evaluated. Bar = 20 µm.
Figure 2.
Quantification of exposed PS on the surface of fertilized eggs.
ZP-free eggs were incubated in the presence or absence of sperm, and at different post-insemination (p.i.) times (1–24 h), they were stained with FITC-ANX5 and Hoechst 33342. Each egg was photographed, and the total fluorescent intensity/area for fertilized eggs relative to that measured in non-inseminated eggs (A) was calculated, as well as calculating the intensity within the labeled areas normalized to the intensity measured in non-labeled areas (B) using the ImageJ software. Each bar represents the mean value ± s.e.m. of at least 3 independent experiments in each of which 6–15 eggs/group were evaluated. The total number of analyzed eggs for each group is presented in brackets. # p<0.05 vs 1, 3 and 12 h; * p<0.001 vs 24 h; ## p<0.05 vs 6 and 24 h; ** p<0.001 vs 1 and 3 h; ### p<0.05 vs 12 hs; *** p<0.001 vs 1, 3 and 6 h.
Figure 3.
Localization of the fluorescent labeling observed at different times after insemination.
Phase contrast, Hoechst staining and FITC-ANX5 labeling of fertilized eggs. ZP-free eggs were inseminated and gametes were co-incubated for 1 (a–c), 3 (d–f), 6 (g–i), 12 (j–l) and 24 h (m–o). FITC-ANX5 was added during the last hour of gamete co-incubation. Eggs were then stained with Hoechst 33342 for DNA visualization, and observed. Arrow: decondensed sperm head. Arrowhead: negative area corresponding to the membrane overlying the meiotic spindle. Asterisk: negative area corresponding to the membrane overlying the sperm entry site. Results are representative of 3 independent experiments in each of which 6–15 eggs/group were evaluated. Bar = 20 µm.
Figure 4.
PS exposure in polyspermic eggs.
Phase contrast, Hoescht staining, and FITC-ANX5 labeling of polyspermic eggs. ZP-free eggs were inseminated with a high sperm concentration, and the gametes were co-incubated for 3 h. FITC-ANX5 (c,f) was added during the last hour and eggs were then stained with Hoechst 33342 (b,e) to determine the number of penetrating sperm (arrows). Note the decrease in the PS exposing surface in an egg penetrated by 4 sperm (d–f) compared to that observed in an egg penetrated by 2 sperm (a–c). Results are representative of 3 independent experiments in each of which 6–15 eggs/group were evaluated. Bar = 20 µm.
Figure 5.
RT-PCR analysis of flippases and scramblases expression in mouse eggs.
Total mouse egg RNA was subjected to RT-PCR using specific primers for each tested enzyme. Products were separated on 2% agarose gels and stained with ethidium bromide (lane C). As positive controls, total RNA from different tissues were used for each tested enzyme (lane A): epididymis for Atp8a1, testis for ATP8a2, liver for Plscr1, kidney for Plscr2, lung for Plscr3 and Plscr4, and cumulus cells for Cd52. As negative controls, water was used as template for the reverse transcription (lane B) and during the amplification reaction (lane D). The expression of Cd52 was evaluated to control contamination with cumulus cells. Results are representative of 3 independent egg RNA isolations.
Figure 6.
Exposure of PS in parthenogenetically activated eggs and Ca2+ requirement.
Wild-type ZP-free eggs were incubated in the absence (a) or presence (b) of 50 µM BAPTA-AM prior to activation with 10 mM SrCl2. (c) Zona-free eggs recovered from CaMKIIγ−/− females were activated with 10 mM SrCl2. (d) Wild-type ZP-free eggs were activated with 100 µM TPEN. In all cases, eggs were then incubated for an additional hour in the presence of FITC-ANX5 (green), fixed, and DNA was stained with TO-PRO3 (red) for confocal imaging. Arrowhead points metaphase II spindles. Note resumption of meiosis in (a) and (d), and exposed PS labeling in (a) and (c). Results are representative of 3 independent experiments in each of which 6–15 eggs/group were evaluated. Bar = 20 µm.
Figure 7.
Exposure of PS in eggs incubated with different activating agents.
(A) ZP-free eggs were parthenogenetically activated with either 10 mM SrCl2 (a), 7% ethanol (b) or 5 µM Ca2+ ionophore A23187 (c). FITC-ANX5 (green) was added during the last hour of incubation. Eggs were then fixed, and DNA was stained with TO-PRO 3 (red) for confocal imaging. Note resumption of meiosis in all cases, and exposed PS labeling only on SrCl2- and ethanol-activated eggs. Results are representative of 5 independent experiments in each of which 6–15 eggs/group were evaluated. (B) ZP-free eggs were incubated with 5 µM Ca2+ ionophore A23187. Those exhibiting a second polar body were inseminated, and the gametes co-incubated for 3 h. FITC-ANX5 was added during the last h of incubation, and eggs were then stained with Hoechst 33342 to evaluate the presence of penetrating sperm. The percentage of eggs presenting exposed PS was determined in each group. Bars represent the mean value ± s.e.m. of 5 independent experiments in each of which 6–15 eggs/group were evaluated. The total number of analyzed eggs in each group is presented in brackets. * p<0.001 vs control (non-inseminated non-activated) eggs; ** p<0.05 vs control and vs inseminated eggs. (C and D) ZP-free eggs were incubated in the absence (a) or presence (b) of SrCl2, or with 5 µM Ca2+ ionophore A23187 (c). (C) Eggs were fixed, permeabilized and stained for cortical granule (CG) content with TRITC-LCA (red). DNA was stained with Hoechst 33342 (blue). Note the decreased CG-labeling in SrCl2- and ionophore- activated eggs compared to control. (D) The cortical granules exudate was stained with TRITC-LCA (red). Eggs were then fixed and stained with Hoechst. Note the increase in CG exudate labeling in SrCl2- and ionophore- activated eggs compared to control. Results are representative of 3 independent experiments in each of which 6–15 eggs/group were evaluated. Bar = 20 µm.
Figure 8.
Effect of cytoskeleton perturbing drugs on activation-induced PS exposure.
ZP-free eggs were incubated in medium alone, or medium containing 10 µM cytD or 0.5 µM Jas prior to activation with 10 mM SrCl2. In each case, eggs were then divided into three groups. One group was fixed, permeabilized, and stained with FITC-phalloidin for analyzing actin distribution (b,d). This staining was not performed for Jas-treated eggs. Another group was fixed, and cortical granule exudates detected by staining with TRITC-LCA (f, h, j). The third group of eggs was incubated with FITC-ANX5 for 1 h, and observed (l, n, p). In all cases, DNA was stained with Hoechst 33342 (a, c, e, g, i, k, m,o). Note resumption of meiosis without cytokinesis in cytD- and Jas-treated eggs, as well as a decrease in CG exocytosis and PS exposure in Jas-treated eggs. Results are representative of 3 independent experiments in each of which 6–15 eggs/group were evaluated. Bar = 20 µm.