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Figure 5


Fig. 5. Dynamics of CD9 and {alpha}6ß1 integrin during fertilization. Intact mouse oocytes were left uninseminated (A,B) or were inseminated (C-M). After incubation, the eggs were fixed, labelled with DAPI, stained with an anti-{alpha}6 integrin mAb (green) and a CD9 mAb (red), and analysed by confocal microscopy as described in the Materials and Methods. Composite images were generated by superimposition of the green and red signals, with areas of overlap appearing yellow. On the left (A,C,E,G,I) are shown superimpositions of a transmission image with the DAPI staining (blue) to identify the stage of the oocytes. (A,B) Non-inseminated metaphase II oocyte. The integrin {alpha}6ß1 and CD9 have a finely punctuate distribution and strongly overlap. (C,D) An inseminated metaphase II oocyte with a sperm attached to the membrane (arrow). The distribution of both molecules is very similar to non-inseminated oocytes. Note the strong labelling where the sperm is attached, seen at a higher magnification in K where the DAPI staining is also shown. (E,F) After fusion has occurred, the integrin {alpha}6ß1 and CD9 are excluded from the region (see the area delimited by short lines) surrounding the sperm entry point (arrow) and start to gather into small clusters. Note in E the sperm DNA starting to decondense (arrow) and the anaphase of the oocyte (arrowheads). The region surrounding the oocyte DNA (*) is poor in microvilli (the amicrovillar region) and poorly expresses CD9 and the integrin {alpha}6ß1. (G,H) An egg at the pronuclei stage. The pronuclei are indicated by arrowheads in G. The disappearance of both molecules from the region surrounding the sperm entry point (delimited by short lines) has continued and the molecules concentrate into heterogeneous patches. Note the concentration of these molecules in the meiotic cleavage furrow. The polar body is indicated (PB). (I,J) In some case the sperm heads fused before the sperm tail had entirely crossed the ZP. A high local concentration of integrin {alpha}6ß1 was observed at the sperm entry site (arrow). L and M show a higher magnification of the region of the sperm entry site. Arrowheads in I indicate pronuclei. Note again the high concentration of these molecules in the meiotic cleavage furrow. Bar 40 µm.





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