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First published online 17 January 2006
doi: 10.1242/jcs.02730

Journal of Cell Science 119, 416-424 (2006)
Published by The Company of Biologists 2006
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CD9 controls the formation of clusters that contain tetraspanins and the integrin {alpha}6ß1, which are involved in human and mouse gamete fusion

Ahmed Ziyyat1,*, Eric Rubinstein2,*, Frédérique Monier-Gavelle1, Virginie Barraud1, Olivier Kulski1, Michel Prenant2, Claude Boucheix2, Morgane Bomsel3 and Jean-Philippe Wolf1,{ddagger}

1 Université Paris 13, Laboratoire de Biologie de la Reproduction, UPRES 3410, UFR SMBH, Bobigny, France; AP-HP, Hôpital Jean Verdier, Service d'Histologie, Embryologie et Cytogénétique, Bondy, France
2 INSERM, U602, Villejuif, France; Université Paris XI, Villejuif, France; Institut André Lwoff, Villejuif, France
3 INSERM, U567, Institut Cochin, Paris, France


Figure 1
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  		Fig. 1. Distribution of integrins and tetraspanins on human eggs. Intact (A-E) or zona pellucida free (F-O) human eggs were labelled with anti-{alpha}6 integrin (A,F,I,L), anti-ß1 integrin (B,G), anti-CD151 (C,J), anti-CD81 (D,M) and anti-CD9 (E,O), mAbs. A merged image (H) of {alpha}6 (F) and ß1 (G) shows that the subunits co-localize on the plasma membrane. Merged images (K and N) of {alpha}6 (I and L) with CD151 (J) or CD81 (M), respectively, show that the integrin {alpha}6ß1 co-localizes with these two tetraspanins on the oolemma. All these molecules are evenly distributed on ZP-intact eggs. On ZP free eggs, ß1 integrin subunit, CD81 and CD151 tetraspanins co-localize with {alpha}6 integrin subunit. CD9 remained evenly distributed. Bar, 50 µm.

 

Figure 2
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  		Fig. 2. Effects of an anti-{alpha}6, anti-CD151 or anti-CD9 mAb on human sperm-egg fusion. Zona pellucida-free human eggs were incubated with the blocking anti-{alpha}6 mAb GoH3 (A) the anti-CD151 mAb 11B1G4 (B) or the anti-CD9 mAbs SYB-1 and ALB-6 (C) before and during insemination with human sperm. Lines represent the mean ± s.d. from five different experiments. The number of eggs used in each condition is given above. *Significantly different from control (P<0.001).

 

Figure 3
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  		Fig. 3. Effects of anti-CD9 mAb added before zona removal. (A) {alpha}6 integrin subunit distribution of a control zona pellucida-free egg (a) or of eggs pretreated before zona pellucida removal with the mAb SYB-1 directed against CD9 (b,c). At 50 µg/ml, the patches were smaller (b) whereas at 100 µg/ml (c) the {alpha}6 distribution was similar to that of ZP intact oocytes (d). Bar, 50 µm. (B) Effect of mAb against CD9 on gamete fusion. Eggs pretreated with 100 µg/ml of SYB-1 prior to zona pellucida removal were tested in the presence of 50 µg/ml of SYB-1 for 18 hours. *The number of fused spermatozoa was significantly reduced (P<0.001). Lines represent the mean ± s.d. from three different experiments. The number of eggs used in each condition is given above. (C) CD151 (e) and CD81(f) tetraspanins distribution on eggs pretreated before zona pellucida removal with the mAb SYB-1 (100 µg/ml). (D) {alpha}6 integrin subunit distribution on eggs pretreated before zona pellucida removal with the mAb anti-CD81, Z81 (100 µg/ml).

 

Figure 4
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  		Fig. 4. Antibody-induced redistribution of {alpha}6ß1 integrin in wild-type (WT) and CD9–/– oocytes. {alpha}6ß1 integrins were aggregated at the surface of WT and CD9–/– ZP intact eggs using a combination of anti-integrin {alpha}6 and goat anti-mouse antibodies and its distribution was studied by confocal microscopy as described in the Materials and Methods. As a control, wild-type eggs that were fixed before labelling were analysed. Note that the {alpha}6ß1 integrin patches were bigger and more heterogeneous in CD9-null oocytes than in WT oocytes. Bar, 40 µm.

 

Figure 5
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  		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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