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Oocyte-triggered dimerization of sperm IZUMO1 promotes sperm-egg fusion in mice.

Inoue N, Hagihara Y, Wright D, Suzuki T, Wada I - Nat Commun (2015)

Bottom Line: Sperm-egg fusion is indispensable for completing mammalian fertilization.Interestingly, JUNO associates with monomeric IZUMO1, which is then quickly removed as tight adhesion of the two cells is subsequently established.We therefore propose that global structural rearrangement of IZUMO1 occurs on JUNO recognition and that this rearrangement may then initiate force generation to overcome repulsion between the juxtaposing membranes, through an unidentified receptor on the egg.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Science, Institutes for Biomedical Sciences, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima City, Fukushima 960-1295, Japan.

ABSTRACT
Sperm-egg fusion is indispensable for completing mammalian fertilization. Although the underlying molecular mechanisms are poorly understood, requirement of two spermatozoon factors, IZUMO1 and SPACA6, and two oocyte factors, CD9 and the IZUMO1 counter-receptor JUNO, has been proven by gene disruption, and the binding of cells to an oocyte can be reconstituted by ectopic expression of IZUMO1. Here we demonstrate that robust IZUMO1-dependent adhesion of sperm with an oocyte accompanies the dimerization of IZUMO1. Despite the intrinsic dimeric property of its N-terminal region, IZUMO1 is monomeric in spermatozoa. Interestingly, JUNO associates with monomeric IZUMO1, which is then quickly removed as tight adhesion of the two cells is subsequently established. We therefore propose that global structural rearrangement of IZUMO1 occurs on JUNO recognition and that this rearrangement may then initiate force generation to overcome repulsion between the juxtaposing membranes, through an unidentified receptor on the egg.

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JUNO disappears from the contact site of not only cell–cell but also sperm–egg adhesion.(a,b) The reconstitution approach in COS-7 cells. COS-7 cells transfected with a mammalian expression vector inserted with Juno and Izumo1 cDNA were mixed and incubated for 24 h before observation using confocal microscopy. Lower magnification images are shown in the left panels. The region of the contact site is shown by a dashed box. (a) COS-7 cells were stained with α-JUNO-Alexa647 (red), Mab18-Alexa488 (green) and Mab125-Alexa546 (magenta). (b) BiFC analysis (green) was performed with α-JUNO-Alexa647 (red) and Mab125-Alexa546 (magenta). Magnification of the orange or blue dashed box is in the right panels. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification. (c,d) BiFC in Izumo1-VNC155-expressing cells. COS-7 cells were transiently co-transfected with Izumo1-VN155 and VC155. After 48-h incubation, they were stained with Mab125-Alexa647 (magenta) and JUNO-FC (c, red) or Mab18-Alexa546 (d, red). After Z-sectioning and 3D reconstitution, magnified images (right panel) were taken from the direction of the arrow. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification. (e,f) The characteristics of each antibody before and after sperm–egg fusion. Zona-free oocytes and spermatozoa were co-incubated with Mab18-Alexa488 (green), Mab125-Alexa546 (magenta) and JUNO (red) antibodies (e,f). Sperm–egg fusion was visualized with Hoechst transfer (blue). Images of before (e) and after (f) fusion were taken after fixation. Highly magnified photographs correspond to the dashed box in the left pictures. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification.
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f5: JUNO disappears from the contact site of not only cell–cell but also sperm–egg adhesion.(a,b) The reconstitution approach in COS-7 cells. COS-7 cells transfected with a mammalian expression vector inserted with Juno and Izumo1 cDNA were mixed and incubated for 24 h before observation using confocal microscopy. Lower magnification images are shown in the left panels. The region of the contact site is shown by a dashed box. (a) COS-7 cells were stained with α-JUNO-Alexa647 (red), Mab18-Alexa488 (green) and Mab125-Alexa546 (magenta). (b) BiFC analysis (green) was performed with α-JUNO-Alexa647 (red) and Mab125-Alexa546 (magenta). Magnification of the orange or blue dashed box is in the right panels. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification. (c,d) BiFC in Izumo1-VNC155-expressing cells. COS-7 cells were transiently co-transfected with Izumo1-VN155 and VC155. After 48-h incubation, they were stained with Mab125-Alexa647 (magenta) and JUNO-FC (c, red) or Mab18-Alexa546 (d, red). After Z-sectioning and 3D reconstitution, magnified images (right panel) were taken from the direction of the arrow. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification. (e,f) The characteristics of each antibody before and after sperm–egg fusion. Zona-free oocytes and spermatozoa were co-incubated with Mab18-Alexa488 (green), Mab125-Alexa546 (magenta) and JUNO (red) antibodies (e,f). Sperm–egg fusion was visualized with Hoechst transfer (blue). Images of before (e) and after (f) fusion were taken after fixation. Highly magnified photographs correspond to the dashed box in the left pictures. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification.

Mentions: The previous report showed that, after 24 h mixing of Izumo1-expressing 293T cells with Juno-expressing cells, both IZUMO1 and JUNO seemed to co-exist at the contact site5. To confirm this, we did a comparable experiment in COS-7 cells and carefully examined the reconstituted image from the Z-stacks. Intriguingly, we could reproduce the exclusion of JUNO at the interface in cell-to-cell adhesion, consistent with the observation described above (Fig. 5a,b). Moreover, Mab125-Alexa546, but not Mab18-Alexa488, intruded into the contact site (Fig. 5a), and the IZUMO1 was BiFC-positive at the adhesive region (Fig. 5b). However, we did not find any fused cells even after 48 h of co-incubation. As shown in Fig. 2b, BiFC also occurred at the contact site of the cells expressing Izumo1-VNC155. During the course of this experiment, we noticed that this dimerization was incomplete or at least distinct from the JUNO-triggered reaction because neither JUNO-FC nor Mab18-Alexa546 were excluded from the contact site of JUNO-negative and IZUMO1-VNC155-positive cells (Fig. 5c,d), indicating that this dimerization essentially differs from the JUNO-triggered dimerization as seen at the contact site (Fig. 5a,b).


Oocyte-triggered dimerization of sperm IZUMO1 promotes sperm-egg fusion in mice.

Inoue N, Hagihara Y, Wright D, Suzuki T, Wada I - Nat Commun (2015)

JUNO disappears from the contact site of not only cell–cell but also sperm–egg adhesion.(a,b) The reconstitution approach in COS-7 cells. COS-7 cells transfected with a mammalian expression vector inserted with Juno and Izumo1 cDNA were mixed and incubated for 24 h before observation using confocal microscopy. Lower magnification images are shown in the left panels. The region of the contact site is shown by a dashed box. (a) COS-7 cells were stained with α-JUNO-Alexa647 (red), Mab18-Alexa488 (green) and Mab125-Alexa546 (magenta). (b) BiFC analysis (green) was performed with α-JUNO-Alexa647 (red) and Mab125-Alexa546 (magenta). Magnification of the orange or blue dashed box is in the right panels. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification. (c,d) BiFC in Izumo1-VNC155-expressing cells. COS-7 cells were transiently co-transfected with Izumo1-VN155 and VC155. After 48-h incubation, they were stained with Mab125-Alexa647 (magenta) and JUNO-FC (c, red) or Mab18-Alexa546 (d, red). After Z-sectioning and 3D reconstitution, magnified images (right panel) were taken from the direction of the arrow. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification. (e,f) The characteristics of each antibody before and after sperm–egg fusion. Zona-free oocytes and spermatozoa were co-incubated with Mab18-Alexa488 (green), Mab125-Alexa546 (magenta) and JUNO (red) antibodies (e,f). Sperm–egg fusion was visualized with Hoechst transfer (blue). Images of before (e) and after (f) fusion were taken after fixation. Highly magnified photographs correspond to the dashed box in the left pictures. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification.
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Related In: Results  -  Collection

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f5: JUNO disappears from the contact site of not only cell–cell but also sperm–egg adhesion.(a,b) The reconstitution approach in COS-7 cells. COS-7 cells transfected with a mammalian expression vector inserted with Juno and Izumo1 cDNA were mixed and incubated for 24 h before observation using confocal microscopy. Lower magnification images are shown in the left panels. The region of the contact site is shown by a dashed box. (a) COS-7 cells were stained with α-JUNO-Alexa647 (red), Mab18-Alexa488 (green) and Mab125-Alexa546 (magenta). (b) BiFC analysis (green) was performed with α-JUNO-Alexa647 (red) and Mab125-Alexa546 (magenta). Magnification of the orange or blue dashed box is in the right panels. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification. (c,d) BiFC in Izumo1-VNC155-expressing cells. COS-7 cells were transiently co-transfected with Izumo1-VN155 and VC155. After 48-h incubation, they were stained with Mab125-Alexa647 (magenta) and JUNO-FC (c, red) or Mab18-Alexa546 (d, red). After Z-sectioning and 3D reconstitution, magnified images (right panel) were taken from the direction of the arrow. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification. (e,f) The characteristics of each antibody before and after sperm–egg fusion. Zona-free oocytes and spermatozoa were co-incubated with Mab18-Alexa488 (green), Mab125-Alexa546 (magenta) and JUNO (red) antibodies (e,f). Sperm–egg fusion was visualized with Hoechst transfer (blue). Images of before (e) and after (f) fusion were taken after fixation. Highly magnified photographs correspond to the dashed box in the left pictures. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification.
Mentions: The previous report showed that, after 24 h mixing of Izumo1-expressing 293T cells with Juno-expressing cells, both IZUMO1 and JUNO seemed to co-exist at the contact site5. To confirm this, we did a comparable experiment in COS-7 cells and carefully examined the reconstituted image from the Z-stacks. Intriguingly, we could reproduce the exclusion of JUNO at the interface in cell-to-cell adhesion, consistent with the observation described above (Fig. 5a,b). Moreover, Mab125-Alexa546, but not Mab18-Alexa488, intruded into the contact site (Fig. 5a), and the IZUMO1 was BiFC-positive at the adhesive region (Fig. 5b). However, we did not find any fused cells even after 48 h of co-incubation. As shown in Fig. 2b, BiFC also occurred at the contact site of the cells expressing Izumo1-VNC155. During the course of this experiment, we noticed that this dimerization was incomplete or at least distinct from the JUNO-triggered reaction because neither JUNO-FC nor Mab18-Alexa546 were excluded from the contact site of JUNO-negative and IZUMO1-VNC155-positive cells (Fig. 5c,d), indicating that this dimerization essentially differs from the JUNO-triggered dimerization as seen at the contact site (Fig. 5a,b).

Bottom Line: Sperm-egg fusion is indispensable for completing mammalian fertilization.Interestingly, JUNO associates with monomeric IZUMO1, which is then quickly removed as tight adhesion of the two cells is subsequently established.We therefore propose that global structural rearrangement of IZUMO1 occurs on JUNO recognition and that this rearrangement may then initiate force generation to overcome repulsion between the juxtaposing membranes, through an unidentified receptor on the egg.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Science, Institutes for Biomedical Sciences, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima City, Fukushima 960-1295, Japan.

ABSTRACT
Sperm-egg fusion is indispensable for completing mammalian fertilization. Although the underlying molecular mechanisms are poorly understood, requirement of two spermatozoon factors, IZUMO1 and SPACA6, and two oocyte factors, CD9 and the IZUMO1 counter-receptor JUNO, has been proven by gene disruption, and the binding of cells to an oocyte can be reconstituted by ectopic expression of IZUMO1. Here we demonstrate that robust IZUMO1-dependent adhesion of sperm with an oocyte accompanies the dimerization of IZUMO1. Despite the intrinsic dimeric property of its N-terminal region, IZUMO1 is monomeric in spermatozoa. Interestingly, JUNO associates with monomeric IZUMO1, which is then quickly removed as tight adhesion of the two cells is subsequently established. We therefore propose that global structural rearrangement of IZUMO1 occurs on JUNO recognition and that this rearrangement may then initiate force generation to overcome repulsion between the juxtaposing membranes, through an unidentified receptor on the egg.

Show MeSH
Related in: MedlinePlus