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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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Distinct profiles of monoclonal antibodies recognizing IZUMO1 in cell–oocyte assay.(a) Immunostaining and cell–oocyte assay of a COS-7 cell expressing Izumo1 and DEL5-113. COS-7 cells that were transfected with mouse IZUMO1 and the truncated DEL5-113 cDNA were stained with Mab17-Alexa546 (red), Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta) simultaneously after dissociation by 10 mM EDTA containing PBS, as described in Methods. The final concentration of all antibodies added was at 0.5 μg ml−1. Scale bars, 20 μm. Right photographs show the transmission images of the cell–oocyte assay. Scale bars, 100 μm. (b) Cell–oocyte assay with IZUMO1-specific antibodies. Izumo1-expressing COS-7 cells and oocytes were incubated for 2 h at 37 °C with the monoclonal antibodies in a (at the same concentrations). Inset shows differential interference contrast (DIC) image of the centre of the Z-stacks. Some attached COS-7 cells are out of focus. The adherent surface of COS-7 cells to the oocyte is indicated by asterisks. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm. The outline of the oocyte is marked by dotted lines. (c) The relative fluorescence intensities at the interface. The × 2 magnified image of the white dashed box in b is shown. The relative fluorescence intensity in the white arrow line of the dashed box in c was analysed with ImageJ. The fluorescence intensities of Mab17-Alexa546, Mab18-Alexa488 and Mab125-Alexa647 are indicated in red, green and purple lines, respectively. The arrow indicates a measurement direction.
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f1: Distinct profiles of monoclonal antibodies recognizing IZUMO1 in cell–oocyte assay.(a) Immunostaining and cell–oocyte assay of a COS-7 cell expressing Izumo1 and DEL5-113. COS-7 cells that were transfected with mouse IZUMO1 and the truncated DEL5-113 cDNA were stained with Mab17-Alexa546 (red), Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta) simultaneously after dissociation by 10 mM EDTA containing PBS, as described in Methods. The final concentration of all antibodies added was at 0.5 μg ml−1. Scale bars, 20 μm. Right photographs show the transmission images of the cell–oocyte assay. Scale bars, 100 μm. (b) Cell–oocyte assay with IZUMO1-specific antibodies. Izumo1-expressing COS-7 cells and oocytes were incubated for 2 h at 37 °C with the monoclonal antibodies in a (at the same concentrations). Inset shows differential interference contrast (DIC) image of the centre of the Z-stacks. Some attached COS-7 cells are out of focus. The adherent surface of COS-7 cells to the oocyte is indicated by asterisks. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm. The outline of the oocyte is marked by dotted lines. (c) The relative fluorescence intensities at the interface. The × 2 magnified image of the white dashed box in b is shown. The relative fluorescence intensity in the white arrow line of the dashed box in c was analysed with ImageJ. The fluorescence intensities of Mab17-Alexa546, Mab18-Alexa488 and Mab125-Alexa647 are indicated in red, green and purple lines, respectively. The arrow indicates a measurement direction.

Mentions: When IZUMO1 was transiently expressed in COS-7 cells, all antibodies reacted only with Izumo1-expressing cells (Fig. 1a). In the truncated mutant lacking Asp5-Leu113 (DEL5-113), neither of the new antibodies showed any reactivity as expected, whereas it was well recognized by the non-inhibitory antibody (Mab125)12 (Fig. 1a), along with the assumed epitope Pro234-Arg298 (Supplementary Fig. 1e). Expression of the truncated mutant in COS-7 cells had no effect on egg binding (Fig. 1a). Consistent with the above in vitro fertilization results, Mab17, but not Mab18, completely inhibited the COS-7 cell–egg association at 10 μg ml−1 as well as in vitro fertilization (Supplementary Fig. 1b). We thought that these results might stress the importance of the helical region in the function of IZUMO57–113.


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)

Distinct profiles of monoclonal antibodies recognizing IZUMO1 in cell–oocyte assay.(a) Immunostaining and cell–oocyte assay of a COS-7 cell expressing Izumo1 and DEL5-113. COS-7 cells that were transfected with mouse IZUMO1 and the truncated DEL5-113 cDNA were stained with Mab17-Alexa546 (red), Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta) simultaneously after dissociation by 10 mM EDTA containing PBS, as described in Methods. The final concentration of all antibodies added was at 0.5 μg ml−1. Scale bars, 20 μm. Right photographs show the transmission images of the cell–oocyte assay. Scale bars, 100 μm. (b) Cell–oocyte assay with IZUMO1-specific antibodies. Izumo1-expressing COS-7 cells and oocytes were incubated for 2 h at 37 °C with the monoclonal antibodies in a (at the same concentrations). Inset shows differential interference contrast (DIC) image of the centre of the Z-stacks. Some attached COS-7 cells are out of focus. The adherent surface of COS-7 cells to the oocyte is indicated by asterisks. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm. The outline of the oocyte is marked by dotted lines. (c) The relative fluorescence intensities at the interface. The × 2 magnified image of the white dashed box in b is shown. The relative fluorescence intensity in the white arrow line of the dashed box in c was analysed with ImageJ. The fluorescence intensities of Mab17-Alexa546, Mab18-Alexa488 and Mab125-Alexa647 are indicated in red, green and purple lines, respectively. The arrow indicates a measurement direction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660202&req=5

f1: Distinct profiles of monoclonal antibodies recognizing IZUMO1 in cell–oocyte assay.(a) Immunostaining and cell–oocyte assay of a COS-7 cell expressing Izumo1 and DEL5-113. COS-7 cells that were transfected with mouse IZUMO1 and the truncated DEL5-113 cDNA were stained with Mab17-Alexa546 (red), Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta) simultaneously after dissociation by 10 mM EDTA containing PBS, as described in Methods. The final concentration of all antibodies added was at 0.5 μg ml−1. Scale bars, 20 μm. Right photographs show the transmission images of the cell–oocyte assay. Scale bars, 100 μm. (b) Cell–oocyte assay with IZUMO1-specific antibodies. Izumo1-expressing COS-7 cells and oocytes were incubated for 2 h at 37 °C with the monoclonal antibodies in a (at the same concentrations). Inset shows differential interference contrast (DIC) image of the centre of the Z-stacks. Some attached COS-7 cells are out of focus. The adherent surface of COS-7 cells to the oocyte is indicated by asterisks. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm. The outline of the oocyte is marked by dotted lines. (c) The relative fluorescence intensities at the interface. The × 2 magnified image of the white dashed box in b is shown. The relative fluorescence intensity in the white arrow line of the dashed box in c was analysed with ImageJ. The fluorescence intensities of Mab17-Alexa546, Mab18-Alexa488 and Mab125-Alexa647 are indicated in red, green and purple lines, respectively. The arrow indicates a measurement direction.
Mentions: When IZUMO1 was transiently expressed in COS-7 cells, all antibodies reacted only with Izumo1-expressing cells (Fig. 1a). In the truncated mutant lacking Asp5-Leu113 (DEL5-113), neither of the new antibodies showed any reactivity as expected, whereas it was well recognized by the non-inhibitory antibody (Mab125)12 (Fig. 1a), along with the assumed epitope Pro234-Arg298 (Supplementary Fig. 1e). Expression of the truncated mutant in COS-7 cells had no effect on egg binding (Fig. 1a). Consistent with the above in vitro fertilization results, Mab17, but not Mab18, completely inhibited the COS-7 cell–egg association at 10 μg ml−1 as well as in vitro fertilization (Supplementary Fig. 1b). We thought that these results might stress the importance of the helical region in the function of IZUMO57–113.

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