Limits...
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.

Show MeSH

Related in: MedlinePlus

Recombinant JUNO binds to spermatozoon where IZUMO1 is monomeric.(a) Binding of recombinant JUNO to IZUMO1. The JUNO-FC fusion protein labelled with α-mouse IgG antibodies-Alexa546 selectively bound to IZUMO1-expressing cells (red). Simultaneously, they were incubated with Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta). (b) Cell–oocyte assay with recombinant JUNO. The same experiment was carried out with oocytes. The interface was devoid of JUNO-FC signal (asterisks). The right graph shows the percentage of stained cells in the cell–oocyte interface. Ten oocytes and fifty-seven attached cells were investigated in this analysis. The image of the oocyte is marked by dotted lines. Inset shows middle DIC image among Z-stacks. (c) Immunostaining of wild-type and IZUMO1- spermatozoa with JUNO-FC. Wild-type fresh spermatozoa were incubated for 2 h in TYH medium with JUNO-FC (red), Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta). Acrosome-reacted spermatozoa were detected with IZUMO1 antibodies (Mab18 and Mab125; shown by arrowheads). To detect the acrosome reaction in IZUMO1- spermatozoa, Acro-GFP spermatozoa, which have green fluorescence that should disappear from the acrosome, were used to distinguish the acrosome reaction after 2 h of incubation in TYH medium with JUNO-FC and Mab125-Alexa647. Acrosome-reacted spermatozoa are shown by arrowheads. (d) IZUMO1-mCherry in spermatozoa showed monomeric brightness. The sperm lysate of IZUMO1-mCherry or COS-7 cell lysates expressing Myocilin-mChery, Cd2-mCherry or mCherry was prepared, and specific brightness of each protein was determined as described in the Methods. The true specific brightness was obtained from the bin time dependence (PCMH, left panel). The ranges of the upper and lower confidence interval (0.95) were also calculated from the five measurements and expressed as error bars of the mean. Brightness of IZUMO1-mCherry was statistically not different from that of mCherry or CD2-mCherry. Error bar represents CI95. (e) Immunostaining of acrosome-intact spermatozoa with JUNO-FC. Fresh spermatozoa collected from the epididymis of wild-type mice were mounted on glass slides, dried up and then immunostained with JUNO-FC (red), Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta). Final concentration of all antibodies used was at 0.5 μg ml−1. JUNO-FC was added at 1 μg ml−1. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4660202&req=5

f3: Recombinant JUNO binds to spermatozoon where IZUMO1 is monomeric.(a) Binding of recombinant JUNO to IZUMO1. The JUNO-FC fusion protein labelled with α-mouse IgG antibodies-Alexa546 selectively bound to IZUMO1-expressing cells (red). Simultaneously, they were incubated with Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta). (b) Cell–oocyte assay with recombinant JUNO. The same experiment was carried out with oocytes. The interface was devoid of JUNO-FC signal (asterisks). The right graph shows the percentage of stained cells in the cell–oocyte interface. Ten oocytes and fifty-seven attached cells were investigated in this analysis. The image of the oocyte is marked by dotted lines. Inset shows middle DIC image among Z-stacks. (c) Immunostaining of wild-type and IZUMO1- spermatozoa with JUNO-FC. Wild-type fresh spermatozoa were incubated for 2 h in TYH medium with JUNO-FC (red), Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta). Acrosome-reacted spermatozoa were detected with IZUMO1 antibodies (Mab18 and Mab125; shown by arrowheads). To detect the acrosome reaction in IZUMO1- spermatozoa, Acro-GFP spermatozoa, which have green fluorescence that should disappear from the acrosome, were used to distinguish the acrosome reaction after 2 h of incubation in TYH medium with JUNO-FC and Mab125-Alexa647. Acrosome-reacted spermatozoa are shown by arrowheads. (d) IZUMO1-mCherry in spermatozoa showed monomeric brightness. The sperm lysate of IZUMO1-mCherry or COS-7 cell lysates expressing Myocilin-mChery, Cd2-mCherry or mCherry was prepared, and specific brightness of each protein was determined as described in the Methods. The true specific brightness was obtained from the bin time dependence (PCMH, left panel). The ranges of the upper and lower confidence interval (0.95) were also calculated from the five measurements and expressed as error bars of the mean. Brightness of IZUMO1-mCherry was statistically not different from that of mCherry or CD2-mCherry. Error bar represents CI95. (e) Immunostaining of acrosome-intact spermatozoa with JUNO-FC. Fresh spermatozoa collected from the epididymis of wild-type mice were mounted on glass slides, dried up and then immunostained with JUNO-FC (red), Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta). Final concentration of all antibodies used was at 0.5 μg ml−1. JUNO-FC was added at 1 μg ml−1. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm.

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)

Recombinant JUNO binds to spermatozoon where IZUMO1 is monomeric.(a) Binding of recombinant JUNO to IZUMO1. The JUNO-FC fusion protein labelled with α-mouse IgG antibodies-Alexa546 selectively bound to IZUMO1-expressing cells (red). Simultaneously, they were incubated with Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta). (b) Cell–oocyte assay with recombinant JUNO. The same experiment was carried out with oocytes. The interface was devoid of JUNO-FC signal (asterisks). The right graph shows the percentage of stained cells in the cell–oocyte interface. Ten oocytes and fifty-seven attached cells were investigated in this analysis. The image of the oocyte is marked by dotted lines. Inset shows middle DIC image among Z-stacks. (c) Immunostaining of wild-type and IZUMO1- spermatozoa with JUNO-FC. Wild-type fresh spermatozoa were incubated for 2 h in TYH medium with JUNO-FC (red), Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta). Acrosome-reacted spermatozoa were detected with IZUMO1 antibodies (Mab18 and Mab125; shown by arrowheads). To detect the acrosome reaction in IZUMO1- spermatozoa, Acro-GFP spermatozoa, which have green fluorescence that should disappear from the acrosome, were used to distinguish the acrosome reaction after 2 h of incubation in TYH medium with JUNO-FC and Mab125-Alexa647. Acrosome-reacted spermatozoa are shown by arrowheads. (d) IZUMO1-mCherry in spermatozoa showed monomeric brightness. The sperm lysate of IZUMO1-mCherry or COS-7 cell lysates expressing Myocilin-mChery, Cd2-mCherry or mCherry was prepared, and specific brightness of each protein was determined as described in the Methods. The true specific brightness was obtained from the bin time dependence (PCMH, left panel). The ranges of the upper and lower confidence interval (0.95) were also calculated from the five measurements and expressed as error bars of the mean. Brightness of IZUMO1-mCherry was statistically not different from that of mCherry or CD2-mCherry. Error bar represents CI95. (e) Immunostaining of acrosome-intact spermatozoa with JUNO-FC. Fresh spermatozoa collected from the epididymis of wild-type mice were mounted on glass slides, dried up and then immunostained with JUNO-FC (red), Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta). Final concentration of all antibodies used was at 0.5 μg ml−1. JUNO-FC was added at 1 μg ml−1. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Recombinant JUNO binds to spermatozoon where IZUMO1 is monomeric.(a) Binding of recombinant JUNO to IZUMO1. The JUNO-FC fusion protein labelled with α-mouse IgG antibodies-Alexa546 selectively bound to IZUMO1-expressing cells (red). Simultaneously, they were incubated with Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta). (b) Cell–oocyte assay with recombinant JUNO. The same experiment was carried out with oocytes. The interface was devoid of JUNO-FC signal (asterisks). The right graph shows the percentage of stained cells in the cell–oocyte interface. Ten oocytes and fifty-seven attached cells were investigated in this analysis. The image of the oocyte is marked by dotted lines. Inset shows middle DIC image among Z-stacks. (c) Immunostaining of wild-type and IZUMO1- spermatozoa with JUNO-FC. Wild-type fresh spermatozoa were incubated for 2 h in TYH medium with JUNO-FC (red), Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta). Acrosome-reacted spermatozoa were detected with IZUMO1 antibodies (Mab18 and Mab125; shown by arrowheads). To detect the acrosome reaction in IZUMO1- spermatozoa, Acro-GFP spermatozoa, which have green fluorescence that should disappear from the acrosome, were used to distinguish the acrosome reaction after 2 h of incubation in TYH medium with JUNO-FC and Mab125-Alexa647. Acrosome-reacted spermatozoa are shown by arrowheads. (d) IZUMO1-mCherry in spermatozoa showed monomeric brightness. The sperm lysate of IZUMO1-mCherry or COS-7 cell lysates expressing Myocilin-mChery, Cd2-mCherry or mCherry was prepared, and specific brightness of each protein was determined as described in the Methods. The true specific brightness was obtained from the bin time dependence (PCMH, left panel). The ranges of the upper and lower confidence interval (0.95) were also calculated from the five measurements and expressed as error bars of the mean. Brightness of IZUMO1-mCherry was statistically not different from that of mCherry or CD2-mCherry. Error bar represents CI95. (e) Immunostaining of acrosome-intact spermatozoa with JUNO-FC. Fresh spermatozoa collected from the epididymis of wild-type mice were mounted on glass slides, dried up and then immunostained with JUNO-FC (red), Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta). Final concentration of all antibodies used was at 0.5 μg ml−1. JUNO-FC was added at 1 μg ml−1. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm.
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