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Characterization of CD46 and β 1 integrin dynamics during sperm acrosome reaction

View Article: PubMed Central - PubMed

ABSTRACT

The acrosome reaction (AR) is a process of membrane fusion and lytic enzyme release, which enables sperm to penetrate the egg surroundings. It is widely recognized that specific sperm proteins form an active network prior to fertilization, and their dynamic relocation is crucial for the sperm-egg fusion. The unique presence of the membrane cofactor protein CD46 in the sperm acrosomal membrane was shown, however, its behaviour and connection with other sperm proteins has not been explored further. Using super resolution microscopy, we demonstrated a dynamic CD46 reorganisation over the sperm head during the AR, and its interaction with transmembrane protein integrins, which was confirmed by proximity ligation assay. Furthermore, we propose their joint involvement in actin network rearrangement. Moreover, CD46 and β1 integrins with subunit α3, but not α6, are localized into the apical acrosome and are expected to be involved in signal transduction pathways directing the acrosome stability and essential protein network rearrangements prior to gamete fusion.

No MeSH data available.


SIM super-resolution microscopy and visualization of mutual position of CD46 and β1 integrin.(A) SIM data show the localization of CD46 (green) on the inner and outer acrosomal membrane and β1 integrin (red) on the plasma and outer acrosomal and plasma membrane of the acrosomal area. Scale bar represents 1 μm. (B) SIM super-resolution image analysed by Huygens software, showing the colocalization area (yellow) of selected proteins in the outer acrosomal membrane. The colocalization map is based on Pearson’s correlation coefficient. Scale bar represents 1 μm. DAPI (blue).
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f8: SIM super-resolution microscopy and visualization of mutual position of CD46 and β1 integrin.(A) SIM data show the localization of CD46 (green) on the inner and outer acrosomal membrane and β1 integrin (red) on the plasma and outer acrosomal and plasma membrane of the acrosomal area. Scale bar represents 1 μm. (B) SIM super-resolution image analysed by Huygens software, showing the colocalization area (yellow) of selected proteins in the outer acrosomal membrane. The colocalization map is based on Pearson’s correlation coefficient. Scale bar represents 1 μm. DAPI (blue).

Mentions: Data collected by 3D super–resolution SIM was used for colocalization analysis of the mutual position of the CD46 and β1 integrin subunit. 3D-SIM was chosen to collect data for colocalization analysis due to its higher resolution in z-axis (in our case approximately 350 nm). The mutual position of the CD46 and β1 integrin subunit was visualised with dual immunofluorescence staining in freshly released epididymal sperm with an intact acrosome (Fig. 8A) and microscopy images were captured with a 3D SIM equipped microscope. Pearson’s correlation coefficient was used for the evaluation of the colocalization of the studied proteins44. Fluorescent images were analysed with Huygens software and the resulting values were statistically evaluated. The value of the average Pearson’s correlation coefficient of CD46 and β1 integrin was 0.784 ± 0.037. The result shows a high rate of colocalization of the studied proteins and it confirms the results obtained with the Duolink Proximity ligation assays (Fig. 4). The colocalization map by Huygens software, representing a visualization of Pearson coefficient, is shown in Fig. 8B and Fig. S9.


Characterization of CD46 and β 1 integrin dynamics during sperm acrosome reaction
SIM super-resolution microscopy and visualization of mutual position of CD46 and β1 integrin.(A) SIM data show the localization of CD46 (green) on the inner and outer acrosomal membrane and β1 integrin (red) on the plasma and outer acrosomal and plasma membrane of the acrosomal area. Scale bar represents 1 μm. (B) SIM super-resolution image analysed by Huygens software, showing the colocalization area (yellow) of selected proteins in the outer acrosomal membrane. The colocalization map is based on Pearson’s correlation coefficient. Scale bar represents 1 μm. DAPI (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036054&req=5

f8: SIM super-resolution microscopy and visualization of mutual position of CD46 and β1 integrin.(A) SIM data show the localization of CD46 (green) on the inner and outer acrosomal membrane and β1 integrin (red) on the plasma and outer acrosomal and plasma membrane of the acrosomal area. Scale bar represents 1 μm. (B) SIM super-resolution image analysed by Huygens software, showing the colocalization area (yellow) of selected proteins in the outer acrosomal membrane. The colocalization map is based on Pearson’s correlation coefficient. Scale bar represents 1 μm. DAPI (blue).
Mentions: Data collected by 3D super–resolution SIM was used for colocalization analysis of the mutual position of the CD46 and β1 integrin subunit. 3D-SIM was chosen to collect data for colocalization analysis due to its higher resolution in z-axis (in our case approximately 350 nm). The mutual position of the CD46 and β1 integrin subunit was visualised with dual immunofluorescence staining in freshly released epididymal sperm with an intact acrosome (Fig. 8A) and microscopy images were captured with a 3D SIM equipped microscope. Pearson’s correlation coefficient was used for the evaluation of the colocalization of the studied proteins44. Fluorescent images were analysed with Huygens software and the resulting values were statistically evaluated. The value of the average Pearson’s correlation coefficient of CD46 and β1 integrin was 0.784 ± 0.037. The result shows a high rate of colocalization of the studied proteins and it confirms the results obtained with the Duolink Proximity ligation assays (Fig. 4). The colocalization map by Huygens software, representing a visualization of Pearson coefficient, is shown in Fig. 8B and Fig. S9.

View Article: PubMed Central - PubMed

ABSTRACT

The acrosome reaction (AR) is a process of membrane fusion and lytic enzyme release, which enables sperm to penetrate the egg surroundings. It is widely recognized that specific sperm proteins form an active network prior to fertilization, and their dynamic relocation is crucial for the sperm-egg fusion. The unique presence of the membrane cofactor protein CD46 in the sperm acrosomal membrane was shown, however, its behaviour and connection with other sperm proteins has not been explored further. Using super resolution microscopy, we demonstrated a dynamic CD46 reorganisation over the sperm head during the AR, and its interaction with transmembrane protein integrins, which was confirmed by proximity ligation assay. Furthermore, we propose their joint involvement in actin network rearrangement. Moreover, CD46 and β1 integrins with subunit α3, but not α6, are localized into the apical acrosome and are expected to be involved in signal transduction pathways directing the acrosome stability and essential protein network rearrangements prior to gamete fusion.

No MeSH data available.