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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.


The study of protein-protein interactions.Interactions of CD46 and β1 integrin in C57BL/6 spermatozoa determined by Duolink proximity ligation assay. (A) freshly released sperm, (B) sperm during the induced AR. Positive control (α tubulin/ β tubulin), negative control (β1 integrin/ α tubulin). The small pictures in the corners represent the immunofluorescent dual staining of CD46 and β1 integrin, CD46 (green), β1 integrin (red). Scale bar represents 4 μm.
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f4: The study of protein-protein interactions.Interactions of CD46 and β1 integrin in C57BL/6 spermatozoa determined by Duolink proximity ligation assay. (A) freshly released sperm, (B) sperm during the induced AR. Positive control (α tubulin/ β tubulin), negative control (β1 integrin/ α tubulin). The small pictures in the corners represent the immunofluorescent dual staining of CD46 and β1 integrin, CD46 (green), β1 integrin (red). Scale bar represents 4 μm.

Mentions: Based on a similar distribution of both the proteins, their parallel time relocation during the AR and published data from the somatic cells17, we decided to perform a specific proximity ligation assay that extends the limits of traditional immunofluorescence assays and enables direct detection of specific protein – protein interactions with a single molecule resolution. Besides the studied proteins (CD46 and β1 integrin subunit), the positive (α tubulin and β tubulin) and negative (β1 integrin and α tubulin) control protein pairs were selected. Using Proximity Ligation Assay Duolink (PLA) the proteins were adequately labelled (please, see methods for a detailed description) and evaluated under fluorescent microscopy (Fig. 4). Based on the obtained results, the CD46 - β1 integrin - protein interaction was proven in both freshly released sperm (Fig. 4A, first column) and sperm after the acrosome reaction (Fig. 4B, first column). The dynamic distribution of studied proteins over the sperm head was also visible by this assay and it is possible to compare it with standard fluorescent results in the small picture in the left hand corner of Fig. 4. A positive (Fig. 4A,B second column) and negative control (Fig. 4A,B third column) gave relevant positive/negative results. The assay was repeated twice with the same outcome.


Characterization of CD46 and β 1 integrin dynamics during sperm acrosome reaction
The study of protein-protein interactions.Interactions of CD46 and β1 integrin in C57BL/6 spermatozoa determined by Duolink proximity ligation assay. (A) freshly released sperm, (B) sperm during the induced AR. Positive control (α tubulin/ β tubulin), negative control (β1 integrin/ α tubulin). The small pictures in the corners represent the immunofluorescent dual staining of CD46 and β1 integrin, CD46 (green), β1 integrin (red). Scale bar represents 4 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: The study of protein-protein interactions.Interactions of CD46 and β1 integrin in C57BL/6 spermatozoa determined by Duolink proximity ligation assay. (A) freshly released sperm, (B) sperm during the induced AR. Positive control (α tubulin/ β tubulin), negative control (β1 integrin/ α tubulin). The small pictures in the corners represent the immunofluorescent dual staining of CD46 and β1 integrin, CD46 (green), β1 integrin (red). Scale bar represents 4 μm.
Mentions: Based on a similar distribution of both the proteins, their parallel time relocation during the AR and published data from the somatic cells17, we decided to perform a specific proximity ligation assay that extends the limits of traditional immunofluorescence assays and enables direct detection of specific protein – protein interactions with a single molecule resolution. Besides the studied proteins (CD46 and β1 integrin subunit), the positive (α tubulin and β tubulin) and negative (β1 integrin and α tubulin) control protein pairs were selected. Using Proximity Ligation Assay Duolink (PLA) the proteins were adequately labelled (please, see methods for a detailed description) and evaluated under fluorescent microscopy (Fig. 4). Based on the obtained results, the CD46 - β1 integrin - protein interaction was proven in both freshly released sperm (Fig. 4A, first column) and sperm after the acrosome reaction (Fig. 4B, first column). The dynamic distribution of studied proteins over the sperm head was also visible by this assay and it is possible to compare it with standard fluorescent results in the small picture in the left hand corner of Fig. 4. A positive (Fig. 4A,B second column) and negative control (Fig. 4A,B third column) gave relevant positive/negative results. The assay was repeated twice with the same outcome.

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.