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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.


3D cartoon summarizing the localization of CD46 and α3, α6 and β1 integrins among different membrane structures of the intact sperm head.(A) Apical acrosomal area, (B) Equatorial segment, (C) Sperm hook. PM – plasma membrane, OAM – outer acrosomal membrane, IAM – inner acrosomal membrane, NM – nuclear membrane. Using our experimental techniques, we are not able to determine, if integrins α3 and β1 are present on both PM and OAM or exclusively on PM in the equatorial segment (panel B).
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f7: 3D cartoon summarizing the localization of CD46 and α3, α6 and β1 integrins among different membrane structures of the intact sperm head.(A) Apical acrosomal area, (B) Equatorial segment, (C) Sperm hook. PM – plasma membrane, OAM – outer acrosomal membrane, IAM – inner acrosomal membrane, NM – nuclear membrane. Using our experimental techniques, we are not able to determine, if integrins α3 and β1 are present on both PM and OAM or exclusively on PM in the equatorial segment (panel B).

Mentions: In order not to ignore α subunits of integrin proteins we decided to characterize the localization of α3 and α6 subunits, which were previously detected in sperm2540. As shown in Fig. 5B, their localization was remarkably different. Based on these results, we can conclude that the α6β1 and/or α6β4 pair could be localized in the plasma membrane covering an intact apical sperm head including the hook. Due to an absence of β1 in the equatorial segment of intact sperm, it is probable that only the α6β4 pair would be localized in the equatorial region (Fig. 5BI, see the arrows), entirely excluding the acrosome vesicle. On the other hand, the α3β1 pair is confined to the plasma and outer acrosome membrane (Fig. 5BII,III, S3a–c left), however, the sperm hook is not labelled by α3 integrins (Fig. 5BII,III), which gives us a clear difference between the α3 and α6 subunit of β1 integrins. The co-staining of the α3 integrin subunit with CD46 supports this observation and confirms that the α3 integrin localizes into the outer, but not inner acrosome membrane in intact sperm. This is clearly visible in Fig. 5BIII, as the CD46 labels the entire acrosome vesicle and its signal outspreads the one given by the α3 integrin subunit (Fig. 5BIII, see the arrow). The Estrogen receptor β (ERβ) labelling, which is excusive to the plasma membrane was used to double confirm the expression of α3 integrin (Fig. S3C right). A positive colocalization of ERβ with α3 integrin and a negative one with CD46, supports the identified α3 integrin plasma membrane localization. All the results addressing localisation of various forms of integrins and CD46 among individual membrane structures in the sperm head are then graphically summarised (Fig. 7).


Characterization of CD46 and β 1 integrin dynamics during sperm acrosome reaction
3D cartoon summarizing the localization of CD46 and α3, α6 and β1 integrins among different membrane structures of the intact sperm head.(A) Apical acrosomal area, (B) Equatorial segment, (C) Sperm hook. PM – plasma membrane, OAM – outer acrosomal membrane, IAM – inner acrosomal membrane, NM – nuclear membrane. Using our experimental techniques, we are not able to determine, if integrins α3 and β1 are present on both PM and OAM or exclusively on PM in the equatorial segment (panel B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: 3D cartoon summarizing the localization of CD46 and α3, α6 and β1 integrins among different membrane structures of the intact sperm head.(A) Apical acrosomal area, (B) Equatorial segment, (C) Sperm hook. PM – plasma membrane, OAM – outer acrosomal membrane, IAM – inner acrosomal membrane, NM – nuclear membrane. Using our experimental techniques, we are not able to determine, if integrins α3 and β1 are present on both PM and OAM or exclusively on PM in the equatorial segment (panel B).
Mentions: In order not to ignore α subunits of integrin proteins we decided to characterize the localization of α3 and α6 subunits, which were previously detected in sperm2540. As shown in Fig. 5B, their localization was remarkably different. Based on these results, we can conclude that the α6β1 and/or α6β4 pair could be localized in the plasma membrane covering an intact apical sperm head including the hook. Due to an absence of β1 in the equatorial segment of intact sperm, it is probable that only the α6β4 pair would be localized in the equatorial region (Fig. 5BI, see the arrows), entirely excluding the acrosome vesicle. On the other hand, the α3β1 pair is confined to the plasma and outer acrosome membrane (Fig. 5BII,III, S3a–c left), however, the sperm hook is not labelled by α3 integrins (Fig. 5BII,III), which gives us a clear difference between the α3 and α6 subunit of β1 integrins. The co-staining of the α3 integrin subunit with CD46 supports this observation and confirms that the α3 integrin localizes into the outer, but not inner acrosome membrane in intact sperm. This is clearly visible in Fig. 5BIII, as the CD46 labels the entire acrosome vesicle and its signal outspreads the one given by the α3 integrin subunit (Fig. 5BIII, see the arrow). The Estrogen receptor β (ERβ) labelling, which is excusive to the plasma membrane was used to double confirm the expression of α3 integrin (Fig. S3C right). A positive colocalization of ERβ with α3 integrin and a negative one with CD46, supports the identified α3 integrin plasma membrane localization. All the results addressing localisation of various forms of integrins and CD46 among individual membrane structures in the sperm head are then graphically summarised (Fig. 7).

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.