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A mouse acrosomal cortical matrix protein, MC41, has ZP2-binding activity and forms a complex with a 75-kDa serine
I Tanii1, T Oh-oka, K Yoshinaga
1Department of Anatomy and Reproductive Cell Biology, Miyazaki Medical College, Kihara 5200, Kiyotake, Miyazaki, 889-1692, Japan. itanii@post1.miyazaki-med.ac.jp
Abstract:
Sperm with a large acrosome such as that of guinea pigs and hamsters have a subdomain structure in the anterior acrosome, but the mouse acrosome looks homogeneous and its matrix has not been precisely analyzed. The intra-acrosomal protein MC41 is localized in the cortical region of the mouse anterior acrosome, suggesting a subdomain structure in the mouse acrosome. Thus, the present study was undertaken to analyze the mouse acrosomal matrix using an anti-MC41 antibody. When mouse sperm were treated with 2% Triton X-100, Triton-insoluble matrix components remained in the acrosomal cortical region. Immunogold for MC41 labeled the Triton X-100 and high-salt-insoluble matrix components, demonstrating that MC41 is a subdomain-specific acrosomal matrix protein. We further examined interactions of MC41 with acrosomal proteases and zona proteins. A serine protease of 75 kDa was associated with MC41 under low-salt conditions, presumably forming a complex. Far Western blotting technique indicated that MC41 bound to both ZP2 and ZP2(f) in the presence of high-salt-soluble sperm proteins. In acrosome-reacting sperm, MC41 was present on the hybrid vesicles formed by the fusion of the plasma and outer acrosomal membranes. Presumably, MC41 has a significant role in secondary sperm-zona binding during the acrosomal reaction.
Insights
The mouse sperm acrosome has a subdomain structure, with protein MC41 located in the outer region. This protein interacts with proteases and zona pellucida proteins, aiding sperm-zona binding during the acrosomal reaction.
Area of Science:
- Reproductive Biology
- Sperm Cell Biology
- Molecular Reproduction
Background:
- The acrosome of sperm cells, particularly in species like guinea pigs and hamsters, exhibits a subdomain structure.
- The mouse acrosome matrix has been poorly characterized, appearing homogeneous and lacking detailed analysis.
- The localization of intra-acrosomal protein MC41 in the anterior acrosome's cortical region suggests a potential subdomain structure in mouse sperm.
Purpose of the Study:
- To investigate the structural organization of the mouse acrosomal matrix.
- To analyze the role of the intra-acrosomal protein MC41 in sperm function.
- To elucidate the interactions of MC41 with other sperm components and the zona pellucida.
Main Methods:
- Treatment of mouse sperm with Triton X-100 to isolate insoluble matrix components.
- Immunogold labeling using an anti-MC41 antibody to identify protein localization.
- Far Western blotting to assess the binding interactions of MC41 with acrosomal proteases and zona pellucida proteins (ZP2 and ZP2(f)).
Main Results:
- Triton X-100 insoluble matrix components were localized to the acrosomal cortical region.
- MC41 was identified as a subdomain-specific acrosomal matrix protein, localized in the Triton X-100 and high-salt insoluble fraction.
- A 75 kDa serine protease was found to associate with MC41 under low-salt conditions.
- MC41 demonstrated binding to ZP2 and ZP2(f) in the presence of high-salt-soluble sperm proteins.
- During the acrosomal reaction, MC41 was observed on hybrid vesicles formed by membrane fusion.
Conclusions:
- The mouse acrosome possesses a subdomain structure, with MC41 being a key component of the cortical region.
- MC41 plays a role in secondary sperm-zona binding, likely by interacting with zona pellucida proteins after the acrosomal reaction.
- The findings contribute to understanding the molecular mechanisms of sperm-egg interaction and fertilization.