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Molecular structure and function of CD4 on murine egg plasma membrane
1Department of Immunology and Pathology, University of Tokyo, Japan.
Abstract:
In the present study, the expression of the CD4 molecule on murine egg plasma membrane was confirmed by the indirect immunofluorescence (IIF) method. The full-length CD4 cDNA from murine eggs was synthesised by the reverse transcriptase-polymerase chain reaction (RT-PCR) method and its authenticity verified by Southern blot hybridisation using an end-labelled internal oligonucleotide. The results of DNA sequencing showed that the nucleotide sequence of the cDNA of CD4 from murine egg mRNA was identical to that of immune T cells. To demonstrate the direct interaction of CD4 from murine egg with murine sperm cells bearing MHC (major histocompatibility complex) class II molecule, we employed a baculovirus expression system to generate CD4 on the surface of Spodoptera frugiperda (Sf9) cells. Expression of CD4 on Sf9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV)-CD4 was demonstrated by IIF and immunoblotting. The CD4-expressing Sf9 cells adhered to MHC class II-bearing sperm cells since the adhesion was specifically blocked by anti-CD4 monoclonal antibody (mAb) or anti-monomorphic region of MHC class II mAb. Taking our previous and present experimental results together, they strongly suggest that intercellular membrane adhesion between two gametes at the fusion step in fertilisation is mediated by the MHC class II molecule located on the posterior region of the sperm head and the CD4 molecule on egg plasma membrane.
Insights
Murine egg plasma membranes express CD4 molecules, which interact with sperm
Area of Science:
- Reproductive Biology
- Immunology
- Molecular Biology
Background:
- Fertilization involves gamete membrane interactions.
- The specific molecules mediating sperm-egg adhesion are not fully understood.
- CD4 and MHC class II molecules are known for cell-cell interactions.
Purpose of the Study:
- To investigate the role of CD4 and MHC class II in murine gamete adhesion.
- To confirm CD4 expression on murine eggs.
- To demonstrate direct interaction between egg CD4 and sperm MHC class II.
Main Methods:
- Indirect immunofluorescence (IIF) for CD4 detection.
- Reverse transcriptase-polymerase chain reaction (RT-PCR) for CD4 cDNA synthesis.
- Southern blot hybridization for cDNA verification.
- Baculovirus expression system to produce CD4 on Sf9 cells.
- Monoclonal antibody (mAb) blocking assays.
Main Results:
- CD4 molecule expression confirmed on murine egg plasma membrane.
- Murine egg CD4 cDNA sequence is identical to that of T cells.
- CD4-expressing Sf9 cells adhered to sperm cells expressing MHC class II.
- Adhesion was specifically blocked by anti-CD4 and anti-MHC class II mAbs.
Conclusions:
- CD4 on murine egg plasma membrane interacts with MHC class II on murine sperm.
- This interaction mediates intercellular membrane adhesion during gamete fusion in fertilization.
- The findings elucidate a novel mechanism in the fertilization process.