| Mammalian fertilization is a multistep process that culminates in the fusion of the sperm and egg plasma membrane. However, until recently, the precise mechanism of sperm-egg fusion has remained unclear. It is widely accepted that the equatorial segment of the acrosome-reacted sperm is important in initiating fusion with the egg plasma membrane during fertilization. There are various proteins known to be distributed only in the equatorial segment of sperm. The role of these proteins must be clarified to understand the membrane fusion process. Therefore we chose sperm equatorial segment protein 1（SPESP1） to study its localization in sperm and interaction with other spern-egg fusion related proteins.In the present study we cloned cDNA of Spespl of sheep, expressed it in E. coli BL21（DE3） and collected the recombinant GST-SPESP1 fusion proteins to immunize mice for induction of the anti-SPESP1 ascites polyclonal antibodies. With the antibodies we examined the expression pattern and localization of SPESP1 in sheep testis and sperm cells by immunohistochemistry and immunocytochemistry. We also identified the interactions between sheep SPESP1 and other sperm proteins, such as IZUMO1, IZUMO2, IZUMO3, IZUMO4, TSSK6, MN9, PDIA3, CRISP1 and SLLP1, by yeast two hybridization（Y2H）.The result shows the cloned sequence of Spespl cDNA is 1095 bp, and alignment with cDNA sequence predicted by GenBank （Accession XM004010268） revealed that it had three bases difference resulting in an amino acid alteration. The best inducing conditions of expressing GST-SPESP1 is at 37 ℃ with 0.005% IPTG for 4 h, and the GST fusion protein was mainly found in the inclusion bodies. The results of immunocytochemistry demonstrated that the SPESP1 was located in the equatorial region before acrosome reaction, but disappeared from there after acrosome reaction. The results of protein interaction experiments by Yeast two-hybrid showed that sheep SPESP1 had no direct interaction with other sperm proteins. |
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