Proteins CD9 and CD81 are members of the tetraspanin superfamily and were detected in mammalian sperm, where they are suspected to form an active tetraspanin web and to participate in sperm⁻egg membrane fusion. The importance of these two proteins during the early stages of fertilization is supported by the complete sterility of CD9/CD81 double null female mice. In this study, the putative mechanism of CD9/CD81 involvement in tetraspanin web formation in sperm and its activity prior to fertilization was addressed. Confocal microscopy and colocalization assay was used to determine a mutual CD9/CD81 localization visualised in detail by super-resolution microscopy, and their interaction was address by co-immunoprecipitation. The species-specific traits in CD9 and CD81 distribution during sperm maturation were compared between mice and humans. A mutual position of CD9/CD81 is shown in human spermatozoa in the acrosomal cap, however in mice, CD9 and CD81 occupy a distinct area. During the acrosome reaction in human sperm, only CD9 is relocated, compared to the relocation of both proteins in mice. The structural modelling of CD9 and CD81 homologous and possibly heterologous network formation was used to propose their lateral Cis as well as Trans interactions within the sperm membrane and during sperm⁻egg membrane fusion.

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 128 43 Prague Czech Republic

Department of Veterinary Sciences Faculty of Agrobiology Food and Natural Resources University of Life Sciences Prague Kamycka 129 165 00 Prague Czech Republic

Department of Zoology Faculty of Science Charles University Vinicna 7 128 44 Prague Czech Republic

Group of Reproductive Biology Institute of Biotechnology Czech Academy of Sciences v v i BIOCEV Prumyslova 595 252 50 Vestec Czech Republic

Laboratory of Reproductive Physiology Institute of Animal Biochemistry and Genetics Centre of Biosciences Slovak Academy of Sciences Dubravska Cesta 9 845 05 Bratislava Slovakia

Laboratory of Structural Bioinformatics of Proteins Institute of Biotechnology Czech Academy of Sciences v v i BIOCEV Prumyslova 595 252 50 Vestec Czech Republic

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