Integrins are transmembrane cell receptors involved in two crucial mechanisms for successful fertilization, namely, mammalian intracellular signaling and cell adhesion. Integrins α6β4, α3β1 and α6β1 are three major laminin receptors expressed on the surface of mammalian cells including gametes, and the presence of individual integrin subunits α3, α6, β1 and β4 has been previously detected in mammalian sperm. However, to date, proof of the existence of individual heterodimer pairs in sperm and their detailed localization is missing. The major conclusion of this study is evidence that the β4 integrin subunit is expressed in mouse sperm and that it pairs with subunit α6; additionally, there is a detailed identification of integrin heterodimer pairs across individual membranes in an intact mouse sperm head. We also demonstrate the existence of β4 integrin mRNAs in round spermatids and spermatogonia by q-RT-PCR, which was further supported by sequencing the PCR products. Using super-resolution microscopy accompanied by colocalization analysis, we located integrin subunits as follows: α6/β4-inner apical acrosomal membrane and equatorial segment; α3, α6/β1, β4-plasma membrane overlaying the apical acrosome; and α3/β1-outer acrosomal membrane. The existence of α6β4, α3β1 and α6β1 heterodimers was further confirmed by proximity ligation assay (PLA). In conclusion, we delivered detailed characterization of α3, α6, β1 and β4 integrin subunits, showing their presence in distinct compartments of the intact mouse sperm head. Moreover, we identified sperm-specific localization for heterodimers α6β4, α3β1 and α6β1, and their membrane compartmentalization and the presented data show a complexity of membranes overlaying specialized microdomain structures in the sperm head. Their different protein compositions of these individual membrane rafts may play a specialized role, based on their involvement in sperm-epithelium and sperm-egg interaction.

Laboratory of Reproductive Biology Institute of Biotechnology of the Czech Academy of Sciences v v i BIOCEV Prumyslova 595 25250 Vestec Czech Republic

Laboratory of Reproductive Biology Institute of Biotechnology of the Czech Academy of Sciences v v i BIOCEV Prumyslova 595 25250 Vestec Czech Republic Department of Biochemistry Faculty of Science Charles University Vinicna 7 12844 Prague 2 Czech Republic

Laboratory of Reproductive Biology Institute of Biotechnology of the Czech Academy of Sciences v v i BIOCEV Prumyslova 595 25250 Vestec Czech Republic Department of Veterinary Sciences Faculty of Agrobiology Food and Natural Resources University of Life Sciences Prague Kamycka 129 16500 Prague 6 Czech Republic

Laboratory of Reproductive Biology Institute of Biotechnology of the Czech Academy of Sciences v v i BIOCEV Prumyslova 595 25250 Vestec Czech Republic Department of Zoology Faculty of Science Charles University Vinicna 7 12844 Prague 2 Czech Republic

Laboratory of Reproductive Biology Institute of Biotechnology of the Czech Academy of Sciences v v i BIOCEV Prumyslova 595 25250 Vestec Czech Republic Group of Cell Biology and Innovative Therapies Centre for Molecular Biophysics University of Orleans UPR4301 45071 Orleans France

Laboratory of Structural Bioinformatics of Proteins Institute of Biotechnology of the Czech Academy of Sciences v v i BIOCEV Prumyslova 595 25250 Vestec Czech Republic

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