ABSTRACT: Integrins are transmembrane cell receptors involved in two crucial mechanisms for successful fertilization, namely, mammalian intracellular signaling and cell adhesion. Integrins ?6?4, ?3?1 and ?6?1 are three major laminin receptors expressed on the surface of mammalian cells including gametes, and the presence of individual integrin subunits ?3, ?6, ?1 and ?4 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 ?4 integrin subunit is expressed in mouse sperm and that it pairs with subunit ?6; 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 ?4 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: ?6/?4-inner apical acrosomal membrane and equatorial segment; ?3, ?6/?1, ?4-plasma membrane overlaying the apical acrosome; and ?3/?1-outer acrosomal membrane. The existence of ?6?4, ?3?1 and ?6?1 heterodimers was further confirmed by proximity ligation assay (PLA). In conclusion, we delivered detailed characterization of ?3, ?6, ?1 and ?4 integrin subunits, showing their presence in distinct compartments of the intact mouse sperm head. Moreover, we identified sperm-specific localization for heterodimers ?6?4, ?3?1 and ?6?1, 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.
