The participation of extracellular vesicles in many cellular processes, including reproduction, is unquestionable. Although currently, the tetraspanin proteins found in extracellular vesicles are mostly applied as markers, increasing evidence points to their role in extracellular vesicle biogenesis, cargo selection, cell targeting, and cell uptake under both physiological and pathological conditions. In this review, we bring other insight into the involvement of tetraspanin proteins in extracellular vesicle physiology in mammalian reproduction. We provide knowledge regarding the involvement of extracellular vesicle tetraspanins in these processes in somatic cells. Furthermore, we discuss the future direction towards an understanding of their functions in the tissues and fluids of the mammalian reproductive system in gamete maturation, fertilization, and embryo development; their involvement in mutual cell contact and communication in their complexity.

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.

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