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.

Sperm–egg interaction and fusion represent a key moment of fertilization. In mammals, it is not possible without the interaction of the tetraspanin superfamily proteins including CD81. A detailed immunohistochemical localization of CD81 was monitored in bovine oocytes during different maturation stages, as well as during early embryogenesis. In addition, characterization of CD81 was carried out in bovine and mouse sperm. In bovine eggs, CD81 was detected on the plasma membrane of the germinal vesicle, metaphase I and metaphase II oocytes. During fertilization, accumulation of CD81 molecules in the perivitelline space of fertilized oocytes, which appeared as vesicles associated with plasma membrane, was observed. In majority of bull-ejaculated sperm and caput, corpus and cauda epididymal sperm, as well as mouse cauda epididymal sperm, CD81 was found on the plasma membrane covering the apical acrosome. Although the process of capacitation did not influence the localization of CD81, it was lost from the surface of the acrosome-reacted spermatozoa in bull, in contrast to mouse sperm where there was a relocalization of the CD81 protein during acrosome reaction across the equatorial segment and later over the whole sperm head. The presented results highlight conservative unifying aspects of CD81 expression between cattle and mouse, together with mouse-specific traits in sperm CD81 behaviour, which emphasizes certain species-specific mechanisms of fertilization to be considered.

The physiological importance of CD151 tetraspanin is known from somatic cells and its outside-in signalling through integrins was described. In male germ cells, two tetraspanins, CD9 and CD81, are involved in sperm-egg membrane fusion, and similarly to integrins, they occupy characteristic regions. We report here on a newly discovered presence of CD151 in sperm, and present its expression and distribution during spermatogenesis and sperm transition during the acrosome reaction. We traced CD151 gene and protein expression in testicular cell subpopulations, with strong enrichment in spermatogonia and spermatids. The testicular and epididymal localization pattern is designated to the sperm head primary fusion site called the equatorial segment and when compared to the acrosome vesicle status, CD151 was located into the inner acrosomal membrane overlying the nucleus. Moreover, we show CD151 interaction with α6 integrin subunit, which forms a dimer with β4 as a part of cis-protein interactions within sperm prior to gamete fusion. We used mammalian species with distinct sperm morphology and sperm maturation such as mouse and bull and compared the results with human. In conclusion, the delivered findings characterise CD151 as a novel sperm tetraspanin network member and provide knowledge on its physiology in male germ cells.

Fertilization process is a very clever and unique process comprising some essential steps resulting in formation of zygote. Tetraspanin CD9 is considered to be a serious candidate molecule participating in these events. The importance of CD9 has been discussed in relation to acrosome reaction, sperm-binding, sperm-penetration, sperm-egg fusion and eventually, egg activation. The abundant expression of CD9 oocyte plasma membrane and the presence of CD9-containing vesicles in the perivitelline space of intact oocytes have been confirmed. Despite the fact that majority of authors analyzed CD9 expressed on oocytes, several studies considered the function of sperm CD9, too. To understand CD9 involvement, various conditions of in vitro fertilization (IVF) assays using polyclonal as well as monoclonal antibodies or knockout mice were carried out. However, ambiguous data have been obtained about the importance of CD9 in sperm-egg binding or fusion. Although the current findings did not prove any hypothesis, the indispensable role of CD9 in fertilization process was not excluded and the precise role of CD9 remains unexplained.