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Abstract
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.
Highlights
CD9 and CD81 are expressed in a large variety of cells [1] and belong to the tetraspanin superfamily (TM4SF), whose members are small (20–50 kDa) proteins [2,3]
The association of CD9 and CD81 with α3β1 [8,9] and α6β1 integrins was confirmed [10,11,12], and a similar association can be expected in sperm, as the presence and favourable localization of these integrin heterodimers in sperm head was shown by Frolikova et al [13]
The putative mechanism of involvement of CD9 and CD81 in tetraspanin web formation and its activity during sperm preparation for fertilization was addressed with a focus on the species-specific differences between mouse and human
Summary
CD9 and CD81 are expressed in a large variety of cells [1] and belong to the tetraspanin superfamily (TM4SF), whose members are small (20–50 kDa) proteins [2,3] Their ability to form homologous partnerships as well as interact heterologously with distinct, non-tetraspanin proteins (including adhesion molecules, receptor and co-receptor molecules, and antigens of major histocompatibility complex or cytoplasmic kinases) represents the key feature of tetraspanins that enables them to create a complex active network on a cell membrane surface called a “tetraspanin web” [4,5,6]. The putative mechanism of involvement of CD9 and CD81 in tetraspanin web formation and its activity during sperm preparation for fertilization was addressed with a focus on the species-specific differences between mouse and human. The structural modelling of CD9 and CD81 homologous and heterologous network formation discussing both lateral Cis interaction within the sperm membrane and Trans interaction during gamete fusion was used to propose the tetraspanin network structure
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