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.

17β-estradiol (estradiol) is a natural estrogen regulating reproduction including sperm and egg development, sperm maturation—called capacitation—and sperm–egg communication. High doses can increase germ cell apoptosis and decrease sperm count. Our aim was to answer the biological relevance of estradiol in sperm capacitation and its effect on motility and acrosome reaction to quantify its interaction with estrogen receptors and propose a model of estradiol action during capacitation using kinetic analysis. Estradiol increased protein tyrosine phosphorylation, elevated rate of spontaneous acrosome reaction, and altered motility parameters measured Hamilton-Thorne Computer Assisted Semen Analyzer (CASA) in capacitating sperm. To monitor time and concentration dependent binding dynamics of extracellular estradiol, high-performance liquid chromatography with tandem mass spectrometry was used to measure sperm response and data was subjected to kinetic analysis. The kinetic model of estradiol action during sperm maturation shows that estradiol adsorption onto a plasma membrane surface is controlled by Langmuir isotherm. After, when estradiol passes into the cytoplasm, it forms an unstable adduct with cytoplasmic receptors, which display a signalling autocatalytic pattern. This autocatalytic reaction suggests crosstalk between receptor and non-receptor pathways utilized by sperm prior to fertilization.