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.

Department of Analytical Chemistry Faculty of Science Charles University Albertov 2030 128 43 Prague Czech Republic

Department of Veterinary Sciences Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Kamycka 129 165 00 Prague Czech Republic

Department of Zoology Faculty of Science Charles University Vinicna 7 128 44 Prague Czech Republic

Institute of Vertebrate Biology v v i Czech Academy of Sciences Kvetna 8 603 65 Brno Czech Republic

Laboratory of Reproductive Biology Institute of Biotechnology CAS v v i BIOCEV Prumyslova 595 252 50 Vestec Czech Republic

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