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Multidisciplinary characterization of interactions between oestrogen hormones and their receptors during sperm capacitation.
Bosáková, Tereza
This doctoral thesis was focused on the multidisciplinary characterizatrion of interactions between hormones and their receptors during sperm capacitation, which is the final step in sperm maturation in mammals and the key event to achieve egg fertilization. Three oestrogenic hormones (17β-estradiol (E2), 17α-estradiol (αE2), 17α-ethynylestradiol (EE2)), which are part of environmental pollution, were selected for the study. These hormones are called endocrine disruptors as they can cause disorders of the hormonal system in animals, thereby adversely affecting physiological processes, including reproduction. A new analytical HPLC-MS/MS method was developed for monitoring of concentration changes of oestrogens (E2, αE2 and EE2) during time-dependent capacitation of mouse and rat sperm in vitro. For individual oestrogens, the concentration dependences of free, unbound hormone on the capacitation time were measured for three concentrations of individual hormones (200, 20 and 2 μg/L). The obtained concentration data were converted into values of relative concentrations, which were subjected to kinetic analysis. Kinetic models were proposed for the interaction of individual hormones with sperm, which were described by a system of differential equations with optimization of rate constants and molar...
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Analysis of biologically active compounds using high performance separation methods
Adamusová, Hana ; Bosáková, Zuzana (advisor) ; Jelínek, Ivan (referee) ; Tůma, Petr (referee)
In the first part of this doctoral thesis, a new analytical HPLC-MS/MS method for monitoring of concentration changes of 17β-estradiol (βE2) during in vitro mouse sperm capacitation was developed. Capacitation was performed for three initial concentrations of βE2 (200, 20 and 2 μg/L). For all the concentrations a similar trend for the total unbound βE2 was observed. In general, the βE2 concentration decreased to reach its minimum and then increased again. The position of the minimum differed for the individual tested βE2 concentrations. Experimentally obtained results were subjected to the kinetic analysis. The curves fitted through the experimentally determined points displayed an autocatalytic character. For the agreement between the curves obtained by fitting through the experimental points and the theoretical calculated curves, it is necessary to assume that the first step is adsorption of βE2 onto the surface of the sperm controlled by Langmuir isotherm. The kinetic study was also used to study the effects of fluorides and aluminium fluoride complexes on the capacitation of mouse sperm. The experimental points were in very good agreement with the shape of the theoretical curves and this fact verifies the mechanism of the mouse sperm capacitation kinetics. In the second part of this work, two...
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Multidisciplinary characterization of interactions between oestrogen hormones and their receptors during sperm capacitation.
Bosáková, Tereza ; Komrsková, Kateřina (advisor) ; Smrček, Stanislav (referee) ; Postlerová, Pavla (referee)
This doctoral thesis was focused on the multidisciplinary characterizatrion of interactions between hormones and their receptors during sperm capacitation, which is the final step in sperm maturation in mammals and the key event to achieve egg fertilization. Three oestrogenic hormones (17β-estradiol (E2), 17α-estradiol (αE2), 17α-ethynylestradiol (EE2)), which are part of environmental pollution, were selected for the study. These hormones are called endocrine disruptors as they can cause disorders of the hormonal system in animals, thereby adversely affecting physiological processes, including reproduction. A new analytical HPLC-MS/MS method was developed for monitoring of concentration changes of oestrogens (E2, αE2 and EE2) during time-dependent capacitation of mouse and rat sperm in vitro. For individual oestrogens, the concentration dependences of free, unbound hormone on the capacitation time were measured for three concentrations of individual hormones (200, 20 and 2 μg/L). The obtained concentration data were converted into values of relative concentrations, which were subjected to kinetic analysis. Kinetic models were proposed for the interaction of individual hormones with sperm, which were described by a system of differential equations with optimization of rate constants and molar...
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Analysis of biologically active compounds using high performance separation methods
Adamusová, Hana ; Bosáková, Zuzana (advisor) ; Jelínek, Ivan (referee) ; Tůma, Petr (referee)
In the first part of this doctoral thesis, a new analytical HPLC-MS/MS method for monitoring of concentration changes of 17β-estradiol (βE2) during in vitro mouse sperm capacitation was developed. Capacitation was performed for three initial concentrations of βE2 (200, 20 and 2 μg/L). For all the concentrations a similar trend for the total unbound βE2 was observed. In general, the βE2 concentration decreased to reach its minimum and then increased again. The position of the minimum differed for the individual tested βE2 concentrations. Experimentally obtained results were subjected to the kinetic analysis. The curves fitted through the experimentally determined points displayed an autocatalytic character. For the agreement between the curves obtained by fitting through the experimental points and the theoretical calculated curves, it is necessary to assume that the first step is adsorption of βE2 onto the surface of the sperm controlled by Langmuir isotherm. The kinetic study was also used to study the effects of fluorides and aluminium fluoride complexes on the capacitation of mouse sperm. The experimental points were in very good agreement with the shape of the theoretical curves and this fact verifies the mechanism of the mouse sperm capacitation kinetics. In the second part of this work, two...
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Dynamics of mouse sperm capacitation and acrosome reaction
Dvořáková-Hortová, Kateřina ; Frolíková, Michaela ; Děd, Lukáš ; Šebková, Nataša
Capacitation followed by the acrosome reaction (AR), is a very complex event of molecular changes, including acrosome matrix rearrangement and actin polymerization, which mammalian sperm must undergo in the female reproductive tract in order to obtain the ability to penetrate and fertilize the egg. CD46 and β1-integrin belong to specific proteins, which are predicted to interact during molecular reorganization of capacitating sperm. The IZUMO1 as the primary fusion protein of the mammalian sperm is also involved in this dynamic network. We investigated the relationship between the Izumo, CD46 and β1 integrin relocation in the sperm head during the capacitation and AR in vitro. We have already successfully monitored by immunofluorescent labelling the dynamics of proteins CD46 and β1-integrin. The changes in the localization of these proteins associated with the AR and their mutual co-localization was observed. The original β1-integrin location in the freshly released epididymal sperm is in the acrosome and it relocates during the AR further through the sperm head compartments into the equatorial segment and over the whole sperm head. Its density over the equatorial segment is decreasing with the extended time of the AR. Also its presence in the perforatorium of the mouse sperm head is very prominent. The pattern for protein CD46 is extremely similar if not identical in both aspects such as compartment localization and time progress during capacitation and AR in vitro. The molecular interaction of CD46 and β1-integrin was investigated using the Proximity Ligation Assay and Super resolution microscopy STED. The data were statistically analysed. The newly obtained results from CD46 and β1-integrin relocation are in correlation with IZUMO1 dynamics and giving a substantial knowledge on the studied protein network rearrangement during capacitation and AR in mouse spermatozoa.
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