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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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Monitoring of selected steroid hormones by HPLC-MS/MS and studying their impact on mouse sperm in vitro.
Bosáková, Tereza ; Komrsková, Kateřina (advisor) ; Pacáková, Věra (referee)
The present project aims were development of a new sensitive separation method using high performance liquid chromatography with tandem mass spectrometric detection, which enables quantification of selected steroid hormones, namely 17α-ethynylestradiol (EE2) and progesterone (PRG) in M2 fertilizing medium. M2 medium is used during sperm capacitation in vitro, which is the physiological process of complex molecular and biochemical changes, essential for obtaining fertilizing ability of male gametes. Optimized separation and detection conditions (column Kinetex EVO C18 with mobile phase consisted of 50/50 (v/v) acetonitrile/water with 0.1% formic acid in both components) were applied on monitoring of concentration changes of each hormone during time dependent sperm capacitation. The concentration tested for EE2 were 200, 20 and 2 µg/l and for PRG 200 µg/l only. It was found that concentration of free, unbounded EE2 firstly decreases, reaches the minimum in about 60 min of capacitation and then increases again. This trend was observed for all three tested concentrations. Concentration (200 µg/l) of free unbounded PRG decreased during whole capacitation. The following kinetic analysis will be based on the concentration dependences of the free unbound hormones on the capacitation time. The results of...
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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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Monitoring of selected steroid hormones by HPLC-MS/MS and studying their impact on mouse sperm in vitro.
Bosáková, Tereza ; Komrsková, Kateřina (advisor) ; Pacáková, Věra (referee)
The present project aims were development of a new sensitive separation method using high performance liquid chromatography with tandem mass spectrometric detection, which enables quantification of selected steroid hormones, namely 17α-ethynylestradiol (EE2) and progesterone (PRG) in M2 fertilizing medium. M2 medium is used during sperm capacitation in vitro, which is the physiological process of complex molecular and biochemical changes, essential for obtaining fertilizing ability of male gametes. Optimized separation and detection conditions (column Kinetex EVO C18 with mobile phase consisted of 50/50 (v/v) acetonitrile/water with 0.1% formic acid in both components) were applied on monitoring of concentration changes of each hormone during time dependent sperm capacitation. The concentration tested for EE2 were 200, 20 and 2 µg/l and for PRG 200 µg/l only. It was found that concentration of free, unbounded EE2 firstly decreases, reaches the minimum in about 60 min of capacitation and then increases again. This trend was observed for all three tested concentrations. Concentration (200 µg/l) of free unbounded PRG decreased during whole capacitation. The following kinetic analysis will be based on the concentration dependences of the free unbound hormones on the capacitation time. The results of...
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Analysis of defensive substances of Diving beetles (Dytiscidae) by ultra-high performance liquid chromatography with mass spectrometry
Bosáková, Tereza ; Kozlík, Petr (advisor) ; Nesměrák, Karel (referee)
Obranný sekret potápníků (Dytiscidae) produkovaný jejich prothorakálními žlázami obsahuje řadu steroidních látek. Pro analýzu obranného sekretu potápníka druhu Ilybius fenestratus byla využita ultra- vysokoúčinná kapalinová chromatografie ve spojení s detekcí diodovým polem a hmotnostní detekcí. Analýza methanolického extraktu sekretu probíhala na koloně Acquity BEH C18 za podmínek gradientové eluce s dvousložkovou mobilní fází tvořenou methanolem s 0,1% obsahem amoniaku a 0,1% vodným roztokem amoniaku. V sekretu byla zjištěna přítomnost dvou steroidních hormonů, a to boldenonu a testosteronu a jejich identifikace byla potvrzena na základě shody jejich retenčních časů i hmotnostních spekter s příslušnými standardy.
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