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Monitoring of protein network dynamics: the role of FcRL proteins during sperm-egg membrane interaction
Bašus, Kryštof ; Komrsková, Kateřina (advisor) ; Šušor, Andrej (referee)
Sperm--egg membrane interaction and fusion is mediated by various molecules of the different protein network that are located on both egg and sperm membrane. So far, many proteins have been selected to be fusion candidates, some of them (Izumo1, CD9, Juno) were proven to be essential, whereas others were discovered to play an unsuspected new active role (CD46, tetraspanins). After the adhesion of sperm to an egg, Juno located on the oolema associates with monomeric Izumo1 on sperm membrane, which is results in Izumo1 dimerization following quick removal of Juno from the egg surface as described in mouse. It implies that additional receptor on the egg membrane is required to play a role in sperm--egg fusion. To find a human fusogenic receptor for IZUMO1 protein we used one--bead--one--compound (OBOC) assay, a random screening approach. A bead, fulfilling all the requirements when interacting with the human sperm, carried a peptide sequence showing homology with the conserved Ig domain of the human specific Fc receptor--like protein 3 (FcRL3). In general, the ...
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Spermatogenesis, Classification and Evaluation of Sperm Morphology for Assisted Reproduction.
Kaňoková, Kristýna ; Komrsková, Kateřina (advisor) ; Pěknicová, Jana (referee)
Spermatogenesis, Classification and Evaluation of Sperm Morphology for Assisted Reproduction Abstract Sperm quality has an important role in determining male fertility. There is an increasing amount of couples searching for assisted reproduction due to the increasing decline in quality of sperm. Morphology of sperm is one of the factors influencing male fertility and defects in sperm morphology are the main subject of many studies. Mammalian sperm are formed during spermatogenesis and defects in spermatogenesis may cause severe defects in sperm morphology. Defects of a sperm head, as well as a mid- piece and principal piece of sperm tail sperm can have a serious impact on fertilizing ability of sperm and it is, therefore, necessary in some cases to use methods of assisted reproduction due to morphological abnormalities. The main objects of this thesis were to describe spermatogenesis, normal and pathogenic sperm morphology and evaluate the use and outcome of assisted reproduction methods based on sperm morphology and apply the evaluation in selecting the best method of assisted reproduction. To some extent, sperm morphology influences the outcome of assisted reproduction and it is a significant factor in predicting fertilization, pregnancy and embryo cleavage/development rate. In some cases of severe...
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The role of integrins in gametes prior to fertilization and during their interaction
Foldynová, Veronika ; Komrsková, Kateřina (advisor) ; Tlapáková, Tereza (referee)
Integrins are heterodimeric transmembrane glycoproteins that represent a large group of cell adhesion receptors involved in cell-cell, cell-extracellular matrix and cell-pathogen interacti- ons. Up to now, 24 different integrin heterodimers have been detected in mammals. They are involved in a wide range of processes such as immune response, lymphocyte homing, platelet aggregation, also in wound healing, cell differentiation, migration, proliferation and even in cell survival. Integrins have also been detected on germ cells and are now known to play an important role in reproductive processes such as fertilization, embryo implan- tation, and embryonic development. The main aim of this thesis is to introduce integrins from the perspective of reproduction, integrin physiology, occurrence and localization of individual subunits in male and female germ cells. A significant part of the work is devoted to a discussion of the role of integrins in gametes, both during maturation processes (egg maturation in the ovary, capacitation and the acrosomal reaction of sperm), in sperm mi- gration, oviductal reservoir formation and their direct and indirect involvement in adhesion and fusion of the gametes during fertilization. Keywords: integrins, sperm, egg, fusion, fertilization
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Methodology of transgenic mouse preparation.
Reslová, Gabriela ; Komrsková, Kateřina (advisor) ; Pěknicová, Jana (referee)
The thesis deals with methods used for preparation of transgenic mice and with comparison of them in terms of efficiency and suitability of applications for research purposes. Technologies for the transfer of gene constructs involve microinjection of DNA into the pronukleus of fertilized oocyte, which seems to be the most reliable one. Another possibility is associated with the use of sperm, which carry the incorporated gene construct into the oocyte during in vitro fertilization, or with the use of retroviral vectors by which the oocytes are transfected. Somatic cell nuclear transfer into enucleated oocyte, the use of embryonic stem cells which are incorporated into strange embryo or transgenesis by liposomes are techniques far less explored and not so often used. The next section of this thesis introduces to the reader individual steps which are necessery for successful transgenesis. It is focused on ovarian stimulation which is necessery to obtain sufficient amount of oocytes, as well as on stimulation of recipient mother in embryo transfer as well as on capacitation of sperm required for in vitro fertilization. The thesis also deals with backward analysis confirming positivity of transgene expression by genotyping of pups and by crossbreeding of transgene positive and compares advantages and...
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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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Function of Zinc finger protein 644 (Zfp644) in mouse organism.
Szczerkowska, Katarzyna Izabela ; Sedláček, Radislav (advisor) ; Komrsková, Kateřina (referee) ; Blahoš, Jaroslav (referee)
ZNF644 (Zinc Finger Protein 644) is a C2H2 zinc finger gene encoding a putative transcription regulator, of which a point mutation (S672G) is associated with inherited high myopia in humans. It is also described to be a partner of the G9a/GLP (G9a- euchromatic histone- lysine N-methyltransferase 2, EHMT2; GLP - euchromatic histone-lysine N-methyltransferase 1, EHMT1) complex, known for its essential role in histone methylation, specifically H3K9me1and H3K9me2. It was reported that another transcription factor, WIZ (Widely-Interspaced Zinc Finger-Containing Protein), can bind to this complex and cooperate in gene silencing simultaneously. In order to study Zfp644 impact on myopia, we generated a mouse model, Zfp644S673G that mimics human mutation. In addition, a mouse with a persuasive truncated form of the protein, Zfp644Δ8 was created. Both mouse models went through an examination of retinal function and morphology. Moreover, with use of ultrasonography, different ocular parameters were examined. We conclude, that Zfp644 gene is causative for myopia in mice. Further examinations of Zfp644Δ8 animals show severe symptoms in metabolism and female fertility. To describe the impact of Zfp644 in mouse fertility we performed various experiments including analysis of expression of Zfp644 in reproductive...
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The study of structure and metabolism of mammalian and fish sperm in relation to the process of reproduction
Koubek, Pavel ; Pěknicová, Jana (advisor) ; Komrsková, Kateřina (referee) ; Flajšhans, Martin (referee)
4. CONCLUSION Duringthe studyof teleosteanand chondrosteanfish spermatozoa,a new complex protocol,optimizedforimmunofluorescencestainingofantigenson carpandsturgeon spermatozoa,was developed lt was derivedfrom the protocolused for the mammalianmodel;however,theprotocolrespectsthesimpleandbreakablestructure offishsperm.Inthisway,possibledamagetospermatozoaduringsamplepreparation is minimizedtogetherwithgenerationof nonspecificartefactswhichcouldinfluence the results.Therefore,it is crucialto createandfollowa suitableprotocolforsemen handlingand immunofluorescencestainingnotonlyforevaluationof semenquality butalsoforexperimentalstudreswhenthereproducibilityofthemethodis essential. In the study of energy metabolismon the model of demembranatedcarp spermatozoa,we provedtheexistenceof PCr shuttleand showedthe localizationof creatinekinaseisoenzymesalongthecarpspermflagellumbytheaidof monoclonal antibody.Moreover,withthe contributionof experimentsusinginhibitionof creatine kinase,we demonstratedthe possibilityof initiationand maintenanceof cAMP- independentflagellarmovementof demembranatedcarpspermatozoaonlyby the componentsofthePCr shuttle The studyof chondrosteanfishwas focusedon thefinestructureand morphologyof sterletspermatozoa,which was examinedby scanningand transmisionelectron...
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Fc receptor-like molecules: biological role and phylogenetic conservation of FcRL multigene family
Böhmová, Helena ; Komrsková, Kateřina (advisor) ; Vinkler, Michal (referee)
Fc receptor-like (FcRL) molecules are novel immunological receptors with intriguing proposed roles in the immune system. Phylogenetic studies revealed that FcRL genes are conserved in jawed vertebrates (Gnathostomata), and likely formed during adaptive system genesis. FcRLs have multiple extracellular immunoglobulin domains, and some representatives have newly documented specific ligands. Several FcRLs contain both ITIM and ITAM cytoplasmatic sequences, which provide them with dual signalling properties. These allow them to regulate intracellular signalling pathways. FcRLs are known to engage in B cell regulation. They are largely expressed on B cells, with some representatives preferentially expressed on cytotoxic T cells and NK cells. Their expression patterns are specific to distinct B cell subpopulations and also show different levels of expression during B cell differentiation, pointing to their possible involvement in this process. FcRL molecules have also been detected on the mammalian eggs, and they may potentially play role in gamete fusion. This thesis aims to introduce FcRL molecules in various aspects, including their relation to classical Fc receptors, their structural properties, genomic organisation, phylogenetic conservation and their roles in organism. Thesis also focuses on...
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Monitoring of protein network dynamics: the role of FcRL proteins during sperm-egg membrane interaction
Bašus, Kryštof ; Komrsková, Kateřina (advisor) ; Šušor, Andrej (referee)
Sperm--egg membrane interaction and fusion is mediated by various molecules of the different protein network that are located on both egg and sperm membrane. So far, many proteins have been selected to be fusion candidates, some of them (Izumo1, CD9, Juno) were proven to be essential, whereas others were discovered to play an unsuspected new active role (CD46, tetraspanins). After the adhesion of sperm to an egg, Juno located on the oolema associates with monomeric Izumo1 on sperm membrane, which is results in Izumo1 dimerization following quick removal of Juno from the egg surface as described in mouse. It implies that additional receptor on the egg membrane is required to play a role in sperm--egg fusion. To find a human fusogenic receptor for IZUMO1 protein we used one--bead--one--compound (OBOC) assay, a random screening approach. A bead, fulfilling all the requirements when interacting with the human sperm, carried a peptide sequence showing homology with the conserved Ig domain of the human specific Fc receptor--like protein 3 (FcRL3). In general, the ...
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