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The role of LINC complex in male germinal cells
Kmoníčková, Věra ; Komrsková, Kateřina (advisor) ; Knop, Filip (referee)
LINC (linker of the nucleoskeleton and cytoskeleton) complex is substantial for the overall function of any nuclear cell. It is a bridge between the cytoplasmic and nuclear structures which mediates transmission of mechanical forces generated on the cytoplasmic side. The main objective of this thesis is the function of distinct variants of the LINC complexes in mammalian male germinal cells throughout their development. The nuclear facing side of the LINC complex is a key player in homolog pairing and rapid chromosome movements during meiosis. The cytoplasmic facing side is necessary for the sperm head formation, proper acrosome and nucleus attachment. Evenly, another complex is essential for the head-to-tail coupling as without this one the sperm cells are found decapitated. If the subunits of the LINC complex are defective, males are afflicted with different kinds of infertilities, which nowadays is one of the important issues of our society. Many things about the role and structure of the LINC complex has been discovered due to modern technologies. These up to date findings are summarized and discussed on the following pages.
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Chemical communication of gametes
Otčenášková, Tereza ; Komrsková, Kateřina (advisor) ; Petr, Jaroslav (referee)
Fertilization is a multiple step process leading to fusion of female and male gametes resulting in a formation of a zygote. Besides direct gamete interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves communication based on chemical molecules triggering various signalling pathways. This work is aimed to characterize chemical communication of gametes of a model organism Mus musculus. For this purpose, modern proteomic and visualisation methods like nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS), selected reaction monitoring (SRM) and immunofluorescent microscopy were used. Lipocalins were identified as candidate proteins involved in communication including those from major urinary proteins (MUPs), LCN lipocalins and fatty acid binding proteins (FABPs). For the first time, we report their presence in the sperm acrosome. Based on lipocalins capacity to bind and transport other molecules, we propose that these proteins have a protective and/or signalling role for gametes. Furthermore, chemical communication between sperm and oocyte is based on chemotaxis which enables their interaction before their fusion. In this work, we detected that spermatozoa show chemotactic responses in the presence of L-glutamate. This amino acid naturally...
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Cytoskeleton-membrane protein interaction network in sperm
Adamová, Zuzana ; Komrsková, Kateřina (advisor) ; Tolde, Ondřej (referee)
In order to fertilize the egg, sperm cell undergoes several subsequent maturation processes. The final one called acrosome reaction is an exocytosis of acrosome vesicle, which is filled with lytic enzymes. Acrosome reaction is crucial for penetration of the sperm cell through the egg surroundings, especially zona pellucida, as well as for reorganization of a membrane protein composition on its surface. This rearrangement leads to the exposure of proteins essential for fertilization, mainly for gamete recognition, binding and fusion in specific compartments of the sperm head. One of such protein is CD46, which is located in the acrosomal membrane of an intact sperm and after acosomal exocytosis it relocates to the equatorial segment of a sperm head, which is known to be the initial site of interaction of sperm with the egg plasma membrane. The relocation of CD46 is disrupted by inhibition of actin, which reorganization within sperm head is known to play a role in onset of acrosome reaction, however, the precise mechanism of CD46 interaction with actin in sperm is unknown. In this thesis, ezrin - a crosslinker of membrane proteins and actin - has been studied in context of CD46 and its relocation across the sperm head. Analysis of the immunofluorescent detection of ezrin revealed its mutual...
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Male infertility in context of testicular cancer
Cimlerová, Markéta ; Komrsková, Kateřina (advisor) ; Truksa, Jaroslav (referee)
This bachelor thesis focuses on male infertility in a connection to testicular cancer. Testicular cancer is the most common malignancy among young men in a reproductive age and the worldwide incidence of testicular cancer is on the rise. A lot of attention is also given to an increasing rate of infertility in a context of testicular cancer. For these reasons, the aim of this thesis is to clarify several non-physiological changes, such as hormone levels, spermatogenesis and sperm parameters, which take place in a male body and influence the chance to become a biological father. These pathological changes can be due to the disease itself but also due to the treatment. A cryopreservation of semen is also going to be discussed as the way to preserve male fertility and as an option for couples with the infertility problems due to male factor which rely on help of the assisted reprodiction. Kye words: testicular cancer, cancer treatment, male infertility, sperm parameters, mitochondria, assisted reproduction
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Biological imaging by super-resolution microscopy
Adamová, Zuzana ; Komrsková, Kateřina (advisor) ; Benda, Aleš (referee)
zuzana_adamova_abstract_eng.txt[11.05.2017 20:26:29] Fluorescence microscopy is one of the most widely used imaging techniques in biological research. Despite its numerous advantages, it can be used only for studies of structures larger than 200 nanometres, due to diffraction limit caused by a wave nature of light. The value of 200 nanometres is the best reachable value of optical resolution, in other words, the smallest distance of two objects, which can be separately recognized by conventional optical systems. Up to the end of the 20th century it was therefore impossible to observe finer details of cells. However, recently several breakthrough imaging techniques, named super-resolution microscopy techniques, managed to bypass the diffraction limit and enabled biologists to study much more delicate structures, such as small organelles, virions, protein complexes or even particular proteins, while still using a visual light. This thesis introduces some selected super-resolution methods, explains briefly their principles and presents some of their applications in biology.
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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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Chemical communication of gametes
Otčenášková, Tereza ; Komrsková, Kateřina (advisor) ; Petelák, Aleš (referee)
Many diverse chemoattractants play a part in sperm guidance towards the oocyte without any difference whether organisms with external or internal fertilization. Upon release, spermatozoa are exposed to various cues providing essential information about their surrounding environment. These molecules are structurally very distinct and they induce via specific receptors, expressed over the sperm cell surface, different signalling pathways influencing the sperm motility and regulating the correct sperm function. The overall understanding of these mechanisms brings interesting and often unexpected points of view on evolutionary relationships between invertebrate and vertebrate species. Some principles believed to be shown exclusively only by mammals are nowadays revealed also by marine invertebrates and vice versa. The current knowledge of human gametes interactions is used in fields of medicine that are connected to the reproduction. Thus, with a help of current knowledge, researches aim to shed more light into causes of infertility, as well as to develop novel contraceptives or enhance the rate of successful in vitro fertilizations.
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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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The influence of estrogens on mouse sperm capacitation and acrosome reaction in vitro
Tejnická, Magda ; Komrsková, Kateřina (advisor) ; Linhart, Otomar (referee)
There are an increasing amount of compounds in the environment that can have a negative effect on reproductive parameters in both male and female organism. There has been a worldwide decline of sperm quality during past decades and this fact lead to an increase of unnatural ways of conception through assisted reproduction techniques in the specialised centres. Natural estrogens are one of these compounds and they get into waste water after being excluded from the body by the urine. They get back into the human body from drinking water or from the food, and they can interfere with function of endogenous hormones in very low concentrations. For these reasons it is up to date to deal with the influence of these compounds on mammalian sperm. For many years, estrogens have been considered typically female sex hormones. It is now certain that they are also very important in the regulation of male reproduction. Endogenous estrogens in mammalian males are an important part of the endocrine system. Estrogens play an important role in the development of germ cells, spermatogenesis and processes leading to successful egg fertilization such as a capacitation or acrosomal reaction. Tyrosine phosphorylation is one of the essential steps for the properly ongoing process of capacitation in sperm followed by a...
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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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