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The effect of oxidative stress on the structure and function of sperm cell cytoplasmic membrane during the fertilization process.
Tojnerová, Kateřina ; Krylov, Vladimír (advisor) ; Šanovec, Ondřej (referee)
Oxidative stress is caused by an imbalance between antioxidants and oxidants, and thus a consequent excess of reactive oxygen species (ROS). It can also be caused by insufficient antioxidant protection, which normally reduces ROS levels and protects the cell from damage. Oxidative stress causes changes in the structure and function of the sperm cytoplasmic membrane during the fertilization process. Here it has a positive effect on physiological functions, where low levels of ROS are necessary for normal sperm development such as sperm capacitation or sperm hyperactivation. However, at higher levels ROS exerts a pathophysiological effect, reducing membrane fluidity due to membrane lipid peroxidation, DNA fragmentation, loss of motility and subsequent inability of the sperm to fertilize can also occur. Keywords: oxidative stress, cytoplasmic membrane, sperm cell, fertilization
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Kvalita spermatu ovlivňuje oplození u sladkovodních ryb
CHENG, Yu
Short-term storage of sperm is a widely used technique during artificial fertilization in hatchery practice due to its convenient, inexpensive and practical value. Chapter 2, three egg incubation techniques were compared to supply one simple and efficiency method to incubate fertilized sterlet eggs. The percentage of cleavage, neurulation and hatching rates of embryos and larvae were examined to compare the effects of incubation systems consisting of placing Petri dishes 1) in the hatchery tank, 2) on a table in small incubation box in an air-conditioned room or 3) in a large thermostatic incubator box. No significant differences were found among these egg incubation systems. The experience gained allowed us to practice this simple method of egg incubation in Petri dishes on other fish species such as carp or zebrafish. Chapter 3, attention was paid to processes of egg activation and fertilization, which are highly important issues in fish reproductive biology. In the sterlet, multiple treatment groups with different gradient of spermatozoa number and volume of activation solution were used to fertilize eggs and further determine the minimum number of spermatozoa with optimal volume of activation water. The artificial fertilization experiments revealed that a total of 50,000 spermatozoa in 8 ml or 32 ml of activation water was sufficient for successful fertilization. It was obtained that a very low concentration of 100-1,300 spermatozoa per ml in activation water can achieve efficient fertilization with good quality sperm. Chapter 4, sperm managements including prevent sperm spontaneous movement, artificial seminal plasma composition, use of natural seminal plasma from good sperm were explored. The fertilization optimizations with different methods, spermatozoa: egg ratio and minimum sperm concentration were also examined in zebrafish. Results indicated that extender E400 was efficient during zebrafish sperm storage; a test tube with a well-defined amount of 6,000,000 zebrafish spermatozoa in E400 extender per 100 eggs and 100 ?L of activation solution has proven to be more successful than using a Petri dish. Sperm of European catfish can be collected and stored in immobilization solution for as long as 1 week for successful fertilization. Sterlet sperm with bad quality can be revitalized by incubation with seminal plasma from good sperm. The results help to establishing a sperm in vitro storage method and fertilization procedures in freshwater fishes. Chapter 5, activation solution is one of the key factors providing sperm and egg performance during fertilization. Different activation solutions were employed to test the practical application efficiency in a hatchery jar with sperm that was aged but diluted in artificial seminal plasma. The large-scale artificial fertilization trials indicated that fertilization and hatching rate were high (78-87%) after one week of sperm storage in a carp artificial seminal plasma in activation solutions as hatchery water and Perchec solution. Results offered the practical basic for the further application of aging sperm in a hatchery. Chapter 6, in this study, aged sperm in common carp was used to evaluate the sperm motility and kinematics, sperm viability, osmolality and pH of seminal plasma sperm short-term in vitro storage. DNA methylation analysis based on the whole-genome bisulfite sequencing was also performed to reveal the molecular changes of aging sperm. The results showed that aging spermatozoa in common carp has a considerable negative impact on their performance and as well as artificial fertilization success. the level of methylation at the CpG sites increased significantly with 24 hours post stripping spermatozoa compared to the fresh group, but then reduced significantly at 96 hours post stripping. The results contribute to investigate the sperm phenotype and functional changes in common carp and providing clues for understanding the molecular changes during sperm storage
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NAD+-dependent histone deacetylase SIRT1 in the process of oogenesis, fertilization and early embryonic development
Valentová, Iveta ; Nevoral, Jan (advisor) ; Drutovič, David (referee)
SIRT1 is a histone deacetylase from the sirtuin family that affects epigenetic and non- epigenetic targets. We can assume that the known SIRT1 substrates are involved in the regulation of gametogenesis and early embryonic development. Our hypotheses say SIRT1 is present in oocytes and early embryos and it plays a physiological role in oocyte maturation, fertilization and early embryonic development. A mouse model of a conditional knock-out line producing Sirt1-deficient oocytes was developed to verify our hypotheses. Oocytes and embryos were analyzed for SIRT1, its selected substrates and other markers by immunocytochemistry. We found out that the presence of SIRT1 contributes to oocyte quality through modulation of the chromatin histone code and stabilization of the spindle. Furthermore, the purely maternal origin of SIRT1 presents in both zygote pronuclei. Last but not least we discovered a significant effect of SIRT1 on early embryonic development, probably mainly due to its role in the activation of the embryonic genome. The results confirm our hypothesis that SIRT1 is present in oocytes and embryos mainly around chromatin. The results show that SIRT1 is a maternal factor determining oocyte quality and it is necessary for the embryonic genome activation.
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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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Species-specific structural differences of mammalian sperm and function of their key proteins during fertilization.
Dobrodinská, Anna ; Frolíková, Michaela (advisor) ; Kuntová, Barbora (referee)
The fertilization is a process during which a male and a female gamete merge so that a new organism may come into being. The sperm-egg fusion is preceded by several essential processes, such as the capacitation, acrosome reaction, the sperm binding to the zona pellucida and oolemma, and membrane fusion of the gametes. Numerous proteins, which are located in both sperm and eggs, are major actors in controlling the listed, essential processes. During the process of fertilization these proteins fulfil one or more functions. In mammalian sperm, significant species-specific differences may be found both in their morphology and at the protein level. A complex understanding of species-specific distinctions in sperm structure and functions of key sperm proteins would contribute to a better insight into the process of fertilization, thereby enabling us to better diagnose and subsequently treat the causes of infertility in humans. This bachelor's thesis summarizes the current knowledge of sperm structure and its key proteins that has been acquired through the studies of the following model mammal species: bull, boar, mouse, and human. Further, this thesis brings an interspecific comparison between the studied species. Keywords: sperm, fertilization, acrosome reaction, capacitation, sperm proteins, bull,...
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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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Methods of sperm selection for human and veterinary assisted reproduction
Pavlová, Hana ; Komrsková, Kateřina (advisor) ; Koubek, Pavel (referee)
Sperm selection is an important process influencing the progress of fertilization and the quality of resulting embryos. Competent sperm cells can be isolated from an ejaculate sample according to different criteria. Standard methods allow for sperm selection based on conventional parameters, particularly motility and density. Using advanced methods, sperm cells are selected according to molecular aspects including for instance membrane charge, presence of specific receptors, molecules indicating ongoing apoptosis, and others. The latter procedures are widely used particularly in human practice. Their use is uneconomical in veterinary practice and standard methods including the centrifugation step in the procedure are primarily used to select the sperm of livestock. Sperm cells of livestock are also often selected according to whether it carries the X or Y chromosome. Thus, the sex of the offspring can be effectively determined in order to increase the efficiency of the production of animal products. This thesis presents the most frequently used methods of sperm selection for the purpose of oocyte fertilization in human and veterinary sectors of assisted reproduction and discusses their impact on the parameters and fertilization potential of isolated cells.
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Sperm centrioles and their role in reproduction
Vlčková, Monika ; Frolíková, Michaela (advisor) ; Liška, František (referee)
Centrioles are evolutionarily conserved protein structures composed of microtubules. In somatic cells, centrioles serve as the basal body of cilia and flagella and allow the assembly of pericentriolar material, thereby creating the centrosome. Without centrosome, animal cells are not capable of nuclear division. Centrioles do not arise de novo and their formation always requires the presence of a preexisting centriole. Since there are no centrioles in the egg at the time of fertilization, unlike spermatozoa, sperm is the carrier of centrioles and therefore all of the centrioles of the emerging organism are of paternal origin. There are two centrioles in the sperm - a cylindrical shape proximal and a distal one, which is perpendicular to the proximal. The sperm centrioles are the basis for the formation of the sperm flagellum and after fertilization form the mitotic spindle of the zygote, necessary for equal DNA and cell distribution. It follows from the above that the presence of centrioles in sperm is essential in mammals and defects in their structure may lead to male sterility or embryo development disorders. However, sperm centrioles differ from somatic centrioles in their structure and behavior and understanding these differences is one of the important tasks of reproductive biology.
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Sperm cell penetration and membrane fusion with oocyte during fertilization in mammals
Klimková, Veronika ; Krylov, Vladimír (advisor) ; Frolíková, Michaela (referee)
Fertilization is a process involving multiple steps, which are in continuity and complement each other. Spermatozoa become competent to fertilize after capacitation, hyperactivation and acrosome reaction. Oocytes are surrounded by a layer of cumulus cells and give a signals for spermatozoa to activation and orientation. This study focuses on the latest knowledges about a roles of cumulus cells, production of chemoattractants and the possibility that cumulus cells induce the acrosome reaction after binding a novel sperm protein NYD-SP8 to the cumulus. It seems that progesterone induce the acrosome reaction and also is the best chemoattractant secreted by cumulus cells. Next I am focus on gamete fusion including the role of surface proteins such as Juno and IZUMO1. Key words: membrane fusion, cumulus cells, acrosome reaction, chemotaxis sperm, oocyte, fertilization
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Immunological causes of female infertility
Bajerová, Kateřina ; Brynychová, Iva (advisor) ; Novák, Jan (referee)
Currently, up to 15 % of reproductive age couples struggle with infertility. Female infertility is the cause of conception issues in nearly 40 % of these cases. The origin of female infertility consists of wide range of causes and other factors. Immunological causes are certainly one of the most significant ones. In pathological conditions, defense mechanisms of female immune system can target alloantigens presented on sperm surface. Moreover, autoimmune response and antibody production can occur as well. Antibodies can target various parts of ovarian follicles and oocytes, phospholipids, nuclear antigens, enzymes etc. These antibodies can inhibit fertilization, maturation of oocytes and embryo, embryo implantation, and also recurrent miscarriages. The aim of this thesis is to summarize various causes of female infertility focusing on immunological pathologies. The thesis also describes functioning of immune mechanisms in the female reproductive tract and briefly touches on oogenesis and female hormonal cycle.
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