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The Molecular mechanisms of sperm-egg fusion in mouse
Klinovská, Karolína ; Hortová, Kateřina (advisor) ; Jonáková, Věra (referee)
The mechanism of gamete fusion remains largely unknown on molecular level despite its indisputable significance. A few molecules requisite for membrane interaction are known, among them IZUMO1 on sperm and tetraspanin CD9 on egg. A concept of a large multitprotein complex on both membranes forming a fusion machinery is recently emerging. CD9 is expected to play a role in organizing egg membrane order and to interact laterally with other factors. On the other hand, IZUMO1 contains one immunoglobulin (Ig) domain, which is known for its interaction with various molecules and thus is expected to have a binding partner on the egg membrane. Ig domains are known for their involvement in interactions with Fc receptors and Fc receptor-like molecules, which makes these a potential partner for sperm in gamete binding and fusion. Recent experiments identified Fc receptor-like 3 as a candidate binding partner for sperm by one-bead one-compound assay. The aim of presented study was to assess the localization of FCRL3 in mouse eggs, together with its potential co-localization with IZUMO1 on complexes of zona-free eggs with bound sperm. In this study, FCRL3 molecule has been found across the cytoplasm, possibly in the intracytoplasmic membrane compartments of the cell, as well as on the egg membrane. Its presence...
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Analysis of spermatogenesis in mice C57BL/6 after Toxoplasma gondii infection.
Šidlová, Adéla ; Hortová, Kateřina (advisor) ; Flegr, Jaroslav (referee)
Toxoplasma gondii is an intracellular parasite that causes a persistent infection in 10- 80% of the world's population, depending on geographic location. Infection with this parasite causes changes in behavior and physiological functions of an infected host, including warm- blooded animals including humans. Recent studies also show influence of infection with T.gondii on male reproductive fitness in mice and rat and to investigate this issue further became a task of my diploma thesis. In this study, detailed histometric screening of testicular function along with levels of a pituitary luteinizing hormone (LH) and screening of epigenetic modification of testicular DNA were analysed in infected and control mice. Testicular function and sperm production was significantly decreased in T. gondii positive group after a 30-day infection. The level of LH in urine of T. gondii infected mice was decreased compares to control and the number of leptotene primary spermatocytes and spermatids was lowered, but also number of Sertoli cells and tubule diameter were elevated. In epigenetic study the testicular epigenom was tested to measure global methylation and methylation of specific genes Hspa1, Crem and Creb1 which are essential for successfully ongoing spermatogenesis. The global level of methylation of...
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Sperm morphology and molecular mechanism associated with sperm fertilizing ability in mice.
Šebková, Nataša ; Hortová, Kateřina (advisor) ; Jonáková, Věra (referee) ; Linhart, Otomar (referee)
The process of sperm capacitation and acrosome reaction (AR) are highly dynamic processes essential for the fertilization, including cytoskeleton proteins in the sperm head. The study of the distribution of actin, spectrin and α-tubulin in the head of representative mammalian sperm indicates that before and after the AR there were changes in the distribution of the cytoskeleton structures in the sperm head of all representatives, mainly in the apical part of the acrosome, but also in the equatorial and postacrosome part of the sperm head. The particularly well-studied actin cytoskeleton plays an important role during the capacitation and before and after the AR. Environmental estrogens can interfere with the function of endogenous hormones in very low concentrations. They can interact with specific receptors affecting several signalling pathways leading to sperm capacitation and AR. The effect of 17β-estradiol, estrone, estriol and synthetic 17α-ethynylestradiol was evaluated by the ratio of tyrosine phosphorylation and the state of the acrosome during in vitro capacitation in the sperm head. This study has provided the evidence that estrogens significantly stimulate capacitation progress in a concentration-dependent manner. Estrogens decrease number of sperm after the induced AR too. The raising...
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Role paternálního H4K12ac při utváření pronukleí a v časné embryogenezi u myší.
Dudková, Barbora ; Hortová, Kateřina (advisor) ; Stopka, Tomáš (referee)
During the process of spermatogenesis, histones are replaced by protamines, basic proteins enabling transmission of DNA to the oocyte during fertilization. In mouse sperm, there is only 1% of remaining histones whose N-terminal tails contain post-translationally modified residues. In this study, I was interested in contribution of paternal histone H4 acetylated on lysine K12 residues (H4K12ac) that is present in mature sperm head in remaining nucleosomes. Physiologically, H4K12ac has an important role in transcription factor accumulation and in regulation of gene expression. The presence and abundance of H4K12ac modification in various pronuclei stages of 1-cell embryo and parthenotes were assessed by imunnoflourescent detection with utilization of anti-H4K12ac antibody. Altogether, the paternal pronucleus exhibits a strong acetylation signal on H4K12 since its formation, while in the maternal one, there is a slow continual increase of H4K12ac getting on the same level as paternal pronucleus till the pronuclei fusion. Simultaneously DNA methylation status in both pronuclei was detected. In paternal pronucleus there is a continual decrease in the DNA methylation detectable as a decrease of 5mC and an increase of 5hmC signal. Meanwhile, the maternal pronucleus stays widely methylated. DNA...
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Dynamics of acrosome reaction during intra-specific sperm competition in rodents.
Veselá, Kateřina ; Hortová, Kateřina (advisor) ; Pěknicová, Jana (referee)
Dynamics of acrosome reaction during intra-specific sperm competition in rodents Sperm acrosome integrity is disturbed in promiscuous species field mice (Apodemus) and more than half of the spermatozoa undergoing spontaneous acrosome reaction (AR) before binding to the zona pellucida. In Muridae it is documented a generally high rate of spontaneous AR, and the percentage increases in promiscuous species up to 60 % in 60 min capacitation in vitro. The acrosome integrity positively corellates with presence of CD46 protein which absence in wood mouse is fenotypicaly same as in CD46 knock-out mouse leading to accelerated spontaneous AR. It is necessary to clarify whether for mouse sperm it is essential the primary binding of intact sperm to zona pellucida of the egg or whether it is preferred secondary sperm binding after spontaneous AR. In this context, the question is whether there is a relocalization of the key fusion protein IZUMO in sperm during spontaneous AR. IZUMO relocalization was monitored by immunofluorescence at specific times of capacitation in vitro during spontaneous and induced AR. IZUMO relocalization as closely connected to actin cytoskeleton, and β1 integrins. Dynamics and localization of β1 integrin during spontaneous and induced AR was also detected by immunofluorescence. Our results...
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The Influence of eco-toxicological factors on spermatogenesis, morphology and function of mammalian sperm
Vašinová, Jitka ; Pěknicová, Jana (referee) ; Hortová, Kateřina (advisor)
An increasing infertility due to pathological changes on sperm has become a serious issue. A rising concentration of fluorides in the environment could be a potentially negative factor affecting the male fertility. Eco-toxicological effect of fluorides can be enhanced in the present of aluminium ions by forming fluoro-metallic complexes, analogues of phosphate groups that interfere with the activity of G-proteins and P-type ATPases, which are part of several signalling pathways during sperm maturation. The aim of this thesis was monitoring the effect of fluorides on mouse sperm capacitation in vitro, analysis of the effect of fluorides on male reproductive organ and on the level of mouse spermatogenesis. Fluorides were administered to mice through table water at the concentrations of 0, 1, 10, 100 ppm and at the same time we monitored their synergic action with aluminium at 10 ppm dose. The level of tyrosine phosphorylation in the sperm head was the main marker for successful capacitation in vitro. The results show that an oversized load of ions, to which mice were exposed, affected negatively spermatogenesis and ability of sperm to capacitate in vitro. Besides that, a multigenerational study in mice demonstrated that even low concentrations of aluminium fluorides can have a negative effect on...
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Effect of cryopreservation on mouse sperm.
Veselá, Kateřina ; Pěknicová, Jana (referee) ; Hortová, Kateřina (advisor)
Cryopreservation or freezing of sperm in the reproductive biology is still actual topic. Today is the only method used for sperm storage, whether for the purposes of assisted reproduction, or for scientific purposes. However, this method has a negative impact on such stored cells and is therefore still a subject of many studies. Among the main causes of sperm damage in rodents there are inappropriately selected cryoprotective agents, poor or no elimination of oxidative stress generated during cryopreservation, as well as a poorly chosen speed and temperature of freezing. Correctly chosen procedures and the composition of the media in which sperm are stored, can significantly affect the quality of sperm. This bachelor theses focuses on the influence of cryopreservation on mouse spermatozoa. The main impact of this method includes the effect of freezing on sperm DNA, plasma membrane, acrosome, and sperm motility.
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Influence of estrogens on mammalian sperm.
Šidlová, Adéla ; Šebková, Nataša (referee) ; Hortová, Kateřina (advisor)
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, whether their effect is positive or negative. Endogenous estrogens in mammalian males are an important part of the endocrine system. Estrogens play an important role in the growth and maintenance of bone mass, as well as in the development of germ cells and in the marches of spermatogenesis. Healthy sperm are essential for successful reproduction. Effect of endogenous estrogens is partly involved in the regulation of proper development of sperm. Besides the positive effect of endogenous estrogens, there is also a negative effect of estrogens from the external environment, so-called environmental estrogens. Between environmental estrogens are man-made chemicals, xenoestrogens, which increasingly contaminate the environment. Hazards of these estrogenic substances in nature is hidden in their ability to act as endocrine disruptors, which cause defects in many aspects of mammalian reproduction. With regards to the fact that there are more and more substances in current environment that can interfere with hormonal path of mammalian reproduction, it is important to look at this issue. Reproductive disorders that may be caused by...
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Principles of ovarian stimulation and oocyte collection in mouse and human.
Klinovská, Karolína ; Svoboda, Petr (referee) ; Hortová, Kateřina (advisor)
This thesis deals with methods of controlled ovarian stimulation and oocyte retrieval on physiological basis. Mouse has been major model organism for wide range of key researches and being able to stimulate its ovarian functions correctly is necessary. That is why mouse stimulation is main topic of this thesis. It summarizes available data and stimulation protocols in relation to factors which can affect the result as a condition, strain, quality of hormones used in administration and others. Potential affections on oocyte and embryo quality are mention according to specific types of administration protocols. Human infertility is slowly becoming common and one of the techniques used to treat it in assisted reproduction is also ovarian stimulation. In the end of this thesis is a chapter dealing with this commonly used method. It is focused on stimulation for oocyte retrieval, as well as on stimulation of recipient mother in embryotransfer. Differences based on hormones, success in In vitro fertilization programme (IVF) and application frequency are mentioned.
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Analysis of gene expression during spermatogenesis in mice
Suchan, Jan ; Stopka, Pavel (advisor) ; Hortová, Kateřina (referee)
Spermatogenesis is very unique and complex process regulating and be amenable to reproductive behaviour equally. These processes are affected by outer factors such as social stimuli, represented by interaction between male and female. Mice belong to mammals with high sensitivity to chemical signals. Hence pheromonal communication takes important part in their life. Pheromones have effect on expression activity in testicular tissue throught hormonal pathways. A target of these regulations are for example proteins participating in immunity or competition actions. Phenotype plasticity might proceed on the level of DNA as well as on the level of gene expression. That's why we take interest if factors like social contacts are able to significantly change expression of genes involved in reproduction pathways. We are also interested in possibility that expression changes of target genes take part in speciation events. In this study we demonstrate the effect of short-term social contact to gene expression of biochemical pathways involved in spermatogenesis and by other pathways linked together with reproductive bahaviour. This involvement might be direct or indirect. We found genes, whose level of expression in testicular tissue is different between species Mus m. musculus and Mus m. domesticus. Here I provide...
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