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Protein synthesis and protein degradation in mammalian oocyte development
Šušor, Andrej ; Motlík, Jan (advisor) ; Pěknicová, Jana (referee) ; Petr, Jaroslav (referee)
Concr,usroNs We anďysed eucaryoticinitiation t,anslation factorsthat are responsiblefor protein qmthesisin mammalianoocýes. As well we examinedthe affinity.of this factorsto.'mCap, Sepharoseduring in útro conďtion...Inour'laboratorywas optimisedthe protocol for generation of poreine parttrenotesthat serve us as a model for anďysis of. cellular processos in .rygotes with biochemical approaches.. : , , - Antibod}r micÍoanay analysis was used to investigate the: regulation of signalling pathways primarily during meiotic maturationof pig oocytesand subsequentlyin comparisonto other species(bovine,frog andseastar). on the base of proteomeanďysis of maturationof oocýes was chosenUCH-LI molebuleandis studiedin detail in mammalian oocyte. Abnost thorougily was analysed function of UCH-LI in porcine and bovine oocyte using specific inhibitors and overexpression. The mechanism that is involved UCH-LI in antipolyspermydefencein mammalian oocyte was unveiled in our laboratory.
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The characterisation of sperm and seminal plasma glycoproteins: Their role within the tetraspanin network and male fertility
Páleníková, Veronika ; Postlerová, Pavla (advisor) ; Ryšlavá, Helena (referee) ; Pěknicová, Jana (referee)
Mgr. Veronika Páleníková only to describe the localization and heterodimerization of α3, α6, αV, β1, and β4 integrins in mentioned integrins. Importantly, we detected the presence of integrin β4 subunit in mouse sperm, and characterised that it forms functional heterodimer with α6 subunit. In tion, we described α6β1 and α3β1integrin pairs, which contribute to the integrity and complexity of sperm membranes. Moreover, we found that α6 integrin subunit interacts with proposed CD9 and CD81 as tetraspanin partners for αV integrin in tetraspanin web. Our goal
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Influence of tetrabromobiphenol A (TBBPA) on reproduction parameters, spermatogenesis and expression of selected genes in mice in vivo.
Žatecká, Eva ; Pěknicová, Jana (advisor) ; Kovář, Jan (referee)
Tetrabromobisphenol A is a substance that is widely used in industry as a fire retardant and is currently among the most widely used; its consumption is 210,000 tons / year and is still growing. It was shown that TBBPA can be released into the environment and it was found even in the human body. TBBPA has negative effects on different cell lines in vitro, including TM4 Sertoli cells. It was shown that it can affect the thyroid hormone system as well it might be able to influence the androgen / estrogen hormone system. Due to these facts, TBBPA was added to the list of potentially dangerous substances to human health. In this study, the effect of TBBPA on the reproductive parameters, spermatogenesis and activity of selected genes in mice in vivo was examined. First, we perform two generation in vivo study with outbred mouse line - CD1. In experimental animals we detected increased number of apoptotic cells in the testes, and also we observed changes in the expression pattern of selected genes encoding proteins that play an important role during spermatogenesis. In contrast, sperm quality and reproduction was not affected by TBBPA. As a complementary study was perform one generational in vivo study with inbred mouse line - C57BL/6. This study mostly confirmed the results obtained from previous in...
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Effect of zearalenone on reproductive parameters and the expression of selected genes in mice.
Dvořáková, Eva ; Pěknicová, Jana (advisor) ; Nedvídek, Josef (referee)
A number of chemicals may have a negative impact on the environment and wildlife. Endocrine disruptors (EDs), which can mimic estrogen, interfere with natural hormones in organism and can have a negative effect on the reproductive system. Such substances include zearalenone (ZEA) - mycotoxin, produced by the fungi Fusarium. Despite the non-steriodal structure of its molecule, ZEA and its derivates possess potent estrogenic activity. The influence of ZEA on reproductive parameters and changes in expression of selected genes were tested in the outbred line of mice. This study showed significant effects of ZEA on number of reproductive performances. This effect was observed at lower examined dose, to which are humans normally exposed, and at higher doses, both showed changes in the tested parameters.
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NK cells and their receptors in immune regulation - possible targets for immunomodulation
Svoboda, Jan ; Fišerová, Anna (advisor) ; Pěknicová, Jana (referee) ; Kročová, Zuzana (referee)
(english) Natural Killers - NK cells play an important role in immune surveilance and regulation either by direct cytotoxicity towards infected, transformed or otherwise damaged cells, or by production of cytokines and chemokines. The resulting response of NK cells is given by the sum of stimulating and inhibiting signals, tranduced by a wide array of receptors. Killer Ig-like receptors KIR2DL4 and LILRB1, which recognize self HLA-G molecules in pregnancy, as well as NKR-P1 receptors, which differ in the number of isotypes, are species-dependent and reduced during phylogenesis. NKG2D, reacting to stress-inducible proteins, and adenosine receptors (AR), which supress the inflamatory reaction, remain evolutionary conserved. The aim of this work was to study the involvement of NK cells and their receptors in several immune disorders and in various species, to provide new insights into their function and posisible immune modulation. We have shown here, that the choice of species in the study of NK cell effector functions may be crucial in some cases. The reaction to glycans, using synthetic GlcNAc-terminated glycomimetics GN8P, exerted opposing effects on NK cell function in humans and C57Bl/6 mice. In humans, the glycomimetic decreased cytotoxic activity of high NKR-P1A expressing NK cells, while in...
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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 Influence of eco-toxicological factors on spermatogenesis, morphology and function of mammalian sperm
Vašinová, Jitka ; Hortová, Kateřina (advisor) ; Pěknicová, Jana (referee)
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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The effect of estrogens on capacitation and acrosome reaction of boar spermatozoa in vitro.
Dostálová, Pavla ; Pěknicová, Jana (advisor) ; Nedvídek, Josef (referee)
Fertilization is a unique biological event where male and female gametes fuse together to produce a new organism. Before the gametes are able to fuse, however, they must undergo a series of controlled changes. For the male gamete, capacitation and acrosome reaction (AR) must occur, which take place during the sperm migration through the female genital tract. Unfortunately, while the process of capacitation has been known for over half a century, the molecular basis and influential factors behind it are not fully understood. Although estrogens have been considered mainly female reproductive hormones, there is increasing evidence suggesting that these steroids have an important role also in regulation of male reproductive functions. Sperm come into the contact with estrogens during their formation in the male and female genital tract, indicating that the hormone may play an important role in sperm maturation. In this study, we examined the importance of three endogenous estrogens (E1 -estron, E2 - 17β estradiol, E3 - estriol) and one synthetic estrogen (EE2 - 17α ethinylestradiol) on sperm maturation during capacitation and AR. Stimulatory effect were observed with all tested estrogens on both capacitation and zona pellucida induced AR. Moreover, we have determinied that the stimulatory effect on...
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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 effect of diabetes mellitus on reproductive parameters and expression of selected testicular genes in diabetic mice
Valášková, Eliška ; Pěknicová, Jana (advisor) ; Kaňka, Jiří (referee)
According to the World Health Organization (WHO), 15% of couples in reproductive age suffer from infertility problems, and up to 60% of cases are caused by male factor. Causes of this condition could be genetic background, environmental factors and various diseases, including diabetes mellitus (DM). The aim of this study was to investigate the effects of DM on reproductive parameters and expression of selected testicular genes using mouse model (FVB inbred mouse strain). DM (type 1) was artificially induced by chemical substance streptozotocin, which causes destruction of pancreatic β cells. These mice were exposed to diabetic condition for 6 weeks and then subjected to analysis. Our results have shown that diabetic condition had an impact on body weight, weight of reproductive organs as well as kidneys and livers. We also observed decreased concentration and viability of diabetic sperm compared to control. Moreover, we noticed increased staining with apoptotic marker annexin V. Further, we evaluated changes of sperm nuclear proteins - protamines. In diabetic animals, we observed higher number of sperm with insufficient protamination. Nevertheless, protamine 1 to protamine 2 ratio (P1/P2), a marker of male fertility, was not altered in sperm of diabetic animals compared to control. Regarding the...
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