Search

The GLUT9 transporter, coded by the SLC2A9 gene, is one of the key proteins enabling the trasnfer of uric acid across the membrane in epithelial cells of the proximal tubule. In humans, this protein is naturally expressed in two variants: long (GLUT9L) and short (GLUT9S), which differ from one another by their N-terminus sequence. Each of these isoforms is localized on a different pole of the epithelial cell. The signal sequence/motif responsible for this difference is presumed to be located in the aforementioned N-terminus domain. Numerous allelic variants influencing the transport properties of the protein have also been described. The first aim of this thesis is to verify the influence of a newly discovered variant, characterized by substituting of valine for leucine in the 114th position (V114L, in the short form its corresponding variant V85L) on the ability to transport uric acid. Second aim is to verify the influence of mutations in selected motifs, which could be responsible for the localization of the protein, thus also changing its transport properties. Two dileucine motifs 12LGL14 and 33LL34 and one tyrosine motif 84YIKA87 were tested. Functional studies using 14 C radiolabeled urate demonstrated significant decrease of transport ability for the V114L/V85L allelic variant in both...

Cumulus cells in the ovarian follicles emerge by differentiation from somatic granulosa cells. They are located around the oocytes in stratified clusters and their innermost layer is called the corona radiata. Cytoplasmic microvilli of cumulus cells pass through zona pellucida to the proximity of the oolema, where formation of gap junctions enables signalling and metabolic codependency. Oocytes produce factors affecting processes in cumulus cells, including their metabolism and viability. By modulating the abundance of cyclic nucleotides in oocytes, cumulus cells allow the maintenance of meiotic arrest, providing time for oocytes to finish their growth and gain competence for maturation. Because oocytes have a very limited ability to process glucose, metabolic cooperation with cumulus cells enables them to gain the energy needed for processes associated with growth and maturation. Several metabolic pathways of glucose processing create energy substrates for the formation of ATP, which they then transport to the oocytes. They also regulate the amount of lipid droplets in oocytes and protect them from cellular toxicity. An increase in the concentration of luteinizing hormone in the follicle just before ovulation induces the transmission of the signal for cumulus expansion. Interruption of the...

Internal fertilization is typical for all mammals. The male transfers sperm cells into the female reproductive tract, where usually one of them reach the oocyte and fertilize it. The female reproductive tract must be adapted to provide a suitable environment for the sperm cells to survive until fertilization. On the other hand, it must also be able to select developmentally competent sperm cells and prevent others to get into the close contact with the oocyte. Physical and molecular mutual communication of sperm cells with the female reproductive tract is ensured by sperm surface molecules and the receptors of the female epithelia lining. It appears that these interactions are required for key transformations, both on the sperm surface and in the cells of the female reproductive tract, where gene expression is affected. As a result of these changes, the most suitable sperm cell with all the required characteristics is allowed to fertilize the matured oocyte.

Left-right asymmetry of the body occurs across a number of organisms from invertebrates to vertebrates, and is mostly exhibited by the asymmetry of internal organs. These asymmetries are established at early stages of embryonic development due to the action of temporary structures called organizers of left-right asymmetry. In ray-finned fishes, the most-studied organizer is the so-called Kupffer's vesicle present in the teleosts. It is a hollow structure composed of monociliary cells. The cilia of these cells rotate and generate a leftward flow of extracellular fluid. The flow subsequently triggers the induction of the Nodal signalling cascade, which is responsible for left-right organ orientation and is considered evolutionarily conserved in vertebrates. The main participants in this pathway are the Nodal, Pitx2 and Lefty factors. In contrast to teleosts, the left-right organiser of non-teleost ray-finned fishes resembles the gastrocoel roof plate present in amphibians, which is apparently ancestral for ray-finned fishes. This bachelor thesis evaluates the origin and function of Kupffer's vesicle, describes the Nodal signalling cascade triggered by this organizer, and compares Kupffer's vesicle with the organizer of non-teleost ray-finned fishes.

A cell culture of common Sertoli and peritubular myoid cells progenitors derived from the testes of male X. tropicalis, called XtiSC, was established in the Laboratory of Developmental Biology. XtiSCs exhibit similar properties as more well-known mesenchymal stem cells that are used in cell therapy for their immunomodulatory and proregenerative properties. Microinjection of XtiSC into the dorsal vein of the tail of the tadpoles has increased macrophage numbers 7 days after tail amputation (dpa), both in controls and in tadpoles after depletion of macrophages by the application of clodrosomes. Macrophage depletion also reduces the migratory ability of XtiSC to the site of tail amputation. Macrophage depletion also led to a reduction in the number of satellite cells 1 dpa. On the seventh day after tail amputation and XtiSC injection, there was a significant increase in their number compared to the control group without XtiSC injection. Using DAF-2DA probe, nitric oxide production was confirmed by injected XtiSC and at the injury site. For future research of the proliferation, differentiation and migration of satellite cells in vivo, a vector expressing EGFP under endogenous Pax7 promoter and Katushka-RFP under gama-crystalline promoter was created and integrated into the X. tropicalis genome using...

Parkinson's disease (PD) is a neurodegenerative disorder caused by the spreading process of loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). DA neurons are a major source of dopamine (DA) in the central nervous system, the release of which plays an important role in controlling many brain functions, including mood and movement control. The induction of DA neurons depends on two signaling centers floor plate (FP) and an isthmic organizer, also known as the midbrain- hindbrain boundary (MHB) of the brain. A signaling cascade is formed by involving a large number of transcription factors that regulate the differentiation of DA neurons through their expression. Subsequent stimulation of fully mature DA neurons allows the release of dopamine in the locomotor networks of the brainstem, which causes movement. New discoveries suggest that there is a detailed consensus among the oldest and current vertebrate representatives in the organization of DA projections. This may indicate that these pathways, which have current representatives of vertebrates, evolved more than 500 million years ago.

Mammalian Target of Rapamycin (mTOR) is serin-threonin kinase, which has become a major topic in many studies in the last decade, leading to new insights into how cell works. This kinase is involved in proteosynthesis, metabolism, cell cycle regulation, proliferation and responses to nutrients and growth factors. There are certain diseases caused by mutations in mTOR gene, which lead to abnormal function of this kinase. These diseases include cancer or fertility disorders. mTOR research is also beneficial due to the search for drugs that could rescue its function and thus provide treatment for these diseases. The best-known drug is inhibitor Rapamycin and its derivates. The aim of this bachelor thesis is to summarize the knowledge about how mTOR can be regulated, the role of its substrates in cell function and to define the role of mTOR in oocyte development, translation and human health. Keywords: Oocyte, mTOR, translation, 4E-BP1, MPF, meiosis, AKT

Body appendages of vertebrates are structures developing during embryonal stages through lateral outgrowth from main body axis. Despite their different composition and high diversity, all these structures are organised along three main axes. Antero-posterior polarization is the most significant in body appendage differentiation. These polarized differentiation changes are best understood in the limbs, where they are controlled by the organizing centre named zone of polarizing activity (ZPA). This centre interacts with another centre named apical ectodermal ridge (AER), which mainly governs limb outgrowth laterally from the body axis. ZPA produces Sonic hedgehog (Shh), along with other signalling molecules participating in the polarization. Shh is a key part of highly conserved signalling pathway common to polarized structures and is influenced by retinoic acid (RA), which plays role in the ZPA creation. ZPA activity stimulates secretion of fibroblast growth factors (Fgf) and bone morphogenic proteins (Bmp). All these signalling pathways ensure correct differentiation of appendages through interaction and concentration-dependent mechanisms. The goal of this thesis is to introduce the mechanisms of polarization in other appendages, such as external genitalia and branchial arch derivates to the reader,...

The model organisms like for example the fish Danio rerio has the ability to regenerate heart muscle during its whole lifespan. Compared to Danio rerio, the ability of heart regeneration differs in amphibians. While the newt has the ability to regenerate its heart tissue throughout its whole life, the Xenopus leavis loses its power when it goes through metamorphosis. The regenerative ability does not only differ between salamanders and claws. We can observe some differences between regeneration of Xenopus tropicalis and Xenopus leavis too. Compared to Xenopus leavis, Xenopus tropicalis has the ability to regenerate its heart tissue even in adulthood. Mammals have a very limited ability to regenerate their heart muscle. We can observe the ability to reverse heart damage in mice and humans for a very limited time of a few days after they are born. In adulhood they repair the heart muscle and the rich collagen scar is formed. It is vital that signaling pathways in regeneration of model organisms is researched further, so that the knowledge gained may help us in the treatment of heart injuries in humans. Key words: regeneration, heart muscle, repair, zebrafish, mammals, amphibians, heart development, vertebrates

Monozygotic twins result from the splitting of one embryo in early embryonic development. The developmental stage, in which the splitting occurs, is the main factor determining the degree of sharing fetal sacs. The etiology and mechanism of monozygotic twinning are still unknown. The human and two species of armadillo, Dasypus novemcinctus and Dasypus hybridus are the only mammals that regularly produce monozygotic multiple pregnancies. The spontaneous occurrence of monozygotic twinning is 0,45 % of all births. The monozygotic twin pregnancies have been reported to occur at a higher rate following the increasing interest of assisted reproduction technologies. In certain consideration, we understand them as a side effect of infertility treatment because monozygotic twins result in a higher rate of prenatal mortality, premature birth and congenital anomalies than singleton pregnancies. This bachelor thesis aims to summarize knowledge about the formation of monozygotic twins in mammals. Also, it tries to discuss potential mechanisms and risk factors which could influence their formation. Monozygotic twins are an important model for explaining the genetic predisposition of some diseases. The thesis also introduces the ways of experimental production of monozygotic twins. Keywords: monozygotic twins,...

Meckel-Gruber syndrome (MKS) is rare multisystemic, autosomal recessive hereditary disorder, which appears in different places around the world. MKS is classified as a ciliopathy. These disorders are caused by defects of primary cilium, cell's signaling organelle, during embryogenesis. Meckel-Gruber syndrome represents the most severe form of ciliopathy in human population. MKS is caused by mutation in several genes, involved in correct formation of primary cilium. Until this day, 13 genes have been confirmed. As a result we distinguish 13 types of MKS. More genes are also included in MKS origin, but they do not define solo type of MKS. They are called MKS-related genes. The syndrome was recognized mainly on the basis of clinical cases. A big amount of cases was described in consanguineous families. The MKS is characterized by occipital encephaloce, polycystic kidney disease and polydactyly of hands and feet. These symptoms are common with all 13 types. Syndrome also goes with disorder of nervous and renal system, face defects or undefined genitals. Key words: Meckel-Gruber syndrome, ciliopathy, primary cilium, autosomal recesive, hereditary disease, consanguineous origin, occipital encephaloce, polycystic kidney disease, polydactyly

Bisphenol A is a widely used chemical in the manufacture of plastics. The presence of BPA in the environment adversely affects human health due to contamination of air, drinking water and food. Growing concerns about the effects of BPA have led to its regulation in production and development of alternative chemicals to BPA, such as bisphenol S (BPS). However, the effects of BPS were not properly tested before its introduction to production and the effects on human reproduction are still unknown. For this reason, it is desirable to test the effect of BPS on mammalian oocyte development. This study hypothesizes that BPS exposure causes inhibition of meiotic maturation of porcine oocytes in vitro. This study aims to investigate the potency of BPS at low concentrations corresponding to normal human exposures to selected porcine oocyte proteins. The results of this study demonstrate the negative effect of BPS on the progression of meiotic maturation and reaching the mature oocyte stage. In addition, the results show an increase in the formation of defective meiotic spindles and a disruption of mitochondrial integrity after exposure to BPS concentrations. However, the effect of BPS on double-strand breaks was not demonstrated in this study, in contrast to the case of BPA. Taken together, the results show...

African clawed frogs (Xenopus) represent an ideal model organism for study of regeneration mechanisms. In frogs, complete regeneration occurs in the tadpole stage. In later stages the regeneration capacity is lost. The Laboratory of Developmental biology was successful in establishment of cell culture called Xenopus tropicalis immature Sertoli cells (XtiSCs) derived from X. tropicalis testes. These cells are common progenitors of Sertoli cells and peritubular myoid cells. XtiSCs show similar characteristics as mesenchymal stem cells. MSCs hold interest of scientists for their immunomodulatory properties and multipotent differential and regeneration potential. In this thesis, we studied regeneration and migration potential of XtiSCs after X. tropicalis tadpole's tail amputation in developmental stage 47 - 50. Transgenic XtiSCs culture expressing RFP was prepared to facilitate transplantation experiments. Transplantation experiments showed preferential migration of XtiSCs into the site of injury. XtiSCs transplantations in X. laevis tadpoles with downregulated NO synthases eNOS and nNOS revealed their migratory dependence on nitric oxide signalization. Imunocytochemical staining of XtiSCs in vitro showed positive iNOS, nNOS and Pax7 expression. Imunohistochemical staining of tadpole's tail vibratome...

Uric acid excretion disorders are the most common cause of primary dysuricemia. The kidneys eliminate two-thirds of uric acid production and the other third is eliminated in the gastrointestinal tract. Renal reabsorption and secretion occur through the polarised epithelial cells in the proximal tubules. Uric acid transporters are expressed on these cell membranes. Reabsorption deficiency leads to hypouricemia and elevated fraction excretion associated with urolithiasis, nephrolithiasis or acute renal injury. Decreased uric acid secretion in the kidneys and small intestine leads to hyperuricemia, which develops into gout in 10% of individuals. Genome wide association studies detected a strong effect of SLC22A12 (URAT1), SLC2A9 (GLUT9) reabsorbing transporters and ABCG2 (ABCG2) secreting transporter on uric acid serum concentration variability. This thesis aimed to map out urate transporter allelic variants in a cohort of primary dysuricemia patients and identification of the variants causing defective uric acid excretion. Six non-synonymous variants were described in SLC22A12 (URAT1) and SLC2A9 (GLUT9) genes in hypouricemic individuals, which had not been identified previously in any population studies. Significant decreases in uric acid transport have been demonstrated experimentally in vitro,...

Vertebrate body appendages include a number of structures such as limbs, paired and median fins, the tail bud, the cloacal region, barbels or pharyngeal arch derivatives. Despite differences in form and function, these appendages share a number of developmental genetic mechanisms. Development of these structures is chiefly regulated by Fgf, Shh, Wnt, Tgf-β and retinoic acid signalling pathways, activity of transcription factors such as Hox or Tbx along with cis-regulatory elements controlling the genes in which these factors are encoded. A recurring feature shared by some of the herein discussed anatomical structures is a transient epithelial ridge whose activity influences production and proliferation of the adjacent mesenchyme. Some are also instructed by a signalling centre, which confers asymmetry to the structure. In this text, the various appendages are compared on the basis of their development in relation to tetrapod limbs and theories evaluating the emergence of vertebrate paired appendages. The fin fold theory and archipterygial theory are discussed in the light of recent data provided by evolutionary developmental biology. All together, this thesis evaluates shared and derived characteristics of vertebrate body appendages with the aim to provide a basis for the upcoming work on the role...

Wound healing of skin in mammals and its regeneration in Amphibians are crucial biomedicine topics in the last few decades. The most important aspect in humans is the scrarring proccess and the effort to substitute it with the regeneration producing functional and differentiated tissues. To modulate the formation of scar it is neccessary to compare both proccesses. The core animal model is axolotl (Caudata) where regeneration takes place during whole lifespam. On the other hand, in frogs (Anurans) this phenomenon is restricted up to metamorphosis. After metamorphosis, the immune system of Anurans is similar if compared to mammals. Similarly, the transition from early embryonic development in mammals, where the fetus was able to completely regenerate damaged tissue to an adult type of healing, goes hand-in-hand with the development of the immune system and structural differentiation of damaged tissue. Thus, the inflammatory cells and their regulation, the formation of ECM, which includes fibroblast proliferation, and the production of appropriate cytokines are key factors that distinguish the process of healing with and without scarring.

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

Uric acid is a main metabolite of purine degradation in humans and in higher primates. Its increased plasmatic level is called hyperuricemia and may be the cause of gout and many other similar diseases. Uricemia is controlled by many transporters, which are located in proximal tubule of human kidney. When some transporter have abnormal function, the physiological plasmatic level of uric acid may be impaired. In genome wide association study (GWAS) it was discovered that some hyperuricemia or gout patients have ABCG2 protein damaged. This protein carries out uric acid from epithelial cell to the urine. The goal of this diploma thesis is the determination of transport capacity of ABCG2 allelic variants found via GWAS (Institute of Rheumatology of 1st medical faculty UK in Prague) in vitro with Xenopus laevis oocyte expression system. Uric acid secretion was compared with wild type variant. Keywords: Uric acid, GWAS study, Xenopus laevis, membrane transport protein, ABCG2

Transdifferentiation induces chromatin reconstructions and epigenetic changes that affect gene expression spectum and cause cell remodeling in general. Direct conversion of mature somatic cell line into another mature cell type occures during the transdifferentiation thereby differences betweeen individual germ layers are eliminated. The aim of the master thesis is transdifferentation of mesenchymal cells - mouse embryonic fibroblast into endodermal cells - hepatocytes in vitro, using combination of transcripion factors Hnf4α and Foxa1. Detection of fibroblasts transformation has been initiated immediately after retroviral transduction and final generation of induced hepatocyte culture was confirmed by morphological and function analysis. The population of mouse induced hepatocytes served as a possible model for human liver disease in case of a pacient whose liver proteins could not be detected immunohistochemically. Genome editing of induced hepatocytes was realized by CRISPR/Cas9 technology which is based on cooperation of guideRNA and Cas9 nuclease followed in addition to generation of DNA-specific double strand breaks. These specific breaks in the Tight junction protein 2 gene were repaired via homologous recombination that induced a missense mutation with amino acid changes in the target...

Thanks to their ability to differentiate into variable cell types and migrate to the site of an injury mesenchymal stem cells (MSC) are broadly used in regenerative medicine. Their relative easy availability together with the property to control the immune system determines them as a cure of autoimmune diseases or a recovery of wounded tissues. Similar features posses Sertoli cells which take place in the seminiferous tubule of testis. Cell culture of testicular stem cells from juvenile male testes of X. tropicalis (XtTSC) was established in supervisor's laboratory. This cell culture showing both MSC's and SeC's properties was transformed to carry red fluorescent protein RFP. The aim of this diploma thesis was to investigate an behavior of transformed XtTSC in living organism, therefore cells were transplanted into the X. tropicalis tadpoles in stage 41. Subsequently, their migration potential was explored. To study of XtTSC's differentiation potential it was necessary to introduce a reliable sectioning techniques for the subsequent immunohistochemical analysis. Based on our experiments, we found that the XtTSC's cell culture contains precursors of SeC and peri-tubular myoid cells, however in vivo these cells turned into the dedifferentiated MSC-like state allowing a strong migration through the...

The PhD thesis is focused on the effect of porcine sperm cell extracellular ubiquitination on early embryonic development up to the blastocyst stage after ICSI. In addition, it also presents a potential improvement of the technique of in vitro fertilization using oocyte incubation with ion channels regulators. To address these aims, we established an entirely novel methodology for sperm cell sorting using flow cytometry and subsequent cryopreservation. We determined the conditions for successful sperm cell sorting based on extracellular ubiquitination rate providing highly specific selection as well as sufficient numbers of viable sperms for fertilization using the ICSI method. Concerning the following cryopreservation, established methods were optimized to enable freezing of a minimal sperm cell suspension volume with low cell numbers. The performed experiments showed a direct relationship between the rate of extracellular ubiquitination and the capability of sperms to give rise to a properly developing embryo. Highly ubiquitinated sperm cells were less successful regarding the embryonic development to the blastocyst stage if compared with the lowly ubiquitinated group (6,2 % vs. 16,7 %, P

The aim of this work was to obtain better insight into the principles of cell structures and organs in the chicken embryo development. To reach this goal special methods of micromanipulations and visualization in vitro, ex vivo, ex ovo and in ovo were implemented and adjusted. These methods were used to study gene expression in neural crest development and eye development. In the course of long term research in our laboratory we observed that oncoprotein v-Myb influences the development of the neural crest and has the capacity to change natural cell fate. We performed a series of experiments to investigate v-Myb protein influence on neural crest cells differentiation, especially melanocyte lineage development, and its influence on gene expression in the neural crest. Therefore we focused on Gremlin 2 (PRDC), the gene upregulated by v-Myb in the neural crest. The established procedure of electroporation in ovo was adjusted to transfect cells of the developing eye and used to study gene expression during lens induction. The results obtained from chicken embryo experiments endorsed the study performed on mouse embryos. Futhermore, the electroporation technique was slightly modified for manipulations of the neural retina in the developing eye in ovo. Thereafter, the retinas were processed ex vivo and...

Nitric oxide (NO) is an interesting molecule, which is involved in many important biological processes such as vasodilatation, neurotransmission, immune response and cell proliferation. This work presents the crucial role of NO during the Xenopus laevis embryonic epidermis development. The outer layer of the embryonic epidermis is composed of 4 cell types (small secretory cells, multi-ciliated cells, ionocytes and goblet cells). Embryonic epidermis composition reflects specialized epithelia such as a respiratory epithelium of mammals. Therefore, Xenopus embryonic epidermis has become a suitable model for the study of human mucosal and mucociliary epithelium and their defects. I found that NO is mainly produced in ionocytes and multi-ciliated cells by using molecular and immunohistochemical approaches. The study of molecular and cellular phenotype changes in embryos with inhibited NO production revealed the necessity of this molecule for correct formation and function of the mucociliary epithelium. NO inhibition caused reduction of specialized epidermal cell types (small secretory cells, ionocytes and multi-ciliated cells) and structural changes in multi-ciliated cells. I also showed that NO affects the development of the embryonic epidermis through the sGC-cGMP-PKG signaling pathway, probably by...

The neural crest is a transient structure formed during the neurulation which undergoes change of phenotype in the process of epithelial-mesenchymal transition. Subsequently, neural crest cells delaminate and migrate collectively and individually to their place of destination, where they differentiate into a broad repertoir of mesenchymal and non-mesenchymal cell types. This thesis aims to examine true differential potential of these cells based on crucial in vitro experiments. Neural crest cells show not only high migration potential, but also stem cells characteristics like multipotency and self-renewal capacity. I also provide answers to questions about cellular potency at the level of neural crest population and along anterior-posterior axis. It was necessary to clarify the essence of events leading to induction, specification, epithelial-mesenchymal transition and migration of neural crest cells, since they are crucial for their differentiation potential. A major role in differentiation as well as in previous processes plays the gene regulatory network which is comprised of mutually affecting signalling pathways. Studying the behaviour of migratory and post-migratory neural crest cells is important for the research of regenerative medicine and even cancer and neurocristopathic treatment.

The mission of assisted reproductive medicine is to achieve the highest success rate of implantation and the subsequent birth of a healthy child. The current standard is the transfer of only one embryo and thus there is a great effort to develop an unbiased and accurate embryo assessment methods. This bachelor thesis is an overview of several selected techniques and their advantages and disadvantages. The selection of methods included in this thesis is based on their potential contribution to embryo assessment and the number and availability of relevant studies. I tried to compare the classical morphological approach with the newly developed methods monitoring vitality biomarkers, including genetic, metabolic and proteomic analysis. Because of the lack of information concerning techniques from clinical practice and large randomized trials, the comparison of new and old methods is questionable. Nevertheless, their principal differences can be compared. Conventional morphological assessment is based on the large extent of the subjective assessment by the assessor. On the other hand biomarker monitoring methods are based on empirical data. The outlook for the future is the complementarity of biomarker monitoring to morphological assessment. Key words: early embryo, human, developmental competence,...

The gonadotropin-induced resumption of meiosis and cumulus expansion in preovulatory follicles is preceded by expression of epidermal growth factor (EGF)-like factors, amphiregulin (AREG) and epiregulin (EREG), in mural granulosa and cumulus cells. In vitro, the EGF-like peptides are also produced in cumulus cells upon stimulation by FSH. Both FSH and the EGF-like peptides stimulate resumption of meiosis and cumulus expansion in vitro via activation of a broad signaling network in cumulus cells. To define signaling pathways that drive FSH- and AREG-induced cumulus expansion and meiotic resumption, in vitro cultured pig cumulus-oocyte complexes (COCs) were treated with specific protein kinase inhibitors. The results document that FSH-stimulated, but not the AREG-stimulated resumption of meiosis, depends on the PKA and MAPK14 activities; both modes of stimulation require activation of EGFR and MAPK3/1. To characterize the effects of FSH and EGF-like peptides on gene expression in cumulus cells, transcriptomes of cumulus cells were analysed using microarray approach. Both FSH and AREG+EREG increased the expression of genes associated with regulation of cell proliferation, blood coagulation and extracellular matrix remodeling. In contrast to AREG+EREG, FSH also increased the expression of genes coding...

There are still many diseases which cannot be successfully cured today. For some of them, the answer might be a cell therapy in the form of stem cells applications. Mesenchymal stem cells (MSCs) are multipotent stem cells which can be most frequently isolated from bone marrow and fat tissues. They have several advantages compared to embryonal stem cells, such as a lower risk of tumorigenesis and fewer ethical issues with their acquisition. MSCs also offer the advantage of immunomodulation abilities and autologous transplantation options. MSCs primarily differentiate into mesodermal lines, i.e. into osteocytes, chondrocytes and adipocytes. However, their differentiation into certain cell types of ectoderm and endoderm origin can also be achieved. This bachelor thesis focuses on the characterisation of mesenchymal stem cells and their differentiation into specific cell types. It addresses the factors which induce and influence differentiation process. Identification of desirable combinations of differentiation agents and factors and establishing safe differentiation protocols is necessary for successful utilization of mesenchymal stem cells in applied medicine. Key words: mesenchymal stem cells (MSCs), differentiation, transcription factor, growth factor, multipotency

Almost 30 thousand species of the ray-finned fishes, which exist in the world today, represent approximately one half of all known vertebrates. The ray-finned fishes are divided into four distinct groups: bichirs, chondrosteans, neopterygians and teleost fishes. These groups differ significantly in their mode of embryogenesis, however, particular details are known only from few model species like danio or medaka, which all belong to the most diversified actinopterygian group, the teleost fishes. In this thesis, I summarize the current knowledge and compare the patterns of embryogenesis of all four groups of ray-finned fishes. Interestingly, their patterns of early ontogenesis are very dissimilar, such as bichirs and sturgeons undergo holoblastic cleavage of the whole egg, similar to amphibians, whereas teleost fishes evolved strictly meroblastic cleavage of the cytoplasm accumulated on the animal pole of yolk mass, which has profound effects on their further development. The meroblastic cleavage, which is described in details in zebrafish, serves as a template for studying early ontogenesis of all other teleost fishes comprising nearly 27 thousand species. The meroblastic cleavage remained preserved in all teleosts, aside from the fact that these fishes have developed many alternative reproductive...

The evolutionary relationships in Xenopus genus are intensively studied for its interspecific variability and high conservation in evolution. These characteristics possess an opportunity for comparative studying of polyploidization phenomenom on interchromosomal level and an occasion to identify the genome-forming mechanisms with cytogenetic methods. XME chromosomes (X. mellotropicalis, 2n=40) were identified via p-/q- arm length ratio in a comparison with morphometric analysis of X. epitropicalis (2n=40) chromosomes. Whole chromosome painting probes were prepared from X. tropicalis (2n=20) microdissected chromosomes and they were applied to XME metaphase spreads via optimalized Zoo-FISH. 10 chromosomal quartets were detected and one balanced non-reciprocal translocation between chromosomes XME 2 and XME 9 which must have occured in a diploid ancestor. Thus, we disprove the theory of Silurana subgenus origin via only one polyploidizatin event. Powered by TCPDF (www.tcpdf.org)

The ubiquitin-proteasomal system which is the major pathway for intracellular protein degradation is also involved in sperm quality control in the mammalian epididymis. Defective sperm become surface- ubiquitinated during epididymal passage. The level of sperm surface ubiquitination negatively correlates with their quality. Hypothetically it is possible that after fertilization, highly ubiquitinated sperm, naturally present in mammalian ejaculates, would be actively recognized by oocyte (probably via 26S proteasomal complex). Subsequent partial or total sperm degradation should negatively affect the development of the potentially defective embryo. In this study, we examined the effect of sperm ubiquitination on the early embryonic development in pig (Sus scrofa f. domestica) using the method of intracytoplasmic sperm injection (ICSI). In vitro embryonic development to the blastocyst stage after ICSI was comparable with other laboratories. In this study, no significant difference was observed in the formation of pronuclei between oocytes fertilized by lower and highly ubiquitinated sperm cells. On the other hand, significantly better embryonic development to the blastocyst stage was observed in oocytes fertilized by sperm with lower surface ubiquitination (17 %) compared with oocytes fertilized by highly...

Asymmetric segregation of cell determinants specific the cell fates in early embryonic development. In C. Elegans somatic and germ cells are separeted in early development. On the basis of the first five asymmetric divisions AB, MS, C, E, D, P4 are found. In this specifications, par genes and genes responsible for the specification of blastomers are involved. P granules are segragated to P4 cells. Xenopus as a representative of amphibians, belonging to a group with regulative embryogenesis , exhibit features of mosaic development like C. Elegans. Maternal RNAs in germ plasm determine germ cells already in oocyte. These RNAs are transported through two distinct pathways. METRO pathways is used for a transportation of mitochondrial cloud RNA. It is specific for Xcat-2, Xpat, XIsirt, Xdazl. Vg1 and VegT RNAs are involved in the second pathway called Vg1 . This RNA contribute to the specification of mesoderm and endoderm. Although mammals reveal regulative development they also exhibit some features of the mosaic embryos. As an example, in the mouse embryo, overexpressing cadherin leads to compaction at the 8 -cell stage. Then the cells of embryo are polarized. Polarized internal cells give rise to ICM. Conversely, the outer nonpolarized cells give rise to TE. Specific factor for TE is Cdx2, for ICM is...

ESCs have been of interest to many research teams since their derivation from the mouse blastocyst 30 years ago. The main reason for studying ESCs is their ability to differentiate into almost all cell types. This feature is known as "pluripotency". The pluripotent state in ESCs is maintained by the control of the gene expression. To maintain their undifferentiated state it is necessary to repress the differentiation genes. This process is controlled primarily by pluripotent transcriptional factors, especially by OCT4, SOX2 and NANOG. Silencing of the differentiation genes is also influenced by the chromatin remodelling complexes. The regulation of the gene expression leading to the cell pluripotency also takes place at the post- transcriptional level via miRNA, lncRNA, hnRNPs or proteins which stabilize pluripotent factors and protect them from degradation. The aim of this thesis is to summarize mechanisms by which pluripotency is maintained in ESC.

GAL4/UAS system is a bipartite gene engineering tool, enabling ectopic expression in temporal and tissue-specific manner in vivo. Design of this technique is based on a mechanism of gene transcription, which was elucidated of large portion by an experimental study of Saccharomyces cerevisiae regulation of metabolic control circuit for processing galactose. It is possible to generate hundreds of stable transgenic lines by independent incorporation of the gene for the transcription factor Gal4p and its binding sequence (UAS), respectively, by using transposible or specific-sequence integration techniques. An individual organism, manifesting ectopic expression in suitable, adjustable conditions, can be obtained by cross breeding individual of GAL4 lines with individual from UAS line. This thesis represents a synthesis of the basic principles of GAL4/UAS system and its variants, particularly adapted to the needs of genetic manipulation of model organisms Drosophila melanogaster and Danio rerio. Additionally, this text summarizes the connection GAL4/UAS system with other techniques and briefly highlights the potential for practical applications mainly in research area of neurology. Powered by TCPDF (www.tcpdf.org)

Acute lymphoblastic leukemia is the most common type of cancer in children. It is a very heterogenous disease in which many recurrent chromosomal abnormalities have been described. The most important chromosomal abnormalities associated with a good prognosis are t(12;21)(p13;q22) which result in ETV6/RUNX1 fusion and hyperdiploidy. On the contrary findings suggesting a poor prognosis are t(9;22)(q34;q11) leading to fusion gene BCR/ABL1, MLL rearrangements or hypodiploidy. Heteroploidy is one of the most frequent findings in childhood ALL. It is characterised by nonrandom gain or loss of chromosomes from diploid cells. One of the most important findings in childhood ALL is hyperdiploidy where a non-random gain of chromosomes is present. Hyperdiploidy has a favorable prognosis and the impact of additional structural aberations requires further research. Another prognostically important group of heteroploidy is hypodiploidy. It is a quite rare finding and has a very poor outcome. There are non-random acquired chromosome losses observed in hypodiploid cells. Hypodiploid cell line may be masked with a doubled hyperdiploid clone which makes it difficult to identify. Proper and early cytogenetical analysis of heteroploid cells is very important as it contributes assigning correct diagnosis and risk stratification,...

Spermatogonial stem cells are unipotent male germ cells which provide spermatogenesis during the whole life. In 2004, an important experiment was conducted. During in vitro cultivation mouse spermatogonial stem cells gained the characteristics of embryonic stem cells, pluripotency by all means. Those cells had the feature to spontaneously differentiate into all three germ layers, endoderm, ectoderm and mesoderm. They also could pass its genetic information to the next generation and they could give rise to teratomas. By this event, experiments started on other vertebrates including rodents, domestic animals and also human. Differentiation of these cells can be directed in vitro to generate specific cell types. On base of these facts, spermatogonial stem cells are alternative source of pluripotent cells which possess many applications in life sciences. The purpose of this thesis is to summarize actual knowledge about differentiation potential in vitro of spermatogonial stem cells in higher vertebrates and try to identify tendencies which they prefer during differentiation, if they exist. Powered by TCPDF (www.tcpdf.org)

The diploid amphibian Xenopus tropicalis represents a significant model organism for studies of early development, genes function and evolution. Such techniques as gynogenesis, injection of morpholino antisense oligonucleotide into fertilized eggs or transgenesis were established. In the recent ten years, many efforts have been made to complete the sequence information. X. tropicalis genome has been sequenced but the completion of its assembly only on the basis of sequence data has been impossible. Therefore, our first work was focused on one of approaches for a genome completing- genetic mapping. First of all, the genetic map of Xenopus tropicalis was established pursuant linkage and physical positions of markers. Since the map contained gaps, we developed a new method for genetic mapping based on the next generation sequencing of laser microdissected arm. Using Illumina next generation sequencing of fifteen copies of a short arm of chromosome 7, we obtained new insights into its genome by localizing previously unmapped genes and scaffolds as well as recognizing mislocalized portions of the genome assembly. This was the first time laser microdissection and sequencing of specific chromosomal regions has been used for the purpose of genome mapping. These data were also used in the evolution study of...

Reptiles, as a group, have a~lot of various types of sex-determining mechanisms. Evolution of sex- deter- mining mechanisms took place in different groups of reptiles by different way and got to different types of sex determination. Gene network underlying gonadal diferenciation has been investigated in many studies, for revealing of principles causing this diversity. Thanks to that, it is very difficult to determine ultimate trigger of switching between male of female fate.Interesting fenomenon is sexreversal, which is enabled by mechanism activated only by unusual circumstances. I have summerized basic facts about sex determination in reptiles in this work. Also, expresion patterns of some genes important for gonadal diferenciation in vertebrates, are recapitulated there. The last things, what this work posses, is summary of knowledge about sex-reversal in reptiles and short treatise about its evotution.

Because of their possible therapeutic potential stem cells are one of the most promising fields of study in biology. The aim of this thesis was to study the migration potential of testicular stem cells of Xenopus tropicalis, whose culture was established from the testes of juvenile males in the laboratory of my supervisor. Due to external fertilization and embryonic development Xenopus represents an ideal model organism for transplantation and microinjection experiments in-vivo. Transplantation of vital marked (PKH26) testicular stem cells into blastula and peritoneum of tadpoles showed their wide migratory potential including intestine (entoderm), heart, pronephros, genital groove (mesoderm) and epidermis (ectoderm). Based on my experiments, I found that the ideal number of cells for transplantation ranges between 250-500 per tadpole. To further characterize the stem cells, I constructed a plasmid vector carrying a gene for a red fluorescent protein. This plasmid was then used for preparation of frogs with whole-body expression of Katushka RFP under control of CAG promoter. The next aim is to gain the RFP positive offspring by crossing the transgenic individuals with the wild type. Male offspring can be used for establishing culture of testicular stem cells stably expressing the reporter gene. In this way...

Xenopus leavis was a favorite model organism during the 20th. century, but nowadays it has been replaced by diploid Xenopus tropicalis, which has not only shorter generation time, but also smaller genom. One of the disadvantages of Xenopus tropicalis is the lack of full physical and linkage map. According to JGI genome database (assembly 4.1) there are unmapped regions on short arm of the chromosome 2 and 7 . Several BAC clones ( with a single or dual-end sequence) has been found to be located within this region, according to a recent assembly 7.1. However , it isn't clear whether 100bp length of BAC ends is enough to place entire BAC clone into the genom of Xenopus tropicalis. In order to prove correct inclusion of these BAC clones into JGI database, several BAC clones, which are supposed to be located on short arm of chromosome 2, were picked. Using fluorescence in situ hybridisation, the signal of these BAC clones was localised on the short arm of chromosome 1 instead of chromosome 2 and in most cases they had opposite orientation. It means that the 100bp lenght of BAC ends propably isn't sufficient to place entire BAC clone on chromosome. New working protocol of BAC DNA isolation and labeling was established.

Spermatogonia, or spermatogonial stem cells are necessary to maintain male fertility. In the complex process of ongoing spermatogenesis in the testes these pluripotent stem cells proliferate and differentiate into sperm cells. In 1994 the first spermatogonial transplantation technique was described in rodents to allow the study of male germ cells. The following series of studies of intra- and inter-species transmission of testicular tissue revealed the regenerative capacity of transplanted spermatogonial stem cells and their possible usage. Recently, spermatogonia transplantation systems are developed in many vertebrates making it possible to study the development of sperm as well as artificial production of male and female gametes derived from germ cell donors. The differentiation potential of spermatogonial stem cells enabled the creation of transgenic organisms by genetic manipulation of isolated spermatogonia and subsequent transplantation into a suitable recipient. Spermatogonial transplantation in the future may find their application in regenerative medicine, the treatment of disorders of spermatogenesis, or serve to preservation of genetic stock of endangered species.

Uric acid is the end product of purine metabolism of humans and some higher primates. In other mammals enzyme urate oxidase is capable to further convert uric acid to allantoin. During evolution of primates several mutations in urate oxidase gene had occured, led the enzyme to decrease its activity or to complete loss of function. What was the initial impetus for the utilization of uric acid and what biochemical traits of uric acid led to a tendency to retain a major of this metabolite production in the bloodstream? Uric acid has antioxidant capabilities and it is scavanger of free radicals. Increased levels of uric acid in blood has an effect on acute and chronic elevations of blood pressure. It is possible that hyperuricemia helped to maintain blood pressure under low-salt dietary conditions of primates during the Miocene. This mechanism probably have allowed to stabilize bipedalism our ancestors. Uric acid has an important role as a neuroprotector. As inhibitor the permeability blood-brain barrier, uric acid limits the infiltration of undesirable substances to the neurons and prevents central nervous system against the formation of inflammatory diseases. Such as neurodegenerative diseases may be caused by reduced serum uric acid levels. Uric acid protects against peroxinitrite damage tissues in the...

The goal of the thesis was to characterize transcriptional profiles of in vivo and in vitro derived embryos during bovine minor and major embryonic genome activation and to identify mRNA transcripts newly synthesized during these stages. In our first work we have concentrated on the study of minor genome activation at the 4-cell stage of embryo. Using SSH, we have identified 31 amplicons homologous with already identified genes. We have selected 5 of these for detailed study of their expression during the whole period of preimplantation development: centromere protein, 350/400 kDa (CENPF, mitosin), splicing factor arginine/serine-rich 3 (SRFS3), high mobility group nucleosomal binding domain 2 (HMGN2) protein and eukaryotic translation initiation factors EIF4A2 a EIF4E. All these genes play an important role in the early embryo development. SRFS3 is the first described gene with an important function in preimplantation development, which is expressed already during bovine minor genome activation, and its transcription is α-amanitin sensitive during this period. We have selected CENPF gene for a more thorough study. By silencing its expression by the injection of CENPF dsRNA into the zygote, we have studied its function throughout the whole preimplantation development of bovine embryo....

In our Laboratory of Developmental Biology there was established a long term culture derived from Xenopus tropicalis testes. It contains pre-Sertoli cells mostly. They compose a feeder layer allowing cultivation of stem cells, revealing the morphology of spermatogonial stem cells. This diploma thesis was focused on a preparation of two growth factors, FGF2 (fibroblast growth factor 2) and GDNF (glial cell line-derived neurotrophic factor), with the subsequent characterization of their influence at cell culture mentioned above. Factors were selected on the basis of the microenvironmental niche theory, according which FGF2 and GDNF are the most important factors for spermatogonial stem cells proliferation and self-renewal. FGF2 recombinant factor was gained using the expression plasmid pET-15b. Its characterization in the testicular culture brought surprising result. Even a low concentration of FGF2 factor (2.5ng/ml) caused cell detaching and dying. Similar result was previously shown in differentiating osteoblast culture only. More experiments need to be done to prove if apoptose take place and why do testicular cells act this way. Key words: Xenopus tropicalis, FGF2, GDNF, SSC, pre-Seroli cells

1 ABSTRACT Genomic imprinting is a form of epigenetic regulation that results in gene expression from a single allele in a parent-of-origin manner. It is a form of monoallelic gene expression, another forms of monoallelic expression are X chromosome inactivation and stochastic monoallelic gene expression, so-called allelic exclusion. Imprinting affects expression of many genes which regulate growth and development. Disruption of imprinting leads to some diseases, such as Prader-Willie and Angelman syndromes, Russel-Silver and Beckwith- Wiedemann syndromes, transient neonatal diabetes, persistent hyperinsulinemic hypoglycemia of infancy and pseudehypoparathyroidism. Disruption of imprinting is implicated at some types of embryonic cancers. Imprinted genes are arranged in clusters and the expression in these domains is regulated by imprinting control regions (ICRs). Expression of imprinted genes is a complex process which underlies a strict regulatory mechanisms. DNA methylation and histone modifications are included in a molecular mechanism of imprinting. The methylation imprints are established during gametogenesis and are maintained during lifetime. Methylation marks at ICRs are essential for activity of genes localized in a cluster. Additional regulatory mechanism contributing to imprinting is non- coding...

The homeostatic self-renewal of tissues in the adult mammal organism is maintained by stem- cell activity. The majority of tissue-specific stem cells are dispersed in a tissue in a low number. The small intestinal epithelium is a suitable model tissue for study of the stem cells because of its regular structure and rapid self-renewal. One of the first knowledge about the intestinal stem cell characteristics was obtained from the experiments with the mice embryonal aggregation chimeras and the transgenic chimeras. There were obtained a crucial role of the Wnt signalization pathway in the control of dynamics of the stem cells and contextual coherent finding of the unique molecular marker protein of the intestinal stem cells Lgr5. Subsequently, the genetically modified mice with "reporter" genes produced from the Lgr5 locus became the main tool in the intestinal epithelial stem cell research. The views on dividing organization and particular identity of the intestinal stem cells are still controversial. keywords: stem cells, crypt base cells, intestinal epithelium, chimera, the Wnt signaling pathway, Lgr5, reporter mouse strains

The intracellular sperm injection (ICSI) technique is a very effective tool for the fertilization research. In the newly established laboratory at the Faculty of Science of the Charles University it was necessary to introduce this method and define the early developmental potential of fertilized oocytes. After fertilization oocytes were incubated to the blastocyst stage with a success comparable with other laboratories (17%). The ubiquitin-proteasome system which plays a major role in a protein degradation within cells is involved in a regulatory mechanism of sperm maturation. It is also responsible for a penetration of a vitelline membrane. In these processes ubiquitin residues are localized extracellulary. High level of sperm ubiquitination correlates with their low quality. Hypotetically it can be expected that the ubiqutination of impaired sperm cells can be used as a negative marker for their recognition and degradation by 26S proteasome complex localized. Experiments in this diploma thesis were designed based on the hypothesis that the executive part of the selective mechanism is the 26S proteasome. Therefore the effect of MG132 peptide inhibition of the 20S proteasome on the pronuclei formation and subsequent early embryonic development after ICSI was studied. Inhibition of 20S proteasome...

Identification of genes that start their expression in different time of embryonal development is crucial for proper understanding of early mammalian embryogenesis. Genes that are differently expressed during this period are supposed to be important for normal preimplantation development. One of these genes is nucleophosmin, the multifunctional protein, which functions among others as a transporting protein between cytoplasm and nucleoli. It is involved in duplication of centrosomes, formation of ribosomes and functions as a chaperoning protein. Nucleophosmin shuttles rapidly between the nucleolus and cytoplasm. Its localization is cell cycle dependent and reflects the development of the nucleoli. For studying the effect of nucleophosmin on the bovine preimplantation development we used RNA interference that causes degradation of the target mRNA after injection of nucleophosmin double-stranded RNA. The average decrease of nucleophosmin mRNA abundance is 86.3 % between nucleophosmin dsRNA injected embryos and control group. We detected no differences in protein expression and localization of nucleophosmin between the groups by immunofluorescence method. Embryos in all groups were able to develop until the blastocyst stage, although there were significant difference between uninjected control group and...

In mammals, the ovarian cycle is controlled by follicle-stimulating hormone (FSH) and luteinizing hormone (LH), secreted by the anterior lobe of the pituitary gland. FSH is assumed to be essential for follicle growth. LH is required for the terminal phases of follicle growth, estrogen synthesis, ovulation, development of the corpus luteum and progesterone synthesis. Although cumulus cells and oocytes lack LH-receptors, LH causes cumulus expansion and meiosis resumption. The effect of LH is mediated via EGF-like peptides - amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC). The precursors of these peptides are present in the mural granulosa cells and EGF-like peptides are released by activation of metalloproteases in response to LH-stimulus. The aim of this thesis is to present EGF-like peptides in the wide context of the molecular mechanisms regulating the late phases of folliculogenesis and meiotic maturation of oocytes.

2. Abstract Xenopus germ plasm originates in previtellogenic oocytes in an area rich in mitochondria - the mitochondrial cloud. After its first appearance, the germ plasm localises to the vegetative pole of the egg and takes the form of small islands. After fertilization, the germ plasm aggregates at the apex of the vegetal pole of the embryo and then it separates into blastomers directly surrounding the vegetal pole. The germ plasm consists of mitochondria, germinal granules, ribozomes and various ribonucleic acids (RNAs). In Xenopus, the germ plasm RNAs are required for the formation of the primordial germ cell line. XDead end is fundamental for the migration, differentiation and survival of germ cells. Embryos depleted of fatvg are defective in primordial germ cell (PGC) formation and cortical rotation and organelle transport are inhibited in these embryos. Xdazl is required for early PGC differentiation and indirectly contributes to the migration of PGCs through the endoderm. Some germ plasm proteins, for example, Fatvg and Xcat-2, also play an important role in the determination of the dorsal/ventral axis.