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Transgenic RNAi in mouse oocytes
Sarnová, Lenka ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee)
RNA interference (RNAi) is double-stranded RNA (dsRNA)-mediated mRNA degradation. RNAi has been widely used to investigate gene functions. Many methods to induce transient or stable RNAi have been developed. Transient RNAi can be induced by delivering of a long dsRNA or short interfering RNAs (siRNAs). Stable RNAi may be induced by introducing plasmids expressing a long or a short hairpin RNA. Both small and long RNAs have been used to induce transient RNAi in mouse oocytes. Nevertheless, only long hairpin-expressing system has been used to trigger stable RNAi in oocytes. Although, this system appeared to be highly efficient and specific, it has several disadvantages as complicated long inverted repeat cloning or limited possibility to test these vectors in the cell culture. Here, we constructed a short hairpin-expressing vector suitable for transgenic RNAi induction in mouse oocytes. The new vector, pTMP_ZP3_sh, was derived from a lentiviral short hairpin vector selected based on comparative study of different short hairpin-expressing plasmids. The pTMP_ZP3_sh vector was tested by targeting Moloney sarcoma oncogene (Mos) mRNA, which is a common model for RNAi in mouse oocytes. We designed several candidate short hairpin sequences and tested their efficacy. Subsequently, the most efficient one was selected...
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Role and regulation of nuclear membrane during meiotic maturation of mammalian oocyte
Končická, Markéta ; Kubelka, Michal (advisor) ; Petr, Jaroslav (referee) ; Binarová, Pavla (referee)
Meiotic division of a female germ cell, an oocyte, is more prone to segregation errors and consequently to aneuploidies than meiosis of a sperm. Aneuploidies and chromosomal aberrations in oocytes increase with higher maternal age in humans and also in mice. Meiotic maturation onset is connected with activity of cyclin dependent kinase 1 (CDK1) that leads to dissociation of nuclear membrane. Moreover regulation of translation of key transcripts is necessary for proper meiotic progression. In thesis findings from four scientific publications are interpreted. We have analyzed the timing of nuclear envelope breakdown (NEBD) and polar body extrusion in mouse oocytes originating from two distinct female age groups: young (2 months old) and aged (12 months old). We found that meiotic maturation happens faster in aged females' oocytes due to early phosphorylation of Lamin A/C, a component of nuclear lamina, and rapid dissociation of nuclear membrane. Moreover aged females' oocytes presented unique characteristic invaginations of nuclear membrane and thus significantly increased circumference of the nuclear envelope compared to the oocytes from young females. These data combined with increased activity of CDK1 and Cyclin B, as well as increased translation of factors that regulate the translation itself,...
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The effects of long double-stranded RNA expression in mammalian cells.
Nejepínská, Jana ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee) ; Štefl, Richard (referee)
Double stranded RNA (dsRNA) is a foreign molecule that arises in the cell either as a by-product of viral replication or it is produced by the intramolecular or intermolecular pairing of complementary RNAs, often originating from repetitive sequences. In mammals, dsRNA can enter one of three pathways: the sequence-specific RNA silencing, the sequence-independent interferon (IFN) response, or editing by adenosine deaminases. The main focus of my PhD project was to comprehensively analyze the effects of the expressed dsRNA in mammals in the context of the whole organism. To follow this aim, we generated a construct expressing dsRNA in a form of an mRNA containing a long perfect hairpin structure. Transgenic mice ubiquitously expressing dsRNA were viable and, in contrast to the previous studies, the IFN response was not activated. In somatic cells, dsRNA was poorly processed into small interfering RNAs, did not cause transcriptional silencing in trans, and underwent low adenosine deamination without the nuclear retention. Consistent results were obtained in human cells transiently transfected with a dsRNA-expressing plasmid. On the other hand, dsRNA expression caused robust RNA interference (RNAi) in oocytes. Thus, we show for the first time that expressed dsRNA, in contrast to many other forms of...
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Phenotypic study Huntington's disease TgHD minipigs: Appearance and progress of reproductive and biochemical changes
Bohuslavová, Božena ; Motlík, Jan (advisor) ; Roth, Jan (referee) ; Petr, Jaroslav (referee) ; Krylov, Vladimír (referee)
Huntington's disease (HD) is one of the incurable and fatal diseases. HD belongs to the monogenic neurodegenerative diseases. According to the number of the CAG repetitions in the gene coding huntingtin, the onset of the disease is in childhood (5%), in the middle age, which is the most common (90%) and in the older age (5%). Beginning of the disease is manifested by changes in behavior; including problems with coordination and movement. Later, there is a psychological change. The disease leads to death. Until now, there is no effective curative treatment. In 2009, we created a model of the transgenic minipigs (TgHD) carrying the N - terminal part of the human mutant huntingtin (mtHtt) at our Institute in Liběchov. The number of offsprings and the resemblance in physiology and morphology between the pig (Sus scrofa) and humans (Homo sapiens) give us opportunities not only to study changes not only in central nerve organs, but also in peripheral tissues. The reproductive problems of TgHD boars were observed as the first phenotypic changes. Therefore, my work focuses at first on a study of the reproduction parameters of TgHD boars as well as ultrastructural, immunocytochemical and biochemical changes in testes and spermatozoa. In PhD thesis, I described in details the reproductive defects in TgHD...
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Testicular Degeneration of Transgenic Porcine Model of Huntington's Disease
Skřivánková, Monika ; Motlík, Jan (advisor) ; Roth, Jan (referee) ; Petr, Jaroslav (referee)
Huntington's disease is an autosomal dominant neurodegenerative disorder caused by an extended (≥36) CAG repeat in the huntingtin gene. Its hallmark is brain athrophy, but huntingtin is widely deposited in all tissues of the body, most notably in the brain and testes. Its pathogenic effect is conditioned by the formation of cytotoxic forms of aggregates and fragments, which occur in both brain and peripheral tissues. Testicular atrophy has been demonstrated in postmortem samples from human patients with Huntington's disease and in transgenic mouse models. We investigated reproductive decline in a large animal model of Huntington's disease. A transgenic (tgHD) minipig model was created by inserting a lentiviral vector into the genome of a pig. Vector contained a truncated form of the N terminal part of huntingtin gene. Boars of this transgenic line showed a reduced ability to produce offspring from 13 months of age. We confirmed apoptosis of seminiferous epithelial cells and Sertoli cells, and a production of morphologically damaged spermatozoa, which were unable to efficiently fertilize the oocyte under in vitro conditions. We found a reduction of mitochondrial metabolism parameters in the sperm of tgHD boars. These changes were not dependent on the age of the boars., It is directly related to the...
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RNA interference in mouse oocytes and somatic cells
Táborská, Eliška ; Svoboda, Petr (advisor) ; O´Connell, Mary Anne (referee) ; Petr, Jaroslav (referee)
RNA interference (RNAi) is a pathway, which employs Dicer to process long double stranded RNAs (dsRNA) from endogenous or exogenous sources into short interfering RNAs (siRNA). siRNAs are loaded onto Argonaute proteins to mediate sequence-specific post-transcriptional RNA targeting resulting in regulation of protein-coding genes and retrotransposons or antiviral immune response. Another small RNA pathway - PIWI-associated RNA (piRNA) pathway is suppressing retrotransposons in the germline. In mice, canonical RNAi pathway activity is negligible in somatic cells where a full-length Dicer produces gene-regulatory microRNAs (miRNA) but RNAi is highly active in oocytes, which express a truncated oocyte-specific Dicer isoform (DicerO ). DicerO lacks an N-terminal DExD helicase domain and has higher cleavage activity of long dsRNAs. Deletion of oocyte specific DicerO promoter leads to transcriptome aberrations, which include upregulation of putative RNAi targets and MT retrotransposons and, consequently, to meiotic spindle defects and female sterility. In contrast, the piRNA pathway is non-essential in mouse oocytes, potentially because of overlapping functions of RNAi. The PhD thesis aims to understand biological significance of mammalian endogenous RNAi and to explore consequences of re-activated RNAi...
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Bisphenol S influence on selected markers of meiotic maturation of porcine oocytes
Černíková, Terezie ; Petr, Jaroslav (advisor) ; Krylov, Vladimír (referee)
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...
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The development of tourism potential in Ghana using social media as a marketing tool
Petr, Jaroslav
The aim of my bachelor thesis is to analyze the development of tourism using social media as a marketing tool. The first part, the literature review, deals with the terms of world tourism, tourism in Africa, tourism in Ghana and types of social media. The second practical part deals with the current situation in which social media help in the development of tourism and which are most suitable for presentation. The practical part is based on the research of states, more specifically cities, which increased the share of tourism thanks to marketing on social networks. The practical part also includes a research questionnaire on local tourism in Ghana. The conclusion and recommendations that Ghana or the agency that represents them on social networks should be implemented based on research and analysis.
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Mechanism and risk factors for the development of monozygotic twins in mammals
Žabková, Světlana ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
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,...
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