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Effect of the sperm ubiquitination in the early embryonic development in pig
Petelák, Aleš ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
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...
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Use of interspecific ICSI method for assessing the quality of the retained biological material from genetic resources
Vacková, Kristýna ; Benc, Michal (advisor) ; Barnetová, Irena (referee)
Intracytoplasmic sperm injection, or ICSI, is a commonly used method in assisted reproduction centers and is also an effective tool for a fertilization research. The interspecific ICSI is the suitable method for production of zygotes, which are formed by association of gametes of different, distantly unrelated species. It can bypass natural interspecies barriers and create so-called interspecific zygotes. This can be used to evaluation properties of genetic material. In this work, this method was used for evaluation of the quality of stored sperm originating from male goats of the white shorthair goat genetic resource. The aim of this work was to evaluate the effect of cryopreservation on the quality of goat sperm, which was frozen in three different ways at two different breeding stations. Freshly collected semen served as a control group. The heads of xenogeneic goat sperm were injected into mature mouse oocytes. The injected oocytes were allowed to develop to the zygote stage, in which both female (mouse) and male (goat) pronuclei was formed. In male pronuclei, DNA damage was assessed by immunofluorescence staining with anti-γH2AX antibody, a marker of DNA breaks. The results obtained by measuring the level of γH2AX fluorescence intensity in the paternal pronuclei show that the values differ...
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The impact of intracytoplasmic sperm injection on succesful development and fertility in selected mammal species
Kaufman, Jonáš ; Petelák, Aleš (advisor) ; Petr, Jaroslav (referee)
5 Abstract This thesis aims to summarize the current knowledge about the development and reproductive capacities of individuals conceived by artificial conception, especially intracytoplasmic sperm injection (ICSI), and to make a comparison with naturally conceived ones. The focus is primarily on human assisted reproduction, even though scientific work with human gametes and embryos faces ethical difficulties. However, the main objective is to determine whether ICSI has a negative effect on offspring, partially using results from animal models. Furthermore, this thesis compares birth outcomes, incidence of birth defects, cognitive and reproductive abilities and both genetic and epigenetic integrity of individuals conceived naturally with those conceived via ICSI. As of yet, according to available information, ICSI appears to be a safe artificial method of reproduction. Poor results may often either be explained by the reduced fertility of parents, or their significance is negligible. Keywords: artificial fertilization, ART, ICSI, reproduction, offspring, mouse, livestock, human
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Effect of sperm ubiquitination on the early embryonic development in pig
Kroumanová, Kristýna ; Krylov, Vladimír (advisor) ; Nevoral, Jan (referee)
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...
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Effect of the sperm ubiquitination in the early embryonic development in pig
Petelák, Aleš ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
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...
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In vitro embryo production in horses
Babická, Dominika ; Šichtař, Jiří (advisor) ; Šimoník, Ondřej (referee)
The aim of this work is to write a literature review covering the latest findings in the field of in vitro embryo production in horses. In order to perform in vitro embryo production, you first need to know the anatomy of a mare and management of sexual activity. Reproductive systém of mares consists of two ovaries, two oviducts, uterus, vagina and vulva. Unlike other livestock, the development of oocytes is in the cortex of the ovary. Sexual activity is driven neurohumoral and production of hormone is ensured by the central nervous system and endocrine glands.
For in vitro embryo production must be obtained first oocytes. These are collected from either the living or the dead mares. For live mares are used ovum pick up (OPU, transvaginal ultrasound guided aspiration), standing flank laparotomy or transcutaneous flank puncture. OPU is a noninvasive method, where each follicle is flushed several times, and in the obtained solution is looked for oocytes. Invasive, but a very successful method for obtaining oocytes is transcutaneous flank puncture. There are three ways for collecting oocytes from abbatoir ovaries - either by slicing ovaries or curettage or aspiration. Very good results in amounts of obtained oocytes was proved by slicing ovaries. First, after collecting oocytes must be sorted according to cumulus-oocyte complex (COC) on compact, expanded or degenerating oocytes. Compact and expanded oocytes are distinguished under a microscope based on the morphology of the edges of this COC. Compact oocytes have a clear line margins, while margins of expanded oocytes are deformed and have heterogeneous cytoplasm. Best results for the production of embryos exhibit expanded oocytes. First, it has to be matured in the medium to the stage of MII. The culture system for oocyte maturation consists of the M199 with Earl's salts, 10% fetal bovine serum and 5 mU - ml of bovine follicle stimulating hormone in a humidified atmosphere of 5% CO2 in air at 38.2 ° C. It was found out that regardless of the duration of cultivation, results of development of blastocysts after 24 or 48 hours were the same.
Standard IVF in horses can not be performed due to the zona pellucida and sperm capacitation. Therefore it is used intracytoplasmic sperm injection (ICSI), when one immobilized sperm is injected directly into the oocyte. Before ICSI sperm capacitation must be done by using the calcium ionophore. Oocytes were further cultured in vitro to the blastocyst stage. Another possibility is to transfer the fertilized oocyte directly to the uterine horn of the mare, or it is used cryopreservation for future.
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Ethical problems of reproductive medicine
STANISLAVOVÁ, Lucie
This thesis is aimed at the ethical aspect of reproductive medicine. I tried to outline particular dilemmas dealt with by individual religions in context with medical knowledge. Elaboration of the religious part was also supported by knowledge of experts in the field. The aim of the thesis was to outline the ethical aspect of reproductive medicine. The research was aimed at showing whether young couples see ethical problems in artificial insemination nowadays and whether the ethical aspect of the issue would discourage them from this step. Quantitative method was used for the research. The method of questioning in the form of non-standardized questionnaire was chosen for the research technique. It was designed for people at productive age (from under 23 years of age through over 41) living in marriages and their partners, who are childless or have just one child. The research has shown that nearly full majority of the pairs agree with homologous insemination; however I was convinced that they might have objections against heterologous variation of artificial insemination. Nevertheless the research as well as the consequent discussion showed that the desire for a child overrides everything else at the moment, including the standpoints of the churches the pairs support and their ethical objections. The research showed the fact that a certain number of couples, if they accepted the heterologous variation of artificial insemination would prefer the donator{\crq}s anonymousness to be eliminated. However there is a question whether the anonymous donators themselves would have the same opinion. I see the contribution of this thesis in showing that desire for a child is so strong that people are ready to put their whole-life religion aside. Another plus in my opinion is at least a basic outline of opinions on the artificial insemination issues and possible objections against its heterologous variant.
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Health and social aspects of infertility and its treatment
ZAVADILOVÁ, Zuzana
Infertility is the inability to achieve a pregnancy. WHO classify infertility into two groups. Primary infertility is the term used to describe a couple that has never been able to conceive a pregnancy, after at least 1 year of unprotected intercourse. Secondary infertility describes couples who have previously been pregnant at least once, but have not been able to achieve another pregnancy. Causes of infertility include a wide range of physical as well as emotional factors. Approximately 30 - 40% of all infertility is due to a "male" factor such as retrograde ejaculation, impotence hormone deficiency, environmental pollutants, scarring from sexually transmitted disease, or decreased sperm count. A "female" factor -- scarring from sexually transmitted disease or endometriosis, ovulation dysfunction, poor nutrition, hormone imbalance, ovarian cysts, pelvic infection, tumor or transport system abnormality from the cervix through the fallopian tubes -- is responsible for 40 - 50% of infertility in couples. The remaining 10 -30% of infertility cases may be caused by contributing factors from both partners, or no cause can be identified. This dissertation has two main goals. The first goal is to describe how a couple face up to their problems with infertility. The second goal is that this dissertation can be used as a study material for social and medical employees. This dessertation is based on a qualitative research. I used a method CAMI {--} communication through the use of computer. The observational goup were people disccusing at www.neplodnost.cz.
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