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Role of sirtuins in pronucleus formation in vitro fertilised porcine oocytes
Maryníková, Veronika ; Žalmanová, Tereza (advisor) ; Miriama , Miriama (referee)
Recently, the increasing importance of reproductive biotechnologies rises. They provide us to get higher performance of livestock or to improve treatment in human medicine. It is neccessary to have a sufficient amount of developmentaly competent oocytes and further healthy liveable embryos for in vitro culture to supply a progress of reproductive technologies. Immediately after fertilization, pronucleus formation is a key moment for
further embryonicdevelopment. Male and female pronuclei have their own pattern of histone code. For development of early embryo, it is neccessary to supply the correct pattern of histone code. NAD+-dependent histon deacetylases, sirtuins, are one of the mechanism which
plays in regulation of histone code. These family contains seven isoforms, SIRT1-7.
Based on current research, we decided for hypothesis that sirtuins are present in porcine fertilized oocytes and regulate the pronucleus formation. In this thesis, porcine COCs were culture in modificated culture medium and after 44 hr. maturation, only oocytes with extruded first polar body were chosen and used for further in vitro fertilization. Presumed zygotes were subsequently cultured with sirtuins inhibitors, nicotinamide or sirtinol. After 22 hr. of in vitro culture, zygotes were subjected by imunocytochemicaly localization of
methylated and acetylated (on lysine K9) histone H3 and image analysis.
Our results show that SIRT1 is localizated in porcine zygotes, especially in pronuclei. There are changes in acetylation and methylation H3K9 after sirtuin inhibition. Significant increase of H3K9 acetylation and decrese in H3K9 methylation are appeared. Sirtinol usage
has confirmed that the changes are result of SIRT1 action. Role of SIRT1 in histone code regulation of pronucleus formation is still not enought described in porcine.
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Histone code and its regulation during early embryonic development in pigs
Jelínková, Pavla ; Žalmanová, Tereza (advisor) ; Miriama, Miriama (referee)
Both pronuclei of the zygote undergo epigenetic changes after fertilization, which determines the quality of the zygote and successful early mammalian embryonic development. Shortly after fertilization epigenetic asymmetry among the pronuclei of the zygote is evident, while the paternal pronucleus undergoes active DNA demethylation, the DNA of the maternal pronucleus remains methylated. The male pronucleus in addition undergoes histone acetylation, whereas the histones of the female pronucleus remain methylated. Asymmetry of pronuclei and their epigenetic status predicts successful reprogramming of the genome, and thus the success of embryonic development. For the successful development of the embryo is therefore required correct formation of both of these pronuclei of the zygote and this formation of pronuclei is regulated by post-translational histone modifications called histone code. It was hypothesized that the histone code is regulated by the activity of NADP+ - dependent histone deacetylases, sirtuins.
In the experiment were used fully grown in vitro maturated pig oocytes that were fertilized with pig spermatozoa in vitro. After isolation of zygotes cultured with addition of the activator sirtuin resveratrol was performed immunofluorescence analysis of acetylated and methylated histone H3 at lysine K9 of pronuclei of the zygotes. From the results of control group asymmetry between the pronuclei of the zygote is evident; wherein the male pronucleus exhibits higher acetylation intensity contrast female pronucleus exhibits higher methylation intensity. After adding resveratrol to all experimental groups female pronucleus showed a significant increase of the methylated histone H3 at lysine K9, and contrary to the male pronucleus significant decrease of acetylated histone H3 at lysine K9.
Sirtuins are involved in the regulation of histone code in porcine zygote and it can be assumed that they also play a role during subsequent embryonic development, which is the subject of further study.
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