Quality and fertilization rate of oocytes matured in vitro conditions is essential for their use in biotechnology. Many morphological and functional changes occurs in oocytes during prolonged cultivation, which result in decreased fertilization rates and higher levels of apoptosis, polyspermy, parthenogenesis and chromosomal anomalies. These changes also have a negative effect on subsequent embryonic development. Understanding the mechanisms of oocyte aging could thus make a major contribution to the development of reproductive biotechnologies in livestock and human assisted reproduction.
The enzyme heme oxygenase (HO) catalyzes the oxidative cleavage of heme to biliverdin, carbon monoxide (CO) and ferrous ions. There are two isoforms of heme oxygenase, i.e. HO 1 and HO 2. HO 1 has cytoprotective properties and in the cells is expressed especially in response to stress factors. HO 2 is responsible for basal production of CO. CO belongs to gasotransmitters, which are endogenously produced signaling gaseous molecules. Meaning of HO/CO system was proven in reproductive system of both males and females. Its importance in oocytes is not known.
The aim of this thesis was to verify the hypothesis that the HO is expressed in porcine oocytes during their prolonged cultivation in vitro conditions and that the expression of both isoforms changes during aging. Oocytes were evaluated at the metaphase stage of the second meiotic division (MII) and three stages of aging according to the length of prolonged cultivation, i.e. 24, 48 and 72 hours. HO 1 and HO 2 were localized by immunocytochemistry method.
By evaluating the experiments it was confirmed that both HO isoforms are expressed during aging in all evaluated areas, i.e. in the perichromosomal area, cortical area and cytoplasm. HO 1 is located mainly in the perichromosomal area and during aging occurs statistically significant increase in signal intensity. In the cortical area and the cytoplasm was detected statistically significant difference only in oocytes aging 72 hours and the signal intensity is weaker in these areas.
In the case of HO 2 signal is evenly distributed throughout the oocyte and compared with the HO 1 the signal is significantly stronger. A statistically significant increase in signal intensity occurred between oocytes at the MII stage and the group of oocytes aging 24 hours. An expression of HO 2 compared with matured oocytes significantly increases during prolonged cultivation.
A scientific hypothesis was confirmed in the case of both HO isoforms, i.e. HO 1 and HO 2 were localized in aging porcine oocytes and at the same time there is a change in expression during prolonged cultivation. From the results of experiments it can be assumed that products of enzymatic activity of HO-1 and HO-2, including CO are also present in oocytes, and therefore HO/CO system is probably related to the oocyte aging. The assumption is that HO 1 acts as a cytoprotective factor, which could have a possitive effect on oocyte quality. HO 2 is responsible for basal production of CO, but it may also play a role in the regulation of oxidative stress in oocytes.
The data obtained should be investigated further with more experiments focused on localization and redistribution of both isoforms during oocyte aging. The subject of further research should also be a mechanism of action and specific function of HO 1 and HO 2 in oocytes.
| Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:258194 |
| Date | January 2016 |
| Creators | Poupalová, Michaela |
| Contributors | Sedmíková, Markéta, Lenka, Lenka |
| Publisher | Česká zemědělská univerzita v Praze |
| Source Sets | Czech ETDs |
| Language | Czech |
| Detected Language | English |
| Type | info:eu-repo/semantics/masterThesis |
| Rights | info:eu-repo/semantics/restrictedAccess |
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