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Změna abundance mRNA, oxidativních produktů a aktivit antioxidačních enzymů spojených se stárnutím rybích oocytů
MOHAGHEGHI SAMARIN, Azadeh
In fish, delayed spawning in nature, delayed egg collection in capture and delayed fertilization after egg stripping lead to excessive oocyte ageing and finally over-ripening. Until now there has been only poor understanding about the processes and underlying mechanism of oocyte ageing in fish as well as other vertebrates. Some studies on other vertebrates have suggested the oxidative stress as initiating factor for the progress of oocyte ageing. The present work investigated possible changes in the mRNA levels of transcripts involved in oxidative damage, mitochondrial function and stress response, with roles in fertilization, embryo development, transcriptional regulation and cell cycling as well as the ones related to the apoptosis during the egg over-ripening. This study investigated the oxidation status of oocytes during post-ovulatory ageing by measuring TBARs as the marker of lipid oxidation and carbonyls which shows the extension of protein oxidation. In addition the role of oxidative stress in the progress of oocyte ageing was assessed by evaluation the activity of antioxidant enzymes, CAT, SOD, GPX and GR. Possible changes in lipid class and fatty acid composition during fish oocyte aging were also examined. Egg viability parameters and larval ploidy levels were examined. Complete loss of egg viability for African catfish (Clarias gariepinus) occurred at 16 and 24 Hours Post Stripping (HPS) when eggs were stored at 25°C and 4°C respectively. Under both storage temperatures, the embryo mortality and larval malformation rates increased significantly over time and were the highest in the most aged oocytes. We determined the time in which the eggs retain their fertilizing ability 10 hours for tench (Tinca tinca) at 20 °C, 18 hours for goldfish (Carassius auratus) at 20 °C and 14 hours Post Ovulation (HPO) and more than 10 Hours Post Stripping (HPS) for common carp (Cyprinus carpio) at 20 °C. Our results demonstrated no significant changes in the mRNA levels of oxidative stress related genes and genes involved in cell cycling during the progress of oocyte ageing in any of species; African catfish, goldfish and common carp. Additionally, with elapsing time following ovulation the amount of TBARs and carbonyls, did not change in any of the oocytes from our experimented species; African catfish, tench, goldfish and common carp. However an increase in the mRNA abundance of apoptotic related genes were observed. Antioxidant enzyme analysis indicated no significant changes in the activity of CAT and SOD during post-ovulatory ageing of common carp, goldfish and tench oocytes. However, a significant decrease in the activity of GPX was observed during post-ovulatory ageing of tench oocytes which could play a major role in the overall drop of egg quality. This observation in GPX activity was not observed in common carp and goldfish oocytes. Besides the direct effects of oxidative stress on oocytes, also changes of fatty acid and lipid composition caused by oxidation could affect the functionality. We observed that post-ovulatory ageing of the oocytes does not change the fatty acids and lipid class composition of the oocytes. Therefore, according to our obtained results, oxidative stress is not the main initiator or promotor of the oocyte ageing process. However, complementary tests and analysis are required to clearly clarify its involvement. Increased mRNA levels of apoptotic related transcripts during oocyte ageing in this study demonstrates that apoptotic pathway might be involved in the molecular changes during the progress of oocyte ageing. Investigation of epigenetic changes associated with fish oocyte ageing seems to be interesting for future research works.
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