Aberrant epigenetic modifications are a hallmark of ageing process. To investigate the involvement of epigenetic modifications in the progress of fish oocyte ageing the present study focused primarily on the epigenetic mechanisms of DNA methylation and histone acetylation. Investigating and understanding the molecular mechanisms underlying oocyte ageing is a research gap in fish and other vertebrates.
Common carp Cyprinus carpio oocytes were aged in vitro at 20 °C and their capacity to fertilize and progress to the larval stage were evaluated. The two important cytosine epigenetic marks, 5-methyl-2´-deoxycytidine (5-mdC) and 5-hydroxymethyl-2´-deoxycytidine (5-hmdC), were analyzed in the whole genome in fresh and different aged oocytes and in the emerging embryos using liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. Additionally, we examined global histone modifications and the probable changes in the acetylation patterns of selected lysine residues on histones H3 and H4 during oocyte ageing by immunoblotting.
No significant difference was detected in the global DNA methylation between the fresh and aged oocytes. However, with the progress of oocyte ageing the genome-wide DNA hydroxymethylation decreased significantly in 30-h embryos originating from aged oocytes, as compared to those produced from freshly ovulated oocytes. Since studies have indicated the diagnostic and prognostic value of the mechanism of decreased 5-hmC in the whole genome, there might be a potential use of 5-hmC in the study of phenotypic failures associated with post-ovulatory oocyte ageing. In vitro oocyte ageing for 28 hours led to significantly increased levels of H4K12 acetylation. Furthermore, the activity of histone acetyltransferases showed an upward trend during oocyte ageing. Since 5-hmdC plays the role as a DNA demethylation intermediate, further studies on TET activity are needed. Investigation of the probable involvement of other epigenetic mechanisms such as histone methylation, the involvement of miRNAs, and the regulation of epigenetic-associated genes in the progress of oocyte ageing is of our interest for future studies.
Acknowledgments: This study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic, project: LRI CENAKVA LM2018099, and by the Czech Science Foundation (GACR No. 20-01251S).