Semester of Graduation
Master of Science (MS)
While oocyte vitrification has become a common practice, it still faces some challenges such as the low survival rates after warming, probably related to cryoinjuries and cryoprotectant (CPA) toxicity. Evidence suggests that vitrification might have an effect on the patterns of some epigenetic marks including DNA methylation and histone acetylation. During fertilization and embryogenesis, key events for healthy and adequate embryo development take place, not only governed by the information contained within the DNA sequence, but also by epigenetic mechanisms.
This study was aimed at determining the effect of vitrification and CPA exposure, using a combination of ethylene glycol (EG) with either DMSO or glycerol (Gly), on DNA methylation and histone acetylation of bovine oocytes. Additionally, the effect of vitrification and cryoprotective solutions on the meiotic spindle was evaluated. To achieve this goal, three experiments were carried out. The first experiment was intended to evaluate the effect of vitrification on DNA methylation of bovine cumulus-oocyte complexes (COCs) at two different maturation stages, germinal vesicle (GV) and metaphase II (MII). The second was designed to determine the effect of CPA exposure on DNA methylation and histone acetylation. The last experiment assessed the impact of vitrification and CPA exposure on microtubule distribution and chromosome arrangement, and if a subsequent incubation period after vitrification could promote the reorganization of the spindle. Results obtained suggest that vitrification of bovine oocytes at the GV stage does not have an effect on DNA methylation patterns. Similar outcomes were obtained when comparing oocytes in the MII vitrified with DMSO and fresh oocytes. However, oocytes vitrified with Gly showed an abnormality presented in the form of DNA fragmentation. On the other hand, exposure to EG + DMSO increases the levels of DNA methylations in comparison with fresh oocytes. CPA exposure does not have an effect on histone acetylation levels. Finally, results of the third experiment indicate that CPA exposure has no impact on the incidence of abnormal meiotic spindles. In contrast, vitrification using DMSO increases the occurrence of abnormal meiotic spindles and the damage seems to be irreversible. The incubation period following vitrification with EG + Gly promotes the reorganization of microtubules.
Gutierrez-Castillo, Emilio Jose, "Effect of Vitrification on Epigenetic Modifications and the Meiotic Spindle of Bovine Oocytes" (2018). LSU Master's Theses. 4673.