Doctor of Philosophy (PhD)
Vitrification is a technique that has become extremely beneficial in human assisted reproduction and has great potential for the indefinite storage of genetics of domestic animals, endangered species, and research germplasm. This cryopreservation technique uses high concentration of cryoprotectants and ultra-rapid cooling rates which have been shown to cause deleterious effects to molecular, physiological, and morphological aspects of oocytes and embryos. This study aimed to improve vitrification outcomes of bovine oocytes through post-warming extended culture, EGTA addition to vitrification solutions, and supplementation of post-warming medium with resveratrol. Additionally, we explored the long term effects of vitrification by analyzing global gene expression through RNA-sequencing of elongated embryos produced from previously vitrified in vitro produced bovine blastocysts.
Results showed that extended culture reduces the incidence of abnormal meiotic spindles and can recover mitochondrial membrane potential, but not ATP content of MII bovine oocytes. The supplementation of post-warming medium with 1 µM of resveratrol reduced the incidence of abnormal meiotic spindles, rescued mitochondrial activity, and decreased ROS levels after warming. However, ATP content remained lower than the control in all oocytes that underwent vitrification. Furthermore, resveratrol supplementation increased the developmental ability of vitrified-warmed oocytes. The beneficial effects of adding EGTA to vitrification solutions for bovine oocytes are yet to be determined.
RNA-seq analysis of whole embryo or isolated trophectoderm (TE) from vitrified and fresh recovered D14 embryos revealed a total of 927 genes changed their expression as a result of vitrification, among them 782 and 145 genes were up- and down-regulated, respectively. In TE isolates, vitrification resulted in 4,096 and 280 up- and down-regulated genes, respectively. In addition, we found 671 and 61 genes commonly up- and down-regulated in both vitrified whole embryo and TE. Commonly up-regulated pathways by vitrification included epithelial adherens junctions, sirtuin signaling, germ cell-sertoli cell junction, ataxia-telangiectasia mutated (ATM) signaling, nucleotide excision repair (NER), and protein ubiquitination pathways. The commonly down-regulated pathways included Eukaryotic Translation Initiation Factor 2 (eIF2) signaling, oxidative phosphorylation, mitochondrial dysfunction, regulation of Eukaryotic Translation Initiation Factor 4 (eIF4) and P70 S6 Kinase signaling, mechanistic target of rapamycin (mTOR) signaling, sirtuin singling, and NER pathways.
Gutierrez Castillo, Emilio Jose, "Vitrification of Bovine Oocytes and Embryos" (2022). LSU Doctoral Dissertations. 5854.
Available for download on Monday, May 14, 2029