Effect of vitrification on the mRNA transcriptome of bovine oocytes

Abstract

Vitrification has been shown to decrease the developmental capacity of mammalian oocytes, and this is closely associated with the abnormal mRNA expressions of vitrified oocytes. However, the effect of vitrification on transcriptional machinery of oocytes examined by RNA sequencing (RNA-seq) has yet to be defined. In the present study, the mRNA transcriptomes of fresh and vitrified bovine oocytes were analysed by Smart-seq2 with the differently expressed genes determined by DEseq2 (an adjusted p-value of .05 and a minimum fold change of 2). The differentially expressed mRNAs were then searched against the Gene Ontology (GO) and Genomes (KEGG) database. Finally, the mRNA expressions of 10 candidate genes were validated using quantitative real-time PCR (qRT-PCR). Approximately 12,000 genes were detected in each sample of fresh or vitrified oocytes. Of these, the expression levels of 102 genes differed significantly in vitrified groups: 12 genes mainly involved in cell cycle, fertilization and glucose metabolism were upregulated, and 90 genes mainly involved in mitochondria, ribosomal protein, cytoskeleton, transmembrane protein, cell cycle and calcium ions were downregulated. GO analysis showed that these genes were mainly enriched in terms of membrane-bounded organelles, macromolecular complex, and intracellular part. The mRNA expression levels of 10 candidate genes selected randomly were in agreement with the results of the RNA-seq. In conclusion, our results showed that vitrification affected the mRNA transcriptome of bovine oocytes by downregulating genes, which contributed to the decreased developmental capacity of vitrified oocytes. Our findings will be useful in determining approaches to improve the efficiency of vitrified oocytes.

Probing lasting cryoinjuries to oocyte-embryo transcriptome

“…Previous studies on slowly frozen-thawed and vitrified MII oocytes revealed that both cryopreservation methods lead to the reduction in the mRNA content of some genes involved in chromosomal structure maintenance, DNA repair, cell-cycle regulation, cellular response to stress, and ubiquitination pathway [34][35][36]. Although there are similarities between the findings of the studies mentioned above and our results, a number of dissimilarities also exist.…”

“…These dissimilarities are probably due to differences in the timing of the transcriptome analysis. The aforementioned three studies [34][35][36] assessed the transcript levels at the MII stage shortly after cryopreservation, whereas our study examined the transcriptome at the four-cell stage after activation of embryonic genome. Since the MII oocytes are normally transcriptionally silent, the up-and downregulated genes reported in the previously published three studies could be explained by the following possibilities: (1) increased degradation of many transcripts by cryopreservation-associated processes (downregulated ones); (2) compared fresh controls, slower degradation of a small number of mRNAs in cryopreserved oocytes resulting in their detection at a higher level (upregulated ones); and (3) demethylation of promoters of some genes by cryopreservation leading to their premature transcription (upregulated ones).…”

Resveratrol improved the developmental potential of oocytes after vitrification by modifying the epigenetics

“…Despite the successful results with vitrification, there are studies indicating various structural, biochemical, and molecular changes caused by vitrification which affect oocyte quality (Bulgarelli, Vireque, Pitangui-Molina, Silva-De-Sá, & Ac, 2017;Liu et al, 2016;Pitchayapipatkul et al, 2017;Wang et al, 2017). Although high survival rates can be achieved by vitrification protocols, these do not necessarily signify developmental competence after fertilization and during embryogenesis (Coticchio, Bonu, Bianchi, Flamigni, & Borini, 2005).…”

RNA-Seq transcriptome profiling of mouse oocytes after in vitro maturation and/or vitrification

“…However, many of the oocytes they used were unfertilized MII stage oocytes after ICSI, and the time of oocyte collection varied. Recently, a study using RNA-seq showed that vitrification of bovine oocytes matured in vitro exhibited changes in the expression of some genes; however, the oocytes they used came from bovine ovaries obtained from a local abattoir 27 , and the different genetic background among these oocytes should not be ignored. In this study, oocytes obtained from CD-1 mice (all of the same genetic background), could better explore the influence of vitrification, and no significant difference in the transcription of oocytes after vitrification was found.…”