6 GENE EXPRESSION ANALYSIS OF IN VIVO- AND IN VITRO-MATURED PORCINE METAPHASE II OOCYTES

Abstract

In vitro-matured (IVM) oocytes lack the same developmental competence as oocytes that are matured in vivo (IVV), yet no compelling explanation for this discrepancy has been provided at the molecular level. The aim of this study was to quantify and compare mRNA levels in IVM and IVV oocytes for genes from a wide variety of functional gene categories, including RNA degradation, pluripotency, epigenome modification, oocyte-specific, and apoptosis. Quantitative real-time PCR (qPCR) was used to evaluate the relative gene expression levels of 70 genes in each of 33 individual IVM oocytes from 4 different collection days and 29 individual IVV oocytes from 4 different donor animals. The qPCR data were analysed using ANOVA and significance was assigned at P < 0.05. After a multiple testing correction was applied, relative transcript abundances for 32 of the 70 genes tested were found to be significantly different (q < 0.05) between the IVM and IVV oocytes. Of these significantly different genes, 23 were higher in the IVM oocytes and only 9 were higher in the IVV oocytes. The 32 significantly differentially expressed genes were then evaluated in relation to their corresponding functional gene categories. Of particular interest, transcripts for 7/14 RNA degradation-related genes (CNOT3, DCP1A, DDX6, LSM1, PABPN1, PABPN1L, PARN) and 3/9 oocyte specific genes (BMP15, YBX2, H1FOO) were significantly more abundant in the IVM oocytes. In contrast, transcripts for 4/8 epigenetic related transcripts (ASH2l, DNMT1, EHMT2, EZH2), 2/2 apoptosis related genes (BCL2, XIAP), and 1/4 pluripotency factors (LIN28) were significantly more abundant in the IVV oocytes. Gene set enrichment analysis confirmed that, within the context of this experimental design, RNA degradation and chromatin remodelling pathways are significantly perturbed in IVM oocytes. We conclude that in vitro maturation has profound effects on transcript populations of metaphase-II oocytes, with most transcripts being higher in IVM oocytes. We expect that this data will lead to a better understanding of how we can improve the quality of oocytes that are matured in vitro as well as provide information to help to identify markers that could be indicative of oocyte quality.