Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

Abstract

Background: Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis.

Results: 8489 transcripts were detected across the two oocyte groups, of which ~25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of α-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation.

Conclusion: Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.

Figure 1

Classification of the differentially expressed transcripts based on gene ontology. (A) Biological process (a and b); (B) Molecular functions (c and d); and (C) Cellular components (e and f)

Figure 2

qPCR analysis of selected genes from the list of over-expressed transcripts. Expression of MII oocytes (black bars) was compared to GV oocytes (white bars). All expression levels are relative to the level of expression in GV oocytes (white bars) which has been arbitrarily set to one-fold. Stars denote statistical difference * = p < 0.05, **= p < 0.01, ***= p < 0.001.

Figure 3

qPCR analysis of selected genes from the list of under-expressed transcripts. Expression of MII oocytes (black bars) was compared to GV oocytes (white bars). All expression levels are relative to the level of expression in MII oocytes (black bars) which has been arbitrarily set to one-fold. Stars denote statistical difference * = p < 0.05, **= p < 0.01, *** = p < 0.001.

Figure 4

Relative abundance of specific transcripts in bovine oocyte matured with or without α-amanitin. The figure shows GV (white), MII (black), MII oocytes matured in the presence of α-amanitin after 3hr of culture (line spotted), MII oocytes matured in α-amanitin for 24 hr (dot spotted). All expression levels are relative to the level of expression in MII oocytes (black bars) which has been arbitrarily set to one-fold

Figure 5

Relative gene expression profiles of in vitro mature bovine oocytes at different time points. In all cases the expression at time 0 h was taken as calibrator against which the relative levels of other time points were calculated. Time points with the same letter are not significantly different (p < 0.05).

Figure 6

Relative gene expression profiles of bovine oocytes, before (GV) and after (MII) in vivo maturation. In all cases the expression at time immature (GV) stage was taken as calibrator against which the relative levels of other time points were calculated. ** indicates significance (p < 0.01) and *** indicates significance (p < 0.0001). Due to the insignificant levels of LUM in immature oocytes an arbitrary 40 Cq values were assigned to calculate the relative fold change.