Potential and limitations of bovine-specific arrays for the analysis of mRNA levels in early development: preliminary analysis using a bovine embryonic array

Abstract

New insights into the early development of large mammals are becoming available through the measurement of differential mRNA levels in oocytes and preimplantation embryos. These advances in knowledge are rapidly picking up in pace, mainly owing to the advantages brought by new molecular biology approaches being developed. The possibility of amplifying the starting material and therefore making measurements in single embryo units is now feasible. With these tools, the evaluation of variations in gene expression patterns during the preimplantation period or the impact of culture on mRNA levels is now possible. However, it is important to keep in mind that these methods still have limitations associated with sample preparation or the use of the appropriate controls. Even proper methods of analysis are very important to achieve the full benefit of the application of these tools. The present paper describes some of the potential, as well as limitations, of mRNA level analysis in early embryos, especially for microarray analysis. We have generated a bovine cDNA array (>2000 clones) that contains expressed sequence tags (ESTs) collected from various preimplantation development stages. Using this chip, we have initiated the characterisation of global mRNA level patterns of several key developmental stages from the immature oocyte to the blastocyst stage. As expected, the hybridisation results indicate very different expression profiles involving hundreds of genes when comparing oocyte and blastocyst samples to a reference mRNA sample made from a pool of ESTs from pooled somatic tissues. Although this array is still in its preliminary stage and the EST bank has not been processed to contain only unigenes, it is already a very useful tool for discovering candidate genes that may play important roles during early embryonic life.

Identification and expression analysis of genes associated with bovine blastocyst formation

“…This kit is optimised for the amplification of cDNA without altering the original expression ratios [63,64] and is widely used in combination with the subtractive hybridisation procedure to study gene expression during embryo development [4,10,30,65,66]. …”

Zygotic Genome Activation During the Maternal-to-Zygotic Transition

“…Time-course experiments have revealed transcriptome-wide changes in gene expression during the MZT in many different species (Aanes et al, 2011; Dobson et al, 2004; Hamatani et al, 2004; Harvey et al, 2013; Heyn et al, 2014; Lee et al, 2013; Paranjpe et al, 2013; Sirard et al, 2005; Tan et al, 2013; Vassena et al, 2011; Wang et al, 2004; Wei et al, 2006; Xie et al, 2010; Xue et al, 2013; Zhang et al, 2009); however, attributing these changes to active zygotic transcription can still be a challenge. Explicit techniques that distinguish bona fide zygotic gene expression from post-transcriptional regulation of the maternal contribution are thus invaluable for understanding the dynamics and extent of ZGA.…”

Gene expression analysis of bovine blastocysts produced by parthenogenic activation or fertilisation

“…Overall, 12 hybridisations, corresponding to the two developmental stages (early and expanded, hybridised separately), were performed using our customised bovine developmental cDNA microarray (BlueChip V1.3). This chip contains more than 2000 ESTs from various bovine embryo developmental stages (Sirard et al 2005). Hybridisations were performed in Slidehyb buffer (Ambion, Austin, TX, USA) at 508C for 18 h in the SlideBooster hybridisation station (Advalytix, San Francisco, CA, USA).…”

“…So we hypothesised that bovine parthenogenesis could generate better synchronisation, especially before the 16-cell stage, and would offer a great model to study developmental competence. In this study, we analysed the gene expression of activated and fertilised bovine embryos with BlueChip, a customised 2000-cDNA array developed in our laboratory and enriched in clones from various stages of bovine embryo development (Sirard et al 2005). This is the first study in cows where microarray analysis has been used to identify genes or gene networks based on different embryo status.…”