122 USE OF A DAY-14 EMBRYONIC ARRAY TO STUDY THE ELONGATION PHASE OF THE BOVINE EMBRYO

Abstract

In cattle, more than 30% of embryonic losses observed after artificial insemination (AI) have an early origin, coincident with a marked elongation of the trophoblast which occurs before implantation, between the 13th and 19th days of pregnancy. During this exponential growth phase, physiological interactions essential for pregnancy are established between the embryo and the uterus. Our work focuses on the identification of transcripts that regulate this key developmental period in several domestic species. For that, we generated a nylon membrane that contained 1920 gridded inserts originating from a Day-14 bovine embryo cDNA library (dbEST ID.15979; Hue et al., in preparation). Gene expression profiles in trophoblasts of increasing sizes were compared using ovoid (10–18-mm), tubular (50–60-mm), and early filamentous (140–150-mm) stages as complex probes.

Trophoblasts were collected and immediately snap-frozen. RNA extractions were performed using RNAplus (Quantum Appligene, Illkirch 67402, France). Due to the scarce amount of mRNA per embryo, amplified material was used to hybridize the array. For that, antisense-RNA (aRNA) and cDNA were generated starting from 1 μg of total RNA, as described by the MessageAmp aRNA kit instructions (Ambion, Rusin, TX 78744, USA) and according to Revel et al. (1995 Zygote 3, 241–250). Five hundred nanograms of aRNA or cDNA were random-primed and labelled with ³³P-alpha-dATP [aRNA, according to the procedure of Decraene et al. 1999 BioTechniques 27, 962–966; cDNA using the Atlas SMART Probe Amplification kit, (Clontech, Osyme, Saint Quentin Yvelines 78053, France)]. For each protocol, two probes were generated independently and each of these probes was hybridized to four identical membranes according to Clontech instructions. These were then exposed to phosphoscreens and scanned after 7 days. Quantifications were done using ImaGene 5.1 (BioDiscovery, El Segundo, CA 90245, USA) and statistically analyzed with the AnovArray package freely available for non-commercial use at http://www.jouy.inra.fr/stat/AnovArray (Piot et al. 2004 Bioinformatics, submitted). Reproducibility of the two protocols used to amplify material (aRNA and cDNA) was confirmed by slot blot quantifications before labelling. The hybridization profiles generated for each protocol (8 membranes per stage) were also highly reproducible (0.95 < r < 0.97), allowing a global statistical analysis with the AnovArray package. The results of the analysis of variance (ANOVA), including the correction for False Discovery Rate (FDR < 0.05), led to the identification of several bovine ESTs with unknown function that are differentially expressed during the rapid phase of trophoblastic elongation. Since genes, already known to be involved during elongation (IFN tau, Kunitz inhibitor), were also found differentially expressed in this study, this genomic approach using amplified complex probes is reliable to search for new markers of early developmental stages in cattle. Additionally, a thorough analysis of those markers may define them as interesting tools to assess the quality of embryonic development after AI, IVF (in vitro fertilisation), or SNT (somatic nuclear transfer).

This work was supported by EU (BOI4-CT95-0190) and INRA-AGENA (AIPP00183) grants.