74 PROTEOMIC PROFILING DURING FETAL DEVELOPMENT OF BOVINE CLONES

Abstract

Previous data have shown that bovine clones display a delay in their muscle differentiation during their first year postnatal (Jurie et al. 2009 Animal 2, 244-250). This delay could originate from perturbations in fetal muscle development as illustrated by lower numbers and degree of organization of the first generation of myotubes at 60 dpc and by their lower energy metabolism and their myosin heavy chain pattern at 260 dpc (Cassar-Malek et al. 2009 Proc. XIth ISRP abst). In order to understand the mechanisms underlying the delay in myogenesis, we have performed a comparative proteomic analysis of the semitendinosus muscle in fetuses derived from somatic nuclear transfer and their control counterparts obtained after AI at these two important developmental stages. Two-dimensional electrophoresis using a 3-10 non-linear pH gradient were performed on samples at 60 dpc (in a group of Holstein animals and a group of Charolais animals, n = 4 fetuses per lot) and at 260 dpc (in a group of Holstein animals, n = 4 fetuses per lot). Gel analysis was conducted using the image analysis SameSpots (Nonlinear Dynamics, Newcastle upon Tyne, UK). As expected, the protein profiles were visually very different between developmental stages. At 60 dpc, 463 spots common to all gels were retained for statistical analysis using the significance analysis of microarrays (SAM) method (FDR <5%; Meunier et al. 2005 Anal. Biochem. 340, 226-230). At 260 dpc, 491 spots were selected for SAM analysis. The statistical analysis revealed a small number of differential spots (9 and 10 spots, respectively, at 60 dpc in Holstein and Charolais, and 10 spots at 260 dpc Holstein). The differential spots were excised from the gels and their identification by mass spectrometry is in progress. Preliminary results are presented in Table 1. In conclusion, subtle changes in the muscle proteome were detected in clones v. controls. Some of them were related to the regulation of cell cycle/apoptosis at 60 dpc and to energy metabolism and chaperone activity at 260 dpc. The relevance of these changes will be further explored using bioinformatics tools.

Table 1.  Examples of identified spots with differential abundance between clones and controls

The authors thank C. Barboiron for excellent technical assistance.