Muscle transcriptomes of Duroc and Pietrain pig breeds during prenatal formation of skeletal muscle tissue using microarray technology

Abstract

Mammalian myogenesis is an exclusive prenatal process regulated by the muscle regulatory factor gene family, which itself is regulated by numerous other genes. We developed a microarray consisting of the clones of two muscle-specific cDNA libraries with the addition of 500 genes with known function in myogenesis and energy metabolism. Tissue samples were collected of Duroc and Pietrain prenatal litters of 14 and 21 days of age (complete embryos) and 35, 49, 63, 77, and 91 days of age (longissimus muscle tissue) and RNA was isolated. Microarrays were hybridised with pools of six RNA samples. For each age comparisons between Duroc and Pietrain breeds were made, and transcriptome profile changes in time were made for Duroc pigs. Comparison of Duroc and Pietrain prenatal muscle transcriptome expression profiles revealed differences in myogenesis regulating genes, suggesting differential timing of myogenesis between the two pig breeds. The differential development of the expression of the muscle structural genes strengthens this conclusion. Furthermore, differences in the expression of the energy metabolism genes were found. The results also suggest that the differential fat content between the Duroc and Pietrain pig breeds already starts to develop during early prenatal development. The changes in the muscle transcriptome expression profiles during Duroc prenatal muscle development shows a profile of waves of expression of (i) myoblast proliferation stimulating genes,(ii) followed by myoblast proliferation inhibiting and differentiation stimulating genes during the primary muscle fibre development, which is repeated with lower magnitude during secondary muscle fibre development. Furthermore, expression of energy metabolism genes reaches a nadir when differentiation of myoblasts into myotubes takes place. Microarray expression profiles were validated with five genes showing differential expression in the Duroc ¿ Pietrain comparison, and in the Duroc development in time studies using 18S rRNA for normalisation. The real time PCR confirmed the microarray result