Histological and transcriptome-wide level characteristics of fetal myofiber hyperplasia during the second half of gestation in Texel and Ujumqin sheep

Hangxing Ren 0 3 Li Li 0 1 Hongwei Su 2 Lingyang Xu 0 Caihong Wei 0 Li Zhang 0 Hongbin Li 0 Wenzhong Liu 2 Lixin Du 0 0 National Center for Molecular Genetics and Breeding of Animal, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences , Beijing, 100193 , China 1 College of Animal Science and Technology, Sichuan Agricultural University , Ya'an, Sichuan, 625014 , China 2 College of Animal Science and Technology, Shanxi Agricultural University , Taigu, Shanxi, 030801 , China 3 Chongqing Academy of Animal Sciences , Chongqing, 402460 , China Background: Whether myofibers increase with a pulsed-wave mode at particular developmental stages or whether they augment evenly across developmental stages in large mammals is unclear. Additionally, the molecular mechanisms of myostatin in myofiber hyperplasia at the fetal stage in sheep remain unknown. Using the first specialized transcriptome-wide sheep oligo DNA microarray and histological methods, we investigated the gene expression profile and histological characteristics of developing fetal ovine longissimus muscle in Texel sheep (high muscle and low fat), as a myostatin model of natural mutation, and Ujumqin sheep (low muscle and high fat). Fetal skeletal muscles were sampled at 70, 85, 100, 120, and 135 d of gestation. Results: Myofiber number increased sharply with a pulsed-wave mode at certain developmental stages but was not augmented evenly across developmental stages in fetal sheep. The surges in myofiber hyperplasia occurred at 85 and 120 d in Texel sheep, whereas a unique proliferative surge appeared at 100 d in Ujumqin sheep. Analysis of the microarray demonstrated that immune and hematological systems' development and function, lipid metabolism, and cell communication were the biological functions that were most differentially expressed between Texel and Ujumqin sheep during muscle development. Pathways associated with myogenesis and the proliferation of myoblasts, such as calcium signaling, chemokine (C-X-C motif) receptor 4 signaling, and vascular endothelial growth factor signaling, were affected significantly at specific fetal stages, which underpinned fetal myofiber hyperplasia and postnatal muscle hypertrophy. Moreover, we identified some differentially expressed genes between the two breeds that could be potential myostatin targets for further investigation. Conclusions: Proliferation of myofibers proceeded in a pulsed-wave mode at particular fetal stages in the sheep. The myostatin mutation changed the gene expression pattern in skeletal muscle at a transcriptome-wide level, resulting in variation in myofiber phenotype between Texel and Ujumqin sheep during the second half of gestation. Our findings provide a novel and dynamic description of the effect of myostatin on skeletal muscle development, which contributes to understanding the biology of muscle development in large mammals. - Background Texel sheep, a typical double muscle breed due to a GDF8 mutation [1-3], are now commercially produced throughout the world, with no adverse effects detected by objective assessments of meat quality [4]. However, evidence for an association between g+6723G > A and decreased intramuscular fat and reduced eating quality has been observed [5]. Compared with Texel sheep, indigenous Chinese Ujumqin sheep, with no GDF8 mutation [6], are less muscular and have a higher fat content, but they are superior in terms of perceived meat quality. Therefore, these two sheep breeds provide a good natural model for studying muscle and fat development, as well as for identifying myostatin genes. Prenatal skeletal muscle development is an important determinant of both muscularity and meat quality [7]. In large precocial species such as sheep [8,9] and cattle [10], the maximum myofiber complement of a muscle is achieved prior to birth. More than three waves of myogenic cells appear in sheep, and most myofibers form during the second half of gestation [11,12]. However, whether the myofibers increase with a pulsed-wave mode at certain developmental stages or whether they augment evenly across developmental stages in fetal sheep remains unclear. Myostatin, a member of the transforming growth factor-b (TGF-b) family, is predominantly expressed and secreted by skeletal muscle and functions as a negative regulator of muscle growth. Mutations in the myostatin gene lead to a hypertrophic phenotype in mice, sheep, cattle, dog, and human [1,3,5,13-18]. The effect of myostatin on gene expression in prenatal muscles at the genome-wide level was recently explored in fetal cattle [19-22], but no studies have been conducted dynamically at multiple fetal stages comparing two pure breeds with extreme phenotypes. A recent mice study demonstrated that myoblasts from embryonic and fetal stages not only had different fusion abilities, proliferation, differentiation and responses to TGF-b, phorbol ester 12O-tetradecanoylphorbol-13-acetate, and bone morphogenetic protein-4 in vitro, but they also differed in gene expression profiles [23], indicating that complicated and obvious changes in physiology and biochemistry occur during the prenatal stage in vivo. Therefore, investigating the subtle changes of the effect of a myostatin mutation on skeletal muscle development at multiple fetal stages using our experimental model is necessary. Here, we examined gene expression and myofiber development in Texel and Ujumqin sheep at the transcriptome and histological levels. Our findings contribute to understanding the dynamic effects of myostatin on the biology of prenatal skeletal muscle development in large mammals. The results also provide clues into human myopathy and obesity during prenatal stages. We also identified putative candidate genes that are valuable for meat-quality traits in farm animals. Results Histological characteristics of ovine fetal skeletal muscle development We examined the number and diameter of myofibers at each developmental stage in Texel and Ujumqin sheep via histological analysis (hematoxylin and eosin [H and E] staining) (Figures 1 and 2). Significant difference was observed in myofiber diameter between Texel and Ujumqin sheep at each development stage, except at 70 Figure 1 Changes in muscle fiber diameter throughout the five developmental stages in Texel and Ujumqin sheep. Longissimus dorsi muscle fiber diameter was examined in cross sections using hematoxylin and eosin staining. At least three animals were used to analyze each development stage in each breed. Photomicrographs of five to eight randomly selected areas were used to calculate the diameter of myofibers in each cross section of muscle examined. d. However, the myofiber diameter patterns of the two breeds were similar throughout the five developmental stages. Unexpectedly, we found that the myofiber hyperplasia profiles differed between Texel and Ujumqin fetuses (Figure 2). The proliferation of myofibers in Texel sheep was fastest at 70 and 85 d, par (...truncated)