A visibly distinct muscular hypertrophy (mh), commonly known as double muscling, occurs with high frequency in the Belgian Blue and Piedmontese cattle breeds. The autosomal recessive mh locus causing double-muscling condition in these cattle maps to bovine chromosome 2 within the same interval as myostatin, a member of the TGF-␤ superfamily of genes. Because targeted disruption of myostatin in mice results in a muscular phenotype very similar to that seen in double-muscled cattle, we have evaluated this gene as a candidate gene for double-muscling condition by cloning the bovine myostatin cDNA and examining the expression pattern and sequence of the gene in normal and double-muscled cattle. The analysis demonstrates that the levels and timing of expression do not appear to differ between Belgian Blue and normal animals, as both classes show expression initiating during fetal development and being maintained in adult muscle. Moreover, sequence analysis reveals mutations in heavy-muscled cattle of both breeds. Belgian Blue cattle are homozygous for an 11-bp deletion in the coding region that is not detected in cDNA of any normal animals examined. This deletion results in a frame-shift mutation that removes the portion of the Myostatin protein that is most highly conserved among TGF-␤ family members and that is the portion targeted for disruption in the mouse study. Piedmontese animals tested have a G-A transition in the same region that changes a cysteine residue to a tyrosine. This mutation alters one of the residues that are hallmarks of the TGF-␤ family and are highly conserved during evolution and among members of the gene family. It therefore appears likely that the mh allele in these breeds involves mutation within the myostatin gene and that myostatin is a negative regulator of muscle growth in cattle as well as mice.[The sequence data for bovine myostatin has been submitted to GenBank under accession no. AF019761.]The muscular hypertrophy (mh), or double-muscle phenotype, is a heritable condition in cattle that primarily results from an increase in number of muscle fibers (hyperplasia) rather than the enlargement of individual muscle fibers (hypertrophy), relative to normal cattle (Hanset et al. 1982). The relative increase in fiber number is observed early in pregnancy (Swatland and Kieffer 1974) and results in a calf possessing nearly twice the number of muscle fibers at the time of birth. The occurrence of double muscling has been observed in several cattle breeds worldwide since it was first documented by Culley in 1807. The breed in which this muscular hypertrophy and its effects have been analyzed most extensively is the Belgian Blue breed, which has been systematically selected for double muscling to the point of fixation in many herds. Domestic animals other than cattle also show dramatic increases in muscle mass. Malignant hyperthermia of pigs with muscular hypertrophy (Brenig and Brem 1992) and muscle hypertrophy of cats associated with a dystrophin deficiency (Gaschen et al. 1992) have been anal...

Myostatin, a member of the transforming growth factor-␤ (TGF-␤) superfamily, has been shown to be a negative regulator of myogenesis. Here we show that myostatin functions by controlling the proliferation of muscle precursor cells. When C 2 C 12 myoblasts were incubated with myostatin, proliferation of myoblasts decreased with increasing levels of myostatin. Fluorescence-activated cell sorting analysis revealed that myostatin prevented the progression of myoblasts from the G 1 -to S-phase of the cell cycle. Western analysis indicated that myostatin specifically up-regulated p21Waf1, Cip1 , a cyclin-dependent kinase inhibitor, and decreased the levels and activity of Cdk2 protein in myoblasts. Furthermore, we also observed that in myoblasts treated with myostatin protein, Rb was predominately present in the hypophosphorylated form. These results suggests that, in response to myostatin signaling, there is an increase in p21 expression and a decrease in Cdk2 protein and activity thus resulting in an accumulation of hypophosphorylated Rb protein. This, in turn, leads to the arrest of myoblasts in G 1 -phase of cell cycle. Thus, we propose that the generalized muscular hyperplasia phenotype observed in animals that lack functional myostatin could be as a result of deregulated myoblast proliferation.

Management, nutrition, production, and genetics are the main reasons for the decline in fertility in the modern dairy cow. Selection for the single trait of milk production with little consideration for traits associated with reproduction in the modern dairy cow has produced an antagonistic relationship between milk yield and reproductive performance. The outcome is a multi-factorial syndrome of subfertility during lactation; thus, to achieve a better understanding and derive a solution, it is necessary to integrate a range of disciplines, including genetics, nutrition, immunology, molecular biology, endocrinology, metabolic and reproductive physiology, and animal welfare. The common theme underlying the process is a link between nutritional and metabolic inputs that support complex interactions between the gonadotropic and somatotropic axes. Multiple hormonal and metabolic signals from the liver, pancreas, muscle, and adipose tissues act on brain centers regulating feed intake, energy balance, and metabolism. Among these signals, glucose, fatty acids, insulin-like growth factor-I, insulin, growth hormone, ghrelin, leptin, and perhaps myostatin appear to play key roles. Many of these factors are affected by changes in the somatotropic axis that are a consequence of, or are needed to support, high milk production. Ovarian tissues also respond directly to metabolic inputs, with consequences for folliculogenesis, steroidogenesis, and the development of the oocyte and embryo. Little doubt exists that appropriate nutritional management before and after calving is essential for successful reproduction. Changes in body composition are related to the processes that lead to ovulation, estrus, and conception. However, better indicators of body composition and measures of critical metabolites are required to form precise nutritional management guidelines to optimize reproductive outcomes. The eventual solution to the reduction in fertility will be a new strategic direction for genetic selection that includes fertility-related traits. However, this will take time to be effective, so, in the short term, we need to gain a greater understanding of the interactions between nutrition and fertility to better manage the issue. A greater understanding of the phenomenon will also provide markers for more targeted genetic selection. This review highlights many fruitful directions for research, aimed at the development of strategies for nutritional management of reproduction in the high-producing subfertile dairy cow.

Myostatin is a secreted growth and differentiating factor (GDF-8) that belongs to the transforming growth factor-beta (TGF-beta) superfamily. Targeted disruption of the myostatin gene in mice and a mutation in the third exon of the myostatin gene in double-muscled Belgian Blue cattle breed result in skeletal muscle hyperplasia. Hence, myostatin has been shown to be involved in the regulation of skeletal muscle mass in both mice and cattle. Previous published reports utilizing Northern hybridization had shown that myostatin expression was seen exclusively in skeletal muscle. A significantly lower level of myostatin mRNA was also reported in adipose tissue. Using a sensitive reverse transcription-polymerase chain reaction (RT-PCR) technique and Western blotting with anti-myostatin antibodies, we show that myostatin mRNA and protein are not restricted to skeletal muscle. We also show that myostatin expression is detected in the muscle of both fetal and adult hearts. Sequence analysis reveals that the Belgian Blue heart myostatin cDNA sequence contains an 11 nucleotide deletion in the third exon that causes a frameshift that eliminates virtually all of the mature, active region of the protein. Anti-myostatin immunostaining on heart sections also demonstrates that myostatin protein is localized in Purkinje fibers and cardiomyocytes in heart tissue. Furthermore, following myocardial infarction, myostatin expression is upregulated in the cardiomyocytes surrounding the infarct area. Given that myostatin is expressed in fetal and adult hearts and that myostatin expression is upregulated in cardiomyocytes after the infarction, myostatin could play an important role in cardiac development and physiology.

Myostatin is a negative regulator of myogenesis, and inactivation of myostatin leads to heavy muscle growth. Here we have cloned and characterized the bovine myostatin gene promoter. Alignment of the upstream sequences shows that the myostatin promoter is highly conserved during evolution. Sequence analysis of 1.6 kb of the bovine myostatin gene upstream region revealed that it contains 10 E-box motifs (E1 to E10), arranged in three clusters, and a single MEF2 site. Deletion and mutation analysis of the myostatin gene promoter showed that out of three important E boxes (E3, E4, and E6) of the proximal cluster, E6 plays a significant role in the regulation of a reporter gene in C 2 C 12 cells. We also demonstrate by band shift and chromatin immunoprecipitation assay that the E6 E-box motif binds to MyoD in vitro and in vivo. Furthermore, cotransfection experiments indicate that among the myogenic regulatory factors, MyoD preferentially upregulates myostatin promoter activity. Since MyoD expression varies during the myoblast cell cycle, we analyzed the myostatin promoter activity in synchronized myoblasts and quiescent "reserve" cells. Our results suggest that myostatin promoter activity is relatively higher during the G 1 phase of the cell cycle, when MyoD expression levels are maximal. However, in the reserve cells, which lack MyoD expression, a significant reduction in the myostatin promoter activity is observed. Taken together, these results suggest that the myostatin gene is a downstream target gene of MyoD. Since the myostatin gene is implicated in controlling G 1 -to-S progression of myoblasts, MyoD could be triggering myoblast withdrawal from the cell cycle by regulating myostatin gene expression.Myostatin is a newly identified member of the transforming growth factor ␤ superfamily, and myostatin-null mice have been found to show a two-to threefold increase in skeletal muscle mass due to an increase in the number of muscle fibers (hyperplasia) and the size of the fibers (hypertrophy) (21). Subsequently, we (15) and others (9, 22) reported that Belgian Blue and Piedmontese breeds of cattle, which are characterized by heavy muscling, have mutations in the myostatin gene coding sequence. Hence, myostatin is considered a negative regulator of muscle growth.Earlier studies have indicated that myostatin gene expression appears to be transcriptionally regulated during development (15, 21). Initially myostatin gene expression is detected in myogenic precursor cells of the myotome compartment of developing somites, and the expression is continued in adult axial and paraxial muscles (21). Different axial and paraxial muscles have been shown to express different levels of myostatin (15). Recent publications have shown that myostatin protein is also detected in heart (33) and mammary gland (14). In addition, myostatin is present in human skeletal muscle, and its expression is increased in the muscles of human immunodeficiency virus-infected men with muscle wasting compared to that in healthy men (8). Recently Wehling ...