Callipyge sheep exhibit extreme postnatal muscle hypertrophy in the loin and hindquarters as a result of a single nucleotide polymorphism (SNP) in the imprinted DLK1-DIO3 domain on ovine chromosome 18. The callipyge SNP up-regulates the expression of surrounding transcripts when inherited in cis without altering their allele-specific imprinting status. The callipyge phenotype exhibits polar overdominant inheritance since only paternal heterozygous animals have muscle hypertrophy. Two studies were conducted profiling gene expression in lamb muscles to determine the down-stream effects of over-expression of paternal allele-specific DLK1 and RTL1 as well as maternal allele-specific MEG3, RTL1AS and MEG8, using Affymetrix bovine expression arrays. A total of 375 transcripts were differentially expressed in callipyge muscle and 25 transcripts were subsequently validated by quantitative PCR. The muscle-specific expression patterns of most genes were similar to DLK1 and included genes that are transcriptional repressors or affect feedback mechanisms in β-adrenergic and growth factor signaling pathways. One gene, phosphodiesterase 7A had an expression pattern similar to RTL1 expression indicating a biological activity for RTL1 in muscle. Only transcripts that localize to the DLK1-DIO3 domain were affected by inheritance of a maternal callipyge allele. Callipyge sheep are a unique model to study over expression of both paternal allele-specific genes and maternal allele-specific non-coding RNA with an accessible and nonlethal phenotype. This study has identified a number of genes that are regulated by DLK1 and RTL1 expression and exert control on postnatal skeletal muscle growth. The genes identified in this model are primary candidates for naturally regulating postnatal muscle growth in all meat animal species, and may serve as targets to ameliorate muscle atrophy conditions including myopathic diseases and age-related sarcopenia.
The callipyge mutation causes postnatal muscle hypertrophy in heterozygous lambs that inherit a paternal callipyge allele (+/CLPG). Our hypothesis was that the up-regulation of one or both of the affected paternally expressed genes (DLK1 or PEG11) initiates changes in biochemical and physiological pathways in skeletal muscle to induce hypertrophy. The goal of this study was to identify changes in gene expression during the onset of muscle hypertrophy to identify the pathways that are involved in the expression of the callipyge phenotype. Gene expression was analysed in longissimus dorsi total RNA from lambs at 10, 20, and 30 days of age using the Affymetrix Bovine Expression Array. An average of 40.6% of probe sets on the array was detected in sheep muscle. Data were normalized and analysed using a two-way anova for genotype and age effects with a false discovery rate of 0.10. From the anova, 13 genes were significant for the effect of genotype and 13 were significant for effect of age (P < 0.10). No significant age-by-genotype interactions were detected (P > 0.10). Of the 13 genes indicating an effect of genotype, quantitative PCR assays were developed for all of them and tested on a larger group of animals from 10 to 200 days of age. Nine genes had significantly elevated transcript levels in callipyge lambs. These genes included phosphofructokinase, a putative methyltransferase protein, a cAMP phosphodiesterase, and the transcription factor DNTTIP1.
The developmental competence of oocytes is progressively attained as females approach puberty. The poor quality of prepubertally derived oocytes suggests that essential processes during cytoplasmic maturation have not been completed. The objective of this experiment was to identify genes in oocytes that are associated with good (cyclic females) and poor (prepubertal females) developmental competence. Development to the blastocyst stage in vitro was significantly decreased in oocytes derived from prepubertal females compared with cyclic females (5.26 and 12.86%, respectively). Approximately 10% of the oocyte transcriptome was differentially expressed between in vitro-matured oocytes derived from cyclic and prepubertal females (P < 0.05); 58% of differentially expressed genes had increased transcript abundance in oocytes derived from cyclic females. Genes involved in the metabolism and regulation of biological processes had increased transcript abundance in oocytes derived from cyclic females, whereas genes involved in translation were increased in prepubertally derived oocytes. Quantitative PCR confirmed differential expression (P < 0.05) for 6 out of 11 selected genes [DPYD (dihydropyrimidine dehydrogenase), RDH11 (retinol dehydrogenase 11), SFRS4 (serine/arginine-rich splicing factor 4), SFRS7 (serine/arginine-rich splicing factor 7), TL4 (transcribed loci 4), and TOP2B (topoisomerase II β)] that were differentially expressed with greater than a 2-fold change by microarray, although 3 of these genes, DPYD, TL4, and TOP2B, were in opposing directions by the 2 methods. In conclusion, expression of multiple genes involved in metabolism and translation was significantly altered in oocytes from prepubertal females compared with cyclic females, which was associated with reduced in vitro development to the blastocyst stage. These genes may represent important cellular mechanisms that regulate oocyte quality.
Members of the Ty3-Gypsy retrotransposon family are rare in mammalian genomes despite their abundance in invertebrates and some vertebrates. These elements contain a gag-pol-like structure characteristic of retroviruses but have lost their ability to retrotranspose into the mammalian genome and are thought to be inactive relics of ancient retrotransposition events. One of these retrotransposon-like elements, PEG11 (also called RTL1) is located at the distal end of ovine chromosome 18 within an imprinted gene cluster that is highly conserved in placental mammals. The region contains several conserved imprinted genes including BEGAIN, DLK1, DAT, GTL2 (MEG3), PEG11 (RTL1), PEG11as, MEG8, MIRG and DIO3. An intergenic point mutation between DLK1 and GTL2 causes muscle hypertrophy in callipyge sheep and is associated with large changes in expression of the genes linked in cis between DLK1 and MEG8. It has been suggested that over-expression of DLK1 is the effector of the callipyge phenotype; however, PEG11 gene expression is also strongly correlated with the emergence of the muscling phenotype as a function of genotype, muscle type and developmental stage. To date, there has been no direct evidence that PEG11 encodes a protein, especially as its anti-sense transcript (PEG11as) contains six miRNA that cause cleavage of the PEG11 transcript. Using immunological and mass spectrometry approaches we have directly identified the full-length PEG11 protein from postnatal nuclear preparations of callipyge skeletal muscle and conclude that its over-expression may be involved in inducing muscle hypertrophy. The developmental expression pattern of the PEG11 gene is consistent with the callipyge mutation causing recapitulation of the normal fetal-like gene expression program during postnatal development. Analysis of the PEG11 sequence indicates strong conservation of the regions encoding the antisense microRNA and in at least two cases these correspond with structural or functional domains of the protein suggesting co-evolution of the sense and antisense genes.
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