Growth characterization of callipyge cells



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Texas Tech University


Callipyge is a mutation that causes post-natal muscle hypertrophy in the loin and hindquarters of domestic sheep. Although much research has been conducted in the past 20 years to explain this mechanism, no clear answer has been reached. This thesis contains three separate studies to try to explain the mutated phenotype of large muscle fibers and leaner carcasses.

Study I consists of two sets of data on the proliferation rates of satellite cells from the muscles of normal and mutated sheep. Satellite cells are mononucleated cells located beneath the basement membrane of skeletal muscles and incorporate themselves into existing muscle fibers for muscle growth or repair. Satellite cells were isolated from the semimembranosus muscles of callipyge CLPG/+ (n = 13), maternal non-expressers +/CLPG (n = 6), homozygous CLPG/CLPG (n = 6), and normal +/-t- (n = 13) lambs ranging from 2 to 8 wks of age. Primary isolated satellite cell growth rates were taken on days five, six, and seven of culture, and calculated as a percent gain from the day five counts to standardize. A genotype effect was seen in primary counts (P = 0.0005), with the non-expressers having the highest growth rates at 231.74 + 111.44%, followed by callipyge 175.57 + 48.72%, normal 137.81 + 42.27%, and homozygous 99.81 + 22.89% gain over the day five counts. Secondary cultured satellite cell plates were made by adding lO^ cells to plates and counting 24, 48, and 72 h after plating. Genotype was also significant in the secondary counts (P = 0.0358). Proliferation rates were non-expressers 404.39 ± 150.40%, homozygous 309.34 + 88.23%, callipyge 236.41 + 49.25%, and normal 193.85+ 51.11%.

Study 2 involved two experiments evaluating the plasma leptin levels of normal, callipyge. and homozygous sheep at differing physiological states. Leptin is a peptide hormone produced in white adipose tissue of mammals. This hormone is involved in a feedback loop to help maintain body composition homeostasis. Large amounts of adipose tissue produce high levels of leptin, which signal the hypothalamus to trigger a decrease in appetite and an increase in physical activity to help reduce the level of fat in the body. Experiment I quantified the plasma leptin levels in callipyge (n = 10) and normal ewes (n = 10) during mid-gestation, as well as 60 d old callipyge (n = 10) and normal (n = 10) lambs. No genotypic effect was observed in either the ewes (P = 0.78) or the lambs (P = 0.95). Experiment 2 involved quantifying the plasma leptin levels of callipyge (n = 6), homozygous (n = 8), and normal (n = 8) ewes through late gestation, lambing, and early lactation. A significant genotype effect was observed in Experiment 2 (P = 0.0005), with the normal ewes having the highest mean leptin levels at 9.13 +.0.93 ng/ml, foUowed by homozygous ewes at 8.11 + 0.70 ng/ml. The callipyge ewes showed the lowest mean leptin levels at 5.41 +_0.40 ng/ml.

Study 3 consisted of quantifying the relative expression levels of five muscle regulatory factors in cultured myoblast and tissue samples from callipyge (n = 3), nonexpresser (n = 3), homozygous (n = 3), and normal (n = 3) lambs at ages 2, 4, and 8 wks. We evaluated the expression level of four positive muscle growth regulators including MyoDl, myf-5, myogenin, and MRF4. One negative regulator, myostatin, was also evaluated. Total RNA was isolated from each sample. Total cDNAs were made via reverse transcription, then specific factors were quantified using Real-Time PCR. Ribosomal RNA levels were used to standardize the amount of total RNA per sample. A significant age x genotype interaction was observed in all factors tested. In the cultured cells, the expression levels of MyoDI (p = 0.0002), myogenin (p < 0.0001), MRF4 (p < O.OOOI), and myostatin (p = 0.0125) were all highest at 4 wks of age. Myf-5 expression was lowest in the 4 wk cells (p < 0.0001). Only a few of the most prominent comparisons are reported between genotypes here. The 4 wk non-expressers showed the highest overall expression levels of MyoDl (p < 0.0001 versus all others), myogenin (p < 0.0001 versus all others), MRF4 (p < 0.0001 versus all others), and myostatin (p < 0.001 versus all others) and showed the overall lowest expression level of myf-5 (p < 0.0001). The callipyge cells showed the overall lowest expression of MyoDI (p < 0.0001 versus nonexpresser, p = 0.0002 versus normal, p = 0.3346 versus homozygous), myogenin (p < 0.0001 versus non-expresser, p = 0.0205 versus homozygous, p = 0.6105 versus normal), MRF4 (p < 0.0001 versus non-expresser and homozygous, p = 0.1717 versus normal) at 4 wks. At 2 wks of age, the callipyge cells showed the highest expression level of myf-5 (p < O.OOOI versus all others) and the lowest expression of myostatin (p < 0.0001 versus non-expresser and normal, p = 0.2803 versus homozygous). For most comparisons, the homozygous and normal cells were intermediate in their expression. In the 2 wk tissue samples, the non-expresser showed the highest expression of myf-5 (P < 0.0001), myogenin (P < 0.0001), MRF4 (P < 0.0001), and myostatin (P < 0.0001). No detectable levels of MyoDl were observed in the 2 wk tissue. No data were obtained from the 4 and 8 wk tissue samples due to low RNA quality.

These three studies do indicate genotypic effects in the sheep possessing the callipyge mutation. In Study 1 with the cell proliferation rates, it is puzzling to see the two non-expressing carriers of the mutation have the highest growth rates. However, the callipyge cells showed their highest growth rates at the youngest age tested, so perhaps the expressers of the phenotype begin their muscle growth earlier in life and exhibit more plasticity throughout their entire development. The leptin data in Experiment 2 of Study 2 indicate that the callipyge ewes possess lower fat stores, even during gestation. This may help explain how these sheep are more efficient in production and muscle accretion, since more of their nutrients would go toward growth versus fat deposition. In Study 3, the differences seen between genotypes in the expression levels of different muscle growth factors may provide further explanation for early post-natal growth in callipyge sheep. The fact that expression levels between callipyge and non-expresser lambs at 4 wks were so different may provide clues toward understanding the phenotypic expression.



Muscles -- Hypertrophy, Sheep -- Genetics, Leptin, Satellite cells, Lambs -- Growth