C. S. Coy

Effects of glypican‐1 on turkey skeletal muscle cell proliferation, differentiation and fibroblast growth factor 2 responsiveness

The heparan sulfate proteoglycan, glypican‐1, is a low affinity receptor for fibroblast growth factor 2 (FGF2). Fibroblast growth factor 2 is a potent stimulator of skeletal muscle cell proliferation and an inhibitor of differentiation. Heparan sulfate proteoglycans like glypican‐1 are required for FGF2 to transduce an intracellular signal. Understanding the role of glypican‐1 in the regulation of FGF2‐mediated signaling is important in furthering the understanding of the biological processes involved in muscle development and growth. In the current study, a turkey glypican‐1 expression vector construct was transfected into turkey myogenic satellite cells resulting in the overexpression of glypican‐1. The proliferation, differentiation, and responsiveness to FGF2 were measured in control and transfected cell cultures. The overexpression of glypican‐1 in turkey myogenic satellite cells increased both satellite cell proliferation and FGF2 responsiveness, but decreased the rate of differentiation. The current data support glypican‐1 modulation of both proliferation and differentiation through an FGF2‐mediated pathway.

Variation in fibroblast growth factor response and heparan sulfate proteoglycan production in satellite cell populations.

Variation in the responsiveness of myogenic satellite cell subpopulations to mitogenic stimuli was examined in cells isolated from the turkey pectoralis major muscle. Faster growing clonal cell populations were more responsive to fibroblast growth factor (FGF-2) and expressed greater levels of FGF-2 and FGF receptor-1 at the onset of proliferation than did slower growing cells. Faster growing clones also expressed higher levels of heparan sulfate proteoglycans (HSPG), especially during differentiation, than did slower growing clones. HSPG, which is important in FGF receptor signaling, increased during proliferation of all clones tested and decreased in all but one of the clones during differentiation. Slower growing clones increased their expression of FGF receptor-1 through proliferation and differentiation. However, expression of the receptor in faster growing clones decreased during differentiation. The FGF receptors-2 and -3 were not detected on turkey satellite cells or myotubes by reverse transcriptase-polymerase chain reaction methodology. These results demonstrate that there is heterogeneity in the properties and responsiveness of satellite cell populations derived from single muscles. Satellite cells that differ in proliferation rates differ in responsiveness to FGF-2, and in the expression of FGF-2, FGF receptor-1, and HSPG.

Effect of the timing of posthatch feed restrictions on the deposition of fat during broiler breast muscle development

The effect of the timing of posthatch feed restriction on adipose deposition and adipogenic gene expression in the broiler pectoralis major muscle was studied by applying a 20% feed restriction either the first or second week after hatch. Broiler chicks at hatch were divided into a full-fed (control) group and a 20% feed restriction group. The expression of adipogenic genes, peroxisome proliferator-activated receptor gamma (PPARγ), and CCAAT/enhancer-binding protein alpha (C/EBPα) were measured. The expression of both PPARγ and C/EBPα was affected by the wk 1 feed restriction with expression significantly increased during the first week posthatch. The deposition of fat within the pectoralis major muscle was affected by the timing of the feed restriction. Extensive fat depots were present by 27 d of age in the pectoralis major muscle of the wk 1 restricted group compared with the control. Fat deposition was eliminated when the 20% feed restriction occurred in wk 2. Taken together, these results demonstrate that the timing of early posthatch feed restrictions in chicks is critical in the deposition of fat in the pectoralis major muscle and expression of adipogenic genes.

Effect of the timing of posthatch feed restrictions on broiler breast muscle development and muscle transcriptional regulatory factor gene expression

The effect of the timing of an immediate posthatch feed restriction on broiler pectoralis major muscle development was studied by applying a 20% feed restriction either the first or second week after hatch. Pectoralis major muscle morphological structure and the expression of the myogenic transcriptional regulatory factors, myogenic determination factor 1 (MyoD), myogenic regulatory factor 4 (MRF4), and myogenin, were measured. Broiler chicks at hatch were divided into a full-fed (control) group and a 20% feed restriction treatment administered either the first or second week posthatch. At the end of the feed restriction, the chicks were placed on a full feed ad libitum diet with no further restrictions. Muscle fiber diameter and fiber bundle size of the pectoralis major muscle were smaller in the wk 1 restricted group than the control group by 7 d of age. By 15 d of age through the duration of the study, d 43, both endomysial and perimysial connective tissue spacing were diminished in the wk 1 feed-restricted group. The expression of MyoD, MRF4, and myogenin was affected by the wk 1 feed restriction. The expression of MyoD and MRF4 was significantly increased during the first week posthatch. Both of the genes have been shown to be expressed during proliferation especially MyoD, which is required for muscle cell proliferation. In contrast, myogenin expression was significantly decreased. Myogenin expression is required for differentiation to occur. The morphological changes and gene expression changes observed with the wk 1 feed restriction were eliminated by moving the 20% feed restriction to wk 2, which is after the period of maximal myogenic satellite cell mitotic activity. Taken together, these results demonstrate that the timing of early posthatch feed restrictions to chicks is critical for the morphological development of the pectoralis major muscle and the expression of genes required for muscle satellite cell proliferation and differentiation.

The effect of temperature on apoptosis and adipogenesis on skeletal muscle satellite cells derived from different muscle types

Satellite cells are multipotential stem cells that mediate postnatal muscle growth and respond differently to temperature based upon aerobic versus anaerobic fiber‐type origin. The objective of this study was to determine how temperatures below and above the control, 38°C, affect the fate of satellite cells isolated from the anaerobic pectoralis major (p. major) or mixed fiber biceps femoris (b. femoris). At all sampling times, p. major and b. femoris cells accumulated less lipid when incubated at low temperatures and more lipid at elevated temperatures compared to the control. Satellite cells isolated from the p. major were more sensitive to temperature as they accumulated more lipid at elevated temperatures compared to b. femoris cells. Expression of adipogenic genes, CCAAT/enhancer‐binding protein β (C/EBPβ) and proliferator‐activated receptor gamma (PPARγ) were different within satellite cells isolated from the p. major or b. femoris. At 72 h of proliferation, C/EBPβ expression increased with increasing temperature in both cell types, while PPARγ expression decreased with increasing temperature in p. major satellite cells. At 48 h of differentiation, both C/EBPβ and PPARγ expression increased in the p. major and decreased in the b. femoris, with increasing temperature. Flow cytometry measured apoptotic markers for early apoptosis (Annexin‐V‐PE) or late apoptosis (7‐AAD), showing less than 1% of apoptotic satellite cells throughout all experimental conditions, therefore, apoptosis was considered biologically not significant. The results support that anaerobic p. major satellite cells are more predisposed to adipogenic conversion than aerobic b. femoris cells when thermally challenged.

Changes in satellite cell proliferation and differentiation during turkey muscle development

Posthatch muscle growth is determined by the activation, differentiation, and fusion of satellite cells. Satellite cells composing an individual muscle are heterogeneous, which will differentially affect muscle growth. The proliferation and differentiation of turkey primary pectoralis major muscle cells were investigated in vitro at 1 d of age and at 4, 8, 16, 26, 35, 45, and 54 wk of age. The turkey was selected for these studies because turkey skeletal muscle fibroblasts do not grow in primary muscle cell cultures. Results from the proliferation analysis showed a decrease in proliferation by 8 wk of age. Differentiation into myotubes was significantly decreased by 4 wk of age and myotube diameter was decreased. The changes in muscle weight relative to total BW were measured for the anterior latissimus dorsi, biceps brachii, pectoralis major, sartorius, biceps femoris, and gastrocnemius muscles to compare the relative growth of different muscles. The age at which the muscles reached their maximum relative weight was muscle-dependent, with the biceps brachii plateauing the earliest at 4 wk and the sartorius the latest at 45 wk of age. These data suggested that changes in myogenic cells begin to occur early in muscle development and the activity of the satellite cells during these initial stages of posthatch growth is critical in overall muscle mass accumulation.

Effects of 17β-estradiol on turkey myogenic satellite cell proliferation, differentiation, and expression of glypican-1, MyoD and myogenin.

The hypothesis of this study was that 17β-estradiol (estradiol) stimulates turkey skeletal muscle growth by influencing myogenic satellite cell proliferation, differentiation, and the gene expression of selected proteins important in regulating growth and development. Increasing levels of estradiol were administered in basal medium containing additional nutrients. Female-derived pectoralis major (PM) satellite cell proliferation was stimulated by estradiol at a level of 10(-9)M following 4days of treatment. Male PM and biceps femoris (BF) satellite cell proliferation was increased at 10(-12)M estradiol. Turkey embryonic myoblast proliferation, however, decreased with 10(-9)M and 10(-5)M estradiol following 3days under these conditions. Estradiol had no effect on the differentiation of any of the 4 groups of cells. Likewise, glypican-1 expression was unaffected by estradiol treatment. MyoD expression decreased in male PM but not BF cells. MyoD expression in female PM cells and embryonic myoblasts were also unaffected by estradiol administration. Estradiol decreased myogenin expression in male satellite cells, but had no effect on female cells. There was a slight decrease in myogenin expression in embryonic myoblasts. The results demonstrate a direct effect of estradiol on avian satellite cell proliferation independent of glypican-1, and decreased expression of MyoD and myogenin in some myogenic cells, coinciding with increased cellular proliferation.

Temperature effect on proliferation and differentiation of satellite cells from turkeys with different growth rates

Poultry selected for growth have an inefficient thermoregulatory system and are more sensitive to temperature extremes. Satellite cells are precursors to skeletal muscle and mediate all posthatch muscle growth. Their physiological functions are affected by temperature. The objective of the current study was to determine how temperature affects satellite cells isolated from the pectoralis major (p. major) muscle (breast muscle) of turkeys selected for increased 16 wk body weight (F line) in comparison to a randombred control line (RBC2) from which the F line originated. Pectoralis major muscle satellite cells were thermally challenged by culturing between 33°C and 43°C to analyze the effects of cold and heat on proliferation and differentiation as compared to control temperature of 38°C. Expression levels of myogenic regulatory factors: myogenic differentiation factor 1 (MYOD1) and myogenin (MYOG) were quantified by quantitative polymerase chain reaction (qPCR). At all sampling times, proliferation increased at a linear rate across temperature in both the RBC2 and F lines. Differentiation also increased at a linear rate across temperature from 33 to 41°C at all sampling times in both the F and RBC2 lines. Satellite cells isolated from F line turkeys were more sensitive to both hot and cold temperatures as proliferation and differentiation increased to a greater extent across temperature (33 to 43°C) when compared with the RBC2 line. Expression of MYOD1 and MYOG increased as temperatures increased from 33 to 41°C at all sampling times in both the F and RBC2 lines. These results demonstrate that satellite cell function is sensitive to both cold and hot temperatures and p. major muscle satellite cells from F line turkeys are more sensitive to temperature extremes than RBC2 satellite cells.

The Effect of Fibroblast Growth Factor 2 on the In Vitro Expression of Syndecan-4 and Glypican-1 in Turkey Satellite Cells

The membrane-associated heparan sulfate proteoglycan families, consisting of the syndecans and glypicans, are low-affinity receptors for fibroblast growth factor 2 (FGF2) that are essential in regulating the cellular response to FGF2. Fibroblast growth factor 2 is a potent stimulator of skeletal muscle cell proliferation and a strong inhibitor of differentiation. The regulation of the expression of the syndecans and glypicans will likely play a role in modulating the effects of FGF2 on cellular growth properties. In the present study, the effect of FGF2 on the expression of syndecan-4 and glypican-1 was measured by real-time PCR during turkey myogenic satellite cell proliferation and differentiation in vitro. Both syndecan-4 and glypican-1 transcription were influenced by the addition of exogenous FGF2. Syndecan-4 mRNA expression was reduced only during proliferation, whereas glypican-1 expression was reduced during both proliferation and differentiation. These results suggest that FGF2 growth factor signaling is, in part, regulated by an autoregulatory loop involving FGF2 regulation of syndecan-4 and glypican-1 expression and will affect the growth of skeletal muscle by modulating the proliferation and differentiation of satellite cells.

The influence of age on maternal inheritance of breast muscle morphology in turkeys

It was apparent in previous studies at our institution using turkeys that measurements of muscle fibers and extracellular spacing were not adequate to explain what was observed in entire pectoralis major muscle sections. A rating system was developed in which muscle sections were rated from 1 (little extracellular matrix and indistinct muscle fibers) to 5 (large extracellular space and distinct muscle fibers). Maternal inheritance was observed at 16 wk of age but not at 8 or 20 wk of age. The purpose of the current study was to determine the effect of age on maternal inheritance. A line (F) selected long-term for increased 16-wk BW, its randombred control (RBC2), and reciprocal crosses between them were compared from 8 through 18 wk of age. Samples of pectoralis major muscle were obtained in a manner to avoid muscle contraction. After being fixed and cross-sectioned, the muscle samples were stained with hematoxylin and eosin and rated by 4 individuals. No significant difference among genetic groups was observed in scores at 8 wk of age. At 10 wk of age, the F line had lower scores than the other genetic groups. Maternal inheritance was suggested at 12 wk of age. The scores for RBC2 were higher than those for F, whereas the F x RBC2 cross did not differ from the pure RBC2 line score at this age. Although the RBC2 x F scores were higher than the pure F-line scores at 12 wk, they were lower than those of the F x RBC2 crosses. From 14 through 18 wk of age, the scores for the RBC2 line were higher than those for the F line and the maternal inheritance was absolute because the value for the individual crosses did not differ from that of the maternal parent. Based on the results, the type of mating used to produce commercial turkeys would have a major effect on breast muscle morphology from 12 through 18 wk of age.