Conference: In Vitro Approaches to Understanding Growth and Development

Cell Culture as a Tool for the Study of Poultry Skeletal Muscle Development12 DOUGLAS

C. McFARLAND3

Department of Animal and Range Sciences, South Dakota State University, Brookings, SD 57007-0392 cognizant of the fact that muscle cells normally do interact/communicate with other cell types in the body and are affected by their presence. Cell culture allows us to separate these interactions and more clearly ex amine the cell's nutritional, hormonal and environ mental requirements for maintenance, proliferation and differentiation.

ABSTRACT Postnatal development of skeletal muscle is the responsibility of the myogenic satellite cells. Sat ellite cells, isolated from the pectoralis major muscle of young growing torn turkeys, have been cultured in vitro to provide a system for studying cellular and hor monal aspects of poultry skeletal muscle development. Satellite cell clones derived from primary cultures have been developed so that in vitro observations would not be confounded by the presence of nonmyogenic cells. Likewise, a serum-free medium that promotes prolif eration of the turkey satellite cell has been developed to provide a hormonally controlled environment for in vitro developmental studies. These two techniques have enabled us to examine the following: 1) factors that influence satellite cell proliferation and differentiation, 2) the interaction of hormones with cellular receptors, 3) secretion of biologically important proteins from cells and 4) the expression of genes important to muscle development. J. Nutr. 122: 818-829, 1992.

EMBRYONIC MYOBLASTS AND SATELLITE CELLS Embryonic myoblasts are responsible for the early prenatal or prehatch development of skeletal muscles. These mononucleated cells fuse to form multinucleated myotubes, which synthesize contractile proteins, acetylcholine receptors and other proteins character istic of their differentiated state, and ultimately de velop into mature muscle fibers. At some point before birth or hatching, myogenic development becomes the

INDEXING KEY WORDS:

•muscle •turkeys •cell culture

1 Presented as part of the 56th Annual Poultry Nutrition Con ference: In Vitro Approaches to Understanding Growth and De velopment, given at the 75th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlanta, GA, April 21, 1991. This conference was sponsored by the American Institute of Nutrition and supported by grants from Archer Daniels Midland Company, Pfizer Inc., SmithKline Beecham Animal Health, Hoechst Roussel Agri-Vet Company, Rhône-Poulenc Animal Nutrition N.A., Campbell Institute for Research &.Technology, Lilly Research Lab oratories, Cuddy Farms, Perdue Farms, Inc., and Merck Sharp and Dohme Res. Labs. Guest editor for this conference was M. S. Lilburn, Poultry Science Department, Ohio State University, Wooster, OH. 1 This research was funded by the South Dakota Agricultural Experiment Station Project H-037, the National Science Foundation EPSCoR program and the South Dakota Poultry Industries Associ ation. Scientific Paper Number 2547. 3 To whom correspondence should be addressed: Department of Animal and Range Sciences, Box 2170, South Dakota State Univer sity, Brookings, SD 57007-0392.

Cell culture has proven to be an invaluable tech nique for researchers exploring mechanisms of animal growth and development. Cells from the developing chick embryo are often used as a tool for this research. These cells are easily obtained and proliferate well in culture. The cultured chick embryonic myoblast cell system was first employed over 40 years ago (1,2) and continues to be a valuable tool. Not only is this system useful for studies of muscle development, it is also useful for a variety of other biochemical and physio logical studies. Certainly included among the greatest advantages of muscle cell culture is the ability to di rectly examine behavior or responses of these cells without confounding interactions of other cell types as occurs in the whole animal. One must always be 0022-3166/92

$3.00 ©1992 American Institute of Nutrition.

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CONFERENCE: UNDERSTANDING

responsibility of another cell type, which is called the satellite cell. Electron microscopic examination of sat ellite cells reveals a spindle or fusiform-shaped cell residing between the basement membrane and the plasmalemma of skeletal muscle fibers (3, 4). The physical dimensions of these cells as well as their nu clear sizes appear to vary between species (see réf. 5 for review). The developmental origins of the satellite cells are not yet known. They may be descendants of embryonic or fetal myoblasts. Moss and LeBlond (6), using autoradiographic methods, observed the incorporation of 3H-thymidine into rat satellite cell nuclei and the passage of the la beled nucleic acid (i.e., nuclei) to the myofibers to be come muscle nuclei. This established the key role of these cells in postnatal DNA accretion. It was previ ously documented by Stockdale and Holtzer (7) that increases in postnatal myofiber DNA were not the re sult of myotube nuclear replication. In fact, most of the nuclei residing in mature muscle fibers are derived from the myogenic satellite cells (8). Although muscle fiber number does not increase appreciably during postnatal muscle growth (9), dramatic increases in muscle cell size and nuclear content occur (8,10). This increase in satellite-cell-derived myonuclei is propor tional to (11), and a prerequisite for (6), muscle protein accumulation, i.e., muscle growth. In addition to their role in normal muscle devel opment, satellite cells appear to be responsible for re generation of traumatized muscles (12-14). Further more, nutritional state appears to affect satellite cell activity because in malnourished children they are fewer in number and predominantly in an inactive or noncycling state (15, 16). Recovery from malnourishment is characterized by a dramatic increase in the number of these cells and the proportions that are ac tivated. Additionally, satellite cells appear to be im portant in the regeneration of load-bearing skeletal muscles of animals subjected to hind-limb suspension (17) and space flight (18). Hind-limb-suspension-in duced muscle atrophy causes a pronounced decrease in myonuclei and satellite cell numbers. These results suggest there is both an increased incorporation of satellite cells into the muscle fibers and a decrease in their proliferative capacity. Satellite cells also appear to be important in exercise-induced increases in skel etal muscle mass (19), and some evidence has been presented indicating that exercise may result in the for mation of new fibers in adult skeletal muscle (20, 21).

GROWTH AND DEVELOPMENT

819

cells from the muscle fibers followed by differential centrifugation to separate the cells from the muscle debris. These cells were capable of proliferating and differentiating to form multinucleated myotubes. Since then, satellite cells have been isolated from hu mans (23) and a number of agriculturally important animals, including chickens (24-26), turkeys (27), sheep (28), cattle (29), swine (Doumit, M. E. & Merkel, R. A., Michigan State University, East Lansing, MI, personal communication) and fish (30), and cultured in vitro. We are using the turkey satellite cell system in our laboratory as a tool to elucidate regulatory factors in volved in the development of turkey skeletal muscles. Although the turkey satellite cell possesses many of the characteristics of other muscle cells in culture, there are some interesting species differences that will be discussed. Satellite cells were isolated from the pectoralis major of young growing toms because this muscle is easily and rapidly accessible and has rela tively little connective tissue compared with other muscles. Visible connective tissue and blood vessels were removed before enzymatic (pronase) liberation of satellite cells to minimize contamination from nonmyogenic cells such as fibroblasts. Birds of one sex (toms) were used in the majority of our studies because it remains unclear how the sex of the donor animal may influence muscle culture characteristics. Our initial studies were designed to optimize the proliferation and differentiation of the turkey satellite cell in a serum-containing media (27). Of the substrata and media tested, the best results were obtained when cells were plated in Dulbecco's Modified Eagle's Me dium (DMEM)4containing 10% horse serum (HS) into gelatin-coated (31) cell culture plates. However, DMEM containing 10 or 15% HS will not support proliferation of the turkey satellite cell, and in fact, DMEM-10% HS will promote extensive differentia tion (32). On the other hand, DMEM-15% HS has been demonstrated to be very adequate for supporting pro liferation of rat (33), ovine (34) and bovine (29) satellite cells. After a 16-24-h attachment period, this medium is typically replaced with a growth-promoting me dium. Of 36 media-sera combinations tested (6 sera X 6 media), McCoy's 5A medium-15% chicken serum (CS) supported the highest levels of proliferation and subsequent myotube formation. Additionally, all cul tures grown in the presence of 15% CS (regardless of media type) showed the highest levels of proliferation and subsequent myotube formation (P < 0.05). Similar results were seen with cultures of turkey embryonic myoblasts (unpublished observations). We have spec-

SATELLITE CELL CULTURE Viable satellite cells were first isolated in 1974 by Bischoff (22) from rat skeletal muscles. The procedure involved a pronase or trypsin treatment to liberate the Downloaded from https://academic.oup.com/jn/article-abstract/122/suppl_3/818/4755378 by Boston University user on 07 July 2018

4 Abbreviations:

DMEM, Dulbecco's Modified Eagle's Medium;

HS, horse serum; CS, chicken serum; PM, pectoralis major; ALD, anterior latissimus dorsi; IGF, insulin-like growth factor; FGF, fibroblast growth factor; TGF-0, transforming growth factor-beta.

820

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48 72 5A-15%

96 CS

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168

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192

216

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FIGURE I Kinetics of turkey satellite cell proliferation and myotube formation. Satellite cell suspensions were ap plied to fibronectin-coated cell culture wells at 0.5 g tissueequivalents/well. Cells were grown in McCoy's 5A medium15% CS for 4 d with one medium change after 2 d. This was followed by a 5-d treatment period with DMEM-1% HS with daily medium changes. Cells were fixed and stained after the 24-h attachment period and at 24-h intervals throughout the experiment. Each bar represents the mean ±SEM.Reproduced with permission from réf.27.

ulated the mitogens in CS that are responsible for tur key satellite cell proliferation are structurally more similar to the turkey muscle mitogen(s) and conse quently interact more effectively with receptors on the cell surface. It is also possible that the levels of mitogen(s) in CS are higher. Media containing CS do not support proliferation of some muscle cell types, in cluding porcine satellite cells (Doumit and Merkel, Michigan State University, East Lansing, MI, personal communication) and the rat L6 embryonic myoblast cell line (White, The Ohio State University, Columbus, OH, personal communication). When turkey satellite cells are plated in McCoy's 5A-15% CS, they attach to substrata poorly. The inclusion of 15% porcine serum in any of the media we tested caused cellular detachment and death within 24 h. Conversely, media containing 10 or 20% porcine serum have been used to support satisfactory growth of rat (33, 35) and por cine (Doumit and Merkel, Michigan State University, East Lansing, MI, personal communication) satellite cells. Myotube formation (differentiation) is maximized in turkey satellite cell cultures after a 4-d treatment with DMEM-1% HS (Fig. 1). These same media con ditions have been used to promote differentiation of the rat (36, 37), bovine (29) and ovine (28) satellite cells. More recently, we have used DMEM-10% HS to promote fusion of our cultures. With this latter me dium, fusion is generally maximized earlier (by 3 d) and there appears to be less detachment of myotubes than with DMEM-1% HS. Certainly, among the great est advantages of the turkey satellite cell system over Downloaded from https://academic.oup.com/jn/article-abstract/122/suppl_3/818/4755378 by Boston University user on 07 July 2018

many of the other satellite cell systems is that primary cultures are often capable of differentiating to form cultures with > 90% fused cells. Such highly myogenic cultures alleviate some of the concerns of the effects of nonmyogenic cells in culture. However, primary cultures of similarly grown turkey embryonic myoblasts are prone to becoming overgrown with fibroblastic cells (unpublished observations). The influence of animal donor age on satellite cell proliferation in culture has been examined in several studies with use of the rat (38, 39) and the muscular dystrophic chicken (40) systems. However, relatively little is known about the in vitro characteristics of satellite cells derived from other species at different stages of growth. Therefore, we conducted several ex periments to determine the influence of age as well as sex of donor turkeys on satellite cell behavior in cul ture (41). Because of the large cell numbers required for our studies and the variable yields from the iso lation procedure, tertiary cultures were established. Satellite cells derived from hens and toms at 3, 9 and 15 wk of age were used in these studies. Figure 2 demonstrates that proliferation rates increased with increasing age of donor bird (P < 0.05). Because there was no detectable difference in the proliferation rates of cells between hens or toms, the sexes were pooled by donor age. This age-associated responsiveness of satellite cells was also seen with our primary cultures (41). Although these differences are most likely pri marily due to differences in lag phase growth, there may be true differences in the proliferation rates of

72

24 Hours

FIGURE 1 Kinetics of satellite cell proliferation in cul ture. Cells were derived from 3-, 9-, and 15-wk-old Nicholas torn and hen turkeys. Tertiary cultures were suspended in DMEM-10% HS and plated on gelatin-coated cell culture wells. After a 24-h attachment period, control cultures (time 0) were fixed and stained. Remaining cultures were grown in McCoy's 5A medium-15% CS with one medium change after 48 h. Replicate treatments were fixed and stained at 24-h intervals and the relative cell number was calculated as the ratio of nuclei on each day to that at time 0. Each bar represents the mean ±SEM. Reproduced with permission from réf.41.

CONFERENCE:

UNDERSTANDING

cells derived from animals of different ages. A possible explanation is that cells from older birds may be better able to adapt to culture conditions than cells from younger birds. Differentiation rates of cultures (as in dicated by cell fusion percentages) were similar be tween the three age groups (Fig. 3). Because the cul tures achieved high fusion percentages (78-82%), the influence of nonmyogenic cells on our results was probably minimal. We also examined the effects of turkey serum source on cell proliferation by using a turkey satellite cell clone bioassay. Half-maximal stimulation of prolif eration with turkey serum occurred with ~3% serum. When cells were treated with sera at this level, there was an age-associated decline in proliferation of cells exposed to hen sera but not torn sera (P < 0.05; Fig. 4). Fusion percentages as induced by turkey sera were generally low and did not correlate with cell density (r = 0.09; P > 0.05). Serum insulin-like growth factor (IGF)-I levels were similar between the groups and overall did not correlate with satellite proliferation (r = 0.15; P > 0.05) or fusion (r = 0.31; P > 0.05). There was, however, a paradoxical positive correlation (r = 0.99; P < 0.05) between serum IGF-I levels and the proliferation-stimulating activity of serum from 15wk-old toms. The same relationship (r = 0.98; P < 0.05) was seen with cultures administered 4% serum. In studies with chicken satellite cells, Matsuda et al. (42) demonstrated that cultures derived from the fast (pectoralis major) and slow (anterior latissimus dorsi) muscles produce myotubes that synthesize isoforms of myofibrillar proteins characteristic of the muscle source. We were interested in determining whether satellite cells derived from these muscles of the turkey differed with respect to proliferation and differentiation characteristics in culture. Under the

120

96

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AND DEVELOPMENT

50

4O

30

2O

Tom Hen

1O

6

9

12

15

18

Bird Age (weeks)

FIGURE 4 Effects of donor age and sex on mitogenic ac tivity of serum. Cloned satellite cells were applied to gelatincoated cell culture wells at ~24 cells/mm2. Cultures were treated for 5 d with McCoy's 5A medium containing 3% turkey serum derived from each bird (n = 27) and media was replaced once after 48 h. The mitogenic activity of serum from four or five birds in each group is shown (mean ±SEM). Reproduced with permission from réf.41.

culture conditions we used (proliferation in McCoy's 5A-15% CS and fusion in DMEM-1% HS), we detected no differences in proliferation or differentiation rates between satellite cells derived from these two muscles of 15-wk-old toms (Fig. 5). In spite of the high myogenic potential of turkey primary (or passaged) satellite cell cultures, we decided to establish several cell clones (43) to avoid any pos sible interferences by nonmyogenic cells, such as fibroblasts, in culture. Fibroblasts from human tissues are capable of producing a number of substances that have been demonstrated to influence the proliferation and differentiation of satellite cells from several spe cies (44, 45). Among these factors are basic fibroblast growth factor (FGF) (46), IGF-I (47, 48) and IGF-binding proteins (49). Our procedure involved the seques tering of individual cells by glass cloning rings and allowing them to proliferate in the presence of a mix ture of conditioned media and fresh media. Successful clones were then passed to increasingly larger vessels. This technique was also used to develop clones of the turkey embryonic myoblast. Clones developed in this manner were used in the IGF receptor studies, in the development of a serum-free medium and in the gene expression studies.

144

Hours

FIGURE 3 Turkey satellite cell myotube formation. Af ter 72-h growth (Fig. 2), satellite cell cultures were induced to fuse by treatment with DMEM-1% HS with daily medium changes. Cells were fixed and stained at 24-h intervals. Each bar represents the mean ±SEM.Reproduced with permission from réf.41. Downloaded from https://academic.oup.com/jn/article-abstract/122/suppl_3/818/4755378 by Boston University user on 07 July 2018

HORMONAL REGULATION OF AVIAN SKELETAL MUSCLE DEVELOPMENT A number of hormones have been identified that have mitogenic effects on muscle cells in culture. These include FGF (50), transferrin (51, 52), "chicken

822

McFARLAND

PM ALD

24

48

72

Hours

100 90 80

PM

70 60

ALD

50 40 30 20 10 O 72

96

120 Hours

FIGURE 5 Proliferation and myotube formation in sat ellite cell cultures from pectoralis major (PM) and anterior latissimus dorsi (ALD) muscles, [a]After a 14-h attachment period, cultures were exposed to McCoy's 5A medium-15% CS for 72 h with one medium change after 48 h. (b) After 72-h growth, fusion was induced by exposure of cells to DMEM-1% HS with daily medium changes. Total and myo tube nuclei were counted and the percentage fusion was de termined for replicate cultures at 24-h intervals. Reproduced with permission from réf. 41.

muscle growth factor" (a protein similar to, if not identical with, FGF; réf. 53), epidermal growth factor (54) and the IGFs (37, 55). In some cell systems, in cluding chick embryonic muscle, IGFs also stimulate differentiation (56, 57). In recent years, IGF-I (58) and both basic and acidic forms of FGF (59) have been shown to be expressed by cultured mammalian satellite cells, suggesting an autocrine role of these hormones in skeletal muscle development. Additionally, IGF-I mRNA expression in skeletal muscle is under growth hormone control (60). Fetal rat myoblasts release IGFI as well as IGF-II in culture (61).Transforming growth factor-beta (TGF-/3)has been demonstrated to be a po tent inhibitor of the differentiation of rat satellite cells (62), rat L6 muscle cells (63) and chick embryonic myoblasts (64). Allen and Boxhorn (57) showed that, by altering the levels of IGF-I, FGF and TGF-/3, cul tured rat satellite cells may be induced to divide, dif ferentiate or remain quiescent. Downloaded from https://academic.oup.com/jn/article-abstract/122/suppl_3/818/4755378 by Boston University user on 07 July 2018

To unravel cellular mechanisms involved in the de velopment of turkey skeletal muscles, our laboratory has approached the problem from three avenues: 1}to identify which hormones influence turkey muscle cell proliferation and differentiation in culture, 2) to ex amine the interaction of hormones with cell surface receptors and 3) to examine the expression of genes that may be important to proliferation and differen tiation of these cells. Our current efforts have concen trated on the role of the IGFs, the IGF receptors and the IGF-binding proteins. A major hindrance to the elucidation of the hor monal and nutritional requirements of many cell types, including muscle, has been the lack of a suitable serum-free or defined medium for such studies. Be cause serum-containing media contain variable levels of known as well as unknown factors that may influ ence cellular activity, they are of limited value in es tablishing cell requirements. Serum-free medium for mulations have been developed for several muscle cell types, including the chick embryonic myoblast (65), rat embryonic L6 cell (66) and rat (67), human (54) and ovine (68) satellite cells. To examine the role of various hormones and nutrients in turkey postnatal skeletal muscle development, we formulated a serum-free me dium that supports proliferation of the turkey satellite cell (Table 1) (69).Additionally, this medium is capable of supporting proliferation of turkey satellite cells plated at low densities. Because there is little condi tioning of the medium under these circumstances, it

TABLE 1 Turkey satellite cell serum-free medium supplements1

Final level in medium Growth factors Insulin Fibroblast growth factor Other proteins Deutsch fetuin Bovine serum albumin Minerals2 FeSO«-7HjO CuSO4• 5H2O (NH4)6Mo70M-4H20 ZnSO4-7H2O MnSO4 Na2Se03 Other organica Dexamethasone Linoleic acid Thymidine Adenine d-a-tocopherol

0.5 g/L 25.0^/L 0.45 g/L 2.0 g/L 4.98 2.48 X 10"* 1.24X IO"3 1.40 1.69X IO-4 5.56 X IO-3 IO"6mol/L 0.75 mg/L 726. 405. 0.129 mg/L

1Added to McCoy's 5A medium containing antibiotics. Repro duced with permission from réf. 69. z Mineral values expressed in mg/L.

CONFERENCE:

UNDERSTANDING

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GROWTH

823

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600 500

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100

1000

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FIGURE 6 Dose-response curve for insulin. Cells were grown in serum-free medium containing increasing levels of insulin for 6 d with medium changes after days 2 and 4. Cells were then fixed and stained. Each point represents the mean ±SEM.

is likely that this medium closely meets the nutritional and hormonal requirements of the turkey satellite cell. Additionally, the medium supports proliferation of primary cultures of turkey satellite cells (unpublished observations), suggesting that the clone used in the development of the medium has similar nutritional and hormonal requirements to freshly harvested tur key satellite cells. However, clonal-derived turkey embryonic myoblasts do not proliferate in this me dium but rather differentiate to form myotubes (un published observations). The development of the serum-free medium has al lowed us to begin examining the effects of various hormones and growth factors on the proliferation and differentiation of the turkey satellite cell. The response of turkey satellite cells to increasing levels of insulin is illustrated in Figure 6. A biphasic increase in pro liferation results with increasing levels of insulin. Similar dose-response curves have been reported with other cell types (70). It has been postulated that the mitogenic effects of insulin at levels in the microgram per liter range are due to insulin binding to its own high affinity receptor, and that the broad dose-response curve in the milligram per liter range is due to spillover of insulin onto IGF-I receptors. The effect of increasing levels of human recombinant IGF-I or IGF-II on sat ellite cell proliferation is illustrated in Figure 7. Our results indicate turkey satellite cells respond equally toward both of these mitogens. In contrast, rat em bryonic L6 cells are much more responsive to IGF-I (71). This medium was also used to evaluate the re sponse of cells to increasing levels of FGF. Figure 8 illustrates that maximum cell proliferation occurred with 25 ng of FGF/L in serum-free medium. This level is considerably less than the levels reported by others (100-300 Mg/L)for optimum proliferation of muscle Downloaded from https://academic.oup.com/jn/article-abstract/122/suppl_3/818/4755378 by Boston University user on 07 July 2018

FIGURE 7 Dose-response curve for IGF-I or IGF-II. Cells were grown in serum-free medium containing increasing levels of IGF-I or IGF-II for 6 d with medium changes after days 2 and 4. (Insulin was included at 5 ¿tg/Lin these studies.) Cells were then fixed and stained. Each point represents the mean ±SEM.

cells from mammalian species (66, 67), as well as the chick embryonic myoblast (65). Studies are currently underway to compare these mitogenic responses of turkey satellite cells with that of turkey embryonic myoblasts.

IGF RECEPTORS

Mammalian cells generally possess two distinct IGF receptor types on their cell surfaces (see réf.72 for review). The type I receptor, which is structurally similar to the insulin receptor, binds IGF-I with greater 4OO

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250

300

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FIGURE 8 Dose-response curve for FGF. Cells were grown in test media for 6 d with media changes after days 2 and 4. Levels of all other components were as indicated in Table 1 except as follows: dexamethasone (10s mol/L), fetuin (0.6 g/L), insulin (0.303 g/L). Each point represents the mean ±SEM.Reproduced with permission from réf.69.

824

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13

12

11

10

9

-LOG [PEPTIDE] Moie*

FIGURE 9 Displacement curves of [125]IGF-Ibinding to (a) turkey satellite cells and (b) satellite cell-derived myotubes. Cultures were incubated 4 h at 15°Cwith [125]IGF-I and increasing concentrations of unlabeled IGF-I, IGF-II (rat multiplication-stimulating activity) or insulin. Each point represents the mean specific radioactivity bound of four rep licate wells; SEMwas < 5% of bound radioactivity. Repro duced with permission from réf.43.

affinity than IGF-II and also interacts weakly with in sulin. The type II receptor is structurally dissimilar from the insulin and type I receptor (73), and binds IGF-II with greater affinity than IGF-I, but does not interact with insulin. In avian tissues, the picture is not yet as clear. Relatively little work has been con ducted to elucidate the mechanisms by which IGFs interact with cellular receptors in avian tissues. Ex ceptions to this are several chicken embryonic cells, particularly fibroblasts, and more recently, chicken liver membranes (74). Schmid et al. (56) reported that, although IGFs had only weak mitogenic effects on chick embryonic myoblasts, they preferentially and equipotently stimulated myoblast differentiation. Bassas and coworkers (75) reported that both IGF-I and IGF-II bind only to a type I receptor on plasma membranes derived from chick embryonic heart, liver, Downloaded from https://academic.oup.com/jn/article-abstract/122/suppl_3/818/4755378 by Boston University user on 07 July 2018

limb buds and brain. Only recently has evidence been presented that suggests the presence of a type II IGF receptor in any avian tissue. Mohan and coworkers (76), using embryonic chick calvarÃ-acells, showed that at least some of the mitogenic effects of IGF-II were mediated through a type II receptor. To examine the interaction of IGFs with turkey skeletal muscles, we performed competitive binding assays and autoradiographic analysis of cross-linked receptor-ligand complexes with turkey satellite cells and satellite-cell-derived myotubes. Figure 9 illus trates the effects of increasing concentrations of un labeled IGF-I, IGF-II (rat MSA) or insulin on [125I]IGFI binding to satellite cell and myotube cultures. Halfmaximal displacement values (IC50) of [125I]IGF-I binding by these hormones are summarized in Table 2. Although the [125I]IGF-Iassociation constants were similar (within the same order of magnitude) among satellite cell and myotube cultures, Scatchard analysis (consistent with that of a single class of noninteracting binding sites) revealed a 64% decrease in the maximum density of binding sites (on a per nuclei basis) after differentiation of satellite cells into myotubes (Fig. 10). Table 3 summarizes the IGF-I receptor data. Turkey satellite cells bind IGF-I with an affinity similar to rat (77), ovine (78) and human (79) satellite cells, L6 myo blasts (80, 81) and chick embryo fibroblasts (82). Fur thermore, the IGF-I binding affinity did not change as turkey satellite cells differentiated into myotubes. This is consistent with results found with rat L6 myoblasts (80). The displacement curves indicated that IGF-I is 20-30-fold more potent than IGF-II or insulin in dis placing IGF-I and strongly suggests the presence of a classical type I IGF receptor on turkey satellite cells and myotubes. The biphasic nature of our displace ment curves is similar to other reported [125I]IGF-I binding studies (83-85). To identify the molecular weight and thus the spe cific type of receptor that interacts with [125I]IGF-Ion the surface of turkey satellite cells, receptors were

Comparison

TABLE 2 of IGF-binding

properties1

Mononucleated turkey satellite cells

Turkey satellite cell-derived myotubes

['"IjIGF-I association constant, (mol/L)'1

3.6 ±0.25 x 10"

1.8±0.23xl08

50% Inhibition constant, mol/L IGF-I IGF-II Insulin

3.7 ±0.60 X IO"9 7.5 ±0.75 x 10-" 8.7 ±2.40 X IO"8

3.1 ±0.60X IO'9 7.5 ±2.10 X IO'8 9.6 ±2.50 X IO"8

1Values represent the mean of four replicate wells ±SEM.Re produced with permission from réf. 43.

CONFERENCE: UNDERSTANDING

825

reduction of the complex with dithiothreitol, a major band appeared at 130 KDa, consistent with the alphasubunit of a type I IGF receptor. Furthermore, excess unlabeled IGF-I (1 X 10~7 mol/L) displaced the 130

B/F

KDa band, whereas equimolar concentrations of un labeled IGF-II or insulin did not. We have recently conducted similar cross-linking studies with [125I]IGFII. Our results suggest that IGF-II also interacts with the type I IGF receptor of both clonal-derived satellite cells and embryonic myoblasts (unpublished obser vations). Autoradiographic bands characteristic of the type II receptor (molecular weight 220 KDa native and 260 KDa reduced) were not observed. These results are similar to those reported by Bassas and coworkers (75) using chick embryonic tissues.

0.021

0.000

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GROWTH AND DEVELOPMENT

5.65E-11

1.13E-10 Bound

1.70E-10

2.26E-10

(M)

B/F 0.015

IGF-BINDING 0.011

0.000 O.OOE+00

2.04E-11

4.09E-11 Bound

6.13E-11

S.1BE-11

(M)

FIGURE 10 Scatchard analysis of [125|IGF-Ibinding to (a) turkey satellite cells and (b) satellite cell-derived myotubes. Assuming a single class of noninteracting binding sites, the ratio of bound/free (B/F) radioactivity is plotted against the total molar concentration of IGF-I bound (M) to satellite cells. Each point represents the mean of four rep licate wells. Reproduced with permission from réf. 43.

chemically cross-linked with this radiolabeled ligand and subjected to gel electrophoresis and autoradiography. Under native (nonreduced) conditions, [125I]IGF-Iwas cross-linked to a receptor complex of apparent molecular weight > 300 KDa (Fig. 11). After

Comparison

of satellite

Nuclei/mm2 Mgprotein/well Maximum density of ¡1Z5I]IGF-I binding sites Receptors/nuclei Receptors/Mg protein

Circulating IGFs are mostly found in association with one of several binding proteins (see réf.86 for review). IGF-binding proteins are produced and se creted by the liver as well as other tissues, including skeletal muscle (87, 88). Although the complete pic ture of the role of IGF-binding proteins is not clear, these proteins significantly increase the half-life of circulating IGFs (86) and modulate the responses of cells to the IGFs ¡88,89). Both inhibitory and stimu latory effects of IGF-binding proteins on IGF action have been reported (see réf. 90 for review). The IGFbinding proteins present in turkey serum (Fig. 12) are similar in molecular weight to that seen in chicken (91) and porcine (92) sera. In a collaborative study with Michael White and Catherine Ernst at The Ohio State University, we measured a 50-80% decrease in the gene expression of IGF-binding protein-2 in turkey satellite cells as they differentiated to form myotubes (32). This differential gene expression corresponded to decreased release of a 30 KDa-binding protein into serum-free medium. During this same period there was also a decrease in IGF-I gene expression by these cells. Further studies are presently underway to de termine how these changes may influence the differ entiation process.

TABLE 3 cell and myotube

Mononucleated

IGF receptor

turkey satellite cells

692.6 ±102.4 173.0 ±14.4 2.3 ±0.12 (X 10-'°mol/L) 207,000 ±25,000 7.9 ±0.54 (X 10»)

1Values represent the mean of four to six replicate wells ±SEM.Reproduced

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PROTEINS

with permission

numbers1 Turkey satellite cell-derived myotubes 709.4 ±77.5 531.5 ±15.7 8.2 ±0.83 (X 10'" mol/L) 73,000 ±6,500 9.3 ±0.23 |x IO7) from réf.43.

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We have developed two powerful tools that now allow us to examine in closer detail the development of turkey skeletal muscles. Clonal-derived cell popu lations provide us with a model system to directly study the hormonal as well as nutritional requirements for maintenance, proliferation and differentiation of turkey satellite cells. Because this system uses a pure population of satellite cells, our observations are not confounded by the presence of nonmyogenic cells in culture. With our recent development of a turkey em bryonic myoblast clone, we have begun to compare these two cell types with respect to differences in hor monal responses and receptor properties, numbers of hormone receptors and other in vitro characteristics. Our serum-free medium formulation has provided us with a method of examining the effects of hormones and growth factors on turkey skeletal muscle prolif eration, differentiation and metabolism in a hormonally controlled environment. The future for investigators of muscle development holds many difficult challenges. These include provid ing a better understanding of the role of IGF-binding proteins and myogenic regulatory genes in muscle de velopment and how different cell types and their se cretions influence satellite cell proliferation and dif ferentiation. Certainly one of the most perplexing questions remains: how does the environment in the skeletal muscle compare with that which we have ar tificially provided for the satellite cell and myotube in culture? Several variations from classical cell culture -DIT Mrx10-3

IGF-I

IGF-II insulin

+DTT IGF-I IGF-II insulin

0

300H

130^

FIGURE 11 Autoradiogram of [125]IGF-Icross-linking to turkey satellite cell receptors with or without reduction by dithiothreitol (DTT). Near-confluent turkey satellite cell cultures were incubated 4 h at 15°Cwith 4.2 X 10~" mol [125]IGF-I/L in the presence or absence of 1 X 10~7 mol IGFI, IGF-II or insulin per liter. The bound ligand was crosslinked with 0.1 m mol disuccinimidyl suberate/L [±100 mmol/L dithiothreitol (DTT)] and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Reproduced with permission from réf.43.

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26.1OO

FIGURE 12 Identification of IGF-binding proteins in turkey serum by ligand blotting. Samples were subjected to polyacrylamide gel electrophoresis with a 7-15% acrylamide continuous gradient, transferred to an Imobilon (Millipore Corporation, Bedford, MA) membrane and probed with [125]IGF-I. The bands were visualized by autoradiography.

are currently being used to study muscle development. These systems were designed to better reproduce the natural environment of myogenic cells. Bischoff (93) used individual skeletal muscle fibers in culture as a model to study satellite cell proliferation and differ entiation. This system has proven useful for examining hormonal influences on satellite cell behavior and in studies of muscle regeneration. A computer-driven device described by Vandenburgh (94), which me chanically stretches muscle cultures, and a method of forming a contracting three-dimensional structure, including an endomysium, perimysium and epimysium described by Strohman and coworkers (95) offer additional tools for the muscle biologist. A clearer un derstanding of hormonal and nutritional regulation of muscle development will be realized as we become better able to reproduce this environment in culture.

LITERATURE

CITED

1. Rinaldini, L. M. (1959) An improved method for the isolation and quantitative cultivation of embryonic cells. Exp. Cell Res. 16: 477-505. 2. Königsberg, I. R. (1960) The differentiation of cross-striated myofibrils in short term cell culture. Exp. Cell Res. 21: 414420. 3. Mauro, A. (1961) Satellite cell of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9: 493-495. 4. Muir, A. R. (1970) The structure and distribution of satellite cells. In: Regeneration of Striated Muscle and Myogenesis, pp. 91-100, Exerpta Medica, Amsterdam, The Netherlands. 5. Campion, D. R. (1987) The muscle satellite cell: A review. Int. Rev. Cytol. 87:225-251. 6. Moss, F. P. & LeBlond, C. P. (1971) Satellite cells as the source of nuclei in muscles of growing rats. Anat. Ree. 170: 421-436.

CONFERENCE: UNDERSTANDING 7. Stockdale, F. E. &.Holtzer, H. (1961 ) DN A synthesis and myogenesis. Exp. Cell Res. 24: 508-520. 8. Allen, R. E, Merkel, R. A. & Young, R. B. (1979) Cellular as pects of muscle growth: Myogenic cell proliferation. J. Anim. Sci. 49: 115-127. 9. Smith, J. H. (1963) Relation of body size to muscle cell size and number in the chicken. Poult. Sci. 42: 283-290. 10. Moss, F. P., Simmonds, R. A. & McNary, H. W. (1964) The growth and composition of skeletal muscle in the chicken. 2. The relationship between muscle weight and the number of nu clei. Poult. Sci. 43: 1086-1091. 11. Moss, F. P. (1978) The relationship between the dimensions of the fibers and the number of nuclei during normal growth of skeletal muscle in the domestic fowl. Am. J. Anat. 122: 555564. 12. Bischoff, R. (1975) Regeneration of single skeletal muscle fibers in vitro. Anat. Ree. 182: 215-236. 13. Carlson, B. M. & Faulkner, J. A. (1983) The regeneration of skeletal muscle fibers following injury: A review. Med. Sci. Sports Exercise 15: 187-198. 14. Schultz, E. (1989) Satellite cell behavior during skeletal muscle growth and regeneration. Med. Sci. Sports Exercise 21(suppl.|: 5181-5186. 15. Hansen-Smith, F. M., Picou, D. & Golden, M. N. H. (1978) Quantitative analysis of nuclear population in muscle from malnourished and recovered children. Pediatr. Res. 12: 167170. l«.Hansen-Smith, F. M., Picou, D. & Golden, M. N. H. (1979) Muscle satellite cells in malnourished and nutritionally reha bilitated children. J. Neurol. Sci. 41: 207-221. 17. Darr, K. C. & Schultz, E. (1989) Hindlimb suspension sup presses muscle growth and satellite cell proliferation. J. Appi. Physiol. 67: 1827-1834. 18. Riley, D. A., Ilyina-Kakuera, E. I., Ellis, S., Bain, J. L. W., Slocum, G. R. & Sedlak, F. A. (1990) Skeletal muscle fiber, nerve and blood vessel breakdown in space-flown rats. FASEB J. 4: 84-91. Õ9. Darr, K. C. & Schultz, E. (1987) Exercise-induced satellite cell activation in growing and mature skeletal muscle. J. Appi. Physiol. 63: 1816-1821. 20. Appell, H.-J.,Forsberg,S. a Hollmann, W. (1988) Satellite cell activation in human skeletal muscle after training: Evidence for muscle fiber neoformation. Int. J. Sports Med. 9: 297-299. 21. Alway,S. E., Davis, M. E., Romans, W. &Gonyea, W. J. (1989) Muscle fiber proliferation and fiber hypertrophy during the first week of stretch in the adult Japanese quail. Med. Sci. Sports Exercise 21(suppl.|: 529 (abs.). 22. Bischoff, R. (1974) Enzymatic liberation of myogenic cells from adult rat muscle. Anat. Ree. 180: 645-661. 23. Blau, H. &. Webster, C. (1981) Isolation and characterization of human muscle cells. Cell Biol. 78: 5623-5627. 24. Yablonka-Reuveni, Z., Quinn, L. S. & Nameroff, M. (1987) Isolation and clonal analysis of satellite cells from chicken pectoralis muscle. Dev. Biol. 119: 252-259. 25. Johnson, D. D., Wilcox, R. & Wenger, B. (1983) Precocious in vitro development of satellite cells from dystrophic chicken muscle. In Vitro 19: 723-729. 26. Matsuda, R., Spector, D. H. &. Strohman, R. C. (1983) Regen erating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms. Dev. Biol. 100: 478-488. 27. McFarland, D. C., Doumit, M. E. & Minshall, R. D. (1988) The turkey myogenic satellite cell: Optimization of in vitro prolif eration and differentiation. Tissue Cell 20: 899-908. 28. Dodson, M. V., McFarland, D. C., Martin, E. L. & Brannon, M. A. (1986) Isolation of satellite cells from ovine skeletal muscle. J. Tissue Culture Meth. 10: 233-237. 29. Dodson, M. V., Martin, E. L., Brannon, M. A., Mathison, B. A. & McFarland, D. C. (1987) Optimization of bovine satellite

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cell derived myotube formation in vitro. Tissue Cell 19: 159166. 30. Powell, R. L., Dodson, M. V. &. Cloud, J. G. (1989) Cultivation and differentiation of satellite cells from skeletal muscle of the rainbow trout Salmo gairdneri. J. Exp. Zool. 250: 333-338. 31. Richler, C. & Yaffe, D. (1970) The in vitro cultivation and dif ferentiation capacities of myogenic cell lines. Dev. Biol. 21:122. 32. Ernst, C. W., McFarland, D. C. & White, M. E. (1990) Gene expression of IGF-1 and the low molecular weight IGF-binding protein (IGF-BP-2) in turkey satellite cells during proliferation and differentiation. J. Anim. Sci. 68(suppl. 1): 285. 33. Allen, R. E., McAllister, P. K. & Masak, K. C. (1980) Myogenic potential of satellite cells in skeletal muscle of old rats, a brief note. Mech. Ageing Dev. 13: 105-109. 34. Dodson, M. V., McFarland, D. C., Martin, E. L. & Brannon, M. A. (1986) Isolation of satellite cells from ovine skeletal muscle. J. Tissue Culture Meth. 10: 233-237. 35. Allen, R. E., Masak, K. C., McAllister, P. K. &. Merkel, R. A. (1983) Effect of growth hormone, testosterone and serum con centration on actin synthesis in cultured satellite cells. J. Anim. Sci. 56: 833-837. 3é.Allen, R. E., Dodson, M. V. & Luiten, L. S. (1984) Regulation of skeletal muscle satellite cell proliferation by bovine pituitary fibroblast growth factors. Exp. Cell Res. 152: 154-160. 37. Dodson, M. V., Allen, R. E. & Hossner, K. L. (1985) Ovine somatomedin, multiplication-stimulating activity and insulin promote skeletal muscle satellite cell proliferation in vitro. En docrinology 117: 2357-2363. 38. Schultz, E. & Lipton, B. H. (1982) Skeletal muscle satellite cells: Changes in proliferation potential as a function of age. Mech. Ageing Dev. 20: 377-383. 39. Dodson, M. V. & Allen, R. E. (1987) Interaction of multipli cation stimulating activity/rat insulin-like growth factor II with skeletal muscle satellite cells during aging. Mech. Ageing Dev. 39: 121-128. 40. Wright, W. E. (1985) Myoblast senescence in muscular dys trophy. Exp. Cell Res. 157: 343-354. 41. Doumit, M. E., McFarland, D. C. & Minshall, R. D. (1990) Satellite cells of growing turkeys: Influence of donor age and sex on proliferation and differentiation in vitro. Exp. Cell Res. 189:81-86. 42. Matsuda, R., Spector, D. H. & Strohman, R. C. (1983) Regen erating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms. Dev. Biol. 100: 478-488. 43. Minshall, R. D., McFarland, D. C. &. Doumit, M. E. (1990) Interaction of insulin-like growth factor I with turkey satellite cells and satellite cell-derived myotubes. Dornest. Anim. Endocrinol. 7: 413-424. 44. Florini, J. R. (1985) Hormonal control of muscle cell growth. J. Anim. Sci. 61(suppl. 2): 21-38. 45. McCusker, R. & Clemmons, D. (1988) Insulin-like growth factor binding protein secretion by muscle cells. Effect of cellular differentiation and proliferation. J. Cell Physiol. 137: 505-512. 4i. Story, M. T. (1989) Cultured human foreskin fibroblasts pro duce a factor that stimulates their growth with properties similar to basic fibroblast growth factor. In Vitro Cell. Dev. Biol. 25: 402-408. 47. Clemmons, D., Underwood, L. & Van Wyk, J. (1981) Hor monal control of immunoreactive somatomedin production by cultured human fibroblasts. J. Clin. Invest. 67: 10-19. 48. Cook, J., Haynes, K. & Werther, G. (1988) Mitogenic effects of growth hormone in cultured human fibroblasts: Evidence for action via local insulin-like growth factor 1 production. J. Clin. Invest. 81:206-212. 49. Clemmons, D., Elgin, R., Han, V., Casella, S., D'Ercole, A. & Van Wyk, J. (1986) Cultured

fibroblast monolayers

secrete a

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protein that alters the cellular binding of somatomedin-C/insulin-like growth factor 1. J. Clin. Invest. 77: 1548-1556. 50. Allen, R. E., Dodson, M. V. & Luiten, L. S. (1984) Regulation of skeletal muscle satellite cell proliferation by bovine pituitary fibroblast growth factors. Exp. Cell Res. 152: 154-160. 51. li, I.,Kimura, I. &.Ozawa, E. |1982) A myotrophic protein from chick embryo extract: Its purification, identity to transferrin and indispensability for avian myogenesis. Dev. Biol. 94: 366377. 52. Matsuda, R., Spector, D. & Strohman, R. C. (1984) There is selective accumulation of a growth factor in chicken skeletal muscle. I. Transferrin accumulation in adult anterior latissimus dorsi. Dev. Biol. 103: 267-275. 53. Strohman, R. C. & Kardami, E. (1986) Muscle regeneration re visited: Growth factor regulation of myogenic cell replication. In: Cellular Endocrinology: Hormonal Control of Embryonic and Cellular Differentiation, pp. 287-296, Alan R. Liss, New York, NY. 54. Ham, R. G., St. Clair, J. A., Webster, C. & Blau, H. M. (1988) Improved media for normal human muscle satellite cells: Serumfree clonal growth and enhanced growth with low serum. In Vitro Cell. Dev. Biol. 24: 833-844. 55. Florini, J. R., Nicholson, M. L. & Dulak, N. C. (1977) Effects of peptide anabolic hormones on growth of myoblasts in culture. Endocrinology 101: 32-41. 56. Schmid, C., Steiner, T. & Froesch, E. R. (1983) Preferential en hancement of myoblast differentiation by insulin-like growth factors (IGF I and IGF II) in primary cultures of chicken em bryonic cells. FEBSLett. 161: 117-121. 57. Allen, R. & Boxhorn, L. (1989) Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-beta, insulin-like growth factor I and fibroblast growth factor. J. Cell. Physiol. 138: 311-315. 58. Jennische, E., Skottner, A. & Hansson, H.-A. (1987) Satellite cells express the trophic factor IGF-I in regenerating skeletal muscle. Acta Physiol. Scand. 129: 9-15. 5?. Alterio, J., Courtois, Y., Robelin, J., Bechet, D. & Martelly, I. (1990) Acidic and basic fibroblast growth factor mRNAs are expressed by skeletal muscle satellite cells. Biochem. Biophys. Res. Commun. 166: 1205-1212. 60. Isgaard,J.,Nilsson,A., Vikman, K. OUsaksson, O. G. P. (1989) Growth hormone regulates the level of insulin-like growth factor-I mRNA in rat skeletal muscle. J. Endocrinol. 120: 107112. él.Hill, J., Grace, C. J., Nissley, S. P., Morrell, D., Holder, A. T. & Milner, D. G. (1985) Fetal rat myoblasts release both rat somatomedin-C (SMC-C)/insulin-like growth factor I (IGF I) and multiplication-stimulating activity in vitro: Partial character ization and biological activity of myoblast-derived SM-C/IGF I. Endocrinology 117: 2061-2072. e2. Allen, R. E. & Boxhorn, L. K. (1987) Inhibition of skeletal muscle satellite cell differentiation by transforming growth fac tor-beta. J. Cell. Physiol. 133: 567-572. 63. Florini, J. R., Roberts, A. B., Ewton, D. Z., Falen, S. L., Flanders, K. C. & Sporn, M. B. (1986) Transforming growth factor-beta. A very potent inhibitor of myoblast differentiation, identical to the differentiation inhibitor secreted by buffalo rat liver cells. J. Biol. Chem. 261: 16,509-16,513. 64. Massague', J., Chiefetz, S., Endo, T. & Nadal-Ginard, B. (1986) Type ß transforming growth factor is an inhibitor of myogenic differentiation. Proc. Nati. Acad. Sci. USA 83: 82068210. 65. Dollenmeier, P., Turner, D. C. & Eppenberger, H. M. (1981) Proliferation and differentiation of chick skeletal muscle cells cultured in a chemically defined medium. Exp. Cell Res. 135: 47-61. 66. Florini, J. R. & Roberts, S. B. (1979) A serum-free medium for the growth of muscle cells in culture. In Vitro 15: 983-992.

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67. Allen, R. E., Dodson, M. V., Luiten, L. S. & Boxhorn, L. K. (1985) A serum-free medium that supports the growth of cul tured skeletal muscle satellite cells. In Vitro Cell Dev. Biol. 21: 636-640. 68. Dodson, M. V. & Mathison, B. A. (1988) Comparison of ovine and rat muscle-derived satellite cells: Response to insulin. Tissue Cell 20: 909-918. 69. McFarland, D. C., Pesali, J. E., Norberg, J. M. & Dvoracek, M. A. (1991) Proliferation of the turkey myogenic satellite cell in a serum-free medium. Comp. Biochem. Physiol. 99A: 163167. 70. Straus, D. S. (1989) Regulation by insulin of cellular growth and proliferation: Relationship to the insulin-like growth factor. In: Insulin Action. (Draznin, B., Melmed, S. & LeRoith, D., eds.) Alan R. Liss, New York, NY. 71. Ewton, D. Z., Falen, S. L. a Fiorini, J. R. (1987) The type II IGF receptor has low affinity for IGF-I analogs: Pleiotypic actions of IGFs on myoblasts are apparently mediated by the type I receptor. Endocrinology 120: 115-124. 72. Nissley, S. P. & Haskell, J. F. (1985) Insulin-like growth factor receptors. J. Cell Sci. 3(suppl.|: 39-51. 73. Massague', J. & Czech, M. P. (1982) The subunit structures of two distinct receptors for insulin-like growth factors I and II and their relationship to the insulin receptor. J. Biol. Chem. 257: 5038-5045. 74. Duelos, M. J. & Goddard, C. (1990) Insulin-like growth factor receptors in chicken liver membranes: Binding properties, spec ificity, developmental pattern and evidence for a single receptor type. J. Endocrinol. 125: 199-206. 75. Bassas, L., Lesniak, M. A., Serrano, J., Roth, J. &. De Pablo, F. (1988) Developmental regulation of insulin and type I in sulin-like growth factor receptors and absence of type II recep tors in chicken embryo tissues. Diabetes 37: 637-644. 76. Mohan, S., Linkhart, T., Rosenfeld, R. & Baylink, D. (1989) Characterization of the receptor for insulin-like growth factor II in bone cells. J. Cell Physiol. 140: 169-176. 77. Dodson, M. V., Allen, R. E., Shimizu, N., Shimizu, S. & Hossner, K. L. (1987) Interaction of ovine somatomedin and multipli cation-stimulating activity/rat insulin-like growth factor II with cultured skeletal muscle satellite cells. Acta Endocrinol. 116: 425-432. 78. Mathison, B. D., Mathison, B. A., McNamara, J. P. & Dodson, M. V. (1989) Insulin-like growth factor I receptor analysis of satellite cell-derived myotube membranes established from two lines of Targhee rams selected for growth rate. Dornest. Anim. Endocrinol. 6: 191-201. 79. Shimizu, M. C., Webster, C., Morgan, D. O., Blau, H. M. & Roth, R. A. (1986) Insulin and insulin-like growth factor re ceptors and responses to cultured human muscle cells. Am. J. Physiol. 250: E611-E615. 80. Beguinot, F., Kahn, C. R., Moses, A. C. & Smith, R. J. (1985) Distinct biologically active receptors for insulin, insulin-like growth factors I and II in cultured skeletal muscle cells. J. Biol. Chem. 260: 15,892-15,898. 81. Burant, C. F., Treutelaar, M. K., Allen, K. D., Sens, D. A. & Buse, M. G. (1987) Comparison of insulin and insulin-like growth factor-1 receptors from rat skeletal muscle and L6 myocytes. Biochem. Biophys. Res. Commun. 147: 100-107. 82. Rechler, M. M., Zapf, J., Nissley, S. P., Froesch, E. R., Moses, A. C., Podskalny, J. M., Schilling, E. E. & Humbel, R. E. (1980) Interactions of insulin-like growth factors I and II and multi plication-stimulating activity with receptors and serum carrier proteins. Endocrinology 107: 1451-1459. 83. Steele-Perkins, G., Turner, J., Edman, J. C., Hari, J., Pierce, S. B., Stover, C., Rutler, W. J. & Roth, R. A. (1988) Expression and characterization of a functional human insulin-like growth factor I receptor. J. Biol. Chem. 263: 11,486-11,492. 84. Ballard, F. J., Ross, M., Upton, F. M. & Francis, G. L. (1988)

CONFERENCE: UNDERSTANDING Specific binding of insulin-like growth factors 1 and 2 to the type 1 and type 2 receptors respectively. Biochem. J. 249: 721726. 85. Conover, C. A., Misra, P., Hintz, R. L. & Rosenfeld, R. G. (1988) Differential binding of '"I-IGF-I preparations to human fibroblast monolayers. Acta Endocrinol. 118: 513-520. 8e. Holly, J. M. P. & Wass, J. A. H. (1989) Insulin-like growth factors; autocrine, paracrine or endocrine? New perspectives of the somatomedin hypothesis in the light of recent developments. J. Endocrinol. 122:611-618. 87. McCusker, R. H. &. Clemmons, D. R. (1988) Insulin-like growth factor binding protein secretion by muscle cells: Effect of cellular differentiation and proliferation. J. Cell. Physiol. 137: 505-512. 88. McCusker, R. H., Camacho-Hubner, C. & Clemmons, D. R. (1989) Identification of the types of insulin-like growth factorbinding proteins that are secreted by muscle cells in vitro. J. Biol. Chem. 264: 7795-7800. 8».Blum, W. F., Jenne, E. W., Reppin, F., Kietzmann, K., Ranke, M. B. & Bierich, J. R. (1989) Insulin-like growth factor I (IGFI)-binding protein complex is a better mitogen than free IGF-I. Endocrinology 125: 766-772.

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H). Adashi, E. Y. (1989) Coming of age of insulin-like growth factor binding proteins: Major players in a complex equation. Am. J. Reprod. Immunol. 20: 97-99. 91. Armstrong, D. G., McKay, C. O., Morrell, D. J. &. Goddard, C. (1989) Insulin-like growth factor-I binding proteins in serum from the domestic fowl. J. Endocrinol. 120: 373-378. 91. McCusker, R. H., Campion, D. R., Jones, W. K. & Clemmons, D. R. (1989) The insulin-like growth factor-binding proteins of porcine serum: Endocrine and nutritional regulation. Endo crinology 125: 501-509. 93. Bischoff, R. (1989) Analysis of muscle regeneration using single myofibers in culture. Med. Sci. Sports Exercise 21(suppl.|: S164S172. >4. Vandenburgh, H. H. (1988) A computerized mechanical cell stimulator for tissue culture: Effects on skeletal muscle organ ogénesis.In Vitro Cell. Dev. Biol. 24: 609-619. 95. Strohman, R. C., Bayne, E., Spector, D., Obinata, T., MicouEastwood, J. & Maniotis, A. (1990) Myogenesis and histogenesis of skeletal muscle on flexible membranes in vitro. In Vitro Cell. Dev. Biol. 26: 201-208.