Induction of Myoblast Proliferation in L6 Myoblast ...

Induction of myoblast proliferation in L6 myoblast cultures by fetal serum of double-muscled and normal cattle D. E. Gerrard and M. D. Judge J ANIM SCI 1993, 71:1464-1470.

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Induction of Myoblast Proliferation in L6 Myoblast Cultures by Fetal Serum of Double-Muscled and Normal Cattle1 D. E. Gerrard2 and M. D. Judge Department of Animal Sciences, Purdue University, West Lafayette, IN 47907

ABSTRACT: Double-muscled (DM ) cattle possess more muscle fibers than do normal-muscled (NM) beef or dairy cattle. Serum-borne growth factors have been shown to modulate myogenesis. Media containing serum from 12 DM and 60 NM fetuses grouped by crown-rump lengths (CRL) of I 2 5 , 26 t o 50, 51 to 75, or > 75 cm were used to test the effect of fetal serum on L6 myoblast proliferation. Because DM and NM fetuses were similar in CRL at ages corresponding to 20- to 60-cm CRL, CRL was used to determine fetal age. Normal-muscled fetal serum-induced thymidine incorporation in L6 myoblasts was greater ( P < .05) at CRL > 50- than at I 50-cm CRL. Mean incorpora-

tion tended to increase with CRL. Thymidine incorporation was 56, 41, and 41% greater ( P < .05) with serum from DM fetuses than with that from NM fetuses at CRL of I 25, 26 to 50, and 51 to 75 cm, respectively. Morphological examination of cross-sections of the semitendinosus muscle showed that apparent muscle fiber number was greater ( P < .05) for DM fetuses than for NM fetuses. These results confirm greater apparent muscle fiber number in DM cattle and show the existence of greater growth factor activity in serum of DM fetuses during early fetal development. This greater growth factor activity may play a role in bovine muscle fiber hyperplasia.

Key Words: Bovine, Fetus, Double Muscling, Myoblast Proliferation, Muscle Fibers, Serum

J. h i m . Sci. 1993. 71:1464-1470

Introduction Fetal muscle fiber development occurs in two phases (Le., formation of primary followed by formation of secondary myofibers; Ontell and Kozeka, 1984). Secondary myofibers develop from a population of myoblasts that attach longitudinally to primary myotubes. Alterations in the ratio of primary:secondary fibers during fetal muscle development have been implicated in muscle hypertrophy (Swatland, 1984). Double-muscled ( DM) cattle develop nearly 40% more muscle fibers than do normal-muscled ( NM) cattle at birth (MacKellar, 1968). Fetal blood-borne growth factors may modulate muscle fiber formation via secondary myofiber-forming myoblasts during development. Alterations in serum growth-factor profiles have been assessed by several researchers using myoblast

'Journal paper no. 13500, Purdue Agric. Exp. Sta., West Lafayette, IN 47907. Supported in part by a grant from Lilly Research Laboratories, Greenfield, IN. Presented in part at the ASAS 84th Annu. Mtg., Laramie, WY, at the Reciprocal Meat Conf., Manhattan, KS, 1991, and at the 38th Int. Congr. of Meat Sci. and Tech., Clermont Ferrand, France, 1992. 2Present address: Dept. of Food Sci. and Nutr., Univ. of Missouri, Columbia 65211. Received August 10, 1992. Accepted February 5, 1993.

cultures as a bioassay (Allen et al., 1982, Kotts et ai., 1987a,b; White et al., 1988, 1989; Konishi, et al., 1989). Therefore, the purpose of this research was to determine the mitogenic activity of serum from developing bovine fetuses of DM and NM cattle using an L6 myoblast culture bioassay.

Experimental Procedures Serum Samples Fetal blood samples from 60 mixed breeds and sexes of beef or Holstein dairy (DM) fetuses were collected from pregnant cows slaughtered in a commercial slaughter plant. Although it is possible that some of the fetuses collected from this facility were DM, they could be only heterozygous for the condition because none of the dams showed the signs of the DM phenotype as described by Kieffer and Cartwright (1980). Blood was also collected from 12 DM Belgian Blue fetuses of pregnant cows slaughtered in the Purdue University abattoir. Intact uteri were retained, fetuses were liberated, and umbilical cords were exposed. Blood was collected from the umbilical cords within 15 min after exsanguination of the dam, placed on ice, and held for 24 h at 4°C to allow clotting. Serum was separated by centrifugation of the

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SERUM-INDUCED MYOBLAST PROLIFERATION

clotted blood samples at 2,000 x g for 2 h at 4°C. Serum samples were sterilized by filtration through .22-pm filters (Fisher Scientific, Pittsburgh, PA), divided into small sample sizes, and stored at -20°C. Fetal age was recorded by crown-rump length (CRL; measured from the ischium to the poll of the cranium). Because calves attain CRL of approximately 100 cm a t birth (Evans and Sack, 19731, four predetermined groups were established representing stage of gestation (< 25, 26 to 50, 51 to 75, > 75 cm).

Muscle Fiber Number Muscle samples were taken from the middle of the intact semitendinosus muscle for estimation of apparent muscle fiber number. Samples were taken within 30 min after exsanguination. Circumference of the semitendinosus muscle as determined using a small, flexible measuring tape. Small groups of muscle fibers were teased apart and clips separated by a copper rod were affixed to exposed muscle fibers. Samples were quickly frozen in liquid nitrogen-cooled freon and were stored in liquid nitrogen until sectioning. Frozen muscle strips were tempered to -2"C, and 10-pm sections were made using a rotary freezing microtome (International Equipment, Needham Heights, MA). Sections were dry-mounted and stained with Harris's hematoxylin and eosin. Quantification of the apparent muscle fiber number was performed using a Quantimet 570 image analyzer (Leica, Deerfield, IL). Five randomly selected fields (approximately 480 pm2 each) were projected onto a monitor and all muscle fibers within the field (excluding the fibers that were partially outside the field) were outlined and counted. Average areas of muscle fibers and connective tissue matrix were calculated and used t o determine mean packing density (muscle fibers/ area). Estimated apparent muscle fiber number was determined for the semitendinosus cross-section using the circumference and mean packing density estimates.

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medium at a concentration of 1, 100, and 100 ngimL, respectively, and served as treatments for characterizing the response of L6 myoblasts to growth factors. Cell numbers were determined using triplicate plates stained with Giemsa and counting nuclei in 10 randomly selected fields per plate.

Labeled Thymidine Uptake After an initial 24-h period for myoblast anchorage, complete medium was removed, monolayers were washed with warmed Hank's Balance Saline Solution (HBSS; Sigma Chemical) and treatment media were added. Treatment media consisted of 10% HS, FBS or serum from an assigned fetus and 90% EMEM. Thymidine ( M e t h ~ l - ~ H60 , to 90 Ciimmol [ICN Biomedicals, Costa Mesa, CAI) was added for a final concentration of .1 pCiimL of media. Myoblasts were allowed to replicate in the presence of treatment media for 18 h. Myoblast replication was halted by addition of cold HBSS. Monolayers were precipitated with cold 10% trichloroacetic acid ( TCA) and washed three times with 1% TCA. Precipitated cells were digested in .5 M sodium hydroxide for 30 min at room temperature. An aliquot was combined with scintillation cocktail (Budget-Solve, Research Products International, Mount Prospect, IL) and assessed by liquid scintillation counting. Mitogenic activity was expressed as nucleotide uptake (counts per minute/ dish).

Statistical Analysis Data were statistically analyzed using the GLM procedure of SAS (1988). Main effects tested were treatment (NM or DM) and age (CRL group: < 25, 26 to 50, 51 to 75, or > 75 cm). Means were separated using Bonferonni t-tests (SAS, 1988).

Results and Discussion

L6 Myoblast Cultures

Fetal Growth and Muscle Histology

A subclone (ELC5; Anderson et al., 1990) of the rat L6 myoblast cell line (Yaffe, 1968) was obtained from C. K. Smith I1 (Lilly Research Laboratories, Greenfield, IN). Myoblasts were plated at 10,000 cells/cm2 in untreated 35-mm sterile, Falcon tissue culture plates (Becton Dickinson, Lincoln Park, N J ) in the presence of complete media (90% Eagle's Minimal Essential Medium, EMEM;10% fetal bovine serum, FBS [Gibco BRL, Life Technologies, Gaithersburg, MDI; in combination with 1 mgimL of penicillinstreptomycin, .5 mg/mL of gentamicin sulphate, .25 mg/mL of amphotericin B [Sigma Chemical, St. Louis, Mol). Cultures were incubated at 37°C in a humidified 5% C02 atmosphere. Transforming growth factorbeta 1 (TGF-81, IGF-11, or fibroblast growth factor (FGF; Sigma Chemical) was included in the culture

A sample of 9 of the 60 NM fetuses of known ages used in this study had CRL (body length [cml = -16.8 + .392 [age]) similar to those reported by Spencer and Coulombe (1965) (body length [cml = -22.7 + .419 [age]). Crown-rump length of DM fetuses was similar to those of NM from 20- to approximately 65-cm CRL, after which CRL was not an indication of age for DM fetuses. This is not surprising because Robelin et al. (1991) showed that data used to establish a regression equation to calculate the age of bovine fetuses had more variation during the later stages of gestation. Because fetal bone growth has a growth coefficient of > 1 during the last stage of gestation (Johnson, 1974) and NM fetuses have a greater percentage of bone than do DM fetuses (Dumont, 19821, it is logical that CRL, which is


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dependent on bone growth, would not be a reliable estimate of fetal age during later stages of development of DM fetuses. Furthermore, it is well documented that skeletal size of DM cattle is less than that of NM cattle (MacKellar, 1968; Vissac, 1968; Ansay and Hanset, 1972, 1974). Therefore, age estimation of bovine fetuses by CRL should be restricted to fetuses that are < 210 d postconception. These data reaffirm CRL as an estimate of fetal age between 90 and 210 d postconception for NM fetuses and show that CRL can be used to determine the age of DM fetuses within this time frame. Histological examination of developing muscle tissue revealed linear decreases in the connective tissue matrix in the cross-section of the semitendinosus ( R 2 = .92 for NM fetuses and .74 for DM fetuses). Muscle fibers accounted for approximately 30 and 90% of the cross-sectional area of the semitendinosus of 15-cm fetuses and near-term fetuses, respectively. These observations are in agreement with those of Robelin et al. (1991). Connective tissue matrix crosssectional area of the semitendinosus muscle of a DM fetus was reduced at the earliest time point observed. The reason for this is not known; however, adult DM cattle have less connective tissue than NM cattle do (Bailey et al., 1982). Further reduction of connective tissue cross-sectional area during fetal development was not observed until the latest time point. However, when data were fitted to a line, DM fetuses tended to have less connective tissue matrix than NM fetuses did. Ontogeny of the semitendinosus muscle in NM fetuses was similar to that reported by others (Ashmore et al., 1974; Swatland and Kieffer, 1974; Robelin et al., 1991). The youngest NM fetuses examined had muscle fibers with a mean area of approximately 100 pm2 (Figure 1).Although histological sections of only one DM fetus of this age were examined, mean fiber area was only approximately 50 pm2. Given that the semitendinosus muscle of DM fetuses has less connective tissue matrix than does that of NM fetuses, DM fetuses have more muscle fibers than do NM fetuses at the earliest time point observed. This observation reaffirms the hypothesis by others (Ashmore et al., 1974; Swatland and Kieffer, 1974) that the DM phenotype manifests itself before 90 d after conception. Mean fiber area decreased greatly from 100 to 50 pm2 between 15- and 38-cm CRL in NM fetuses. Simultaneously, a large increase in apparent muscle fiber number was observed at approximately 38-cm CRL. Swatland and Kieffer (1974) showed similar increases in muscle fiber number between 10 and 42 cm. Data from fetuses of < 13.3-cm CRL were not available in this study, but these data suggest that a major phase of myofiber hyperplasia occurs between 13.3- and 38-cm CRL in bovine fetuses. Mean area of myotubes from DM fetuses changed little between 20- and 38-cm CRL compared with the

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area of myotubes from NM fetuses. Because the decrease of myofiber area observed in NM fetuses was due to increased presence of smaller secondary fibers, a possible explanation for this observation is that, at 20-cm CRL, DM fetuses already have begun secondary myofiber formation. This phase of myofiber formation in DM fetuses continued past that of NM fetuses that leads t o approximately 1 x lo6 more muscle fibers in the cross-section of the semitendinosus muscle of a full-term fetus. This observed increase in apparent muscle fiber number of DM cattle compares favorably to the increase observed by MacKeller (1968) in the extensor digitorum longus muscle of South Devon cattle. Larger differences between NM and DM fetuses could be expected, but estimation of muscle fiber number is more difficult to determine in the fetal stage due to the presence of intrafascicularly terminating muscle fibers, which would account for a higher percentage of the total number of muscle fibers in fetal muscle tissue than in mature muscle. For the period of gestation studied in this experiment, estimated apparent muscle fiber number increased linearly (NM fiber number = 287480[CRLl - 3900210, R2 = .958; DM fiber number = 337102rCRLl t 1359989, R2 = 885).

L6 Myoblast Proliferation For L6 myoblast cultures, plating efficiencies of > 90% were observed with trypan blue exclusion across all studies. In preliminary studies, the use of a myosin



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Figure 2. Means (reported in thousands) and standard errors for cell number and incorporation of [3H]thymidine into replicating L6 myoblasts after 24 and 72 h in medium containing fetal bovine serum (FBS) or horse serum {HS).Cell number after 24 h (solid bars). Cell number after 72 h (open bars). Thymidine incorporation after 72 h (hatched bars). Each bar represents the mean of three cultures. Means within a time bearing letters do not differ ( P < .05].

Figure 3. Means (reported in thousands) and standard errors for incorporation of [3H]thymidine into replicating L6 myoblasts cultured in the presence of different sera and growth factors (HS, horse serum; IGF-11, insulin-like growth factor-11; FGF, fibroblast growth factor; TGF-P, transforming growth factor-beta]. Each bar represents the mean of three cultures. Means within serum type bearing the same letters do not differ (P > .05).

heavy-chain antibody as described by Bader et al. (1982) indicated that < 10% of the total nuclei in L6 myoblast cultures, before treatment application, were in myotubes. These results suggest that myoblast cultures were viable and > 90% of the nuclei were in undifferentiated cells. Linear increases in tritiated thymidine incorporation were observed with cell number (data not shown). Tritiated thymidine incorporation was greater for myoblasts cultured in the presence of 10% FBS than for those cultured in 10% horse serum ( HS) (Figure 2). This increase was similar to an increase in Giemsa-stained myoblasts of sister cultures allowed to replicate for 72 h after addition of thymidine. These data indicate that incorporation of tritiated thymidine increases with cell number and serum type and may be used as an indicator of cell replication in L6 myoblast cultures. Addition of 100 ng of IGF-II/mL of medium increased ( P < .05) the incorporation of tritiated thymidine compared with control 1%HS (Figure 3 ) . Similar increases in proliferation of myoblasts in the presence of IGF-I1 have been observed by Ewton and Florini (1980). Myoblasts cultured in the presence of 10% FBS exhibited increased thymidine uptake over control media, whereas addition of FGF to HSsupplemented medium did not elicit ( P > .05) a response. This observation complements research of

Clegg et al. (19871, who were unable to detect FGF receptors on L6 myoblasts. Furthermore, addition of TGF-/3 to L6 myoblast cultures only slightly decreased thymidine uptake ( P > .05) compared with control 10% FBS. This observation is in agreement with others who claim that L6 myoblasts do not respond to a significant extent to TGF-p (Florini et al., 1986; Massague et al., 1986; Florini and Ewton, 1988; Florini and Magri, 1989). Conversely, Pampusch et al. (1990) showed that as little as .08 ng of TGF-OgImL of medium causes a 50% reduction in L6 myoblast proliferation. The lack of inhibition in this system may represent differences in L6 clones, lower plating densities, or the length of time that cells were exposed to TGF-0, compared with those of the other studies. These results confirm that the L6 myoblast clone used in this study responds to growth factors similar to L6 myoblast clones reported in the literature. Although not statistically significant at all time points, serum-stimulated myoblast replication tended to increase with CRL for both NM and DM fetuses (Figure 4 1. Normal-muscled fetal serum-induced thymidine incorporation in L6 myoblasts was greatest at CRL > 50, least at CRL 5 25, and intermediate for 26- to 50-cm CRL ( P < .05). Thymidine uptake was greater with serum from DM fetuses than with serum from NM fetuses at CRL 5 25, 26 to 50, and 51 to 75 cm ( P < .05) and tended to be greater at > 75 cm.


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Figure 4. Means (reported in thousands) and standard errors for incorporation of [3H]thymidine into replicating L6 myoblasts cultured in the presence of medium containing 10% sera from developing double-muscled (hatched bars) and normal [open bars) fetuses. Means bearing the same letters do not differ (I' > .05).

General Discussion During time points at which secondary myofiber formation was greatest, serum-stimulated myoblast replication was greater for DM than for NM fetuses. It is tempting to suggest that serum-borne growth factors play a role in mediating muscle fiber hyperplasia; however, direct evidence for this phenomenon is not available from these studies. Whether the population of myoblasts that form the secondary fibers is already positioned at these young ages is not yet known; however, it is difficult t o imagine that all nuclei needed to establish the amount of muscle found in the newborn calf are present during these early stages of development. Furthermore, Ansay and Hanset (1985) reported that from 3 to 7 mo after conception, muscle weight is paralleled by increases in DNA concentration in developing bovine fetuses. Quinn et al. (1990) showed that myoblasts from DM fetuses replicate more than do those from NM fetuses. Although the mechanism controlling the increase in DNA concentration is now known, a paracrine (autocrine) production of growth factors is likely, but production of growth factors remote to their site of action cannot be ruled out (Daughaday and Rotwein, 1989). Greater myoblast proliferation observed in the presence of serum from DM fetuses over NM fetuses may result from the presence of a DM-specific serum factor that is responsible for the expression of bovine muscle fiber hyperplasia. More likely, an aberrant level of growth factor is present during a critical stage

of myogenesis. The L6 myoblasts used in this study did not respond to two well-known mitogens, FGF and TGF-P (Figure 2). This does not eliminate the possibility of TGF-/3 and(or) FGF mediating prenatal muscle development, but it indirectly suggests that other growth factors are responsible for increased myoblast replication in this system. Florini et al. (1991) showed that IGF-I1 expression is mandatory for myogenesis in several muscle cell lines. Even though IGF-I1 is required for the expression of myogenin during differentiation (Florini et al., 19911, IGF-I1 stimulates myoblast proliferation at high concentrations (Florini et al., 1986). Hypothetically, the paracrine (autocrine) production of muscle IGF-I1 may be responsible for the increased mitogenic activity observed in the L6 myoblast bioassay. Florini et al. (1991) showed increased IGF-I1 mRNA and IGF-I1 protein during myoblast differentiation. Because DM fetuses have more muscle fibers differentiating at all times observed, the amount of IGF-I1 that the developing muscle of DM fetuses would contribute to the serum may be greater than that contributed by fetuses with less muscle mass. Assuming identical contributions from the liver of both types of fetuses, circulating IGF-I1 in DM fetuses would be greater than that in NM fetuses. If this is true, then the increased serum-induced myoblast proliferation by DM fetuses may be from additional IGF-I1 produced in muscle tissue; however, experiments to delineate the source of circulating IGF-I1 are presently difficult to conduct and none has been reported. Evaluation of serum concentrations of both IGF-I and -11 would provide information concerning the ontogeny of the growth factors during fetal development; however, cross-reaction of IGF-I1 antibodies to IGF-I has prevented estimates of serum IGF-I1 concentrations in bovine fetuses (Honegger and Humbel, 1986) but may be available using the procedure shown by Vega et al. (1991). Data on the expression of IGF-I1 in muscle and liver tissues would be helpful in supporting or refuting this hypothesis because abundance of IGF-I1 mRNA in the liver is correlated to concentrations in the serum (Romanus et al., 1988). Serum-stimulated mitogenic activity has been correlated to birth weight in humans (Underwood and D'Ercole, 19841, whereas birth weight is correlated to serum IGF-I collected from the umbilical cords of developing humans (Bennett et al., 1983; Gluckman et al., 1983; Ashton et al., 1985). The majority of increase in fetal bovine weight occurs during the last trimester of gestation (Robelin et al., 19911, which corresponds to 51- to 75- and > 75-cm CRL in this study. Therefore, serum-induced myoblast proliferation increases with bovine fetal body weight. Collectively, these observations suggest that age-associated changes in serum-induced L6 myoblast proliferation may be a consequence of a larger amount of developing tissue. Curiously, the third trimester of gestation coincides with muscle cell hypertrophy, not hyperplasia (Hammond, 1962; Ashmore et al., 1972; Swatland



and Kieffer, 1974). Thus, a direct effect of serum to stimulate the development of additional fetal muscle fibers is unlikely; however, the time of gestation during which satellite cell replication occurs is not known but may be dependent on serum growth factors. Age-related increases in serum-induced myoblast replication could be attributed to increased presence of circulating IGF-I. Although concentrations were not evaluated in this study, evidence is mounting that implicates IGF-I as a growth factor that occurs in late gestation and postnatally. In support of this hypothesis, liver IGF-I mRNA and circulating IGF-I concentrations increase during the later stages of fetal development (Daughaday et al., 1980; Vileisis and DErcole, 1986; Rotwein et al., 1987). Furthermore, Kotts et al. (1987a) artificially increased circulating IGF-I concentrations in pigs by administration of growth hormone and were successful in stimulating myoblast proliferation in cultures with sera from these pigs. Therefore, it is reasonable to postulate that the increase in mitogenic activity of serum from older fetuses is from the presence of greater concentrations of circulating IGF-I. Together, these data show that DM fetuses have a greater number of muscle fibers by approximately 90 d of gestation and that serum from DM fetuses stimulates replication of cultured L6 myoblasts more than does serum from NM fetuses. Serum-borne growth factors may play a role in the regulation of prenatal muscle growth.

Implications Mechanisms controlling the development of an animal with 40% more muscle represent a biological phenomenon worth exploiting. A large portion of muscle cell hyperplasia in bovine fetuses occurs between 85 and 210 d after conception. Serum-induced mitogenic activity is greater in double-muscled fetuses than in normal-muscled fetuses during this time. Serum-borne growth factors may play a role in development of the double-muscled phenotype. Further work to identify the source and type of growth factors present during the development of doublemuscled fetuses that result in increased myoblast proliferation may lead to better understanding of the complex mechanisms controlling myogenesis.

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