| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Animal Growth and Development Laboratory, Department of Animal Science, University of Minnesota, St. Paul 55108 and
and
Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506
3 Correspondence:
348 ABLMS, 1334 Eckles Ave. (phone: 612-624-2234; fax: 612-624-3677; E-mail:
wdayton{at}umn.edu).
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
Key Words: Estradiol • Growth Promoters • Insulin-Like Growth Factor • Muscles • Steers • Trenbolone
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
The University of Minnesota Animal Care Committee approved all experimental procedures. Fourteen crossbred yearling steers, matched for age and frame size, were used in this study. Average weight of the steers at the beginning of the experiment was 382 kg. Prior to implantation, steers were maintained on a high-roughage backgrounding diet (80% corn silage, 15% cracked corn, and 5% supplement). Beginning 14 d before implantation, steers were fed a step-up diet, so that by the day of implantation, they were consuming a high-concentrate (85% cracked corn, 10% corn silage, and 5% supplement) diet. This diet (77% DM; 13.2% CP) was fed ad libitum for the duration of the study. Steers were allocated into four pens (two pens of four steers/pen and two pens of three steers/pen), and steers in two of the pens (one pen containing four steers and one pen containing three steers) were implanted with Revalor-S on d 0 of the study. Steers were weighed weekly and feed intake of each pen was determined daily. Blood samples for IGF-I radioimmunoassays were taken from the left jugular vein on d 0, 6, 14, 20, 31, and on the day of slaughter. Insulin-like growth factor-I RIA data obtained from this study have been reported previously (Johnson et al., 1998a). Because facilities allowed preparation of satellite cells from two steers per day, one randomly selected implanted steer and one randomly selected nonimplanted steer were sacrificed on d 32 through 38 and satellite cells and RNA were isolated from the SM of each animal.
Ribonuclease Protection Assays (RPA) for IGF-I, IGFBP-3, IGFBP-5 Myostatin, and HGF. Semimembranosus muscle and liver tissues were obtained immediately after death from steers used for satellite cell isolation and flash-frozen in liquid nitrogen. Total RNA was isolated using the single-step guanidinium thiocyanate procedure (Chomczynski and Sacchi, 1987). Briefly, total muscle tissue RNA (30 µg) or total liver RNA (10 µg) was hybridized with 5 x 105 counts/min of a bovine IGF-I, IGFBP-3, IGFBP-5 myostatin, or HGF antisense complementary RNA (cRNA) probe at 45°C overnight in hybridization solution (80% deionized formamide, 100 mM sodium citrate, 300 mM sodium acetate, pH 6.4, 1 mM EDTA). Following hybridization, samples were treated with a mixture of RNase A and T1 (Ambion, Inc., Austin, TX) for 30 min at 37°C. Protected RNA fragments were precipitated, dissolved in loading buffer, denatured at 90°C for 5 min, and resolved by electrophoresis on a 5% acrylamide, 8 M urea denaturing gel. The gel was then dried and subjected to autoradiography. In order to control for variation in RNA loading in the RPA, a labeled cRNA fragment of bovine cyclophilin was included in each hybridization reaction and the cyclophilin signal intensity was used to normalize the IGF-I, IGFBP-3, IGFBP-5, HGF, myostatin, and HGF signal intensities. The relative signal intensities of the protected fragment bands were measured using a Biorad GS-670 imaging densitometer in conjunction with Biorad Molecular Analyst software (Biorad Laboratories, Hercules, CA).
Bovine IGF-I, IGFBP-3, IGFBP-5, and Myostatin cDNA. Bovine IGF-I, IGFBP-3, and IGFBP-5 cDNA were prepared in our laboratory by reverse transcription-PCR (RT-PCR) from bovine liver total RNA, and bovine myostatin cDNA was prepared by RT-PCR from bovine semimembranosus muscle total RNA.
Insulin-like growth factor-I primers were designed to amplify a 348-nucleotide (nt) sequence (209 to 556, accession number X15726) of the coding region of IGF-I and to add the T3 (5' end) and T7 (3' end) RNA polymerase promoter sequences to the amplified sequence. The forward primer was 5'-TTATTAACCCTCACTAAAGGGAAGGCTTTTATTTCAACAAGCCC-3' and the reverse primer was 5'-TAATACGACTCACTATAGGGCGATTTTGGTA-GGTCTTCTGGTG-3'. The sequence of the IGF-I cDNA amplicon was verified to be a 100% match with the appropriate section of the published bovine IGF-I cDNA (accession number X15726). The amplicon was then inserted into a TA cloning vector (pT-Adv, Clontech, Palo Alto, CA), and a clone with inverted insertion was isolated and cut with StyI (Promega Corp., Madison, WI) to remove an unwanted portion of the IGF-I sequence. The resulting plasmid, containing the T7 polymerase site, nucleotides 389 to 556 from the IGF-I sequence, and a portion of the vector sequence, was allowed to self-ligate. To generate an antisense IGF-I cRNA probe, this construct was cut with AvaII and transcribed with T7 polymerase. This yielded a 257-nt antisense probe that protected a 168-nt portion of IGF-I mRNA.
Bovine IGFBP-3 cDNA (nt 321 to 841, accession number M76478) was produced by RT-PCR of bovine liver total RNA using the forward primer 5'-GGCTGCGGTTGCTGTCTCA-3' and the reverse primer 5'-TCGCAGTTGGGAATGTGGATG-3'. The amplicon was inserted into a PCR cloning vector PT-Adv (Clontech) in the antisense orientation. When cut with StyI and transcribed with T7 polymerase, this construct yielded a 248-nt cRNA probe that protects a 180-nt (662 to 841) fragment of the bovine IGFBP-3 mRNA.
Bovine IGFBP-5 cDNA (corresponding closely to nt 351 to 680 of human IGFBP-5 cDNA) was produced by RT-PCR of bovine liver total RNA using the forward primer 5'-GGTTTGCCTCAACGAAAAGAG-3' and reverse primer 5'-GCGTGGGCTGGCTTTG -3' designed from the human cDNA sequence (accession number M62782). The amplicon was cut with BglII (Promega Corp.) and PstI (Promega Corp.) to yield a 199-nt fragment (nt 402 to 600) that was inserted into pBluescript SK+. When cut with SpeI (Promega Corp.) and transcribed with T7 polymerase, this construct yielded a 264-nt cRNA probe that protected a 199-nt fragment of the bovine IGFBP-5 mRNA.
Bovine myostatin cDNA was produced using RT-PCR of bovine SM total RNA. Primers were chosen from the published bovine myostatin cDNA sequence (accession number AF019620) to yield a 945-nt amplicon (nt 56 to 1,000). The forward primer was 5'-GCCCAGTGGATCTGA-ATGAGA-3' and the reverse primer was 5'-CTCTGGGGTTTGCTTGGTG-3'. The amplicon, whose nucleotide sequence was identical to the published sequence of bovine myostatin cDNA, was inserted into a TA cloning vector (pT-Adv, Clontech.). This construct was then cut with SpeI and the resulting 581-nt fragment was ligated into the SpeI site of pBluescript. When cut with Bgl4 (Promega Corp.) and transcribed with T7 polymerase, this construct yielded a 293-nt antisense cRNA probe that protected a 208-nt fragment of the myostatin mRNA (nt 793 to 1,000).
Bovine Hepatocyte Growth Factor cDNA. Bovine HGF cDNA was produced using RT-PCR of total RNA isolated from bovine liver. Primers were chosen from the published genomic sequence of bovine ovarian thecal cell HGF (accession number S72476). Comparison of the published bovine genomic HGF sequence with the human HGF cDNA sequence (M60718) revealed that the human sequence contained 40 nt (656 to 695) that were not present in the bovine sequence. Consequently, we chose forward and reverse primers from regions of the bovine sequence that were present in the human cDNA sequence. The forward primer was 5'-TGCTATCGGGGTAAAGACCTAC-3' and the reverse primer was 5'-CCCATCAAAGCCCTTGT-3'. Based on the published bovine sequence and the location of the primers, we anticipated an amplicon of 235 nt. Instead, the amplicon we obtained contained 275 nt. The additional nucleotides in the amplicon corresponded to the 40 nt present in the human HGF cDNA sequence, but missing from the published bovine genomic sequence; in addition, nt 3 to 275 in the amplicon were identical to nucleotides 569 to 841 in the human HGF cDNA. Whereas it is unclear why there is a discrepancy between our sequence and the published bovine genomic sequence, our sequence agrees closely with the human HGF cDNA sequence. The amplicon was inserted into a TA cloning vector (pT-Adv, Clontech) and this construct was then cut with XhoI (Promega Corp.) and Kpn1 (Promega Corp.) to yield a restriction fragment that was ligated into the multiple cloning site of pBluescript. When cut with XhoI (Promega Corp.) and transcribed with T7 polymerase, this construct yielded a 296-nt antisense cRNA probe that protected a 233-nt fragment of the HGF mRNA.
Statistical Analysis. Data were analyzed by ANOVA with the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). Total RNA isolated from each steer was analyzed in three separate ribonuclease protection assays (RPA) for each growth factor. Data shown for each growth factor are from one of these assays that was representative of the results obtained in each individual RPA. In cases where main effects were significant, differences between means for preplanned comparisons were tested using Fisher’s LSD test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
Since serum IGF-I levels were elevated in implanted steers, and the liver was thought to be the source of much of the circulating IGF-I, we were interested in determining if hepatic steady-state IGF-I mRNA levels were also elevated by implantation. Ribonuclease protection assays were used to measure the steady-state IGF-I mRNA concentrations in total RNA isolated from liver samples collected from control and implanted steers on d 32 to38 postimplantation. Figure 1
shows the full length and protected fragments of the probes used in the RPA. Figure 2A shows the results of an IGF-I ribonuclease protection assay of total RNA isolated from the liver of representative implanted and nonimplanted steers. Densitometric analysis of RPA data (Figure 2B) from seven control and seven implanted steers showed that steady-state IGF-I mRNA levels were 69% higher (P < 0.01, n = 7) in the livers of implanted steers than in the livers of nonimplanted animals. This suggests that the liver may be a source of at least part of the increased circulating IGF-I in steroid-implanted animals.
|
|
Several studies have established that IGF-I is produced locally in muscle tissue and that it may function in an autocrine or paracrine manner to stimulate muscle growth. Additionally, our previous studies with the same steers used in the current study have shown that more satellite cells can be isolated from the SM of implanted steers than from the SM of nonimplanted steers. Consequently, it was of interest to determine if TBA/E2 implantation increased steady-state concentration of IGF-I mRNA in muscle tissue. Figure 3A shows the results of an IGF-I ribonuclease protection assay of total RNA isolated from the semimembranosus muscles of a representative implanted and nonimplanted steers. Densitometric analysis of RPA data from seven control and seven implanted steers showed that IGF-I mRNA concentrations were higher in semimembranosus (50%, P < 0.05, n = 7) muscles of implanted steers than in the corresponding muscles of nonimplanted steers (Figure 3B). Viewed in light of reports that increased local IGF-I levels increase muscle mass, and in light of results from the same steers used in the current study showing that the SM from implanted steers contained more satellite cells than the SM from nonimplanted steers, these data suggest that TBA/E2-induced increases in muscle IGF-I may be at least partially responsible for the increased cell number and muscle growth observed in implanted steers.
|
Steady-state IGFBP-5, HGF and myostatin mRNA levels in the SM of implanted and nonimplanted steers were compared. There were no significant differences in the levels of these mRNA in the muscle of implanted vs. nonimplanted steers; however, muscle HGF mRNA tended to be lower (P < 0.1, n = 7) in implanted steers (Figure 4). Additionally, the levels of hepatic IGFBP-5 and HGF mRNA were not different in implanted vs. nonimplanted steers (Figure 5).
|
|
Since we have previously shown that circulating IGFBP-3 levels are increased in implanted vs. nonimplanted steers, we measured steady-state IGFBP-3 mRNA levels in SM and livers of implanted and nonimplanted steers in order to determine the source of this increased circulating IGFBP-3. Steady-state hepatic IGFBP-3 mRNA levels are 24% higher (P < 0.07, n = 7) in implanted steers than in nonimplanted steers (Figure 5
). Steady-state IGFBP-3 mRNA levels in the SM of implanted and nonimplanted steers are not different (Figure 4).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
Myostatin is a member of the transforming growth factor (TGF)-ß super family and has been shown to suppress muscle growth in mice (McPherron et al., 1997). Additionally, mutations in the myostatin gene are responsible for the double muscling phenomenon observed in beef cattle (Grobet et al., 1997). Recent reports indicate that the level of myostatin protein and/or mRNA is altered in muscle tissue undergoing hypertrophy, repair, or atrophy (Carlson et al., 1999; Kirk et al., 2000; Sakuma et al., 2000). These results led us to speculate that myostatin mRNA levels might be altered (decreased) by Revalor-S implantation, thus allowing increased proliferation of muscle satellite cells. However, our results show that after 32 to 38 d of implantation, the myostatin mRNA levels in the SM of implanted steers are not significantly different than those in the SM of nonimplanted steers.
We have shown previously that a greater number of actively proliferating satellite cells can be isolated from the SM of implanted steers than from the corresponding muscles of nonimplanted steers. The greater yield of proliferating satellite cells could result from increased activation of quiescent satellite cells or from maintaining satellite cells in an actively proliferating state. Because HGF has been reported to be a crucial element in the activation of quiescent satellite cells (Allen et al., 1995; Tatsumi et al., 1998; Sheehan et al., 2000), our finding that HGF mRNA levels are not increased and may even be lower in the SM of Revalor-S-implanted steers than in the corresponding muscles of nonimplanted steers could be interpreted to mean that activation of quiescent cells does not play a significant role in the Revalor-S-induced increase in proliferating satellite cells. However, several factors need to be considered before drawing this conclusion. Our study measured HGF mRNA after 32 to 38 d of implantation. It is possible that transient elevations in HGF mRNA and protein levels that are sufficient to activate quiescent satellite cells have already occurred by this time. Alternatively, it is possible that HGF levels are increased by releasing HGF that is stored in muscle tissue or by increasing the translation rate of HGF mRNA. Neither of these alternatives would require increases in HGF mRNA levels. Consequently, although our current results raise questions as to the role of increased HGF and satellite cell activation in the Revalor-S-induced increase in actively proliferating satellite cells in the SM, these results do not unequivocally eliminate activation of quiescent satellite cells as a mechanism. Further studies evaluating the time course of changes in muscle HGF and myostatin mRNA levels and satellite cell response to Revalor-S implantation will be required to ascertain the role of these growth factors and satellite cell activation in the Revalor-S-induced increase in actively proliferating satellite cells.
Hepatic IGFBP-3 mRNA levels are slightly elevated in implanted steers vs. nonimplanted controls. Although this elevation is slight, it is consistent with our previous observation that circulating IGFBP-3 levels are increased by Revalor-S implantation (Johnson et al., 1996b). The biological significance of this increase in circulating IGFBP-3 is not clear.
Steady-state IGF-I mRNA levels are significantly increased in the livers of steers implanted for 32 to 38 d with Revalor-S. Previous studies with these same steers have shown that circulating IGF-I concentrations are 33% higher (P < 0.01, n = 7) in steers implanted for 30 d with Revalor-S than in nonimplanted steers (Johnson et al., 1998a). These data are consistent with previous studies showing that hepatic IGF-I mRNA levels and circulating IGF-I concentrations are elevated (40% and 68%, respectively) in Revalor-S-implanted lambs (Johnson et al., 1998b). Thus, in both lambs and steers, Revalor-S implantation increases both hepatic IGF-I mRNA levels and circulating IGF-I levels. Since liver is thought to be a major source of circulating IGF-I, it is likely that increased production of IGF-I by the liver is at least partially responsible for increased circulating IGF-I concentrations in Revalor-S-implanted animals. However, recent reports of normal growth in mice in which the hepatic IGF-I gene has been specifically knocked out, put in question the role of circulating IGF-I in growth (Sjogren et al., 1999).
In addition to a Revalor-S-induced increase in steady-state hepatic IGF-I mRNA, our current study has shown a Revalor-S-induced increase in IGF-I mRNA in the SM of implanted steers. These data are consistent with our previous results demonstrating increased levels of IGF mRNA in the longissimus muscles of Revalor-S-implanted steers (Johnson et al., 1998b). The mechanism by which Revalor-S elevates muscle and liver IGF-I mRNA is not clear at present. Based on our current knowledge of how IGF-I levels are regulated, it is reasonable to speculate that Revalor-S-induced changes in GH levels or GH receptor levels/affinities may be responsible for the increased muscle and liver IGF-I mRNA (Breier et al., 1988). Growth hormone has been shown to play a major role in regulating serum IGF-I levels. Additionally, GH treatment of pigs raises the level of IGF-I mRNA in the liver and SM, but not in the longissimus muscle (Grant et al., 1991; Coleman et al., 1994; Brameld et al., 1996). However, since GH levels are reportedly not increased by Revalor-S treatment of steers (Hunt et al., 1991; Hayden et al., 1992), it appears unlikely that alterations in GH levels are responsible for Revalor-S-induced increases in muscle IGF-I mRNA level. This does not preclude the possibility that Revalor-S alters the number and/or affinity of GH receptors in liver and muscle tissues. These alterations could enhance the responsiveness of liver and muscle to GH. However, the fact that GH levels are not increased by Revalor-S implantation also raises the possibility that Revalor-S may increase liver and muscle IGF-I mRNA level via a mechanism that does not involve GH.
Since we have observed Revalor-S-induced increases in IGF-I mRNA in both the SM and longissimus muscles, we believe that increased muscle production of IGF-I may be a general response to Revalor-S implantation of steers. Additionally, we have previously shown that Revalor-S implantation increased the number of actively proliferating satellite cells in the SM of these same steers (Johnson et al., 1998a). Both the IGF-I and satellite cell results are very significant in view of a recent report that virally induced overexpression of IGF-I in the muscle tissue of mice resulted in a 15% increase in muscle mass in young adult mice and maintenance of muscle mass in old mice that would otherwise be losing muscle mass (Barton-Davis et al., 1998). These workers hypothesized that increased muscle growth resulted from increased activation and proliferation of satellite cells in muscles that are producing higher levels of IGF-I. This hypothesis is supported by a recent report that overexpression of IGF-I in muscle tissue increases the proliferative capacity of satellite cells (Chakravarthy et al., 2000). Other researchers also have suggested that IGF-I acts in an autocrine and/or paracrine manner to enhance muscle growth (Grant et al., 1991; Czerwinski et al., 1994; Brameld et al., 1996). It is possible that the increased IGF-I level in muscle of implanted steers stimulates satellite cell proliferation and maintains a high number of proliferating satellite cells at a point in the growth curve where satellite cell numbers and activity are normally dropping off. This would prolong the period of rapid muscle growth resulting in the observed increased rate and efficiency of muscle deposition in implanted steers. Whereas there is evidence that elevated IGF-I levels may increase the proliferative life of satellite cells, several studies have shown that IGF-I alone is not able to activate quiescent satellite cells (Allen et al., 1995). Thus, it is unlikely that the increased number of actively proliferating satellite cells in the semimembranosus muscle of implanted steers results from IGF-I stimulated activation of quiescent cells. Time course studies are needed to determine whether muscle levels of other growth factors such as HGF, myostatin, or fibroblast growth factor are elevated immediately after implantation.
|
Implications |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
|
Footnotes |
|---|
2 This research was funded in part by NRI Competitive Grants Program/USDA Grant No. 99-03256 and by a gift from Intervet, Inc.
Received for publication June 17, 2002. Accepted for publication December 3, 2002.
|
Literature Cited |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
|---|
This article has been cited by other articles:
|
|
 |
|
  W. R. Dayton and M. E. White Cellular and molecular regulation of muscle growth and development in meat animals J Anim Sci, April 1, 2008; 86(14_suppl): E217 - E225. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Y. Chung and B. J. Johnson Application of cellular mechanisms to growth and development of food producing animals J Anim Sci, April 1, 2008; 86(14_suppl): E226 - E235. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Gonzalez, J. N. Carter, D. D. Johnson, S. E. Ouellette, and S. E. Johnson Effect of ractopamine-hydrochloride and trenbolone acetate on longissimus muscle fiber area, diameter, and satellite cell numbers in cull beef cows J Anim Sci, August 1, 2007; 85(8): 1893 - 1901. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. K. Sissom, C. D. Reinhardt, and B. J. Johnson Melengestrol acetate alters muscle cell proliferation in heifers and steers J Anim Sci, November 1, 2006; 84(11): 2950 - 2958. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. T. Oliver, J. Rosenberger, R. Lopez, A. Gomez, K. K. Cummings, and M. L. Fiorotto The Local Expression and Abundance of Insulin-Like Growth Factor (IGF) Binding Proteins in Skeletal Muscle Are Regulated by Age and Gender But Not Local IGF-I in Vivo Endocrinology, December 1, 2005; 146(12): 5455 - 5462. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. G. Mateescu and M. L. Thonney Effect of testosterone on insulin-like growth factor-I, androgen receptor, and myostatin gene expression in splenius and semitendinosus muscles in sheep J Anim Sci, April 1, 2005; 83(4): 803 - 809. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Dunn, B. J. Johnson, J. P. Kayser, A. T. Waylan, E. K. Sissom, and J. S. Drouillard Effects of flax supplementation and a combined trenbolone acetate and estradiol implant on circulating insulin-like growth factor-I and muscle insulin-like growth factor-I messenger RNA levels in beef cattle J Anim Sci, December 1, 2003; 81(12): 3028 - 3034. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Pampusch, B. J. Johnson, M. E. White, M. R. Hathaway, J. D. Dunn, A. T. Waylan, and W. R. Dayton Time course of changes in growth factor mRNA levels in muscle of steroid-implanted and nonimplanted steers J Anim Sci, November 1, 2003; 81(11): 2733 - 2740. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Davis, S. L. Boyles, S. J. Moeller, and R. C. M. Simmen Genetic parameter estimates for serum insulin-like growth factor-I concentration and ultrasound measurements of backfat thickness and longissimus muscle area in Angus beef cattle J Anim Sci, September 1, 2003; 81(9): 2164 - 2170. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
