AJP - Endo Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 285: E197-E205, 2003. First published April 1, 2003; doi:10.1152/ajpendo.00370.2002
0193-1849/03 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
285/1/E197    most recent
00370.2002v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (37)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sinha-Hikim, I.
Right arrow Articles by Bhasin, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sinha-Hikim, I.
Right arrow Articles by Bhasin, S.

Testosterone-induced muscle hypertrophy is associated with an increase in satellite cell number in healthy, young men

Indrani Sinha-Hikim,1 Stephen M. Roth,2 Martin I. Lee,1 and Shalender Bhasin1

1Division of Endocrinology, Metabolism, and Molecular Medicine, Charles R. Drew University of Medicine and Science, Los Angeles, California 90059; and 2Department of Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvamia 15261

Submitted 22 August 2002 ; accepted in final form 26 March 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Testosterone (T) supplementation in men induces muscle fiber hypertrophy. We hypothesized that T-induced increase in muscle fiber size is associated with a dose-dependent increase in satellite cell number. We quantitated satellite cell and myonuclear number by using direct counting and spatial orientation methods in biopsies of vastus lateralis obtained at baseline and after 20 wk of treatment with a gonadotropin-releasing hormone agonist and a 125-, 300-, or 600-mg weekly dose of T enanthate. T administration was associated with a significant increase in myonuclear number in men receiving 300- and 600-mg doses. The posttreatment percent satellite cell number, obtained by direct counting, differed significantly among the three groups (ANCOVA P < 0.000001); the mean posttreatment values (5.0 and 15.0%) in men treated with 300- and 600-mg doses were greater than baseline (2.5 and 2.5%, respectively, P < 0.05 vs. baseline). The absolute satellite cell number measured by spatial orientation at 20 wk (1.5 and 4.0/mm) was significantly greater than baseline (0.3 and 0.6/mm) in men receiving the 300- and 600-mg doses (P < 0.05). The change in percent satellite cell number correlated with changes in total (r = 0.548) and free T concentrations (r = 0.468). Satellite cell and mitochondrial areas were significantly higher and the nuclear-to-cytoplasmic ratio lower after treatment with 300- and 600-mg doses. We conclude that T-induced muscle fiber hypertrophy is associated with an increase in satellite cell number, a proportionate increase in myonuclear number, and changes in satellite cell ultrastructure.

anabolic steroids; anabolic effects of androgens; mechanisms of androgen action; testosterone effects on muscle stem cells

TESTOSTERONE SUPPLEMENTATION increases muscle mass in healthy hypogonadal men (7, 11, 25, 43, 48, 49), older men with low testosterone levels (26, 44, 45), and men with chronic illnesses and low testosterone levels (5, 8, 14). The anabolic effects of testosterone on muscle mass are dose and concentration dependent (9). Testosterone-induced increase in muscle mass is associated with a dose-dependent increase in cross-sectional areas of both type I and type II muscle fibers but is not attended by a change in muscle fiber number (40). However, the mechanisms by which testosterone increases muscle mass are not well understood (4). The prevalent dogma for the past 50 years has been that testosterone increases muscle mass by stimulating fractional muscle protein synthesis (11, 13, 28, 29, 37, 47, 52). Indeed, lowering of circulating testosterone concentrations by administration of a gonadotropin-releasing hormone (GnRH) agonist is associated with a reduction in fractional muscle protein synthesis (29); conversely, testosterone replacement in healthy hypogonadal men increases muscle protein synthesis (11). However, in a recent study (40), we noted that testosterone-induced muscle fiber hypertrophy is associated with a dose-dependent increase in myonuclear number. It would be difficult to explain this increase in myonuclear number if the sole effect of testosterone were on muscle protein synthesis. Muscle growth during postnatal development or hypertrophy is dependent on the addition of myonuclei to muscle fibers (1, 2, 30, 35, 36). Because the nuclei within the muscle fibers are postmitotic, new myonuclei must be contributed by the satellite cells that are outside the muscle fiber. Inhibition of satellite cell proliferation by gamma irradiation at doses that do not produce overt cellular damage prevents the muscle growth and increase in myonuclear number that follow muscle atrophy due to hindlimb suspension (1). Muscle remodeling and repair following injury often involve satellite cell replication and recruitment of new stem cells into the myogenic cell lineage (35, 36). Similarly, the hypertrophy of levator ani muscle in the female rat induced by exogenous testosterone administration is associated with satellite cell entry into the cell cycle and proliferation (20–22, 31, 32, 46). Taken together, these animal data suggest that an increase in satellite cell number is an important antecedent of an increase in myonuclear number and muscle fiber adaptation leading to hypertrophy. However, we do not know whether similar changes in satellite cell number are also observed in testosterone-treated humans. Furthermore, previous reports studied satellite cells in the levator ani muscle of rodents, which differs significantly from skeletal muscle in the magnitude of response to castration and testosterone supplementation. Therefore, in this study, we tested the hypothesis that testosterone-induced muscle fiber hypertrophy would be associated with an increase in satellite cell number in the skeletal muscle of testosterone-treated men. Because the gains in muscle mass during testosterone supplementation are correlated with testosterone dose and concentrations (9), we hypothesized that the change in satellite cell number would also be correlated with testosterone dose and concentrations.

Accordingly, we measured the number of satellite cells in muscle biopsies obtained from healthy men in whom endogenous testosterone production was suppressed by administration of a GnRH agonist, and different levels of serum testosterone concentrations were created by weekly intramuscular injections of graded doses of testosterone enanthate. The design and the main findings of this study have been published (9, 40). Because significant increases in type I and type II muscle fiber cross-sectional areas were observed only in men receiving the 600-mg weekly doses of testosterone enanthate (40), and to a lesser extent in those receiving the 300-mg dose, we focused our analysis of satellite cells on these two groups. In addition, we included the 125-mg dose group as a control, because men in this group did not experience demonstrable muscle fiber hypertrophy or an increase in myonuclear number.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects. This was a double-blind, randomized study. The details of the study design have been previously published (9, 40). Briefly, the participants were 61 healthy, eugonadal men, 18–35 yr of age. Each participant provided informed consent, and the study was approved by the institutional review boards of Charles R. Drew University and Harbor-UCLA Research and Education Institute. Participants were randomly assigned to one of five experimental groups to receive monthly injections of a long-acting GnRH agonist (decapeptyl; Debio Pharm, Geneva, Switzerland) to suppress endogenous testosterone production and weekly injections of 25, 50, 125, 300, or 600 mg of testosterone enanthate for 20 wk. In our previous study, we had noted muscle fiber hypertrophy in men receiving the 600-mg dose and to a lesser extent in men in the 300-mg dose group (40); therefore, we focused our analysis on these groups. We included as a control group one additional group that had not demonstrated a significant increase in muscle fiber cross-sectional area. In this investigation, muscle biopsies were available for electron microscopy before and after testosterone treatment in six men receiving 125 mg, eight men receiving 300 mg, and five men receiving 600 mg of testosterone enanthate weekly.

Nutritional intake and exercise stimulus were controlled at the outset of the study, as previously described (9, 40). Two weeks before the initiation of the study, subjects were prescribed a diet standardized for energy intake at 150 kJ·kg-1·day-1 and protein intake at 1.3 g·kg-1·day-1. These instructions were reinforced every 4 wk during a meeting with the dietitian in which each subject's actual nutrient intake was verified by analysis of 3-day food records.

Hormone assays. Serum total testosterone was measured by a previously reported radioimmunoassay (5–7, 9, 10). Free testosterone was separated by an equilibrium dialysis procedure and measured in the dialysate by radioimmunoassay (41). The sensitivity of total testosterone assay was 0.6 ng/dl; intra-assay coefficient of variation was 8.2%, and interassay coefficient of variation was 13.2%. For the free testosterone assay, the sensitivity was 0.22 pg/ml, and intra- and inter-assay coefficients of variation were 4.2 and 12.3%, respectively.

Muscle biopsy and tissue fixation. Percutaneous needle muscle biopsies were obtained from the midbelly of the vastus lateralis muscle in the quadriceps muscle group before and within 5 days after the final testosterone enanthate injection. Biopsy specimens were immediately fixed in 5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) overnight and processed for epoxy embedding (38). After fixation, the tissues were washed in cacodylate buffer, postfixed in 1% osmium tetroxide, dehydrated in graded series of ethanol, and embedded in Epon. During embedding, the tissue blocks were oriented longitudinally. Embedded tissue blocks were sectioned with an LKB ultramicrotome. Thin sections showing pale gold interference color were stained with uranyl acetate and lead citrate and examined with a Hitachi 600 electron microscope.

Spatial distribution of myonuclei and satellite cells. All samples were processed and micrographed by an observer who was unaware of the treatment assignment. Parts of longitudinally oriented, healthy muscle fibers were selected randomly and photographed using an electron microscope at x3,500 magnification. The electron micrographs were further magnified three times to obtain a total magnification of x10,500. Montages of the fibers were constructed, and total numbers of myonuclei and satellite cells were counted and expressed per unit length (mm) of muscle fiber. Six montages were constructed for each subject before and after treatment (averaging 0.9–1.2 mm of muscle fiber in each sample).

Identification of satellite cells. The satellite cells were identified on the basis of their characteristic ultrastructural morphology (34, 42) by using the following criteria (Fig. 1). 1) The satellite cells are mononucleated cells with an independent cytoplasm that are inside the basal lamina of the muscle fibers. 2) The satellite cells are located outside the sarcolemma of the muscle fiber.



View larger version (161K):
[in this window]
[in a new window]
  		Fig. 1. Electron micrograph of a typical myonucleus (A) and a muscle satellite cell (B). Muscle satellite cells (S) were identified by their location inside the basal lamina (arrowheads) and outside the sarcolemma (arrows) and an independent cytoplasm. In contrast, a myonucleus (M) is located inside the sarcolemma of the muscle fiber and does not contain an independent cytoplasm. Bar, 1 µm.

 

Determination of satellite cell proportion by direct counting and by spatial distribution. We used two separate counting procedures to determine the number of satellite cells and myonuclear number in muscle fibers. In the direct-counting procedure, the numbers of myonuclei and satellite cells were counted in randomly selected photomicrographs from each subject. A total of 620–1,189 myonuclei were photographed in each of the three groups (97–210 myonuclei per biopsy) at each time point, and the number of satellite cells was expressed as a percentage of myonuclear number.

In addition, all myonuclei and satellite cells were counted in the reconstructed fiber montages (6 per biopsy) to obtain the satellite cell percentage by spatial distribution. Percent satellite cells equaled the number of satellite cells divided by the sum of myonuclear number and the number of satellite cells and was expressed as a percentage (19, 34).

Morphometry of the satellite cells. The electron micrographs of the satellite cells were evaluated to identify possible differences in morphology before and after treatment in each group. The point counting method was used to obtain the volume densities [volume of a given component per unit volume of the cell (Vv)] of the satellite cell components (3, 18, 50).

The total cellular, nuclear, and mitochondrial areas were calculated by the equation: A = P·U2 (15, 16, 38), where A denotes area, P denotes the number of points falling on the satellite cell or its nucleus, and U represents the distance between two neighboring points in the grid, taking into account the magnification used. Because no significant changes were observed in either muscle volume or satellite cell number in men in the 125-mg dose group, we performed the ultrastructural analysis only in the 300- and 600-mg dose groups. Also, because the number of satellite cells in baseline biopsies was very small, we pooled the baseline data on ultrastructural analysis.

Statistical analyses. All data are presented as means ± SE for each of the three treatment groups. The main outcome measures were absolute and percent satellite cell number and myonuclear number. We used analysis of covariance (ANCOVA) to compare posttreatment values, adjusting for baseline values for each outcome measure. If the overall ANCOVA revealed a significant effect, the statistical significance of the between-group differences was tested by the Tukey-Kramer multiple comparison procedure. We also compared the change from baseline in each outcome measure by one-way ANOVA. The significance of the change from baseline to posttreatment within a dose group was evaluated using the paired t-test. For all statistical analyses, P values <0.05 were considered significant. The NCSS2001 (version 5/02, Kaysville, UT) and Sigma-Stat programs (SPSS, Chicago, IL) were used for all statistical analyses. The correlations between serum testosterone concentrations and various outcomes were evaluated using the Pearson correlation coefficient and were tested for significance from zero with an appropriate t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Of the 61 men who enrolled in the main study, 54 completed the treatment (9). Of these, sufficient tissue was available for evaluation of satellite cells by electron microscopy in 19 men: six in the 125-mg group, eight in the 300-mg group, and five in the 600-mg group. We selected the 300- and 600-mg groups for evaluation of satellite cells because men in these groups had experienced significant increases in muscle fiber area and myonuclear number (40); in addition, we selected the 125-mg group as a reference for comparison because men in this group had not demonstrated significant changes in muscle fiber cross-sectional area.

A detailed description of the hormonal changes in all 61 treated men has been published (7). Among the 19 men who were evaluated by electron microscopy, the three groups did not significantly differ in their baseline characteristics, including age, body weight, and height, or in serum total and free testosterone concentrations (Table 1). Combined administration of GnRH agonist and graded doses of testosterone enanthate resulted in dose-dependent changes in serum total and free testosterone concentrations and muscle volumes (change in thigh muscle volumes by MRI, +21, +43, and +68 cm3, overall P = 0.001), as previously reported (9). Consistent with our previous report (40), testosterone administration was associated with dose- and concentration-dependent increases in cross-sectional area of type I and type II muscle fibers (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 1. Baseline characteristics of subjects in the 3 treatment groups

 

Myonuclear number. When we used the spatial distribution method, the myonuclear number per millimeter of muscle fiber length did not differ significantly among the three groups at baseline (Table 2). However, posttreatment myonuclear number after combined administration of a GnRH agonist and graded doses of testosterone was significantly different among the three groups, after control for baseline values (AN-COVA P = 0.003). The change from baseline in myonuclear number in the three groups was significantly different (ANOVA, P < 0.001; Fig. 2). The change in myonuclear number was greater in men receiving the 600-mg dose than in those receiving the 125- or the 300-mg dose of testosterone enanthate (P < 0.05 for each of the two comparisons based on the global significance level of the Tukey-Kramer procedure). The number of myonuclei was significantly greater after treatment compared with baseline in men receiving the 300-mg (P < 0.05 vs. baseline) and 600-mg (P < 0.05 vs. baseline) doses of testosterone enanthate. The change in myonuclear number was significantly correlated with serum total (r = 0.53, P = 0.02) testosterone concentrations.


View this table:
[in this window]
[in a new window]
  		Table 2. Myonuclear number per millimeter of muscle fiber assessed by spatial orientation method before and after treatment with GnRH agonist and graded doses of testosterone

 


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 2. Effect of testosterone administration on myonuclear number (A) and absolute satellite cell number (B). Number of myonuclei and satellite cells per mm of muscle fiber length were computed by spatial distribution. Change was calculated as the difference between posttreatment and baseline values. *Values significantly different from 0. X-axis: weekly dose of testosterone enanthate is shown. *P < 0.05 vs. 0 change.

 

Satellite cell number. By use of the spatial distribution method, the number of satellite cells at baseline in all three groups varied from 0 to 2 satellite cells/mm of muscle fiber and did not differ significantly among the three groups (Table 3). By ANCOVA, the mean posttreatment values for satellite cell number per millimeter of muscle fiber measured by spatial orientation were significantly different among the three groups, after control for the baseline values (P = 0.0002). The posttreatment satellite cell number was greater in men receiving the 600-mg dose than in those receiving the 125- or the 300-mg doses (P < 0.05 for each comparison by use of the global significance level of the Tukey-Kramer procedure). The increase in the number of satellite cells per millimeter of muscle fiber from baseline was statistically significant in men receiving the 300- and 600-mg testosterone doses (P < 0.05 vs. baseline for each comparison; Fig. 2). The change in satellite cell number was significantly correlated with the change in total (r = 0.548, P = 0.015) and free testosterone concentrations (r = 0.468, P = 0.043; Fig. 3A).


View this table:
[in this window]
[in a new window]
  		Table 3. Satellite cell number before and after treatment with GnRH agonist and graded doses of testosterone

 


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 3. Correlation of change in satellite cell number with change in total (A) and free (B) testosterone concentrations.

 

The mean posttreatment values for satellite cell number, measured by direct counting and expressed as a percentage of myonuclear number (Table 3), were also significantly different by ANCOVA, after control for the baseline values (P < 0.000001). The change from baseline in satellite cell number was greater in the 600-mg dose compared with the 125- and 300-mg dose groups (P < 0.05 for each comparison). The satellite cell number after treatment was significantly greater compared with baseline in men treated with the 300- and 600-mg doses of testosterone (P < 0.05 for each comparison).

Satellite cell morphology and ultrastructure. The mean satellite cell area was significantly greater after treatment with 300- and 600-mg doses compared with baseline area (ANOVA P < 0.05; Table 4 and Fig. 4). The mean absolute nuclear area was not significantly different after treatment with the 300- or 600-mg dose compared with baseline (P = 0.40); however, the ratio of the nuclear to total cellular area decreased significantly in men receiving the 300- and 600-mg doses (P < 0.005 by ANOVA). The increase in cytoplasmic area compared with the nuclear area during treatment was statistically significant (P < 0.005 by ANOVA). The ratio of Vv% nucleus to Vv% cytoplasm was significantly different after treatment with the 300- and 600-mg doses (P < 0.01). The mean satellite cell mitochondrial area increased significantly from baseline in men receiving the 300 and 600 mg of testosterone enanthate weekly (P < 0.05). The heterochromatin volume, expressed as a percentage of satellite cell nuclear volume (P = 0.45), did not differ significantly among the groups.


View this table:
[in this window]
[in a new window]
  		Table 4. Morphometric analysis of satellite cell ultrastructure

 


View larger version (170K):
[in this window]
[in a new window]
  		Fig. 4. Satellite cell ultrastructure in 1 subject at baseline and after 20 wk of treatment with gonadotropin-releasing hormone (GnRH) agonist and 600 mg of testosterone enanthate weekly. A: electron micrograph of a muscle biopsy obtained at baseline. B, C, and D: electron micrographs of biopsies obtained after 20 wk of treatment with 600 mg of testosterone enanthate and GnRH agonist. Arrowheads, basilar lamina; upright thin arrows, sarcolemma; *, lamellopodia; V, pinocytic vesicles; m, mitochondria. Bar, 1 µm.

 

We noted additional qualitative changes in satellite cell morphology after testosterone treatment, including the presence of lamellopodia in one-half of the posttreatment biopsies in the 300- and 600-mg groups, a greater amount of endoplasmic reticulum, and a greater number of pinocytic vesicles compared with the baseline biopsies (Fig. 4).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our data demonstrate that testosterone-induced muscle fiber hypertrophy is associated with increases in the numbers of myonuclei and satellite cells. The satellite cell number, measured by spatial orientation as well as by direct counting methods, increased significantly in proportion to the testosterone dose. The increase in satellite cell number during testosterone administration was dose dependent: higher doses of testosterone were associated with a greater increase in satellite cell number than lower doses. Testosterone supplementation was also associated with ultrastructural changes in satellite cells, including increased cellular and mitochondrial areas, a decrease in the nuclear-to-cytoplasmic ratio, and an increased number of lamellopodia compared with baseline.

In this study, significant increases in satellite cell number were observed only in men who were treated with supraphysiological doses of testosterone enanthate. We do not know whether physiological replacement doses of testosterone when administered to older men with low testosterone concentrations or to men with chronic illness would produce similar anabolic effects.

Because of the difficulties inherent in obtaining multiple muscle biopsies from volunteers and the labor-intensive nature of electron microsopic analysis, the sample size was relatively small. The number of satellite cells in the adult muscle is relatively small and constitutes a very small fraction of the myonuclear number. Not surprisingly, in baseline samples, we were able to locate only one or two, and in some subjects no satellite cells. Although this increases the imprecision in quantitation of satellite cell number in baseline biopsies, the remarkable increases in satellite cell numbers following testosterone treatment observed in the present study are particularly striking. Similarly, the ultrastructural details were evaluated in a small number of satellite cells that were identified and should be viewed cautiously. The satellite cells were identified by electron microscopy using previously established histological criteria (19, 34); the satellite cells recognized by these criteria might not represent a monomorphic cell population. There is growing evidence that these cells represent myogenic cells at more than one stage of differentiation (17).

Although testosterone's effects on muscle have generated enormous controversy for over five decades (10, 51), a number of recent studies are in agreement that testosterone supplementation increases muscle mass in healthy hypogonadal men (7, 11, 25, 43, 48, 49), older men with low or low normal testosterone concentrations (13, 26, 44, 45), and men with chronic illnesses (5, 8, 14). Our dose-response study demonstrated that testosterone's anabolic effects on fat-free mass and muscle size are dose and concentration dependent (9). The mechanisms by which testosterone induces muscle mass accretion are not well understood. The prevalent hypothesis that testosterone stimulates muscle protein synthesis (11, 13, 47) emerged from observations made 50 years ago that testosterone promotes nitrogen retention in castrated males of many mammalian species (26), in prepubertal boys, and in women (27). Lowering of testosterone concentrations by administration of a GnRH agonist is associated with decreased muscle protein synthesis (29). Testosterone supplementation in healthy hypogonadal men (11, 29) and older men with low normal or low testosterone levels (47) increases fractional muscle protein synthesis. Indeed, muscle fiber hypertrophy could not possibly occur without an increase in muscle protein synthesis or a decrease in muscle protein degradation. However, this hypothesis cannot explain our observations that testosterone administration is associated with increases in satellite cell and myonuclear number. Our data lead us to conclude that an increase in satellite cell number and the subsequent fusion of satellite cells with muscle fibers resulting in an increase in myonuclear number and muscle fiber hypertrophy are likely involved in mediating androgen-induced muscle hypertrophy. However, our data cannot exclude the important role that changes in muscle protein synthesis and degradation might play in the process of muscle hypertrophy associated with testosterone supplementation. We do not know whether the increase in satellite cell number is the primary event that is subsequently associated with increased muscle protein synthesis or whether the increases in myonuclear and satellite cell numbers occur secondarily to maintain the myonuclear domain (2).

Substantial evidence supports a role for the modulation of myonuclear number during muscle remodeling in response to injury or disease (1, 2, 19–23, 33, 35, 36). Muscle hypertrophy involves the addition of newly formed myonuclei via the fusion of myogenic cells to the adult myofibers (2). Therefore, we speculate that muscle fiber hypertrophy and the increase in myonuclear number observed here were preceded by testosterone-induced increases in satellite cell number and fusion of satellite cells with muscle fibers. The hypertrophy of levator ani muscle in the female rat induced by exogenous testosterone administration is associated with satellite cell proliferation (20–22, 31, 32, 46). Muscle remodeling and repair following injury or hormone and growth factor stimulation often involve satellite cell replication and recruitment of new stem cells into the myogenic cell lineage (2, 20, 22–24, 30–33, 35, 36, 42, 46).

The mechanisms by which testosterone increases satellite cell number are not known. An increase in satellite cell number could occur by an increase in satellite cell replication, inhibition of satellite cell apoptosis, and/or increased differentiation of stem cells into the myogenic lineage. We do not know which of these processes is the site of regulation by testosterone. On the basis of the observations that testosterone administration induces a rapid proliferative response in muscle satellite cells of prepubertal rats, Jubert and Tobin (22) inferred that androgen receptors might be present on the satellite cells. Doumit et al. (12) demonstrated the presence of androgen receptors in cultured porcine satellite cell nuclei and myotubes and reported that testosterone exposure increased androgen receptor immunostaining in satellite cells. The authors further demonstrated that the cultured satellite cells exhibited lower differentiation in response to testosterone exposure without an effect on proliferation. Taken together, these data point to a direct role for testosterone in satellite cell regulation.

Our observations that testosterone promotes muscle fiber hypertrophy by increasing the number of satellite cells might have clinical and commercial applications. Testosterone effects on satellite cells could serve as the platform for the development of in vitro assays for anabolic agents and have implications for the discovery of selective androgen receptor modulators. Because of the constraints inherent in obtaining multiple biopsy specimens in humans, the effects of testosterone on satellite cell replication and stem cell recruitment into the myogenic lineage would be more easily studied in an animal model that demonstrates androgen responsiveness and sexual dimorphism in skeletal muscle mass similar to what is observed in humans. It is possible that testosterone might promote entry of satellite cells into the cell cycle (22); however, it is equally plausible that androgens might promote myogenesis by stimulating stem cell differentiation into the myogenic lineage. The molecular mechanisms by which testosterone increases muscle satellite cell number should be examined.


   ACKNOWLEDGMENTS
 
Support for this project was provided by a research grant from the National Institute of Aging, 1RO1 AG-14369–01. Additional support was provided by National Institutes of Health Grants 1RO1 DK-59627–01, 2RO1 DK-49296–02A, 5F32 AG-05893–02 (S. M. Roth), the Clinical Trials Unit Grant U01 DK-54047, Research Center in Minority Institutions (RCMI) Clinical Research Infrastructure Initiative (P20 RR-11145), RCMI Grants G12 RR-03026 and U54 RR-14616, General Clinical Research Center Grant MO1 RR-00425, and a Harbor-UCLA University of California AIDS Research Program DrewCares HIV Center grant. BioTechnology General Corporation provided the testosterone enanthate, and DebioPharm Inc. (Geneva, Switzerland) provided the long-acting GnRH agonist for these studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Shalender Bhasin, UCLA School of Medicine, Division of Endocrinology, Metabolism, and Molecular Medicine, Charles R. Drew Univ. of Medicine and Science, 1731 E. 120th St., Los Angeles, CA 90059 (E-mail: sbhasin{at}ucla.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Adams GR, Caiozzo VJ, Haddad F, and Baldwin KM. Cellular and molecular responses to increased skeletal muscle loading after irradiation. Am J Physiol Cell Physiol 283: C1182–C1195, 2002.[Abstract/Free Full Text]
  2. Allen DL, Roy RR, and Edgerton VR. Myonuclear domains in muscle adaptation and disease. Muscle Nerve 22: 1350–1360, 1999.[ISI][Medline]
  3. Baranska W, Baran W, Skopinski P, and Ziemba H. Morphometric analysis of satellite cells in rat skeletal muscles: soleus and extensor digitorum longus. Acta Anat (Basel) 160: 88–94, 1997.[ISI][Medline]
  4. Bhasin S, Fielder T, Peacock N, Sod-Moriah UA, and Swerdloff RS. Dissociating antifertility effects of GnRH-antagonist from its adverse effects on mating behavior in male rats. Am J Physiol Endocrinol Metab 254: E84–E91, 1988.[Abstract/Free Full Text]
  5. Bhasin S, Storer TW, Asbel-Sethi N, Kilbourne A, Hays R, Sinha-Hikim I, Shen R, Arver S, and Beall G. Effects of testosterone replacement with a nongenital, transdermal system, Androderm, in human immunodeficiency virus-infected men with low testosterone levels. J Clin Endocrinol Metab 83: 3155–3162, 1998.[Abstract/Free Full Text]
  6. Bhasin S, Storer TW, Berman N, Callegari C, Clevenger B, Phillips J, Bunnell TJ, Tricker R, Shirazi A, and Casaburi R. The effects of supraphysiologic doses of testosterone on muscle size and strength in normal men. N Engl J Med 335: 1–7, 1996.[Abstract/Free Full Text]
  7. Bhasin S, Storer TW, Berman N, Phillips J, Clevenger B, and Casaburi R. Testosterone replacement increases fat-free mass and muscle size in hypogonadal men. J Clin Endocrinol Metab 82: 407–413, 1997.[Abstract/Free Full Text]
  8. Bhasin S, Storer TW, Javanbakht M, Yarasheski K, Phillips J, Berman N, and Beall G. Testosterone replacement and resistance exercise in HIV-infected men with weight loss and low testosterone levels. JAMA 283: 763–770, 2000.[Abstract/Free Full Text]
  9. Bhasin S, Woodhouse L, Casaburi R, Singh AB, Bhasin D, Berman N, Chen X, Yarasheski KE, Magliano L, Dzekov C, Dzekov J, Bross R, Phillips J, Sinha-Hikim I, Shen R, and Storer TW. Testosterone dose-response relationships in healthy young men. Am J Physiol Endocrinol Metab 281: E1172–E1181, 2001.[Abstract/Free Full Text]
  10. Bhasin S, Woodhouse L, and Storer TW. Proof of the effect of testosterone on skeletal muscle. J Endocrinol 170: 27–38, 2001.[Abstract]
  11. Brodsky IG, Balagopal P, and Nair KS. Effects of testosterone replacement on muscle mass and muscle protein synthesis in hypogonadal men—a clinical research center study. J Clin Endocrinol Metab 81: 3469–3475, 1996.[Abstract]
  12. Doumit ME, Cook DR, and Merkel RA. Testosterone up-regulates androgen receptors and decreases differentiation of porcine myogenic satellite cells in vitro. Endocrinology 137: 1385–1394, 1996.[Abstract]
  13. Ferrando AA, Sheffield-Moore M, Yeckel CW, Gilkison C, Jiang J, Achacosa A, Lieberman SA, Tipton K, Wolfe RR, and Urban RJ. Testosterone administration to older men improves muscle function: molecular and physiological mechanisms. Am J Physiol Endocrinol Metab 282: E601–E607, 2002.[Abstract/Free Full Text]
  14. Grinspoon S, Corcoran C, Askari H, Schoenfeld D, Wolf L, Burrows B, Walsh M, Hayden D, Parlman K, Anderson E, Basgoz N, and Klibanski A. Effects of androgen administration in men with the AIDS wasting syndrome. A randomized, double-blind, placebo-controlled trial. Ann Intern Med 129: 18–26, 1998.[Abstract/Free Full Text]
  15. Gundersen HJ and Jensen EB. The efficiency of systematic sampling in stereology and its prediction. J Microsc 147: 229–263, 1987.[Medline]
  16. Gundersen HJ, Jensen EB, Kieu K, and Nielsen J. The efficiency of systematic sampling in stereology—reconsidered. J Microsc 193: 199–211, 1999.[Medline]
  17. Hawke TJ and Garry DJ. Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 91: 534–551, 2001.[Abstract/Free Full Text]
  18. Hikim AP, Amador AG, Bartke A, and Russell LD. Structure/function relationships in active and inactive hamster Leydig cells: a correlative morphometric and endocrine study. Endocrinology 125: 1844–1856, 1989.[Abstract]
  19. Ishimoto S, Goto I, Ohta M, and Kuroiwa Y. A quantitative study of the muscle satellite cells in various neuromuscular disorders. J Neurol Sci 62: 303–314, 1983.[ISI][Medline]
  20. Joubert Y and Lebart MC. Testosterone induced masculinization of the rat levator ani muscle during puberty. Dev Biol 162: 104–110, 1994.[ISI][Medline]
  21. Joubert Y and Tobin C. Satellite cell proliferation and increase in the number of myonuclei induced by testosterone in the levator ani muscle of the adult female rat. Dev Biol 131: 550–557, 1989.[ISI][Medline]
  22. Joubert Y and Tobin C. Testosterone treatment results in quiescent satellite cells being activated and recruited into cell cycle in rat levator ani muscle. Dev Biol 169: 286–96, 1995.[ISI][Medline]
  23. Kadi F, Eriksson A, Holmner S, and Thornell LE. Effects of anabolic steroids on the muscle cells of strength-trained athletes. Med Sci Sports Exerc 31: 1528–1534, 1999.[ISI][Medline]
  24. Kadi F and Thornell LE. Concomitant increases in myonuclear and satellite cell content in female trapezius muscle following strength training. Histochem Cell Biol 113: 99–103, 2000.[ISI][Medline]
  25. Katznelson L, Finkelstein JS, Schoenfeld DA, Rosenthal DI, Anderson EJ, and Klibanski A. Increase in bone density and lean body mass during testosterone administration in men with acquired hypogonadism. J Clin Endocrinol Metab 81: 4358–4365, 1996.[Abstract]
  26. Kenny AM, Prestwood KM, Gruman CA, Marcello KM, and Raisz LG. Effects of transdermal testosterone on bone and muscle in older men with low bioavailable testosterone levels. J Gerontol A Biol Sci Med Sci 56: M266–M272, 2001.[Abstract/Free Full Text]
  27. Kenyon AT, Knowlton K, Sandiford I, Kock FC, and Lotwin G. A comparative study of the metabolic effects of testosterone propionate in normal men and women and in eunuchoidism. Endocrinology 26: 26–45, 1940.
  28. Kochakian CD. Comparison of protein anabolic properties of various androgens in the castrated rat. Am J Physiol 60: 553–558, 1950.
  29. Mauras N, Hayes V, Welch S, Rini A, Helgeson K, Dokler M, Veldhuis JD, and Urban RJ. Testosterone deficiency in young men: marked alterations in whole body protein kinetics, strength, and adiposity. J Clin Endocrinol Metab 83: 1886–1892, 1998.[Abstract/Free Full Text]
  30. Mitchell PO and Pavlath GK. A muscle precursor cell-dependent pathway contributes to muscle growth after atrophy. Am J Physiol Cell Physiol 281: C1706–C1715, 2001.[Abstract/Free Full Text]
  31. Mulvaney DR, Marple DN, and Merkel RA. Proliferation of skeletal muscle satellite cells after castration and administration of testosterone propionate. Proc Soc Exp Biol Med 188: 40–45, 1988.[Abstract]
  32. Nnodim JO. Testosterone mediates satellite cell activation in denervated rat levator ani muscle. Anat Rec 263: 19–24, 2001.[Medline]
  33. Reimann J, Irintchev A, and Wernig A. Regenerative capacity and the number of satellite cells in soleus muscles of normal and mdx mice. Neuromuscul Disord 10: 276–282, 2000.[ISI][Medline]
  34. Roth SM, Martel GF, Ivey FM, Lemmer JT, Metter EJ, Hurley BF, and Rogers MA. Skeletal muscle satellite cell populations in healthy young and older men and women. Anat Rec 260: 351–358, 2000.[Medline]
  35. Schultz E. Satellite cell behavior during skeletal muscle growth and regenration. Med Sci Sports Med 21: S181–S186, 1989.
  36. Schultz E and McCormick KM. Skeletal muscle satellite cells. Rev Physiol Biochem Pharmacol 123: 213–257, 1994.[ISI][Medline]
  37. Sheffield-Moore M, Urban RJ, Wolf SE, Jiang J, Catlin DH, Herndon DN, Wolfe RR, and Ferrando AA. Short-term oxandrolone administration stimulates net muscle protein synthesis in young men. J Clin Endocrinol Metab 84: 2705–2711, 1999.[Abstract/Free Full Text]
  38. Sinha Hikim AP, Rajavashisth TB, Sinha Hikim I, Lue Y, Bonavera JJ, Leung A, Wang C, and Swerdloff RS. Significance of apoptosis in the temporal and stage-specific loss of germ cells in the adult rat after gonadotropin deprivation. Biol Reprod 57: 1193–1201, 1997.[Abstract]
  39. Sinha-Hikim AP and Swerdloff RS. Temporal and stage-specific changes in spermatogenesis of rat after gonadotropin deprivation by a potent gonadotropin-releasing hormone antagonist treatment. Endocrinology 133: 2161–2170, 1993.[Abstract]
  40. Sinha-Hikim I, Artaza J, Woodhouse L, Gonzalez-Cadavid N, Shen R, and Bhasin S. Testosterone-induced increase in muscle size is associated with muscle fiber hypertrophy. Am J Physiol Endocrinol Metab 283: E154–E164, 2002.[Abstract/Free Full Text]
  41. Sinha-Hikim I, Arver S, Beall G, Shen R, Guerrero M, Sattler F, Shikuma C, Nelson JC, Landgren BM, Mazer NA, and Bhasin S. The use of a sensitive equilibrium dialysis method for the measurement of free testosterone levels in healthy, cycling women and in human immunodeficiency virus-infected women [published erratum in J Clin Endocrinol Metab 83: 2959, 1998]. J Clin Endocrinol Metab 83: 1312–1318, 1998.[Abstract/Free Full Text]
  42. Snow M. A quantitative ultrastructural analysis of satellite cells in denervated fast and slow muscle of the mouse. Anat Rec 207: 593–604, 1983.[Medline]
  43. Snyder PJ, Peachey H, Berlin JA, Hannoush P, Haddad G, Dlewati A, Santanna J, Loh L, Lenrow DA, Holmes JH, Kapoor SC, Atkinson LE, and Strom BL. Effects of testosterone replacement in hypogonadal men. J Clin Endocrinol Metab 85: 2670–2677, 2000.[Abstract/Free Full Text]
  44. Snyder PJ, Peachey H, Hannoush P, Berlin JA, Loh L, Lenrow DA, Holmes JH, Dlewati A, Santanna J, Rosen CJ, and Strom BL. Effect of testosterone treatment on body composition and muscle strength in men over 65 years of age. J Clin Endocrinol Metab 84: 2647–2653, 1999.[Abstract/Free Full Text]
  45. Tenover JS. Effects of testosterone supplementation in the aging male. J Clin Endocrinol Metab 75: 1092–1098, 1992.[Abstract]
  46. Thompson SH, Boxhorn LK, Kong WY, and Allen RE. Trembolone alters the responsiveness of skeletal muscle satellite cells to fibroblast growth factor and insulin-like growth factor I. Endocrinology 124: 2110–2117, 1989.[Abstract]
  47. Urban RJ, Bodenburg YH, Gilkison C, Foxworth J, Coggan AR, Wolfe RR, and Ferrando A. Testosterone administration to elderly men increases skeletal muscle strength and protein synthesis. Am J Physiol Endocrinol Metab 269: E820–E826, 1995.[Abstract/Free Full Text]
  48. Wang C, Eyre DR, Clark R, Kleinberg D, Newman C, Iranmanesh A, Veldhuis J, Dudley RE, Berman N, Davidson T, Barstow TJ, Sinow R, Alexander G, and Swerdloff RS. Sublingual testosterone replacement improves muscle mass and strength, decreases bone resorption, and increases bone formation markers in hypogonadal men—a clinical research center study. J Clin Endocrinol Metab 81: 3654–3662, 1996.[Abstract]
  49. Wang C, Swedloff RS, Iranmanesh A, Dobs A, Snyder PJ, Cunningham G, Matsumoto AM, Weber T, and Berman N. Transdermal testosterone gel improves sexual function, mood, muscle strength, and body composition parameters in hypogonadal men. Testosterone Gel Study Group. J Clin Endocrinol Metab 85: 2839–2853, 2000.[Abstract/Free Full Text]
  50. Weibel E. Stereological Methods: Practical Methods for Biological Morphometry. New York: Academic, vol. 1, 1979.
  51. Wilson JD. Androgen abuse by athletes. Endocr Rev 9: 181–199, 1988.[ISI][Medline]
  52. Wolfe R, Ferrando A, Sheffield-Moore M, and Urban R. Testosterone and muscle protein metabolism. Mayo Clin Proc 75 Suppl: S55–S60, 2000.



This article has been cited by other articles:


Home page
 J ANIM SCIHome page
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]

Home page
 J. Appl. Poult. Res.Home page
J. Cao, W. Liu, Z. Wang, D. Xie, L. Jia, and Y. Chen
Green and Blue Monochromatic Lights Promote Growth and Development of Broilers Via Stimulating Testosterone Secretion and Myofiber Growth
J. Appl. Poult. Res., January 1, 2008; 17(2): 211 - 218.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
K. Aizawa, M. Iemitsu, T. Otsuki, S. Maeda, T. Miyauchi, and N. Mesaki
Sex differences in steroidogenesis in skeletal muscle following a single bout of exercise in rats
J Appl Physiol, January 1, 2008; 104(1): 67 - 74.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
M. I. Lewis, M. Fournier, T. W. Storer, S. Bhasin, J. Porszasz, S.-G. Ren, X. Da, and R. Casaburi
Skeletal muscle adaptations to testosterone and resistance training in men with COPD
J Appl Physiol, October 1, 2007; 103(4): 1299 - 1310.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
R. S. O'Connor and G. K. Pavlath
Point:Counterpoint: Satellite cell addition is/is not obligatory for skeletal muscle hypertrophy
J Appl Physiol, September 1, 2007; 103(3): 1099 - 1100.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
K. Aizawa, M. Iemitsu, S. Maeda, S. Jesmin, T. Otsuki, C. N. Mowa, T. Miyauchi, and N. Mesaki
Expression of steroidogenic enzymes and synthesis of sex steroid hormones from DHEA in skeletal muscle of rats
Am J Physiol Endocrinol Metab, February 1, 2007; 292(2): E577 - E584.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
L. B. Verdijk, R. Koopman, G. Schaart, K. Meijer, H. H. C. M. Savelberg, and L. J. C. van Loon
Satellite cell content is specifically reduced in type II skeletal muscle fibers in the elderly
Am J Physiol Endocrinol Metab, January 1, 2007; 292(1): E151 - E157.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
A M Solomon and P M G Bouloux
Modifying muscle mass - the endocrine perspective.
J. Endocrinol., November 1, 2006; 191(2): 349 - 360.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
A. S. Brack, H. Bildsoe, and S. M. Hughes
Evidence that satellite cell decrement contributes to preferential decline in nuclear number from large fibres during murine age-related muscle atrophy
J. Cell Sci., October 15, 2005; 118(20): 4813 - 4821.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
J. M. McClung, K. A. Mehl, R. W. Thompson, L. L. Lowe, and J. A. Carson
Nandrolone decanoate modulates cell cycle regulation in functionally overloaded rat soleus muscle
Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2005; 288(6): R1543 - R1552.
[Abstract] [Full Text] [PDF]

Home page
 J. Gerontol. A Biol. Sci. Med. Sci.Home page
S. Asthana, S. Bhasin, R. N. Butler, H. Fillit, J. Finkelstein, S. M. Harman, L. Holstein, S. G. Korenman, A. M. Matsumoto, J. E. Morley, et al.
Masculine Vitality: Pros and Cons of Testosterone in Treating the Andropause
J. Gerontol. A Biol. Sci. Med. Sci., May 1, 2004; 59(5): M461 - M465.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
285/1/E197    most recent
00370.2002v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (37)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sinha-Hikim, I.
Right arrow Articles by Bhasin, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sinha-Hikim, I.
Right arrow Articles by Bhasin, S.

HOME HELP