Sexual dimorphism is associated with decreased expression of processed myostatin in males

Christopher D. McMahon, Ljiljana Popovic, Ferenc Jeanplong, Jenny M. Oldham, Sonnie P. Kirk, Claire C. Osepchook, Karen W. Y. Wong, Mridula Sharma, Ravi Kambadur, and John J. Bass

Animal Genomics, AgResearch Ltd., Ruakura Agricultural Centre, Hamilton 2001, New Zealand


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Myostatin inhibits skeletal muscle development. Therefore, we sought to determine whether larger body and muscle mass in male mice was associated with lower mRNA and protein expression of myostatin compared with females. Ten male and ten female mice of the C57 strain were killed at 16-18 wk of age, and their biceps femoris, gastrocnemius, and quadriceps femoris muscles were collected. Body and muscle masses were 40% heavier (P < 0.001) in males than in females. Northern analysis showed no difference in mRNA between males and females. In contrast, Western analysis showed that processed myostatin (26 kDa) was 40-60% lower (P < 0.001) in males compared with females. These data show first that decreased processed myostatin is a posttranscriptional and posttranslational event and, second, that decreased abundance of processed myostatin is associated with increased body mass and skeletal muscle mass in male compared with female mice.

sex; body mass


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MYOSTATIN IS A MUSCLE-SPECIFIC growth factor that inhibits muscle development (15). Remarkable increases (2- to 3-fold) in skeletal muscle occur in myostatin-null (mstn-/-) mice and in cattle with natural mutations in the myostatin gene that render the protein inactive (11, 15, 16). Therefore, myostatin may have a critical role in regulating the amount of muscle mass in individuals.

Myostatin is a 376-amino acid peptide in mice (52 kDa) belonging to the transforming growth factor-beta (TGF-beta ) family and, as such, is suggested to be secreted as a latent complex consisting of a 40-kDa latency-associated peptide (LAP) that, after proteolytic cleavage at a specific RSRR sequence (amino acids 263-266), is noncovalently attached to the processed COOH-terminal portion, the size of which varies in reports from 12.5 to 30 kDa, depending on species and posttranslational modifications (6, 13, 21, 22). Based on TGF-beta , homodimers of LAP and processed myostatin protein are noncovalently linked to form a latent complex (7, 17, 18). Activation enables processed myostatin to separate from LAP to stimulate activin type II receptors (13).

Male mammals are generally larger than females, and this sexual dimorphism (5) is attributed to the presence of androgens (1), insulin-like growth factor I (14) and growth hormone (23). However, given that myostatin inhibits muscle development, it is possible that myostatin also contributes to sexually dimorphic growth. If true, this would suggest that expression of myostatin is lower in skeletal muscles of males compared with those of females.

The purpose of the present study was to test the hypothesis that larger body and muscle mass in adult male mice is related to decreased expression of myostatin in skeletal muscles of male compared with female mice.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Ten male and ten female C57 mice were maintained under a photoperiod of 14:10-h light-dark cycle and fed mouse chow (Diet 86, Sharps Grain and Seed, Carterton, NZ). Mice were killed between 16 and 18 wk by CO2 asphyxiation followed by cervical dislocation. At death, mice were weighed, a sample of blood was collected via cardiac puncture, and the following three hindlimb muscles were dissected free, weighed, and then frozen in liquid nitrogen: biceps femoris, gastrocnemius, and quadriceps femoris. Plasma was harvested and stored at -20°C before analysis by Western blot. This study was approved by the Ruakura Animal Ethics Committee.

Protein extraction and Western blot analysis. One milliliter of lysis buffer (PBS, pH 7.2) with 0.5% IGEPAL detergent (Sigma Chemical, St. Louis, MO) and an enzyme inhibitor (Complete, Roche Diagnostics NZ, Auckland, NZ) was added to 100 mg of muscle from each animal. Samples were homogenized on ice and then centrifuged at 11,000 g for 10 min. Supernatant was recovered, mixed with Laemmli loading buffer (12), boiled for 5 min, and then stored at -20°C until analysis. The protein concentration of the supernatant was determined using the bicinchoninic acid assay (Sigma Chemical).

Twenty micrograms of protein from each muscle sample were loaded and separated in a 10% SDS-polyacrylamide gel under reducing conditions and then transferred to a nitrocellulose membrane. After transfer, membranes were stained with Ponceau S to verify transfer of protein. Membranes were incubated with rabbit anti-myostatin antibody (19) overnight (1:3,000), washed in 0.05 M Tris-buffered saline with 0.05% Tween 20 (TBST, pH 7.6), and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Sigma Chemical) at 1:10,000 for 2 h and then washed again in TBST. Bound-HRP activity was detected with enhanced chemiluminescence, and then blots were exposed to XOMAT AR film (Eastman Kodak, Rochester, NY), after which the relative optical densities of myostatin precursor, LAP, and processed bands were determined using a densitometer (GS 800, Bio-Rad Laboratories, Auckland, NZ) and Quantity One software (Bio-Rad Laboratories). Membranes were then stripped (0.2 M Tris, pH 7.6, 2% SDS, and 0.05 M beta -mercaptoethanol, at 50°C for 30 min) and then exposed to rabbit anti-actin (Sigma Chemical) at a dilution of 1:10,000 and developed as above to assess uniformity of loading.

RNA extraction and Northern blot analyses. Frozen muscle samples were homogenized on ice in TRIzol reagent (GIBCO-BRL, Gaithersburg, MD) for 30 s at 13,500 rpm using an Ultra Turrax homogenizer. Debris was removed by centrifugation for 10 min at 10,000 g, and total RNA was isolated using the TRIzol protocol (GIBCO-BRL). RNA was resuspended in diethyl pyrocarbonate-treated water, and the final concentration was determined by measuring absorbance at 260 nm. Ten micrograms of total RNA were separated in a 1.2% formaldehyde-agarose gel and then transferred to an uncharged nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Auckland, NZ) by capillary action. Membranes were cross-linked using ultraviolet irradiation and stained with methylene blue to verify the uniformity of loading and transfer. The primers to generate myostatin-specific DNA probe were 5'-GGTATTTGGCAGAGTATTGAT-3'and 5'-ATCTACTACCATGGCTGGAAT-3'. First-strand cDNA synthesis was performed using a Superscript II Pre-Amplification kit (GIBCO-BRL) and 5 µg of total RNA from mouse skeletal muscle, according to the manufacturer's protocol. PCR was carried out with 2 µl of the reverse transcriptase reaction (94°C for 30 s, 55°C for 1 min, and 72°C for 1 min) for 35 cycles and a final extension of 5 min at 72°C. Radiolabeled cDNA probes were prepared using [alpha -32P]dCTP and the Rediprime II Labeling Kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Membranes were prehybridized in Church and Gilbert buffer for 2 h (0.5 M Na2HPO4, pH 7.2, 7% SDS, 1 mM EDTA) at 55°C. Membranes were then hybridized at 55°C overnight in fresh buffer with the radiolabeled probe. After hybridization, they were washed at 55°C for 15 min in each of 2× SSC-0.5% SDS and 1× SSC-0.5% SDS and exposed against BioMax X-ray film (Eastman Kodak). The intensity of each band was measured and analyzed as per Western blot analysis.

Statistical analysis. Data were subjected to t-tests and are presented as means ± SE or the pooled SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Body and muscle masses were over 40% heavier (P < 0.001) in male than in female mice (Table 1). A representative Northern blot is shown for biceps femoris, gastrocnemius, and quadriceps femoris muscles from two male and two female mice (Fig. 1). There was no difference between sexes in steady-state mRNA expression of myostatin, although there was a tendency (P = 0.07) for lower expression of myostatin mRNA in female gastrocnemius (Fig. 2). Three immunoreactive bands were identified for myostatin protein on Western blots that corresponded to precursor, LAP, and processed myostatin. A representative Western blot is shown for biceps femoris, gastrocnemius, and quadriceps femoris muscles for three male and three female mice (Fig. 3). Protein expression of processed myostatin was consistently lower (P < 0.001) by 40-60% in all three muscles collected from males than from females despite there being no difference in expression of LAP (Fig. 4). Expression of precursor myostatin was more variable, being higher in male gastrocnemius and quadriceps femoris muscles, but not different between sexes in biceps femoris (Fig. 4). To determine whether the reduced abundance of processed myostatin in muscles of males was associated with increased secretion into blood, we measured processed myostatin in plasma by use of Western blot analysis. A representative Western blot is shown for processed myostatin in plasma (Fig. 5A), and abundance was not different (P = 0.17) between sexes (Fig. 5B).

                              
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Table 1.   Mean body mass and wet mass of biceps femoris, gastrocnemius, and quadriceps femoris muscles collected from 10 male and 10 female mice of the C57 strain



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Fig. 1.   Representative Northern blot showing the 2.9-kb myostatin mRNA transcript in biceps femoris (B), gastrocnemius (G), and quadriceps femoris (Q) from 2 representative male and female C57 mice. Uniformity of loading was assessed by staining membranes with methylene blue to expose the 28S and 18S ribosomal RNA bands.



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Fig. 2.   Mean (±SE) optical density of myostatin mRNA for biceps femoris, gastrocnemius, and quadriceps femoris muscles in male and female C57 mice (n = 10/sex; dagger P = 0.07).



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Fig. 3.   Representative Western blot of myostatin forms [precursor (Pre), latency-associated peptide (LAP), and processed (Proc)] in biceps femoris, gastrocnemius, and quadriceps femoris muscles for 3 male and 3 female C57 mice. Protein (20 µg) from each muscle extract was run under reducing conditions. Uniformity of loading was assessed by immunoblotting for actin.



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Fig. 4.   Mean (±SE) optical density for precursor, LAP, and processed myostatin in biceps femoris (A), gastrocnemius (B), and quadriceps femoris (C). * Significant differences between male and female C57 mice for each myostatin form (n = 10/sex; *P < 0.05, ***P < 0.001).



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Fig. 5.   A: representative Western blot of processed myostatin in plasma from 5 male and 5 female C57 mice. Processed myostatin is present at 26 kDa in both sexes. B: mean (±SE) optical density of the 26-kDa processed myostatin band in male and female C57 mice was not different (P = 0.17) between sexes (n = 10/sex).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We demonstrate here that increased body and muscle mass in male compared with female mice is associated with decreased expression of processed myostatin. Interestingly, this decreased expression of processed myostatin occurs after translation and, presumably, after secretion, because there was no difference in steady-state mRNA or in the LAP form of myostatin, the latter of which is cosecreted with the processed COOH-terminal fragment of myostatin in equimolar proportions.

This is the first report to our knowledge showing that processed myostatin is reduced in skeletal muscle of adult male compared with female mammals that exhibit sexually dimorphic growth. Others have reported that myostatin mRNA does not differ between sexes in humans (9) or in swine (10). Our data are consistent with these findings, but we also observed a tendency for lower mRNA in female gastrocnemius compared with male. This difference is apparent in the precursor form but does not affect the abundance of the LAP form of myostatin. There is also a higher abundance of the precursor form of myostatin in male quadriceps femoris, whereas there is no difference in the LAP form. These data suggest to us that there are other sex-related differences in transcription and translation of myostatin than processed myostatin, and their function is not immediately apparent.

At present, it is unclear what happens to processed myostatin after it is secreted. On the basis of TGF-beta as the best studied member of this superfamily, the processed moiety is proteolytically cleaved from LAP in the Golgi apparatus and then recombines in a noncovalent manner with LAP before secretion (2-4). The binding of the processed form of myostatin to the LAP form and to large latent TGF-beta -binding proteins is crucial for correct folding and secretion of TGF-beta (7, 18). Therefore, the decrease in abundance of processed myostatin in muscle likely occurs after secretion and could have resulted from a higher rate of secretion from muscles to blood of males. One problem with this possibility is that processed myostatin circulates bound to the LAP form (24), and we observed that the LAP form remained in muscle in similar abundance between sexes, whereas the processed myostatin was 50% lower in muscle. Therefore, without increased abundance of processed myostatin in blood, it is more likely that processed myostatin is degraded in skeletal muscle at a higher rate in males than in females. We cannot, however, rule out the possibility that processed myostatin is cleared faster from blood of males than females. Whatever mechanism is involved, a reduction in the abundance of processed myostatin in the skeletal muscles of males suggests reduced bioactivity and, thereby, reduced inhibition on myogenesis. It should be noted that although myostatin-null mice have two to three times more muscle mass than wild-type controls, males remain heavier than females (15). Therefore, a reduction in myostatin does not regulate sexual dimorphism per se. Rather, it may enable anabolic factors to enhance growth. In this regard, sexually dimorphic growth of males also requires insulin-like growth factor I (14), growth hormone mediated by signal transducer and activator of transcription 5b (23), and testosterone (1).

Growth rates are similar for male and female mice up to 3 wk of age, after which time growth rates accelerate in males. Growth rates decrease in both sexes after 60 days and reach a plateau in females after 100 days, but males continue growing slowly from 100 to 200 days (data not recorded after 200 days) (8). Thus, at 16-18 wk (112-126 days), the age at which muscles were collected from mice in the present study, most growth had ceased. Therefore, it is not clear whether reduced processed myostatin is important for maintaining the greater absolute muscle mass in adult males or contributes to accelerated growth in male compared with female adolescent mice. Furthermore, increased muscle mass in males is relative to the increased skeletal frame rather than increased mass on the same-sized skeletal frame and is attributed to the greater duration and rate of growth compared with females before skeletal maturation (8, 20). These data suggest that reduced abundance of processed myostatin in male mice may play a role in sexually dimorphic growth and/or maintaining the greater muscle mass at maturity. We are currently examining the expression of myostatin during various stages of postnatal growth from birth to adulthood and through to old age in male and female mice.

In summary, skeletal muscles of adult male C57 mice have a reduced abundance of processed myostatin compared with females. We suggest that a reduced abundance of processed myostatin reflects reduced inhibition on myogenesis and contributes to the greater muscle mass in males compared with females.


    ACKNOWLEDGEMENTS

We thank Rick Broadhurst and the staff of the Small Animal Colony at AgResearch, Ruakura, for care and maintenance of mice during this study. We also thank Dr. Neil Cox for statistical advice.


    FOOTNOTES

Address for reprint requests and other correspondence: C. D. McMahon, AgResearch Ltd., Ruakura Agricultural Centre, Private Bag 3123, Hamilton, 2001 New Zealand (E-mail: chris.mcmahon{at}agresearch.co.nz).

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.

First published October 15, 2002;10.1152/ajpendo.00282.2002

Received 26 June 2002; accepted in final form 10 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bardin, CW, and Catterall JF. Testosterone: a major determinant of extragenital sexual dimorphism. Science 211: 1285-1294, 1981[ISI][Medline].

2.   Dubois, CM, Blanchette F, Laprise M-H, Leduc R, Grondin F, and Seidah NG. Evidence that furin is an authentic transforming growth factor-beta 1-converting enzyme. Am J Pathol 158: 305-316, 2001[Abstract/Free Full Text].

3.   Gentry, LE, Lioubin MN, Purchio AF, and Marquardt H. Molecular events in the processing of recombinant type 1 pre-pro-transforming growth factor beta to the mature polypeptide. Mol Cell Biol 8: 4162-4168, 1988[ISI][Medline].

4.   Gentry, LE, and Nash BW. The pro domain of pre-pro-transforming growth factor beta 1 when independently expressed is a functional binding protein for the mature growth factor. Biochemistry 29: 6851-6857, 1990[ISI][Medline].

5.   Glucksmann, A. Sexual dimorphism in mammals. Biol Rev Camb Philos Soc 49: 423-475, 1974[ISI][Medline].

6.   Gonzalez-Cadavid, NF, Taylor WE, Yarasheski K, Sinha-Hikim I, Ma K, Ezzat S, Shen R, Lalani R, Asa S, Mamita M, Nair G, Arver S, and Bhasin S. Organisation of the human myostatin gene and expression in healthy man and HIV-infected men with muscle wasting. Proc Natl Acad Sci USA 95: 14938-14943, 1998[Abstract/Free Full Text].

7.   Gray, AM, and Mason AJ. Requirement for activin A and transforming growth factor beta 1 pro-regions in homodimer assembly. Science 247: 1328-1330, 1990[ISI][Medline].

8.   Griffin, GE, and Goldspink G. The increase in skeletal muscle mass in male and female mice. Anat Rec 177: 465-470, 1973[ISI][Medline].

9.   Ivey, FM, Roth SM, Ferrell RE, Tracy BL, Lemmer JT, Hurlbut DE, Martel GF, Siegel EL, Fozard JL, Jeffrey Metter E, Fleg JL, and Hurley BF. Effects of age, gender, and myostatin genotype on the hypertrophic response to heavy resistance strength training. J Gerontol A Biol Sci Med Sci 55: M641-M648, 2000[Abstract/Free Full Text].

10.   Ji, S, Losinski RL, Cornelius SG, Frank GR, Willis GM, Gerrard DE, Depreux FF, and Spurlock ME. Myostatin expression in porcine tissues: tissue specificity and developmental and postnatal regulation. Am J Physiol Regul Integr Comp Physiol 275: R1265-R1273, 1998[Abstract/Free Full Text].

11.   Kambadur, R, Sharma M, Smith TPL, and Bass JJ. Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle. Genome Res 7: 910-916, 1997[Abstract/Free Full Text].

12.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

13.   Lee, S-J, and McPherron AC. Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci USA 98: 9306-9311, 2001[Abstract/Free Full Text].

14.   Liu, J-L, and LeRoith D. Insulin-like growth factor I is essential for post-natal body growth in response to growth hormone. Endocrinology 140: 5178-5184, 1999[Abstract/Free Full Text].

15.   McPherron, AC, Lawler AM, and Lee S-J. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387: 83-90, 1997[ISI][Medline].

16.   McPherron, AC, and Lee S-J. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA 94: 12457-12461, 1997[Abstract/Free Full Text].

17.   Miyazono, K, Ichijo H, and Heldin C-H. Transforming growth factor-beta : latent forms, binding proteins and receptors. Growth Factors 8: 11-22, 1993[ISI][Medline].

18.   Miyazono, K, Olofsson A, Colosetti P, and Heldin C-H. A role of the latent TGF-beta 1-binding protein in the assembly and secretion of TGF-beta 1. EMBO J 10: 1091-1101, 1991[Abstract].

19.   Sharma, M, Kambadur R, Matthews KG, Somers WG, Devlin GP, Conaglen JV, Fowke PJ, and Bass JJ. Myostatin, a transforming growth factor beta  superfamily member, is expressed in heart muscle and is upregulated in cardiomyocytes after infarct. J Cell Physiol 180: 1-9, 1999[ISI][Medline].

20.   Stewart, SA, and German RZ. Sexual dimorphism and ontogenetic allometry of soft tissues in Rattus norvegicus. J Morphol 242: 57-66, 1999[ISI][Medline].

21.   Taylor, WE, Bhasin S, Artaza J, Byhower F, Azam M, Willard DH, Jr, Kull FC, Jr, and Gonzalez-Cadavid N. Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells. Am J Physiol Endocrinol Metab 280: E221-E228, 2001[Abstract/Free Full Text].

22.   Thomas, M, Langley B, Berry C, Sharma M, Kirk S, Bass JJ, and Kambadur R. Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. J Biol Chem 275: 40235-40243, 2000[Abstract/Free Full Text].

23.   Udy, GB, Towers RP, Snell RG, Wilkins RJ, Park S-H, Ram PA, Waxman DJ, and Davey HW. Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression. Proc Natl Acad Sci USA 94: 7239-7244, 1997[Abstract/Free Full Text].

24.   Zimmers, TA, Davies MV, Koniaris LG, Haynes P, Esquela AF, Tomkinson KN, McPherron AC, Wolfman NM, and Lee S-J. Induction of cachexia in mice by systemically administered myostatin. Science 296: 1486-1488, 2002[Abstract/Free Full Text].


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