Introduction

Myostatin (MSTN), originally identified as a growth differentiation factor 8 (GDF-8), is a recently discovered member of the transforming growth factor-β (TGF-β) superfamily (McPherron et al., 1997). The TGF-β superfamily includes a number of secreted factors that mediate key events in tissue growth and development through signal transduction cascades. In mammals, MSTN is expressed initially in the myotome compartment of the developing somites and continues to be expressed in the myogenic lineage throughout the development and in adult animals (McPherron and Lee, 1997; Lee and McPherron, 2001). The following two lines of evidence have led to the conclusion that MSTN functions as a negative regulator of skeletal muscle growth: (1) MSTN knockout mice display increased muscle mass (McPherron et al., 1997; Lee and McPherron, 2001); and (2) mutations of the MSTN gene in cattle are associated with muscle hyperplasia and hypertrophy (Grobet et al., 1997; McPherron and Lee, 1997).

MSTN gene has been cloned and identified from a wide variety of vertebrates including human (Gonzalez-Cadavid et al., 1998), mice (McPherron and Lee, 1997), cattle (McPherron and Lee, 1997), chicken (McPherron and Lee, 1997; Kocamis et al., 1999), and several fish species such as zebrafish (Danio rerio; Xu et al., 2003; Amali et al., 2004; Biga et al., 2005), Atlantic salmon (Salmo salar; Ostbye et al., 2001), rainbow trout (Oncorhynchus mykiss; Rescan et al., 2001), brook trout (Salvelinus fontinalis; Roberts and Goetz, 2001), striped bass (Morone saxatilis; Rodgers and Weber, 2001), tilapia (Oreochromis mossambicus; Rodgers et al., 2001), gilthead seabream (Sparus aurata; Maccatrozzo et al., 2001a,b), channel catfish (Ictalurus punctatus; Kocabas et al., 2002a), and European seabass (Dicentrarchus labrax; Terova et al., 2005). While MSTN gene is expressed primarily in myogenic linage cells in mammals, MSTN gene in fish is expressed in a variety of tissues including muscle, gill, eyes, tongue, spleen, heart, stomach, intestine, kidney, liver, ovaries, brain, and testes (Maccatrozzo et al., 2001a,b; Ostbye et al., 2001; Rescan, 2001; Rescan et al., 2001; Roberts and Goetz, 2001; Rodgers and Weber, 2001; Kocabas et al., 2002b; Radaelli et al., 2003; Gregory et al., 2004; Terova et al., 2005). Studies conducted by Amali et al. (2004) showed that downregulation of myostatin-1 gene in zebrafish embryos by antisense morpholino-specific to myostatin resulted in upregulation of muscle-specific transcription factors during embryonic somatogenesis. Because fish MSTN gene is expressed in many cell types other than myogenic linage in fish, this suggests that fish MSTN gene may be involved in additional functions other than regulating muscle development. In most salmonids and zebrafish, there are two MSTN transcripts that are products of separate genes. These two transcripts are differentially regulated, and tissues expressing these two MSTN genes appear to be paralog-dependent (Maccatrozzo et al., 2001a; Rescan et al., 2001; Roberts and Goetz, 2001; Xu et al., 2003; Amali et al., 2004; Biga et al., 2005).

The orange spotted grouper, a protogynous hermaphroditic fish, is of great interest for fisheries as well as aquaculture because of its excellent meat quality and its high commercial value. This fish species has become one of the most commercially important marine fish species, and is popularly cultured in Taiwan. One of the bottlenecks in commercial aquaculture of grouper is its slow somatic growth and prone to disease infection. To overcome these problems, development of strategies for acceleration of somatic growth and disease resistant would be desirable. As a step toward this direction, we have initiated studies to clone and characterize genes related to growth of this fish species. In this article we report cloning and characterization of MSTN gene in the orange spotted grouper and the tissue-specific and developmental stage-specific expression of the gene in 1-year-old fish. Because the only fish species that MSTN gene and its expression patterns have been studied is gilthead seabream (Maccatrozzo et al., 2001a,b) and European seabass (Terova et al., 2005), results of our study reported here for the orange spotted grouper will provide valuable information for comparative studies of MSTN gene in marine fish species.

Materials and Methods

Animal and Sample Collection

One-year-old orange spotted grouper, ranging from 250 to 350 g body weights and 20–25 cm body lengths, were obtained from a farm in Tainan, Taiwan, and acclimated in fiberglass containers with full strength seawater (35 ppt) for 3 days. The fish were fed daily with fish or shrimp meat at 10% of body weight. Various tissues for determining the distribution of MSTN mRNA were collected from 1-year-old fish. Unfertilized eggs, embryos, and larval grouper from 1 to 60 days postfertilization (dpf) were collected during the reproductive season of 2005 (from May to June). All tissue samples were frozen immediately in liquid nitrogen and stored at –80°C until RNA extraction.

Isolation and Characterization of MSTN Gene

Total RNA was extracted from various tissues of adult grouper using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendation. The concentration of the total RNA was estimated by measuring the absorbance at 260 nm.

Degenerate primers used for reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of grouper MSTN cDNA are shown in Table 1. These primers were designed based on the highly conserved sequences of MSTN of zebrafish (AF019626, AY614000), brook trout (AF247650), tilapia (AF197193), white (AF197194), striped sea-bass (AF290910), white perch (AF290911), Atlantic salmon S. salar (ASA297267, ASA344158), rainbow trout (AF273035, AF273036), gilthead seabream (AF258447, AY046314), channel catfish (AF396747), blue catfish (AY540992), white catfish (AY540994), and European sea bass (AY839106) in the GenBank database (Benson et al., 1994).

Table 1 Sequences of Degenerate and Gene Specific Primers Used in This Study
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For the isolation of a grouper MSTN cDNA fragment, total RNA (5 μg) was reverse transcribed using Superscript™ III RNase H− reverse transcriptase (Invitrogen) and oligo (T)18 primer to obtain first-strand cDNA. The generated cDNA was used to amplify MSTN gene using degenerative primers shown in Table 1. Initially, a partial cDNA fragment of 291 bp was amplified using a pair of degenerative primers: MSTN-F, 5′-CGGTGYTGCMGSTAYCCNCTY-3′ and MSTN-R, 5′-TCABGAGCABCCRCADCGGTC-3′. The resulting PCR products were ligated into pGEM-T Easy sequencing vector (Promega, Madison, WI) and transformed into Escherichia coli (JM109) competent cells (Promega). Positive clones were isolated by blue/white screening and were grown for plasmid preparation. The resulting cDNAs were sequenced using a modified dideoxy chain termination method with Big Dye Terminator (Applied Biosystems, Foster City, CA). Sequencing reactions were precipitated and pellets resuspended in Hi-Di Formamide with EDTA (Applied Biosystems) and analyzed using a 3730 Sequencer (Applied Biosystems).

To clone the entire cDNA of MSTN, 5 μg of total RNA were reverse transcribed using GeneRacer Kit (Invitrogen). To obtain the full-length MSTN cDNA, nested 3′- and 5′-rapid amplification of cDNA ends (RACE) PCR was performed. For 3′-RACE PCR, gene-specific sense primers (MSTN 3-1F and MSTN 3-2F, Table 1) and antisense primers (GeneRacer Kit 3′ primers, Table 1) were used as amplification primers and for 5′-RACE PCR, two specific antisense primers (MSTN 5-1R and MSTN 5-2R, Table 1) and GeneRacer Kit 5′ first primer were used as amplification primers. PCR was carried out in a 50-μl final volume that contains 5 μl of 10 × PCR buffer with 200 mM Tris-HCl (pH 8.4), 500 mM KCl, 1.5 μl of 50 mM MgCl2, 1 μl of 10 mM dNTP mix, 1 μl of 10 μM each primer, 36.3 μl of sterile deionized water and 0.2 μl of Platinum Taq DNA Polymerase (5U μl−1) (Invitrogen), and 4 μl of a 1:100 dilution of RACE-ready first-strand cDNA as template. The amplification is: 2 min initial denaturation at 94°C for one cycle, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and elongation at 72°C for 1 min, followed by a 10-min extension at 72°C and cooling to 4°C. The PCR product of the predicted size was gel-separated, purified, and inserted into a pGEM-T Easy Vector, and positive clones were isolated and nucleotide sequence determined as described in the previous section.

DNA was extracted from muscle of orange spotted grouper using Gene-Spin™ Genomic DNA Isolation Kit following instruction provided by the supplier (Protech, Taiwan). The MSTN gene was amplified from the genomic DNA by PCR, using gene-specific oligomers, MSTN-GF (5′-CAGTGTGGGACATTAATCC-3′) and MSTN-GR (5′-CTCACCAGGATCTCCGTCCC-3′) designed from the orange spotted grouper MSTN full-length cDNA as amplification primers. The positions of the primers used for the PCR are indicated by arrows in Figure 1. The amplified product was cloned into the pGEM-T Easy vector (Promega) and nucleic acid sequence determined.

Fig. 1
figure 1

Schematic diagram showing the strategy of PCR cloning of grouper MSTN gene. The names and relative positions of forward and reverse primers of grouper MSTN are shown in the diagram. Primers MSTN-F and MSTN-R were used to amplify genomic DNA. Primers MSTN RT-F and MSTN RT-R were used for quantitative real-time RT-PCR analysis.

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Nucleotide Sequence Analysis

The cDNA sequence and the deduced amino acid sequence of grouper MSTN were compared with default settings on the complete nonredundant GenBank database using BLAST program available for the NCBI Internet Web site (http://www.ncbi.nlm.nih.gov/blast/). Sequences were translated into predicted amino acid sequences and both nucleotide and predicted protein sequences were aligned with various known MSTN sequences. Multiple alignments of cDNA sequences and amino acid sequences were performed using the programs of BioEdit and CLUSTAL W, and the same software was used to analyze similarity of the aligned sequences using a neighbor joining (NJ) algorithm. Based on these multiple sequence alignments, Poisson-corrected distances were estimated for all possible pairs. A phylogenetic tree was constructed using the neighbor-joining method based on the obtained distance matrix, and node robustness was assessed using the bootstrap method (N = 1000 replications). All phylogenetic analyses were conducted using the Phylip program and the phylogenetic tree of MSTN was constructed with the programs CLUSTAL W, Bioedit, Phylip, and Treeview. Unreliable alignment regions and gap-containing regions were excluded from the analysis. Pairwise comparisons between various MSTN sequences were conducted using CLUSTAL X 1.8 program (Thompson et al., 1997). The phylogenetic tree based on the obtained distance matrix was reconstructed by the neighbor-joining algorithm using Neighbor program in the Phylip 3.6 package (Saitou and Nei, 1987). The reliability of the branching was assessed by bootstrap resampling method using 1000 bootstrap replications (Felsenstein, 1985). A phylogenetic tree was depicted using Treeview 1.6.6 program (Page, 1996) (Table 2).

Table 2 Organisms, Genbank Accession Numbers, Abbreviations, Proteins, and References of MSTNs
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Quantitative Real-Time RT-PCR

Eye, brain, gill, heart, muscle, head kidney, stomach, intestine, spleen, and liver were dissected from 1-year-old fish, or embryos and larvae of different stages, and total RNA was extracted in the TRIzol reagent (Invitrogen) following the protocol provided by the supplier. The total RNA was digested by the addition of RNAse-free DNAse (Promrga) to remove any genomic DNA contaminant. Five micrograms of total RNA was used for synthesizing the first strand cDNA with Superscript™ III RNase H- reverse transcriptase (Invitrogen).

The standard curve of MSTN gene was prepared according to Bustin (2000). Synthetic MSTN RNA was prepared by in vitro transcription with T7 RNA polymerase (Promega) using a plasmid containing MSTN cDNA as the template, and the product was digested with RNase-free DNase (Promega) and the resulting synthetic RNA was quantified with a spectrophotometer. The synthetic RNA diluted to different concentrations with diethyl pyrocarbonate (DEPC)-treated water was used as RNA standards. The RNA was reverse transcribed as described in the preceding text and stored at –80°C. Two gene-specific primers, MSTN RT-F and MSTN RT-R, were used to amplify the MSTN transcript (Table 1) for quantitative real-time RT-PCR.

Levels of MSTN mRNA expression in adult tissues or in embryos of different developmental stages were determined by quantitative real-time RT-PCR in the LightCycler 1.0 Continuous Fluorescence Detection System (Roche Diagnostics, Indianapolis, IN). The amplification primers for MSTN mRNA are MSTN RT-F (5′-GACGTGCTGGGAGATG-3′) and MSTN RT-R (5′-AGCTGAGCTCGGACTA-3′) which were designed across an exon: intron boundary to ensure that any contaminating DNA present was not amplified. Both reverse transcription and PCR amplification were carried out in one tube of 20 μl as the final reaction volume, which contained 0.2 μM forward primer, 0.2 μM reverse primer, 5 mM MgSO4, and 0.5 μg of total RNA. The reaction programs were as follows: initial RT incubation at 50°C for 30 min, initial denaturation step for 5 min at 94°C, 40 cycles of denaturing (94°C for 5 s), annealing (60°C for 10 s), and extending (72°C for 20 s). All samples were run in duplicate with a standard curve of serially diluted synthetic RNA. For all real-time assays, ΔC t values were converted to relative values based on their respective standard curves and were normalized to the corresponding β-actin RNA values. Data analysis was performed via LightCycler software V3.5 (Roche, Indianapolis, IN). Data are presented as means ± SD (n = 3).

Statistical Analysis

A multiple comparison (Tukey) test was conducted to compare the significant differences among levels of MSTN gene expression in different tissues and different developmental stages using SAS software (SAS Institute, Cary, NC). A significance level of p ≤ 0.05 was chosen.

Results

Cloning and Characterization of the Orange Spotted Grouper MSTN cDNA

To obtain the sequence of a full-length MSTN cDNA from the orange spotted grouper, RACE PCR was performed in the 3′ and 5′ directions. The 3′ RACE PCR generated a single cDNA fragment of 1648 bp, including a partial ORF (open reading frame) and the entire portion of the 3′-untranslated region (Figure 1). The 5′-RACE PCR extended the cDNA sequence in the 5′ direction with a single cDNA fragment of 1209 bp and provided the complete precursor protein sequence (Figure 1). The MSTN cDNA obtained from grouper is 2608 bp in length and has an ORF of 1128 bp encoding a prepro-MSTN of 376 amino acid residues (Figure 2). The 5′-untranslated region is 99 bp and the 3′-untranslated region is 1381 bp with three consensus AATAAA polyadenylation signals. Like other members of the TGF-β superfamily, grouper MSTN peptide has nine conserved cysteine residues and a RVRR proteolytic cleavage site (Figure 2). The grouper MSTN cDNA sequence and the deduced amino acid sequence have been submitted to the NCBI GenBank (accession no. DQ493889).

Fig. 2
figure 2

Nucleotide sequence of grouper MSTN gene with the deduced amino acid. The deduced amino acid sequence of grouper MSTN is reported below the corresponding coding region (upper case), which is delimited by the putative start and stop codons (in boldface). Nucleotides are numbered from the first base at the 5′ end (transcription start site). Amino acids are numbered from the initiating methionine. The noncoding sequences are typed in lower case. The (AC) repeat region (see text) is underlined. The proteolytic processing site (RVRR) is indicated double underlined with gray shading. The nine conserved cysteine residues, characteristic of MSTN, are indicated in bold with gray shading. The asterisk indicates the stop codon. The polyadenylation signals are enclosed in solid lines at the 3´ downstream region. The sequence was submitted to GenBank with accession no. DQ493889.

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Two gene specific primers (MSTN-GF and MSTN-GR) were used to amplify a DNA fragment of 3598 bp from the genomic DNA. Nucleotide sequence analysis revealed that this DNA fragment contains 2 introns (Intron I, 363 bp; Intron II, 811 bp) and three exons (Exon I, 379 bp. Exon II, 371 bp and Exon III, 381 bp). In comparison with the MSTN gene in mammals, the sizes of Intron I and Intron II of grouper MSTN gene are much shorter than those of the mammalian MSTN gene.

Comparison of MSTN Amino Acid Sequences Among Molluscs, Fish, Chicken, and Mammals

Results presented in Figures 3 and 4 and Table 3 show that the amino acid sequence of the predicted grouper prepro-MSTN shared a high degree of homology with other MSTNs reported to date. Grouper MSTN shared about 96% homology with that of Morone saxatilis, Morone americana, and Dicentrarchus labrax, and had a lower homology with Danio rerio MSTN-II (62%), Sparus aurata MSTN-b (66%), Ictalurus punctatus (75%), Ictalurus furcatus (74%) or Ameiurus catus (74%). With Homo sapiens, Papio hamadryas, Bos taurus, Sus scrofa, Ovis aries, Mus musculus, Rattus norvegicus, Gallus gallus, Meleagris gallopavo, or Argopecten irradians, grouper prepro-MSTN shares a homology of 64%, 64%, 62%, 64%, 63%, 63%, 63%, 64%, and 63% with each other, respectively (Table 3). Figure 5 presents the phylogenetic relationship of prepro-MSTN with molluscs, fish, chicken, and mammals. As shown in Figure 5, the grouper MSTN belongs to the cluster of teleostean MSTN and was closely related to Morone saxatilis, Morone americana, and Dicentrarchus labrax.

Fig. 3
figure 3

Multiple alignment of the predicted amino acid sequence of grouper MSTN with amino acid sequences of MSTN in mammalian and avian species. MSTN proteins compared by CLUSTAL W multiple sequence alignments and their associated accession numbers are as follows: Epinephelus coioides (EpC-MSTN, DQ493889), Homo sapiens (HoS-MSTN, AF019627), Papio hamadryas (PaH-MSTN, AF019619), Bos taurus (BoT-MSTN, AF019761), Sus scrofa (SuS-MSTN, AF019623), Ovis aries (OvA-MSTN, AF019622), Mus musculus (MuM-MSTN, NM_010834), Rattus norvegicus (RaN-MSTN, AF019624), Gallus gallus (GaG-MSTN, AF019621), Meleagris gallopavo (MeG-MSTN, AF019625), and Argopecten irradians (ArI-MSTN, AY553362). Dashes indicate insertion–deletions; shading refers to different degree of overall conservation for each site (black, identical; gray, conservative). The putative RXXR proteolytic processing site of MSTN is enclosed in solid lines. The nine conserved cysteine residues in MSTN C-terminus are indicated with an asterisk.

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Fig. 4
figure 4

Multiple alignment of the predicted amino acid sequence of grouper (Epinephelus coioides) MSTN with other piscine MSTN amino acid sequences. Piscine MSTN proteins compared by CLUSTAL W multiple sequence alignments and their associated accession numbers are as follows: Danio rerio (DaR-MSTN-I, AF019626; DaR-MSTN-II, AY693972), Salvelinus fontinalis (SaF-MSTN, AF247650), Oreochromis mossambicus (OrM-MSTN, AF197193), Morone chrysops (MoC-MSTN, AF197194), Morone saxatilis (MoS-MSTN, AF290910), Morone americana (MoA-MSTN, AF290911), Salmo salar (SaS-MSTN-I, ASA297267;SaS-MSTN-II, ASA344158), Oncorhynchus mykiss (OnM-MSTN-I, AF273035;OnM-MSTN-II, AF273036), Sparus aurata (SpA-MSTN-a, AF258447;SpA-MSTN-b, AY046314), Ictalurus punctatus (IcP-MSTN, AF396747), Ictalurus furcatus (IcF-MSTN, AY540992), Ameiurus catus (AmC-MSTN, AY540994), and Dicentrarchus labrax (DiL-MSTN, AY839106). Dashes indicate insertion–deletions; shading refers to different degree of overall conservation for each site (black, identical; gray, conservative). The putative RXXR proteolytic processing site of MSTN is enclosed in solid lines. The nine conserved cysteine residues in MSTN C-terminus are indicated with an asterisk.

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Table 3 Amino Acid Sequence Percent Similarity (%) of Orange Spotted Grouper Prepro-MSTN Compared to Different Molluscan, Teleostean, Avian and Mammalian Species
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Fig. 5
figure 5

Neighbor-joining phylogetic tree of MSTN amino acid sequences of molluscan, teleostean, avian, and mammalian species, based on Poisson-corrected protein distances. Phylogenetic tree of MSTNs obtained using Phylip software via the neighbor-joining method. The tree was generated via CLUSTAL X1.8 and depicted visually via TreeView1.6.6. Positions containing gaps were excluded from the analysis. Numbers at tree nodes refer to bootstrap values after 1000 replicates. The scale bar refers to a phylogenetic distance of 0.1 amino acid substitutions per site.

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Tissue-Specific and Developmental Stage-Specific Expression of Grouper MSTN Gene

To examine tissue-specific expression of grouper MSTN gene in 1-year-old fish, central nervous system and various tissues (eyes, brain, gill, heart, muscle, head kidney, stomach, intestine, spleen, and liver) were collected from 1-year-old fish and RNA samples extracted from these tissues for measuring levels of MSTN mRNA by quantitative real-time RT-PCR analysis. As shown in Figure 6, MSTN mRNA was detectable in all of these tissues. The highest level of MSTN mRNA was observed in the gill and muscle tissues, medium levels in eyes, brain, heart, head kidney, stomach, and intestine tissues, and low levels in spleen and liver tissues. These results showed that grouper MSTN gene is expressed in many tissues other than gill and muscle in 1-year-old fish.

Fig. 6
figure 6

Levels of MSTN mRNA in various tissues of adult grouper. RNA samples were isolated from adult tissues and levels of MSTN mRNA were measured by real-time RT-PCR analysis following procedures described in Materials and Methods. Each data point is the average of three independent determinations and each sample contains tissues from three animals. The data points are expressed as mean ± SD, and were analyzed by one-way analysis of variance (ANOVA). Different letters indicate means with significant differences (P < 0.05). E, eyes; B, brain; G, gills, H, heart; M, muscle; HK, head kidney; S, stomach; I, intestine, SL, spleen; and L, liver.

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The expression of grouper MSTN gene in different embryonic stages and larval stages was also assessed by quantitative real-time RT-PCR analysis. MSTN mRNA was detected in the unfertilized eggs, the newly fertilized eggs, 16-cell stage, and morula stage embryos (Figure 7a). Although lower levels of MSTN mRNA were detected in the blastula, gastrula, and neurula stages, higher levels of mRNA were detected during the lens formation stage and then gradually decreased to lower levels again in day 60 larval stage (Figure 7b).

Fig. 7
figure 7

Levels of MSTN mRNA in grouper of different embryonic (A) and larval (B) stages. RNA samples were isolated grouper of different developmental stages and levels of MSTN were determined by real-time RT-PCR as described in Materials and Methods. The data points are expressed as mean ± SD, and were analyzed by one-way analysis of variance (ANOVA). Different letters indicate means with significant differences (P < 0.05). (A) 1, unfertilized eggs; 2, fertilized eggs; 3, 16 cells stage; 4, morula stage; 5, blastula stage; 6, gastrula stage; 7, neurula stage; 8, lens formation stage; 9, somite stage; and 10, hatching. (B) 1, 1-day-old larvae (newly hatched larvae with yolk); 2, 2-day-old larvae (fry); 3, 5-day-old larvae (mouth formation); 4, 10-day-old larvae (pigment formation); 5, 15-day-old larvae (fin rays formation); 6, 20-day-old larvae (fin rays maximum); 7, 25-day-old larvae (fin rays reduction); 8, 30-day-old larvae (5 to 25 mm total length (TL)); 9, 45-day-old larvae (25 to 35 mm TL); and 10, 60-day-old larvae (35 to 45 mm TL).

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Discussion

In this article we report the isolation of a full-length cDNA encoding the MSTN precursor from the muscle of orange spotted grouper using RACE-PCR. The grouper MSTN cDNA sequence is 2608 bp in length and has an ORF of 1128 bp encoding a prepro-MSTN with 376 amino acid residues. The deduced amino acid sequence contains a potential proteolytic site (RVRR), matching with the RXRR consensus site, and nine conserved cysteine residues at the carboxyl-terminus, like all of the previously described MSTN orthologues (Hu et al., 1998). It is believed that these nine cysteine residues found in the MSTN orthologues of all TGF-β superfamily members participated in the formation of the characteristic cysteine knot through intramolecular disulfide linkages (Rodgers et al., 2001). Comparison of the amino acid sequence of grouper MSTN with those of known MSTN reported to date revealed that grouper MSTN shared a higher degree of homology with those of other fish MSTN, but a lower degree of homology with those of mammalian MSTN. Results of phylogenetic analysis showing that grouper MSTN is closest to Morone saxatilis, Morone americana, and Dicentrarchus labrax in the dandrogram further support the conclusion that grouper MSTN is closer to MSTN of other fish than to higher vertebrates (Kerr et al., 2005). Because grouper MSTN is well conserved through evolution, it suggests that this peptide may be important in controlling growth and development, and the biological action may be conserved as well.

While the function of MSTN in controlling muscle development and growth has been well documented in higher mammals (McPherron and Lee, 1997; McPherron et al., 1997; Lee and McPherron, 2001), the function of fish MSTN is awaiting elucidation. Two recent studies have been attempted unsuccessfully to reproduce the enhanced muscle growth phenotype in zebrafish by overexpressing the prodomain of MSTN which will inhibit the function of MSTN (Xu et al., 2003) or by reducing the level of MSTN mRNA production with an antisense morpholinos (Amali et al., 2004). In both studies, only an increased number of myofibers in skeletal muscles was observed, suggesting that fish MSTN may control only the hypertrophy of the skeletal muscle. Therefore, detail studies on the tissue-specific and developmental stage-specific expression of MSTN gene in different fish species may shed insight on the understanding of the biological function(s) of MSTN in fish. Quantitative real-time RT-PCR analysis on the tissue distribution of MSTN mRNA in 1-year-old grouper revealed that several tissues other than myogenic lineage cells expressed MSTN gene. Although the highest level of grouper MSTN mRNA was detected in the gill and muscle, lower levels in the eyes, brain, heat, head kidney, stomach, and intestine, and much lower levels in the spleen and liver. These results are in good agreement with those reported for other fish species (Maccatrozzo et al., 2001a,b; Roberts and Goetz, 2001; Rodgers and Weber, 2001; Rodgers et al., 2001; Radaelli et al., 2003), even though we did not determine the presence of myostatin protein in these tissues. In contrast to fish, MSTN mRNA is predominantly expressed in skeletal muscle, though there have been reports of MSTN protein detected in cardiomyocytes, Purkinje fibers of the heart, adipose tissue (Sharma et al., 1999), and in the mammary gland (Ji et al., 1998) in higher vertebrates.

In this study, the expression of MSTN gene in grouper during embryonic and larval development was also determined by quantitative real-time PCR analysis. The results of the study showed that low levels of MSTN mRNA were detected in the unfertilized eggs, the newly fertilized eggs, 16-cell stage, and morula stage, while higher levels were detected in the blastula stage, gastrula stage, neurula stage, lens formation stage, and somite stage. Further, the levels of MSTN mRNA continue to increase during development and peaks at 30-day-old larvae. These results are somewhat consistent with the expression pattern of MSTN in mammals in which MSTN is expressed in the myotome compartment of the somite during embryogenesis and in limb muscle during fetal development (McPherron et al., 1997). Variation on the ontogeny of MSTN gene expression in fish has been observed in catfish [detected at 1 day postfertilization (Kocabas et al., 2002b)], and in zebrafish [detected at 4 days postfertilization via in situ hybridization (Xu et al., 2003)]. However, one distinct difference between the expression of MSTN gene in grouper and in mammals is that grouper MSTN mRNA is found as a maternal mRNA and is also detectable in very early stages of embryogenesis. Because grouper MSTN gene is expressed in early embryogenesis as well as in multiple tissues, we, therefore, believe that, in addition to controlling skeletal muscle growth and differentiation, MSTN gene might also play other roles in fish. Recently Mitchell et al. (2006) reported that treatment of human term placental explants with myostatin protein resulted in an increase in deoxyglucose uptake compared with controls. Our findings of MSTN mRNA in mature oocytes as well as during early stages of embryogenesis might suggest its involvement in the mobilization of carbohydrates and proteins in these stages of development. Further in-depth studies are required to unveil the biological function(s) of MSTN in fish.