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Biology of Reproduction 67, 401-408 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Development of Skeletal Muscle and Expression of Candidate Genes in Bovine Fetuses from Embryos Produced In Vivo or In Vitro1

Adrienne E. Crosiera, Charlotte E. Farina, Karina F. Rodrigueza, Patrick Blondin3,,a, Joseph E. Alexandera, and Peter W. Farin2,,b

a Departments of Animal Science, b Farm Animal Health and Resource Management, North Carolina State University, Raleigh, North Carolina 27606


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objectives of this study were to determine the effects of in vitro embryo production on histological development and gene expression in the skeletal muscle of bovine fetuses during late gestation. Blastocysts produced in vivo were obtained from superovulated Holstein cows. Blastocysts produced in vitro were obtained from oocytes of Holstein cows that were matured and fertilized in vitro. Single blastocysts were transferred into heifers at a synchronized estrous and fetuses were recovered at Day 222 of gestation (n = 12 each for in vivo and in vitro). Samples of semitendinosus muscle were obtained for histological analysis and assessment of gene expression. Individual muscle sections were stained for the assessment of primary muscle fibers, secondary muscle fibers, or total muscle fibers. Semiquantitative reverse transcription-polymerase chain reaction assays were performed for 5 different candidate genes. The ratio of secondary-to-primary fiber number was greater in fetuses from embryos produced in vitro compared with fetuses from embryos produced in vivo. Similarly, the ratio of secondary-to-primary fiber volume density tended to be greater in fetuses from embryos produced in vitro. The proportional volume of tissue present between myofibrils was greater in fetuses from embryos produced in vitro. The expression of mRNA for myostatin was decreased in skeletal muscle of fetuses in the in vitro group compared with controls. The expression of mRNA for glyceraldehyde-3-phosphate dehydrogenase tended to be increased in skeletal muscle of fetuses in the in vitro treatment group. There was no effect of treatment on the expression of mRNAs for myf-5, myoD, or myogenin. In conclusion, in vitro production of embryos resulted in fetuses with altered development of skeletal muscle fibers. Myostatin was identified as the candidate gene whose expression may contribute to the observed changes in muscle development of these fetuses.

conceptus, developmental biology, embryo, fetus, gene regulation, in vitro fertilization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Manipulation of bovine and ovine embryos during the preimplantation period can affect the development of the resulting fetuses and offspring [1–7]. For example, in late gestation, bovine fetuses resulting from embryos produced in vitro were heavier and had altered development of the musculoskeletal system compared with fetuses from embryos produced in vivo [1]. Similarly, calves resulting from in vitro-produced embryos were heavier [2–4] and yielded more salable meat at slaughter [3] compared with control calves. Finally, the transfer of ovine embryos into an advanced uterine environment resulted in increased total skeletal muscle fiber number and increased ratio of secondary-to-primary fiber number in fetuses at late gestation compared with that of control fetuses [5].

In cattle, primary skeletal muscle fibers (type I) differentiate from primary myotubes between Days 39 and 180 of gestation [8]. Secondary, or type II, muscle fibers are derived from a separate population of myotubes and begin differentiation between Days 100 and 120 of gestation [8]. Differentiation of both primary and secondary fibers is complete in the bovine fetus at the time of parturition [9]. As the fetus develops, muscle fiber bundles become more organized and the connective tissue surrounding the muscle fibers decreases in total area [10].

Differentiation of skeletal muscle fibers during development is coordinated by the myogenic determining factor family of proteins (MDFs; [11]). In vertebrates, this family of proteins consists of the transcription factors myoD, myf-5, myogenin, and MRF4, which are expressed in a temporal-spatial pattern during fetal development [12]. Each of these factors can convert nonmuscle cells cultured in vitro into cells with myogenic properties [13]. MyoD and myf-5 mRNAs are expressed in proliferating myoblasts prior to differentiation [14] and in newly differentiated muscle fibers [11]. Myogenin regulates normal differentiation of skeletal muscle fibers [14] after the establishment of myotubes. The myostatin gene is not considered to be a member of the MDF family; however, it does regulate skeletal muscle fiber development in cattle [15]. Myostatin acts as an inhibitor of muscle development by limiting the muscle fiber number [16]. Double-muscling commonly occurs when the myostatin gene is altered by an 11-base pair (bp) deletion causing a frame-shift in the amino acid sequence [15]. This deletion results in an increased muscle fiber number and a 20–25% increase in muscle mass [17], primarily in the secondary-fiber population [18].

The influence of in vitro embryo production on skeletal muscle differentiation in bovine fetuses has not been described. Therefore, the overall objective of this study was to determine the effect of in vitro embryo production on the development and regulation of skeletal muscle in bovine fetuses at late gestation. The following 2 specific objectives were pursued: 1) to compare the histological development of skeletal muscle and 2) to analyze the expression of candidate genes that likely contribute to the regulation of skeletal muscle development in bovine fetuses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Hormones

Tissue culture medium (TCM-199 with Earle salts) was purchased from Gibco BRL (Grand Island, NY). Equine pituitary LH (11.5 NIH LH-S1 U/mg) and porcine pituitary FSH (50 mg/vial Armour FSH standard) preparations were obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents and media supplements were of tissue culture grade and were purchased from Sigma. Fatty-acid-free BSA was purchased from Boehringer Mannheim (Indianapolis, IN). Prostaglandin F2{alpha} was obtained from Pharmacia and Upjohn Co. (Lutalyse; Kalamazoo, MI). Folltropin was obtained from Vetrepharm Canada, Inc. (London, ON, Canada). Muscle biopsy clamps were purchased from the Allegiance Medical Products Co. (McGaw Park, IL). TriReagent was purchased from Molecular Research Center, Inc. (Cincinnati, OH). DNase was purchased from Roche Molecular Biochemicals (Mannheim, Germany). SuperScript II reverse transcriptase, random hexamers, dNTPs, and rabbit globin mRNA were purchased from Gibco. The Gene-Clean II purification kit was obtained from Bio 101 (Carlsbad, CA). Taq polymerase was purchased from either Roche or Qiagen, Inc. (Valencia, CA). All primers for PCR were custom synthesized by Sigma-Genosys (Woodlands, TX).

Production of Embryos

For production of blastocysts in vivo, 14 Holstein cows were given a total of 400 mg Folltropin by i.m. injection over a 3- or 4-day period to induce superovulation. Estrus was induced by i.m. administration of 25 mg prostaglandin F2{alpha} on the morning and evening of the third day of FSH treatment. Estrus detection was performed twice daily beginning 24 h after the first prostaglandin F2{alpha} injection. Cows were inseminated at 12 and 24 h after first standing estrus with semen from a proven Holstein sire. Embryos were recovered from cows by nonsurgical lavage of the uterus on either Day 7 or 8 after estrus (Day 0 = day of first standing estrus and first insemination).

For production of blastocysts in vitro, cumulus oocyte complexes (COCs) were aspirated, matured, and fertilized in vitro as described by Farin and Farin [1]. Briefly, ovaries from Holstein cattle were collected at a local abattoir and held in saline with 0.75 µg/ml penicillin for 4–6 h at an ambient temperature. COCs were aspirated from 2- to 7-mm follicles and washed 5 times in modified Tyrode medium (TL-Hepes; [19]). COCs were matured in groups of 20–30 for approximately 22 h in 1 ml TCM-199 with 10% heat-inactivated estrus cow serum (ECS), 10 µg/ml LH, 5 µg/ml FSH, 1 µg/ml estradiol, 200 µM pyruvate sodium salt, and 50 µg/ml gentamicin. Cultures were maintained in an atmosphere of 5% CO2 in air with 100% humidity. At the end of the 22 h maturation period, COCs were washed once and placed in fertilization medium consisting of heparin-supplemented tyrode albumin lactate pyruvate medium with 6 mg/ml fatty acid-free BSA [19, 20]. Thawed frozen semen from the same Holstein sire was used for production of all in vivo and in vitro embryos. Motile spermatozoa were collected by swim-up procedure [19] and used at a final concentration of 1 x 106 spermatozoa per milliliter. Gametes were coincubated for 18–20 h, after which time presumptive zygotes with their cumulus investments were washed 6 times in TL-Hepes. Embryos were cultured in groups of 20–30 in wells containing 1 ml TCM-199 with 10% ECS and 50 µg/ml gentamicin in an atmosphere of 5% CO2 in air with 100% humidity. Culture medium was changed at 48-h intervals throughout the 168-h culture period.

Transfer of Embryos

Angus heifers (n = 42) were given 2 injections of 25 mg prostaglandin F2{alpha} by i.m. administration 10–12 days apart to synchronize estrous cycles. Based on evaluation of embryos at 60x, grade 1 [21] blastocysts from in vivo or in vitro production were transferred in TL-Hepes medium singly into the uterine horn ipsilateral to the ovary bearing the CL of recipient heifers.

Recovery of Fetuses and Muscle Biopsy

At Day 222 of gestation (215 days after embryo transfer), pregnant recipients were killed and fetuses were recovered. Fetuses (in vivo, n = 12; in vitro, n = 12) were weighed, necropsied, and biopsies were taken of semitendinosus muscle. For histology, the central muscle belly was biopsied using a stainless steel muscle clamp with a jaw width of 8 mm. The muscle sample was then excised from the surrounding tissue. Muscle samples were frozen in liquid nitrogen-cooled isopentane for 1 min and stored at -80°C. For analysis of gene expression, samples of muscle were snap frozen in liquid nitrogen and stored at -80°C.

Histology

Individual muscle sections were stained with either acidic myofibrillar ATPase, alkaline myofibrillar ATPase, or hematoxylin and eosin for assessment of primary fibers, secondary fibers, or total muscle fibers, respectively [22]. Computer-assisted image analysis (Optimas Visual Imaging System 6.1; Optimas Corporation, Bothell, WA) was used to determine the number of primary, secondary, or total muscle fibers per fixed sampling area. For each fetus, 4 randomly selected views totaling 400 µm2 were used for assessment of fiber number. The proportional volume between myofibrils (intermyofibril volume density) as well as the volume densities of primary and secondary fibers were also determined. Approximately 690 µm2 were evaluated from each muscle sample for assessment of fiber volume density. To determine the average area of individual muscle fibers, a subset of 50 primary and 50 secondary fibers were measured for each fetus.

wcRNA Extraction and Reverse Transcription

Semitendinosus muscle samples from 2 bovine fetuses at approximately Day 225 of gestation (as determined by crown-rump length and body weight) were snap frozen in liquid nitrogen and used as laboratory controls. Whole-cell RNA (wcRNA) was extracted as previously described [23]. Briefly, frozen muscle tissue was weighed, placed in a mortar, and crushed to a fine powder. TriReagent was added to the frozen powder (1 ml/100 mg tissue), and samples were briefly homogenized (Brinkmann Homogenizer PT 10/35; Westbury, NY). wcRNA was extracted according to the manufacturer's protocol and resuspended in diethyl pyrocarbonate-treated water. The concentration of wcRNA was determined based on absorbance at 260 nm. After verifying the quality of wcRNA by visualization of ethidium bromide-stained 28S and 18S rRNA bands on 2% agarose gels, 6-µg aliquots of wcRNA were snap frozen in liquid nitrogen and stored at -80°C prior to analysis.

Individual wcRNA aliquots were thawed on ice and 2 µg of each sample was treated with DNase (15 units) for 20 min at 37°C. DNase-free wcRNA was then reverse transcribed using random hexamers and SuperScript II reverse transcriptase under conditions recommended by the manufacturer. A sample containing all reaction components except wcRNA was included in each group of reverse transcription reactions as a negative control. Samples of cDNA were then cleaned using the Gene Clean II kit as recommended by the manufacturer.

PCR Reaction Conditions

Unless otherwise noted, primer sequences were generated utilizing Genebank mRNA sequences, Oligo 4.0.2 primer analysis software (Plymouth, MA), and Gene Amplify 1.2 software (Madison, WI). Specific primer sequences and annealing temperatures used are indicated in Table 1. For each PCR assay, the final reaction mix consisted of 200 ng each of the appropriate forward and reverse primers, 16 µM dNTPs, and 2.5 units of Taq polymerase in a 20 µl reaction. In addition, the reaction mixes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), myf-5, myogenin, myoD, and rabbit globin each included 2 µl of 10x PCR buffer (Qiagen) and 1.5 µl of Q solution (Qiagen). The final reaction mix for myostatin included 3 µl of 10x PCR buffer (Roche). All reactions contained 125 ng cDNA. In addition to the samples from the 12 fetuses, a sample containing the master mix and no cDNA was included in each PCR assay as a negative control. All PCR reactions were overlaid with oil and briefly spun before being placed into a PTC-100 thermocycler (MJ Research Inc., Watertown, MA) at 92°C for 2 min. The standard PCR program used included denaturation for 30 sec at 94°C, 30 sec at the appropriate annealing temperature, and 60 sec at 72°C for primer extension. The last cycle of this program for each primer set was followed by 5 min at 72°C for maximum primer extension.


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  		TABLE 1. Primer sequences used for PCR analysis of bovine fetal skeletal muscle

Determination of Linear Phase of Amplification

For determination of the linear phase of amplification and optimal conditions for each primer pair, PCR was performed using cDNA from the 2 laboratory control fetuses. Reactions were conducted for a total of either 40 or 45 cycles, and 2 reaction tubes were removed every 5 cycles beginning with cycle 20. PCR products were visualized on ethidium bromide-stained 2% agarose gels. The signal intensity of individual bands was determined using the Optimas visual imaging system. A response curve was generated for each of the 5 primer pairs, and the exponential phase of product amplification was determined.

Semiquantitative PCR Analysis

A subset of 10 fetuses (n = 5 in vivo and n = 5 in vitro) were used for assessment of gene expression of GAPDH, myostatin, myf-5, myoD, and myogenin in semitendinosus muscle. Each assay was designed to assess expression of 1 mRNA type and included all experimental fetal samples (n = 10 combined from both treatments) as well as laboratory control fetuses (n = 2). cDNA was prepared as described above and duplicate samples from each fetus were subjected to PCR amplification for the cycle number determined to be within the exponential phase. PCR products were visualized and the signal intensity of the resulting bands was determined. The signal intensity for each PCR product from each fetus was calculated based on the average of the duplicate reactions. For each mRNA examined, the entire assay was replicated twice. Intraassay coefficients of variation of duplicate samples for the 2 laboratory control fetuses for GAPDH, myostatin, myf-5, myoD, and myogenin were 4.44%, 1.53%, 1.24%, 6.57%, and 2.68%, respectively. Based on analysis of the 2 laboratory control fetal samples, the interassay coefficients of variation for expression of mRNA for GAPDH, myostatin, myf-5, myoD, and myogenin were 7.16%, 0.76%, 7.26%, 7.52%, and 7.66%, respectively. To control for potential variation between reverse transcription reactions, 2 pg of exogenous rabbit globin mRNA was added to each wcRNA sample prior to reverse transcription [24] in 1 of the 2 replicate assays performed for each mRNA analysis. Amplification signals for rabbit globin mRNA were determined in a separate assay and were used to calculate ratios of relative expression for each mRNA of interest. Because of the small variation in the expression of mRNA for the laboratory control fetuses between replicate assays and the inclusion of rabbit globin mRNA in the second assay, data from only the second assay are presented.

Statistical Analysis

All data were analyzed using general linear model procedures [25], and means were considered statistically different at P < 0.05. Values between P = 0.055 and P = 0.10 are presented as tendencies (P < 0.10). No significant interaction of treatment by sex was found for the body weight of fetuses. In addition, the main effect of sex on fetal body weight was not significant. Therefore, the final statistical model included only the main effect of treatment (in vivo, in vitro) on body weight and eviscerated weight. The effect of treatment on muscle fiber measurements was analyzed using a model that included the effects of treatment, sex of fetus, the interaction of treatment by sex of fetus, and the covariate of fetal body weight. No significant interactions of treatment by sex were found for any parameters. Therefore, the final statistical model included only the effects of treatment, sex of the fetus, and the covariate of fetal body weight.

The statistical model used to analyze the effect of treatment on the expression of mRNA (signal intensities) for GAPDH, myostatin, myf-5, myogenin, and myoD included the main effects of treatment, sex of fetus, and reverse transcription reaction, as well as the continuous effect of fetal body weight. The model used for analysis of data on the ratio of mRNA expression for GAPDH, myostatin, myf-5, myoD, and myogenin to expression of rabbit globin mRNA contained the effects of treatment, sex of fetus, reverse transcription reaction, the interaction of treatment by sex of fetus, and the covariate of fetal body weight.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Histological Analysis

Fetuses from embryos produced in vitro had an increased (P < 0.05) body weight as well as an increased (P < 0.05) eviscerated weight compared with fetuses from embryos produced in vivo (Table 2). Figure 1 illustrates transverse sections of semitendinosus muscle of bovine fetuses from embryos produced in vivo (Fig. 1A) or in vitro (Fig. 1, B and C). Hematoxylin- and eosin-stained muscle sections for assessment of muscle fibers is shown in Figure 1A. Figure 1B depicts acidic myofibrillar ATPase-stained muscle for assessment of primary fibers. An alkaline myofibrillar ATPase-stained muscle section for evaluation of secondary fibers is illustrated in Figure 1C.


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  		TABLE 2. Effect of treatment on the weight (kg) of bovine fetuses from embryos produced in vivo or in vitro.a



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  		FIG. 1. Transverse sections of semitendinosus muscle of fetuses from bovine embryos produced in vivo or in vitro. A) Hematoxylin and eosin staining for assessment of total fibers. B) Acidic myofibrillar ATPase staining for assessment of primary fibers. C) Alkaline myofibrillar ATPase staining for assessment of secondary fibers. In B and C, examples of fibers considered to be positively stained are indicated (arrow). Bar in each micrograph is 20 µm

Fetuses in the in vitro group had a greater (P < 0.05) ratio of secondary-to-primary muscle fiber number compared with that for fetuses in the in vivo group (Fig. 2A). The ratio of the proportional volumes occupied by secondary-to-primary muscle fibers tended to be greater (P < 0.07) for fetuses from embryos produced in vitro compared with that for controls (Fig. 2B). There was an increase (P < 0.01) in the proportional volume of tissue present between the myofibrils (intermyofibril volume density) for fetuses in the in vitro group compared with that for fetuses from embryos produced in vivo (Fig. 3). No differences in embryo production method were found for the numbers of primary, secondary, or total muscle fibers in skeletal muscle of fetuses (Table 3). In addition, there were no differences in the volume density of primary or secondary muscle fiber types between treatment groups. Finally, there were no differences between treatment groups in the areas of individual primary or secondary muscle fibers (Table 3).



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  		FIG. 2. A) Ratio of secondary-to-primary muscle fiber number for bovine fetuses from embryos produced in vivo or in vitro (a,bP < 0.05; least-squares means ± SEM). B) Ratio of secondary-to-primary muscle fiber volume density for bovine fetuses from embryos produced in vivo or in vitro (a,bP < 0.07; least-squares means ± SEM)



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  		FIG. 3. Volume density of intermyofibril tissue for bovine fetuses from embryos produced in vivo or in vitro (a,bP < 0.01; least-squares means ± SEM)


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  		TABLE 3. Effect of treatment on muscle fiber area and fiber number for bovine fetuses from embryos produced in vivo or in vitro.a

Semiquantitative RT-PCR Assay

A subset of fetuses were chosen from each treatment group (n = 5 each for in vivo and in vitro treatments) for analysis of expression of mRNAs for GAPDH, myostatin, myf-5, myoD, and myogenin. Fetuses from embryos produced in vitro were heavier (P < 0.05) compared with fetuses from embryos produced in vivo (17.1 ± 0.4 kg and 20.6 ± 0.4 kg for in vivo and in vitro, respectively). The various primer sequences used for PCR analyses, their expected product length, annealing temperature, and cycle number used for linear amplification are listed in Table 1. Figure 4 (inset) presents an example of 1 of the ethidium-bromide-stained agarose gels used in the determination of the linear amplification range for the myf-5 primer pair. Data based on analysis of the signal intensities of the amplification products from the complete reaction series (20–40 cycles) are also depicted in Figure 4. Figure 5 shows a representative ethidium-bromide-stained agarose gel depicting 228-bp products from duplicate PCR reactions utilizing the myf-5 primer pair.



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  		FIG. 4. Inset: A representative ethidium bromide-stained agarose gel depicting PCR products in duplicate from cycles 30 and 35 for determination of the exponential phase of amplification utilizing the myf-5 primer pair and cDNA from the 2 laboratory control fetuses (A and B). Graph: Data based on complete analysis of the average signal intensities for the duplicate PCR products for the myf-5 primer pair and control cDNA samples. The SEM associated with the mean of each duplicate ranged from 0% to 2%. Cycle number 33 was determined to be within the exponential phase of amplification and was utilized for all subsequent PCR reactions using the myf-5 primer pair



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  		FIG. 5. A representative ethidium bromide-stained agarose gel depicting 228-bp PCR products from reactions utilizing the myf-5 primer pair. All individual samples (in vivo, in vitro, and laboratory controls [LC]) were assayed in duplicate (lanes 1–10). Lane 11 contains the negative PCR control sample (C). Lane 12 contains the molecular base pair markers (M)

Expression of mRNA for GAPDH tended to be higher (P < 0.07) in skeletal muscle of fetuses from embryos produced in vitro compared with that for those produced in vivo (Fig. 6A). The expression of mRNA for myostatin was lower (P < 0.05) in skeletal muscle of fetuses in the in vitro group compared with that for fetuses in the control group (Fig. 6B). There was no effect of treatment on the expression of mRNA for the MDFs myf-5, myogenin, and myoD (Fig. 6, C–E) or for expression of mRNA for rabbit globin (Fig. 6F).



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  		FIG. 6. Expression of mRNA for GAPDH (A, a,bP < 0.07), myostatin (B, a,bP < 0.05), myf-5 (C), myoD (D), and myogenin (E) in the skeletal muscle of bovine fetuses from embryos produced in vivo or in vitro. Expression of mRNA is pictured for exogenously added rabbit globin (F). All values are least-squares means ± SEM

When amplification signals were expressed as a ratio to signals obtained for exogenously added rabbit globin mRNA, expression of mRNA for GAPDH was significantly higher (P < 0.01) in skeletal muscle of fetuses from embryos produced in vitro compared with that for fetuses from embryos produced in vivo (Fig. 7A). In addition, the expression of mRNA for myostatin tended to be lower (P <= 0.10) for fetuses in the in vitro group compared with that for fetuses from the in vivo group (Fig. 7B). Consistent with the analysis of unadjusted amplification signal intensities, there was no effect of treatment on the expression of mRNA for myf-5, myogenin, or myoD when data were expressed as a ratio to rabbit globin mRNA amplification signal intensity (Fig. 7, C–E).



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  		FIG. 7. Ratio of the expression of mRNA for GAPDH (A, a,bP < 0.01), myostatin (B, a,bP <= 0.10), myf-5 (C), myoD (D), and myogenin (E) to the expression of mRNA for rabbit globin in skeletal muscle of bovine fetuses from embryos produced in vivo or in vitro. All values are least-squares means ± SEM


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Manipulation of preimplantation-stage mammalian embryos can result in divergent patterns of fetal growth [1, 5–7]. For example, bovine fetuses from embryos produced in vitro were heavier [1, 7] and had disproportionate growth of the musculoskeletal system [1] at Day 222 of gestation compared with fetuses from embryos produced in vivo. Similarly, development of skeletal muscle in ovine fetuses [5, 6] and calves [3] may be altered during embryogenesis by in vitro production. To our knowledge, this study represents the first analysis of bovine fetal muscle development following transfer of embryos produced in vitro.

In the present study, bovine fetuses from embryos produced in vitro had an increased ratio of secondary-to-primary fiber number and an increased ratio of the secondary-to-primary fiber volume density compared with that for fetuses produced in vivo. These data are consistent with the observation of an increased ratio of secondary-to-primary fiber number in fetuses from asynchronously transferred ovine embryos [5]. This altered pattern of muscle fiber development was suggested to result from an increase in myoblast hyperplasia during early fetal development [5]. It is plausible that this proposed mechanism also could be the cause of the increased ratios of secondary-to-primary fiber number and secondary-to-primary fiber volume density observed in the present study.

An increased occurrence of hydroallantois has been associated with bovine [26] and ovine [6] pregnancies resulting from transfer of embryos produced in vitro. Based on data in the present study, fetuses from embryos produced in vitro had an increased intermyofibril volume density compared with fetuses from embryos produced in vivo. Thus it is possible that the increased area between myofibrils resulted from an increased amount of fluid within the muscle tissue at the time of sample collection. Alternatively, the increase in intermyofibril volume density could have resulted from increased connective tissue within the semitendinosus muscle of fetuses from in vitro-produced embryos. During normal development of bovine fetuses, the proportion of semitendinosus muscle occupied by connective tissue decreases rapidly from Day 70 through Day 260 of gestation [10]. Therefore, it is plausible that the normal pattern of connective tissue development may be different in fetuses resulting from embryos produced in vitro.

There was no difference in the average individual fiber area for fetuses from embryos produced in vivo or in vitro in the present study. For bovine fetuses resulting from artificial insemination, the diameter of individual muscle fibers increased throughout gestation [10]. Based on the data from that study [10], an approximate area for individual muscle fibers at Day 225 of gestation can be calculated as 324 µm2 [10]. The values obtained for individual fiber area of both primary and secondary fibers for fetuses in the present study are in good agreement with this estimate.

Although myostatin is not a member of the MDF family [11], myostatin does act as an inhibitor of muscle development by limiting muscle fiber number [18]. Double-muscling in cattle occurs when the myostatin gene is altered resulting in an increase in muscle fiber number and a 20%–25% increase in muscle mass [17, 27]. This increase occurs primarily in the secondary-fiber population [18, 28]. Thus the decreased level of expression for myostatin mRNA in semitendinosus muscle of fetuses in the in vitro group is consistent with the increased ratio of secondary-to-primary fiber number and the increased ratio of secondary-to-primary fiber volume density observed in histological sections of muscle from these same fetuses.

Specific imprinted alleles, which are involved in fetal, placental, and/or neonatal growth, may be differentially expressed because of manipulation of embryos produced in vitro [29]. Specifically, altered expression of growth factors in fetuses has been associated with in vitro production of embryos [23, 30, 31]. For example, male bovine fetuses from embryos produced in vitro in a serum-supplemented medium had an increased expression of mRNA for insulin-like growth factor II (IGF-II) in liver compared with fetuses from embryos produced in vivo [23]. In addition, overgrowth of ovine fetuses from embryos produced in vitro was suggested to result from reduced expression of the IGF-II receptor [30]. Myostatin is a member of the transforming growth-factor ß family [16], and, interestingly, IGF-II may be involved in the negative control of mRNA expression for transforming growth-factor ß early in myogenesis [32].

GAPDH is a critical enzyme in the glycolytic pathway [33]. Although this enzyme does not contribute to muscle fiber development, it is involved in cellular metabolism in skeletal muscle [34]. Secondary muscle fibers produce increased amounts of ATP through glycolysis [34]. This, in turn, is a result of increased activity of glycolytic enzymes [34], including GAPDH [33], within these fibers. The observed increase in expression of mRNA for GAPDH in the skeletal muscle of fetuses from embryos produced in vitro, therefore, is consistent with the increase in the ratio of secondary-to-primary fiber number and volume density seen in semitendinosus muscle of these fetuses.

Asynchronous transfer of ovine embryos resulted in an increased level of the myf-5 protein in skeletal muscle at late gestation compared with fetuses produced from synchronously transferred control embryos [5]. In contrast, there was no difference in the expression of mRNA for myf-5 in skeletal muscle of bovine fetuses in the present study. Differences in species, as well as differences in the systems used for the production of embryos (asynchronous transfer [5] vs. in vitro culture [present study]) may have resulted in different mechanisms of fetal muscle development. Alternatively, this apparent discrepancy could reflect actual differences in the level of protein versus expression of mRNA for myf-5 in the skeletal muscle of fetuses.

Expression of mRNA for myoD in the skeletal muscle of bovine fetuses was maintained at peak levels in fetuses between 30 and 46 cm in length [35], or approximately Days 120 and 180 of gestation [10]. Most recently, the expression of both myoD and myogenin mRNAs did not differ in semitendinosus muscle of normal or double-muscled bovine fetuses during late gestation (Day 210 through birth) [36]. Thus the lack of treatment differences in the expression of myoD and myogenin mRNAs observed at Day 222 of gestation in the present study may have resulted from collection of samples outside the time-period of gestation when differences in expression of myoD or myogenin are apparent.

In summary, bovine fetuses from embryos produced in vitro had increased ratios of both secondary-to-primary fiber number and volume density, an increased volume density of tissue between myofibrils, an increased expression of mRNA for GAPDH, and a decreased expression of mRNA for myostatin compared with fetuses from embryos produced in vivo. In conclusion, the development of skeletal muscle in bovine fetuses during late gestation is altered by the production of embryos in vitro. Furthermore, myostatin has been identified as a candidate gene that may contribute to the observed changes in fetal muscle development.


   ACKNOWLEDGMENTS
 
The authors wish to thank Drs. William Flowers, John Gadsby, and Robert Petters for critical review of this manuscript, and Mr. Jeffrey Sommer for excellent technical assistance.


   FOOTNOTES
 
First decision: 25 October 2001.

1 Supported by the NCSU-CVM Competitive Research Grants Program and USDA grant 9602482. Back

2 Correspondence: Peter W. Farin, Department of Farm Animal Health and Resource Management, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606. FAX: 919 513 6464; peter_farin{at}ncsu.edu Back

3 Current address: Office of Technology Transfer, McGill University, Montreal, QC, Canada H3A 2A7 Back

Accepted: February 20, 2002.

Received: September 17, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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