Real-time reverse transcription–PCR expression profiling of porcine troponin I family in three different types of muscles during development

In this study, the expression profiling of three troponin I isoforms (TNNI1, TNNI2 and TNNI3) was investigated in two pig breeds differing in muscularity (Yorkshire and Meishan) at six stages (fetal 60 days and postnatal 3, 35, 60, 120, and 180 days) and three types of muscles (longissimus dorsi muscle, LD; semitendinosus, ST; cardiac muscle, CM) using relative real-time quantitative PCR. Significant differences of troponin I expression in three muscles were found between Yorkshire and Meishan breeds at some stages. The expression peak of TNNI1 and TNNI2 in LD and ST was at postnatal 35 or 60 days in Yorkshire and at postnatal 120 or 180 days in Meishan pigs, while it occurred in CM at postnatal 3 days in two pig breeds. The relative expression values of TNNI1 and TNNI2 were significantly higher in LD than ST at most of stages after birth. The expression ratio of TNNI2 versus TNNI1 favoured TNNI2 expression in ST and LD, but on the contrary in CM. The expression peak of TNNI3 occurred at postnatal 60 and 120 days in Yorkshire and Meishan pigs, respectively. TNNI1 and TNNI3 were co-expressed in CM during the fetal and earlier stages after birth.     

Association of 3 polymorphisms in porcine troponin I genes (TNNI1 andTNNI2) with meat quality traits

The contractile protein troponin I (TnI), a constituent protein of the troponin complex located on the thin filaments of striated muscle, is involved in inhibition of calcium-induced myosin AT Pase activity (and thus contraction). TnI-slow (slow-twitch skeletal muscle isoform, named TNNI1) and TnI-fast (fast-twitch skeletal muscle isoform, named TNNI2) are muscle-fiber-type-specific proteins, and expression of their genes may affect the composition of muscle fiber, thereby influencing the meat quality traits. Thus, the TnI genes are potential candidate genes for traits related to meat quality in animals. Association of 2 SNPs (EU743939:g.5174T>C in intron 4, and EU743939:g.8350C>A in intron 7) of theTNNI1 gene and a SNP (EU696779:g.1167C>T in intron 3) of theTNNI2 gene with 11 meat quality traits were studied on 334 Large White × Meishan F₂ pigs. In theTNNI1 gene, g.5174T>C and g.8350C>A were found to be significantly associated with intramuscular fat content and meat color value of biceps femoris. The g.5174T>C also showed significant effects on meat color value and marbling score of longissimus dorsi, as well as pH of longissimus dorsi and semispinalis capitis. The g.1167C>T polymorphism in theTNNI2 gene affected significantly the pH of longissimus dorsi, meat color value of longissimus dorsi and semispinalis capitis, marbling score of longissimus dorsi, and intramuscular fat.     

Proteomic Analysis of Fetal Ovary Reveals That Ovarian Developmental Potential Is Greater in Meishan Pigs than in Yorkshire Pigs

Time-dependent expression of functional proteins in fetal ovaries is important to understand the developmental process of the ovary. This study was carried out to enhance our understanding of the developmental process of porcine fetal ovaries and to better address the differences in fetal ovary development of local and foreign pigs. The objective of the present study is to test the expression of key proteins that regulate the growth and development of fetal ovaries in Meishan and Yorkshire porcine breeds by using proteomics technology. Six Meishan and 6 Yorkshire pregnant gilts were used in this experiment. Fetal ovaries were obtained from Yorkshire and Meishan gilts on days 55 and 90 of the gestation period. Using 2D-DIGE (two dimensional-difference in gel electrophoresis) analysis, the results showed that there are about 1551 and 1400 proteins in gilt fetal ovaries on days 55 and 90, respectively of the gestation. Using MALDI TOF-TOF MS analysis, 27 differentially expressed proteins were identified in the fetal ovaries of the 2 breeds on day 55 of gestation, and a total of 18 proteins were identified on day 90 of gestation. These differentially expressed proteins were involved in the regulation of biological processes (cell death, stress response, cytoskeletal proteins) and molecular functions (enzyme regulator activity). We also found that alpha-1-antitrypsin, actin, vimentin, and PP2A proteins promote the formation of primordial follicles in the ovaries of Yorkshire pigs on day 55 of gestation while low expression heat shock proteins and high expression alpha-fetoproteins (AFP) may promote Meishan fetal ovarian follicular development on day 90 of gestation. These findings provide a deeper understanding of how reduced expression of heat shock proteins and increased expression of AFP can significantly reduce the risk of reproductive disease in obese Meishan sows. Our study also shows how these proteins can increase the ovulation rate and may be responsible for the low reproductive efficiency reported in other obese breeds. The ovarian developmental potential was found to be greater in Meishan pigs than in Yorkshire pigs.     

Age at puberty, ovulation rate, uterine length, prenatal survival and litter size in Chinese Meishan and Yorkshire females

Studies on the ovulation rate, prenatal survival and litter size of Chinese Meishan pigs have given widely divergent results depending on the extent of inbreeding of the animals, their original genetic diversity, the age and parity, and the conditions of management. To obtain meaningful results, it is necessary to characterize the population under study. The following report characterizes populations of Meishan and Yorkshire of a widely diverse background. First farrowing data were collected on 21 Meishan and 20 Yorkshire gilts. Meishan gilts had 12.4 fully formed piglets and Yorkshire gilts had 7.4 fully formed piglets (P < 0.01). Meishan gilts averaged 1.86 mummified fetuses per litter vs 0.05 per Yorkshire litter (P < 0.01). Yorkshire piglets averaged 1.3 kg body weight at birth vs 0.9 kg for Meishan piglets (P < 0.01). At 47 days of second gestation, 19 Meishan and 12 Yorkshire sows averaged 22.7 and 16.3 corpora lutea (CL), respectively (P < 0.01). Uterine length and number of fetuses were not different (P > 0.40) in the two breeds. Daily estrous detection of 50 Meishan and 34 Yorkshire gilts began at 60 and 120 days of age, respectively. Meishan gilts reached sexual maturity at 95 days of age, which was 105 days earlier than Yorkshire gilts (P < 0.01). Meishan gilts were in estrus nearly 1 day longer than Yorkshire gilts at first, second and third estrus (P < 0.05). No differences in cycle length between breeds were detected for the first or second estrous cycle (P > 0.60). Nineteen Meishan gilts were slaughtered at 51 days of gestation and their reproductive tracts were recovered. The mean number of dissected CL (17.0), number of fetuses (13.1), total uterine length (396 cm), spacing per fetus (29.9 cm), allantoic (124.9 ml) and amniotic (32.2 ml) volumes, crown-rump length (82.8 mm), weight (35.4 g), sex, and direction of each fetus were determined. Chinese Meishan gilts reached puberty much earlier and were in estrus longer than Yorkshire gilts and Meishan sows had more CL than Yorkshire sows.     

Aberrant expression of myosin isoforms in skeletal muscles from mice lacking the rev-erbAalpha orphan receptor gene

The rev-erbAalpha orphan protein belongs to the steroid nuclear receptor superfamily. No ligand has been identified for this protein, and little is known of its function in development or physiology. In this study, we focus on 1) the distribution of the rev-erbAalpha protein in adult fast- and slow-twitch skeletal muscles and muscle fibers and 2) how the rev-erbAalpha protein influences myosin heavy chain (MyHC) isoform expression in mice heterozygous (+/-) and homozygous (-/-) for a rev-erbAalpha protein null allele. In the fast-twitch extensor digitorum longus muscle, rev-erbAalpha protein expression was linked to muscle fiber type; however, MyHC isoform expression did not differ between wild-type, +/-, or -/- mice. In the slow-twitch soleus muscle, the link between rev-erbAalpha protein and MyHC isoform expression was more complex than in the extensor digitorum longus. Here, a significantly higher relative amount of the beta/slow (type I) MyHC isoform was observed in both rev-erbAalpha -/- and +/- mice vs. that shown in wild-type controls. A role for the ratio of thyroid hormone receptor proteins alpha1 to alpha2 in modulating MyHC isoform expression can be ruled out because no differences were seen in MyHC isoform expression between thyroid hormone receptor alpha2-deficient mice (heterozygous and homozygous) and wild-type mice. Therefore, our data are compatible with the rev-erbAalpha protein playing an important role in the regulation of skeletal muscle MyHC isoform expression.     

cDNA sequence, tissue-specific expression, and chromosomal mapping of the human slow-twitch skeletal muscle isoform of troponin I

Troponin I (TnI) is a myofibrillar protein involved in the calcium-mediated regulation of striated muscle contraction. Three isoforms of TnI are known and each is expressed in a muscle fiber-type-specific manner. TnI-fast and TnI-slow are expressed exclusively in fast-twitch and slow-twitch skeletal muscle myofibers, respectively, while a third isoform, TnI-card, is expressed in both the atrium and the ventricle of the heart. An explanation of the myofiber-type-restricted expression of the troponin I multigene family will further aid in understanding how various types of striated muscle fibers are established. To initiate the study of TnI isoform gene expression, we have isolated a full-length cDNA representing the human slow-twitch skeletal muscle isoform of troponin I. Sequence comparisons demonstrate that the TnI-slow protein is highly conserved between species. Therefore, the cDNA was used as a probe to investigate the tissue-specific and developmental regulation of the TnI-slow gene in both rodent and human myogenic cells. TnI-slow message appears to be restricted to muscle tissue containing slow-twitch skeletal muscle myofibers. TnI-slow gene expression is induced in differentiated cultures of primary human muscle cells and several (but not all) myogenic cell lines. In addition, a human-specific probe prepared from the 3' untranslated region of the cDNA has been used to probe a panel of human/mouse somatic cell hybrid lines, resulting in the assignment of the human TnI-slow gene to the q12----qter region of chromosome 1. The locus is designated TNNI1.     

Mutations in genes encoding fast-twitch contractile proteins cause distal arthrogryposis syndromes

The distal arthrogryposes (DAs) are a group of disorders characterized by multiple congenital contractures of the limbs. We previously mapped a locus for DA type 2B (DA2B), the most common of the DAs, to chromosome 11. We now report that DA2B is caused by mutations in TNNI2 that are predicted to disrupt the carboxy-terminal domain of an isoform of troponin I (TnI) specific to the troponin-tropomyosin (Tc-Tm) complex of fast-twitch myofibers. Because the DAs are genetically heterogeneous, we sought additional candidate genes by examining modifiers of mutant Drosophila isoforms of TnI. One of these modifiers, Tm2, encodes tropomyosin, another component of the Tc-Tm complex. A human homologue of Tm2, TPM2, encodes beta-tropomyosin and maps to the critical interval of DA type 1 (DA1). We discovered that DA1 is caused by substitution of a highly conserved amino acid residue in beta-tropomyosin. These findings suggest that DAs, in general, may be caused by mutations in genes encoding proteins of the contractile apparatus specific to fast-twitch myofibers. This provides a new opportunity to directly study the etiology and pathogenesis of multiple-congenital-contracture syndromes.     

Effects of active immunization against porcine Sox6 on meat quality and myosin heavy chain isoform expression in growing-finishing pigs

A feeding trial for 91 days was conducted to investigate effects of active immunization against porcine Sox6 (pSox6) on meat quality and myosin heavy chain (MyHC) isoform expression in growing-finishing pigs. Twenty-four castrated Duroc?×?Landrace?×?Yarkshire pigs were randomly divided into three groups: (1) Control group; (2) 1?mg/head pSox6 active immunity group; (3) 4?mg/head pSox6 active immunity group (4?mg/head group). The results showed that pigs in 4?mg/head group had a greater a* (Redness) and a higher marbling score, while no significant effect was observed in L* (Lightness), b* (Yellowness), intramuscular fat and cooking loss. Muscle succinic dehydrogenase activity in pSox6 active immunization groups was significantly increased, and muscle lactate dehydrogenase activity was significantly reduced. Meanwhile, active immunization against pSox6 upregulated the mRNA expression of MyHC I, while no effect was observed on the mRNA expressions of MyHC IIa, MyHC IIx, MyHC IIb. In addition, pigs in the 4?mg/head group exhibited lower Sox6 mRNA level and higher MyHC I protein level, while no significant influence was observed on MyHC IIb protein level. Together, our data imply that active immunization against pSox6 could improve the pork quality and promote the MyHC I expression in growing-finishing pigs.     

Differential expression of the vascular endothelial growth factor-receptor system in the gravid uterus of yorkshire and Meishan pigs

Litter size in the pig is limited by uterine capacity, which is dependent on uterine size, placental size, and vascularity. Placentae of U.S. pig breeds, such as the Yorkshire, exhibit marked growth from mid to late gestation, increasing their surface area of endometrial attachment. In contrast, placentae of the prolific Chinese Meishan pig exhibit little growth from mid to late gestation; instead, they exhibit a marked and progressive increase in the density of placental blood vessels. Vascular endothelial growth factor (VEGF) is a potent angiogenic and permeability-enhancing factor that is produced and secreted by placentae of several species, including the pig. The activity of VEGF is mediated through two specific receptors (VEGF-R1 and VEGF-R2), both of which are expressed by placental and endometrial tissues in pigs and are thought to play a role in mediating increased vascularization and/or permeability at the fetal-maternal interface. The objectives of the present study were to determine concentrations of VEGF in fetal blood and placental fluids as well as placental and adjacent endometrial mRNA expression of VEGF, VEGF-R1, and VEGF-R2 on Days 30, 50, 70, 90, and 110 of gestation in Yorkshire and Meishan pigs. Day 90 Meishan conceptuses exhibited marked increases (P < 0.05) in placental VEGF mRNA expression as well as fetal blood and allantoic fluid concentrations of VEGF, which remained elevated through Day 110. In contrast, Yorkshire conceptuses failed to exhibit increases in placental VEGF mRNA expression or concentrations of VEGF in fetal blood or allantoic fluid until Day 110. Receptor mRNA expression patterns differed between Meishan and Yorkshire conceptuses, but no difference was found in their expression levels. Placental efficiency (fetal weight/placental weight) was higher (P < 0.05) on Days 90 and 110 in Meishan than in Yorkshire conceptuses. The earlier increase in VEGF protein and mRNA expression in the Meishan versus the Yorkshire conceptus may explain the previously reported increased vascularity and increased placental efficiency of this breed compared the Yorkshire breed.     

Persistent Expression of Developmental Myosin Heavy Chain Isoforms in the Tapered Ends of Adult Pigeon Pectoralis Muscle Fibres

We have shown previously that in addition to the adult myosin heavy chain (MyHC) isoform present throughout the length of each fast-twitch glycolytic muscle fibre within the pectoralis of the mature chicken, the neonatal isoform is retained in the tapered ends of these fibres. This work, however, has been the only published report of this phenomenon. Here, we tested the hypothesis that similar to the chicken, the ends of mature pigeon pectoralis muscle fibres contain developmental MyHC isoform(s). A histological stain was used to visualize endomysium to assist in the analysis of transverse sections of pectoralis muscle from four mature pigeons. Immunocytochemical techniques were used to localize MyHC isoform(s) characteristic of pigeon pectoralis development. We show that within mature pigeon pectoralis, the ends of both fast-twitch glycolytic and fast-twitch oxidative-glycolytic fibre types express MyHC isoform(s) characteristic of their earlier development. Thus, we extend our findings on chicken to another species and an additional muscle fibre type. Retention of developmental MyHC isoform(s) within the tapered ends of mature muscle fibres may be more widespread than is currently appreciated.     

Postnatal myosin heavy chain isoform expression in normal mice and mice null for IIb or IId myosin heavy chains

The patterns of myosin heavy chain (MyHC) isoform expression in the embryo and in the adult mouse are reasonably well characterized and quite distinct. However, little is known about the transition between these two states, which involves major decreases and increases in the expression of several MyHC genes. In the present study, the expression of seven sarcomeric MyHCs was analyzed in the hindlimb muscles of wild-type mice and in mice null for the MyHC IIb or IId/x genes at several time points from 1 day of postnatal life (dpn) to 20 dpn. In early postnatal life, the developmental isoforms (embryonic and perinatal) comprise >90% of the total MyHC expression, while three adult fast isoforms (IIa, IIb, and IId) comprise <1% of the total MyHC protein. However, between 5 and 20 dpn their expression increases to comprise >90% of the total MyHC. Expression of each of the three adult fast isoforms occurs in a spatially and temporally distinct manner. We also show that alpha MyHC, which is almost exclusively expressed in the heart, is expressed in scattered fibers in all hindlimb muscles during postnatal development. Surprisingly, the timing and localization of expression of the MyHC isoforms is unchanged in IIb and IId/x null mice, although the magnitude of expression is altered for some isoforms. Together these data provide a comprehensive overview of the postnatal expression pattern of the sarcomeric MyHC isoforms in the mouse hindlimb.     

Age-related changes and nutritional regulation of myosin heavy-chain composition in longissimus dorsi of commercial pigs

The objective of this study is to investigate the age-related changes of and the effects of dietary conjugated linoleic acid (CLA) on muscle-fibre types in commercial pigs. We divided 25 crossbred male pigs into five age groups (7, 30, 60, 100 and 180 days) and 30 finishing pigs into two dietary groups (one fed a CLA-enriched diet and the other fed a control diet for 30 days). We analysed the composition (%) of myosin heavy-chain (MyHC) mRNA according to the absolute copies of each MyHC (I, IIa, IIb and IIx) mRNA, and the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) in the longissimus muscle. From days 7 to 180, the MyHC I mRNA abundance and SDH and MDH activities presented a decreasing trend, the MyHC IIb mRNA abundance presented a steady trend and the MyHC IIa and IIx mRNA abundances presented an increasing trend. On day 30, MyHC I and IIb mRNA abundances were at their lowest (P < 0.05), and the MyHC IIa and IIx mRNA abundances were at their highest (P < 0.05). In the CLA group, the MyHC I mRNA abundance and the activities of SDH and MDH were improved in the longissimus muscle, whereas pressure loss, drip loss and average back fat depth significantly decreased (P < 0.01) and shear force significantly increased (P < 0.01). Loin eye area, feed conversion rate and meat colour showed some tendency to be improved. These results indicated that more oxidative fibres might convert to glycolytic fibres with increasing age or weight, and that the early developmental stage might be a key stage for this conversion. During the finishing stage, the proportion of oxidative fibres might be increased by dietary CLA supplementation, which may contribute to the water-holding capacity of meat. The results would provide an important basis for the application of muscle-fibre types in the improvement of pork quality.     

猪MyoG基因的PCR-RFLP多态性分析

以杜洛克、长白、大约克、南昌白、二花脸、梅山猪、玉山黑猪、乐平花猪、金华两头乌及上高两头乌等中外10个猪种共计561头猪为研究材料,采用3对引物（PCR1、PCR2、PCR3）分别扩增猪肌细胞生成素（MyoG）基因的不同区域,扩增产物经限制性核酸内切酶MspⅠ酶切后发现：（1）在PCR1 MspⅠ-RFLP位点上,外来品种杜洛克、长白、大约克及培育品种南昌白中极大多数个体表现为AA型,个别为BB型;而6个中国地方猪种除乐平花猪外均以BB型居多。（2）在PCR2 MspⅠ-RFLP位点上,6个中国地方猪种除一头玉山黑猪表现为MN型外,其余均为MM型;而外来品种以NN型占大多数,培育品种南昌白更趋向于外来品种。（3）在PCR3 MspⅠ-RFLP位点上,所有猪种均可得到扩增产物,但无MspⅠ酶切位点。（4）在梅山猪及与其亲缘关系较近的二花脸猪中,没有发现Soumillion等(1997)报道的梅山猪特异性MspⅠ多态性酶切位点。     

Molecular Cloning and Comparative Characterization of the Porcine Troponin I Family

Troponin I (TnI) is a family of three muscle-specific myofibrillar proteins involved in calcium-sensitive regulation of contraction in cardiac and skeletal muscle. In this study, the full-length cDNA and genomic sequence of three genes of porcine TnI family were cloned and sequenced. The full-length cDNA of TNNI1, TNNI2, and TNNI3 genes were 989 bp, 734 bp, and 831 bp in length, which contained an open reading frame of 564, 549, and 636 nucleotides, respectively. Three Troponin I shared 54.4 ～ 58.3% similarity with each other in their predicted amino acid sequences. The TNNI1, TNNI2, and TNNI3 displayed the same genomic structure as other vertebrates and spanned over 9785 bp, 2373 bp, and 3648 bp genomic regions, respectively. The regulatory elements in the proximal promoter of TNNI2 and TNNI3 were conserved among human, mouse, and pig, but regulatory element differences existed in the TNNI1 promoter among them. Expression profiling showed that TnI genes were widely expressed in the tissues studied, with the highest expression level of TNNI1 and TNNI2 in skeletal muscle, and TNNI3 in cardiac muscle.     

Sexually differentiated,androgen-regulated,larynx-specific myosin heavy-chain isoforms in <Emphasis Type="Italic">Xenopus tropicalis</Emphasis>; comparison to <Emphasis Type="Italic">Xenopus laevis</Emphasis>

We have shown that the sarcoplasmic myosin heavy-chain (MyHC) isoform xtMyHC-101d is highly and specifically expressed in the larynx of the aquatic anuran, Xenopus tropicalis. In male larynges, the predominant MyHC isoform is xtMyHC-101d, while in females, another isoform, xtMyHC-270c, predominates. The X. tropicalis genome has been sequenced in its entirety, and xtMyHC-101d is part of a specific array of xtMyHC genes expressed otherwise in embryonic muscles (Nasipak and Kelley, Dev Genes Evol, in press, 2008). The administration of the androgen dihydrotestosterone increases the expression of xtMyHC-101d in juvenile larynges of both sexes. Using ATPase histochemistry, we found that in adults, X. tropicalis male laryngeal muscle contains only fast-twitch fibers, while the female laryngeal muscle contains a mix of fast- and slow-twitch fibers. Juvenile larynges are female-like in fiber type composition (44% slow twitch, 56% fast twitch); androgen treatment increases the percentage of fast-twitch fibers to 86%. xtMyHC-101d predominates in larynges of dihydrotestosterone-treated juveniles but not in larynges of untreated juveniles. We compared the larynx-specific expression of xtMyHC genes in X. tropicalis to the MyHC gene expressed in X. laevis larynx (xlMyHC-LM) by sequencing the entire xlMyHC-LM gene. The androgen-regulated xtMyHC that predominates in the male larynx of X. tropicalis is not the gene phylogenetically most similar to xlMyHC-LM at the nucleotide level but is instead a similar isoform found in the same MyHC array and expressed in the embryonic muscle.     

Dynamics of myosin heavy chain isoform transition in the longissimus muscle of domestic and wild pigs during growth: a comparative study

Dynamics of myofiber differentiation/maturation in porcine skeletal muscle is associated with domestication, breeding and rearing conditions. This study was aimed to comparatively elucidate the age-dependent myosin heavy chain (MyHC) isoform expression and transition pattern in domestic and wild pig (WP) skeletal muscle from birth until adulthood. Domestic pigs (DPs) of Large White breed raised in conventional production system were compared with WPs reared in a large hunting enclosure. Muscle samples for immuno/enzyme histochemistry were taken from the longissimus dorsi muscle within 24 h postmortem at 24 to 48 h, 21 to 23 days, 7 months and ~2 years postpartum. Based on the antibody reactivity to MyHCs (NCL-MHCs, A4.74, BF-F3) and succinate dehydrogenase activity, myofibers were classified into I, I/IIa, IIa, IIx and IIb types. In addition, foetal MyHC expression was determined with the use of F158.4C10 antibody. Maturation of the longissimus dorsi muscle in the WP was characterized by an accelerated transformation of the fast to slow MyHC during the first hours postpartum, followed by differentiation towards oxidative myofibers in which type I, IIa and IIx MyHCs predominated. In the DP, the transformation shifted towards glycolytic myofibers that expressed MyHC-IIb. The expression of foetal MyHC was higher in the DP than in the WP at 1 day of age, and the decline in the foetal MyHC during the first 3 weeks was more rapid in the WP than in the DP denoting an accelerated early postnatal muscle maturation in WP than DP piglets. All foetal MyHC-positive myofibers co-expressed IIa isoform, but not vice versa. The intense myofiber hypertrophy was evident from 3 weeks until 7 months of age. In this period, the myofiber cross-sectional area increased up to 10- and 20-fold in the WP and the DP, respectively. In the DP, the hypertrophy of all myofiber types was more pronounced than in the WP, particularly the hypertrophy of IIx and IIb myofibers. To summarize, the comparison between growing DP with wild ancestors showed that genetic selection and rearing conditions lead to substantial changes in the direction and intensity of postnatal MyHC transformation as evidenced by different proportion of individual myofiber types and differences in their hypertrophic potential.     

Quantifying the temporospatial expression of postnatal porcine skeletal myosin heavy chain genes.

Postnatal skeletal muscle fiber type is commonly defined by one of four major myosin heavy chain (MyHC) gene isoforms (slow/I, 2a, 2x, and 2b) that are expressed. We report on the novel use of combined TaqMan quantitative real-time RT-PCR and image analysis of serial porcine muscle sections, subjected to in situ hybridization (ISH) and immunocytochemistry (IHC), to quantify the mRNA expression of each MyHC isoform within its corresponding fiber type, termed relative fiber type-restricted expression. This versatile approach will allow quantitative temporospatial comparisons of each MyHC isoform among muscles from the same or different individuals. Using this approach on porcine skeletal muscles, we found that the relative fiber type-restricted expression of each postnatal MyHC gene showed wide spatial and temporal variation within a given muscle and between muscles. Marked differences were also observed among pig breeds. Notably, of the four postnatal MyHC isoforms, the 2a MyHC gene showed the highest relative fiber type-restricted expression in each muscle examined, regardless of age, breed, or muscle type. This suggests that although 2a fibers are a minor fiber type, they may be disproportionately more important as a determinant of overall muscle function than was previously believed.     

Related expression of MyoD and Myf5 with myosin heavy chain isoform types in bovine adult skeletal muscles

Skeletal muscles are characterized as fast and slow muscles, according to the expression pattern of myosin heavy chain (MyHC) isoforms in the muscle fibers. To investigate the relationships between MyHC isoforms and myogenic regulatory factors (MRFs) including MyoD, Myf5, myogenin, and MRF4 in adult skeletal muscles, expressions of these MRFs in the ten muscles of three cows were analyzed by a semi-quantitative RT-PCR. The results showed that MyoD expression was significantly lower in the lingual muscles (TN), masseter (MS) and diaphragm (DP), which lack MyHC-2x (fast glycolytic) expression and abound with MyHC-slow (slow oxidative) and/or MyHC-2a (fast oxidative), than it was in the pectoralis (PP), psoas major (PM), longissimus thoracis (LT), spinnalis (SP), semitendinosus (ST), semimembranosus (SM), and biceps femoris (BF). In contrast, the Myf5 expression in TN, MS, and DP was significantly higher than in PM, LT, ST, SM, and BF. No significant difference was observed in myogenin and MRF4 expression among the muscles tested. The results suggest that MyoD and Myf5 influence the MyHC isoform expression, although the effects are not decisive in specifying the phenotypes of adult muscles.     

猪ATF4基因多态性与生产性状的关联及基因表达分析

为进一步了解和认识ATF4基因的功能,揭示ATF4对猪脂肪代谢的影响,寻找与肉质性状相关联的分子标记,文章采用PCR方法扩增了ATF4基因部分序列,通过序列比对发现在翻译起始密码子ATG下游159 bp处存在A159G转换,通过PCR-AluⅠ-RFLP对大白猪、长白猪、梅山猪和通城猪进行酶切分型,发现在大白猪和长白猪中均为AA基因型,在梅山猪和通城猪中均为GG基因型。进一步对大白猪×梅山F2群体资源家系进行了酶切分型,并分析该位点的多态性与生产性状的关系。结果表明,ATF4的多态性与臀部平均膘厚存在极显著相关(P<0.01),与胸腰椎间膘厚、平均膘厚、眼肌高、眼肌面积存在显著相关(P<0.05)。采用Real-time PCR分析了ATF4基因在大白猪与梅山猪背最长肌不同发育阶段的表达模式。结果表明,ATF4基因在大白猪和梅山猪胚胎期65 d和出生后3 d中的表达水平相对都比较低,且在两品种间无明显差异;而在出生后60 d和120 d,ATF4基因在大白猪中与梅山猪均出现了上调表达,并且在梅山猪中的相对表达水平要显著高于大白猪。研究结果为进一步深入研究猪ATF4基因在脂肪代谢中的分子机理奠定了基础。