52a Reunião Anual da Sociedade Brasileira de
Zootecnia
Zootecnia: Otimizando Recursos e Potencialidades
Belo Horizonte – MG, 19 a 23 de Julho de 2015
Expressão de miogenes durante o desenvolvimento pré-natal em longissimus dorsi de suínos comercial
e local Piau1
Evelyze Pinheiro dos Reis2, Elcer Albenis Zamora Jerez2, Débora Martins Paixão2, Walmir Silva2,
Fabyano Fonseca e Silva2, Otávio José Bernardes Brustolini3, Simone Eliza Facioni Guimarães2
1
2
3
Parte da tese de doutorado do primeiro autor, financiada pelo CNPq. email: [email protected]
Laboratório de Biotecnologia Animal, Universidade Federal de Viçosa, Departamento de Zootecnia, Viçosa, Minas Gerais, Brasil
Universidade Federal de Viçosa, Departamento de Bioquímica Agrícola, Viçosa, Minas Gerais, Brasil.
Resumo: Este estudo foi realizado para analisar a expressão de 13 miogenes por qRT-PCR em quatro idades
pré-natais (21, 40, 70 e 90 dias pós-concepção/dpc) em suínos de linha comercial (Duroc x Landrace x
Large-White) e local Piau, que diferem quanto à musculosidade, para se compreender diferenças na
expressão durante a miogênese. Os dados de expressão foram analisados usando a macro %QPCR_MIXED
no SAS. A expressão relativa foi calculada e os dados foram analisados por agrupamento hierárquico.
Verificou-se que CHD8, EID2B, HIF1AN, IKBKB, RSPO3, SOX7 e SUFU apresentaram expressão
constante nos períodos e raças. Em suínos comercial e Piau, CSRP3, MAP2K1, MRAS, MYOG e RBM24
apresentaram expressão similares. A expressão de CSRP3 foi alta, sendo maior aos 40, 70 e 90 dpc.
MAP2K1 apresentou expressão alta, com maior expressão aos 40 e 70 dpc. MRAS apresentou expressão alta
aos 21 e 40 dpc, e expressão baixa nos outros períodos. MYOG apresentou expressão baixa aos 21 dpc e
expressão alta aos 40, 70 e 90 dpc. RBM24 apresentou baixa expressão aos 21 dpc e alta expressão aos 40,
70 e 90 dpc. Por outro lado, LEF1 apresentou alta expressão aos 21 e 40 dpc em comercial e 21 dpc em Piau,
e baixa expressão nos outros períodos. Assim, os resultados sugerem que LEF1 é candidato primário para
explicar as diferenças de musculosidade entre as raças, já que sua expressão pode favorecer a uma maior
fusão de mioblastos em suínos de linha comercial comparados à Piau.
Palavras–chave: expressão gênica, miogênese, PCR em tempo real, expressão relativa
Expression of myogenes during prenatal development in longissimus dorsi of commercial and local
Piau pigs
Abstract: This study was conducted to analyze the expression of 13 myogenes by qRT-PCR at four prenatal
periods (21, 40, 70 and 90 days post-conception/dpc) in commercial line (Duroc x Landrace x Large-White)
and local Piau pigs, which differ in muscularity, in order to understand differences in gene expression during
myogenesis. Expression data were analyzed using the macro %QPCR_MIXED on SAS. Relative expression
was calculated and data were analyzed by hierarchical clustering. It was possible to verify that CHD8,
EID2B, HIF1AN, IKBKB, RSPO3, SOX7 and SUFU had constant expression along all periods and breeds.
In commercial and Piau pigs, CSRP3, MAP2K1, MRAS, MYOG and RBM24 presented similar expression.
CSRP3 had high expression, with a greater expression at 40, 70 and 90 dpc. MAP2K1 had high expression,
with a greater expression at 40 and 70 dpc. MRAS had higher expression at 21 and 40 dpc, and lower
expression in other periods. MYOG presented low expression at 21 dpc and higher expression at 40, 70 and
90 dpc. RBM 24 presented low expression at 21 dpc and high expression at 40, 70 and 90 dpc. On the other
hand, LEF1 had high expression at 21 and 40 dpc in commercial and 21 dpc in Piau, and low expression in
other periods. Thus, the results suggest that LEF1 gene is primary candidate to explain differences in
muscularity between these breeds, since its expression could lead to a greater myoblast fusion in commercial
line animals compared to Piau pigs.
Keywords: gene expression, myogenesis, real-time PCR, relative expression
Introduction
Myogenesis is a prenatal process characterized by formation of muscle fibers and is influenced by
genetic and environmental factors. The number of muscle fibers formed and their hypertrophy in postnatal
periods influence muscle growth and mass. Myoblasts fuse to form primary fibers in early stages of
myoblast fusion (first wave of differentiation) and form secondary fibers using the primary fibers as template
(second wave of differentiation) (Rehfeldt et al., 2000). In pigs, muscle fiber formation waves have
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52a Reunião Anual da Sociedade Brasileira de
Zootecnia
Zootecnia: Otimizando Recursos e Potencialidades
Belo Horizonte – MG, 19 a 23 de Julho de 2015
relatively long periods of time at approximately 30 - 60 days and 54 - 90 days of gestation (Wigmore and
Stickland, 1983).
Studies investigating gene expression in pigs have been conducted in order to understand expression
changes during muscle development, using longissimus dorsi samples of different breeds of pigs in prenatal
periods. Thus, this study was conducted to analyze expression of myogenes in embryo and longissimus dorsi
muscle at 21, 40, 70 and 90 days of gestation/dpc in Piau and commercial line (Duroc x Landrace x LargeWhite) of pigs by qRT-PCR, in order to understand differences in gene expression during muscle formation.
Thus, this study allow to conclude about myogenesis in commercial and Piau pigs, which differ in
muscularity, having gene expression data during the period of somite formation in whole embryos at 21 dpc,
of primary and secondary fiber development in fetuses at 40 dpc and at 70-90 dpc, respectively.
Material and Methods
Twenty-four pregnant gilts of local Piau and commercial line (Landrace and Large-White type
females inseminated by Duroc type males) were used in this study. Embryos and fetuses were obtained by
cesarean of three pregnant gilts inseminated by unrelated males for each breed at prenatal ages (21, 40, 70
and 90 dpc) (CEUA-UFV 85/2013). Longissimus dorsi (LD) muscle was collected except for 21 dpc
embryos, in which whole individual was collected and used at RNA extraction.
Thirteen genes were selected from a RNAseq experiment to have their expression evaluated by qRTPCR (ABI Prism 7300). The genes were related to myogenesis based on gene ontology (GO), identification
of metabolic pathways and their function. Information on GO were obtained at ToppCluster. Metabolic
pathways maps were obtained on DAVID. Cytoscape software was used to view and edit biological
processes, molecular functions and metabolic pathways. qPCR primers were designed using PrimerQuest®,
using nucleotide sequences obtained from Sus scrofa at GenBank. Only RSPO3 sequence was not available
in pigs, and in this case it was used a homologous sequence from Homo sapiens. GAPDH (glyceraldehyde3-phosphate dehydrogenase) was the most stable of three reference genes and then used in statistical
analysis.
Experimental design was completely randomized in factorial design 2 (breeds) x 4 (prenatal ages)
with 6 repetitions (3 biological and 2 technical replicates) per treatment. qRT-PCR data were analysed using
the %QPCR_MIXED macro (Steibel et al., 2009) in SAS with 5% significance level. Moreover, contrast
estimate values were used for assessment of relative expression. Additionally, ΔCt values (Ct target gene Ct reference gene) for the two breeds and four periods were analyzed by a hierarchical clustering, based on
complete linkage method with Pearson correlation as distance in Hierarchical Clustering Explorer.
Results and Discussion
In order to understand the function of 13 selected genes, we collected information about gene
ontology and metabolic pathways, obtaining relevant functional metabolic pathways for muscle development
(Figure 1).
Figure 1. Functional gene networks and interactions. It describes the relationship between 13 genes ( ),
there are 12 important subnets related to muscle development included in biological process ( ),
molecular function ( ) and metabolic pathway ( ).
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52a Reunião Anual da Sociedade Brasileira de
Zootecnia
Zootecnia: Otimizando Recursos e Potencialidades
Belo Horizonte – MG, 19 a 23 de Julho de 2015
qRT-PCR reactions were performed using 13 genes along four periods. Seven genes (CHD8 chromodomain helicase DNA binding protein 8, EID2B - EP300 interacting inhibitor of differentiation 2B,
HIF1AN - hypoxia inducible factor 1, alpha subunit inhibitor, IKBKB - inhibitor of kappa light polypeptide
gene enhancer in B-cells, kinase beta, RSPO3 - R-spondin 3, SOX7 -SRY (sex determining region Y)-box 7
e SUFU - suppressor of fused homolog) showed constant and similar expression along prenatal ages and
breeds by Student t-test, however CHD8 presented null expression at 21 dpc, high at 40 dpc and low at 70
and 90 dpc. EID2B, IKBKB and SUFU showed low expression in all periods. HIF1AN had high expression
at 21 and 40 dpc, null at 70 dpc and low at 90 dpc. RSPO3 presented high expression at 21, 40 and 70 dpc
and null at 90 dpc. SOX7 had low expression at 21 and 70 dpc, and null at 40 and 90 dpc. In commercial and
Piau pigs, CSRP3 (cysteine and glycine-rich protein 3), MAP2K1 (mitogen-activated protein kinase kinase
1), MRAS (muscle RAS oncogene homolog), MYOG (myogenin - myogenic factor 4) and RBM24 (RNA
binding motif protein 24) presented similar expression in both breeds. CSRP3 had high expression in all
periods, with a greater expression during the two waves of myoblast fusion (40, 70 and 90 dpc). MAP2K1
showed high expression along prenatal ages with a greater expression at 40 (period of primary wave) and 70
dpc (period of secondary wave). MRAS presented null expression at 21 dpc, high at 40 dpc and low at 70
and 90 dpc, however expression at 21 and 40 dpc showed non-significant difference by Student t-test.
MYOG had low expression at 21 dpc, null at 40 dpc and high at 70 and 90 dpc (periods of secondary wave),
however expression at 40, 70 and 90 dpc showed non-significant difference by Student t-test. RBM24
presented low expression. at 21 dpc and high at 40 dpc, 70 and 90 dpc. On the other hand, LEF1 (lymphoid
enhancer-binding factor 1) had high expression at 21 dpc and 40 dpc in commercial pigs, and presented high
expression only at 21 dpc in Piau, and in other periods showed low expression. Thus, the results suggest that
the additional peak of LEF1 expression at 40 dpc in commercial can be related to a greater fusion of
myoblasts, which could be responsible for a greater number of primary fiber number in commercial pigs,
once in this period occurs primary fiber formation.
Conclusions
LEF1 gene is a primary candidate to explain muscularity differences between commercial and Piau
pigs. This study is helpful to understand molecular mechanisms during muscle development in commercial
(Duroc x Landrace x Large-White) and Piau pigs, providing novel expression information of some
myogenes in pigs by qRT-PCR.
Acknowledgements
The authors thank trainees and employees of the Swine Breeding Farm at UFV for help in sampling
biological material. EPR was supported by a scholarship from Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
This work was supported by a grant from CNPq.
References
Rehfeldt C, Fiedler I, Dietl G and Ender K (2000). Myogenesis and postnatal skeletal muscle cell growth as
influenced by selection. Livestock Production Science 66: 177-188.
Steibel JP, Poletto R, Coussens PM and Rosa GJ (2009). A powerful and flexible linear mixed model
framework for the analysis of relative quantification RT-PCR data. Genomics 94: 146-152.
Wigmore PM and Stickland NC (1983). Muscle development in large and small pig fetuses. Journal of
anatomy 137 (Pt 2): 235-245.
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