Yuanzhu Xiong

Microarray-Based Approach Identifies Differentially Expressed MicroRNAs in Porcine Sexually Immature and Mature Testes

Background MicroRNAs (miRNAs) are short non-coding RNA molecules which are proved to be involved in mammalian spermatogenesis. Their expression and function in the porcine germ cells are not fully understood. Methodology We employed a miRNA microarray containing 1260 unique miRNA probes to evaluate the miRNA expression patterns between sexually immature (60-day) and mature (180-day) pig testes. One hundred and twenty nine miRNAs representing 164 reporter miRNAs were expressed differently (p<0.1). Fifty one miRNAs were significantly up-regulated and 78 miRNAs were down-regulated in mature testes. Nine of these differentially expressed miRNAs were validated using quantitative RT-PCR assay. Totally 15919 putative miRNA-target sites were detected by using RNA22 method to align 445 NCBI pig cDNA sequences with these 129 differentially expressed miRNAs, and seven putative target genes involved in spermatogenesis including DAZL, RNF4 gene were simply confirmed by quantitative RT-PCR. Conclusions Overall, the results of this study indicated specific miRNAs expression in porcine testes and suggested that miRNAs had a role in regulating spermatogenesis.

Selection of reference genes for gene expression studies in porcine skeletal muscle using SYBR green qPCR.

Quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA. Internal controls such as reference genes are used to normalize mRNA levels between different samples for an exact comparison of gene transcription level. However, the expression levels of these reference genes may vary between cell types, developmental stages, species and experimental conditions, thus proper normalization strategy is an important precondition for reliable conclusions. In this study, we explored 10 commonly used reference genes in porcine skeletal muscle using SYBR green qPCR. We used both geNorm and NormFinder to analyze the expression stability and found that PPIA, HPRT and eEF-1γ were suitable internal controls for porcine skeletal muscle. However, PPIA, HPRT and SDHA were suitable for skeletal muscle of western pigs while PPIA, eEF-1γ and HPRT for indigenous Chinese pigs. Normalized qPCR data of ROCK2 were compared with microarray data to evaluate our developed set of reference genes.

Identification of genome-wide copy number variations among diverse pig breeds by array CGH

BackgroundRecent studies have shown that copy number variation (CNV) in mammalian genomes contributes to phenotypic diversity, including health and disease status. In domestic pigs, CNV has been catalogued by several reports, but the extent of CNV and the phenotypic effects are far from clear. The goal of this study was to identify CNV regions (CNVRs) in pigs based on array comparative genome hybridization (aCGH).ResultsHere a custom-made tiling oligo-nucleotide array was used with a median probe spacing of 2506 bp for screening 12 pigs including 3 Chinese native pigs (one Chinese Erhualian, one Tongcheng and one Yangxin pig), 5 European pigs (one Large White, one Pietrain, one White Duroc and two Landrace pigs), 2 synthetic pigs (Chinese new line DIV pigs) and 2 crossbred pigs (Landrace × DIV pigs) with a Duroc pig as the reference. Two hundred and fifty-nine CNVRs across chromosomes 1–18 and X were identified, with an average size of 65.07 kb and a median size of 98.74 kb, covering 16.85 Mb or 0.74% of the whole genome. Concerning copy number status, 93 (35.91%) CNVRs were called as gains, 140 (54.05%) were called as losses and the remaining 26 (10.04%) were called as both gains and losses. Of all detected CNVRs, 171 (66.02%) and 34 (13.13%) CNVRs directly overlapped with Sus scrofa duplicated sequences and pig QTLs, respectively. The CNVRs encompassed 372 full length Ensembl transcripts. Two CNVRs identified by aCGH were validated using real-time quantitative PCR (qPCR).ConclusionsUsing 720 K array CGH (aCGH) we described a map of porcine CNVs which facilitated the identification of structural variations for important phenotypes and the assessment of the genetic diversity of pigs.

Differential proteome and transcriptome analysis of porcine skeletal muscle during development.

To gain further insight into the molecular mechanism of porcine skeletal muscle development, we combined MS characterization of proteins with high-throughput screening of differential mRNAs obtained from purebred Meishan longissimus dorsi muscle (LM) at four stages of 65 days post conception, 3, 60 and 120 days after birth. Strikingly, the dramatic differences were observed in embryo and newborn pigs, whereas 60 and 120 days pigs exhibited similar patterns in protein and mRNA expression. At the protein level, 66 differentially expressed proteins were identified. The development-dependent alterations in protein abundance indicated dramatic changes in metabolism, myofibrillar filaments, cytoskeleton, contractile activity and stress response, and signal transduction. At the transcript level, gene expression was measured with the Affymetrix Porcine Genechip, and 338 genes, representing approximately 1.7% of the chromosome, differed by two fold or more between the neighboring growth phases. Analysis of one such comparison, the expression patterns of most differential proteins showed a positive correlation with their gene expression at the transcript level during skeletal muscle development. Overall, many proteins or genes were previously unrecognized as differentially expressed during growth stages, and they represented novel starting points for understanding the developmental characteristics of biochemical and physiological properties in porcine skeletal muscle.

Microarray profiling for differential gene expression in PMSG-hCG stimulated preovulatory ovarian follicles of Chinese Taihu and Large White sows

BackgroundThe Chinese Taihu is one of the most prolific pig breeds in the world, which farrows at least five more piglets per litter than Western pig breeds partly due to a greater ovulation rate. Variation of ovulation rate maybe associated with the differences in the transcriptome of Chinese Taihu and Large White ovaries. In order to understand the molecular basis of the greater ovulation rate of Chinese Taihu sows, expression profiling experiments were conducted to identify differentially expressed genes in ovarian follicles at the preovulatory stage of a PMSG-hCG stimulated estrous cycle from 3 Chinese Taihu and 3 Large White cycling sows by using the Affymetrix Porcine Genechip™.ResultsOne hundred and thirty-three differentially expressed genes were identified between Chinese Taihu and Large White sows by using Affymetrix porcine GeneChip (p ≤ 0.05, Fold change ≥ 2 or ≤ 0.5). Gene Ontology (GO) analysis revealed that these genes belonged to the class of genes that participated in regulation of cellular process, regulation of biological process, biological regulation, developmental process, cell communication and signal transduction and so on. Significant differential expression of 6 genes including WNT10B and DKK2 in the WNT signaling pathway was detected. Real-time RT-PCR confirmed the expression pattern in seven of eight selected genes. A search of chromosomal location revealed that 92 differentially expressed transcripts located to the intervals of quantitative trait loci (QTLs) for reproduction traits. Furthermore, SNPs of two differentially expressed genes- BAX and BMPR1B were showed to be associated with litter size traits in Large White pigs and Chinese DIV line pigs (p ≤ 0.1 or p ≤ 0.05).ConclusionsOur study detected many genes that showed differential expression between ovary follicles of two divergent breeds of pigs. Genes involved with regulation of cellular process, regulation of biological process, in addition to several genes not previously associated with ovarian physiology or with unknown function, were differentially expressed between two breeds. The suggestive or significant associations of BAX and BMPR1B gene with litter size indicated these genetic markers had the potentials to be used in pig industry after further validation of their genetic effects. Taken together, this study reveals many potential avenues of investigation for seeking new insights into ovarian physiology and the genetic control of reproduction.

NNAT and DIRAS3 genes are paternally expressed in pigs

Although expression and epigenetic differences of imprinted genes have been extensively characterised in man and the mouse, little is known on livestock species. In this study, the polymorphism-based approach was used to detect the imprinting status of NNAT and DIRAS3 genes in five heterozygous pigs (based on SNP) of Large White and Meishan F1 hybrids. The results show that both genes were paternally expressed in all the tested tissues (heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, uterus, ovary and pituitary). In addition, the NNAT gene had two transcripts in all tested tissues, which is consistent with its counterpart in man and cattle.

Molecular characterization and expression patterns of AMP deaminase1 (AMPD1) in porcine skeletal muscle.

AMPD1 is the muscle-specific form of the AMPD multigene families in mammals and plays an important role in the purine nucleotide cycle and energy metabolism in skeletal muscle. In this study, we cloned and characterized AMPD1 from Sus scrofa muscle. The promoter of porcine AMPD1 contained several putative muscle-specific transcription factor binding sites (E box, myogenin, MEF2, Spl-CTF/NF-l), one RORalpha2 binding motif and NF-kappaB site. The deduced amino acid sequence of porcine AMPD1 contains an AMP deaminase signature sequence (SLSTDDP). RT-PCR analyses showed that AMPD1 was expressed specifically in skeletal muscle. Expression of AMPD1 was up-regulated during the muscle development and was higher in Yorkshire than in Meishan pigs. AMPD1 gene was expressed at higher levels in longissimus dorsi and bicepsfemoris muscles compared with soleus and masseter muscles in both Yorkshire and Meishan pigs. Moreover, we found that a single nucleotide polymorphism (SNP, T/C(426)) in exon12 of the AMPD1 gene was significantly associated with loin muscle area trait (p<0.01), loin muscle height (p<0.01) and average backfat thickness (p<0.05). This result suggests that the AMPD1 gene might be a candidate gene of meat production trait and provides useful information for further studies on its roles in porcine skeletal muscle.

Discovery of Potential piRNAs from Next Generation Sequences of the Sexually Mature Porcine Testes

Piwi- interacting RNAs (piRNAs), a new class of small RNAs discovered from mammalian testes, are involved in transcriptional silencing of retrotransposons and other genetic elements in germ line cells. In order to identify a full transcriptome set of piRNAs expressed in the sexually mature porcine testes, small RNA fractions were extracted and were subjected to a Solexa deep sequencing. We cloned 6,913,561 clean reads of Sus Scrofa small RNAs (18–30 nt) and performed functional characterization. Sus Scrofa small RNAs showed a bimodal length distribution with two peaks at 21 nt and 29 nt. Then from 938,328 deep-sequenced small RNAs (26–30 nt), 375,195 piRNAs were identified by a k-mer scheme and 326 piRNAs were identified by homology searches. All piRNAs predicted by the k-mer scheme were then mapped to swine genome by Short Oligonucleotide Analysis Package (SOAP), and 81.61% of all uniquely mapping piRNAs (197,673) were located to 1124 defined genomic regions (5.85 Mb). Within these regions, 536 and 501 piRNA clusters generally distributed across only minus or plus genomic strand, 48 piRNA clusters distributed on two strands but in a divergent manner, and 39 piRNA clusters distributed on two strands in an overlapping manner. Furthermore, expression pattern of 7 piRNAs identified by homology searches showed 5 piRNAs displayed a ubiquitous expression pattern, although 2 piRNAs were specifically expressed in the testes. Overall, our results provide new information of porcine piRNAs and their specific expression pattern in porcine testes suggests that piRNAs have a role in regulating spermatogenesis.

Detection of quantitative trait loci associated with several internal organ traits and teat number trait in a pig population.

Quantitative trait loci (QTL) were detected for 8 internal organ traits, 3 carcass length traits, and teat number trait in 214 pigs in a resource population that included 180 F(2) individuals. A total of 39 microsatellite markers were examined on SSC4, SSC6, SSC7, SSC8, and SSC13. The genetic traits included heart weight (HW), lung weight (LW), liver and gallbladder weight (LGW), spleen weight (SPW), stomach weight (STW), small intestine weight (SIW), large intestine weight (LIW), kidney weight (KW), carcass length to the first cervical vertebra (CL1), carcass length to the first thoracic vertebra (CL2), rib numbers (RNS), and teat numbers (TNS). Results indicated that, 3 highly significant QTL (P <or= 0.01 at chromosome-wise level) for HW (at 30 cM on SSC6), RNS (at 115 cM on SSC7), TNS (at 110 cM on SSC7), and 6 significant QTL (P <or= 0.05 at chromosome-wise level) for LW (at 119 cM on SSC13), LGW (at 94 cM on SSC6), SPW (at 106 cM on SSC8), SIW (0 cM on SSC4), LIW (170 cM on SSC 4), and TNS (at 95 cM on SSC6) were detected. The phenotypic variances for which these QTL were accounted ranged from 0.04 % to 14.06 %. Most of these QTL had not been previously reported.

The formation of brown adipose tissue induced by transgenic over-expression of PPARγ2.

Brown adipose tissue (BAT) is specialized to dissipate energy as heat, therefore reducing fat deposition and counteracting obesity. Brown adipocytes arise from myoblastic progenitors during embryonic development by the action of transcription regulator PRDM16 binding to PPARγ, which promotes BAT-like phenotype in white adipose tissue. To investigate the capability of converting white adipose tissue to BAT or browning by PPARγ in vivo, we generated transgenic mice with over-expressed PPARγ2. The transgenic mice showed strong brown fat features in subcutaneous fat in morphology and histology. To provide molecular evidences on browning characteristics of the adipose tissue, we employed quantitative real-time PCR to determine BAT-specific gene expressions. The transgenic mice had remarkably elevated mRNA level of UCP1, Elovl3, PGC1α and Cebpα in subcutaneous fat. Compared with wild-type mice, UCP1 protein levels were increased significantly in transgenic mice. ATP concentration was slightly decreased in the subcutaneous fat of transgenic mice. Western blotting analysis also confirmed that phosphorylated AMPK and ACC proteins were significantly (P<0.01) increased in the transgenic mice. Therefore, this study demonstrated that over-expression of PPARγ2 in skeletal muscle can promote conversion of subcutaneous fat to brown fat formation, which can have beneficial effects on increasing energy metabolisms and combating obesity.