Runjun Yang

A comparative profile of the microRNA transcriptome in immature and mature porcine testes using Solexa deep sequencing.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that play key roles in many diverse biological processes such as spermatogenesis. However, no study has been performed on the miRNA transcriptome of developing porcine testes. Here, we employed Solexa deep sequencing technology to extend the repertoire of porcine testis miRNAs and extensively compare the expression patterns of sexually immature and mature porcine testes. Solexa sequencing of two small RNA libraries derived from immature (30 days) and mature (180 days) pig testis samples yielded over 25 million high‐quality reads. Overall, the two developmental stages had significantly different small RNA compositions. A custom data analysis pipeline identified 398 known and/or homologous conserved porcine miRNAs, 15 novel pig‐specific miRNAs and 56 novel candidate miRNAs. We further observed multiple mature miRNA variants and identified a new bidirectional transcribed miRNA locus, ssc‐mir‐181a. A total of 122 miRNAs were differentially expressed in the immature and mature testes, and 10 were validated using quantitative RT‐PCR. Furthermore, GO and KEGG pathway analyses of the predicted miRNA targets further illustrate the likely roles for these differentially expressed miRNAs in spermatogenesis. This study is the first comparative profile of the miRNA transcriptome in immature and mature porcine testes using a deep sequencing approach, and it provides a useful resource for future studies on the role of miRNAs in spermatogenesis and male infertility treatment.

Analysis of DNA Methylation in Various Swine Tissues

DNA methylation is known to play an important role in regulating gene expression during biological development and tissue differentiation in eukaryotes. In this study, we used the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) method to assess the extent and pattern of cytosine methylation in muscle, heart, liver, spleen, lung, kidney and stomach from the swine strain Laiwu, and we also examined specific methylation patterns in the seven tissues. In total, 96,371 fragments, each representing a recognition site cleaved by either or both EcoRI + HpaII and EcoRI + MspI, the HpaII and MspI are isoschizomeric enzymes, were amplified using 16 pairs of selective primers. A total of 50,094 sites were found to be methylated at cytosines in seven tissues. The incidence of DNA methylation was approximately 53.99% in muscle, 51.24% in the heart, 50.18% in the liver, 53.31% in the spleen, 51.97% in the lung, 51.15% in the kidney and 53.39% in the stomach, as revealed by the incidence of differential digestion. Additionally, differences in DNA methylation levels imply that such variations may be related to specific gene expression during tissue differentiation, growth and development. Three types of bands were generated in the F-MSAP profile, the total numbers of these three types of bands in the seven tissues were 46,277, 24,801 and 25,293, respectively. In addition, different methylation patterns were observed in seven tissues from pig, and almost all of the methylation patterns detected by F-MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrated that the F-MSAP technique can be adapted for use in large-scale DNA methylation detection in the pig genome.

Association of the leptin gene E2-169T>C and E3-299T>A mutations with carcass and meat quality traits of the Chinese Simmental-cross steers.

Leptin is a hormone affecting the regulation of body composition, energy balance, and meat quality in mammals. The objective of this study was to evaluate the association of novel single nucleotide polymorphisms in coding region for leptin gene with carcass and meat quality traits of Chinese Simmental-cross steers. Two SNPs (E2-169 T>C and E3-299 T>A) were genotyped on 135 crossbred bulls. The 45 traits being measured included dressing percentage, dressed weight, marbling score, muscle color score, backfat thickness, fatty acid content, etc. Statistical analysis revealed that two SNPs in the exon of leptin gene were associated with the carcass and meat quality traits. The C-bearing genotypes (CC or TC) of E2-169 T>C (C57R) showed higher dressed weight, thickness of loin, MCS, FCS, intramuscular fat content, and polyunsaturated fatty acid content (P<0.05). E3-299 >A(S100T) also showed a significant association with the carcass traits (dressing percentage, living QIB) and fatty acid content in Simmental-cross steers(P<0.05). Our findings suggested that polymorphisms in leptin might be one of the important genetic factors that influence carcass yield and meat quality in beef cattle, and it may be a useful marker for meat quality traits in future marker-assisted selection programs in beef cattle breeding and production.

Deep Sequencing and Screening of Differentially Expressed MicroRNAs Related to Milk Fat Metabolism in Bovine Primary Mammary Epithelial Cells

Milk fat is a key factor affecting milk quality and is also a major trait targeted in dairy cow breeding. To determine how the synthesis and the metabolism of lipids in bovine milk is regulated at the miRNA level, primary mammary epithelial cells (pMEC) derived from two Chinese Holstein dairy cows that produced extreme differences in milk fat percentage were cultured by the method of tissue nubbles culture. Small RNA libraries were constructed from each of the two pMEC groups, and Solexa sequencing and bioinformatics analysis were then used to determine the abundance of miRNAs and their differential expression pattern between pMECs. Target genes and functional prediction of differentially expressed miRNAs by Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes analysis illustrated their roles in milk fat metabolism. Results show that a total of 292 known miRNAs and 116 novel miRNAs were detected in both pMECs. Identification of known and novel miRNA candidates demonstrated the feasibility and sensitivity of sequencing at the cellular level. Additionally, 97 miRNAs were significantly differentially expressed between the pMECs. Finally, three miRNAs including bta-miR-33a, bta-miR-152 and bta-miR-224 whose predicted target genes were annotated to the pathway of lipid metabolism were screened and verified by real-time qPCR and Western-blotting experiments. This study is the first comparative profiling of the miRNA transcriptome in pMECs that produce different milk fat content.

Technical note: Selection of suitable reference genes for studying gene expression in milk somatic cell of yak (Bos grunniens) during the lactation cycle

Quantitative real-time PCR is the most sensitive technique for gene expression analysis. Data normalization is essential to correct for potential errors incurred in all steps from RNA isolation to PCR amplification. The commonly accepted approach for normalization is the use of reference gene. Until now, no suitable reference genes have been available for data normalization of gene expression in milk somatic cells of lactating yaks across lactation. In the present study, we evaluated the transcriptional stability of 10 candidate reference genes in milk somatic cells of lactating yak, including ACTB, B2M, GAPDH, GTP, MRPL39, PPP1R11, RPS9, RPS15, UXT, and RN18S1. Four genes, RPS9, PPP1R11, UXT, and MRPL39, were identified as being the most stable genes in milk somatic cells of lactating yak. Using the combination of RPS9, PPP1R11, UXT, and MRPL39 as reference genes, we further assessed the relative expression of 4 genes of interest in milk somatic cells of yak across lactation, including ELF5, ABCG2, SREBF2, and DGAT1. Compared with expression in colostrum, the overall transcription levels of ELF5, ABCG2, and SREBF2 in milk were found to be significantly upregulated in early, peak, and late lactation, and significantly downregulated thereafter, before the dry period. A similar pattern was observed in the relative expression of DGAT1, but no significant difference was revealed in its expression in milk from late lactation compared with colostrum. Based on these results, we suggest that the geometric mean of RPS9, PPP1R11, UXT, and MRPL39 can be used for normalization of real-time PCR data in milk somatic cells of lactating yak, if similar experiments are performed.

Differential Expression of PPARγ, FASN, and ACADM Genes in Various Adipose Tissues and Longissimus dorsi Muscle from Yanbian Yellow Cattle and Yan Yellow Cattle

The objective of this study was to investigate the correlation between cattle breeds and deposit of adipose tissues in different positions and the gene expressions of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FASN), and Acyl-CoA dehydrogenase (ACADM), which are associated with lipid metabolism and are valuable for understanding the physiology in fat depot and meat quality. Yanbian yellow cattle and Yan yellow cattle reared under the same conditions display different fat proportions in the carcass. To understand this difference, the expression of PPARγ, FASN, and ACADM in different adipose tissues and longissimus dorsi muscle (LD) in these two breeds were analyzed using the Real-time quantitative polymerase chain reaction method (qRT-PCR). The result showed that PPARγ gene expression was significantly higher in adipose tissue than in LD in both breeds. PPARγ expression was also higher in abdominal fat, in perirenal fat than in the subcutaneous fat (p<0.05) in Yanbian yellow cattle, and was significantly higher in subcutaneous fat in Yan yellow cattle than that in Yanbian yellow cattle. On the other hand, FASN mRNA expression levels in subcutaneous fat and abdominal fat in Yan yellow cattle were significantly higher than that in Yanbian yellow cattle. Interestingly, ACADM gene shows greater fold changes in LD than in adipose tissues in Yan yellow cattle. Furthermore, the expressions of these three genes in lung, colon, kidney, liver and heart of Yanbian yellow cattle and Yan yellow cattle were also investigated. The results showed that the highest expression levels of PPARγ and FASN genes were detected in the lung in both breeds. The expression of ACADM gene in kidney and liver were higher than that in other organs in Yanbian yellow cattle, the comparison was not statistically significant in Yan yellow cattle.

Technical note: Identification of suitable normalizers for microRNA expression analysis in milk somatic cells of the yak (Bos grunniens)

MicroRNA are approximately 18- to 22-nucleotide nonprotein coding molecules that play important roles in the regulation of gene expression at the posttranscriptional level. In the present study, we assessed the suitability of 8 noncoding small RNA as normalizers for microRNA (miR) quantitative analysis in milk somatic cells of lactating yaks, including 3 small nuclear RNA (snRNA; RNU1A, RNU5A, and RNU6B), 3 small nucleolar RNA (snoRNA; SNORA73A, Z30, and SNORA74A), 1 rRNA (5S), and 1 transfer RNA (Met-tRNA). The snRNA RNU1A, RNU5A, and SNORA73A were identified as the most stable references in milk somatic cells of lactating yaks. Also, a minimum of 3 reference RNA (RNU1A, RNU5A, and SNORA73A) were required for the normalization of microRNA expression data in milk somatic cells of the lactating yak. We further evaluated the suitability of the combination of RNU1A, RNU5A, and SNORA73A as reference RNA in milk somatic cells of lactating yaks via detecting the relative expression of miR 16b, miR 21-5p, miR 145, and miR 155 as microRNA of putative interest. In comparison to the colostrum period, on the whole, the expressions of the 4 microRNA were found to be upregulated at an early period and, thereafter, a declining pattern was exhibited from early to final periods in all microRNA investigated. Based on the results from this study, we recommend that the combination of RNU1A, RNU5A, and SNORA73A can be used as normalizers for microRNA quantitative analysis in future longitudinal studies on milk somatic cells of lactating yaks in relation to lactation.

Bovine lipid metabolism related gene GPAM: Molecular characterization, function identification, and association analysis with fat deposition traits

Glycerol-3-phosphate acyltransferase mitochondrial (GPAM) is the enzyme that catalyzes the initial and committed step of glycerolipid synthesis in animal lipometabolism related pathway. In the present study, the correlation of GPAM expression level with adipogenesis was examined in vitro by RNAi and gene expression to silencing and over-expression of GPAM gene in BEF cells. And then, 2 novel polymorphisms were identified within key functional domain of bovine GPAM gene by sequencing and the relationship between variants of GPAM gene with fat deposition traits of Chinese Simmental-cross steers was analyzed using statistical methods. The result showed that the knockdown of GPAM expression significantly reduced the synthesis of triglycerides and expression of lipid metabolism-related gene in BEF cells (p<0.05), and the over-expression of GPAM could significantly increased the levels of triglyceride and expression of lipid metabolism-related gene (p<0.05). The results of the correlation analysis also showed that cattle with TC genotype of GPAM E20-2823C>T was significantly associated with fatty acid composition of intramuscular fat (had higher content of arachidic acid, eicosenoic acid, and arachidonic acid), individuals with A-allele homozygotes and AG-allele heterozygotes of E20-3386G>A both had higher content of myristic acid, α-linolenic acid, and linoleic acid in intramuscular fat than those with G-allele homozygotes (p<0.05). The individuals with AA genotype of E20-3386G>A was significantly associated with higher marbling score, and the individuals with CC homozygotes of E20-2823C>T had higher GFW and omasum fat weight in beef cattle population. GPAM play a pivotal role in the regulation of cellular triacylglycerol and phospholipid levels, and its mutations could serve as a useful molecular marker used for marker assisted selection in beef cattle breeding process.

Fas Expression and Mediated Activation of an Apoptosis Programme in Bovine Follicular Granulosa Cells In Vitro

The Fas antigen is a transmembrane receptor that can trigger apoptosis in a variety of somatic cells. Ovarian follicular atresia and luteolysis are thought to occur by apoptosis. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, Fas gene without the stop codon was amplified in the present study using RT-PCR and directly cloned into the eukaryotic expression vector pAcGFP-N1. The resultant recombinant plasmid pAcGFP-bFas was then transfected into bovine follicular granulosa cells. Expression of AcGFP was observed under fluorescent microscopy, and the transcription and translation of Fas were detected by RT-PCR and western blot analysis. The methyl-tetrazolium (MTT) assay, Hoechst33342 staining and DNA ladder method were performed to determine the growth inhibition and apoptosis of the cells. The results showed that GFP expression was detected as early as 24 h after transfection. The Fas fusion gene was successfully expressed in granulosa cells as evidenced by the detection of a 994-bp fragment corresponding to the Fas mRNA by RT-PCR and a 64.5-kD band corresponding to the Fas fusion protein by western blot. Granulosa cell viability decreased significantly at 72 h after transfection, and the apoptosis rate of the cells transfected with pAcGFP-Fas was significantly higher than that of the control group. Cells in the Fas transfection group showed ladder patterns characteristic of apoptosis, and the nuclei were shrunken and densely hyperchromatic or fragmented, suggesting that Fas is capable of inhibiting the proliferation of bovine follicular granulosa cells and inducing cell apoptosis when over-expressed. This study will aid in further understanding the mechanism of regulation of Fas on bovine oocyte formation and development.

Fas ligand expression and mediated activation of an apoptosis program in bovine follicular granulosa cells.

Fas ligand (FasL) is a cytokine that may be expressed as a transmembrane ligand at the cell surface, and induces apoptosis by binding to the Fas. Ovarian follicular atresia and luteolysis are thought to occur by apoptosis. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, FasL gene without the stop codon was amplified and directly cloned into pAcGFP-N1. The resultant recombinant plasmid pAcGFP-bFasL was then transfected into bovine follicular granulosa cells. The transcription and translation of FasL were detected by RT-PCR and Western blot analysis. The methyl-tetrazolium (MTT) assay, Hoechst33342 staining, and DNA Ladder method were performed to determine the growth inhibition and apoptosis of the cells. The real-time quantitative PCR assay was performed to measure the expression of FasL in vivo in granulosa cells collected from diverse stage of dominant and atretic follicles. The results showed that the FasL fusion gene was successfully expressed in granulosa cells as evidenced by the detection of a 847 bp fragment corresponding to the FasL mRNA by RT-PCR and a 59 kDa band corresponding to the FasL fusion protein by Western blot. Granulosa cell viability decreased significantly at 72 h after transfection, and the apoptosis rate of the cells transfected with pAcGFP-FasL was significantly higher than that of the control group. Cells in the FasL transfection group showed ladder patterns characteristic of apoptosis, and the nuclei were shrunken and densely hyperchromatic or fragmented. In addition, FasL was highly expressed in granulosa cells of atretic follicle than dominant follicle in vivo. We found that FasL is capable of inhibiting the proliferation of bovine follicular granulosa cells and inducing cell apoptosis in vitro and in vivo when over-expressed. This study will aid in further understanding the mechanism of regulation of FasL on bovine oocyte formation and development.