Search

Principal component regression (PC regression) is a useful method developed for prediction based on a dimension-reducing strategy. Generally, the principal components (PCs) are added to the regression model one by one based on the eigenvalue (PC-Eigen). Considering that some PCs with large eigenvalues may be poorly associated with the response variable, the PC-Eigen may not be the best framework. Researchers previously tried to add PCs to the model based on their contribution to the sum of squares of the regression (PC-SS) and they found that the performance of PC-SS is generally lower than that of the PC-Eigen. A standard approach for selecting the optimal set of PCs remains a challenge. Here, from the cosine similarity theory, we postulated that we could rank the PCs by dot product, and this framework (we called PC-DOT) could help to preferentially extract PCs that are highly correlated with the response variable and meanwhile have a large eigenvalue. Based on one simulated and three real genomic datasets (a total of 15 traits), we tested the prediction ability of different frameworks. In general, the PC-DOT method showed a better performance than both PC-Eigen and PC-SS. To facilitate the application of PC, we attached a series of R codes for different frameworks (https://github.com/SUNHAO-JLU/Genome_Prediction-PC_DOT). In addition, the HAT matrix was used to reduce the compute complex in reference data during the cross-validation process. Our work may help researchers to better understand and carry out the PC regression model.

Glycine is a well-known free radical scavenger in the cellular antioxidant system that prevents oxidative damage and apoptosis. Excessive fluoride exposure is associated with multiple types of cellular damage in humans and animals. The objective of the present study was to investigate the protective effects of glycine on sodium fluoride (NaF) exposure and the possible underlying mechanisms in a porcine testicular Sertoli cell line model. Cellular viability and proliferation were examined following NaF exposure and glycine supplementation, and glycine dramatically ameliorated the decreases in NaF-induced porcine testicular Sertoli cell viability and proliferation. Further investigations revealed that glycine decreased NaF-induced intracellular reactive oxygen species production, DNA fragment accumulation and the apoptosis incidence in the porcine testicular Sertoli cell line; in addition, glycine improved mitochondrial function and ATP production. Notably, results of the SPiDER-β-Gal analysis suggested that glycine alleviated NaF-induced cellular senescence and downregulated P53, P21, HMGA2 and P16INK4a gene expression in the porcine testicular Sertoli cell line. Collectively, the beneficial effects of glycine alleviate NaF-induced oxidative stress, apoptosis and senescence, and together with our previous findings, support the hypothesis that glycine plays an important role in protecting against NaF exposure-induced impairments in the porcine testicular Sertoli cell line.

As a pentacyclic triterpene in Centella asiatica, asiatic acid (AA) is a powerful antioxidant with many bioactivities. In the present research, we investigated whether AA has the potential to rescue the decrease in porcine oocyte quality that occurs during in vitro aging (IVA). Mature porcine oocytes were collected and then continuously cultured for an additional 24 h or 48 h with or without AA in maturation medium as an IVA model. The results revealed that AA supplementation reduced the percentage of abnormal aged porcine oocytes during IVA. Furthermore, AA supplementation effectively maintained aged porcine oocyte developmental competence, both parthenogenetic activation and in vitro fertilization. The number of sperm that bound to the zona pellucida on aged porcine oocytes was higher in the AA-supplemented group than in the non-supplemented group. Moreover, AA supplementation not only blocked IVA-induced oxidative stress but also maintained intracellular GSH levels and reduced the percentage of early apoptosis aged porcine oocytes. Mitochondrial functions were disordered during the IVA process. The intracellular ATP levels and mitochondrial membrane potential in aged porcine oocytes were dramatically increased by AA supplementation. Therefore, AA has beneficial effects on porcine oocyte quality and developmental potential maintenance during IVA.

Asiatic acid is a pentacyclic triterpene enriched in the medicinal herb Centella asiatica, and it has been suggested to possess free radical scavenging and anti-apoptotic properties. The purpose of the current study was to explore the effects of asiatic acid on porcine early-stage embryonic development and the potential mechanisms for any observed effects. The results showed that 10 μM asiatic acid supplementation during the in vitro culture period dramatically improved developmental competence in porcine embryos derived from parthenogenetic activation (PA), somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). Further analysis revealed that asiatic acid attenuated H2O2-induced intracellular reactive oxygen species (ROS) generation. Notably, asiatic acid not only enhanced intracellular GSH levels but also attenuated mitochondrial dysfunction. Gene expression analysis revealed that asiatic acid upregulated expression of the antioxidant-related gene Sod-1 and the blastocyst formation related gene Cox-2, while downregulating expression of the apoptosis-related gene Caspase-9 in SCNT blastocysts. These results suggest that asiatic acid exerts beneficial effects on early embryonic development in porcine embryos and that asiatic acid may be useful for improving the in vitro production of porcine embryos.

4,4′-(9-Fluorenylidene) diphenol (BPFL, also known as BHPF and fluorene-9-bisphenol) is a novel bisphenol A substitute that is used in the plastics industry as an organic synthesis intermediate and is a potential endocrine disruptor. However, the deleterious effects of BPFL on porcine Sertoli cells (SCs) and the possible underlying mechanisms are still unclear. Chlorogenic acid (CA) is a free radical scavenger in the cellular antioxidant system that prevents oxidative damage and apoptosis. In the present research, we found that BPFL induced impairments in porcine SCs in a dose-dependent manner and that CA protected porcine SCs against BPFL exposure-induced impairments. Cell viability, proliferation and apoptosis assay results revealed that BPFL exposure could inhibit porcine SC proliferation and induce apoptosis, while CA supplementation ameliorated the effects of BPFL. Further analysis revealed that BPFL exposure induced oxidative stress, mitochondrial membrane potential dysfunction and DNA damage accumulation. Transcriptome analysis and further real-time quantitative PCR and Western blot results showed that BPFL exposure induced endoplasmic reticulum stress and apoptosis. Supplementation with CA dramatically ameliorated these phenotypes in BPFL-exposed porcine SCs. Overall, the present research reveals the possible underlying mechanisms by which BPFL exposure induced impairments and CA supplementation protected against these impairments in porcine SCs.

Accumulating evidence indicates that ferroptosis is an iron-dependent form of regulated cell death. This type of iron-dependent programmed cell death is different from traditional forms of regulated cell death, such as apoptosis and autophagy. However, the role of ferroptosis in porcine oocyte maturation and the associated mechanism remain unclear. In the present research, we investigated the effects of ferric ammonium citrate (FAC), a specific ferroptosis inducer, on porcine oocyte meiotic maturation and quality and subsequent embryonic developmental competence. FAC treatment caused obvious accumulation of intracellular ferrous ions in porcine oocytes. At the end of the in vitro maturation (IVM) period, there was a significant decrease in the polar body (PB) extrusion rate and an increase in the percentage of abnormal oocytes in the FAC treatment groups, indicating that iron overload-induced ferroptosis may suppress the meiotic process during porcine oocyte maturation. We also found that after FAC treatment, the subsequent two-cell rate, four-cell rate and blastocyst formation rate were significantly decreased in porcine parthenogenetic activation (PA) embryos, indicating that iron overload-induced ferroptosis decreased porcine oocyte quality. Further analysis revealed that FAC treatment not only enhanced intracellular reactive oxygen species (ROS) generation, decreased intracellular free thiol levels and induced mitochondrial dysfunction but also triggered autophagy in porcine oocytes. Taken together, these findings suggest that iron overload-induced ferroptosis impairs porcine oocyte meiosis and decreases porcine oocyte quality, possibly by increasing oxidative stress, inducing mitochondrial dysfunction and triggering autophagy.

HIGD1A can reduce ROS and apoptosis in cells under low-glucose or hypoxic conditions, and HIGD1A is one of the target genes for miR-375, according to our previous studies. However, it is not known whether miR-375 can indirectly regulate ROS and apoptosis in porcine Sertoli cells. To answer this question, HIGD1A and miR-375 were overexpressed in porcine Sertoli cells, and ROS and apoptosis were assayed. The results showed that ROS levels and expression levels of CASPASE3 in HIGD1A-overexpressing cells were significantly lower than those in the control cells. However, ROS levels and CASPASE3 expression in miR-375-overexpressing cells were significantly higher than those in the control cells. The rate of apoptosis in HIGD1A-overexpressing cells was significantly lower than that in miR-375-overexpressing cells. Considering that the HIGD1A gene is a target of miR-375, these findings suggest that miR-375 can induce an increase in ROS levels and apoptosis by inhibiting HIGD1A in porcine ST cells.

Asiatic acid is a natural triterpene found in Centella asiatica that acts as an effective free radical scavenger. Our previous research showed that asiatic acid delayed porcine oocyte ageing in vitro and improved preimplantation embryo development competence in vitro; however, the protective effects of asiatic acid against oxidative stress in porcine oocyte maturation are still unclear. Here, we investigated the effects of asiatic acid on porcine oocyte in vitro maturation (IVM) and subsequent embryonic development competence after parthenogenetic activation (PA) and in vitro fertilization (IVF). The results of the present research showed that 10 μM asiatic acid supplementation did not affect the expansion of cumulus cells or polar body extrusion of porcine oocytes, while asiatic acid application significantly increased the subsequent blastocyst formation rate and quality of porcine PA and IVF embryos. Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) that induces oxidative stress in porcine oocytes. As expected, asiatic acid supplementation not only decreased intracellular ROS levels but also attenuated H2O2-induced intracellular ROS generation. Further analysis revealed that asiatic acid supplementation enhanced intracellular glutathione production, mitochondrial membrane potential, and ATP generation at the end of IVM. In summary, our results reveal that asiatic acid supplementation exerts beneficial effects on porcine oocytes by regulating oxidative stress during the IVM process and could act as a potential antioxidant in porcine oocytes matured in vitro production systems.

Cypermethrin (CYP) is a widely used insecticide that may be harmful to nontarget species. However, the toxicity of CYP to porcine Sertoli cells (SCs) and its associated mechanism is not known. We investigated the toxicity of CYP and showed that CYP induced cytotoxicity in porcine SCs in a dose-dependent manner. Mechanistic investigations revealed that CYP induced oxidative stress and DNA damage in porcine SCs, which provoked mitochondria-associated apoptosis. CYP also stimulated the phosphorylation of c-Jun N-terminal kinase (JNK) to induce porcine SC apoptosis and inhibited cell proliferation via the inhibition of nuclear factor kappa B (NFκB) expression. The natural antioxidant melatonin had an obvious protective effect against CYP-induced porcine SC toxicity. Overall, our results reveal that the mechanism underlying CYP-induced toxicity in porcine SCs involves oxidative stress, DNA damage, and apoptosis and suggest that melatonin may be used as a highly effective protective agent against oxidative stress.

Objective This study aimed to screen and identify the target genes of miR-375 in pig Sertoli (ST) cells and to elucidate the effect of miR-375 on the proliferation of ST cells. Methods In this study, bioinformatics software was used to predict and verify miR-375 target genes. Quantitative polymerase chain reaction (PCR) was used to detect the relationship between miR-375 and its target genes in ST cells. Enzyme-linked immunosorbent assay (ELISA) of rearranged L-myc fusion (RLF) and hypoxia-induced gene domain protein 1A (HIGD1A) was performed on porcine ST cells, which were transfected with a miR-375 mimics and inhibitor to verify the results. Dual luciferase reporter gene assays were performed to assess the interactions among miR-375, RLF, and HIGD1A. The effect of miR-375 on the proliferation of ST cells was analyzed by CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS). Results Five possible target genes of miR-375, including RLF, HIGD1A, colorectal cancer associated 2, POU class 3 homeobox 1, and WW domain binding protein 1 like, were found. The results of quantitative PCR suggested that mRNA expression of RLF and HIGD1A had a negative correlation with miR-375, indicating that RLF and HIGD1A are likely the target genes of miR-375. The ELISA results revealed that RLF and HIGD1A were negatively correlated with the miR-375 protein level. The luminescence results for the miR-375 group co-transfected with wild-type RLF and HIGD1A vector were significantly lower than those of the miR-375 group co-transfected with the blank vector or mutant RLF and HIGD1A vectors. The present findings suggest that RLF and HIGD1A are target genes of miR-375 and that miR-375 inhibits ST cell proliferation according to MTS analysis. Conclusion It was speculated that miR-375 affects cell proliferation through its target genes, which play an important role in the development of testicular tissue.

Residual feed intake (RFI) is a measure of feed efficiency. Pigs with low RFI have reduced feed costs without compromising their growth. For marker-assisted selection, it is helpful to identify genes or genetic markers associated with RFI in animals with improved feed efficiency at an early age. Using Illumina's PorcineSNP60 BeadChip, we performed a pilot genome-wide association study of 217 Junmu No. 1 white male pigs phenotyped for RFI. Two-step and one-step methods were used separately to identify associated SNPs. Both methods obtained similar results. Twelve SNPs were identified as significantly associated with RFI at a Bonferroni adjusted P-level

The RNA binding protein quaking (QKI), a key member of the STAR family, as an upstream gene could involve in much process including cell proliferation, apoptosis, differentiation and so on. However, the roles of QKI in germ cell, especially in swine testis (ST) cells, was not clear currently. And apoptosis plays important roles in the growth and development. The purpose of the present study was to clarify the relationship between QKI and apoptosis in ST cells. Firstly, our results showed that pEF1α-QKI and shQKI3 have clear effects on expression levels of QKI. Secondly, we established that QKI directly binds to WT1 3′UTR by binding with QRE-1 (2046–2052 bp, ACTAAC) only. Furthermore, QKI overexpression significantly increased the expression levels of WT1 and Bcl-2. QKI also has the effect on delaying the degradation of WT1 mRNA. In addition, we verified that QKI had a significantly suppressed apoptosis in ST cells. Finally, pBI-WT1 could make up for shQKI3-induced decrease in WT1, Bcl-2 mRNA levels and suppress apoptosis in ST cells. The results demonstrated that QKI was an important regulatory factor that affects apoptosis by targeting WT1 gene.

microRNAs are involved in various biological processes by regulating the degradation or repressing the translation of target genes. In this study, the target genes of miR-29b were analyzed and predicted by bioinformatics. And lipoprotein lipase (LPL) and thymine DNA glycosylase (TDG) were selected for further validation by dual luciferase reporter assay. In addition, we investigated the effects of miR-29b on triglyceride synthesis and mammary epithelial cell (MEC) apoptosis. The result showed that luciferase activity was significantly lower in cells that miR-29b cotransfected with LPL and TDG gene reporter vectors (pmiR-RB-REPORT-LPL-WT, pmiR-RB-REPORT-TDG-WT) than in cells of miR-29b cotransfected with gene reporter vectors (pmiR-RB-REPORT-LPL-mut and pmiR-RB-REPORT-LPL-si; pmiR-RB-REPORT-TDG-mut and pmiR-RB-REPORT-TDG-si) (p 0.05), indicating that target sites existed in 3'UTR of LPL and TDG. Furthermore, the expressions of miR-29b were negatively correlated with levels of mRNA and protein of LPL and TDG gene using quantitative real-time polymerase chain reaction and western blot analysis, suggesting that miR-29b might play an important role in regulation of LPL and TDG gene expression. Finally, miR-29b promoted triglyceride production and suppressed apoptosis in MECs, which might be attributed to the expressions of target genes LPL and TDG.

miRNAs are involved in various biological processes, such as host-virus interactions and antiviral immunity. In this study, we investigated the role of miR-29 on porcine reproductive and respiratory syndrome virus (PRRSV) replication and its target genes. At first, miR-29a/b-1/c expression was detected when porcine alveolar macrophages (PAMs) were infected with PRRSV at different infective doses by real time-quantitative polymerase chain reaction (RT-qPCR). The result showed that miR-29a/b-1 expression significantly increased after 6 h (p 0.01), with the peak around 24 h, miR-29c expression in each period of PRRSV infection was very low. Then, pre-miR-29a/b-1 lentiviral vectors were constructed. Absolute RT-qPCR analysis showed that PAMs transfected with pre-miR-29a/b-1 lentiviral vectors significantly promoted PRRSV replication in PAM within 24 h (p 0.01). The expression of the target genes (AKT3, TP53INP1, and RPS6KB1) of miR-29a significantly reduced (p 0.01). Western blot analysis showed that AKT3 and TP53INP1 are reduced at miR-29a overexpression. To further validate the interaction between miR-29a and its target gene sites, the luciferase assay results demonstrated that miR-29a interacted with AKT3 3'UTR 1676 and 1261 sites, leading the inhibition of luciferase expression. Our findings support that miR-29a could promote PRRSV replication during early stage of virus infection in vitro and AKT3 could be the target gene of miR-29a.

Our previous study showed that the expression of miR-196a was significantly higher in immature porcine testes than in mature porcine testes. However, the role of miR-196a in immature Sertoli cells remains unclear. In this study, miR-196a mimics, miR-196a inhibitor, and microRNA-small hairpin negative control (miRNA-ShNC) were transfected into immature Sertoli cells, respectively. Subsequently, the cell cycle and apoptosis rate of the immature Sertoli cells were measured by flow cytometry, and the viability of the Sertoli cells was measured by the MTS assay. Furthermore, the candidate target genes of miR-196a were analyzed by bioinformatics, and the target genes were validated by dual luciferase reporter assays, then the expression of target genes was detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assays. The results showed that miR-196a promotes the proliferation and inhibits the apoptosis of immature Sertoli cells. miR-196a directly binds the 3' untranslated region (3' UTR) of RCC2 and ABCB9. The expression of miR-196a was shown to be negatively correlated with the messenger RNA and protein levels of the RCC2 and ABCB9 genes. The study demonstrates that miR-196a regulates immature Sertoli cell proliferation and apoptosis and inhibits the expression of RCC2 and ABCB9.

Mammary epithelial cells (MECs) affect milk production capacity during lactation and are critical for the maintenance of tissue homeostasis. Our previous studies have revealed that the expression of miR-152 was increased significantly in MECs of cows with high milk production. In the present study, bioinformatics analysis identified ACAA2 and HSD17B12 as the potential targets of miR-152, which were further validated by dual-luciferase repoter assay. In addition, the expressions of miR-152 was shown to be negatively correlated with levels of mRNA and protein of ACAA2, HSD17B12 genes by qPCR and western bot analysis. Furthermore, transfection with miR-152 significantly up-regulated triglyceride production, promoted proliferation and inhibited apoptosis in MECs. Furthermore, overexpression of ACAA2 and HSD17B12 could inhibit triglyceride production, cells proliferation and induce apoptosis; but sh234-ACAA2-181/sh234-HSD17B12-474 could reverse the trend. These findings suggested that miR-152 could significantly influence triglyceride production and suppress apoptosis, possibly via the expression of target genes ACAA2 and HSD17B12.

microRNAs (miRNAs) represent a newly identified class of nonprotein-coding ∼22 nt small RNA that plays important roles in multiple biological processes by degrading targeted mRNA or repressing mRNA translation. This study observed the morphology of swine testicular tissue at different developmental stages (including 1-day old, 1–7 month old) by Hematoxylin-eosin staining. We also examined the expression of miR-499 and its target genes (CYLC1, DMRT1, QKI, XRN2, ZNF313) in samples of tissue slices using quantitative reverse-transcription polymerase chain reaction, which showed that miR-499 had a significant negative correlation with QKI gene. Then, the proteins of QKI gene expression were determined by western blot, which were consistent with results of quantitative polymerase chain reaction (qPCR) detection. Therefore, the luciferase reporter gene system was used to verify correlation between miR-499 and QKI gene. Activity of luciferase was significantly lower in miR-499 co-transfected with pmiR-RB-REPORT-QKI-WT group than the miR-499 co-transfected with pmiR-RB-REPORT-QKI-mut/si groups, indicating that target sequence of miR-499 existed in 3′UTR of QKI gene. Furthermore, the expressions of miR-499 and QKI were detected in testicular cells that were transfected with miR-499, miR-499 negative control and untreated. The results showed that the diameter of convoluted seminiferous tubule growth increased with age. Significantly different expressions of miR-499 and its target genes were found in swine testicular tissue at different developmental stages (p

Interferon-gamma-inducible protein 30 (IFI30) is an IFN-γ-inducible protein that is involved in MHC class II-restricted antigen processing and MHC class I-restricted cross-presentation pathways of adaptive immunity. The present study aimed to investigate the effects of porcine IFI30 expression on PRRSV proliferation in host cells. MARC-145 cells and pig Sertoli (ST) cells were infected with PRRSV after transfection with porcine IFI30 expression vectors and an empty vector. PRRSV copy numbers were analyzed by absolute real-time quantitative PCR, and the results showed that porcine IFI30 expression could significantly inhibit PRRSV transcription. Western blot analysis also determined that IFI30 expression could reduce the production of PRRSV M protein. Flow cytometric analysis indicated that the apoptosis of MARC-145 cells, which are non-porcine but highly permissive to PRRSV cells, was significantly decreased in the IFI30 expression group. In porcine ST cells, apoptosis was significantly increased in IFI30 knockdown cells but not in IFI30-overexpressing cells (**p < 0.01). In conclusion, porcine IFI30 expression may inhibit PRRSV proliferation and host cell apoptosis in vitro.

The dominant white coat colour of farmed blue fox is inherited as a monogenic autosomal dominant trait and is suggested to be embryonic lethal in the homozygous state. In this study, the transcripts of KIT were identified by RT-PCR for a dominant white fox and a normal blue fox. Sequence analysis showed that the KIT transcript in normal blue fox contained the full-length coding sequence of 2919 bp (GenBank Acc. No KF530833), but in the dominant white individual, a truncated isoform lacking the entire exon 12 specifically co-expressed with the normal transcript. Genomic DNA sequencing revealed that a single nucleotide polymorphism (c.1867+1G>T) in intron 12 appeared only in the dominant white individuals and a 1-bp ins/del polymorphism in the same intron showed in individuals representing two different coat colours. Genotyping results of the SNP with PCR-RFLP in 185 individuals showed all 90 normal blue foxes were homozygous for the G allele, and all dominant white individuals were heterozygous. Due to the truncated protein with a deletion of 35 amino acids and an amino acid replacement (p.Pro623Ala) located in the conserved ATP binding domain, we propose that the mutant receptor had absent tyrosine kinase activity. These findings reveal that the base substitution at the first nucleotide of intron 12 of KIT gene, resulting in skipping of exon 12, is a causative mutation responsible for the dominant white phenotype of blue fox.

DNA methylation is an important epigenetic modification in eukaryotes, which plays a significant role in regulating gene expression. When the host is invaded by the influenza virus, gene expression is regulated via changes in DNA methylation levels or patterns, leading to the activation or suppression of relevant signaling pathways or networks, triggering a series of immune responses against viral invasion. Here, we investigated the changes in genomic DNA methylation in the immune organs of chicken infected with H5N1 influenza virus. Genome-wide DNA methylation levels in the spleen, thymus, and bursa of Fabricius of specific pathogen-free (SPF) chicken infected with the Guangdong (G-H5N1) and Anhui (A-H5N1) H5N1 strains, and water (control) were analyzed by fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP). The results indicated that total DNA methylation levels did not differ between spleen genomic DNA in chicken treated with different viral strains and the control (P > 0.05). However, the total DNA methylation levels were significantly upregulated in the thymus (P < 0.01) and bursa (P < 0.05) of chicken in the A-H5N1 group compared to those in the G-H5N1 and control groups. These results provide a basis for the screening of avian influenza-resistance genes or methylation markers, analyzing the epigenetic regulation mechanisms of avian influenza, and performing selective breeding for disease resistance.

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.

Endothelial cells are the main target of classical swine fever virus during infection, and extensive hemorrhage is the most typical clinical sign of classical swine fever. To investigate the molecular mechanism of hemorrhagic pathogenesis, two-dimensional difference gel electrophoresis with fluorescent dyes (2D-DIGE) was used to analyze the proteomic profile of primary porcine umbilical vein endothelial cells (PUVECs) following CSFV infection. Of 15 protein spots with differential expression, 8 were characterized by MALDI-TOF-MS/MS in infected PUVECs at 48h p.i.: moesin, peroxiredoxin 6, stathmin-1, a protein similar to nascent polypeptide-associated complex alpha subunit isoform 2, phosphoglycerate kinase 1, glucosidase II, transketolase and alpha-tubulin. These could be sorted into 5 functional groups: glycometabolism, cell proliferation, anti-oxidative stress, inflammatory response and cytoskeleton. Western blot and real-time RT-PCR analysis confirmed the down-regulation of phosphoglycerate kinase 1 (PGK1) and up-regulation of moesin identified by 2D-DIGE. Pathway analysis of these 15 differentially expressed proteins showed that CSFV infection altered the metabolism, cytoskeleton and cell proliferation of PUVECs, and that consequently an inflammatory response was induced.

Amelogenins, the most abundant proteins in tooth enamel extracellular matrix (ECM), are essential for tooth amelogenesis. The nucleotide sequence of amelogenin gene (AMEL) for rabbit, as an important member of mammals and good continuously growing incisor model, is important for comparative and evolutional study. Previous studies about rabbit amelogenin proteins got no consensus yet even as to their existence or size. In this study, with combined usage of in silico and molecular cloning technologies, we identified sequences of two transcripts of rabbit amelogenin, resulting from the alternative splicing of the 45-bp exon 4. The coding regions of the two transcripts are of 567- and 522-bp, encoding 188 and 173 amino acids including a 17-residue signal peptide, respectively. Sequence analysis revealed that rabbit amelogenin features in extremely high GC-content in nucleotide sequence and Alanine content in protein sequence. Detailed comparison of amino acid sequence with other mammals showed that the rabbit amelogenin protein is conserved in the sites and regions important for protein functions. Overall, our results uncovered the mysteries about rabbit amelogenin and revealed its sequence peculiarities.

DGAT2 (acyl-CoA: diacylglycerol acyltransferase, EC2.3.1.20) is a member of acyl-CoA: monoacylglycerol acyltransferase (MGAT) family, which catalyzes one fatty acyl-CoA and diacylglycerol (DG) molecule to form triacylglycerols (TG) and is the final and rate-limiting step in the reaction of TG synthesis pathways. We previously showed that, during pig development, the fold change of DGAT2 mRNA in backfat tissue is much higher than that of DGAT1, implying that DGAT2 is more important in regulating porcine fat deposition. In this study, a 13 bp indel polymorphism located at 905 bp downstream from the stop codon (TGA) of porcine DGAT2 was found and two alleles of A (with 13 bp insertion) and B (no insertion) were designated. Allele A is dominant in all pig populations investigated. The backfat thickness of individuals with genotype AA is significantly lower than those with genotype AB (p

To verified the target genes of miR-34c, bioinformatics software was used to predict the targets of miR-34c. Three possible target genes of miR-34c related to spermatogenesis and male reproductive development: zinc finger protein 148 (ZNF148), kruppel-like factor 4 (KLF4), and platelet-derived growth factor receptor alpha (PDGFRA) were predicted. Then, the expression of miR-34c and its target genes were detected in swine testicular tissue at different developmental stages by quantitative polymerase chain reaction. The results suggested that the expression of PDGFRA has the highest negative correlation with miR-34c. Then immunohistochemical staining was done to observe the morphology of swine testicular tissue at 2-days and 3, 4, 5-months of age, which indicated that PDGFRA was mainly expressed in the support cells near the basement membrane during the early development stages of testicular tissue, but that the expression of PDGFRA was gradually reduced in later stages. Therefore, western blot analyzed that the highest expression of PDGFRA was generated in 2-days old testicular tissues and the expression levels reduced at 3 and 4-months old, which correlated with the results of immunohistochemical staining. In conclusion, PDGFRA is a target gene of miR-34c.

The growth of muscle fibers can be negatively regulated by bovine myostatin. The first two exons of myostatin gene code for the N-propeptide and its third exon codes for the C-polypeptide. Myostatin is secreted as a latent configuration formed by dimerization of two matured C peptides non-covalently linked with the N terminal pro-peptide. Pro-peptide has two distinct functions in guiding protein folding and regulating biological activity of myostatin. When the structure of the leader peptide is altered via mutations resulting in more tight binding with the mature peptide, myostatin function is inhibited, resulting in the changes of P21 and CDK2 expression levels which are relatedto the regulation of cell cycle. In the present study, the coding region of b MSTN (bovine myostatin) gene was amplified and mutated (A224C and G938A) through fusion PCR, and the mutated b MSTN gene (b MSTN - mut ) was inserted in frame into the pEF1a-IRES-DsRed-Express2 vector and transfected into bovine fibroblast cells. The expression levels of b MSTN - mut , P21 and CDK2 (cyclin dependent kinase 2) were examined with qPCR and Western-blotting. Changes in cell cycle after transfection were also analyzed with flow cytometry. The results indicated that leader peptide mutation resulted in down-regulation of P21 expression levels and up-regulation of CDK2 expression levels. The flow cytometry results showed that the proportion of cells in the G0/G1-phase was lower and that of cells in the S-phase was higher in bMSTN-mut transfected group than that in the control group. The proliferation rate of b MSTN - mut transfected cells was also significantly higher than that of the control cells. In conclusion, the studies have shown that the pEF1a-IRES-DsRed-Express2–b MSTN-mut recombinant plasmid could effectively promote the proliferation of bovine fibroblast cells. The site-directed mutagenesis of b MSTN gene leader peptide and in vitro expression in bovine fibroblast cells could be helpful to further the studies of b MSTN in regulating bovine muscle cell growth and development.

This study was designed to investigate a single nucleotide polymorphism in intron 1 of the liver fatty acid-binding protein (L-FABP) gene in 156 Junmu No. 1 white swine using PCR-single-strand conformational polymorphism. The association between the polymorphism and meat quality traits was also studied. The cloning and sequencing results indicated that the polymorphism in intron 1 was due to a T→C mutation at position 1740 of L-FABP, yielding three genotypes (TT, TC, and CC). Association analysis revealed that the polymorphism had a significant effect on marbling (P0.05): genotype CC had more marbling than TC, and TC had more marbling than TT. The polymorphism also had a highly significant effect on intramuscular fat content (P0.01). Genotypes CC and TC had higher intramuscular fat content than TT; there was no significant difference between CC and TC (P0.05). However, no significant conclusions concerning other traits could be drawn. We tentatively conclude that L-FABP is a candidate gene or a quantitative trait locus-linked gene associated with meat quality traits.

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.

This study was designed to investigate the single nucleotide polymorphism (SNP) in intron 1 of the gene A-FABP in 127 Junmu No. 1 white swine using PCR-SSCP. The association between the polymorphism and meat quality traits was also studied. The cloning and sequencing results indicated that the polymorphism of intron 1 was due to a point mutation in position 3481bp of A-FABP, giving 3 genotypes (CC, CD and DD). Association analysis indicated that the polymorphism had a significant effect on marbling (P

Chickens are susceptible to the highly pathogenic H5N1 strain of avian influenza virus (HPAIV), whereas ducks are not. Here, we used high-throughput sequencing to analyse the microRNA expression in the spleen, thymus and bursa of Fabricius of H5N1-HPAIV-infected and non-infected chickens and ducks. We annotated the genomic positions of duck microRNAs and we compared the microRNA repertoires of chickens and ducks. Our results showed that the microRNA expression patterns in the homologous immune organs of specific-pathogen-free (SPF) chickens and ducks diverge substantially. Moreover, there was larger divergence between the microRNA expression patterns in immune organs of HPAIV-infected chickens than HPAIV-infected ducks. Together, our results might help to elucidate the roles of microRNAs in the divergent immunity of chickens and ducks against H5N1 HPAIV.