Yinghui Ling

Characterization and differential expression of microRNAs in the ovaries of pregnant and non-pregnant goats (Capra hircus)

BackgroundOvarian follicular development and hormone secretion are complex and coordinated biological processes which will usually be altered during pregnancy. Ovarian function is tightly regulated by a multitude of genes, and also by some specific miRNAs. It is necessary to identify the differentially expressed miRNAs in the ovaries of pregnant and non-pregnant mammals, in order to further understand the role of miRNA-mediated post-transcriptional regulation in mammalian reproduction. Here, we performed a comprehensive search for hircine miRNAs using two small RNA sequencing libraries prepared from the ovaries of pregnant and non-pregnant goats.Results617 conserved and 7 putative novel miRNAs were identified in the hircine ovaries. A total of 471 conserved miRNAs (76.34%) were co-expressed in both pregnant and non-pregnant libraries, and 90 pregnancy-specific and 56 non-pregnancy-specific conserved miRNAs were identified. Additionally, 407 unique miRNAs (65.96%) were significantly differentially expressed in the pregnant and non-pregnant libraries, of which 294 were upregulated and 113 were downregulated in the pregnant library compared to the non-pregnant library. Further analysis showed that miR-143 was predicted to bind to the target sequences of Frizzled-6 and -3 receptor genes in the Wnt/beta-catenin signaling pathway, and let-7b may target the Activin receptor I and Smad 2/3 genes in the TGF-beta signaling pathway. The expression level of 5 randomly selected miRNAs were analyzed by quantitative real-time PCR (q-PCR), and the results demonstrated that the expression patterns were consistent with the Solexa sequencing results.ConclusionsThe identification and characterization of differentially expressed miRNAs in the ovaries of pregnant and non-pregnant goats provides important information on the role of miRNA in the regulation of the ovarian development and function. This data will be helpful to facilitate studies on the regulation of miRNAs during mammalian reproduction.

Identification and characterization of microRNAs in the ovaries of multiple and uniparous goats (Capra hircus) during follicular phase

BackgroundSuperior kidding rate is an important economic trait in production of meat goat, and ovulation rate is the precondition of kidding rate. MicroRNAs (miRNAs) play critical roles in almost all ovarian biological processes, including folliculogenesis, follicle development, follicle atresia, luteal development and regression. To find out the different ovarian activity and follicle recruitment with miRNA-mediated posttranscriptional regulation, the small RNAs expressed pattern in the ovarian tissues of multiple and uniparous Anhui White goats during follicular phase was analyzed using Solexa sequencing data.Results1008 miRNAs co-expressed, 309 and 433 miRNAs specifically expressed in the ovaries of multiple and uniparous goats during follicular phase were identified. The 10 most highly expressed miRNAs in the multiple library were also the highest expressed in the uniparous library, and there were no significantly different between each other. The highest specific expressed miRNA in the multiple library was miR-29c, and the one in the uniparous library was miR-6406. 35 novel miRNAs were predicted in total. GO annotation and KEGG Pathway analyses were implemented on target genes of all miRNA in two libraries. RT-PCR was applied to detect the expression level of 5 randomly selected miRNAs in multiple and uniparous hircine ovaries, and the results were consistent with the Solexa sequencing data.ConclusionsIn the present study, the different expression of miRNAs in the ovaries of multiple and uniparous goats during follicular phase were characterized and investigated using deep sequencing technology. The result will help to further understand the role of miRNAs in kidding rate regulation and also may help to identify miRNAs which could be potentially used to increase hircine ovulation rate and kidding rate in the future.

Transcriptome differences between Cry1Ab resistant and susceptible strains of Asian corn borer.

BackgroundAsian corn borer (ACB), Ostrinia furnacalis (Guenée), is the major insect pest of maize in China and countries of East and Southeast Asia, the Pacific and Australasia. ACB can develop strong resistance to the transgenic Bt maize expressing Cry1Ab, the most widely commercialized Bt maize worldwide. However, the molecular basis for the resistance mechanisms of ACB to Cry1Ab remained unclear. Two biological replicates of the transcriptome of Bt susceptible (ACB-BtS) and Cry1Ab resistant (ACB-AbR) strains of ACB were sequenced using Solexa/Illumina RNA-Seq technology to identify Cry1Ab resistance-relevant genes.ResultsThe numbers of unigenes for two biological replications were 63,032 and 53,710 for ACB-BtS and 57,770 and 54,468 for ACB-AbR. There were 35,723 annotated unigenes from ACB reads found by BLAST searching NCBI non-redundant, NCBI non-redundant nucleotide, Swiss-prot protein, Kyoto Encyclopedia of Genes and Genomes, Cluster of Orthologous Groups of proteins, and Gene Ontology databases. Based on the NOISeq method, 3,793 unigenes were judged to be differentially expressed between ACB-BtS and ACB-AbR. Cry1Ab resistance appeared to be associated with change in the transcription level of enzymes involved in growth regulation, detoxification and metabolic/catabolic process. Among previously described Bt toxin receptors, the differentially expressed unigenes associated with aminopeptidase N and chymotrypsin/trypsin were up-regulated in ACB-AbR. Whereas, other putative Cry receptors, cadherin-like protein, alkaline phosphatase, glycolipid, actin, V-type proton ATPase vatalytic, heat shock protein, were under-transcripted. Finally, GPI-anchor biosynthesis was found to be involved in the significantly enriched pathway, and all genes mapped to the pathway were substantially down-regulated in ACB-AbR.ConclusionTo our knowledge, this is the first comparative transcriptome study to discover candidate genes involved in ACB Bt resistance. This study identified differentially expressed unigenes related to general Bt resistance in ACB. The assembled, annotated transcriptomes provides a valuable genomic resource for further understanding of the molecular basis of ACB Bt resistance mechanisms.

Genome-Wide Dynamic Profiling of Histone Methylation during Nuclear Transfer-Mediated Porcine Somatic Cell Reprogramming

The low full-term developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos is mainly attributed to imperfect epigenetic reprogramming in the early embryos. However, dynamic expression patterns of histone methylation involved in epigenetic reprogramming progression during porcine SCNT embryo early development remain to be unknown. In this study, we characterized and compared the expression patterns of multiple histone methylation markers including transcriptionally repressive (H3K9me2, H3K9me3, H3K27me2, H3K27me3, H4K20me2 and H4K20me3) and active modifications (H3K4me2, H3K4me3, H3K36me2, H3K36me3, H3K79me2 and H3K79me3) in SCNT early embryos from different developmental stages with that from in vitro fertilization (IVF) counterparts. We found that the expression level of H3K9me2, H3K9me3 and H4K20me3 of SCNT embryos from 1-cell to 4-cell stages was significantly higher than that in the IVF embryos. We also detected a symmetric distribution pattern of H3K9me2 between inner cell mass (ICM) and trophectoderm (TE) in SCNT blastocysts. The expression level of H3K9me2 in both lineages from SCNT expanded blastocyst onwards was significantly higher than that in IVF counterparts. The expression level of H4K20me2 was significantly lower in SCNT embryos from morula to blastocyst stage compared with IVF embryos. However, no aberrant dynamic reprogramming of H3K27me2/3 occurred during early developmental stages of SCNT embryos. The expression of H3K4me3 was higher in SCNT embryos at 4-cell stage than that of IVF embryos. H3K4me2 expression in SCNT embryos from 8-cell stage to blastocyst stage was lower than that in the IVF embryos. Dynamic patterns of other active histone methylation markers were similar between SCNT and IVF embryos. Taken together, histone methylation exhibited developmentally stage-specific abnormal expression patterns in porcine SCNT early embryos.

Exploring differentially expressed genes in the ovaries of uniparous and multiparous goats using the RNA-Seq (Quantification) method

Fecundity improvement is one of the most important objectives for goat breeders as it greatly increases production efficiency. To investigate the genes associated with litter sizes in the Anhui White goat (AWG), gene expression differences in the ovaries of uniparous and multiparous AWG were assessed using the RNA-Seq (Quantification) method. This analysis generated 6,027,714 and 5,884,062 clean reads in uniparous and multiparous libraries, respectively. A total of 2201 differentially expressed genes (DEGs) were thereby identified (FDR≤0.001, |log₂Ratio|≥1). There were 1583 up-regulated and 618 down-regulated genes in the multiparous samples compared with the uniparous samples. A large number of these DEGs were related to the terms cellular process, cell & cell part and binding. Twelve genes which may be associated with the high prolificacy of AWG were identified using a bioinformatic screen. In addition, pathway analysis revealed that these DEGs were significantly enriched in 11 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including ECM-receptor interactions, focal adhesion, and regulation of the actin cytoskeleton among others. This suggested a role for these pathways in the prolificacy of AWG. These results provide a list of new candidate genes for goat prolificacy.

Screening and evaluating of long noncoding RNAs in the puberty of goats

BackgroundLong noncoding RNAs (lncRNAs) are involved in regulating animal development, however, their function in the onset of puberty in goats remain largely unexplored. To identify the genes controlling the regulation of puberty in goats, we measured lncRNA and mRNA expression levels from the hypothalamus.ResultsWe applied RNA sequencing analysis to examine the hypothalamus of pubertal (case; n = 3) and prepubertal (control; n = 3) goats. Our results showed 2943 predicted lncRNAs, including 2012 differentially expressed lncRNAs, which corresponded to 5412 target genes. We also investigated the role of lncRNAs that act cis and trans to the target genes and found a number of lncRNAs involved in the regulation of puberty and reproduction, as well as several pathways related to these processes. For example, oxytocin signaling pathway, sterol biosynthetic process, and pheromone receptor activity signaling pathway were enriched as Kyoto Encyclopedia of Genes and Genomes (KEGG) or gene ontology (GO) analyses showed.ConclusionOur results clearly demonstrate that lncRNAs play an important role in regulating puberty in goats. However, further research is needed to explore the functions of lncRNAs and their predicted targets to provide a detailed expression profile of lncRNAs on goat puberty.

Identification of differentially expressed microRNAs between Bacillus thuringiensis Cry1Ab-resistant and -susceptible strains of Ostrinia furnacalis.

The Asian corn borer (ACB), Ostrinia furnacalis (Guenée), can develop strong resistance to Cry1Ab, the most widely commercialized Cry toxin for Bt maize worldwide. It is essential to understand the mechanism of resistance for management of this species, but information on the post-transcriptional regulation of Bt resistance in this target insect is limited. In the present study, RNA was extracted from the ACB in various larval stages (1–5 instar) from Cry1Ab-sensitive (ACB-BtS) and -resistant (ACB-AbR) strains, each of which included two biological replicates. Using Illumina sequencing, a total of 23,809,890 high-quality reads were collected from the four ACB libraries. The numbers of known microRNAs (miRNAs) were 302 and 395 for ACB-BtS and 268 and 287 for ACB-AbR. Using Mireap software, we identified 32 and 16 potential novel miRNAs for ACB-BtS and 18 and 22 for ACB-AbR. Among them, 21 known and 1 novel miRNAs had significantly different expression between ACB-BtS and ACB-AbR. Several miRNAs were observed to target potential Bt receptor genes, such as aminopeptidase N and cadherin-like protein. The glycosylphosphatidylinositol-anchor biosynthetic process and ABC transporters pathway were identified through Gene Ontology and KEGG pathway analysis of target genes of the differentially expressed miRNAs.

DNA Methylation Patterns in the Hypothalamus of Female Pubertal Goats.

Female pubertal development is tightly controlled by complex mechanisms, including neuroendocrine and epigenetic regulatory pathways. Specific gene expression patterns can be influenced by DNA methylation changes in the hypothalamus, which can in turn regulate timing of puberty onset. In order to understand the relationship between DNA methylation changes and gene expression patterns in the hypothalamus of pubertal goats, whole-genome bisulfite sequencing and RNA-sequencing analyses were carried out. There was a decline in DNA methylation levels in the hypothalamus during puberty and 268 differentially methylated regions (DMR) in the genome, with differential patterns in different gene regions. There were 1049 genes identified with distinct expression patterns. High levels of DNA methylation were detected in promoters, introns and 3′-untranslated regions (UTRs). Levels of methylation decreased gradually from promoters to 5′-UTRs and increased from 5′-UTRs to introns. Methylation density analysis demonstrated that methylation level variation was consistent with the density in the promoter, exon, intron, 5′-UTRs and 3′-UTRs. Analyses of CpG island (CGI) sites showed that the enriched gene contents were gene bodies, intergenic regions and introns, and these CGI sites were hypermethylated. Our study demonstrated that DNA methylation changes may influence gene expression profiles in the hypothalamus of goats during the onset of puberty, which may provide new insights into the mechanisms involved in pubertal onset.

Role of Nesfatin-1 in the Reproductive Axis of Male Rat

Nesfatin-1 is an important molecule in the regulation of reproduction. However, its role in the reproductive axis in male animals remains to be understood. Here, we found that nesfatin-1 was mainly distributed in the arcuate nucleus (ARC), paraventricular nucleus (PVN), periventricular nucleus (PeN), and lateral hypothalamic area (LHA) of the hypothalamus; adenohypophysis and Leydig cells in male rats. Moreover, the concentrations of serum nesfatin-1 and its mRNA in hypothalamo-pituitary-gonadal axis (HPGA) vary with the age of the male rat. After intracerebroventricular injection of nesfatin-1, the hypothalamic genes for gonadotrophin releasing hormone (GnRH), kisspeptin (Kiss-1), pituitary genes for follicle-stimulate hormone β(FSHβ), luteinizing hormone β(LHβ), and genes for testicular steroidogenic acute regulatory (StAR) expression levels were decreased significantly. Nesfatin-1 significantly increased the expression of genes for 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and cytochrome P450 cleavage (P450scc) in the testis of pubertal rats, but their levels decreased in adult rats (P < 0.05), along with the serum FSH, LH, and testosterone (T) concentrations. After nesfatin-1 addition in vitro, T concentrations of the supernatant were significantly higher than that in the control group. These results were suggestive of the role of nesfatin-1 in the regulation of the reproductive axis in male rats.

Genetic Differentiation of Chinese Indigenous Meat Goats Ascertained Using Microsatellite Information

To investigate the genetic diversity of seven Chinese indigenous meat goat breeds (Tibet goat, Guizhou white goat, Shannan white goat, Yichang white goat, Matou goat, Changjiangsanjiaozhou white goat and Anhui white goat), explain their genetic relationship and assess their integrity and degree of admixture, 302 individuals from these breeds and 42 Boer goats introduced from Africa as reference samples were genotyped for 11 microsatellite markers. Results indicated that the genetic diversity of Chinese indigenous meat goats was rich. The mean heterozygosity and the mean allelic richness (AR) for the 8 goat breeds varied from 0.697 to 0.738 and 6.21 to 7.35, respectively. Structure analysis showed that Tibet goat breed was genetically distinct and was the first to separate and the other Chinese goats were then divided into two sub-clusters: Shannan white goat and Yichang white goat in one cluster; and Guizhou white goat, Matou goat, Changjiangsanjiaozhou white goat and Anhui white goat in the other cluster. This grouping pattern was further supported by clustering analysis and Principal component analysis. These results may provide a scientific basis for the characteristization, conservation and utilization of Chinese meat goats.