Identification and characterization of mRNAs and lncRNAs in the uterus of polytocous and monotocous Small Tail Han sheep (Ovis aries)

Background Long non-coding RNAs (lncRNAs) regulate endometrial secretion and uterine volume. However, there is little research on the role of lncRNAs in the uterus of Small Tail Han sheep (FecB++). Herein, RNA-seq was used to comparatively analyze gene expression profiles of uterine tissue between polytocous and monotocous sheep (FecB++) in follicular and luteal phases. Methods To identify lncRNA and mRNA expressed in the uterus, the expression of lncRNA and mRNA in the uterus of Small Tail Han sheep (FecB++) from the polytocous group (n = 6) and the monotocous group (n = 6) using RNA-sequencing and real-time polymerase chain reaction (RT-PCR). Identification of differentially expressed lncRNAs and mRNAs were performed between the two groups and two phases . Gene ontology (GO) and pathway enrichment analyses were performed to analyze the biological functions and pathways for the differentially expressed mRNAs. LncRNA-mRNA co-expression network was constructed to further analyses the function of related genes. Results In the follicular phase, 473 lncRNAs and 166 mRNAs were differentially expressed in polytocous and monotocous sheep; in the luteal phase, 967 lncRNAs and 505 mRNAs were differentially expressed in polytocous and monotocous sheep. GO and KEGG enrichment analysis showed that the differentially expressed lncRNAs and their target genes are mainly involved in ovarian steroidogenesis, retinol metabolism, the oxytocin signaling pathway, steroid hormone biosynthesis, and the Foxo signaling pathway. Key lncRNAs may regulate reproduction by regulating genes involved in these signaling pathways and biological processes. Specifically, UGT1A1, LHB, TGFB1, TAB1, and RHOA, which are targeted by MSTRG.134747, MSTRG.82376, MSTRG.134749, MSTRG.134751, and MSTRG.134746, may play key regulatory roles. These results offer insight into molecular mechanisms underlying sheep prolificacy

An Immediate Innate Immune Response Occurred In the Early Stage of E. granulosus Eggs Infection in Sheep: Evidence from Microarray Analysis.

Cystic Echinococcosis(CE), caused by infection with the larval stage of the cestode Echinococcus granulosus (E. granulosus), is a chronic parasitic zoonosis, with highly susceptible infection in sheep. However, the comprehensive molecular mechanisms that underlie the process of E. granulosus infection in the early stage remain largely unknown. The objective of this present study was to gain a cluster of genes expression profiles in the intestine tissue of sheep infected with CE.Nine healthy sheep were divided into infection group and healthy controls, with six infected perorally 5000 E. granulosus eggs suspended in 1000 μl physiological saline and three controls perorally injected 1000 μl physiological saline. All animals were sacrificed at 4 hours post-infection, respectively. The intestine tissue was removed and the RNA was extracted. In the infection group, the biology replicates were designed to make sure the accuracy of the data. The ovine microarrays were used to analyze changes of gene expression in the intestine tissue between CE infected sheep and healthy controls. Real-time PCR was used to assess reliability of the microarray data.By biology repeats, a total of 195 differentially expressed genes were identified between infected group and controls at 4 hours post-infection, with 105 genes related to immune responses, while 90 genes associated with functions including energy metabolism, fat soluble transport, etc. Among the 105 immunity genes, 72 genes showed up-regulated expression levels while 33 showed down-regulation levels. Function analysis showed that most of up-regulated genes were related to innate immune responses, such as mast cell, NK cell, cytokines, chemokines and complement. In addition, Real-time PCR analysis of a random selection of nine genes confirmed the reliability of the microarray data.To our knowledge, this is the first report describing gene expression profiles in the intestine tissue of CE infection sheep. These results suggested that the innate immune system was activated to elicit immediate defense in the intestine tissue where E. granulosus invaded in at 4 hour-post infection. Furthermore, future interest will also focus on unraveling similar events, especially for the function of adaptive immunity, but at late stage infection

Expression Analysis of the Prolific Candidate Genes, BMPR1B, BMP15, and GDF9 in Small Tail Han Ewes with Three Fecundity (FecB Gene) Genotypes

The expression characteristics of the prolific candidate genes, BMPR1B, BMP15, and GDF9, in the major visceral organs and hypothalamic–pituitary–gonadal (HPG) axis tissues of three FecB genotypes (FecB BB, FecB B+, and FecB ++) were explored in STH ewes using RT-PCR and qPCR. The results were as follows, BMPR1B was expressed in all FecB BB genotype (Han BB) tissues, and GDF9 was expressed in all selected tissues, but BMP15 was specifically expressed in the ovaries. Further study of ovarian expression indicated that there was no difference in BMPR1B expression between genotypes, but the FecB B+ genotype (Han B+) had greater expression of GDF9 and BMP15 than Han BB and FecB ++ genotype (Han ++) (p < 0.05, p < 0.01). BMP15 expression was lower in the ovaries of Han BB than in Han ++ sheep, but the reverse was shown for GDF9. The gene expression in non-ovarian tissues was also different between genotypes. Therefore, we consider that the three genes have an important function in ovine follicular development and maturation. This is the first systematic analysis of the tissue expression pattern of BMPR1B, BMP15, and GDF9 genes in STH sheep of the three FecB genotypes. These results contribute to the understanding of the molecular regulatory mechanism for ovine reproduction

Passively Q-switching induced by the smallest single-walled carbon nanotubes

We report a passively Q-switched erbium-doped fiber laser (EDFL) by using the smallest single-walled carbon nanotubes (SWNTs) with a diameter of 0.3 nm as the saturable absorber. These small SWNTs are fabricated in the nanochannels of a ZnAPO-11 (AEL) single crystal. By inserting one of the AEL crystal into an EDFL cavity pumped by a 980 nm laser diode, stable passive Q-switching is achieved for a threshold pump power of 206.2 mW, and 4.73 mu s pulses with a repetition rate of 41.78 kHz and an average output power of 3.75 mW are obtained for a pump power of 406 mW. (C) 2014 AIP Publishing LLC

Comparative Transcriptomics Reveal Key Sheep (<em>Ovis aries</em>) Hypothalamus LncRNAs that Affect Reproduction

The diverse functions of long noncoding RNAs (lncRNAs), which execute their functions mainly through modulating the activities of their target genes, have been have been widely studied for many years (including a number of studies involving lncRNAs in the ovary and uterus). Herein, for the first time, we detect lncRNAs in sheep hypothalami with FecB++ through RNA Sequencing (RNA-Seq) and identify a number of known and novel lncRNAs, with 622 and 809 found to be differentially expressed in polytocous sheep in the follicular phase (PF) vs. monotocous sheep in the follicular phase (MF) and polytocous sheep in the luteal phase (PL) vs. monotocous sheep in the luteal phase (ML), respectively. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed based on the predicted target genes. The most highly enriched GO terms (at the molecular function level) included carbonyl reductase (NADPH), 15-hydroxyprostaglandin dehydrogenase (NADP+), and prostaglandin-E2 9-reductase activity in PF vs. MF, and phosphatidylinositol-3,5-bisphosphate binding in PL vs. ML was associated with sheep fecundity. Interestingly, the phenomena of valine, leucine, and isoleucine degradation in PL vs. ML, and valine, leucine, and isoleucine biosynthesis in PF vs. MF, were present. In addition, the interactome of lncRNA and its targets showed that MSTRG.26777 and its cis-targets ENSOARG00000013744, ENSOARG00000013700, and ENSOARG00000013777, and MSTRG.105228 and its target WNT7A may participate in the sheep reproductive process at the hypothalamus level. Significantly, MSTRG.95128 and its cis-target Forkhead box L1 (FOXG1) were shown to be upregulated in PF vs. MF but downregulated in PL vs. ML. All of these results may be attributed to discoveries of new candidate genes and pathways related to sheep reproduction, and they may provide new views for understanding sheep reproduction without the effects of the FecB mutation

Comparative Transcriptomics Identify Key Hypothalamic Circular RNAs that Participate in Sheep (Ovis aries) Reproduction

Circular RNA (circRNA), as an emerging class of noncoding RNA, has been found to play key roles in many biological processes. However, its expression profile in the hypothalamus, a powerful organ initiating the reproductive process, has not yet been explored. Therefore, we used RNA sequencing to explore the expression of circRNAs in the hypothalamus of sheep with the FecB ++ genotype. We totally identified 41,863 circRNAs from sheep hypothalamus, in which 333 (162 were upregulated, while 171 were downregulated) were differentially expressed in polytocous sheep in the follicular phase versus monotocous sheep in the follicular phase (PF vs. MF), moreover, 340 circRNAs (163 were upregulated, while 177 were downregulated) were differentially expressed in polytocous sheep in the luteal phase versus monotocous sheep in the luteal sheep (PL vs. ML). We also identified several key circRNAs including oar_circ_0018794, oar_circ_0008291, oar_circ_0015119, oar_circ_0012801, oar_circ_0010234, and oar_circ_0013788 through functional enrichment analysis and oar_circ_0012110 through a competing endogenous RNA network, most of which may participate in reproduction by influencing gonadotropin-releasing hormone (GnRH) activities or affecting key gene expression, indirectly or directly. Our study explored the overall expression profile of circRNAs in sheep hypothalamus, which potentially provides an alternative insight into the mechanism of sheep prolificacy without the effects of FecB mutation

Differential Expression of Circular RNAs in Polytocous and Monotocous Uterus during the Reproductive Cycle of Sheep

CircRNA plays important roles in cell proliferation, differentiation, autophagy and apoptosis during development. However, there are few reports on circRNAs related to livestock reproduction. In this study, we identified circRNAs by deep sequencing and analyzed their expression in the uteri of polytocous and monotocous sheep (FecB++) during follicular and luteal phases. There were 147 and 364 circRNAs with differential expression in the follicular and luteal phases, respectively. GO and KEGG enrichment analysis was performed for the host genes of the circRNAs to predict the functions of differentially expressed circRNAs. These source genes were mainly involved in the estrogen signaling pathway, TGF&beta; signaling pathway, GnRH signaling pathway, oxytocin signaling pathway, pentose phosphate pathway, and starch and sucrose metabolism related to reproduction and energy metabolism. CircRNA expression patterns were validated by RT-qPCR. Our findings provide a solid foundation for the identification and characterization of key important circRNAs involved in reproduction

Low Expression of Sirtuin 1 in the Dairy Cows with Mild Fatty Liver Alters Hepatic Lipid Metabolism

Dairy cows usually experience negative energy balance coupled with an increased incidence of fatty liver during the periparturient period. The purpose of this study was to investigate the effect of hepatic steatosis on the expression of the sirtuin 1 (SIRT1), along with the target mRNA and protein expressions and activities related to lipid metabolism in liver tissue. Control cows (n = 6, parity 3.0 &plusmn; 2.0, milk production 28 &plusmn; 7 kg/d) and mild fatty liver cows (n = 6, parity 2.3 &plusmn; 1.5, milk production 20 &plusmn; 6 kg/d) were retrospectively selected based on liver triglycerides (TG) content (% wet liver). Compared with the control group, fatty liver cows had greater concentrations of cholesterol and TG along with the typically vacuolated appearance and greater lipid droplets in the liver. Furthermore, fatty liver cows had greater mRNA and protein abundance related to hepatic lipid synthesis proteins sterol regulatory element binding proteins (SREBP-1c), long-chain acyl-CoA synthetase (ACSL), acyl-CoA carbrolase (ACC) and fatty acid synthase (FAS) and lipid transport proteins Liver fatty acid binding protein (L-FABP), apolipoprotein E (ApoE), low density lipoprotein receptor (LDLR) and microsomal TG transfer protein (MTTP) (p &lt; 0.05). However, they had lower mRNA and protein abundance associated with fatty acid &beta;-oxidation proteins SIRT1, peroxisome proliferator-activated receptor co-activator-1 (PGC-1&alpha;), peroxisome proliferator&ndash;activated receptor-&alpha; (PPAR&alpha;), retinoid X receptor (RXR&alpha;), acyl-CoA 1 (ACO), carnitine palmitoyltransferase 1 (CPT1), carnitine palmitoyltransferase 2 (CPT2) and long- and medium-chain 3-hydroxyacyl-CoA dehydrogenases (LCAD) (p &lt; 0.05). Additionally, mRNA abundance and enzyme activity of enzymes copper/zinc superoxide dismutase (Cu/Zn SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and manganese superoxide dismutase (Mn SOD) decreased and mRNA and protein abundance of p45 nuclear factor-erythroid 2 (p45 NF-E2)-related factor 1 (Nrf1), mitochondrial transcription factor A (TFAM) decreased (p &lt; 0.05). Lower enzyme activities of SIRT1, PGC-1&alpha;, Cu/Zn SOD, CAT, GSH-Px, SREBP-1c and Mn SOD (p &lt; 0.05) and concentration of reactive oxygen species (ROS) were observed in dairy cows with fatty liver. These results demonstrate that decreased SIRT1 associated with hepatic steatosis promotes hepatic fatty acid synthesis and inhibits fatty acid &beta;-oxidation. Hence, SIRT1 may represent a novel therapeutic target for the treatment of the fatty liver disease in dairy cows

Primer sequence for qRT-PCR analysis of gene transcripts.

<p>Primer sequence for qRT-PCR analysis of gene transcripts.</p

Andrographolide Inhibits Inflammatory Cytokines Secretion in LPS-Stimulated RAW264.7 Cells through Suppression of NF-κB/MAPK Signaling Pathway

Andrographolide, the main active component extracted from Andrographis paniculata (Burm.f.) Wall. ex Nees, exerts anti-inflammatory effects; however, the principal molecular mechanisms remain unclear. The objective of this study was to investigate the molecular mechanisms of Andrographolide in modifying lipopolysaccharide- (LPS-) induced signaling pathway in RAW264.7 cells. An in vitro model of inflammation was induced by LPS in mouse RAW264.7 cells in the presence of Andrographolide. The concentration and expression levels of proinflammatory cytokines were determined by an enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The nuclear level of NF-κB was measured by an electrophoretic mobility shift assay (EMSA). The expression levels of NF-κB, p38, ERK, and JNK were determined by western blot. Andrographolide dose-dependently inhibited the release and mRNA expression of TNF-α, IL-6, and IL-1β in LPS-stimulated RAW264.7 cells. The nuclear level of p65 protein was decreased in Andrographolide treatment group. Western blot analysis showed that Andrographolide suppressed LPS-induced NF-κB activation and the phosphorylation of IkBa, ERK1/2, JNK, and p38. These results suggest that Andrographolide exerts an anti-inflammatory effect by inhibiting the activation of NF-κB/MAPK signaling pathway and the induction of proinflammatory cytokines