Miniature Hierarchical DNA Hybridization Circuit for Amplified Multiplexed MicroRNA Imaging.

Accurate diagnosis requires the development of multiple-guaranteed DNA circuits. Nevertheless, for reliable multiplexed molecular imaging, existing DNA circuits are limited by poor cell-delivering homogeneity due to their cumbersome and dispersive reactants. Herein, we developed a compact-yet-efficient hierarchical DNA hybridization (HDH) circuit for in situ simultaneous analysis of multiple miRNAs, which could be further exploited for specifically discriminating cancer cells from normal ones. By integrating the traditional hybridization chain reaction and catalytic hairpin assembly reactants into two highly organized hairpins, the HDH circuit is fitted with condensed components and multiple response domains, thus permitting the programmable multiple microRNA-guaranteed sequential activations and the localized cascaded signal amplification. The synergistic multi-recognition and amplification features of the HDH circuit facilitate the magnified detection of multiplex endogenous miRNAs in living cells. The in vitro and cellular imaging experimental results revealed that the HDH circuit displayed a reliable sensing performance with high selective cell-identification capacity. We anticipate that this compact design can provide a powerful toolkit for accurate diagnostics and pathological evolution.

An Autocatalytic DNA Circuit Based on Hybridization Chain Assembly for Intracellular Imaging of Polynucleotide Kinase.

Polynucleotide kinase (PNK) plays an essential role in various cellular events by regulating phosphorylation processes, and abnormal homeostasis of PNK could cause many human diseases. Herein, we proposed an autocatalytic hybridization system (AHS) through the elaborate integration of hybridization chain assembly (HCA) and catalytic DNA assembly (CDA) that enables a highly efficient positive feedback amplification. The PNK-targeting AHS biosensor is composed of three modules: a recognition module, an HCA amplification module, and a CDA autocatalytic module. In the presence of PNK, the recognition module could transform the PNK input into an exposed nucleic acid initiator (I). Then the initiator strand I could trigger the autonomous HCA process in the amplification module, and the resulted HCA products could reassemble the split CDA trigger strand T, subsequently inducing the CDA process in the autocatalytic module to form abundant DNA duplex products. Consequently, the embedded initiator strand I was liberated from the CDA duplex product to autonomously trigger the new rounds of HCA circuit. The rational integration and cooperative cross-activation between the HCA and CDA module could prominently accelerate the reaction and realize the exponential amplification efficiency by initiator regeneration. As a result, the self-sustainable AHS amplifier could implement the sensitive detection of PNK in vitro and in biological samples and further fulfill accurate monitoring of the intracellular PNK activity and the effective screening of PNK inhibitors. This work paves a way for exploiting highly efficient artificial DNA circuits to analyze low-abundance biomarkers, holding great potential in biochemical research and clinical diagnosis.

An Isothermal Autocatalytic Hybridization Reaction Circuit for Sensitive Detection of DNA Methyltransferase and Inhibitors Assay.

Abnormal DNA methylation contributes to the annoying tumorigenesis and the elevated expression of methylation-related methyltransferase (MTase) is associated with many diseases. Hence DNA MTase could serve as a promising biomarker for cancer-specific diagnosis as well as a potential therapeutic target. Herein, we developed an isothermal autocatalytic hybridization reaction (AHR) circuit for the sensitive detection of MTase and its inhibitors by integrating the catalytic hairpin assembly (CHA) converter with the hybridization chain reaction (HCR) amplifier. The initiator-mediated HCR amplifier could generate amplified fluorescent readout, as well as numerous newly activated triggers for motivating the CHA converter. The CHA converter is designed to expose the identical sequence of HCR initiators that reversely powered the HCR amplifier. Thus, the trace amount of target could produce exponentially amplified fluorescent readout by the autocatalytic feedback cycle between HCR and CHA systems. Then an auxiliary hairpin was introduced to mediate the assay of Dam MTase via the well-established AHR circuit. The Dam MTase-catalyzed methylation of auxiliary hairpin leads to its subsequent efficient cleavage by DpnI endonuclease, thus resulting in the release of HCR initiators to initiate the AHR circuit. The programmable nature of the auxiliary hairpin allows its easy adaption into other MTase assay by simply changing the recognition site. This proposed AHR circuit permits a sensitive, robust, and versatile analysis of MTase with the limit of detection (LOD) of 0.011 U/mL. Lastly, the AHR circuit could be utilized for MTase analysis in real complex samples and for evaluating the cell-cycle-dependent expression of MTase. This developed MTase-sensing strategy holds promising potential for biomedical analysis and clinical diagnosis.

Integrative Genome-Wide DNA Methylome and Transcriptome Analysis of Ovaries from Hu Sheep with High and Low Prolific.

DNA methylation plays an important role in biological processes by affecting gene expression. However, how DNA methylation regulates phenotypic variation in Hu sheep remains unclear. Therefore, we generated genome-wide DNA methylation and transcriptomic profiles in the ovaries of Hu sheep with different prolificacies and genotypes (FecBB and FecB+). Results showed that ovary DNA methylome and transcriptome were significantly different between high prolificacy and low prolificacy Hu sheep. Comparative methylome analyses identified 10,644, 9,594, and 12,214 differentially methylated regions and 87, 1,121, and 2,375 genes, respectively, showing differential expression levels in three different comparison groups. Female reproduction-associated differentially methylated regions-related genes and differentially expressed genes were enriched, thereby the respective interaction networks were constructed. Furthermore, systematical integrative analyses revealed a negative correlation between DNA methylation around the transcriptional start site and gene expression levels, which was confirmed by testing the expression of integrin ß2 subunit (ITGB2) and lysosome-associated protein transmembrane-4 beta (LAPTM4B) in vivo and in vitro. These findings demonstrated that DNA methylation influences the propensity for prolificacy by affecting gene expression in the ovaries, which may contribute to a greater understanding of the epigenome and transcriptome that will be useful for animal breeding.

Real-Time Investigation of Intracellular Polynucleotide Kinase Using a Cascaded Amplification Circuit.

Polynucleotide kinase (PNK) shows an in-depth correlationship with DNA repair and metabolism processes. The in situ visualization of intracellular PNK revealed an extremely biological significance in supplementing reliable and quantitative information on its spatiotemporal distribution in live cells. Herein, we developed a versatile cascaded DNA amplification circuit through the integration of catalytic DNA assembly and hybridization chain reaction circuits and realized the accurate evaluation of intracellular PNK activity via the Förster resonance energy transfer (FRET) principle. Initially, without PNK, trigger T was firmly caged in the PNK-recognizing hairpin HT, resulting in no disturbance of the concatenated circuit. However, with the introduction of PNK, the 5'-OH terminal of PNK-addressing HT was phosphorylated, then the phosphorylated HT could be subsequently digested by λ exonuclease (λ Exo) to produce trigger T of the cascaded DNA circuit. As a result, the integrated circuit was stimulated to produce an amplified FRET signal for quantitatively monitoring the activity of PNK. Due to the λ Exo-specific digestion of 5'-phosphate DNA and the high signal gain of the cascade circuit, our proposed strategy enables the sensitive analysis of PNK activity in vitro and in complex biological samples. Furthermore, our PNK-sensing platform was extensively explored in HeLa cells for realizing reliable intracellular PNK imaging and thus showed high potential in the future diagnosis and treatment of kinase-related diseases.

Construction of an Enzyme-Free Initiator-Replicated Hybridization Chain Reaction Circuit for Amplified Methyltransferase Evaluation and Inhibitor Assay.

The enzyme-free nucleic acid amplification circuit, for example, hybridization chain reaction (HCR), has paved a broad avenue for evaluating various enzyme-involved biotransformations, including DNA methyltransferases (MTases). The nonenzymatic MTase-sensing platform has supplemented a versatile toolbox for monitoring aberrant methylation in intricate biological samples, yet their amplification efficiency is always constrained by the initiator-depletion paradigm. Herein, the autonomously initiator-replicated HCR (IR-HCR) was developed as a versatile amplification system for detecting MTase with ∼100-fold sensitivity of the conventional HCR system. The initiator I-triggered HCR leads the assembly of a tandem DNAzyme concatemer that cleaves its substrate. This leads to the cyclic replication of a new initiator I for reversely motivating the initial HCR circuit, resulting in a dramatic Förster resonance energy transfer (FRET) readout. Without M.SssI MTase, hairpin HM can be recognized and digested by restriction endonuclease HpaII to release initiator I for stimulating a high FRET signal. While the M.SssI-methylated HM prohibits the HpaII-mediated cleavage of HM, the caged initiator I fails to trigger the IR-HCR circuit. Based on a systematic investigation, the IR-HCR circuit readily achieves selective and sensitive analysis of M.SssI MTase and its inhibitors. As a general MTase-sensing platform, the IR-HCR principle was further applied to analyze another MTase (Dam) by redesigning HM with the Dam recognition sequence. Overall, the versatile homogeneous MTase sensing platform was achieved via an efficient and robust initiator replication amplification circuit and may have enormous potential for early disease diagnosis.

Effect of CREB1 promoter non-CpG island methylation on its differential expression profile on sheep ovaries associated with prolificacy.

This study aimed to investigate the effect of different methylated regions of cyclic-AMP response element binding protein 1 (CREB1) by comparing the high prolificacy (HP) group and low prolificacy (LP) group, which was detected in our previous study. The expression level of CREB1 mRNA in the ovaries of the HP group was higher than in the LP group (P < 0.05). The differential methylated region (DMR) had 4 methylated CG dinucleotides(CGs): -1546, -1544, -1494 and -1464. The DNA methylation levels of -1546 CGs and -1464 CGs were significantly higher in the HP group than in the LP group (P < 0.05). The activity from -1296 to +26 (without DMR) was significantly higher than the activity from -1598 to +26 (with DMR) (P < 0.05). The result of 5-aza-2'-deoxycytidine treatment indicated that the inhibition DNA methylation of DMR reduced the transcription of CREB1. The bioinformatics predictive analysis were found that the -1546 CG site was located in the CCAAT/enhancer-binding protein alpha (CEBPA) binding site and the -1464 CG site was located in the Sp1 binding site. Finally, this study revealed the relationship between the methylation of non-CpG sites of the promoter and transcription of CREB1. This study will provide a theoretical basis of the Hu sheep ovaries associated with DNA methylation.

Comprehensive analysis of long noncoding RNA and mRNA expression patterns in sheep testicular maturation.

Long noncoding RNAs (LncRNAs) have been identified as important regulators of testis development; however, their expression patterns and roles in sheep are not yet clear. Thus, we used stranded specific RNA-seq to profile the testis transcriptome (lncRNAs and mRNAs) in premature and mature sheep. Hormone levels and the testis index were examined, and histological analyses were performed at five stages of testis development, 5-day-old (D5), 3-month-old (3M), 6-month-old (6M), 9-month-old (9M), and 2-year-old (2Y), the results of which indicate a significant difference in hormone levels and testis morphometries between the 3M and 9M stages (P < 0.05). Based on the comparison between 3M and 9M samples, we found 1,118 differentially expressed (DE) lncRNAs and 7,253 DE mRNAs in the testes, and qRT-PCR analysis showed that the results correlated well with the transcriptome data. Furthermore, we constructed lncRNA-protein-coding gene interaction networks. Forty-seven DE lncRNA-targeted genes enriched for male reproduction were obtained by cis- and trans-acting; 51 DE lncRNAs and 45 cis-targets, 2 DE lncRNAs and 2 trans-targets were involved in this network. Of these, 5 lncRNAs and their targets, PRKCD, NANOS3, SERPINA5, and CYP19A1, were enriched for spermatogenesis and male gonad development signaling pathways. We further examined the expression levels of 5 candidate lncRNAs and their target genes during testis development. Lastly, the interaction of lncRNA TCONS__00863147 and its target gene PRKCD was validated in vitro in sheep Leydig cells. This study provides a valuable resource for further study of lncRNA function in sheep testis development and spermatogenesis.

Genome-wide differential expression profiling of mRNAs and lncRNAs associated with prolificacy in Hu sheep.

Reproductive ability, especially prolificacy, impacts sheep profitability. Hu sheep, a unique Chinese breed, is recognized for its high prolificacy (HP), early sexual maturity, and year-round estrus. However, little is known about the molecular mechanisms underlying HP in Hu sheep. To explore the potential mRNAs and long non-coding RNAs (lncRNAs) involved in Hu sheep prolificacy, we performed an ovarian genome-wide analysis of mRNAs and lncRNAs during the follicular stage using Hu sheep of HP (litter size = 3; three consecutive lambings) and low prolificacy (LP, litter size = 1; three consecutive lambings). Plasma luteinizing hormone (LH) concentration was higher in the HP group than in the LP group (P<0.05) during the follicular stage. Subsequently, 76 differentially expressed mRNAs (DE-mRNAs) and five differentially expressed lncRNAs (DE-lncRNAs) were identified by pairwise comparison; quantitative real-time PCR (qRT-PCR) analysis of ten randomly selected DE genes (mRNA and lncRNA) were consistent with the sequencing results. Gene Ontology (GO) analysis of DE-mRNAs revealed significant enrichment in immune response components, actin filament severing and phagocytosis. Pathway enrichment analysis of DE-mRNAs indicated a predominance of immune function pathways, including phagosomes, lysosomes, and antigen processing. We constructed a co-expression network of DE-mRNAs and mRNA-lncRNAs, with C1qA, CD53, cathepsin B (CTSB), CTSS, TYROBP, and AIF1 as the hub genes. Finally, the expression of lysosomal protease cathepsin genes, CTSB and cathepsin D (CTSD), were significantly up-regulated in sheep ovaries in the HP group compared with the LP group (P<0.05). These differential mRNAs and lncRNAs may provide information on the molecular mechanisms underlying sheep prolificacy.

Age-associated expression of vitamin D receptor and vitamin D-metabolizing enzymes in the male reproductive tract and sperm of Hu sheep.

The cellular response to 1,25-dihydroxyvitamin D3 (Vit D3; biologically active form of Vitamin D) is complex and depends not only on Vitamin D receptor (VDR) expression but also on cellular uptake of circulating Vit D3 and the presence and activity of Vitamin D-metabolizing enzyme. This study evaluated the expression of VDR and Vitamin D-metabolizing enzymes in the ram reproductive tract at different developmental stages and in spermatozoa. Nearly all cell types in the testes and epithelial cells of the caput, corpus, and cauda expressed VDR, CYP27B1, and CYP24A1 proteins. The mRNA and protein expression of CYP2R1, CYP27A1, and CYP27B1 in the testes and cauda increased significantly with increasing age (P < 0.05). However, epididymal VDR mRNA and protein expression showed no significant difference (P < 0.05) between adult (9- and 24-month-old) and prepubertal (3-month-old) rams. Furthermore, VDR and CYP24A1 were mainly concentrated in the mid-piece of ejaculated or cauda epididymis spermatozoa or both. Additionally, VDR and CYP27B1 mRNA and protein expression levels were significantly higher in ejaculated spermatozoa than in cauda epididymal spermatozoa (P < 0.05). Moreover, VDR and CYP24A1 expression was significantly higher in high-motility than in low-motility spermatozoa (P < 0.05). The diverse expression patterns of VDR and Vitamin D-metabolizing enzymes in the ram reproductive tract at different developmental stages and spermatozoa suggest it plays a potential role in spermatogenesis.

Influences of different dietary energy level on sheep testicular development associated with AMPK/ULK1/autophagy pathway.

Energy balance is an important feature for spermatozoa production in the testis. The 5'-AMP-activated protein kinase (AMPK) is a sensor of cell energy, has been implicated as a mediator between gonadal function and energy balance. Herein, we intended to determine the physiological effects of AMPK on testicular development in feed energy restricted and compensated pre-pubertal rams. Lambs had restricted feeding for 2 months and then provided compensatory feeding for another 3 months. Feed levels were 100%(control), 15% and 30% of energy restriction (ER) diets, respectively. The results showed that lambs fed the 30% ER diet had significantly lower testicular weight (P < .05) and spermatids number in the seminiferous tubules, but there were no differences between control and 15% ER groups. Meanwhile, 15% ER and 30% ER diets induced testis autophagy and apoptosis through activating AMPK-ULK1(ULK1, Unc-51 like autophagy activating kinase) signal pathway with characterization of increased Beclin-1 and Light chain 3-Ⅱ/Light chain 3-Ⅰ (LC3-II/LC3-I) ratio, up-regulated the ratio of pro-apoptotic Bcl-2-associated X protein (BAX) and anti-apoptotic B-cell lymphoma 2 (Bcl-2), as well as activated AMPK, phosphorylated AMPK(p-AMPK) and ULK1. Furthermore, a compensation of these parameters occurred when the lambs were re-fed with normal energy requirement after restriction. Taken together, dietary energy levels influence testicular development through autophagy and apoptosis interplay mediated by AMPK-ULK1 signal pathway, which also indicates the important role of the actions of AMPK in the testis homeostasis.

Expression and localization of polypeptide N-acetylgalactosaminyltransferase-like protein 5 in the reproductive organs and sperm of Hu sheep.

The polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5) is involved in male fertility; however, its involvement in the reproduction and fertility of females remains unclear. Therefore, the present study aimed to examine the presence of GALNTL5 in the reproductive organs of ewes during the estrus period and to investigate its expression in cauda epididymal and ejaculated sperm. Results showed that GALNTL5 mRNA and protein were present in some reproductive organs of ewes during the estrus period. The highest levels of GALNTL5 mRNA and protein occurred in the uterine horn and oviductal ampulla and the lowest in the uterine cervix and oviductal infundibulum. Immunohistochemical analysis revealed that GALNTL5 protein was mainly located in luminal and glandular epithelial cells of the uterus and oviduct, and in the theca and granulosa cells of the ovary. GALNTL5 gene expression was significantly higher in ejaculated sperm than in cauda epididymal sperm. The amount of GALNTL5 protein in seminal plasma was significantly higher than in ejaculated sperm. Additionally, GALNTL5 was strongly localized in the mid-piece and head of ejaculated sperm, and in the head-tail coupling apparatus and acrosome of cauda epididymal sperm. This is the first evidence that GALNTL5 might play an important role in a range of reproductive functions as well as in sperm motility and capacitation. Further studies are required to evaluate the function of GALNTL5 in reproduction.

Genome-wide analysis of DNA Methylation profiles on sheep ovaries associated with prolificacy using whole-genome Bisulfite sequencing.

BACKGROUND: Ovulation rate and litter size are important reproductive traits in sheep with high economic value. Recent work has revealed a potential link between DNA methylation and prolificacy. However, a genome-wide study that sought to identify potential DNA methylation sites involved in sheep prolificacy indicated that it is still unknown. Here, we aimed to investigate the genome-wide DNA methylation profiles of Hu sheep ovaries by comparing a high-prolificacy group (HP, litter size of three for at least 2 consecutive lambings) and low prolificacy group (LP, litter size of one for at least 2 consecutive lambings) using deep whole-genome bisulfite sequencing (WGBS). RESULTS: First, our results demonstrated lower expression levels of DNA methyltransferase (DNMT) genes in the ovaries of the HP group than that in the ovaries of the LP group. Both groups showed similar proportions of methylation at CpG sites but different proportions at non-CpG sites. Subsequently, we identified 70,899 differential methylated regions (DMRs) of CG, 16 DMRs of CHG, 356 DMRs of CHH and 12,832 DMR-related genes(DMGs). Gene Ontology (GO) analyses revealed that some DMGs were involved in regulating female gonad development and ovarian follicle development. Finally, we found that 10 DMGs, including BMP7, BMPR1B, CTNNB1, FST, FSHR, LHCGR, TGFB2 and TGFB3, are more likely to be involved in prolificacy of Hu sheep, as assessed by correlation analysis and listed in detail. CONCLUSIONS: This study revealed the global DNA methylation pattern of sheep ovaries associated with high and low prolificacy groups, which may contribute to a better understanding of the epigenetic regulation of sheep reproductive capacity.

Genome-Wide Analysis Reveals Extensive Changes in LncRNAs during Skeletal Muscle Development in Hu Sheep.

As an important type of noncoding RNA molecules, long non-coding RNAs (lncRNAs) act as versatile players in various biological processes. However, little is known about lncRNA regulators during sheep muscle growth. To explore functional lncRNAs during sheep muscle growth, we systematically investigated lncRNAs using strand-specific Ribo-Zero RNA sequencing at three key developmental stages in Hu sheep. A total of 6924 lncRNAs were obtained, and the differentially expressed lncRNAs and genes were screened from (control vs. experiment) fetus vs. lamb, lamb vs. adult, and fetus vs. adult comparisons, respectively. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis results correlated well with the sequencing data. Moreover, functional annotation analysis based on the Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) databases showed that the target genes of the differentially expressed lncRNAs were significantly enriched in organ morphogenesis, skeletal system development as well as response to stimulus and some other terms related to muscle. Furthermore, a co-expression network of the differentially expressed target genes and lncRNAs was constructed and well-known muscle growth regulators such as retrotransposon-like 1 and Junctophilin-2 were included. Finally, we investigated the expression profiles of seven lncRNAs and their target genes, and found that they played vital roles in muscle growth. This study extends the sheep muscle lncRNA database and provides novel candidate regulators for future genetic and molecular studies on sheep muscle growth, which is helpful for optimizing the production of mutton.

Bisphenol A affects cell viability involved in autophagy and apoptosis in goat testis sertoli cell.

Bisphenol A (BPA) is shown to be the endocrine disruptor that induces reproductive dysfunction in male animals. In this study, we aim to probe the effects of BPA exposure on induction of autophagy in goat Sertoli Cells (gSCs), as well as the relationship between autophagy and apoptosis. Results indicated that exposure to BPA (100, 200, 300, 400, 500 and 600µM) decreased the cell viability in a concentration-dependent manner. Exposure of gSCs to 500µM BPA for 12h resulted in in vitro triggered loss of mitochondrial membrane potential (ΔΨm) and increased reactive oxygen species (ROS) production. Apoptosis with an increase in Bax:Bcl-2 ratio and higher rates of autophagy, such as autophagosome formation and increased expression of autophagy-related markers were also induced in gSCs exposed to 500µM BPA. Furthermore, treatment with 350nM Rapamycin (Rap, autophagy activator) alleviated a decrease in cell viability, intracellular ROS production, and reduction of ΔΨm, as well as decreasing apoptosis. Collectively, our results indicated that gSCs viability was disrupted after BPA treatment through affecting ROS production, mitochondrial membrane potential and inducing autophagy/apoptosis.

Long noncoding RNA expression profile changes associated with dietary energy in the sheep testis during sexual maturation.

Spermatogenesis can be affected by nutrition, which operates through normal physiological processes by changing the testicular mass and hormone levels profoundly. However, little is known regarding how testis development is regulated by long noncoding RNA (lncRNA). In this study, we investigated the effects of high-grain (HG) feeding on testis development during sexual maturation mediated by lncRNA. The HG diet group showed an increase in growth hormone (GH), insulin-like growth factor-1 (IGF-1) and testosterone (T) levels, and in the number of sperm in the seminiferous tubules compared with the hay-fed group (p < 0.05). Moreover, we found 59 differentially expressed (DE) lncRNAs and 229 DE mRNAs in sheep testis between the two groups. qRT-PCR results of 20 randomly selected DE lncRNAs and mRNAs were also consistent with the RNA-seq data. Through functional enrichment analysis and lncRNA-mRNA interaction network analysis, we screened several lncRNAs that may be enriched for male reproduction such as spermatogenesis, sperm motility, steroid hormones, MAPK and ErbB signaling pathways. This study provides a first insight into the development of the testis with HG feeding in sheep and shows that these changes are associated with alterations in lncRNA expression.