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In mammals, a new life starts with the fusion of an oocyte and a sperm cell. Parthenogenesis, a way of generating offspring solely from female gametes, is limited because of problems arising from genomic imprinting. Here, we report live mammalian offspring derived from single unfertilized oocytes, which was achieved by targeted DNA methylation rewriting of seven imprinting control regions. Oocyte coinjection of catalytically inactive Cas9 (dCas9)-Dnmt3a or dCpf1-Tet1 messenger RNA (mRNA) with single-guide RNAs (sgRNAs) targeting specific regions induced de novo methylation or demethylation, respectively, of the targeted region. Following parthenogenetic activation, these edited regions showed maintenance of methylation as naturally established regions during early preimplantation development. The transfer of modified parthenogenetic embryos into foster mothers resulted in significantly extended development and finally in the generation of viable full-term offspring. These data demonstrate that parthenogenesis can be achieved by targeted epigenetic rewriting of multiple critical imprinting control regions. [ABSTRACT FROM AUTHOR]

Mammalian oocytes carry specific nongenetic information, including DNA methylation to the next generation, which is important for development and disease. However, evaluation and manipulation of specific methylation for both functional analysis and therapeutic purposes remains challenging. Here, we demonstrate evaluation of specific methylation in single oocytes from its sibling first polar body (PB1) and manipulation of specific methylation in single oocytes by microinjection-mediated dCas9-based targeted methylation editing. We optimized a single-cell bisulfite sequencing approach with high efficiency and demonstrate that the PB1 carries similar methylation profiles at specific regions to its sibling oocyte. By bisulfite sequencing of a single PB1, the methylation information regarding agouti viable yellow (Avy)-related coat color, as well as imprinting linked parthenogenetic development competency, in a single oocyte can be efficiently evaluated. Microinjection-based dCas9-Tet/Dnmt-mediated methylation editing allows targeted manipulation of specific methylation in single oocytes. By targeted methylation editing, we were able to reverse Avy-related coat color, generate full-term development of bimaternal mice, and correct familial Angelman syndrome in a mouse model. Our work will facilitate the investigation of specific methylation events in oocytes and provides a strategy for prevention and correction of maternally transmitted nongenetic disease or disorders. [ABSTRACT FROM AUTHOR]

Background: Our study aim was to evaluate the therapeutic efficacy and mechanisms of miR-133-overexpressing mesenchymal stem cells (MSCs) on acute myocardial infarction. Methods: Rat MSCs were isolated and purified by whole bone marrow adherent culturing. After transfection with the agomir or antagomir of miR-133, MSCs were collected for assay of cell vitality, apoptosis and cell cycle progression. At the same time, exosomes were isolated from the supernatant to analyze the paracrine miR-133. For in-vivo studies, constitutive activation of miR-133 in MSCs was achieved by lentivirus-mediated miR-133 overexpression. A rat myocardial infarction model was created by ligating the left anterior descending coronary artery, while control MSCs (vector-MSCs) or miR-133-overexpressed MSCs (miR-133-MSCs) were injected into the zone around the myocardial infarction. Subsequently, myocardial function was evaluated by echocardiography on days 7 and 28 post infarction. Finally the infarcted hearts were collected on days 7 and 28 for myocardial infarct size measurement and detection of snail 1 expression. Results: Hypoxia-induced apoptosis of MSCs obviously reduced, along with enhanced expression of total poly ADP-ribose polymerase protein, after miR-133 agomir transfection, while the apoptosis rate increased in MSCs transfected with miR-133 antagomir. However, no change in cell viability and cell-cycle distribution was observed in control, miR-133-overexpressed, and miR-133-interfered MSCs. Importantly, rats transplanted with miR-133-MSCs displayed more improved cardiac function after acute myocardial infarction, compared with those that received vector-MSC injection. Further studies indicated that cardiac expression of snail 1 was significantly repressed by adjacent miR-133-overexpressing MSCs, and both the inflammatory level and the infarct size decreased in miR-133-MSC-injected rat hearts. Conclusions: miR-133-MSCs obviously improved cardiac function in a rat model of myocardial infarction. Transplantation of miR-133-overexpressing MSCs provides an effective strategy for cardiac repair and modulation of cardiac-related diseases. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases are the leading causes of death in the world. The limited regenerative capacity of adult cardiomyocytes is the major barrier for heart regeneration. After myocardial infarction, myofibroblasts are the dominant cell type in the infarct zone. Therefore, it is a good idea to reprogram terminally differentiated myofibroblasts into cardiomyocyte-like cells directly, providing a good strategy to simultaneously reduce scar tissue and increase functional cardiomyocytes. Transcription factors were first identified to reprogram myofibroblasts into cardiomyocytes. Thereafter, microRNAs and/or small molecules showed great potential to optimize the reprogramming process. Here, we systemically summarize and compare the major progress in directed cardiac reprogramming including transcription factors and miRNAs, especially the small molecules. Furthermore, we discuss the challenges needed to be overcome to apply this strategy clinically. [ABSTRACT FROM AUTHOR]

Mammalian early embryos maintain accurate genome integrity for proper development within a programmed timeline despite constant assaults on their DNA by replication, DNA demethylation and genetic defects transmitted from germcells. However, how genome integrity is safeguarded during mammalian early embryonic development remains unclear. BCAS2 (breast carcinoma amplified sequence 2), a core component of thePRP19 complex involved in pre-mRNAsplicing, plays an important role in the DNA damage response through the RPA complex, a key regulator in the maintenance of genome integrity. Currently, the physiological role of BCAS2 in mammals is unknown. We now report that BCAS2 responds to endogenous and exogenous DNA damage in mouse zygotes. Maternal depletion of BCAS2 compromises the DNA damage response in early embryos, leading to developmental arrest at the two- to four-cell stage accompanied by the accumulation of damaged DNA and micronuclei. Furthermore, BCAS2 mutants that are unable to bind RPA1 fail in DNA repair during the zygotic stage. In addition, phosphorylated RPA2 cannot localise to the DNA damage sites in mouse zygotes with disrupted maternal BCAS2. These data suggest that BCAS2 might function through the RPA complex during DNA repair in zygotes. Together, our results reveal that maternal BCAS2 maintains the genome integrity of early embryos and is essential for female mouse fertility. [ABSTRACT FROM AUTHOR]

The global prevalence of prediabetes and type 2 diabetes (T2D) is increasing, and it is contributing to the susceptibility to diabetes and its related epidemic in offspring. Although the impacts of paternal impaired fasting blood glucose and glucose intolerance on the metabolism of offspring have been well established, the exact molecular and mechanistic basis that mediates these impacts remains largely unclear. Here we show that paternal prediabetes increases the susceptibility to diabetes in offspring through gametic epigenetic alterations. In our findings, paternal prediabetes led to glucose intolerance and insulin resistance in offspring. Relative to controls, offspring of prediabetic fathers exhibited altered gene expression patterns in the pancreatic islets, with down-regulation of several genes involved in glucose metabolism and insulin signaling pathways. Epigenomic profiling of offspring pancreatic islets revealed numerous changes in cytosine methylation depending on paternal prediabetes, including reproducible changes in methylation over several insulin signaling genes. Paternal prediabetes altered overall methylome patterns in sperm, with a large portion of differentially methylated genes overlapping with that of pancreatic islets in offspring. Our study uniquely revealed that prediabetes can be inherited transgenerationally through the mammalian germ line by an epigenetic mechanism. [ABSTRACT FROM AUTHOR]

Background: Junctional adhesion molecule 2 (Jam2) is a member of the JAM superfamily. JAMs are localized at intercellular contacts and participated in the assembly and maintenance of junctions, and control of cell permeability. Because Jam2 is highly expressed in the luminal epithelium on day 4 of pregnancy, this study was to determine whether Jam2 plays a role in uterine receptivity and blastocyst attachment in mouse uterus. Methodology/Principal Findings: Jam2 is highly expressed in the uterine luminal epithelium on days 3 and 4 of pregnancy. Progesterone induces Jam2 expression in ovariectomized mice, which is blocked by progesterone antagonist RU486. Jam2 expression on day 4 of pregnancy is also inhibited by RU486 treatment. Leukemia inhibitory factor (LIF) up-regulates Jam2 protein in isolated luminal epithelium from day 4 uterus, which is blocked by S3I-201, a cell-permeable inhibitor for Stat3 phosphorylation. Under adhesion assay, recombinant Jam2 protein increases the rate of blastocyst adhesion. Both soluble recombinant Jam2 and Jam3 can reverse this process. Conclusion: Jam2 is highly expressed in the luminal epithelium of receptive uterus and up-regulated by progesterone and LIF via tyrosine phosphorylation of Stat3. Jam2 may play a role in the interaction between hatched blastocyst and receptive uterus. [ABSTRACT FROM AUTHOR]