Reconstruction of artificial nuclei with nuclear import activity in living mouse oocytes.

In eukaryotes, DNA is housed within the cell nucleus. Molecules required for the formation of a nucleus have been identified using in vitro systems with frog egg extracts and in vivo imaging of somatic cells. However, little is known about the physicochemical factors and conditions required for nuclear formation in mouse oocytes. In this study, using a reconstitution approach with purified DNA, we aimed to determine factors, such as the amount and timing of DNA introduction, required for the formation of nuclei with nuclear transport activity in mouse oocytes. T4 phage DNA (~166 kbp) was microinjected into strontium-activated oocytes to evaluate the conditions appropriate for nuclear formation. Microinjection of 100-500 ng/µL of T4 DNA, but not 20 ng/µL, was sufficient for the formation of nucleus-like structures. Furthermore, microinjection of DNA during metaphase II to telophase II, but not during interphase, was sufficient. Electron and fluorescence microscopy showed that T4 DNA-induced nucleus-like structures had nuclear lamina and nuclear pore complex structures similar to those of natural nuclei, as well as nuclear import activity. These results suggest that exogenous DNA can form artificial nuclei with nuclear transport functions in mouse oocytes, regardless of the sequence or source of the DNA.

Intraperitoneal administration of mouse kisspeptin-10 to mice during estrus stage induces pseudopregnancy.

A simple method for producing pseudopregnant mice supports pup production. In this study, pregnant ICR were obtained mice without mating with vasectomized mice via administration of mouse Kisspeptin-10 (mKp-10) and transferring blastocysts to the uterus. Blastocyst transfer after mKp-10 administration to mice with gapping and reddish pink vagina resulted in 65.2% (15/23) pregnancies, and 39.1% (34/87) of the transferred blastocysts showed full-term growth. Vaginal smears were observed for accurate estrus cycle determination, and subsequent administration of mKp10 to mice during the estrus stage and blastocyst transfer resulted in 95.2% (20/21) pregnancies and 50.7% (104/205) birth rates. Regarding 2-cell transfer after administration of mKp-10, 100% (8/8) of the mice became pregnant, and 45.0% (36/80) of the embryos were born. Administration of mKp-10 to mice during the estrus stage is a convenient way to generate pseudopregnant mice.

Visualizing histone H4K20me1 in knock-in mice expressing the mCherry-tagged modification-specific intracellular antibody.

During development and differentiation, histone modifications dynamically change locally and globally, associated with transcriptional regulation, DNA replication and repair, and chromosome condensation. The level of histone H4 Lys20 monomethylation (H4K20me1) increases during the G2 to M phases of the cell cycle and is enriched in facultative heterochromatin, such as inactive X chromosomes in cycling cells. To track the dynamic changes of H4K20me1 in living cells, we have developed a genetically encoded modification-specific intracellular antibody (mintbody) probe that specifically binds to the modification. Here, we report the generation of knock-in mice in which the coding sequence of the mCherry-tagged version of the H4K20me1-mintbody is inserted into the Rosa26 locus. The knock-in mice, which ubiquitously expressed the H4K20me1-mintbody, developed normally and were fertile, indicating that the expression of the probe does not disturb the cell growth, development, or differentiation. Various tissues isolated from the knock-in mice exhibited nuclear fluorescence without the need for fixation. The H4K20me1-mintbody was enriched in inactive X chromosomes in developing embryos and in XY bodies during spermatogenesis. The knock-in mice will be useful for the histochemical analysis of H4K20me1 in any cell types.

Effect of ovarian stimulation on developmental speed of preimplantation embryo in a mouse model.

Ovarian stimulation protocols are widely used to collect oocytes in assisted reproductive technologies (ARTs). Although the influence of ovarian stimulation on embryo quality has been described, this issue remains controversial. Here, we analyzed the influence of ovarian stimulation on developmental speed and chromosome segregation using live cell imaging. Female mice at the proestrus stage were separated by the appearance of the vagina as the non-stimulation (-) group, and other mice were administered pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as the stimulation (+) groups. The cumulus-oocyte complexes from both groups were inseminated with sperm suspensions from the same male mice. Fertilization rates and developmental capacities were examined, and the developmental speed and frequency of chromosome segregation errors were measured by live-cell imaging using a Histone H2B-mCherry probe. The number of fertilized oocytes obtained was 1.4-fold more frequent in the stimulation (+) group. The developmental rate and chromosome stability did not differ between the groups. Image analysis showed that the mean speed of development in the stimulation (+) group was slightly higher than that in the non-stimulation (-) group. This increase in speed seemed to arise from the slight shortening of the 2- and 4-cell stages and third division lengths and consequent synchronization of cleavage timing in each embryo, not from the emergence of an extremely rapidly developing subpopulation of embryos. In conclusion, ovarian stimulation does not necessarily affect embryo quality but rather increases the chances of obtaining high-quality oocytes in mice.

Chromosome counting in the mouse zygote using low-invasive super-resolution live-cell imaging.

In preimplantation embryos, an abnormal chromosome number causes developmental failure and a reduction in the pregnancy rate. Conventional chromosome testing methods requiring biopsy reduce the risk of associated genetic diseases; nevertheless, the reduction in cell number also reduces the pregnancy rate. Therefore, we attempted to count the chromosomes in mouse embryos using super-resolution live-cell imaging as a new method of chromosome counting that does not reduce the cell number or viability. We counted the 40 chromosomes at the first mitosis by injecting embryos with histone H2B-mCherry mRNA under conditions by which pups could be obtained; however, the results were often an underestimation of chromosome number and varied by embryo and time point. Therefore, we developed a method to count the chromosomes via CRISPR/dCas-mediated live-cell fluorescence in situ hybridization targeting the sequence of the centromere region, enabling us to count the chromosomes more accurately in mouse embryos. The methodology presented here may provide useful information for assisted reproductive technologies, such as those used in livestock animals/humans, as a technique for assessing the chromosomal integrity of embryos prior to transfer.

Fatty acids act on vascular endothelial cells and influence the development of cardiovascular disease.

Endothelial cells (ECs) maintain the health of blood vessels and prevent the development of cardiovascular disease (CVD). Free saturated fatty acids (FAs) induce EC damage and increase the risk of CVD by promoting arteriosclerosis. Conversely, polyunsaturated FAs (PUFAs), such as docosahexaenoic acid, are thought to suppress EC damage induced during the early stages of CVD. This review describes the effects of multiple dietary FAs on EC disorders involved in the development of CVD. The roles of FAs in atherosclerosis and CVD were analyzed by evaluating articles published in PubMed, Science Direct, and Web of Science. Saturated FAs were found to induce EC damage by reducing the production and action of EC-derived nitric oxide. Oxidative stress, inflammation, and the renin-angiotensin system were found to be involved in EC disorder. Furthermore, n-3 PUFAs were found to reduce EC dysfunction and prevent the development of EC disorder. These results indicate that FAs may affect EC failure induced during the early stages of CVD and reduce the risk of developing the disease.

Docosahexaenoic acid inhibits ischemic stroke to reduce vascular dementia and Alzheimer's disease.

Stroke and dementia are global leading causes of neurological disability and death. The pathology of these diseases is interrelated and they share common, modifiable risk factors. It is suggested that docosahexaenoic acid (DHA) prevents neurological and vascular disorders induced by ischemic stroke and also prevent dementia. The purpose of this study was to review the potential preventative role of DHA against ischemic stroke-induced vascular dementia and Alzheimer's disease. In this review, I analyzed studies on stroke-induced dementia from the PubMed, ScienceDirect, and Web of Science databases as well as studies on the effects of DHA on stroke-induced dementia. As per the results of interventional studies, DHA intake can potentially ameliorate dementia and cognitive function. In particular, DHA derived from foods such as fish oil enters the blood and then migrates to the brain by binding to fatty acid binding protein 5 that is present in cerebral vascular endothelial cells. At this point, the esterified form of DHA produced by lysophosphatidylcholine is preferentially absorbed into the brain instead of free DHA. DHA accumulates in nerve cell membrane and is involved in the prevention of dementia. The antioxidative and anti-inflammatory properties of DHA and DHA metabolites as well as their ability to decrease amyloid beta (Aß) 42 production were implicated in the improvement of cognitive function. The antioxidant effect of DHA, the inhibition of neuronal cell death by Aß peptide, improvement in learning ability, and enhancement of synaptic plasticity may contribute to the prevention of dementia induced by ischemic stroke.

Micronucleus formation during early cleavage division is a potential hallmark of preimplantation embryonic loss in cattle.

In assisted reproductive technology (ART)-derived embryos of non-rodent mammals, including humans and cattle, chromosome segregation errors are highly likely to occur during early cleavage division, resulting in aneuploidy, including mosaicism. However, the relationship between chromosomal segregation errors during early cleavage and subsequent embryonic development has not been detailed in these mammals. In the present study, we developed non-invasive live-cell imaging of chromosome segregation dynamics using a histone H2B-mCherry mRNA probe in bovine preimplantation embryos. Chromosome segregation errors in early cleavage affected blastocyst formation. Especially, embryos that underwent abnormal chromosome segregation (ACS) with multiple or large micronucleus formation rarely developed into blastocysts. Embryos with the severe ACS had prolonged cell cycle duration. After transfer of blastocysts with live-cell imaging of chromosome segregation to ten cows, six became pregnant and four of them gave full-term offspring. Interestingly, two of them were derived from blastocysts with ACS. Hence, chromosomal segregation errors with micronucleus formation during early cleavage can be a fatal hallmark of preimplantation embryogenesis in cattle. This technique has shown potential for understanding the relationship between chromosome segregation error and subsequent embryo development, and for selecting viable ART-derived embryos for medical and livestock production.

Defective Function of the Blood-Brain Barrier in a Stroke-Prone Spontaneously Hypertensive Rat: Evaluation in an In Vitro Cell Culture Model.

The blood-brain barrier (BBB) comprises three cell types: brain capillary endothelial cells (BECs), astrocytes, and pericytes. Abnormal interaction among these cells may induce BBB dysfunction and lead to cerebrovascular diseases. The stroke-prone spontaneously hypertensive rat (SHRSP) harbors a defective BBB, so we designed the present study to examine the role of these three cell types in a functional disorder of the BBB in SHRSP in order to elucidate the role of these cells in the BBB more generally. To this end, we employed a unique in vitro model of BBB, in which various combinations of the cells could be tested. The three types of cells were prepared from both SHRSPs and Wistar Kyoto rats (WKYs). They were then co-cultured in various combinations to construct in vitro BBB models. The barrier function of the models was estimated by measuring transendothelial electrical resistance and the permeability of the endothelial monolayer to sodium fluorescein. The in vitro models revealed that (1) BECs from SHRSPs had an inherent lower barrier function, (2) astrocytes of SHRSPs had an impaired ability to induce barrier function in BECs, although (3) both pericytes and astrocytes of SHRSPs and WKYs could potentiate the barrier function of BECs under co-culture conditions. Furthermore, we found that claudin-5 expression was consistently lower in models that used BECs and/or SHRSP astrocytes. These results suggested that defective interaction among BBB cells-especially BECs and astrocytes-was responsible for a functional disorder of the BBB in SHRSPs.

Astrocyte-induced synapse formation and ischemic stroke.

Astrocytes are closely associated with the regulation of synapse formation and function. In addition, astrocytes have been shown to block certain brain impairments, including synaptic damage from stroke and other diseases of the central nervous system (CNS). Although astrocytes do not completely prevent synaptic damage, they appear to be protective and to restore synaptic function following damage. The purpose of this study is to discuss the role of astrocytes in synaptogenesis and synaptic damage in ischemic stroke. I detail the mechanism of action of the multiple factors secreted by astrocytes that are involved in synapse formation. In particular, I describe the characteristics and role in synapse formation of each secreted molecule related to synaptic structure and function. Furthermore, I discuss the effect of astrocytes on synaptogenesis and repair in ischemic stroke and in other CNS diseases. Astrocytes release molecules such as thrombospondin, hevin, secreted protein acidic rich in cysteine, etc., due to activation by ischemia to induce synaptic structure and function, an effect associated with protection of the brain from synaptic damage in ischemic stroke. In conclusion, I show that astrocytes may regulate synaptic transmission while having the potential to block and repair synaptic dysfunction in stroke-associated brain damage.

Dietary docosahexaenoic acid inhibits neurodegeneration and prevents stroke.

Stroke severely impairs quality of life and has a high mortality rate. On the other hand, dietary docosahexaenoic acid (DHA) prevents neuronal damage. In this review, we describe the effects of dietary DHA on ischemic stroke-associated neuronal damage and its role in stroke prevention. Recent epidemiological studies have been conducted to analyze stroke prevention through DHA intake. The effects of dietary intake and supply of DHA to neuronal cells, DHA-mediated inhibition of neuronal damage, and its mechanism, including the effects of the DHA metabolite, neuroprotectin D1 (NPD1), were investigated. These studies revealed that DHA intake was associated with a reduced risk of stroke. Moreover, studies have shown that DHA intake may reduce stroke mortality rates. DHA, which is abundant in fish oil, passes through the blood-brain barrier to accumulate as a constituent of phospholipids in the cell membranes of neuronal cells and astrocytes. Astrocytes supply DHA to neuronal cells, and neuronal DHA, in turn, activates Akt and Raf-1 to prevent neuronal death or damage. Therefore, DHA indirectly prevents neuronal damage. Furthermore, NDP1 blocks neuronal apoptosis. DHA, together with NPD1, may block neuronal damage and prevent stroke. The inhibitory effect on neuronal damage is achieved through the antioxidant (via inducing the Nrf2/HO-1 system) and anti-inflammatory effects (via promoting JNK/AP-1 signaling) of DHA.

Altered Properties of Neurons and Astrocytes and the Effects of Food Components in Stroke-Prone Spontaneously Hypertensive Rats.

ABSTRACT: In stroke-prone spontaneously hypertensive rats (SHRSP), stroke induces neuronal vulnerability and neuronal death, while astrocytes show a weakened support function toward neurons. Moreover, certain food components have been demonstrated to prevent the occurrence of stroke. This review aims to explain the stroke-related properties of SHRSP-derived neurons and astrocytes. In addition, it describes the effects of particular dietary phytochemicals on SHRSP. In this study, we obtained information using PubMed, ScienceDirect, and Web of Science. We searched for the functions of neurons and astrocytes and the molecular mechanism of ischemic stroke induction. We summarized the recent literature on the underlying mechanisms of stroke onset in SHRSP and the alleviating effects of typical food-derived phytochemical components. Neuronal death in SHRSP is induced by hypoxia-reoxygenation, suggesting the involvement of oxidative stress. Furthermore, the production of lactate, l-serine, and glial cell line-derived neurotrophic factor in SHRSP-derived astrocytes was reduced compared with that in control Wistar-Kyoto rats. Vitamin E exerts an inhibitory effect on hypoxia-reoxygenation-induced neuronal death in SHRSP. Curcumin, epigallocatechin gallate, resveratrol, and carotenoids can prevent the development of stroke in SHRSP. In particular, the properties of SHRSP-derived neurons and astrocytes affect stroke-induced neuronal death. This review suggests the potential and therapeutic applications of dietary phytochemicals in reducing stroke risk and lowering blood pressure in SHRSP, respectively, by targeting various processes, including oxidative stress, apoptosis, and inflammation. Thus, future research on SHRSP brain cells with a genetic predisposition to stroke can consider using these food ingredients to develop approaches for stroke prevention.

Erratum: Yamazaki et al. Editing DNA Methylation in Mammalian Embryos. Int. J. Mol. Sci. 2020, 21, 637.

The authors wish to make the following corrections to our previously published paper [...].

Potential Effects of Soy Isoflavones on the Prevention of Metabolic Syndrome.

Isoflavones are polyphenols primarily contained in soybean. As phytoestrogens, isoflavones exert beneficial effects on various chronic diseases. Metabolic syndrome increases the risk of death due to arteriosclerosis in individuals with various pathological conditions, including obesity, hypertension, hyperglycemia, and dyslipidemia. Although the health benefits of soybean-derived isoflavones are widely known, their beneficial effects on the pathogenesis of metabolic syndrome are incompletely understood. This review aims to describe the association between soybean-derived isoflavone intake and the risk of metabolic syndrome development. We reviewed studies on soy isoflavones, particularly daidzein and genistein, and metabolic syndrome, using PubMed, ScienceDirect, and Web of Science. We describe the pathological characteristics of metabolic syndrome, including those contributing to multiple pathological conditions. Furthermore, we summarize the effects of soybean-derived daidzein and genistein on metabolic syndrome reported in human epidemiological studies and experiments using in vitro and in vivo models. In particular, we emphasize the role of soy isoflavones in metabolic syndrome-induced cardiovascular diseases. In conclusion, this review focuses on the potential of soy isoflavones to prevent metabolic syndrome by influencing the onset of hypertension, hyperglycemia, dyslipidemia, and arteriosclerosis and discusses the anti-inflammatory effects of isoflavones.

Protective Effect of Epigallocatechin Gallate on Endothelial Disorders in Atherosclerosis.

Healthy vascular endothelial cells regulate vascular tone and permeability, prevent vessel wall inflammation, enhance thromboresistance, and contribute to general vascular health. Furthermore, they perform important functions including the production of vasoactive substances such as nitric oxide (NO) and endothelium-derived hyperpolarizing factors, as well as the regulation of smooth muscle cell functions. Conversely, vascular endothelial dysfunction leads to atherosclerosis, thereby enhancing the risk of stroke, myocardial infarction, and other cardiovascular diseases (CVDs). Observational studies and randomized trials showed that green tea intake was inversely related to CVD risk. Furthermore, evidence indicates that epigallocatechin gallate (EGCG) found in green tea might exert a preventive effect against CVDs. EGCG acts as an antioxidant, inducing NO release and reducing endothelin-1 production in endothelial cells. EGCG enhances the bioavailability of normal NO by reducing levels of the endogenous NO inhibitor asymmetric dimethylarginine. Furthermore, it inhibits the enhanced expression of adhesion molecules such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 and attenuates monocyte adhesion. In addition, EGCG prevents enhanced oxidative stress through the Nrf2/HO-1 pathway. These effects indicate that it might prevent the production of reactive oxygen species, inhibit inflammation, and reduce endothelial cell apoptosis during the initial stages of atherosclerosis. The current review summarizes recent research in this area and discusses novel findings regarding the protective effect of EGCG on endothelial dysfunction and CVDs in general.

Inhibition of Endothelial Dysfunction by Dietary Flavonoids and Preventive Effects Against Cardiovascular Disease.

Cardiovascular disease (CVD), such as stroke, ischemic heart disease, and heart failure, accounts for many deaths, and its increasing incidence is a worldwide concern. Accumulating evidence suggests that the elevated risk of CVD caused by dysfunction of vascular endothelial cells and resultant arteriosclerosis can be mitigated by increased consumption of fruits and vegetables. These foods contain phytochemicals such as polyphenols and carotenoids, as well as dietary fiber. Flavonoids of the polyphenol class are found in vegetables, fruits, grains, bark, roots, stems, flowers, tea, and wine. Several studies have indicated that flavonoids reduce CVD mortality by inhibiting endothelial dysfunction. Flavonoids have a common carbon skeleton and are classified as flavonols, flavones, flavanols, flavanones, anthocyanidins, and isoflavones. In this review, we discuss recent progress in identifying the mechanisms by which dietary flavonoids improve vascular endothelial cell function. Furthermore, we describe the beneficial role that these flavonoids may play in preventing CVD caused by endothelial dysfunction-related atherosclerosis.

Editing DNA Methylation in Mammalian Embryos.

DNA methylation in mammals is essential for numerous biological functions, such as ensuring chromosomal stability, genomic imprinting, and X-chromosome inactivation through transcriptional regulation. Gene knockout of DNA methyltransferases and demethylation enzymes has made significant contributions to analyzing the functions of DNA methylation in development. By applying epigenome editing, it is now possible to manipulate DNA methylation in specific genomic regions and to understand the functions of these modifications. In this review, we first describe recent DNA methylation editing technology. We then focused on changes in DNA methylation status during mammalian gametogenesis and preimplantation development, and have discussed the implications of applying this technology to early embryos.

Reconstitution of the oocyte nucleolus in mice through a single nucleolar protein, NPM2.

The mammalian oocyte nucleolus, the most prominent subcellular organelle in the oocyte, is vital in early development, yet its key functions and constituents remain unclear. We show here that the parthenotes/zygotes derived from enucleolated oocytes exhibited abnormal heterochromatin formation around parental pericentromeric DNAs, which led to a significant mitotic delay and frequent chromosome mis-segregation upon the first mitotic division. A proteomic analysis identified nucleoplasmin 2 (NPM2) as a dominant component of the oocyte nucleolus. Consistently, Npm2-deficient oocytes, which lack a normal nucleolar structure, showed chromosome segregation defects similar to those in enucleolated oocytes, suggesting that nucleolar loss, rather than micromanipulation-related damage to the genome, leads to a disorganization of higher-order chromatin structure in pronuclei and frequent chromosome mis-segregation during the first mitosis. Strikingly, expression of NPM2 alone sufficed to reconstitute the nucleolar structure in enucleolated embryos, and rescued their first mitotic division and full-term development. The nucleolus rescue through NPM2 required the pentamer formation and both the N- and C-terminal domains. Our findings demonstrate that the NPM2-based oocyte nucleolus is an essential platform for parental chromatin organization in early embryonic development.

Live imaging of X chromosome reactivation dynamics in early mouse development can discriminate naïve from primed pluripotent stem cells.

Pluripotent stem cells can be classified into two distinct states, naïve and primed, which show different degrees of potency. One difficulty in stem cell research is the inability to distinguish these states in live cells. Studies on female mice have shown that reactivation of inactive X chromosomes occurs in the naïve state, while one of the X chromosomes is inactivated in the primed state. Therefore, we aimed to distinguish the two states by monitoring X chromosome reactivation. Thus far, X chromosome reactivation has been analysed using fixed cells; here, we inserted different fluorescent reporter gene cassettes (mCherry and eGFP) into each X chromosome. Using these knock-in 'Momiji' mice, we detected X chromosome reactivation accurately in live embryos, and confirmed that the pluripotent states of embryos were stable ex vivo, as represented by embryonic and epiblast stem cells in terms of X chromosome reactivation. Thus, Momiji mice provide a simple and accurate method for identifying stem cell status based on X chromosome reactivation.

Soy Isoflavones Inhibit Endothelial Cell Dysfunction and Prevent Cardiovascular Disease.

Soybeans are among the most popular foods worldwide, and intake of soy-containing foods has been associated with many health benefits in part because of it structure similar to estrogen. Epidemiologic studies have demonstrated that soy consumption improves serum profiles of hypercholesterolemic patients. Several studies have also indicated an inverse relationship between the consumption of soy isoflavones and the incidence of cardiovascular diseases (CVD). Soy is a rich dietary source of isoflavones. The main soy isoflavones are daidzein and genistein; equol, another isoflavone and a major intestinal bacterial metabolite of daidzein, is generated by enterobacterial effects. Many isoflavones have antioxidative effects and anti-inflammatory actions, as well as induce nitric oxide production to maintain a healthy endothelium and prevent endothelial cell dysfunction. These effects may limit the development of atherosclerosis and CVD and restore healthy endothelial function in altered endothelia. Although the evidence supporting the benefits of soy isoflavones in CVD prevention continues to increase, the association between soy isoflavones and disease is not fully understood. This review summarized recent progress in identifying the preventive mechanisms of action of dietary soybean isoflavones on vascular endothelial cells. Furthermore, it describes the beneficial roles that these isoflavones may have on endothelial dysfunction-related atherosclerosis.