Evaluating histone modification analysis of individual preimplantation embryos.

BACKGROUND: We previously reported a modification of the CUT&Tag method (NTU-CAT) that allows genome-wide histone modification analysis in individual preimplantation embryos. In the present study, NTU-CAT was further simplified by taking advantage of the Well-of-the-Well (WOW) system, which enables the processing of multiple embryos in a shorter time with less reagent and cell loss during the procedure (WOW-CUT&Tag, WOW-CAT). RESULTS: WOW-CAT allowed histone modification profiling from not only a single blastocyst but also from a portion of it. WOW-CAT generated similar H3K4me3 profiles as NTU-CAT, but they were closer to the profiles produced by chromatin immunoprecipitation-sequencing, such as a valley-like trend and relatively lower false positive rates, indicating that WOW-CAT may attenuate the bias of Tn5 transposase to cut open chromatin regions. Simultaneous WOW-CAT of two halves of single blastocysts was conducted to analyze two different histone modifications (H3K4me3 and H3K27ac) within the same embryo. Furthermore, trophectoderm cells were biopsied and subjected to WOW-CAT in anticipation of preimplantation diagnosis of histone modifications. WOW-CAT allowed the monitoring of epigenetic modifications in the main body of the embryo. For example, analysis of H3K4me3 modifications of XIST and DDX3Y in trophectoderm biopsies could be used to sex embryos in combination with quantitative PCR, but without the need for deep sequencing. CONCLUSIONS: These results suggest the applicability of WOW-CAT for flexible epigenetic analysis of individual embryos in preimplantation epigenetic diagnosis.

Chimeric PRMT6 protein produced by an endogenous retrovirus promoter regulates cell fate decision in mouse preimplantation embryos†.

Murine endogenous retrovirus with leucine tRNA primer, also known as MERVL, is expressed during zygotic genome activation in mammalian embryos. Here we show that protein arginine N-methyltransferase 6 (Prmt6) forms a chimeric transcript with MT2B2, one of the long terminal repeat sequences of murine endogenous retrovirus with leucine tRNA primer, and is translated into an elongated chimeric protein (PRMT6MT2B2) whose function differs from that of the canonical PRMT6 protein (PRMT6CAN) in mouse preimplantation embryos. Overexpression of PRMT6CAN in fibroblast cells increased asymmetric dimethylation of the third arginine residue of both histone H2A (H2AR3me2a) and histone H4 (H4R3me2a), while overexpression of PRMT6MT2B2 increased only H2AR3me2a. In addition, overexpression of PRMT6MT2B2 in one blastomere of mouse two-cell embryos promoted cell proliferation and differentiation of the blastomere into epiblast cells at the blastocyst stage, while overexpression of PRMT6CAN repressed cell proliferation. This is the first report of the translation of a chimeric protein (PRMT6MT2B2) in mouse preimplantation embryos. Our results suggest that analyzing chimeric transcripts with murine endogenous retrovirus with leucine tRNA primer will provide insight into the relationship between zygotic genome activation and subsequent intra- and extra-cellular lineage determination.

NSUN5 is essential for proper cell proliferation and differentiation of mouse preimplantation embryos.

In brief: Proper early embryonic development in mammals relies on precise cellular signaling pathways. This study reveals that NSUN5 is crucial for the regulation of the Hippo pathway, ensuring normal proliferation and differentiation in mouse preimplantation embryos. Abstract: NOL1/NOP2/Sun domain family, member 5 (NSUN5) is an enzyme belonging to the 5-methylcytosine (m5C) writer family that modifies rRNA and mRNA. Our data revealed an upregulation of Nsun5 at the two-cell stage of mouse preimplantation development, suggesting its significance in early embryonic development. Given m5C's important role in stabilizing rRNA and mRNA and the Hippo signaling pathway's critical function in lineage segregation during embryogenesis, we hypothesized that NSUN5 controls cell differentiation by regulating the expression of components of the Hippo signaling pathway in mouse early embryos. To examine this hypothesis, we employed Nsun5-specific small interfering RNAs for targeted gene silencing in mouse preimplantation embryos. Nsun5 knockdown resulted in significant developmental impairments including reduced blastocyst formation, smaller size of blastocysts, and impaired hatching from the zona pellucida. Nsun5 knockdown also led to decreased cell numbers and increased apoptosis in embryos. We also observed diminished nuclear translocation of yes-associated protein 1 (YAP1) in Nsun5 knockdown embryos at the morula stage, indicating disrupted cell differentiation. This disruption was further evidenced by an altered ratio of CDX2-positive to OCT4-positive cells. Furthermore, Nsun5 depletion was found to upregulate the Hippo signaling-related key genes, Lats1 and Lats2 at the morula stage. Our findings underscore the essential role of Nsun5 in early embryonic development by affecting cell proliferation, YAP1 nuclear translocation, and the Hippo pathway.

Incremental value of tricuspid annular enlargement to progressive tricuspid regurgitation in patients with longstanding persistent atrial fibrillation.

Tricuspid annular enlargement in patients with atrial fibrillation (AF) can induce tricuspid regurgitation (TR). However, risk factors associated with TR progression in patients with AF have not been defined. This study aimed to clarify an association between tricuspid annular diameter (TAD) and TR progression in patients with longstanding persistent AF. We retrospectively analyzed data from 228 patients who had longstanding persistent AF for > 1 year and mild or less TR on baseline echocardiograms. We defined significant TR as moderate or greater TR, graded according to the jet area and vena contracta. The optimal cut-off value of the TAD index (TADI), based on body surface area for predicting progression to significant TR, was estimated using receiver operating characteristic (ROC) curves. The independence and incremental value of the TADI were evaluated using multivariate Cox proportional hazard regression analysis and likelihood ratio tests. Over a median follow-up of 3.7 years, 55 (24.1%) patients developed significant TR. The optimal cut-off value of 21.1 mm/m2 for the TADI at baseline and ROC curves predicted TR progression with 70.4% sensitivity and 86% specificity. Furthermore, TADI was an independent predictor of TR progression (hazard ratio, 1.32; 95% confidence interval, 1.17-1.49, P < 0.001) and had a significant incremental value that exceeded that of models constructed using clinical parameters. In conclusion, TADI was significantly associated with TR progression and was an independent predictor of TR progression in longstanding persistent AF.

Analysis of histone H3K4me3 modifications in bovine placenta derived from different calf-production methods.

In ruminants, the overgrowth of offspring produced by in vitro fertilization (IVF) is a common problem. Abnormal epigenetic modifications caused by environmental factors during the early embryonic period are suspected as an aetiology of overgrowth. In this study, we investigated the genome-wide histone H3K4me3 profiles of bovine placentae that play a pivotal role in foetal development and compared their characteristics between artificial insemination (AI)- and IVF-derived samples. Cotyledons were harvested from the placentae obtained at parturition of 5 AI- and 13 IVF-derived calves, and chromatin immunoprecipitation sequencing was performed for H3K4me3. We confirmed no significant maternal tissue contamination in the samples we used. The revealed H3K4me3 profiles reflected the general characteristics of the H3K4me3 modification, which is abundantly distributed in the promoter region of active genes. By extracting common modifications from multiple samples, the genes involved in placenta-specific biological processes could be enriched. Comparison with the H3K4me3 modifications of blastocyst samples was also effective for enriching the placenta-specific features. Principal component analysis suggested the presence of differential H3K4me3 modifications in AI- and IVF-derived samples. The genes contributing to the difference were related to the developmental biological processes. Imprinted genes such as BEGAIN, ZNF215 and DLX5 were among the extracted genes. Principal component and discriminant analyses using only male samples categorized the samples into three groups based on foetal weight and calf-production methods. To our knowledge, this is the first study to profile the genome-wide histone modifications of bovine foetal placentae and reveal their differential characteristics between different calf-production methods.

An Increase in the Ratio of Brain Natriuretic Peptide to Peak Transvalvular Pressure Gradient Suggests Coexistence of Cardiovascular Complications in Elderly Aortic Stenosis Patients.

The aim of this study was to differentiate between elderly aortic stenosis (AS) patients with and without cardiovascular complications (CCs).In total, 156 consecutive patients with AS aged ≥ 70 years were enrolled. Patients were divided into 2 groups as follows: AS without CCs (group I; n = 110) and AS with CCs (group II; n = 46). Routine electrocardiographic and echocardiographic parameters, peak and mean transvalvular pressure gradients (TPGs), aortic valve area (AVA), brain natriuretic peptide (BNP) levels, and BNP/peak TPG ratio were measured.The mean ages in groups I and II were 80.4 ± 5.5 and 82.5 ± 7.2 years. Left ventricular hypertrophy was greater in group II than in group I. Left ventricular end-diastolic and end-systolic dimensions and left ventricular fractional shortening were normal in both groups. Peak and mean TPGs were greater in group II (67.2 ± 39.3 and 40.2 ± 26.4 mmHg) than in group I (52.0 ± 23.0 and 30.2 ± 13.9, both P < 0.005); however, the AVA showed no significant difference between the 2 groups. The median BNP levels were 65.9 and 433.7 pg/mL in groups I and II (P < 0.0001). A correlation between peak TPG and BNP levels was observed in both groups. The BNP/peak TPG ratio was < 3.0 in all patients of group I and ≥ 3.0 in almost all patients of group II (P< 0.0001). The area under the curve using BNP/peak TPG ratio was 0.9883.BNP and BNP/peak TPG ratio could differentiate between AS with and without CCs in elderly patients.

Premature atrial contraction immediately after catheter ablation was associated with late recurrence of atrial fibrillation.

BACKGROUND: Although premature atrial contractions (PACs) just after catheter ablation (CA) for atrial fibrillation (AF) are common, their clinical significance is uncertain. This study aimed to evaluate whether the PAC burden after an initial CA for AF was associated with late recurrence. METHODS: We enrolled 346 patients with AF (median age, 65 years; 30% female; 57% with paroxysmal AF) who underwent an initial radiofrequency CA and a 24-h Holter monitoring the day after the procedure. PAC was defined as supraventricular complexes occurring ≥30% earlier than expected compared with a previous RR interval, and the number of PAC/24 h during post-procedural Holter monitoring was analyzed. RESULTS: AF recurred in 106 patients (31%) during a median follow-up of 19 months. These patients had significantly more PAC/24 h than those without (median [interquartile range], 891 [316-4351] beats vs. 409 [162-1,303] beats; p < 0.01). The number of PACs was independently associated with AF recurrence after adjustment for clinical parameters and left atrial (LA) enlargement. Receiver operating characteristic (ROC) curve analysis revealed that 1431 PAC/24 h was the optimal cut-off value for predicting AF recurrence. Adding the PAC/24 h to the prediction model with LA diameter appeared to correctly reclassify patients who were thought to be at high risk for AF recurrence into the low-risk group and vice versa. CONCLUSIONS: The number of PACs was an independent risk factor for AF recurrence. A 24-h Holter recording the day after an initial CA is a simple and beneficial tool for the risk stratification of AF recurrence.

A more accurate analysis of maternal effect genes by siRNA electroporation into mouse oocytes.

Maternal RNA and proteins accumulate in mouse oocytes and regulate initial developmental stages. Sperm DNA combines with protamine, which is exchanged after fertilization with maternal histones, including H3.3; however, the effect of H3.3 on development post-fertilization remains unclear. Herein, we established an electroporation method to introduce H3.3 siRNA into germinal vesicle (GV)-stage oocytes without removing cumulus cells. Oocyte-attached cumulus cells need to be removed during the traditional microinjection method; however, we confirmed that artificially removing cumulus cells from oocytes reduced fertilization rates, and oocytes originally free of cumulus cells had reduced developmental competence. On introducing H3.3 siRNA at the GV stage, H3.3 was maintained in the maternal pronucleus and second polar body but not in the paternal pronucleus, resulting in embryonic lethality after fertilization. These findings indicate that H3.3 protein was not incorporated into the paternal pronucleus, as it was repeatedly translated and degraded over a relatively short period. Conversely, H3.3 protein incorporated into the maternal genome in the GV stage escaped degradation and remained in the maternal pronucleus after fertilization. This new method of electroporation into GV-stage oocytes without cumulus cell removal is not skill-intensive and is essential for the accurate analysis of maternal effect genes.

Current status of genome-wide epigenetic profiling of mammalian preimplantation embryos.

Background: Genome-wide information on epigenetic modifications in mammalian preimplantation embryos was an unexplored sanctuary of valuable research insights protected by the difficulty of its analysis. However, that is no longer the case, and many epigenome maps are now available for sightseeing there. Methods: This review overviews the current status of genome-wide epigenetic profiling in terms of DNA methylome and histone modifications in mammalian preimplantation embryos. Main findings: As the sensitivity of methods for analyzing epigenetic modifications increased, pioneering work began to explore the genome-wide epigenetic landscape in the mid-2010s, first for DNA methylation and then for histone modifications. Since then, a huge amount of data has accumulated, revealing typical epigenetic profiles in preimplantation development and, more recently, changes in response to environmental interventions. Conclusions: These accumulating data may be used to improve the quality of preimplantation embryos, both in terms of their short-term developmental competence and their subsequent long-term health implications.

Synthesis and maintenance of lipid droplets are essential for mouse preimplantation embryonic development.

Lipid droplets (LDs), which are ubiquitous organelles consisting of a neutral lipid core coated with a phospholipid monolayer, play key roles in the regulation of cellular lipid metabolism. Although it is well known that mammalian oocytes and embryos contain LDs and that the amount of LDs varies among animal species, their physiological functions remain unclear. In this study, we have developed a method based on two-step centrifugation for efficient removal of almost all LDs from mouse MII oocytes (delipidation). We found that delipidated MII oocytes could be fertilized in vitro, and developed normally to the blastocyst stage even when the embryos were cultured in the absence of a fatty acid supply. LDs were newly synthesized and accumulated soon after delipidation, but chemical inhibition of long chain acyl-CoA synthetases (ACSLs) blocked this process, resulting in severe impairment of early embryonic development. Furthermore, we found that overabundance of LDs is detrimental to early embryonic development. Our findings demonstrate the importance of synthesis and maintenance of LDs, mediated in part by ACSL activity, during preimplantation embryonic development.