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In mammals, DNA 5-hydroxymethylcytosine (5hmC) is involved in methylation reprogramming during early embryonic development. Yet, to what extent 5hmC participates in genome-wide methylation reprogramming remains largely unknown. Here, we characterize the 5hmC landscapes in mouse early embryos and germ cells with parental allele specificity. DNA hydroxymethylation was most strongly correlated with DNA demethylation as compared with de novo or maintenance methylation in zygotes, while 5hmC was targeted to particular de novo methylated sites in postimplantation epiblasts. Surprisingly, DNA replication was also required for 5hmC generation, especially in the female pronucleus. More strikingly, aberrant nuclear localization of Dnmt1/Uhrf1 in mouse zygotes due to maternal deficiency of Nlrp14 led to defects in DNA-replication-coupled passive demethylation and impaired 5hmC deposition, revealing the divergency between genome-wide 5-methylcytosine (5mC) maintenance and Tet-mediated oxidation. In summary, our work provides insights and a valuable resource for the study of epigenetic regulation in early embryo development. DNA 5-hydroxymethylcytosine (5hmC) is mapped in mouse preimplantation embryos, postimplantation epiblasts, primordial germ cells (PGCs) and gametes. Nlrp14 maternal depletion perturbs DNA-replication-coupled passive demethylation and impairs 5hmC deposition.

Background Embryos derived from in vitro fertilization (IVF) single pronucleus (1PN) zygotes can achieve live births. The pronucleus size of 1PN zygotes is related to their embryonic development potential. However, these studies cannot draw clear conclusions because of the limited sample size. Currently, methods for accurately predicting the development potential of 1PN blastocysts on the basis of the pronucleus diameter are lacking. In this study, we aimed to assess the effects of the pronucleus size of human 1PN zygotes derived from conventional IVF on embryological and pregnancy outcomes. Methods This retrospective cohort study included 629 IVF–1PN cycles from 514 individuals between May 2023 and April 2024 to observe embryonic development. Pregnancy outcomes were assessed in 36 patients who received their first transfer of single vitrified–warmed blastocysts derived from IVF–1PN zygotes. Human IVF–1PN zygotes were classified into six groups by PN diameter (≤ 20, 25, 30, 35, 40, and ≥ 45 μm), with the cutoff value determined by examining the receiver operating characteristic curve. Results Compared with 1PN zygotes with smaller PN diameters, 1PN zygotes with larger PN diameters had significantly greater cleavage rates, blastocyst formation rates, and blastocyst qualities ( P  < 0.05). The diameter of 1PN zygotes is a valuable indicator for predicting blastocyst formation (AUC = 0.605, cutoff = 32.5 μm). Moreover, the transfer of IVF–1PN blastocysts with different PN diameters resulted in similar pregnancy outcomes (biochemical pregnancy, ongoing pregnancy, and live birth rates). Conclusions Our results suggest that 1PN zygotes derived from conventional IVF with larger PN diameters have better embryonic development and could be used as predictors of blastocyst formation (> 32.5 μm). However, once the zygotes reach the blastocyst stage, the PN diameter is not associated with pregnancy outcomes.

Fusion of egg and sperm combines the genetic material of both parents in one cell. In mammals, including humans, each parental genome is initially confined in a separate pronucleus. For the new organism to develop, the two genomes must be spatially coordinated so that the first embryonic division can create two cells that combine both genomes in one nucleus. Reichmann et al. found that at the beginning of the first division, two microtubule spindles organize the maternal and paternal chromosomes and subsequently align to segregate the parental genomes in parallel (see the Perspective by Zielinska and Schuh). Failure of spindle alignment led to two-celled embryos with more than one nucleus per cell. Dual-spindle assembly in the zygote thus offers a potential mechanistic explanation for division errors frequently observed in human embryos in the fertility clinic. Science , this issue p. 189 ; see also p. 128 After fertilization, two spindles form around pronuclei in mammalian zygotes and keep the parental genomes apart. At the beginning of mammalian life, the genetic material from each parent meets when the fertilized egg divides. It was previously thought that a single microtubule spindle is responsible for spatially combining the two genomes and then segregating them to create the two-cell embryo. We used light-sheet microscopy to show that two bipolar spindles form in the zygote and then independently congress the maternal and paternal genomes. These two spindles aligned their poles before anaphase but kept the parental genomes apart during the first cleavage. This spindle assembly mechanism provides a potential rationale for erroneous divisions into more than two blastomeric nuclei observed in mammalian zygotes and reveals the mechanism behind the observation that parental genomes occupy separate nuclear compartments in the two-cell embryo.

Following fertilization in mammals, the gametes are reprogrammed to create a totipotent zygote, a process that involves de novo establishment of chromatin domains. A major feature occurring during preimplantation development is the dramatic remodelling of constitutive heterochromatin, although the functional relevance of this is unknown. Here, we show that heterochromatin establishment relies on the stepwise expression and regulated activity of SUV39H enzymes. Enforcing precocious acquisition of constitutive heterochromatin results in compromised development and epigenetic reprogramming, which demonstrates that heterochromatin remodelling is essential for natural reprogramming at fertilization. We find that de novo H3K9 trimethylation (H3K9me3) in the paternal pronucleus after fertilization is catalysed by SUV39H2 and that pericentromeric RNAs inhibit SUV39H2 activity and reduce H3K9me3. De novo H3K9me3 is initially non-repressive for gene expression, but instead bookmarks promoters for compaction. Overall, we uncover the functional importance for the restricted transmission of constitutive heterochromatin during reprogramming and a non-repressive role for H3K9me3.Burton et al. show that H3K9me3 deposition catalysed by SUV39H2 and regulated by pericentromeric RNAs in the mouse paternal pronucleus does not suppress gene expression, but bookmarks promoters for compaction.

Objective To evaluate the developmental potential of zygotes without pronucleus (0PN) and to examine the pregnancy outcomes of euploid blastocysts derived from these zygotes. Methods A total of 4580 fresh autologous preimplantation genetic testing for aneuploidy (PGT-A) cycles from 2634 patients at a single academic center from April 2016 to December 2022 with at least one 0PN at the time of fertilization check (16–19 hours post insemination) were included in the study. Results Of all 0PNs ( n  = 9345), 70.4% reached the cleavage stage, while only 5.3% of the cleaved embryos reached blastocyst stage and met biopsy criteria. These rates were significantly lower than those from the 2PNs ( n  = 32086, 98.7% cleavage, and 43.6% biopsied blastocyst rates, p  < 0.05). Logistic regression model showed that 2PNs were 19.6 times more likely to result in biopsied blastocysts than 0PNs ( p  < 0.05). Of the biopsied blastocysts, 39% and 45% were euploid from the 0PN ( n  = 349) and 2PN ( n  = 13,975) group, respectively ( p  < 0.05). After single euploid frozen blastocyst transfers ( n  = 27 and 1695 in the 0PN and 2PN groups, respectively), no statistical significance was observed in live birth, clinical pregnancy, biochemical pregnancy, and spontaneous pregnancy loss rates between these two groups. Conclusion Compared to 2PN zygotes, the 0PNs showed lower developmental potential, including lower blastocyst formation and euploidy rates. However, euploid blastocysts from either cohort resulted in similar live birth rates, indicating a small percentage of 0PN zygotes can result in normal blastocysts and live births presenting additional reproductive opportunities for patients lacking alternatives.

PurposeTo study associations between novel WEE2 mutations and patients with fertilization failure or poor fertilization.MethodsThirty-one Chinese patients who underwent treatment with assisted reproductive technology and suffered from repeated (at least two times) total fertilization failure (TFF) or a low fertilization rate were enrolled. Genomic DNA was extracted from patients for whole-exome sequencing. Suspicious mutations were validated by Sanger sequencing. WEE2 protein levels in oocytes from affected patients were examined by immunofluorescence. Disruptive effects of mutations on WEE2 protein stability, subcellular localization, and kinase function were analyzed through western blotting, immunofluorescence, and flow cytometry in HeLa cells.ResultsThree of thirty-one (9.6%) enrolled patients had six compound heterozygous mutations of the WEE2 gene, and three of them were reported here for the first time (c.115_116insT, c.756_758delTGA, and c.C1459T). Oocytes from affected patients showed decreased WEE2 immunofluorescence signals. In vitro experiments showed that the mutant WEE2 gene caused reduced WEE2 protein levels or cellular compartment translocation in HeLa cells, leading to decreased levels of the phosphorylated Cdc2 protein. Compared with the wild-type WEE2 protein, the mutant WEE2 proteins were also found to have different effects on the cell cycle.ConclusionThree novel compound heterozygous WEE2 variants were found in patients with pronucleus formation failure. This study provides new evidence that WEE2 mutations result in loss of function, which could result in fertilization failure.

Chromatin remodeling is essential for epigenome reprogramming after fertilization. However, the underlying mechanisms of chromatin remodeling remain to be explored. Here, we investigated the dynamic changes in nucleosome occupancy and positioning in pronucleus-stage zygotes using ultra low-input MNase-seq. We observed distinct features of inheritance and reconstruction of nucleosome positioning in both paternal and maternal genomes. Genome-wide de novo nucleosome occupancy in the paternal genome was observed as early as 1 h after the injection of sperm into ooplasm. The nucleosome positioning pattern was continually rebuilt to form nucleosome-depleted regions (NDRs) at promoters and transcription factor (TF) binding sites with differential dynamics in paternal and maternal genomes. NDRs formed more quickly on the promoters of genes involved in zygotic genome activation (ZGA), and this formation is closely linked to histone acetylation, but not transcription elongation or DNA replication. Importantly, we found that NDR establishment on the binding motifs of specific TFs might be associated with their potential pioneer functions in ZGA. Further investigations suggested that the predicted factors MLX and RFX1 played important roles in regulating minor and major ZGA, respectively. Our data not only elucidate the nucleosome positioning dynamics in both male and female pronuclei following fertilization, but also provide an efficient method for identifying key transcription regulators during development.

Controlled mRNA storage and stability is essential for oocyte meiosis and early embryonic development. However, how to regulate mRNA storage and stability in mammalian oogenesis remains elusive. Here we showed that LSM14B, a component of membraneless compartments including P-body-like granules and mitochondria-associated ribonucleoprotein domain (MARDO) in germ cell, is indispensable for female fertility. To reveal loss of LSM14B disrupted primordial follicle assembly and caused mRNA reduction in non-growing oocytes, which was concomitant with the impaired assembly of P-body-like granules. 10× Genomics single-cell RNA-sequencing and immunostaining were performed. Meanwhile, we conducted RNA-seq analysis of GV-stage oocytes and found that Lsm14b deficiency not only impaired the maternal mRNA accumulation but also disrupted the translation in fully grown oocytes, which was closely associated with dissolution of MARDO components. Moreover, Lsm14b -deficient oocytes reassembled a pronucleus containing decondensed chromatin after extrusion of the first polar body, through compromising the activation of maturation promoting factor, while the defects were restored via WEE1/2 inhibitor. Together, our findings reveal that Lsm14b plays a pivotal role in mammalian oogenesis by specifically controlling of oocyte mRNA storage and stability.

Genome-wide erasure of DNA cytosine-5 methylation has been reported to occur along the paternal pronucleus in fertilized oocytes in an apparently replication-independent manner, but the mechanism of this reprogramming process has remained enigmatic. Recently, considerable amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5-methylcytosine (5mC) by TET proteins, have been detected in certain mammalian tissues. 5hmC has been proposed as a potential intermediate in active DNA demethylation. Here, we show that in advanced pronuclear-stage zygotes the paternal pronucleus contains substantial amounts of 5hmC but lacks 5mC. The converse is true for the maternal pronucleus, which retains 5mC but shows little or no 5hmC signal. Importantly, 5hmC persists into mitotic one-cell, two-cell, and later cleavage-stage embryos, suggesting that 5mC oxidation is not followed immediately by genome-wide removal of 5hmC through excision repair pathways or other mechanisms. This conclusion is supported by bisulfite sequencing data, which shows only limited conversion of modified cytosines to cytosines at several gene loci. It is likely that 5mC oxidation is carried out by the Tet3 oxidase. Tet3, but not Tet1 or Tet2, was expressed at high levels in oocytes and zygotes, with rapidly declining levels at the two-cell stage. Our results show that 5mC oxidation is part of the early life cycle of mammals.

The oxidation product of methylated cytosine is passively lost from DNA in the zygote as cell division progresses. Although global erasure of DNA methylation has been observed in zygotes and primordial germ cells, the responsible enzyme(s) have been elusive. The demonstration that members of the Tet (ten eleven translocation) family of proteins are capable of catalyzing conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC) raises the possibility that Tet proteins may participate in this process. Indeed, recent studies have implicated the involvement of Tet3 in the conversion of 5mC to 5hmC in zygotes. This result, combined with the demonstration that Tet proteins can further oxidize 5hmC to 5-carboxylcytosine followed by excision by thymine-DNA glycosylase, raises the possibility that active demethylation may take place in a process that involves Tet3-mediated oxidation followed by base excision repair. We demonstrated by immunostaining of mitotic chromosome spreads of preimplantation embryos that the 5hmC associated with the paternal genome in zygotes is gradually lost during preimplantation development. Our study suggests that, although the conversion of 5mC to 5hmC in zygotes is an enzyme-catalyzed process, loss of 5hmC during preimplantation appears to be a DNA replication–dependent passive process.