Two-cell Stage Research Articles

Tetraploid embryo aggregation produces high-quality blastocysts with an increased trophectoderm in pigs.

Tetraploid complementation is an ideal method for demonstrating the differentiation potential of pluripotent stem cells. In this study, we selected the most efficient tetraploid production method for porcine embryos and investigated whether tetraploid blastomere aggregation could enhance the quality of tetraploid embryos. Three methods were investigated to produce tetraploid embryos: First, tetraploid embryos were produced using electro-fusion of two-cell stage parthenogenetic blastomere (FUTP). Second, somatic cell was injected into the mature oocyte and fused to produce tetraploid embryos. Third, oocytes were matured with Cytochalasin B (CB) for the late 22h of in vitro maturation to inhibit the first polar body (PB1). Following that, non-PB1 oocytes were treated with CB for 4h after parthenogenetic activation. There was no significant difference in the blastocyst development rate and tetraploid production rate of the embryos produced through the three methods. However, FUTP-derived blastocysts had a significantly lower percentage of apoptotic cells compared to other methods. The developmental competence of embryos, expression of trophectoderm cell marker genes, and distribution of YAP1 protein were investigated in tetraploid embryos produced using the FUTP method. The FUTP method most effectively prevented apoptosis during porcine tetraploid embryo formation. Tetraploid aggregation-derived blastocysts have a high proportion of trophectoderm with increased expression of the CDX2 mRNA and high YAP1 intensity. High-quality blastocysts derived from a tetraploid embryo aggregation can serve as suitable source material for testing the differentiation potential of pluripotent stem cells for blastocyst complementation in pigs.

Loss of function of ribosomal protein L13a blocks blastocyst formation and reveals a potential nuclear role in gene expression.

Ribosomal proteins play diverse roles in development and disease. Most ribosomal proteins have canonical roles in protein synthesis, while some exhibit extra-ribosomal functions. Previous studies in our laboratory revealed that ribosomal protein L13a (RPL13a) is involved in the translational silencing of a cohort of inflammatory proteins in myeloid cells. This prompted us to investigate the role of RPL13a in embryonic development. Here we report that RPL13a is required for early development in mice. Crosses between Rpl13a+/- mice resulted in no Rpl13a-/- offspring. Closer examination revealed that Rpl13a-/- embryos were arrested at the morula stage during preimplantation development. RNA sequencing analysis of Rpl13a-/- morulae revealed widespread alterations in gene expression, including but not limited to several genes encoding proteins involved in the inflammatory response, embryogenesis, oocyte maturation, stemness, and pluripotency. Exvivo analysis revealed that RPL13a was localized to the cytoplasm and nucleus between the two-cell and morula stages. RNAi-mediated depletion of RPL13a phenocopied Rpl13a-/- embryos and knockdown embryos exhibited increased expression of IL-7 and IL-17 and decreased expression of the lineage specifier genes Sox2, Pou5f1, and Cdx2. Lastly, a protein-protein interaction assay revealed that RPL13a is associated with chromatin, suggesting an extra ribosomal function in transcription. In summary, our data demonstrate that RPL13a is essential for the completion of preimplantation embryo development. The mechanistic basis of the absence of RPL13a-mediated embryonic lethality will be addressed in the future through follow-up studies on ribosome biogenesis, global protein synthesis, and identification of RPL13a target genes using chromatin immunoprecipitation and RNA-immunoprecipitation-based sequencing.

Knockdown of Y-box binding protein 1 induces autophagy in early porcine embryos.

Y-box binding protein 1 (YBX1) plays important roles in RNA stabilization, translation, transcriptional regulation, and mitophagy. However, its effects on porcine preimplantation embryos remain unclear. In this study, we knocked down YBX1 in the one-cell (1C) stage embryo via small interfering RNA microinjection to determine its function in porcine embryo development. The mRNA level of YBX1 was found to be highly expressed at the four-cell (4C) stage in porcine embryos compared with one-cell (1C) and two-cell (2C) stages. The number of blastocysts was reduced following YBX1 knockdown. Notably, YBX1 knockdown decreased the phosphatase and tensin homolog-induced kinase 1 (PINK1) and parkin RBR E3 ubiquitin protein ligase (PRKN) mRNA levels. YBX1 knockdown also decreased PINK1, active mitochondria, and sirtuin 1 levels, indicating reduced mitophagy and mitochondrial biogenesis. Furthermore, YBX1 knockdown increased the levels of glucose-regulated protein 78 (GRP78) and calnexin, leading to endoplasmic reticulum (ER) stress. Additionally, YBX1 knockdown increased autophagy and apoptosis. In conclusion, knockdown of YBX1 decreases mitochondrial function, while increasing ER stress and autophagy during embryonic development.

MYC–MAX heterodimerization is essential for the induction of major zygotic genome activation and subsequent preimplantation development

In mouse preimplantation development, zygotic genome activation (ZGA), which synthesizes new transcripts in the embryo, begins in the S phase at the one-cell stage, with major ZGA occurring especially at the late two-cell stage. Myc is a transcription factor expressed in parallel with ZGA, but its direct association with major ZGA has not been clarified. In this study, we found that developmental arrest occurs at the two-cell stage when mouse embryos were treated with antisense oligonucleotides targeting Myc or MYC-specific inhibitors from the one-cell stage. To identify when MYC inhibition affects development, we applied time-limited inhibitor treatment and found that inhibition of MYC at the one-cell, four-cell, and morula stages had no effect on preimplantation development, whereas inhibitor treatment at the two-cell stage arrested development at the two-cell stage. Furthermore, transcriptome analysis revealed that when MYC function was inhibited, genes expressed in the major ZGA phase were suppressed. These results suggest that MYC is essential for the induction of major ZGA and subsequent preimplantation development. Revealing the function of MYC in preimplantation development is expected to contribute to advances in assisted reproductive technology.

CHK1-CDC25A-CDK1 regulate cell cycle progression and protect genome integrity in early mouse embryos.

After fertilization, remodeling of the oocyte and sperm genomes is essential to convert these highly differentiated and transcriptionally quiescent cells into early cleavage-stage blastomeres that are transcriptionally active and totipotent. This developmental transition is accompanied by cell cycle adaptation, such as lengthening or shortening of the gap phases G1 and G2. However, regulation of these cell cycle changes is poorly understood, especially in mammals. Checkpoint kinase 1 (CHK1) is a protein kinase that regulates cell cycle progression in somatic cells. Here, we show that CHK1 regulates cell cycle progression in early mouse embryos by restraining CDK1 kinase activity due to CDC25A phosphatase degradation. CHK1 kinase also ensures the long G2 phase needed for genome activation and reprogramming gene expression in two-cell stage mouse embryos. Finally, Chk1 depletion leads to DNA damage and chromosome segregation errors that result in aneuploidy and infertility.

The rate of de novo structural variation is increased in in vitro-produced offspring and preferentially affects the paternal genome.

Assisted reproductive technologies (ARTs), including in vitro maturation and fertilization (IVF), are increasingly used in human and animal reproduction. Whether these technologies directly affect the rate of de novo mutation (DNM), and to what extent, has been a matter of debate. Here we take advantage of domestic cattle, characterized by complex pedigrees that are ideally suited to detect DNMs and by the systematic use of ART, to study the rate of de novo structural variation (dnSV) in this species and how it is impacted by IVF. By exploiting features of associated de novo point mutations (dnPMs) and dnSVs in clustered DNMs, we provide strong evidence that (1) IVF increases the rate of dnSV approximately fivefold, and (2) the corresponding mutations occur during the very early stages of embryonic development (one- and two-cell stage), yet primarily affect the paternal genome.

Bulk stiffness and recovery time – A measure of development of mouse embryos

Objectives: Understanding the onset of embryonic arrest by studying the morphology of the embryos at different stages of their development is a relatively well-understood subject. The qualitative nature of the study makes it difficult to determine and quantify the causes behind the arrest. For successful embryonic development, it is essential to know, non-destructively, and the developmental viability of the embryos at a particular stage. Material and Methods: We investigate the bulk stiffness and recovery time as the viability quantifiers for grading or classifying embryos at the two-cell stage or after the formation of the first cleavage. A simple cantilever beam-based experimental procedure is established to investigate two-cell mouse embryos in two distinct orientations. Results: Bulk stiffness measurement varies with the embryo’s orientation and deformation. A good quality two-cell embryo shows a significant decrease in stiffness ratio with the deformation. Also, the embryo’s recovery time after releasing deformation may indicate a quality-dependent correlation. Conclusion: Our study concludes that the bulk stiffness measurements in the two principal orientations, their ratio, and recovery time correlate with the underlying morphology of two-cell stage mouse embryos.

Maternal TDP-43 interacts with RNA Pol II and regulates zygotic genome activation

Zygotic genome activation (ZGA) is essential for early embryonic development. However, the regulation of ZGA remains elusive in mammals. Here we report that a maternal factor TDP-43, a nuclear transactive response DNA-binding protein, regulates ZGA through RNA Pol II and is essential for mouse early embryogenesis. Maternal TDP-43 translocates from the cytoplasm into the nucleus at the early two-cell stage when minor to major ZGA transition occurs. Genetic deletion of maternal TDP-43 results in mouse early embryos arrested at the two-cell stage. TDP-43 co-occupies with RNA Pol II as large foci in the nucleus and also at the promoters of ZGA genes at the late two-cell stage. Biochemical evidence indicates that TDP-43 binds Polr2a and Cyclin T1. Depletion of maternal TDP-43 caused the loss of Pol II foci and reduced Pol II binding on chromatin at major ZGA genes, accompanied by defective ZGA. Collectively, our results suggest that maternal TDP-43 is critical for mouse early embryonic development, in part through facilitating the correct RNA Pol II configuration and zygotic genome activation.

Absence of a molecular circadian clock in the preimplantation embryo is a conserved characteristic in the mammal.

It is not known when a functional circadian clock is established in the developing embryo. Lack of expression of key genes involved in the clock mechanism is indicative that a functional circadian clock mechanism is absent in the mammalian preimplantation embryo through the blastocyst stage of development. An embryonic circadian clock could conceivably organize cellular and developmental events temporally and in synchrony with other circadian rhythms in the mother. The hypothesis that a functional molecular clock exists in the preimplantation bovine, pig, human, and mouse embryo was tested by using publicly available RNAseq datasets to examine developmental changes in expression of the core genes responsible for the circadian clock - CLOCK, ARNTL, PER1, PER2, CRY1, and CRY2. In general, the transcript abundance of each gene decreased as development advanced to the blastocyst stage. The most notable exception was for CRY2, where transcript abundance was low and constant from the two-cell or four-cell to the blastocyst stage. Developmental patterns were generally the same for all species although there were some species-specific patterns such as an absence of PER1 expression in the pig, an increase in ARNTL expression at the four-cell stage in human, and an increase in expression of Clock and Per1 from the zygote to two-cell stage in the mouse. Analysis of intronic reads (indicative of embryonic transcription) for bovine embryos indicated an absence of embryonic transcription. Immunoreactive CRY1 was not detected in the bovine blastocyst. Results indicate that the preimplantation mammalian embryo lacks a functional intrinsic clock although specific components of the clock mechanism could conceivably play a role in other functions in the embryo.

P-117 The reason why direct cleavage and rapid cleavage should be differentiated

Abstract Study question Direct cleavage (DC) and Rapid cleavage (RaC) both appear to be 1 cell divided into 3 (or more) cells, should they be distinguished? Summary answer Since blastomeres by RaC had higher developmental potential than blastomeres by DC, and since many RaC embryos had “normal” blastomere, the two should be distinguished. What is known already DC, in which one cell divides into three (or more) cells without the two-cell stage, and RaC, in which one or two cells divide rapidly after the short two-cell stage (The former lead to segregation of chromosomes into three cells, while the latter leads to inadequate DNA replication), are both often recorded as “DC”, but when classified in detail, there are reports that the blastocyst development rate is higher in RaC embryos. However, the reason for this has not been clarified. Study design, size, duration This was a retrospective study of 643 embryos collected and cultured for at least 5 days in 2020 at our clinic. The embryos were time-lapse monitored by EmbryoScope (Vitrolife, Sweden) and classified into three groups according to the style of first division. Participants/materials, setting, methods The embryos were classified as follows: DC group: embryos that have divided into three (or more) cells without a two-cell stage; RaC group: embryos that rapidly (<5 hours) divided one (or both) cells after the 2-cell stage; Normal cleavage (NC) group: embryos that had a 2-cell stage for more than 5 hours. The subsequent development of blastomeres of all embryos was observed and whether they participated in blastocysts or not was recorded. Main results and the role of chance Of the embryos, 63 were in the DC group and 199 were in the RaC group (173 of which had one rapidly dividing blastomere and 26 of which had two). The blastocyst participation rate of blastomeres was 4.4% in the DC group, 19.5% of rapidly dividing blastomeres and 61.6% of normal blastomeres in the RaC group, with significant differences between all groups (p < 0.01). The good blastocyst (4BC or higher in Gardner grade) development rate was 4.8% (3/63) in the DC group, 40.5% (70/173) in the RaC group with one rapidly dividing blastomere (RaC1 group), 19.2% (5/26) in the RaC group with two such blastomeres (RaC2 group), showing a significant difference between the DC and RaC1 groups (P < 0.01). The live birth rate for single blastocyst transfer was 50% (1/2) in the DC group, 27.8% (5/18) in the RaC1 group, and 100% (1/1) in the RaC2 group. (Note that there were 381 in the NC group, and the above rates were 76.5%, 63.3% (241/381), and 29.3% (22/75), respectively) Limitations, reasons for caution This study examined only the style of first division and did not consider second and subsequent divisions. In addition, as of 2020, the clinical use of PGT-A is not approved in principle in Japan, and the subject embryos have not undergone chromosome analysis. Wider implications of the findings The reason for the higher blastocyst development rate in RaC embryos was indicated by the difference in blastocyst participation rates of the respective blastomeres and the presence of normal blastomeres, but since live births were obtained in both embryos, so these irregular divisions would not completely compromise the blastocyst euploidy. Trial registration number not applicable

P-244 Nucleation errors at four-cell stage reduces chances of live births in single euploid-blastocyst transfers

Abstract Study question What is the impact of nucleation errors (NE) and morphokinetic parameters on live birth rates in single euploid blastocyst transfers? Summary answer Nucleation errors (NE) at the four-cell stage was the only parameter associated with decreased live birth (LB) rates. What is known already Morphokinetic studies using time-lapse monitoring (TLM) systems have proposed algorithms to select embryos with the highest implantation potential. However, no single biomarker can reliably predict live-birth so far. Morphokinetics can deselect embryos for Preimplantation-Genetic-Testing for Aneuploidies (PGT-A). However, it is unclear whether morphokinetic markers can be used for selecting embryos with highest chance of LB among the euploid blastocysts. Performing morphokinetics enable detection of features such as nuclear errors (NE) which could be underdiagnosed with intermittent evaluation. It remains to be determined to which extend NE, next to morphokinetic parameters, are associated with LB potential of euploid blastocysts. Study design, size, duration Retrospective observational study including 383 single euploid blastocyst transfers in FET cycles between October 2017 to June 2021. These blastocysts were graded ≥BL3CC (Gardner criteria) and underwent TE biopsy for PGT-A by Next Generation Sequencing (NGS). Only ICSI with fresh autologous cycles using ejaculated sperm were considered. Outcomes of euploid blastocyst transfers were recorded and as pregnant (P): biochemical pregnancy (BP) (n = 17), clinical miscarriage (CM) (n = 35), live birth (LB) (n = 205); or not pregnant (NP) (n = 126). Participants/materials, setting, methods Patient baseline characteristics (Age, AMH, BMI) and infertility duration were considered. Morphokinetics were annotated with time-lapse-imaging (KID-score) for time (t) of PB2 (tPB2), tPNa (PN-appearance), tPNf (PN-fading), t2-t9, CP (compaction), tM (Morula), tSB (start blastulation), tB (blastocyst) and timings between each developmental event. Nuclear errors (NE) such as micronucleation, binucleation, multinucleation were annotated when present in at least one blastomere at two- and four-cell stages between 19.4-51.4 hours and 22.1-63.1 hours, respectively, post-insemination. Main results and the role of chance Median (IQR) values of Age and AMH of female partner, and infertility duration were similar among the P and NP groups. BMI was significantly higher in patients who miscarried with Median (IQR) values of 28.4kg/m2 (26.1-33.4) vs < 27.9kg/m2 in all other groups (P < 0.01). Significantly more blastocysts with type A inner cell mass quality resulted in LB (24.9%) than to CM (14.3%), BP (5.9%) and NP (13.5%) (P < 0.001). Concerning TLM morphokinetics evaluation, none of the timed parameters evaluated, neither the timings between the different morphokinetics events resulted in significant differences among the LB and the other P and NP groups. However, LB group had significantly less euploid embryos presenting NE at four-cell stage (7.8%) compared to CM (17.0%), BP (23.5%) and NP (17.5%) (P = 0.02). In a logistic regression model, NE when present at four-cell stage but not at two-cell stage, had a negative impact in LB rates (OR:0.39, CI: 0.2-0.72; P = 0.003). Limitations, reasons for caution The retrospective nature of the study and the fact that NE evaluation was performed manually based on TLM videos by a single observer. Wider implications of the findings NE-constituted embryos can give rise to euploid blastocysts and have an implantation potential. Since these embryos are associated with lower LB, it is relevant to include NE as an evaluation parameter to deprioritize multinucleated four-cell embryos for transfer, even when they result in euploid blastocysts. Trial registration number not applicable

Live-cell imaging reveals redox metabolic reprogramming during zygotic genome activation.

Metabolic programming is deeply intertwined with early embryonic development including zygotic genome activation (ZGA), the polarization of zygotic cells, and cell fate commitment. It is crucial to establish a noninvasive imaging technology that spatiotemporally illuminates the cellular metabolism pathways in embryos to track developmental metabolism in situ. In this study, we used two high-quality genetically encoded fluorescent biosensors, SoNar for NADH/NAD+ and iNap1 for NADPH, to characterize the dynamic regulation of energy metabolism and redox homeostasis during early zygotic cleavage. Our imaging results showed that NADH/NAD+ levels decreased from the early to the late two-cell stage, whereas the levels of the reducing equivalent NADPH increased. Mechanistically, transcriptome profiling suggested that during the two-cell stage, zygotic cells downregulated the expression of genes involved in glucose uptake and glycolysis, andupregulated the expression of genes for pyruvate metabolism in mitochondria and oxidative phosphorylation, with a decline in the expression of two peroxiredoxin genes, Prdx1 and Prdx2. Collectively, with the establishment of in situ metabolic monitoring technology, our study revealed the programming of redox metabolism during ZGA.

PGC7 regulates H3K27me3 modification by inhibiting the interaction of YY1 with PRC2.

Primordial germ cell 7 (PGC7)(Dppa3 or Stella) is a small inherently disordered protein that is mainly expressed in oocytes and plays a vital role in the regulation of DNA methylation reprogramming in imprinted loci through interaction with other proteins. Most of PGC7-deficient zygotes are blocked at two-cell stage with an increased tri-methylation at lysine 27 of histone H3 (H3K27me3) level in the nucleus. Our previous work has indicated that PGC7 interacts with yin-yang1 (YY1) that is essential for the recruitment of enhancer of zeste homolog 2 (EZH2)-containing Polycomb repressive complex 2 (PRC2) to H3K27me3 modification sites. Here, we found that the presence of PGC7 weakened the interaction between YY1 and PRC2 without disrupting the assembly of core subunits of the PRC2 complex. In addition, PGC7 promoted AKT to phosphorylate serine 21 of EZH2, resulting in inhibition of EZH2 activity and the dissociation of EZH2 from YY1, thereby decreasing H3K27me3 level. In zygotes, the PGC7-deficient and AKT inhibitor MK2206 both promoted EZH2 to enter the pronuclei but without disturbing the subcellular localization of YY1 and caused an increase in the level of H3K27me3 in the pronuclei, as well as inhibition of the expression of zygote-activating genes regulated by H3K27me3 in two-cell embryos. In summary, PGC7 could affect zygotic genome activation during early embryonic development by regulating the level of H3K27me3 through regulation of PRC2 recruitment, EZH2 activity, and subcellular localization.NEW & NOTEWORTHY PGC7 and YY1 interaction inhibits recruitment of PRC2 by YY1. PGC7 promotes AKT and EZH2 interaction to increase pEZH2-S21 level, which weakens YY1 and EZH2 interaction, thereby decreasing H3K27me3 level. In zygotes, the PGC7-deficient and AKT inhibitor MK2206 promote EZH2 to enter the pronuclei, and increase H3K27me3 level in the pronuclei, as well as inhibition of the expression of zygote-activating genes regulated by H3K27me3 in two-cell embryos, which ultimately affects early embryo development.

Nucleolus assembly impairment leads to two-cell transcriptional repression via NPM1-mediated PRC2 recruitment.

The nucleolus is a compartmentalized organelle in eukaryotic cells known to form during embryogenesis, yet how its layered architecture is transformed from homogenous precursor bodies is unclear, and any impacts of this formation on embryonic cell fate determination remain unknown. Here, we demonstrate that lncRNA LoNA tethers granular-component-enriched NPM1 to dense-fibrillar-component-enriched FBL and drives the formation of compartmentalized nucleolus via facilitating liquid-liquid phase separation of those two nucleolar proteins. Phenotypically, LoNA-deficient embryos show developmental arrest at the two-cell (2C) stage. Mechanistically, we demonstrate that LoNA deficiency leads to nucleolar formation failure, resulting in mislocalization and acetylation of NPM1 in the nucleoplasm. Acetylated NPM1 recruits and guides PRC2 complex to 2C genes, where PRC2 complex trimethylates H3K27, leading to transcriptional repression of these genes. Collectively, our findings reveal that lncRNA is required for the establishment of nucleolar structure, and this process has an impact on two-cell embryonic development via 2C transcriptional activation.

Sperm-borne microRNA-34c regulates maternal mRNA degradation and preimplantation embryonic development in mice

BackgroundStudies have shown that sperm-borne microRNAs (miRNAs) are involved in mammalian preimplantation embryonic development. In humans, spermatozoan miR-34c levels are correlated with in vitro fertilization outcomes, such as embryo quality and the clinical pregnancy and live birth rates. In rabbits and cows, miR-34c improves the developmental competence of embryos generated by somatic cell nuclear transfer. However, the mechanisms underlying the regulation of embryonic development by miR-34c remain unknown.MethodsFemale C57BL/6 mice (6–8 weeks old) were superovulated, and pronucleated zygotes were collected and microinjected with an miR-34c inhibitor or a negative-control RNA. The embryonic development of the microinjected zygotes was evaluated, and the messenger RNA (mRNA) expression profiles of the embryos at the two-cell, four-cell and blastocyst stages (five embryos per group) were determined by RNA sequencing analysis. Gene expression levels were verified by reverse transcription–quantitative polymerase chain reaction. Cluster analysis and heat map visualization were performed to detect differentially expressed mRNAs. Pathway and process enrichment analyses were performed using ontology resources. Differentially expressed mRNAs were systematically analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins database to determine their biological functions.ResultsEmbryonic developmental potential was significantly reduced in zygotes microinjected with the miR-34c inhibitor compared with those microinjected with a negative-control RNA. Two-cell stage embryos microinjected with an miR-34c inhibitor presented altered transcriptomic profiles, with upregulated expression of maternal miR-34c target mRNAs and classical maternal mRNAs. Differentially expressed transcripts were mainly of genes associated with lipid metabolism and cellular membrane function at the two-cell stage, with cell-cycle phase transition and energy metabolism at the four-cell stage; and with vesicle organization, lipid biosynthetic process and endomembrane system organization at the blastocyst stage. We also showed that genes related to preimplantation embryonic development, including Alkbh4, Sp1, Mapk14, Sin3a, Sdc1 and Laptm4b, were significantly downregulated after microinjection of an miR-34c inhibitor.ConclusionsSperm-borne miR-34c may regulate preimplantation embryonic development by affecting multiple biological processes, such as maternal mRNA degradation, cellular metabolism, cell proliferation and blastocyst implantation. Our data demonstrate the importance of sperm-derived miRNAs in the development of preimplantation embryos.

GND-PCA Method for Identification of Gene Functions Involved in Asymmetric Division of C. elegans

Due to the rapid development of imaging technology, a large number of biological images have been obtained with three-dimensional (3D) spatial information, time information, and spectral information. Compared with the case of two-dimensional images, the framework for analyzing multidimensional bioimages has not been completely established yet. WDDD is an open biological image database. It dynamically records 3D developmental images of 186 samples of nematodes C. elegans. In this study, based on WDDD, we constructed a framework to analyze the multidimensional dataset, which includes image segmentation, image registration, size registration by the length of main axes, time registration by extracting key time points, and finally, using generalized N-dimensional principal component analysis (GND-PCA) to analyze the phenotypes of bioimages. As a data-driven technique, GND-PCA can automatically extract the important factors involved in the development of P1 and AB in C. elegans. A 3D bioimage can be regarded as a third-order tensor. Therefore, GND-PCA was applied to the set of third-order tensors, and a set of third-order tensor bases was iteratively learned to linearly approximate the set. For each tensor base, a corresponding characteristic image is built to reveal its geometric meaning. The results show that different bases can be used to express different vital factors in development, such as the asymmetric division within the two-cell stage of C. elegans. Based on selected bases, statistical models were built by 50 wild-type (WT) embryos in WDDD, and were applied to RNA interference (RNAi) embryos. The results of statistical testing demonstrated the effectiveness of this method.

Investigation of the Effects of Some Cardiovascular Drugs on Angiogenesis by Transgenic Zebrafish.

Angiogenesis contributes to the pathophysiology of cardiovascular disease (CVD). Some cardiovascular drugs used in the treatment of CVD have an effect on the process of angiogenesis. Transgenic Tg (flk1: EGFP) zebrafish embryos were used to identify the effects of some cardiovascular drugs on angiogenesis during vertebral development in vivo. Zebrafish embryos at a one-cell stage or two-cell stage were cultured with embryo medium containing cardiovascular drugs at a final solvent concentration of 0.5% (V/V) dimethyl sulfoxide (DMSO) for 24 hours in 24-well plates. We found that 6 drugs including isosorbide mononitrate, amlodipine, bisoprolol fumarate, carvedilol, irbesartan, and rosuvastatin calcium may affect angiogenesis by vascular endothelial growth factor (VEGF) signaling pathway. These new findings of some cardiovascular drugs should improve the treatment of cardiovascular diseases.

The RNA m6A landscape of mouse oocytes and preimplantation embryos.

Despite the significance of N6-methyladenosine (m6A) in gene regulation, the requirement for large amounts of RNA has hindered m6A profiling in mammalian early embryos. Here we apply low-input methyl RNA immunoprecipitation and sequencing to map m6A in mouse oocytes and preimplantation embryos. We define the landscape of m6A during the maternal-to-zygotic transition, including stage-specifically expressed transcription factors essential for cell fate determination. Both the maternally inherited transcripts to be degraded post fertilization and the zygotically activated genes during zygotic genome activation are widely marked by m6A. In contrast to m6A-marked zygotic ally-activated genes, m6A-marked maternally inherited transcripts have a higher tendency to be targeted by microRNAs. Moreover, RNAs derived from retrotransposons, such as MTA that is maternally expressed and MERVL that is transcriptionally activated at the two-cell stage, are largely marked by m6A. Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development.

Successful Production of Offspring Derived from Phospholipase C Zeta-Deficient Sperm by Additional Artificial Activation.

During mammalian fertilization, repetitive rises of intracellular calcium called calcium oscillations are required for full activation of oocytes. Therefore, oocytes such as round spermatid injected or somatic cell nuclear transferred require additional artificial activation which mimics the calcium oscillations. It is well recognized that sperm specific phospholipase C (PLCζ) is a strong candidate as the sperm factor which can induce calcium oscillations and, at least in mammals, the genetic mutation of PLCζ in human causes male infertility due to the lack of calcium oscillations in the oocytes. Recent studies showed that the sperm lacking PLCζ (Plcz1-/-) still could induce rise(s) of intracellular calcium in the oocytes after IVF but not intracytoplasmic sperm injection (ICSI). In the ICSI oocytes, no pronuclear formation or development to the two-cell stage was observed. However, it is still unclear whether additional activation treatment can rescue the low developmental ability of Plcz1-/--sperm-derived oocytes after ICSI. In this study, we examined whether oocytes injected with a Plcz1-/- sperm can develop to term by additional artificial activation. In oocytes injected a Plcz1-/- sperm and Plcz1-/- and eCS (another candidate of the sperm factor) double knockout sperm (Plcz1-/-eCS-/-), the rates of pronuclear formation were very low (2.0 ± 2.3% and 6.1 ± 3.7%, respectively) compared to control (92.1 ± 2.6%). However, these rates were dramatically improved by additional procedures of PLCζ-mRNA injection or SrCl2 treatment (Plcz1-/- sperm + PLCζ mRNA, Plcz1-/- sperm + SrCl2 and Plcz1-/-eCS-/- sperm + PLCζ mRNA; 64.2 ± 10.8%, 89.2 ± 2.4% and 72.6 ± 5.4%, respectively). Most of the oocytes were developed to the two-cell stage. After embryo transfer, healthy pups were obtained in all these groups (Plcz1-/- sperm + PLCζ mRNA:10.0 ± 2.8%, Plcz1-/- sperm + SrCl2:4.0 ± 4.3% and Plcz1-/-eCS-/- sperm + PLCζ mRNA: 10.0 ± 5.7%). The rate in Plcz1-/- sperm + SrCl2 group was significantly lower than that in control (26.0 ± 2.4%). Taken together, our present results show that additional activation treatment such as SrCl2 and PLCζ mRNA can fully support to develop to term even in oocyte injected Plcz1-/- sperm. In addition, PLCζ-induced oocyte activation is more suitable for successful development to term compared to that such as phenomenon induced by SrCl2. These findings will contribute to improvement for male-dependent human infertility and reproductive technologies in other mammalian species.

A change in biophysical properties accompanies heterochromatin formation in mouse embryos.

The majority of our genome is composed of repeated DNA sequences that assemble into heterochromatin, a highly compacted structure that constrains their mutational potential. How heterochromatin forms during development and how its structure is maintained are not fully understood. Here, we show that mouse heterochromatin phase-separates after fertilization, during the earliest stages of mammalian embryogenesis. Using high-resolution quantitative imaging and molecular biology approaches, we show that pericentromeric heterochromatin displays properties consistent with a liquid-like state at the two-cell stage, which change at the four-cell stage, when chromocenters mature and heterochromatin becomes silent. Disrupting the condensates results in altered transcript levels of pericentromeric heterochromatin, suggesting a functional role for phase separation in heterochromatin function. Thus, our work shows that mouse heterochromatin forms membrane-less compartments with biophysical properties that change during development and provides new insights into the self-organization of chromatin domains during mammalian embryogenesis.