Nr5a2 is dispensable for zygotic genome activation but essential for morula development.

Early embryogenesis is driven by transcription factors (TFs) that first activate the zygotic genome and then specify the lineages constituting the blastocyst. Although the TFs specifying the blastocyst's lineages are well characterized, those playing earlier roles remain poorly defined. Using mouse models of the TF Nr5a2, we show that Nr5a2-/- embryos arrest at the early morula stage and exhibit altered lineage specification, frequent mitotic failure, and substantial chromosome segregation defects. Although NR5A2 plays a minor but measurable role during zygotic genome activation, it predominantly acts as a master regulator at the eight-cell stage, controlling expression of lineage-specifying TFs and genes involved in mitosis, telomere maintenance, and DNA repair. We conclude that NR5A2 coordinates proliferation, genome stability, and lineage specification to ensure correct morula development.

The TLI system can help day-3 single cleavage embryo transfer to obtain comparative clinical outcomes to day-4 or day-5.

The aim was to explore whether the time-lapse imaging system can help day-3 single cleavage embryo transfer to obtain comparative clinical outcomes to day-4 or 5. The data of 1237 patients who underwent single embryo transfer from January 1, 2018, to September 30, 2020, in our reproductive medicine centre were retrospectively analysed. They were divided into the day-3 single cleavage-stage embryo transfer (SCT) group (n = 357), day-4 single morula transfer (SMT) group (n = 129) and day-5 single blastocyst transfer (SBT) group (n = 751) according to the different embryo transfer stage. The clinical and perinatal outcomes of the three groups were analysed and compared. The clinical pregnancy rates of the patients in the day-3 SCT group, day-4 SMT group and day-5 SBT group were 68.07, 70.54 and 72.04%, respectively. The live birth rates were 56.86, 61.24 and 60.99%, respectively. The monozygotic twin (MZT) rate in the day-3 SCT group was significantly lower than that in the day-5 SBT group (P = 0.049). Regarding perinatal outcomes, only the secondary sex ratio had a significant difference (P < 0.05). After age stratification, no improvement was found in the pregnancy outcomes of patients >35 years of age receiving blastocyst transfer. Our findings suggest that for patients with multiple high-quality embryos on day-3, prolonging the culture time can improve the pregnancy outcome to some extent, but it will bring risks. For centres that have established morphodynamic models, day-3 SCT can also achieve an ideal pregnancy outcome and reduce the rate of monozygotic twins and sex ratio.

Comparison of pregnancy outcomes between 4th day morula and 5th day blastocyst after embryo transfer: a retrospective cohort study.

BACKGROUND: This study was designed to evaluate pregnancy outcomes between morulae transferred on day 4 (D4) and blastocysts transferred on day 5 (D5). METHODS: From September 2017 to September 2020, 1963 fresh transfer cycles underwent early follicular phase extra-long protocol for assisted conception in our fertility center were divided into D4 (324 cases) and D5 (1639 cases) groups, and the general situation and other differences of patients in both groups were compared. To compare the differences in pregnancy outcomes, the D4 and D5 groups were further divided into groups A and B based on single and double embryo transfers. Furthermore, the cohort was divided into two groups: those with live births (1116 cases) and those without (847 cases), enabling a deeper evaluation of the effects of D4 or D5 transplantation on assisted reproductive outcomes. RESULTS: In single embryo transfer, there was no significant difference between groups D4A and D5A (P > 0.05). In double embryo transfer, group D4B had a lower newborn birthweight and a larger proportion of low birthweight infants (P < 0.05). The preterm delivery rate, twin delivery rate, cesarean delivery rate, and percentage of low birthweight infants were lower in the D5A group than in the D5B group (P < 0.05). Analysis of factors influencing live birth outcomes further confirmed the absence of a significant difference between D4 and D5 transplantation in achieving live birth (P > 0.05). CONCLUSION: When factors such as working life and hospital holidays are being considered, D4 morula transfer may be a good alternative to D5 blastocyst transfer. Given the in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) success rate and risk of twin pregnancy, D4 morula transfer requires an adapted decision between single and double embryo transfer, although a single blastocyst transfer is recommended for the D5 transfer in order to decrease the twin pregnancy rate. In addition, age, endometrial thickness and other factors need to be taken into account to personalize the IVF program and optimize pregnancy outcomes.

Branching topology of the human embryo transcriptome revealed by Entropy Sort Feature Weighting.

Analysis of single cell transcriptomics (scRNA-seq) data is typically performed after subsetting to highly variable genes (HVGs). Here, we show that Entropy Sorting provides an alternative mathematical framework for feature selection. On synthetic datasets, continuous Entropy Sort Feature Weighting (cESFW) outperforms HVG selection in distinguishing cell-state-specific genes. We apply cESFW to six merged scRNA-seq datasets spanning human early embryo development. Without smoothing or augmenting the raw counts matrices, cESFW generates a high-resolution embedding displaying coherent developmental progression from eight-cell to post-implantation stages and delineating 15 distinct cell states. The embedding highlights sequential lineage decisions during blastocyst development, while unsupervised clustering identifies branch point populations obscured in previous analyses. The first branching region, where morula cells become specified for inner cell mass or trophectoderm, includes cells previously asserted to lack a developmental trajectory. We quantify the relatedness of different pluripotent stem cell cultures to distinct embryo cell types and identify marker genes of naïve and primed pluripotency. Finally, by revealing genes with dynamic lineage-specific expression, we provide markers for staging progression from morula to blastocyst.

Time-lapse imaging of morula compaction for selecting high-quality blastocysts: a retrospective cohort study.

PURPOSE: Before blastocyst development, embryos undergo morphological and metabolic changes crucial for their subsequent growth. This study aimed to investigate the relationship between morula compaction and blastocyst formation and the subsequent chromosomal status of the embryos. METHODS: This retrospective cohort study evaluated embryo development (n = 371) using time-lapse imaging; 94 blastocysts underwent preimplantation genetic testing for aneuploidy (PGT-A). RESULTS: The embryos were classified as fully (Group 1, n = 194) or partially (Group 2, n = 177) compacted. Group 1 had significantly higher proportions of good- and average-quality blastocysts than Group 2 (21.6% vs. 3.4%, p = 0.001; 47.9% vs. 26.6%, p = 0.001, respectively). The time from the morula stage to the beginning and completion of compaction and blastocyst formation was significantly shorter in Group 1 than in Group 2 (78.6 vs. 82.4 h, p = 0.001; 87.0 vs. 92.2 h, p = 0.001; 100.2 vs. 103.7 h, p = 0.017, respectively). Group 1 embryos had larger surface areas than Group 2 embryos at various time points following blastocyst formation. Group 1 blastocysts had significantly higher average expansion rates than Group 2 blastocysts (653.6 vs. 499.2 µm2/h, p = 0.001). PGT-A revealed a higher proportion of euploid embryos in Group 1 than in Group 2 (47.2% vs. 36.6%, p = 0.303). CONCLUSION: Time-lapse microscopy uncovered a positive relationship between compaction and blastocyst quality and its association with embryo ploidy. Hence, compaction evaluation should be prioritized before blastocyst selection for transfer or cryopreservation.

Protocol for generating mouse morula-like cells resembling 8- to 16-cell stage embryo cells.

Generating cell types with properties of embryo cells with full developmental potential is of great biological importance. Here, we present a protocol for generating mouse morula-like cells (MLCs) resembling 8- to 16-cell stage embryo cells. We describe steps for induction, via increasing Stat3 activation, and the isolation of MLCs. We then detail procedures for segregating MLCs into blastocyst cell fates and how to create embryo-like structures from them. This system provides a stem-cell-based embryo model to study early embryo development. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Maternal Ezh1/2 deficiency impairs the function of mitochondria in mouse oocytes and early embryos.

Maternal histone methyltransferase is critical for epigenetic regulation and development of mammalian embryos by regulating histone and DNA modifications. Here, we reported a novel mechanism by revealing the critical effects of maternal Ezh1/2 deletion on mitochondria in MII oocytes and early embryos in mice. We found that Ezh1/2 knockout in mouse MII oocytes impaired the structure of mitochondria and decreased its number, but membrane potential and respiratory function of mitochondrion were increased. The similar effects of Ezh1/2 deletion have been observed in 2-cell and morula embryos, indicating that the effects of maternal Ezh1/2 deficiency on mitochondrion extend to early embryos. However, the loss of maternal Ezh1/2 resulted in a severe defect of morula: the number, membrane potential, respiratory function, and ATP production of mitochondrion dropped significantly. Content of reactive oxygen species was raised in both MII oocytes and early embryos, suggesting maternal Ezh1/2 knockout induced oxidative stress. In addition, maternal Ezh1/2 ablation interfered the autophagy in morula and blastocyst embryos. Finally, maternal Ezh1/2 deletion led to cell apoptosis in blastocyst embryos in mice. By analyzing the gene expression profile, we revealed that maternal Ezh1/2 knockout affected the expression of mitochondrial related genes in MII oocytes and early embryos. The chromatin immunoprecipitation-polymerase chain reaction assay demonstrated that Ezh1/2 directly regulated the expression of genes Fxyd6, Adpgk, Aurkb, Zfp521, Ehd3, Sgms2, Pygl, Slc1a1, and Chst12 by H3K27me3 modification. In conclusion, our study revealed the critical effect of maternal Ezh1/2 on the structure and function of mitochondria in oocytes and early embryos, and suggested a novel mechanism underlying maternal epigenetic regulation on early embryonic development through the modulation of mitochondrial status.

Blastocysts from partial compaction morulae are not defined by their early mistakes.

RESEARCH QUESTION: Is partial compaction during morula formation associated with an embryo's developmental ability and implantation potential? DESIGN: Retrospective analysis of data from 196 preimplantation genetic testing for aneuploidy (PGT-A) cycles. Embryos starting compaction were grouped according to the inclusion or not of all the blastomeres in the forming morula (full compaction or partial compaction). The possible effect of maternal age and ovarian response on compaction was analysed. Morphokinetic characteristics, blastocyst formation rate, morphology and cytogenetic constitution of the obtained blastocysts were compared. Comparisons of reproductive outcomes after the transfer of euploid blastocysts from both groups were established. Finally, in a subset of embryos, the chromosomal constitution concordance of the abandoned cells and the corresponding blastocyst through trophectoderm biopsies was assessed. RESULTS: A total of 430 embryos failed to include at least one cell during compaction (partial compaction group [49.3%]), whereas the 442 remaining embryos formed a fully compacted morula (full compaction group [50.7%]). Neither female age nor the number of oocytes collected affected the prevalence of partial compaction morulae. Morphokinetic parameters were altered in embryos from partial compaction morulae compared with full compaction. Although an impairment in blastocyst formation rate was observed in partial compaction morulae (57.2% versus 70.8%, P < 0.001), both chromosomal constitution (euploidy rate: partial compaction [38.4%] versus full compaction [34.2%]) and reproductive outcomes (live birth rate: partial compaction [51.9%] versus full compaction [46.2%]) of the obtained blastocysts were equivalent between groups. A high ploidy correlation of excluded cells-trophectoderm duos was observed. CONCLUSIONS: Partial compaction morulae show a reduced developmental ability compared with full compaction morulae. Resulting blastocysts from both groups, however, have similar euploidy rates and reproductive outcomes. Cell exclusion might be a consequence of a compromised embryo development regardless of the chromosomal constitution of the excluded cells.

Induction of Transient Morula-Like Cells in Mice Through STAT3 Activation.

Developing in vitro cell models that faithfully replicate the molecular and functional traits of cells from the earliest stages of mammalian development presents a significant challenge. The strategic induction of signal transducer and activator of transcription 3 (STAT3) phosphorylation, coupled with carefully defined culture conditions, facilitates the efficient reprogramming of mouse pluripotent cells into a transient morula-like cell (MLC) state. The resulting MLCs closely mirror their in vivo counterparts, exhibiting not only molecular resemblance but also the ability to differentiate into both embryonic and extraembryonic lineages. This reprogramming approach provides valuable insights into controlled cellular fate choice and opens new opportunities for studying early developmental processes in a dish.

Association of early cleavage, morula compaction and blastocysts ploidy of IVF embryos cultured in a time-lapse system and biopsied for genetic test for aneuploidy.

IVF embryos have historically been evaluated by morphological characteristics. The time-lapse system (TLS) has become a promising tool, providing an uninterrupted evaluation of morphological and dynamic parameters of embryo development. Furthermore, TLS sheds light on unknown phenomena such as direct cleavage and incomplete morula compaction. We retrospectively analyzed the morphology (Gardner Score) and morphokinetics (KIDScore) of 835 blastocysts grown in a TLS incubator (Embryoscope+), which were biopsied for preimplantation genetic testing for aneuploidy (PGT-A). Only the embryos that reached the blastocyst stage were included in this study and time-lapse videos were retrospectively reanalysed. According to the pattern of initial cleavages and morula compaction, the embryos were classified as: normal (NC) or abnormal (AC) cleavage, and fully (FCM) or partially compacted (PCM) morulae. No difference was found in early cleavage types or morula compaction patterns between female age groups (< 38, 38-40 and > 40 yo). Most of NC embryos resulted in FCM (≅ 60%), while no embryos with AC resulted in FCM. Aneuploidy rate of AC-PCM group did not differ from that of NC-FCM group in women < 38 yo, but aneuploidy was significantly higher in AC-PCM compared to NC-FCM of women > 40 yo. However, the quality of embryos was lower in AC-PCM blastocysts in women of all age ranges. Morphological and morphokinetic scores declined with increasing age, in the NC-PCM and AC-PCM groups, compared to the NC-FCM. Similar aneuploidy rates among NC-FCM and AC-PCM groups support the hypothesis that PCM in anomalous-cleaved embryos can represent a potential correction mechanism, even though lower morphological/morphokinetic scores are seen on AC-PCM. Therefore, both morphological and morphokinetic assessment should consider these embryonic development phenomena.

In vitro generation of mouse morula-like cells.

Generating cells with the molecular and functional properties of embryo cells and with full developmental potential is an aim with fundamental biological significance. Here we report the in vitro generation of mouse transient morula-like cells (MLCs) via the manipulation of signaling pathways. MLCs are molecularly distinct from embryonic stem cells (ESCs) and cluster instead with embryo 8- to 16-cell stage cells. A single MLC can generate a blastoid, and the efficiency increases to 80% when 8-10 MLCs are used. MLCs make embryoids directly, efficiently, and within 4 days. Transcriptomic analysis shows that day 4-5 MLC-derived embryoids contain the cell types found in natural embryos at early gastrulation. Furthermore, MLCs introduced into morulae segregate into epiblast (EPI), primitive endoderm (PrE), and trophectoderm (TE) fates in blastocyst chimeras and have a molecular signature indistinguishable from that of host embryo cells. These findings represent the generation of cells that are molecularly and functionally similar to the precursors of the first three cell lineages of the embryo.

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.

Characterization of the long noncoding RNA transcriptome in human preimplantation embryo development.

PURPOSE: Infertility remains a human health burden globally. Only a fraction of embryos produced via assisted reproductive technologies (ARTs) develop to the blastocyst stage in vitro. lncRNA abundance changes significantly during human early embryonic development, indicating vital regulatory roles of lncRNAs in this process. The aim of this study is to obtain insights into the transcriptional basis of developmental events. METHODS: scRNA-seq data and SUPeR-seq data were used to investigate the lncRNA profiles of human preimplantation embryos. The top 50 highly expressed unique and shared lncRNAs in each stage of preimplantation development were identified. Comparative analysis of the two datasets was used to verify the consistent expression patterns of the lncRNAs. Differentially expressed lncRNAs were identified and subjected to functional enrichment analysis. RESULTS: The lncRNA profiles of human preimplantation embryos in the E-MTAB-3929 dataset were similar to those in the GSE71318 dataset. The ratios of overlap among the top 50 highly expressed lncRNAs between two pairs of stages (2-cell stage vs. 4-cell stage and 8-cell stage vs. morula) were aberrantly low compared with those between other stages. Each stage of preimplantation development exhibited unique and shared lncRNAs among the top 50 highly expressed lncRNAs. Among the between-group comparisons, the 2-cell stage vs. 4-cell stage showed the highest number of differentially expressed lncRNAs. Functional enrichment analysis revealed that differentially expressed lncRNAs and their associated super enhancers and RNA binding proteins (RBPs) are closely involved in regulating embryonic development. These lncRNAs could function as important cell markers for distinguishing fetal germ cells. CONCLUSIONS: Our study paves the way for understanding the regulation of developmental events, which might be beneficial for improved reproductive outcomes.

Maternal Kdm2a-mediated PI3K/Akt signaling and E-cadherin stimulate the morula-to-blastocyst transition revealing crucial roles in early embryonic development.

Histone methylation plays an essential role in oocyte growth and preimplantation embryonic development. The modification relies on histone methyl-transferases and demethylases, and one of these, lysine-specific demethylase 2a (Kdm2a), is responsible for modulating histone methylation during oocyte and early embryonic development. The mechanism of how Kdm2a deficiency disrupts early embryonic development and fertility remains elusive. To determine if maternally deposited Kdm2a is required for preimplantation embryonic development, the expression profile of Kdm2a during early embryos was detected via immunofluorescence staining and RT-qPCR. The Kdm2a gene in oocytes was specifically deleted with the Zp3-Cre/LoxP system and the effects of maternal Kdm2a loss were studied through a comprehensive range of female reproductive parameters including fertilization, embryo development, and the number of births. RNA transcriptome sequencing was performed to determine differential mRNA expression, and the interaction between Kdm2a and the PI3K/Akt pathway was studied with a specific inhibitor and activator. Our results revealed that Kdm2a was continuously expressed in preimplantation embryos and loss of maternal Kdm2a suppressed the morula-to-blastocyst transition, which may have been responsible for female subfertility. After the deletion of Kdm2a, the global H3K36me2 methylation in mutant embryos was markedly increased, but the expression of E-cadherin decreased significantly in morula embryos compared to controls. Mechanistically, RNA-seq analysis revealed that deficiency of maternal Kdm2a altered the mRNA expression profile, especially in the PI3K/Akt signaling pathway. Interestingly, the addition of a PI3K/Akt inhibitor (LY294002) to the culture medium blocked embryo development at the stage of morula; however, the developmental block caused by maternal Kdm2a loss was partially rescued with a PI3K/Akt activator (SC79). In summary, our results indicate that loss of Kdm2a influences the transcriptome profile and disrupts the PI3K/Akt signaling pathway during the development of preimplantation embryo. This can result in embryo block at the morula stage and female subfertility, which suggests that maternal Kdm2a is a potential partial redundancy with other genes encoding enzymes in the dynamics of early embryonic development. Our results provide further insight into the role of histone modification, especially on Kdm2a, in preimplantation embryonic development in mice.

Involvement of linker histone variant H1a in the regulation of early preimplantation development in mice.

Linker histone variants regulate higher-order chromatin structure and various cellular processes. It has been suggested that linker histone variant H1a loosens chromatin structure and activates transcription. However, its role in early mouse development remains to be elucidated. We investigated the functions of H1a during preimplantation development using H1a gene-deleted mice. Although H1a homozygous knockout (KO) mice were born without any abnormalities, the number of offspring were reduced when the mothers but not fathers were homozygous KO animals. Maternal H1a KO compromised development during the morula and blastocyst stages, but not differentiation of the inner cell mass or trophectoderm. Thus, maternal linker histone H1a is important in early development.

Maternal age affects pronuclear and chromatin dynamics, morula compaction and cell polarity, and blastulation of human embryos.

STUDY QUESTION: Does maternal ageing impact early and late morphokinetic and cellular processes of human blastocyst formation? SUMMARY ANSWER: Maternal ageing significantly affects pronuclear size and intra- and extra-nuclear dynamics during fertilization, dysregulates cell polarity during compaction, and reduces blastocoel expansion. WHAT IS KNOWN ALREADY: In ART, advanced maternal age (AMA) affects oocyte yield, fertilization, and overall developmental competence. However, with the exception of chromosome segregation errors occurring during oocyte meiosis, the molecular and biochemical mechanisms responsible for AMA-related subfertility and reduced embryo developmental competence remain unclear. In particular, studies reporting morphokinetics and cellular alterations during the fertilization and pre-implantation period in women of AMA remain limited. STUDY DESIGN, SIZE, DURATION: A total of 2058 fertilized oocytes were stratified by maternal age according to the Society for Assisted Reproductive Technology classification (<35, 35-37, 38-40, 41-42, and >42 years) and retrospectively analysed. AMA effects were assessed in relation to: embryo morphokinetics and morphological alterations; and the presence and distribution of cell polarity markers-Yes-associated protein (YAP) and protein kinase C-ζ (PKC-ζ)-involved in blastocyst morphogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 1050 cycles from 1050 patients met the inclusion criteria and were analysed. Microinjected oocytes were assessed using a time-lapse culture system. Immature oocytes at oocyte retrieval and mature oocytes not suitable for time-lapse monitoring, owing to an excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage patterns and embryo quality were annotated and compared among groups. Furthermore, 20 human embryos donated for research by consenting couples were used for immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: Static microscopic observation revealed that blastocyst formation and expansion were impaired in the 41-42 and >42-year groups (P < 0.0001). The morphological grades of the inner cell mass and trophectoderm were poorer in the >42-year group than those in the <35-year group (P = 0.0022 and P < 0.0001, respectively). Time-lapse microscopic observation revealed a reduction in nucleolus precursor body alignment in female pronuclei in the 41-42 and >42-year groups (P = 0.0010). Female pronuclear area decreased and asynchronous pronuclear breakdown increased in the >42-year group (P = 0.0027 and P < 0.0122, respectively). Developmental speed at cleavage stage, incidence of irregularity of first cleavage, type and duration of blastomere movement, and number of multinucleated cells were comparable among age groups. Delayed embryonic compaction and an increased number of extruded blastomeres were observed in the >42-year group (P = 0.0002 and P = 0.0047, respectively). Blastulation and blastocyst expansion were also delayed in the 41-42 and >42-year groups (P < 0.0001 for both). YAP positivity rate in the outer cells of morulae and embryo PKC-ζ immunoflourescence decreased in the >42-year group (P < 0.0001 for both). LIMITATIONS, REASONS FOR CAUTION: At the cellular level, the investigation was limited to cell polarity markers. Cell components of other developmental pathways should be studied in relation to AMA. WIDER IMPLICATIONS OF THE FINDINGS: The study indicates that maternal ageing affects the key functions of embryo morphogenesis, irrespective of the well-established influence on the fidelity of oocyte meiosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the participating institutions. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

The proteomic analysis of bovine embryos developed in vivo or in vitro reveals the contribution of the maternal environment to early embryo.

BACKGROUND: Despite many improvements with in vitro culture systems, the quality and developmental ability of mammalian embryos produced in vitro are still lower than their in vivo counterparts. Though previous studies have evidenced differences in gene expression between in vivo- and in vitro-derived bovine embryos, there is no comparison at the protein expression level. RESULTS: A total of 38 pools of grade-1 quality bovine embryos at the 4-6 cell, 8-12 cell, morula, compact morula, and blastocyst stages developed either in vivo or in vitro were analyzed by nano-liquid chromatography coupled with label-free quantitative mass spectrometry, allowing for the identification of 3,028 proteins. Multivariate analysis of quantified proteins showed a clear separation of embryo pools according to their in vivo or in vitro origin at all stages. Three clusters of differentially abundant proteins (DAPs) were evidenced according to embryo origin, including 463 proteins more abundant in vivo than in vitro across development and 314 and 222 proteins more abundant in vitro than in vivo before and after the morula stage, respectively. The functional analysis of proteins found more abundant in vivo showed an enrichment in carbohydrate metabolism and cytoplasmic cellular components. Proteins found more abundant in vitro before the morula stage were mostly localized in mitochondrial matrix and involved in ATP-dependent activity, while those overabundant after the morula stage were mostly localized in the ribonucleoprotein complex and involved in protein synthesis. Oviductin and other oviductal proteins, previously shown to interact with early embryos, were among the most overabundant proteins after in vivo development. CONCLUSIONS: The maternal environment led to higher degradation of mitochondrial proteins at early developmental stages, lower abundance of proteins involved in protein synthesis at the time of embryonic genome activation, and a global upregulation of carbohydrate metabolic pathways compared to in vitro production. Furthermore, embryos developed in vivo internalized large amounts of oviductin and other proteins probably originated in the oviduct as soon as the 4-6 cell stage. These data provide new insight into the molecular contribution of the mother to the developmental ability of early embryos and will help design better in vitro culture systems.

High-resolution ribosome profiling reveals translational selectivity for transcripts in bovine preimplantation embryo development.

High-resolution ribosome fractionation and low-input ribosome profiling of bovine oocytes and preimplantation embryos has enabled us to define the translational landscapes of early embryo development at an unprecedented level. We analyzed the transcriptome and the polysome- and non-polysome-bound RNA profiles of bovine oocytes (germinal vesicle and metaphase II stages) and early embryos at the two-cell, eight-cell, morula and blastocyst stages, and revealed four modes of translational selectivity: (1) selective translation of non-abundant mRNAs; (2) active, but modest translation of a selection of highly expressed mRNAs; (3) translationally suppressed abundant to moderately abundant mRNAs; and (4) mRNAs associated specifically with monosomes. A strong translational selection of low-abundance transcripts involved in metabolic pathways and lysosomes was found throughout bovine embryonic development. Notably, genes involved in mitochondrial function were prioritized for translation. We found that translation largely reflected transcription in oocytes and two-cell embryos, but observed a marked shift in the translational control in eight-cell embryos that was associated with the main phase of embryonic genome activation. Subsequently, transcription and translation become more synchronized in morulae and blastocysts. Taken together, these data reveal a unique spatiotemporal translational regulation that accompanies bovine preimplantation development.

Knockdown of Toe1 causes developmental arrest during the morula-to-blastocyst transition in mice.

The target of EGR1 protein 1 (TOE1) is evolutionarily conserved from Caenorhabditis elegans to mammals, which plays a critical role in the maturation of a variety of small nuclear RNAs. Mutation in human TOE1 has been reported to cause pontocerebellar hypoplasia type 7, a severe neurodegenerative syndrome. However, the role of TOE1 in early embryonic development remains unclear. Herein, we found that Toe1 mRNA and protein were expressed in mouse preimplantation embryos. Silencing Toe1 by siRNA led to morula-to-blastocyst transition failure. This developmental arrest can be rescued by Toe1 mRNA microinjection. EdU incorporation assay showed a defect in blastomere proliferation within developmentally arrested embryos. Further studies revealed that Toe1 knockdown caused increased signals for γH2AX and micronuclei, indicative of sustained DNA damage. Moreover, mRNA levels of cell cycle inhibitor p21 were significantly upregulated in Toe1 knockdown embryos before developmental arrest. Together, these results suggest that TOE1 is indispensable for mouse early embryo development potentially through maintaining genomic integrity. Our findings provide further insight into the role of TOE1 in mouse preimplantation embryonic development.

Establishment of 3D chromatin structure after fertilization and the metabolic switch at the morula-to-blastocyst transition require CTCF.

The eukaryotic genome is organized in 3D at different scales. This structure is driven and maintained by different chromatin states and by architectural factors, such as the zinc finger protein CTCF. Zygotic genome structure is established de novo after fertilization, but its impact during the first stages of mammalian development is unclear. We show that deletion of Ctcf in mouse embryos impairs the establishment of chromatin structure, but the first cell fate decision is unperturbed and embryos are viable until the late blastocyst. Furthermore, maternal CTCF is not necessary for development. Gene expression changes in metabolic and protein homeostasis programs that occur during the morula-to-blastocyst transition depend on CTCF. However, these changes do not correlate with disruption of chromatin but with binding of CTCF to the promoter of downregulated genes. Our results show that CTCF regulates both 3D genome organization and transcription during mouse preimplantation development, but as independent processes.