Single cell RNA sequencing of preimplantation bovine embryos grown under different oxygen levels
Ontology highlight
ABSTRACT: We utilised STRTN-seq, a newly optimised single cell 5' targeted RNA sequencing method to understand the development of bovine preimplantation embryos under different oxygen levels. Single embryos from the zygote, 4-cell, 8-cell, 16-cell and blastocyst stages were sampled for experiments. Here, FASTQ files can be found. The raw data (BCL files) is available in DOI 10.5281/zenodo.13384651
Project description:DNA methylation reprogramming plays important roles in mammalian embryogenesis. Mammalian somatic cell nuclear transfer (SCNT) embryos with reprogramming defects fail to develop. Thus, we compared DNA methylation reprogramming in preimplantation embryos from bovine SCNT and in vitro fertilization (IVF) and analyzed the influence of vitamin C (VC) on the reprogramming of DNA methylation. The results showed that global DNA methylation followed a typical pattern of demethylation and remethylation in IVF preimplantation embryos; however, the global genome remained hypermethylated in SCNT preimplantation embryos. Compared with the IVF group, locus DNA methylation reprogramming showed three patterns in the SCNT group. First, some pluripotency genes (POU5F1 and NANOG) and repeated elements (satellite I and α-satellite) showed insufficient demethylation and hypermethylation in the SCNT group. Second, a differentially methylated region (DMR) of an imprint control region (ICR) in H19 exhibited excessive demethylation and hypomethylation. Third, some pluripotency genes (CDX2 and SOX2) were hypomethylated in both the IVF and SCNT groups. Additionally, VC improved the DNA methylation reprogramming of satellite I, α-satellite and H19 but not that of POU5F1 and NANOG in SCNT preimplantation embryos. These results indicate that DNA methylation reprogramming was aberrant and that VC influenced DNA methylation reprogramming in SCNT embryos in a locus-specific manner.
Project description:The development of next generation sequencing (NGS) platform-based single-cell RNA sequencing (scRNA-seq) techniques has tremendously changed biological researches, while there are still many questions that cannot be addressed by them due to their short read lengths. We developed a novel scRNA-seq technology based on third-generation sequencing (TGS) platform (single-cell amplification and sequencing of full-length RNAs by Nanopore platform, SCAN-seq). SCAN-seq exhibited high sensitivity and accuracy comparable to NGS platform-based scRNA-seq methods. Moreover, we captured thousands of unannotated transcripts of diverse types, with high verification rate by reverse transcription PCR (RT-PCR)-coupled Sanger sequencing in mouse embryonic stem cells (mESCs). Then, we used SCAN-seq to analyze the mouse preimplantation embryos. We could clearly distinguish cells at different developmental stages, and a total of 27,250 unannotated transcripts from 9,338 genes were identified, with many of which showed developmental stage-specific expression patterns. Finally, we showed that SCAN-seq exhibited high accuracy on determining allele-specific gene expression patterns within an individual cell. SCAN-seq makes a major breakthrough for single-cell transcriptome analysis field.
Project description:We developed the Rainbow-seq technology to trace cell division history and reveal single-cell transcriptomes. With distinct fluorescent protein genes as lineage markers, Rainbow-seq enables each single-cell RNA sequencing (RNA-seq) experiment to simultaneously decode the lineage marker genes and read single-cell transcriptomes. We triggered lineage tracking in each blastomere at the 2-cell stage, observed microscopically inequivalent contributions of the progeny to the two embryonic poles at the blastocyst stage, and analyzed every single cell at either 4- or 8-cell stage with deep paired-end sequencing of full-length transcripts. Although lineage difference was not marked unequivocally at a single-gene level, it became clear when the transcriptome was analyzed as a whole. Moreover, several groups of novel transcript isoforms with embedded repeat sequences exhibited lineage difference, suggesting a possible link between DNA demethylation and cell fate decision. Rainbow-seq bridged a critical gap between division history and single-cell RNA-seq assays.
Project description:During maternal-to-embryonic transition control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome activation (EGA). Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts. Using the bovine model system, we established by RNA sequencing a comprehensive catalogue of transcripts in germinal vesicle and metaphase II oocytes, and in embryos at the four-cell, eight-cell, 16-cell, and blastocyst stages. These were produced by in vitro fertilization of Bos taurus taurus oocytes with sperm from a Bos taurus indicus bull to facilitate parent-specific transcriptome analysis. Transcripts from 12.4 to 13.7 × 10(3) different genes were detected in the various developmental stages. EGA was analyzed by (i) detection of embryonic transcripts, which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. These strategies revealed (i) 220, (ii) 937, and (iii) 6,848 genes to be activated from the four-cell to the blastocyst stage. The largest proportion of gene activation [i.e., (i) 59%, (ii) 42%, and (iii) 58%] was found in eight-cell embryos, indicating major EGA at this stage. Gene ontology analysis of genes activated at the four-cell stage identified categories related to RNA processing, translation, and transport, consistent with preparation for major EGA. Our study provides the largest transcriptome data set of bovine oocyte maturation and early embryonic development and detailed insight into the timing of embryonic activation of specific genes.
Project description:Lysosomal cathepsin, in particular cathepsin B (CTSB), plays an important role in implantation, pregnancy, and embryonic development. However, little is known about the mechanism related to the dynamic status of lysosomal cathepsins in bovine oocytes and preimplantation embryos. In the present study, we investigated the dynamics of gene expression, activity, and immunolocalization of CTSB, as well as the activities of lysosome, in bovine oocytes and preimplantation embryos. After gene expression analysis of several cathepsin-related genes, transcript levels of CTSB, CTSD and CTSZ were highest in Metaphase II (MII) oocytes followed by a significant decrease from the 8-cell embryo stage. Activity of CTSB showed a significant increase in 1-cell and morula stage embryos. Lysosomal activity was also significant higher in 1-cell and morula stages, which was consistent with CTSB activities. However, immunolocalization of CTSB did not show the similar pattern of CTSB and lysosomal activities. We also found significantly higher expression levels of CTSB transcript in the trophectoderm (TE) compared to inner cell mass (ICM), whereas activity and immunolocalization of CTSB showed an opposite pattern, i.e. significantly higher in ICM than TE. These patterns were confirmed by the same analysis using separated ICM and TE. Our results suggest that lysosomal CTSB has a pivotal role during embryonic development and differentiation, especially fertilization and the differentiation period.
Project description:Global activation of the embryonic genome is the most critical event in early mammalian development. After fertilization, a rich supply of maternal proteins and RNAs support development whereas a number of zygotic and embryonic genes are expressed in a stage-specific manner leading to embryonic genome activation (EGA). However, the identities of embryonic genes expressed and the mechanism(s) of EGA are poorly defined in the bovine. Using the Affymetrix bovine-specific DNA microarray as the biggest available array at present, we analyzed gene expression at two key stages of bovine development, matured oocytes (MII) and 8-cell-stage embryos, constituting the ultimate reservoir for life and a stage during which EGA takes place, respectively. Key genes in regulation of transcription, chromatin-structure cell adhesion, and signal transduction were up-regulated at the 8-cell stage as compared with 8-cell embryos treated with alpha-amanitin and MII. Genes controlling DNA methylation and metabolism were up-regulated in MII. These changes in gene expression, related to transcriptional machinery, chromatin structure, and the other cellular functions occurring during several cleavage stages, are expected to result in a unique chromatin structure capable of maintaining totipotency during embryogenesis and leading to differentiation during postimplantation development. Dramatic reprogramming of gene expression at the onset of development also has implications for cell plasticity in somatic cell nuclear transfer, genomic imprinting, and cancer.
Project description:DNA methylation is an important epigenetic modification that undergoes dynamic changes in mammalian embryogenesis, during which both parental genomes are reprogrammed. Despite the many immunostaining studies that have assessed global methylation, the gene-specific DNA methylation patterns in bovine preimplantation embryos are unknown. Using reduced representation bisulfite sequencing, we determined genome-scale DNA methylation of bovine sperm and individual in vivo developed oocytes and preimplantation embryos. We show that (1) the major wave of genome-wide demethylation was completed by the 8-cell stage; (2) promoter methylation was significantly and inversely correlated with gene expression at the 8-cell and blastocyst stages; (3) sperm and oocytes have numerous differentially methylated regions (DMRs)-DMRs specific for sperm were strongly enriched in long terminal repeats and rapidly lost methylation in embryos; while the oocyte-specific DMRs were more frequently localized in exons and CpG islands (CGIs) and demethylated gradually across cleavage stages; (4) DMRs were also found between in vivo and in vitro matured oocytes; and (5) differential methylation between bovine gametes was confirmed in some but not all known imprinted genes. Our data provide insights into the complex epigenetic reprogramming of bovine early embryos, which serve as an important model for human preimplantation development.
Project description:Assisted reproductive technologies impact transcriptome and epigenome of embryos and can result in long-term phenotypic consequences. Whole-genome DNA methylation profiles from individual bovine blastocysts in vivo- and in vitro-derived (using three sources of protein: reproductive fluids, blood serum and bovine serum albumin) were generated. The impact of in vitro culture on DNA methylation was analyzed, and sex-specific methylation differences at blastocyst stage were uncovered. In vivo embryos showed the highest levels of methylation (29.5%), close to those produced in vitro with serum, whilst embryos produced in vitro with reproductive fluids or albumin showed less global methylation (25-25.4%). During repetitive element analysis, the serum group was the most affected. DNA methylation differences between in vivo and in vitro groups were more frequent in the first intron than in CpGi in promoters. Moreover, hierarchical cluster analysis showed that sex produced a stronger bias in the results than embryo origin. For each group, distance between male and female embryos varied, with in vivo blastocyst showing a lesser distance. Between the sexually dimorphic methylated tiles, which were biased to X-chromosome, critical factors for reproduction, developmental process, cell proliferation and DNA methylation machinery were included. These results support the idea that blastocysts show sexually-dimorphic DNA methylation patterns, and the known picture about the blastocyst methylome should be reconsidered.
Project description:The effects of the increased soil copper (Cu) on fruit quality due to the overuse of Cu agents have been a hot social issue. Seven representative citrus orchards in Guangxi province, China, were investigated to explore the fruit quality characteristics under different soil Cu levels and the relationship between soil-tree Cu and fruit quality. These results showed that pericarp color a value, titratable acid (TA), and vitamin C (Vc) were higher by 90.0, 166.6, and 22.4% in high Cu orchards and by 50.5, 204.2, and 55.3% in excess Cu orchards, compared with optimum Cu orchards. However, the ratio of total soluble solids (TSS)/TA was lower by 68.7% in high Cu orchards and by 61.6% in excess Cu orchards. With the increase of soil Cu concentrations, pericarp color a value and Vc were improved, TA with a trend of rising first then falling, and TSS/TA with a trend of falling first then rising were recorded. As fruit Cu increased, pericarp color a value and TSS reduced and as leaf Cu increased, TSS/TA decreased while Vc was improved. Moreover, a rise in soil Cu enhanced leaf Cu accumulation, and a rise in leaf Cu improved fruit Cu accumulation. Fruit Cu accumulation reduced fruit quality by direct effects, leaf Cu improved fruit quality by direct and indirect effects. Soil Cu affected fruit quality by indirect effects by regulating leaf Cu and fruit Cu. Therefore, reasonable regulation and control of soil Cu concentrations can effectively increase pericarp color, sugar, and acid accumulation in citrus fruit.
Project description:Intracellular calcium homeostasis is essential for proper cell function. We investigated the effects of heat shock on the development of and the intracellular Ca2+ levels in bovine preimplantation embryos in vitro and the effects of calcitonin (CT), a receptor-mediated Ca2+ regulator, on heat shock-induced events. Heat shock (40.5 C for 10 h between 20 and 30 h postinsemination) of in vitro-produced bovine embryos did not affect the cleavage rate; however, it significantly decreased the rates of development to the 5- to 8-cell and blastocyst stages as compared with those of the control cultured for the entire period at 38.5 C (P < 0.05). The relative intracellular Ca2+ levels at the 1-cell stage (5 h after the start of heat shock), as assessed by Fluo-8 AM, a fluorescent probe for Ca2+, indicated that heat shock significantly lowered the Ca2+ level as compared with the control level. Semiquantitative reverse transcription PCR and western blot analyses revealed the expression of CT receptor in bovine preimplantation embryos. The addition of CT (10 nM) to the culture medium ameliorated the heat shock-induced impairment of embryonic development beyond the 5- to 8-cell stage. The Ca2+ level in the heat-shocked embryos cultured with CT was similar to that of the control embryos, suggesting that heat shock lowers the Ca2+ level in fertilized embryos in vitro and that a lower Ca2+ level is implicated in heat shock-induced impairment of embryonic development. Intracellular Ca2+ -mobilizing agents, e.g., CT, may effectively circumvent the detrimental effects of heat shock on early embryonic development.