scholarly journals Whole-Genome DNA Methylation Dynamics of Sheep Preimplantation Embryo Investigated by Single-Cell DNA Methylome Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Zijing Zhang ◽  
Jiawei Xu ◽  
Shijie Lyu ◽  
Xiaoling Xin ◽  
Qiaoting Shi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Preimplantation Embryo ◽  
Preimplantation Embryos ◽  
Whole Genome ◽  
Cell Stage ◽  
Preimplantation Embryo Development ◽  
Developmental Block

The early stages of mammalian embryonic development involve the participation and cooperation of numerous complex processes, including nutritional, genetic, and epigenetic mechanisms. However, in embryos cultured in vitro, a developmental block occurs that affects embryo development and the efficiency of culture. Although the block period is reported to involve the transcriptional repression of maternal genes and transcriptional activation of zygotic genes, how epigenetic factors regulate developmental block is still unclear. In this study, we systematically analyzed whole-genome methylation levels during five stages of sheep oocyte and preimplantation embryo development using single-cell level whole genome bisulphite sequencing (SC-WGBS) technology. Then, we examined several million CpG sites in individual cells at each evaluated developmental stage to identify the methylation changes that take place during the development of sheep preimplantation embryos. Our results showed that two strong waves of methylation changes occurred, namely, demethylation at the 8-cell to 16-cell stage and methylation at the 16-cell to 32-cell stage. Analysis of DNA methylation patterns in different functional regions revealed a stable hypermethylation status in 3′UTRs and gene bodies; however, significant differences were observed in intergenic and promoter regions at different developmental stages. Changes in methylation at different stages of preimplantation embryo development were also compared to investigate the molecular mechanisms involved in sheep embryo development at the methylation level. In conclusion, we report a detailed analysis of the DNA methylation dynamics during the development of sheep preimplantation embryos. Our results provide an explanation for the complex regulatory mechanisms underlying the embryo developmental block based on changes in DNA methylation levels.

scholarly journals Inhibition of DRP1 Impedes Zygotic Genome Activation and Preimplantation Development in Mice

2021 ◽  
Vol 9 ◽  
Author(s):  
Yuanyuan Li ◽  
Ning-Hua Mei ◽  
Gui-Ping Cheng ◽  
Jing Yang ◽  
Li-Quan Zhou
Keyword(s):  
Embryo Development ◽  
Preimplantation Embryo ◽  
Preimplantation Embryos ◽  
Dependent Manner ◽  
Zygotic Genome Activation ◽  
Preimplantation Embryo Development ◽  
Cell Embryo ◽  
Embryo Stage ◽  
Genome Activation ◽  
Zygotic Genome

Mitochondrion plays an indispensable role during preimplantation embryo development. Dynamic-related protein 1 (DRP1) is critical for mitochondrial fission and controls oocyte maturation. However, its role in preimplantation embryo development is still lacking. In this study, we demonstrate that inhibition of DRP1 activity by mitochondrial division inhibitor-1, a small molecule reported to specifically inhibit DRP1 activity, can cause severe developmental arrest of preimplantation embryos in a dose-dependent manner in mice. Meanwhile, DRP1 inhibition resulted in mitochondrial dysfunction including decreased mitochondrial activity, loss of mitochondrial membrane potential, reduced mitochondrial copy number and inadequate ATP by disrupting both expression and activity of DRP1 and mitochondrial complex assembly, leading to excessive ROS production, severe DNA damage and cell cycle arrest at 2-cell embryo stage. Furthermore, reduced transcriptional and translational activity and altered histone modifications in DRP1-inhibited embryos contributed to impeded zygotic genome activation, which prevented early embryos from efficient development beyond 2-cell embryo stage. These results show that DRP1 inhibition has potential cytotoxic effects on mammalian reproduction, and DRP1 inhibitor should be used with caution when it is applied to treat diseases. Additionally, this study improves our understanding of the crosstalk between mitochondrial metabolism and zygotic genome activation.

60 TELOMERE LENGTH DYNAMICS DURING BOVINE PREIMPLANTATION EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 142
Author(s):  
C. de Frutos ◽  
P. Bermejo-Alvarez ◽  
D. Rizos ◽  
A. Gutierrez-Adan
Keyword(s):  
Developmental Stage ◽  
Telomere Length ◽  
Embryo Development ◽  
Relative Abundance ◽  
Preimplantation Embryo ◽  
Telomerase Activity ◽  
Alternative Mechanism ◽  
Cell Stage ◽  
Preimplantation Embryo Development ◽  
Early Embryos

The establishment of telomere length (TL) during embryogenesis determines telomere reserves in newborn mammals. However, limited information is available on TL dynamics during preimplantation in contrast to the extensive existing data on telomerase activity in germ cells and embryogenesis. Telomerase activity is high in the male germ line, low or absent in oocytes and early stage embryos and high in blastocysts (Bl). Mechanisms allowing early embryos to reset TL remain poorly understood. The documented telomere lengthening at the morula/Bl transition in mice and bovines is dependent on telomerase activity. A recombinant-based mechanism termed alternative lengthening of telomeres (ALT) has been postulated to be responsible for the lengthening of telomeres in early embryos. The aims of the present study were to analyse the telomere dynamics during preimplantation embryo development in 2 species of known different TL: mice and bovines and the relative expression of 2 components of telomerase [telomerase reverse transcriptase (Tert; the key factor that controls the activity of the telomerase) and telomerase RNA component (Terc)]. Twenty samples for each developmental stage with an equivalent number of cells (matured oocytes/zygotes: 8 and 32; 2-cell embryos: 4 and 16; 4-cell embryos: 2 and 8; 8-cell embryos: 1 and 4; 16-cell embryos: 2 only for bovine; morulae: 1 and 1; and Bl: 1 and 1 for mice and bovines, respectively) were analysed for relative TL measurement using the real-time quantitative PCR method described previously (Bermejo-Alvarez et al. 2008 Physiol. Genomics 32, 264272). For measuring the mRNA, 3 groups of 10 oocytes/embryos for each developmental stage were used. Data were analysed by 1-way ANOVA. In mice, matured oocytes had the shortest telomeres of all stages examined (P < 0.01); a slight increase up to the 4-cell stage and a decrease at the 8-cell and morula stages was noted (P < 0.05), while a marked increase was observed in Bl, as expected (P < 0.01). In contrast, bovine matured oocytes had longer telomeres than zygotes and this length gradually decreased up to the 4-cell stage and increased again at the 16-cell stage (P < 0.05). Then, telomeres shortened at the morula stage (P < 0.05) and a significant increase was observed at the Bl stage like in mice (P < 0.01). The relative abundance of mTerc increased throughout development with a marked up-regulation at the morula and Bl stages (P < 0.01). On the other hand, the relative abundance of mTert was significantly higher in the mature oocytes and zygotes compared to later stages (P < 0.01); however, it should be noted that there was a gradual increase from the 2-cell stage up to Bl. In conclusion, in contrast to mice, bovine oocytes have longer telomeres than zygotes. Knowing that the telomerase activity is low or absent until the Bl stage (indicated by the low expression of Tert), the TL increase detected in 16-cell bovine embryos indicates an alternative mechanism for telomere elongation during early development, like that observed in mice. Understanding how telomeres reset during early embryo development has implications for the study of stem cells and regenerative biology.

scholarly journals Phosphatidylinositol 3-kinase signaling in mammalian preimplantation embryo development

Reproduction ◽  
2008 ◽  
Vol 136 (2) ◽  
pp. 147-156 ◽  
Author(s):  
Chris O'Neill
Keyword(s):  
Embryo Development ◽  
Normal Development ◽  
Preimplantation Embryo ◽  
Preimplantation Embryos ◽  
External Information ◽  
Ph Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Preimplantation Embryo Development ◽  
Wide Range ◽  
Phosphatidylinositol 3

The development of the preimplantation mammalian embryo is an autopoietic process; once initiated development proceeds without an absolute requirement for external information or growth cues. This developmental autonomy is partly explained by the generation of autocrine trophic ligands that are released and act back on the embryo via specific receptors. Several embryotrophic ligands cause receptor-dependent activation of 1-o-phosphatidylinositol 3-kinase. This enzyme phosphorylates phosphatidylinositol-4,5-bisphosphate to form phosphatidylinositol-3,4,5-trisphosphate. Genetic or pharmacological ablation of this enzyme activity disrupts normal development of preimplantation embryos. Phosphatidylinositol-3,4,5-trisphosphate is a membrane lipid that acts as a docking site for a wide range of proteins possessing the pleckstrin homology (PH) domain. Such proteins are important regulators of cell survival, proliferation, and differentiation. RAC-α serine/threonine protein kinase is an important PH domain protein and its activity is required for normal preimplantation embryo development and survival. The activity of a range of PH domain proteins is also implicated in the normal development of the embryo. This review critically examines the evidence for the activation of 1-o-phosphatidylinositol 3-kinase in the generation of pleiotypic trophic response to embryotrophins in the autopoietic development of the preimplantation embryo.

scholarly journals Intracellular oxygen metabolism during bovine oocyte and preimplantation embryo development

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Paul J. McKeegan ◽  
Selina F. Boardman ◽  
Amy A. Wanless ◽  
Grace Boyd ◽  
Laura J. Warwick ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Oxygen Consumption ◽  
Oxygen Concentration ◽  
Embryo Development ◽  
Preimplantation Embryo ◽  
Complex Iii ◽  
Preimplantation Embryos ◽  
Mitochondrial Oxygen Consumption ◽  
Preimplantation Embryo Development

AbstractWe report a novel method to profile intrcellular oxygen concentration (icO2) during in vitro mammalian oocyte and preimplantation embryo development using a commercially available multimodal phosphorescent nanosensor (MM2). Abattoir-derived bovine oocytes and embryos were incubated with MM2 in vitro. A series of inhibitors were applied during live-cell multiphoton imaging to record changes in icO2 associated with mitochondrial processes. The uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) uncouples mitochondrial oxygen consumption to its maximum, while antimycin inhibits complex III to ablate mitochondrial oxygen consumption. Increasing oxygen consumption was expected to reduce icO2 and decreasing oxygen consumption to increase icO2. Use of these inhibitors quantifies how much oxygen is consumed at basal in comparison to the upper and lower limits of mitochondrial function. icO2 measurements were compared to mitochondrial DNA copy number analysed by qPCR. Antimycin treatment increased icO2 for all stages tested, suggesting significant mitochondrial oxygen consumption at basal. icO2 of oocytes and preimplantation embryos were unaffected by FCCP treatment. Inner cell mass icO2 was lower than trophectoderm, perhaps reflecting limitations of diffusion. Mitochondrial DNA copy numbers were similar between stages in the range 0.9–4 × 106 copies and did not correlate with icO2. These results validate the MM2 probe as a sensitive, non-toxic probe of intracellular oxygen concentration in mammalian oocytes and preimplantation embryos.

scholarly journals Assessing heterogeneity among single embryos and single blastomeres using open microfluidic design

2020 ◽  
Vol 6 (17) ◽  
pp. eaay1751
Author(s):  
Elisabet Rosàs-Canyelles ◽  
Andrew J. Modzelewski ◽  
Alisha Geldert ◽  
Lin He ◽  
Amy E. Herr
Keyword(s):  
Single Cell ◽  
Preimplantation Embryo ◽  
Single Cell Protein ◽  
Cell Protein ◽  
Molecular Heterogeneity ◽  
Cell Stage ◽  
Multimodal Data ◽  
Preimplantation Embryo Development ◽  
Specificity And Sensitivity ◽  
Single Blastomeres

The process by which a zygote develops from a single cell into a multicellular organism is poorly understood. Advances are hindered by detection specificity and sensitivity limitations of single-cell protein tools and by challenges in integrating multimodal data. We introduce an open microfluidic tool expressly designed for same-cell phenotypic, protein, and mRNA profiling. We examine difficult-to-study—yet critically important—murine preimplantation embryo stages. In blastomeres dissociated from less well-studied two-cell embryos, we observe no significant GADD45a protein expression heterogeneity, apparent at the four-cell stage. In oocytes, we detect differences in full-length versus truncated DICER-1 mRNA and protein, which are insignificant by the two-cell stage. Single-embryo analyses reveal intraembryonic heterogeneity, differences between embryos of the same fertilization event and between donors, and reductions in the burden of animal sacrifice. Open microfluidic design integrates with existing workflows and opens new avenues for assessing the cellular-to-molecular heterogeneity inherent to preimplantation embryo development.

257. Activation of a calcium-activated chloride channel by paf is required for normal preimplantation mouse embryo development in vitro

2008 ◽  
Vol 20 (9) ◽  
pp. 57
Author(s):  
Y. Li ◽  
M. L. Day ◽  
C. O.'Neill
Keyword(s):  
Embryo Development ◽  
Mouse Embryo ◽  
Preimplantation Embryo ◽  
Platelet Activating Factor ◽  
Outward Current ◽  
Normal Embryo ◽  
Cell Stage ◽  
Preimplantation Embryo Development ◽  
Cl Channel

Platelet activating factor (paf) is an autocrine survival factor for preimplantation embryo. Binding of paf to its receptor activates PI3kinase, causing an IP3-dependent release of Ca2+ from intracellular stores as well as activation of Ca2+ influx via a dihydropyridine-sensitive Ca2+ channel. These actions result in the generation of a defined intracellular calcium ([Ca2+]i) transient in the 2-cell embryo[1]. By using combined whole-cell patch-clamp and real-time [Ca2+]i analyses, we have shown that paf also induces a concomitant hyperpolarisation of the membrane potential in 2-cell embryos, accompanied by an increased net outward ion current. Both the membrane hyperpolarisation and outward current were dependent upon the occurrence of the paf-induced [Ca2+]i transient[2]. The aim of this study was to investigate the characteristics of the paf-induced outward current in 2-cell embryos and to assess whether it has a role in normal mouse preimplantation development. We show that: (1) removal of extracellular anions or treatment with niflumic acid (NFA, 100 μM, a Ca2+-activated Cl- channel blocker) prevented activation of the outward current by paf but had no effect on the paf-induced [Ca2+]i transient; and (2) The culture of embryos with NFA (100 μM) from the 1-cell to late 2-cell stage significantly reduced their development to the blastocyst stage (P < 0.001), but treatment with NFA from the late 2-cell stage had no effect on development. The results show that paf induces an increase in [Ca2+]i which in turn activates a Ca2+-activated Cl- channel. The activity of this NFA-sensitive channel during the zygote to 2-cell stage is required for normal embryo development. (1) C. O’Neill (2008) The potential roles of embryotrophic ligands in preimplantation embryo development. Hum Reprod Update 14:275–288. (2) Y. Li, M.L. Day & C. O’Neill (2007) Autocrine activation of ions currents in the two-cell mouse embryo. Exp Cell Res. 313:2785–2794.

Exposure to mono-n-butyl phthalate disrupts the development of preimplantation embryos

2013 ◽  
Vol 25 (8) ◽  
pp. 1174 ◽  
Author(s):  
Da-Peng Chu ◽  
Shi Tian ◽  
Da-Guang Sun ◽  
Chan-Juan Hao ◽  
Hong-Fei Xia ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Dna Methylation ◽  
Dibutyl Phthalate ◽  
Preimplantation Embryo ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Oxygen Species ◽  
Preimplantation Embryo Development ◽  
Immunofluorescent Analysis ◽  
Butyl Phthalate

Dibutyl phthalate (DBP), a widely used phthalate, is known to cause many serious diseases, especially in the reproductive system. However, little is known about the effects of its metabolite, mono-n-butyl phthalate (MBP), on preimplantation embryo development. In the present study, we found that treatment of embryos with 10–3 M MBP impaired developmental competency, whereas exposure to 10–4 M MBP delayed the progression of preimplantation embryos to the blastocyst stage. Furthermore, reactive oxygen species (ROS) levels in embryos were significantly increased following treatment with 10–3 M MBP. In addition, 10–3 M MBP increased apoptosis via the release of cytochrome c, whereas immunofluorescent analysis revealed that exposure of preimplantation embryos to MBP concentration-dependently (10–5, 10–4 and 10–3 M) decreased DNA methylation. Together, the results indicate a possible relationship between MBP exposure and developmental failure in preimplantation embryos.

Corrigendum to: Exposure to mono-n-butyl phthalate disrupts the development of preimplantation embryos

2014 ◽  
Vol 26 (3) ◽  
pp. 491 ◽  
Author(s):  
Da-Peng Chu ◽  
Shi Tian ◽  
Da-Guang Sun ◽  
Chan-Juan Hao ◽  
Hong-Fei Xia ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Dna Methylation ◽  
Dibutyl Phthalate ◽  
Preimplantation Embryo ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Oxygen Species ◽  
Preimplantation Embryo Development ◽  
Immunofluorescent Analysis ◽  
Butyl Phthalate

Dibutyl phthalate (DBP), a widely used phthalate, is known to cause many serious diseases, especially in the reproductive system. However, little is known about the effects of its metabolite, mono-n-butyl phthalate (MBP), on preimplantation embryo development. In the present study, we found that treatment of embryos with 10–3M MBP impaired developmental competency, whereas exposure to 10–4M MBP delayed the progression of preimplantation embryos to the blastocyst stage. Furthermore, reactive oxygen species (ROS) levels in embryos were significantly increased following treatment with 10–3M MBP. In addition, 10–3M MBP increased apoptosis via the release of cytochrome c, whereas immunofluorescent analysis revealed that exposure of preimplantation embryos to MBP concentration-dependently (10–5, 10–4 and 10–3M) decreased DNA methylation. Together, the results indicate a possible relationship between MBP exposure and developmental failure in preimplantation embryos.

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