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Reproduction & Fertility Nov 2023The refinement of embryo culture media is essential in improving embryo viability and in vitro production efficiency. Our previous work demonstrated that the nutrients...
The refinement of embryo culture media is essential in improving embryo viability and in vitro production efficiency. Our previous work demonstrated that the nutrients (carbohydrates, amino acids, and vitamins) in traditional culture media far exceed the need for an embryo and producing developmentally competent embryos in a reduced nutrient environment is feasible. Here, we aim to evaluate the impact of exogenous lipid and L-carnitine supplementation on bovine blastocyst development and refine our RN condition further. Zygotes were cultured in the control medium (100% nutrients) and reduced nutrient media containing 6.25% of the standard nutrient concentrations supplemented with L-carnitine and lipid free or lipid rich BSA. Increased blastocyst development was observed in the reduced nutrient lipid rich medium compared to the other two groups. However, in both reduced nutrient conditions, blastocyst cell numbers were lower than those obtained in the control condition. We then examined the expression level of 18 transcripts correlated with lipid metabolism, glucose metabolism, redox balance, and embryo quality, along with mitochondrial DNA copy numbers, ATP productions, and lipid profile. The results indicated lipid metabolism, embryo quality, and redox enzyme related genes were upregulated while glucose related gene was downregulated in embryos derived from reduced nutrient lipid rich condition Finally, we identified that the lipid rich BSA has enriched linoleic, stearic, oleic, palmitic, and alpha-linoleic fatty acids, a lipid profile that may contribute to the increased lipid metabolism and improved blastocyst development of the bovine embryos under the reduced nutrient condition.
PubMed: 37971749
DOI: 10.1530/RAF-23-0057 -
International Journal of Molecular... Oct 2023Germline manipulation at the zygote stage using the CRISPR/Cas9 system has been extensively employed for creating genetically modified animals and maintaining... (Review)
Review
Germline manipulation at the zygote stage using the CRISPR/Cas9 system has been extensively employed for creating genetically modified animals and maintaining established lines. However, this approach requires a long and laborious task. Recently, many researchers have attempted to overcome these limitations by generating somatic mutations in the adult stage through tail vein injection or local administration of CRISPR reagents, as a new strategy called "in vivo somatic cell genome editing". This approach does not require manipulation of early embryos or strain maintenance, and it can test the results of genome editing in a short period. The newborn is an ideal stage to perform in vivo somatic cell genome editing because it is immune-privileged, easily accessible, and only a small amount of CRISPR reagents is required to achieve somatic cell genome editing throughout the entire body, owing to its small size. In this review, we summarize in vivo genome engineering strategies that have been successfully demonstrated in newborns. We also report successful in vivo genome editing through the neonatal introduction of genome editing reagents into various sites in newborns (as exemplified by intravenous injection via the facial vein), which will be helpful for creating models for genetic diseases or treating many genetic diseases.
Topics: Animals; Gene Editing; CRISPR-Cas Systems; Animals, Newborn; Zygote
PubMed: 37894981
DOI: 10.3390/ijms242015301 -
Nature Communications Apr 2024During embryonic development, pluripotent cells assume specialized identities by adopting particular gene expression profiles. However, systematically dissecting the...
During embryonic development, pluripotent cells assume specialized identities by adopting particular gene expression profiles. However, systematically dissecting the relative contributions of mRNA transcription and degradation to shaping those profiles remains challenging, especially within embryos with diverse cellular identities. Here, we combine single-cell RNA-Seq and metabolic labeling to capture temporal cellular transcriptomes of zebrafish embryos where newly-transcribed (zygotic) and pre-existing (maternal) mRNA can be distinguished. We introduce kinetic models to quantify mRNA transcription and degradation rates within individual cell types during their specification. These models reveal highly varied regulatory rates across thousands of genes, coordinated transcription and destruction rates for many transcripts, and link differences in degradation to specific sequence elements. They also identify cell-type-specific differences in degradation, namely selective retention of maternal transcripts within primordial germ cells and enveloping layer cells, two of the earliest specified cell types. Our study provides a quantitative approach to study mRNA regulation during a dynamic spatio-temporal response.
Topics: Animals; Zebrafish; Single-Cell Gene Expression Analysis; Embryonic Development; Transcription, Genetic; RNA, Messenger; Gene Expression Regulation, Developmental
PubMed: 38600066
DOI: 10.1038/s41467-024-47290-9 -
Nature Cell Biology Apr 2024The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if...
The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if and how transcription bodies affect gene expression. Here we disrupted the formation of two prominent endogenous transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analysed the effect on gene expression using enriched SLAM-seq and live-cell imaging. We find that the disruption of transcription bodies results in the misregulation of hundreds of genes. Here we focus on genes that are upregulated. These genes have accessible chromatin and are poised to be transcribed in the presence of the two transcription bodies, but they do not go into elongation. Live-cell imaging shows that disruption of the two large transcription bodies enables these poised genes to be transcribed in ectopic transcription bodies, suggesting that the large transcription bodies sequester a pause release factor. Supporting this hypothesis, we find that CDK9-the kinase that releases paused polymerase II-is highly enriched in the two large transcription bodies. Overexpression of CDK9 in wild-type embryos results in the formation of ectopic transcription bodies and thus phenocopies the removal of the two large transcription bodies. Taken together, our results show that transcription bodies regulate transcription by sequestering machinery, thereby preventing genes elsewhere in the nucleus from being transcribed.
Topics: Animals; Chromatin; Gene Expression; Positive Transcriptional Elongation Factor B; RNA Polymerase II; Transcription, Genetic; Zebrafish
PubMed: 38589534
DOI: 10.1038/s41556-024-01389-9 -
European Journal of Medical Research Jul 2023Polycystic ovary syndrome (PCOS) women have high incidences of dyslipidemia, obesity, impaired glucose tolerance (IGT), diabetes, and insulin resistance (IR) and are...
Adiposity and lipid metabolism indicators mediate the adverse effect of glucose metabolism indicators on oogenesis and embryogenesis in PCOS women undergoing IVF/ICSI cycles.
BACKGROUND
Polycystic ovary syndrome (PCOS) women have high incidences of dyslipidemia, obesity, impaired glucose tolerance (IGT), diabetes, and insulin resistance (IR) and are fragile to female infertility. Obesity and dyslipidemia may be the intermediate biological mechanism for the associations between glucose metabolism dysfunction and abnormal oogenesis and embryogenesis.
METHODS
This retrospective cohort study was performed at a university-affiliated reproductive center. A total of 917 PCOS women aged between 20 and 45 undergoing their first IVF/ICSI embryo transfer cycles from January 2018 to December 2020 were involved. Associations between glucose metabolism indicators, adiposity and lipid metabolism indicators, and IVF/ICSI outcomes were explored using multivariable generalized linear models. Mediation analyses were further performed to examine the potential mediation role of adiposity and lipid metabolism indicators.
RESULTS
Significant dose-dependent relationships were found between glucose metabolism indicators and IVF/ICSI early reproductive outcomes and between glucose metabolism indicators and adiposity and lipid metabolism indicators (all P < 0.05). Also, we found significant dose-dependent relationships between adiposity and lipid metabolism indicators and IVF/ICSI early reproductive outcomes (all P < 0.05). The mediation analysis indicated that elevated FPG, 2hPG, FPI, 2hPI, HbA1c, and HOMA2-IR were significantly associated with decreased retrieved oocyte count, MII oocyte count, normally fertilized zygote count, normally cleaved embryo count, high-quality embryo count, or blastocyst formation count after controlling for adiposity and lipid metabolism indicators. Serum TG mediated 6.0-31.0% of the associations; serum TC mediated 6.1-10.8% of the associations; serum HDL-C mediated 9.4-43.6% of the associations; serum LDL-C mediated 4.2-18.2% of the associations; and BMI mediated 26.7-97.7% of the associations.
CONCLUSIONS
Adiposity and lipid metabolism indicators (i.e., serum TG, serum TC, serum HDL-C, serum LDL-C, and BMI) are significant mediators of the effect of glucose metabolism indicators on IVF/ICSI early reproductive outcomes in PCOS women, indicating the importance of preconception glucose and lipid management and the dynamic equilibrium of glucose and lipid metabolism in PCOS women.
Topics: Humans; Female; Polycystic Ovary Syndrome; Sperm Injections, Intracytoplasmic; Fertilization in Vitro; Retrospective Studies; Adiposity; Cholesterol, LDL; Lipid Metabolism; Embryonic Development; Oogenesis; Obesity; Dyslipidemias
PubMed: 37400924
DOI: 10.1186/s40001-023-01174-8 -
Genes & Development Mar 2024Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo...
A maternal-effect variant causes nuclear and cytoplasmic abnormalities in oocytes, as well as failure of epigenetic reprogramming and zygotic genome activation in embryos.
Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo lethality. In humans, SCMC gene variants were found in the healthy mothers of children affected by multilocus imprinting disturbances (MLID). However, how the SCMC controls the DNA methylation required to regulate imprinting remains poorly defined. We generated a mouse line carrying a missense variant that was identified in a family with Beckwith-Wiedemann syndrome and MLID. If homozygous in female mice, this variant resulted in interruption of embryo development at the two-cell stage. Single-cell multiomic analyses demonstrated defective maturation of mutant oocytes and incomplete DNA demethylation, down-regulation of zygotic genome activation (ZGA) genes, up-regulation of maternal decay genes, and developmental delay in two-cell embryos developing from mutant oocytes but little effect on genomic imprinting. Western blotting and immunofluorescence analyses showed reduced levels of UHRF1 in oocytes and abnormal localization of DNMT1 and UHRF1 in both oocytes and zygotes. Treatment with 5-azacytidine reverted DNA hypermethylation but did not rescue the developmental arrest of mutant embryos. Taken together, this study demonstrates that PADI6 controls both nuclear and cytoplasmic oocyte processes that are necessary for preimplantation epigenetic reprogramming and ZGA.
Topics: Animals; Child; Female; Humans; Mice; CCAAT-Enhancer-Binding Proteins; Cytoplasm; DNA Methylation; Embryonic Development; Genomic Imprinting; Oocytes; Ubiquitin-Protein Ligases; Zygote
PubMed: 38453481
DOI: 10.1101/gad.351238.123 -
PLoS Biology Feb 2024Alternative splicing is an essential regulatory mechanism for development and pathogenesis. Through alternative splicing one gene can encode multiple isoforms and be...
Alternative splicing is an essential regulatory mechanism for development and pathogenesis. Through alternative splicing one gene can encode multiple isoforms and be translated into proteins with different functions. Therefore, this diversity is an important dimension to understand the molecular mechanism governing embryo development. Isoform expression in preimplantation embryos has been extensively investigated, leading to the discovery of new isoforms. However, the dynamics of isoform switching of different types of transcripts throughout the development remains unexplored. Here, using single-cell direct isoform sequencing in over 100 single blastomeres from the mouse oocyte to blastocyst stage, we quantified isoform expression and found that 3-prime partial transcripts lacking stop codons are highly accumulated in oocytes and zygotes. These transcripts are not transcription by-products and might play a role in maternal to zygote transition (MZT) process. Long-read sequencing also enabled us to determine the expression of transposable elements (TEs) at specific loci. In this way, we identified 3,894 TE loci that exhibited dynamic changes along the preimplantation development, likely regulating the expression of adjacent genes. Our work provides novel insights into the transcriptional regulation of early embryo development.
Topics: Female; Pregnancy; Animals; Mice; DNA Transposable Elements; Embryonic Development; Protein Isoforms; Zygote; Single-Cell Analysis
PubMed: 38363809
DOI: 10.1371/journal.pbio.3002505 -
Biophysical Reports Dec 2023During zygotic mitosis in many species, forces generated at the cell cortex are required for the separation and migration of paternally provided centrosomes, pronuclear...
During zygotic mitosis in many species, forces generated at the cell cortex are required for the separation and migration of paternally provided centrosomes, pronuclear migration, segregation of genetic material, and cell division. Furthermore, in some species, force-generating interactions between spindle microtubules and the cortex position the mitotic spindle asymmetrically within the zygote, an essential step in asymmetric cell division. Understanding the mechanical and molecular mechanisms of microtubule-dependent force generation and therefore asymmetric cell division requires identification of individual cortical force-generating units . There is no current method for identifying individual force-generating units with high spatiotemporal resolution. Here, we present a method to determine both the location and the relative number of microtubule-dependent cortical force-generating units using single-molecule imaging of fluorescently labeled dynein. Dynein behavior is modeled to classify trajectories of cortically bound dynein according to whether they are interacting with a microtubule. The categorization strategy recapitulates well-known force asymmetries in zygote mitosis. To evaluate the robustness of categorization, we used RNAi to deplete the tubulin subunit TBA-2. As predicted, this treatment reduced the number of trajectories categorized as engaged with a microtubule. Our technique will be a valuable tool to define the molecular mechanisms of dynein cortical force generation and its regulation as well as other instances wherein anchored motors interact with biopolymers (e.g., actin, tubulin, DNA).
PubMed: 37811483
DOI: 10.1016/j.bpr.2023.100130 -
Nature Structural & Molecular Biology May 2024Gene expression during natural and induced reprogramming is controlled by pioneer transcription factors that initiate transcription from closed chromatin. Nr5a2 is a key...
Gene expression during natural and induced reprogramming is controlled by pioneer transcription factors that initiate transcription from closed chromatin. Nr5a2 is a key pioneer factor that regulates zygotic genome activation in totipotent embryos, pluripotency in embryonic stem cells and metabolism in adult tissues, but the mechanism of its pioneer activity remains poorly understood. Here, we present a cryo-electron microscopy structure of human NR5A2 bound to a nucleosome. The structure shows that the conserved carboxy-terminal extension (CTE) loop of the NR5A2 DNA-binding domain competes with a DNA minor groove anchor of the nucleosome and releases entry-exit site DNA. Mutational analysis showed that NR5A2 D159 of the CTE is dispensable for DNA binding but required for stable nucleosome association and persistent DNA 'unwrapping'. These findings suggest that NR5A2 belongs to an emerging class of pioneer factors that can use DNA minor groove anchor competition to destabilize nucleosomes and facilitate gene expression during reprogramming.
Topics: Humans; Cryoelectron Microscopy; DNA; Models, Molecular; Nucleic Acid Conformation; Nucleosomes; Protein Binding; Protein Conformation; Receptors, Cytoplasmic and Nuclear
PubMed: 38409506
DOI: 10.1038/s41594-024-01239-0 -
Cell Reports Mar 2024In mammals, many retrotransposons are de-repressed during zygotic genome activation (ZGA). However, their functions in early development remain elusive largely due to...
In mammals, many retrotransposons are de-repressed during zygotic genome activation (ZGA). However, their functions in early development remain elusive largely due to the challenge to simultaneously manipulate thousands of retrotransposon insertions in embryos. Here, we applied CRISPR interference (CRISPRi) to perturb the long terminal repeat (LTR) MT2_Mm, a well-known ZGA and totipotency marker that exists in ∼2,667 insertions throughout the mouse genome. CRISPRi robustly perturbed 2,485 (∼93%) MT2_Mm insertions and 1,090 (∼55%) insertions of the closely related MT2C_Mm in 2-cell embryos. Remarkably, such perturbation caused downregulation of hundreds of ZGA genes and embryonic arrest mostly at the morula stage. Mechanistically, MT2 LTRs are globally enriched for open chromatin and H3K27ac and function as promoters/enhancers downstream of OBOX/DUX proteins. Thus, we not only provide direct evidence to support the functional importance of MT2 activation in development but also systematically define cis-regulatory function of MT2 in embryos by integrating functional perturbation and multi-omic analyses.
Topics: Mice; Animals; Regulatory Sequences, Nucleic Acid; Zygote; Chromatin; Retroviridae; Retroelements; Terminal Repeat Sequences; Embryonic Development; Gene Expression Regulation, Developmental; Mammals
PubMed: 38381606
DOI: 10.1016/j.celrep.2024.113775