Adenine ; Animals ; Gene Editing ; Mice ; Zygote ; metabolism

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
Blastocyst ; CRISPR-Cas Systems ; Hemoglobins, Abnormal ; genetics ; metabolism ; Humans ; Zygote

Cell Cycle Checkpoints/genetics* ; Checkpoint Kinase 1/metabolism* ; Heterozygote ; Humans ; Mitosis/genetics* ; Mutation ; Zygote/metabolism*

Male germ cells are particularly susceptible to DNA damage by genotoxic agents during spermiogenesis and spermatozoal maturation, and meanwhile lack an effective repair system to eliminate the lesions. Because the DNA damaged sperm still has fertilizability and developmental potentiality, damage repair may occur after fertilization, but its mechanism remains unknown. Histone H2AX phosphorylation (gammaH2AX) is reportedly involved in the repair of damaged sperm DNA after fertilization. This review aims to summarize the present knowledge on the mechanism of gammaH2AX-mediated repair of DNA damaged sperm in the zygote.
DNA Damage ; DNA Repair ; Histones ; metabolism ; Humans ; Male ; Phosphorylation ; Spermatozoa ; pathology ; Zygote

OBJECTIVETo investigate the relationship between human embryo implantation rates and the zygote morphology and establish zygote morphologic indices that indicate the embryo implantation potential after in vitro fertilization and embryo transfer (IVF-ET).

METHODSSixty-two patients with IVF-ET were enrolled in this study, who were below 35 years of age with endometrium thickness no greater than 8 mm on the day of HCG injection. Embryo transfer was performed on day 3 after oocyte retrieval, and 30 patients with successful implantation of all the embryos transferred (implantation rate of 100%) were allocated into the implantation group, and the other 32 patients with implantation failure (implantation rate of 0) served as the control group. The zygote morphologic characteristics were analyzed for the pronuclei, nucleolar precursor bodies (NPB), polar body, cytoplasmic halo, color and granulation of the cytoplasm, and the results were compared between the two groups.

RESULTSThe implantation rate was significantly higher for embryos with the two pronuclei in close vicinity, central position of the pronuclei in the cytoplasm and comparable size of the two pronuclei. Embryos developed from zygotes with linear arrangement of 3 to 7 NPB in moderate sizes tented to have a higher implantation rate (P<0.05). The implantation rate could be obviously lowered when the cytoplasm contained excessive cytoplasmic granularity (P<0.05). The other morphologic characteristics of the embryos such as the polar bodies, color of the cytoplasm, cytoplasm halo, or vacuoles in the cytoplasm did not significantly impact on the implantation rate.

CONCLUSIONThe morphology of the two pronuclei reflects the quality of the zygote and may help predict the developmental potential of the embryo chosen for transfer on day 3 in IVF.

Adult ; Cell Nucleolus ; metabolism ; Cytoplasmic Granules ; metabolism ; Embryo Implantation ; Embryo Transfer ; Female ; Fertilization in Vitro ; Humans ; Zygote ; cytology

Aging ; genetics ; Embryo Research ; ethics ; Embryo, Mammalian ; metabolism ; Fertilization in Vitro ; Humans ; Morals ; Preimplantation Diagnosis ; RNA Editing ; genetics ; Zygote ; metabolism

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28's role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.
Animals ; Cell Differentiation ; Embryo, Mammalian/metabolism* ; Embryonic Development ; Mice ; Pluripotent Stem Cells/metabolism* ; RNA, Messenger/genetics* ; RNA, Ribosomal ; RNA-Binding Proteins/metabolism* ; Transcription Factors/metabolism* ; Zygote/metabolism*

The active DNA demethylation in early embryos is essential for subsequent development. Although the zygotic genome is globally demethylated, the DNA methylation of imprinted regions, part of repeat sequences and some gamete-specific regions are maintained. Recent evidence has shown that multiple proteins and biological pathways participate in the regulation of active DNA demethylation, such as TET proteins, DNA repair pathways and DNA methyltransferases. Here we review the recent understanding regarding proteins associated with active DNA demethylation and the regulatory networks controlling the active DNA demethylation in early embryos.
Animals ; DNA Methylation ; Embryo, Mammalian ; cytology ; embryology ; metabolism ; Gene Expression Regulation, Developmental ; Gene Regulatory Networks ; genetics ; Genome ; genetics ; Humans ; Mice ; Models, Genetic ; Zygote ; cytology ; growth & development ; metabolism

The efficiency of the exogenous DNA transfecting mouse sperm was studied by the DIG end labeled and immunohistochemistry technology. The results suggested that: the efficiency of transfecting positive rate of individual mouse sperm was distinct difference (P < 0.01), and the average rate was 13%. The acrosomal reaction was evaluated using the technology of Coomassie brilliant blue stained, and the appropriate in vitro fertilization (IVF) medium TYH was elected. Mouse sperms were transferred with GFP gene in vitro, and the mature oocytes were fertilized using IVF, and then the zygotes were cultured in vitro. The embryos were observed using the fluorescence microscopy, and the transgenic rate was 4.7%. The results suggested that sperm mediated gene transfer (SMGT) was an effective and feasible method.
Animals ; Embryo, Mammalian ; cytology ; metabolism ; Feasibility Studies ; Female ; Fertilization in Vitro ; Gene Expression ; Gene Transfer Techniques ; Green Fluorescent Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Mice, Transgenic ; Microscopy, Fluorescence ; Oocytes ; cytology ; metabolism ; Spermatozoa ; cytology ; metabolism ; Zygote ; cytology ; metabolism

To investigate the effect of Protein kinase B on the expression and location of p21 in mouse early development. Immunopreciptation technology was used to detect the localization of p21 and Western blotting was used to analyze the expression of p21 after microinjecting mRNA of WT-PKB, myt-PKB and PKB-KD to mouse eggs. There was no obvious difference between the three kinds of mRNA in the expression of p21. But the cell localization altered. The p21 retain in cytoplasm after microjecting myt-PKB. In mouse fertilized egg PKB/Akt controls the cell cycle by changing the cell localization of p21.
Animals ; Cell Nucleus ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; Cytoplasm ; metabolism ; Female ; Fluorescent Antibody Technique ; Male ; Mice ; Microinjections ; Mutation ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA, Messenger ; administration & dosage ; genetics ; metabolism ; Time Factors ; Zygote ; cytology ; growth & development ; metabolism