Dataset Information

Calcium-Free and Cytochalasin B Treatment Inhibits Blastomere Fusion in 2-Cell Stage Embryos for the Generation of Floxed Mice via Sequential Electroporation.

ABSTRACT: The generation of conditional knockout mice using the Cre-loxP system is advantageous for the functional analysis of genes. Flanked by two loxP sites (floxed) mice can be directly obtained from fertilized eggs by the CRISPR/Cas9 genome editing system. We previously reported that sequential knock-in (KI) of each loxP site by electroporation (EP) at the 1- and 2-cell embryonic stages increases the number of mice with floxed alleles compared with simultaneous KI. However, EP at the 2-cell stage frequently induced blastomere fusion. These fused embryos cannot develop to term because they are tetraploidized. In this study, we examined the following three conditions to inhibit blastomere fusion by EP at the 2-cell stage: (1) hypertonic treatment, (2) Calcium (Ca²⁺)-free treatment, and (3) actin polymerization inhibition. Hypertonic treatment of 2-cell stage embryos prevented blastomere fusion and facilitated blastocyst development; however, KI efficiency was decreased. Ca²⁺-free treatment and actin polymerization inhibition by cytochalasin B (CB) reduced fusion rate, and did not have negative effects on development and KI efficiency. These results suggest that Ca²⁺-free and CB treatment at the 2-cell stage is effective to generate floxed mice in combination with a sequential EP method.

SUBMITTER: Horii T

PROVIDER: S-EPMC7290713 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Calcium-Free and Cytochalasin B Treatment Inhibits Blastomere Fusion in 2-Cell Stage Embryos for the Generation of Floxed Mice via Sequential Electroporation.

Horii Takuro T Kobayashi Ryosuke R Kimura Mika M Morita Sumiyo S Hatada Izuho I

Cells 20200428 5

The generation of conditional knockout mice using the Cre-loxP system is advantageous for the functional analysis of genes. Flanked by two loxP sites (floxed) mice can be directly obtained from fertilized eggs by the CRISPR/Cas9 genome editing system. We previously reported that sequential knock-in (KI) of each loxP site by electroporation (EP) at the 1- and 2-cell embryonic stages increases the number of mice with floxed alleles compared with simultaneous KI. However, EP at the 2-cell stage fre ...[more]

PMID: 32354036

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Project description:Blastomere cytofragmentation in mammalian embryos poses a significant problem in applied and clinical embryology. Mouse two-cell-stage embryos display strain-dependent differences in the rate of cytofragmentation, with a high rate observed in C3H/HeJ embryos and a lower rate observed in C57BL/6 embryos. The maternally inherited genome exerts the strongest effect on the process, with lesser effects mediated by the paternally inherited genome and the ooplasm. The effect of the maternal genome is transcription dependent and independent of the mitochondrial strain of origin. To identify molecular mechanisms that underlie cytofragmentation, we evaluated transcriptional activities of embryos possessing maternal pronuclei (mPN) of different origins. The mPN from C57BL/6 and C3H/HeJ strains directed specific transcription at the two-cell stage of mRNAs corresponding to 935 and 864 Affymetrix probe set IDs, respectively. Comparing transcriptomes of two-cell-stage embryos with different mPN revealed 64 transcribed genes with differential expression (1.4-fold or greater). Some of these genes occupy molecular pathways that may regulate cytofragmentation via a combination of effects related to apoptosis and effects on the cytoskeleton. These results implicate specific molecular mechanisms that may regulate cytofragmentation in early mammalian embryos. The most striking effect of mPN strain of origin on gene expression was on adenylate cyclase 2 (Adcy2). Treatment with dibutyryl cAMP (dbcAMP) elicits a high rate and severe form of cytofragmentation, and the effective dbcAMP concentration varies with maternal genotype. An activator of exchange proteins directly activated by cAMP (EPACs, or RAPGEF 3 and 4) 8-pCPT-2'-O-methyl-cAMP, elicits a high level of fragmentation while the PKA-specific activator N6-benzoyl-cAMP does not. Inhibition of A kinase anchor protein activities with st-Ht31 induces fragmentation. Inhibition of phosphatidylinositol 3-kinase signaling also induces fragmentation. These results reveal novel mechanisms by which maternal genotype affects cytofragmentation, including a system of opposing signaling pathways that most likely operate by controlling cytoskeletal function.

Project description:BackgroundDespite limited information on neonatal safety, the transfer of frozen-thawed cleavage-stage embryos with blastomere loss is common in women undergoing in vitro fertilization. We aimed to evaluate the pregnancy outcomes and safety of frozen-thawed cleavage-stage embryos with blastomere loss.MethodsThis prospective, multicenter, cohort study included all frozen-thawed cleavage-stage embryo transfer (FET) cycles between 2002 and 2012. Pregnancy outcomes and subsequent neonatal outcomes were compared between FET cycles with intact embryos and those with blastomere loss.ResultsA total of 12,105 FET cycles were included in the analysis (2259 cycles in the blastomere loss group and 9846 cycles in the intact embryo group). The blastomere loss group showed significantly poorer outcomes with respect to implantation, pregnancy, and live birth rates than the intact embryo group. However, following embryo implantation, the two groups were similar with respect to live birth rates per clinical pregnancy. Among multiple pregnancies (4229 neonates), neonates from the blastomere loss group were at an increased risk of being small for gestational age (aOR = 1.50, 95% CI 1.00-2.25) compared to those from the intact group. A similar trend was observed among singletons (aOR = 1.84, 95% CI 0.99-3.37). No associations were found between blastomere loss and the subsequent occurrence of congenital anomalies or neonatal mortality. However, neonates from the blastomere loss group were at an increased risk of transient tachypnea of the newborn (aOR = 5.21, 95% CI 2.42-11.22).ConclusionsThe transfer of embryos with blastomere loss is associated with reduced conception rates. Once the damaged embryos have implanted, pregnancies appear to have the same probability of progressing to live birth but with an increased risk of small for gestational age neonates and transient tachypnea of the newborn.Study registrationThis study was retrospectively registered at Chinese Clinical Trial Registry. Registration number: ChiCTR-OOC-16007753 . Registration date: 13 January 2016.

Dataset Information

Calcium-Free and Cytochalasin B Treatment Inhibits Blastomere Fusion in 2-Cell Stage Embryos for the Generation of Floxed Mice via Sequential Electroporation.

Publications

Calcium-Free and Cytochalasin B Treatment Inhibits Blastomere Fusion in 2-Cell Stage Embryos for the Generation of Floxed Mice via Sequential Electroporation.

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