Development of an Efficient Method for Obtaining Metaphase Chromosomes in Individual Blastomeres of Mouse and Human Preimplantaion Embryos: Effect of Microtubule Depolymerizing Agents / 대한불임학회지

OBJECTIVES: The development of an useful method for obtaining metaphase chromosomes from a biopsied blastomere would allow differentiation between embryos with balanced and normal chromosome complements in the preimplantation genetic diagnosis for chromosomal translocations. This study was performed to evaluate the effects of microtubule depolymerizing agents (MTDAs) on the blastomeres of mouse and human preimplantation embryos, and to establish an effective method for obtaining metaphase chromosomes of biopsied blastomeres in human early embryos. MATERIALS AND MEHTODS: Early embryos (2-4 cell stage) from superovulated mice (ICR strain) were collected and treated with single or mixture MTDAs, such as vinblastine, nocodazole and colcemid. After the treatment of MTDAs for 16 hours, the metaphase aquisition (MA) rates were evaluated by the observation of chromosome status with bis-benzimide or DAPI staining. The optimal condition from the above experiment was applied to human embryos, which were developed from abnormal fertilization (3-pronuclei). Fluorescence in-situ hybridization (FISH) with whole chromosome probes was conducted on the human metaphase chromosomes by the MTDAs. RESULTS: In mouse embryos, the effective concentrations of each MTDAs for obtaining metaphase chromosomes were 1.0 micrometer of vinblastine (20.3%), 5.0 micrometer of nocodazole (28.1%) and 1.0 micrometer colcemid (55.6%), respectively. The highest MA rate (91.2%) in the mouse embryos was obtained by a mixture of vinblastine (1.0 micrometer) and nocodazole (1.0 micrometer). In the human embryos, the metaphase chromosomes of blastomeres were obtained in 44 of 113 blastomeres (38.9%) by treatment of the mixture of vinblastine and nocodazole. FISH signals of the metaphase chromosomes were successfully observed in human individual blastomeres. CONCLUSIONS: The treatment of a mixture MTDAs for obtaining metaphase chromosomes was an efficient method, and the MA rate was above 90% in the mouse embryos. However, only a relatively small proportions of the blastomeres yielded metaphase chromosomes by the MTDAs in the human embryos. The inconsistent effects of MTDAs may be related to the variation of different species and the poor developmental potency of abnormally fertilized human embryos. We should develop more reliable and efficient methods for obtaining the metaphase chromosomes in the biopsied blastomeres of human preimplantation embryos.

Development of an Efficient Method for Obtaining Metaphase Chromosomes in Individual Blastomeres of Mouse and Human Preimplantaion Embryos: Effect of Microtubule Depolymerizing Agents / 대한불임학회지

OBJECTIVES: The development of an useful method for obtaining metaphase chromosomes from a biopsied blastomere would allow differentiation between embryos with balanced and normal chromosome complements in the preimplantation genetic diagnosis for chromosomal translocations. This study was performed to evaluate the effects of microtubule depolymerizing agents (MTDAs) on the blastomeres of mouse and human preimplantation embryos, and to establish an effective method for obtaining metaphase chromosomes of biopsied blastomeres in human early embryos. MATERIALS AND MEHTODS: Early embryos (2-4 cell stage) from superovulated mice (ICR strain) were collected and treated with single or mixture MTDAs, such as vinblastine, nocodazole and colcemid. After the treatment of MTDAs for 16 hours, the metaphase aquisition (MA) rates were evaluated by the observation of chromosome status with bis-benzimide or DAPI staining. The optimal condition from the above experiment was applied to human embryos, which were developed from abnormal fertilization (3-pronuclei). Fluorescence in-situ hybridization (FISH) with whole chromosome probes was conducted on the human metaphase chromosomes by the MTDAs. RESULTS: In mouse embryos, the effective concentrations of each MTDAs for obtaining metaphase chromosomes were 1.0 micrometer of vinblastine (20.3%), 5.0 micrometer of nocodazole (28.1%) and 1.0 micrometer colcemid (55.6%), respectively. The highest MA rate (91.2%) in the mouse embryos was obtained by a mixture of vinblastine (1.0 micrometer) and nocodazole (1.0 micrometer). In the human embryos, the metaphase chromosomes of blastomeres were obtained in 44 of 113 blastomeres (38.9%) by treatment of the mixture of vinblastine and nocodazole. FISH signals of the metaphase chromosomes were successfully observed in human individual blastomeres. CONCLUSIONS: The treatment of a mixture MTDAs for obtaining metaphase chromosomes was an efficient method, and the MA rate was above 90% in the mouse embryos. However, only a relatively small proportions of the blastomeres yielded metaphase chromosomes by the MTDAs in the human embryos. The inconsistent effects of MTDAs may be related to the variation of different species and the poor developmental potency of abnormally fertilized human embryos. We should develop more reliable and efficient methods for obtaining the metaphase chromosomes in the biopsied blastomeres of human preimplantation embryos.

Cell cycle and DNA content of mitotic cells in brain ganglia of Drosophila larvae.

The programmes of replication of hetero- and euchromatin regions, mitotic cell cycle and the DNA content in metaphases in brain ganglia from late third instar larvae of Drosophila melanogaster (wild type and a tumour bearing mutant, 1(2)gl, strain) and of Drosophila nasuta were examined by autoradiography of [3H]thymidine labelled (continuous or pulse) cells and by cytophotometry, respectively. Brain ganglia labelled continuously with [3H]thymidine for 24 h in vitro showed a significantly high proportion of cells with incorporation of radioactivity restricted to heterochromatin only. Pulse labelling of brain ganglia from larvae of Drosophila melanogaster and Drosophila nasuta followed by chase for different time intervals showed that (i) the frequency of labelled metaphases was more than 50% within 15 to 30 min of chase and remained higher than 50% in nearly all the chase samples till 24 h, (ii) euchromatin labelled metaphases appeared with a low frequency within 1 to 4 h chase period but the heterochromatin labelled metaphases continued to be more common in the later chase samples also, (iii) single chromatid labelled second cycle metaphases were seen within 1 to 4 h after the pulse, but their frequency did not increase in the later samples. Cytophotometry of feulgen-DNA and Hoechst 33258 stained metaphases in late third instar larval brain ganglia revealed a greater variation in the DNA content of individual metaphases, although the means were close to the expected 4 C content. It appears that in relation to the known asymmetric cell divisions of neuroblast and other neural cells, the mitotically active cells in brain ganglia comprise a heterogenous population with widely varying lengths of the different phases of cell cycle; some of them may not cycle regularly and may possibly have a discontinuous S-phase.