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C.C. Chang1, Z.P. Nagy2, X.C. Tian1, R. Abdelmassih3, X. Yang11University of Connecticut, Animal Science, Storrs, USA2Reproductive Biology Associates, Atlanta, USA3Clínica e Centro de Pesquisa em Reprodução Humana Roger Abdelmassih, São Paulo, Brasil
Introduction: Preliminary reports suggest that haploidization technique may provide the possibility to obtain artificial gametes through reductive segregation of somatic cell nucleus in the ooplast. However, key elements such as spindle formation and accuracy of chromosome separation during haploidization have not been investigated. Therefore our objective was to study chromosome alignment and microtubule dynamics in the course of artificial meiosis, after transferring G2/M (4N) somatic cell nuclei into GV ooplasts.
Materials and Methods: To obtain cells at G2/M phase, mouse skin fibroblast cells were cultured with 0.125 µg/ml nocodazole for 18 hr in DMEM with 10 % FCS prior to nuclear transfer. 103 enucleated mouse GV ooplasts were transferred with large somatic cells (25-30 µm - ~70% at G2/M phase by FACS analysis) by micromanipulation and electrofusion. The nuclear transferred oocytes were matured in vitro in HTF and 20% FCS. To investigate the chromosomes and microtubule networks, the oocytes were fixed and stained with propidium iodide and FITC-conjugated anti-(-tubulin antibody at 4-6 h (expected MI stage) and 17 h (expected MII stage) of in vitro maturation and examined using laser scanning confocal microscopy.
Results: A total of 50 NT oocytes that fused were examined. During in vitro maturation, microtubule networks formed in the vicinity of condensed somatic chromosomes in reconstructed oocytes (31/50). However, at the expected MI stage, the somatic chromosomes were not able to align properly around the microtubule networks (17/17). At the expected MII stage, most of the meiotic II spindles were not assembled correctly in the reconstructed oocytes showing various abnormal array formations (12/14). In all oocytes examined at this stage, misalignment of somatic chromosomes were observed (14/14).
Conclusions: This study demonstrated that GV ooplasts can provide a suitable cytoplasmic environment to induce somatic cells at G2/M stage to undergo artificial meiosis. It also revealed a high frequency of alterations in the meiotic spindle integrity, chromosome alignment and separation during the haploidization process, indicating the deficiency of cytoplasmic factors controlling the meiotic cell-cycle checkpoints as a possible consequence of incompatibility between mitotic chromosomes introduced into the maturing meiotic ooplasm.
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