Proliferative characteristics and chromosomal stability of bovine donor cells for nuclear transfer

Few studies have characterized donor cell lines in terms of proliferative capacity and chromosomal stability. Abnormal phosphorylation patterns of the histones during metaphase could lead to abnormal chromosome segregation and extensive chromosome loss during mitosis. Suboptimal culture conditions may lead to abnormal histone H3 phosphorylation patterns, ultimately inducing missegregation and loss of chromosomes. The objective of the present study was to determine proliferative characteristics, chromosomal stability, and level of histone phosphorylation in cell lines established by explants and enzymatic dissociation. Proliferative characteristics, percentage of aneuploid cells, and relative levels of phosphorylated histone H3 (ser10) were determined at different population doublings (PD) by cell counting, karyotyping, and flow cytometry, respectively. The level of aneuploidies was high and remained elevated throughout the study independent of the technique used to establish the primary culture. Some cell lines had up to 50% of aneuploid cells during early passages. Multinucleated cells and abnormal spindle configurations were observed after prolonged time in culture (60 and 41%, respectively). An increase in the relative level of phosphorylated histone occurred after extended time in culture (55.7 during early passages vs. 102.6 at late passages). These data demonstrate the importance of determining chromosome content and the selection of healthy cell lines to decrease the percentage of aneuploid reconstructed embryos and increase the efficiency of nuclear transfer (NT).