Effect of the number of passages of fetal and adult fibroblasts on nuclear remodelling and first embryonic division in reconstructed horse oocytes after nuclear transfer.

This study explores how repeated use of fetal and adult fibroblast cells (cells from connective tissue) in horses influences the transformation of nuclei and first embryonic division during oocyte reconstruction. It also investigates reasons for developmental stagnation in the progression to the two-cell stage. The results imply that fibroblast cells that have undergone fewer passages are the best for donation, although both types of cells have a limited ability to reactivate the first embryonic development following nuclear transfer than cells of other species.

Methodology and Results

• The researchers performed their investigation using a total of 463 metaphase II oocytes, which are mature egg cells at a specific stage of meiosis. These cells were manipulated to produce 427 fibroblast-cytoplasm couplings via nuclear transfer and ultimately resulted in 319 reconstructed oocytes.
• The researchers found that with an increased number of passages, or transfers of fibroblast cells in vitro, the rates of nuclear remodelling in the oocytes decreased. About half of the cells still showed S-G2-prometaphase stages of the first embryonic division 18-20 hours after the fusion treatment, irrespective of the number of cell passages.
• The rates of first embryonic division in the reconstructed oocytes declined with the increasing age of the donor cells. This decrease was reportedly statistically significant when compared to metaphase II oocytes that were parthenogenetically activated.
• Microscopic analysis of the oocytes that were developmentally blocked revealed most of these cells could not form the mitotic spindle and the metaphase plate of chromosomes, which are crucial components in cell division process.

Implications

• These results suggest that in the process of horse oocyte reconstruction either fetal or adult fibroblasts that have undergone relatively few passages are the ideal donors.
• However, regardless of the type of fibroblast used, both have shown a lesser potential to restart the first embryonic development after nuclear transfer compared to equivalent cells in other species.
• The researchers concluded that to successfully produce cloned embryos in horses, it is essential to improve the rate of donor cell nuclear progression from S-G2-prometaphase to beyond the metaphase stage and organize the first embryonic development in reconstructed horse oocytes normally.