Many factors influence the production of cloned animals when using the technique of nuclear transfer.
One is the remodeling of the donor cell nucleus within the cytoplasm of the recipient oocyte to organize the first embryonic division.
Usually, greater extents of donor cell nuclear remodeling and embryonic development can be achieved when transferring embryonic rather than somatic cell nuclei into the cytoplasm of metaphase II oocytes, although this general rule does not hold true for all species.
The number of donor cell passages is another significant factor in cloning by nuclear transfer.
Most reports of successful cloning in domestic animal species have used cells of limited passage (3-9) as sources of donor nuclei.
Roh et al. (2000) reported that nuclei from both early passage (8-16) and late passage (17-32) donor cells were capable of supporting in vitro development after nuclear transfer in cattle, although the rate of blastocyst formation was lower when using the late passage cells.
It has been compared the competence of two types of horse cell (FFC and AFC) to undergo nuclear remodeling and the first embryonic division after nuclear transfer.
Results showed average rates of cell fusion and nuclear remodeling of about 70-78% when using both fetal and adult fibroblasts, with no significant differences between the two types of cell in their ability to act as donor nuclei to reconstruct enucleated oocytes.
This finding is similar to those in other domestic species used for nuclear transfer experiments, and it establishes that the nuclei of both fetal and adult horse fibroblasts have similar potential to induce remodeling in oocytes matured in vitro.
Furthermore, after 28-30 h of culture after cell fusion, the average rates of embryonic cleavage to the two-cell stage in the reconstructed oocytes were again not different between those made with fetal versus adult fibroblasts (27% and 25%, respectively).
However, when comparing these rates of first embryonic division in the reconstructed horse oocytes with those of other domestic species, large differences are evident, as 25-27% is only about half the rate achieved routinely in cattle, sheep, goats and pigs.
The remodeling of the donor cell nucleus and the first embryonic division in the reconstructed oocytes has been characterized in mice, rabbits and cattle, and this has indicated that the state of the cytoplasm in the recipient oocyte is a key factor in the production of cloned embryos and normal offspring.
Co-culture of horse COCs with oviduct epithelial cells has improved cytoplasmic maturation to support fertilization and embryonic development better after intracytoplasmic sperm injection.
Therefore, this study used the same in vitro culture system to provide the metaphase II oocytes used as recipient cytoplasts.
In addition, induction of parthenogenesis showed that 79% of metaphase II oocytes that had matured under the same conditions used in nuclear transfer progressed to two-cell stage embryos.
This result indicates the presence of different requirements for horse cytoplasmic competence in fertilization, parthenogenesis and nuclear remodeling.
The molecular mechanisms, which are involved in reprogramming donor nuclei after somatic cell nuclear transfer, have not been fully elucidated.
Thus, the question remains as to how best to provide more physiologically mature metaphase II oocytes from abattoir ovaries for horse nuclear transfer studies.
Donor cell nuclear remodelling in reconstructed horse oocytes 18-20 h after cell fusion after nuclear transfer.
(a) Original donor cells (G0-G1 stage); (b) swelling nucleus; (c) prometaphase stage; (d) metaphase stage; (e) anaphase stage (chromosomes separating); (f) two nuclei.
Arrows indicate the position of the chromatin or nucleus.
Scale bar represents 20 μm.
The number of donor cell passages is another significant factor when attempting to clone by nuclear transfer.
Kubota et al. (2000) found no differences in the development of nuclear transfer embryos when using adult fibroblasts that ranged in passage numbers from five to 15.
In the present study, the rate of nuclear remodeling decreased significantly when using fetal fibroblasts of increasing passage number.
During cell passage, both genetic and epigenetic alterations that might affect nuclear remodeling would be expected to accumulate in the cultured cells, and other possible disruptions to the regulation of imprinted genes could also be induced by repeated culture, thereby leading to perturbations in embryonic and fetal development.
Furthermore, it would be sensible to try other types of cell obtained from different somatic tissues for use as cell donors in this species.
Successful cell division requires the formation of the metaphase spindle after DNA replication during the normal mitotic cell cycle.
By confocal microscopic analysis of non-cleaved oocytes in the present study, it was possible to demonstrate that the microtubules in 78% of FFC-constructed oocytes, and in 92% of AFC constructed oocytes, did not form a mitotic spindle-like structure and so were unable to progress to later stages of the first cell cycle.
Furthermore, even the 22% of the FFC-constructed oocytes and 8% of the AFC-constructed oocytes in which the chromosomes were at the metaphase stage, or were beginning to separate, could not complete the first embryonic division due either to structural defects of the spindle, or the distribution of the chromosomes, or both.
Tremoleda et al. (2001) reported that changes in the microtubules and the organization of the microfilaments during in vitro maturation of horse oocytes are similar to those seen in other species.
Thus, it can be speculated that the underlying cause of the first embryonic division block in the reconstructed horse oocytes created by nuclear transfer may be a deficiency of the cytoplasmic transition factors in the recipient cytoplasm, such as those in the cell cyclin family and other cyclin-dependent factors, that are involved specially in the progression of the introduced donor nucleus from S-G2-prometaphase to metaphase.
In addition, in contrast to the situation in mitotic cells, meiotic spindles in mammalian oocytes lack centrioles.
Hence, the introduction of a foreign centrosome during somatic cell nuclear transfer results in the presence of the centrioles which play an indispensable role in reorganization of the first embryonic division in the reconstructed oocytes.
Whether these factors are relevant to horse nuclear transfer remains to be clarified.