Resumen de Oocyte chemical enucleation and histone deacetylase inhibitors: strategies to improve mammalian cloning by somatic cell nuclear transfer
Somatic cell nuclear transfer (SCNT) requires the erasing the epigenetic program characteristic of a somatic cell to allow for a switch in gene expression and enable the conversion of the transferred nucleus from a differentiated into an embryonic state. These reprogramming events, performed by the recipient oocyte, constitute the main principles that make of SCNT a powerful technology with innumerous possible reproductive and therapeutic applications.
However, while the current efficiency of nuclear transfer-derived embryonic stem (ntES) cells derivation in experimental species such as the mouse is moderately high, the proportion of cloned embryos able to develop to term is still extremely low and the cloned animals frequently exhibit a variety of health abnormalities. These problems have been attributed to a lack of competence of the oocyte to correctly reprogram the transferred nucleus and to promote its subsequent embryonic development, as oocytes are naturally designed to reprogram sperm chromatin, and not somatic nuclei. Besides, the competence of the oocytes may be compromised if the enucleation procedure used to prepare recipient cytoplasts for SCNT depletes the oocyte from cytoplasmic factors involved in nuclear reprogramming or cell division mechanisms, as may be the case of the mechanical enucleation (ME) method routinely employed in current SCNT protocols.
In this sense, studies aimed to improve the oocyte's ability to reverse a somatic nucleus into a totipotent embryonic state and to promote the subsequent development of the cloned embryo are important to increase SCNT efficiency, as well as to elucidate the key mechanisms that regulate dedifferentiation and redifferentiation of the transferred nucleus.
This thesis is focused on the establishment of new protocols of chemically-assisted (AE) and induced (IE) with the expectancy that they could represent better alternatives to traditional ME in the preparation of competent cytoplasts for SCNT. The first two studies were designed to determine the best conditions to achieve high enucleation rates with both chemical enucleation methods as well as to analyze different aspects that could interfere with the quality of the cytoplasts produced, so that we could evaluate the major advantages and disadvantages of each method with regards to traditional ME. In particular, we tested different antimitotic drugs and treatment times for both AE and IE methods and we compared the enucleation efficiencies, the amount of cytoplasmic volume removed from the oocytes and the distribution of spindle associated factors in the resultant cytoplasts after the application of the three enucleation procedures. With the purpose to further assist the enucleated oocytes with the difficult task of reprogramming the transferred nucleus, in a third study we explored the effects of treating cloned embryos prepared by ME with HDACis. Finally, in the last study, cloned embryos prepared by ME, AE and IE were treated with HDACis with the expectancy that the combination of the two strategies developed along this thesis could result in an improvement of SCNT rates in the mouse species.
