Millora de la reprogramació del nucli somàtic en procediments de transferència nuclear en ratolí

Abstract

Somatic cell nuclear transfer (SCNT) success depends on a wide range of factors which limit its efficiency. On one hand, the vast nuclear reprogramming that the somatic nucleus transferred must undergo for gene expression to change from a differentiated to an embryonic pattern is often defective. It is well known that treatment of SCNT embryos with histone deacetylase inhibitors (HDACis) corrects the hipoacetylation and hipermethylation of their genome, and this helps reprogramming factors in the ooplasm to gain access to the transferred nucleus, improving nuclear reprogramming. But the role of other epigenetic modifiers, such as histone demethylases (HDMs), is unclear. On the other hand, the manipulations performed on the oocyte during the SCNT protocol affect its viability. In this sense, the use of latrunculin A (LatA) instead of cytochalasin B for actin polymerization inhibition significantly improves the development to term of HDACitreated SCNT embryos. However, its effect on in vitro development and on untreated embryos is unknown. In addition, SCNT embryos are especially sensible to in vitro culture conditions and a non-invasive method for the prediction of their developmental potential, which could increase success rates, is lacking. In this thesis, the cloning efficiency of B6CBAF1 mice has been improved from 0-0.5 %, in untreated embryos, to 3-5 % in embryos treated with psammaplin A (PsA), an HDACis never used in nuclear reprogramming studies before, and/or vitamin C (VitC), an antioxidant reported ti act as a cofactor of Jumonji-domain-containing HDMs and ten eleven translocation enzymes. Both epigenetic modifiers have also proved to improve embryonic stem cell derivation from SCNT embryos. However, the cloning efficiency previously achieved treating SCNT embryos with the HDACi valproic acid (VPA) has not been improved, in spite of the increase of its concentration and exposure time tested. PsA and VPA have been shown to improve nuclear reprogramming through the increase of histone acetylation levels in SCNT embryos, but an effect of VitC on the nuclear reprogramming of SCNT embryos has not been detected. In addition, it has been shown here that LatA improves in vitro developmental rates, especially of those embryos non-treated with epigenetic modifiers. However, in order to improve full-term development, LatA has to be combined with an epigenetic modifier such as PsA or VitC. Thus, LatA could be involved in the nuclear reprogramming of SCNT embryos and might exert a synergistic effect with epigenetic modifiers. Finally, the development of SCNT embryos has been assessed with a time-lapse system that does not reduce their viability. We observed that SCNT embryos are delayed, from the second division, in comparison with fertilized embryos, and that the epigenetic modifier treatment has an effect on embryo kinetics. In particular, when embryos are treated with PsA the delay is more pronounced and appears earlier. In contrast, VitC accelerates the developmental kinetics of SCNT embryos, making them more similar to fertilized ones. A prediction model for the developmental potential of SCNT embryos (both untreated and treated) has been established, based on the time of division to the four-cell stage, alone or combined with compaction duration and/or fragmentation to increase prediction accuracy. The validation of this model would allow the selection of SCNT embryos with higher developmental potential prior to their transfer to recipient females.

Date of Award	17 Nov 2014
Original language	Catalan
Supervisor	Elena Ibáñez (Director) & Josep Santalo Pedro (Director)