27 PSAMMAPLIN A INCREASES DEVELOPMENT AND QUALITY OF MOUSE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS
Authors: Mallol A. , Santaló J. , Ibáñez E. 
Published: December 2012

Among the many biological and technical factors affecting the success rate of mouse somatic cell nuclear transfer (SCNT), faulty reprogramming of the differentiated donor nucleus to a totipotent embryonic state by the recipient oocyte seems key. Accordingly, treatment of SCNT embryos with epigenetic modifiers such as valproic acid (VPA), a histone deacetylase inhibitor (HDACi), enhances cloning efficiency. Psammaplin A (PsA) is a natural and potent DNA methyltransferase inhibitor and HDACi that has never been used in nuclear reprogramming studies. The purpose of our study was to determine the effect of PsA on the development and quality of mouse SCNT embryos, and to compare it to that of VPA. To this aim, mechanically enucleated oocytes from B6CBAF1 female mice were reconstructed with cumulus cell nuclei, activated, and cultured in the presence of the epigenetic modifier. Embryos that reached the blastocyst stage were differentially stained for counting inner cell mass (ICM) and trophectoderm cells. Alternatively, 2-cell embryos were transferred to CD1 recipient females to assess full-term development. In a first set of experiments, embryos were exposed to different concentrations of PsA (5, 10, and 20 µM) or VPA (2 and 4 mM) for 1 to 2 h after reconstruction and 6 h of activation (total 8–9 h). We found that 10 µM PsA and 2 mM VPA significantly increased blastocyst rates (37.3 and 31 v. 23.3% for the control group), although no differences were found in blastocyst quality (10.4–13.6 ICM cells). In a second set of experiments, we studied the effect of treatment duration by incubating the embryos in 10 µM PsA or 2 mM VPA for 8 to 9, 16, or 24 h after reconstruction. With PsA, all treatments showed equivalent blastocyst rates (35.2–43.3%), which were significantly higher than in the control group (20%), but only treatments for 16 and 24 h yielded blastocysts with significantly higher numbers of ICM cells (16.3 and 18.5 v. 10 for the control group). With VPA, treatments for 8 to 9 h and 16 h were equivalent in terms of blastocyst rates (34.0 and 32.5%) and significantly higher than the control group, but only VPA 16 h yielded blastocysts with a significantly higher number of ICM cells (15.6). In a third set of experiments, we studied the full-term development of embryos treated with 10 µM PsA or 2 mM VPA for 16 h and we found that both treatments, but especially the PsA treatment, resulted in higher birth rates than those obtained in the control group, although the differences were not statistically significant (1.79 and 0.86 v. 0.46%). Finally, when the actin polymerization inhibitor latrunculin A was used instead of cytochalasin B in the SCNT protocol during oocyte micromanipulation and activation, we obtained a 3-fold increase in the birth rate of embryos treated with PsA (5.29%). In conclusion, PsA enhances development and quality of mouse SCNT embryos, to a greater extent than VPA, and when combined with the use of latrunculin A instead of cytochalasin B, it results in an 11.5-fold increase in full-term development.
Support from MEC AGL-2011-23784, 2009-SGR-282, and PIF-UAB Fellowships is acknowledged.