41 DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFERRED RAT EMBRYOS TO THE BLASTOCYST BY A HISTONE DEACETYLASE INHIBITOR, TRICHOSTATIN A

Abstract

Although cloned animals have been reproduced in many species via somatic cell nuclear transfer (SCNT), only 1 previous report showed successful generation of cloned rats. Some researchers evaluated developmental competence of reconstructed rat embryos and showed that these reconstructed embryos were never progressed beyond the 2-cell stage because of inadequate formation of the mitotic assembly and nuclear organisation or improper transcription of cytoskeleton genes. Even though our group also improved the proportion of 2 pronuclear formation in rat reconstructed embryos by the treatment with a proteasome inhibitor, MG132 (Nakajima et al. 2008 Cloning and Stem Cells 10, 461–468), no embryos were developed beyond the 2-cell stage. Recently, it has been demonstrated that a histone deacetylase inhibitor, trichostatin A (TSA) treatment dramatically improves efficiency of cloning in the mouse, although the precise effect of TSA is unclear in SCNT. However, in the case of rats, availability of TSA has not been tested. The aim of the present study was to clarify whether TSA treatment was also effective in rat SCNT. According to our previous reports, rat oocytes were collected and the spindles of the oocytes were removed in medium supplemented with MG132. After enucleation, nuclei of cumulus cells were injected in the oocytes and the reconstructed embryos were cultured in the medium supplemented with 0 (control), 5, 50, or 500 nM TSA. More than 100 embryos are used for each treatment group. Pronuclear formation, development to the 2-cell stage and blastocyst formation were evaluated. Our results demonstrated that TSA treatment affected neither the proportion of pronuclear formation nor development to the 2-cell stage in rat reconstructed embryos. However, the treatment with higher concentrations of TSA treatment (50 nM and 500 nM) compared to the concentration (5 nM) which was usually used for mouse SCNT enabled the reconstructed embryos to develop to the blastocyst stage but at low rate (1.4 to 2.2%). In both the 5 nM treatment and control groups, none of the rat reconstructed embryos developed to blastocyst. Taken together, our data suggests that TSA treatment also seems to be effective in rat SCNT. These findings will be useful for improvement of the protocol in rat SCNT. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan, Grant-in-Aid for Matching Fund Subsidy for Private Universities to J. I. and N. K.