45 EFFECT OF TRICHOSTATIN A TREATMENT ON THE TERM DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER RABBIT EMBRYOS

Abstract

The efficiency of somatic cell nuclear transfer (SCNT) is low in the rabbit. So far, there have been few live births reported and most clones died within the first 3 weeks after birth. It has been shown that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, improved cloning efficiency in cattle (Enright et al. 2003 Biol. Reprod. 69, 896–901) and mice (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). Although a recent report indicated that TSA treatment could increase the cell number of rabbit SCNT blastocysts (Xu et al. 2007 Reprod. Fertil. Dev. 19, 165), term development of TSA-treated cloned embryos in this species has not been reported. In this study we investigated the effect of TSA treatment on the term development of somatic cell nuclear transfer (SCNT) rabbit embryos. The oocytes and cumulus cells were collected from superovulated Hycole hybrid rabbits. After staining with Hoechst 33342 and locating following 1–2 s of UV illumination, the nuclei of oocytes were removed by micromanipulation, A cumulus cell was then inserted into the perivitelline space and fused with the cytoplast with three 20 μs 3.2 kV cm^–1 DC pulses. Fused embryos were activated using the same electrical parameters 1 h later, treated with 2 mm 6-dimethylaminopurine and 5 μg mL^–1 cycloheximide for 1 h, and then subsequently cultured in Earles Balanced Salt Solution (EBSS) with or without 5 nm TSA for 10 h. The embryos were then cultured in EBSS either overnight (before ET) or for 4.5 days. Embryos were transferred at the 2- to 4-cell stages to the recipients 22 h after collection of the oocytes from the donors. Caesarean sections were performed on Day 30 post-ET. In vitro developmental data (Table 1) showed no differences in the cleavage, blastocyst rates, and blastocyst cell numbers between the TSA-treated or untreated cloned embryos. After ET in the TSA group, one pregnant female delivered 7 live and 3 stillborn pups, but all of the live pups died within 1 h to 19 days later. In the untreated group, one pregnant female gave birth to 2 live and 1 stillborn pup. One pup died within 1 h after birth; the other survived (2.5 months old when this abstract was submitted). In conclusion, the results indicate that TSA treatment has a limited effect on in vitro development of SCNT embryos, and both TSA-treated and untreated SCNT clones can develop to term in rabbit. The effects of TSA treatment on the health of clones need further investigation.

Table 1. In vitro and in vivo development of SCNT rabbit embryos with or without TSA treatment

This study was supported by Wellcome Trust (Grant No. 070246), EU FP6 (MEXT-CT-2003-509582, MRTN-CT-2006-035468), and Chinese-Hungarian Bilateral projects (TET CHN-28/04, CHN-41/05).