54 THE EFFECT OF TREATMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS WITH VALPROIC ACID ON THEIR SUBSEQUENT IN VITRO DEVELOPMENT

Abstract

Recently, trichostatin A (TSA) and some other histone deacetylase inhibitors (HDACi) were reported to enhance the development of mouse somatic cell nuclear transfer (SCNT) embryos. Previously, we have succeeded in improving in vitro development of bovine SCNT embryos significantly with inhibitors of class I, IIa, and IIb HDACs such as TSA or Scriptaid (SCR). In this study, we examined the effect of valproic acid (VPA), a selective inhibitor of class I and IIa HDACi, on in vitro development of bovine SCNT embryos. 3 cell lines (adult male, adult female, and fetal female fibroblast cells) were used as donor cells. SCNT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). Reconstructed embryos were chemically activated by treatments with 10 μM calcium ionophore for 5 min and 10 μg mL–1 cycloheximide for 5 h. Then SCNT embryos were cultured in serum-free medium for 7 days. Embryo development data were analyzed by chi-square test. Differences were considered significant at P < 0.05. Each experiment was replicated at least 3 times. At first, to determine the suitable concentration of VPA, bovine SCNT embryos derived from adult female fibroblast were treated with various concentrations of VPA (0, 0.01, 0.1, 1, 10 mM) for 20 h from the start of chemical activation and embryo development was examined. There was no difference in blastocyst formation rates based on the number of cleaved embryos among the groups [46% (166/357), 53% (49/92), 46% (51/112), 55% (56/102), and 44% (44/100), respectively], and the cell numbers of blastocysts were also similar. Next, we examined the effect of duration of VPA treatment on development of SCNT embryos obtained from adult male, female, and fetal female fibroblast cells. Based on the results of previous experiment, bovine SCNT embryos were treated with 1 mM VPA for 20 h or 40 h and those without treatment were used as control. Neither 20 h nor 40 h VPA treatment affected blastocyst formation rates of SCNT embryos from adult male, control: 38% (41/107), 20 h: 45% (47/104), 40 h: 46% (39/85); adult female, control: 47% (166/357), 20 h: 55% (56/102), and 40 h: 36% (12/33); and fetal female fibroblast cells, control: 7% (4/58), 20 h: 13% (7/54), and 40 h: 16% (8/51). In the present study, treatment of bovine SCNT embryos with VPA did not improve in vitro development significantly. Comparing these results with our previous results on TSA or SCR treatment, inhibition of HDAC class IIb may be a key factor to improve development of bovine SCNT embryos.