Incidence of apoptosis and effects of apoptosis inhibitors in a somatic cell nuclear transfer program

Abstract

This study was conducted to examine the incidence of apoptosis in bovine embryos cloned with adult ear fibroblasts and to evaluate whether the treatment of donor somatic cells and reconstructed embryos with hemoglobin (Hb) and/or β-mercaptoethanol (ME) could improve preimplantation development after somatic cell nuclear transfer (SCNT). Furthermore, the effect of treatment of oocytes provided for recipients during in vitro maturation with such substrates were also examined. Apoptosis was detected by a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling assay, and Hb and/or ME effects were evaluated by morphogenic and cytological analysis. Apoptosis was first detected as early as from the 4-cell stage in clone embryos, while from the 8-cell in in vitro fertilized (IVF) embryos of a control group. In the first series of experiment, clone embryos had more apoptotic blastomere than IVF embryos and significant (p<0.05) difference was found at the 4- to 8-cell (1.3 vs. 0 cells/embryo) and blastocyst (8.9 vs. 6.2 cells/embryo) stages. In the second series of experiment, when the donor cells were cultured in ME (10 μM)- containing medium before cloning, apoptosis index of clone embryos showing the ratio of apoptotic to normal blastomere number was significantly decreased (0.058 vs. 0.083). In this treatment, improved blastocyst formation (41% vs. 34%) with increasing blastomere (131 vs. 123 cells/blastocyst) and inner cell mass (ICM) cell (31.9 vs. 27.6 cells/blastocyst) numbers and ICM to trophectoderm (TE) cell ratio (0.24 vs. 0.21) was achieved compared with no treatment. Provided ME-treated donor cells, culture of clone embryos in ME+Hb-containing medium further reduced apoptotic index (0.085 to 0.069) and increased all parameters of cell number (142 to 155, 27 to 41, 98 to 114 and 0.22 to 0.26 in total blastomere, ICM, TE and ICM to TE ratio, respectively). In the third series of experiment performed in the optimal system, the treatment of oocytes during in vitro maturation with both substrates did not further provide clone embryo development. In conclusion, the incidence of apoptosis was significantly increased in clone than in IVF embryos and the treatment of donor somatic cell and clone embryos before and after SCNT greatly promoted preimplantation development.