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

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서울대학교 대학원
Thesis (master`s)--서울대학교 대학원 :농생명공학부 발생공학 전공,2003.
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.
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Food and Animal Biotechnology (식품·동물생명공학부)Theses (Master's Degree_식품·동물생명공학부)
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