62 EXPRESSION AND DISTRIBUTION OF Oct-4 IN INTERSPECIES-CLONED LONG-TAILED MONKEY (MACACA FASCICULARIS) EMBRYOS

Abstract

The transcription factor Oct-4 is one of the important factors for early embryonic development and differentiation in several animal species such as monkey (Mitalipov et al. 2003 Biol. Reprod. 69, 1785–1792) and bovine (Kurosaka et al. 2004 Biol. Reprod. 71, 1578–1582). However, Oct-4 expression and distribution in interspecies somatic cell nuclear transfer (iSCNT) embryo have not yet been studied. Therefore, this study was undertaken to evaluate the Oct-4 distribution in monkey iSCNT embryos at the 2-cell (2C), 4-cell (4C), 8-cell (8C), 16-cell (16C)/morula, hatching (Day 7), and hatched blastocyst stages (Day 8), compared with bovine SCNT embryos. The skin fibroblasts from adult monkey (Macaca fascicularis) and bovine were used as donor cells, and bovine enucleated oocytes were used as recipient cytoplasts. SCNT procedures were performed as previously reported (Laowtammathron et al. 2005 Theriogenology 64, 1185–1196). The result was that monkey iSCNT embryos have cleavage (93.8%; 76/81) and development rates up to the 8-cell stage (76.5%; 62/81) similar to those in bovine SCNT (96.0%; 96/100, and 89.0%; 89/100, respectively). Bovine SCNT developed to morulae (61.5%; 59/96) and blastocysts (41.7%; 40/96) at a higher rate than monkey iSCNT (43.4%; 33/76, and 11.8%; 9/76, respectively). Moreover, bovine SCNT blastocysts showed inner cell mass (ICM) and trophectoderm (TE), but monkey iSCNT embryos formed a blastocoel and ICM and TE could not be clearly seen. Five embryos from each group were used to study Oct-4 expression and distribution by immunocytochemistry. The embryos were incubated with 1 : 1000 anti-mouse Oct-4 polyclonal antibodies (Chemicon International, Inc., Temecula, CA, USA), then rinsed in PBS, and incubated with 1 : 200 FITC-labeled antimouse IgG (Sigma-Aldrich Corp., St Louis, MO, USA), followed by 2 µg mL^-1 422-6-Diamidino-2-phenylindole (DAPI) (Sigma); the embryos were then mounted and examined by fluorescence microscopy. The results showed Oct-4 staining in every nucleus of bovine and monkey iSCNT embryos at the 2C, 4C, 8C, and 16C/morula stages but was varied in blastocysts. The Oct-4 staining was reduced in TE cells when half of the embryonic cells hatched out of the zona pellucida (60.0%; 3/5). The Oct-4 staining continued to decrease in the TE of hatched blastocysts (Day 8) but did not affect the ICM. The total cell number of bovine SCNTs at blastocyst stage were 140.9 ± 13.2 and, of these, 13.5% (19/140) showed apoptotic nuclei morphologies, whereas the cell numbers of monkey iSCNT early blastocysts had only 15.6 ± 2.3 cells and, of these, 19.2% (3/15) showed apoptotic nuclei. It can be concluded from this study that monkey iSCNT embryos show Oct-4 staining in every intact nucleus.