83 THE ENDOMETRIUM AND EXTRAEMBRYONIC TISSUES RESPOND DIFFERENTLY TO CLONED v. NATURAL MATING EMBRYOS ON EARLY PREGNANCY

Abstract

Porcine cloning by somatic cell nuclear transfer (SCNT) has limitations because of the high incidence of fetal failure after embryo transfer to recipient. The fetal failure may originate from disturbed embryo-uterine interactions in the early implantation period. In this study, we compared apoptotic-related gene expression and cytokines using real-time RT-PCR and protein chip array. Ovaries were obtained from prepubertal crossbred gilts at a local slaughterhouse. Oocytes were matured for 40 to 44 h at 38.5°C under 5% CO2 in air. As donor cells, fibroblast cells were cultured from ear skin tissue of 8-month-old MHC inbred miniature pig. After the enucleation and injection process, eggs were held in TCM-199. For fusion, two DC pulses of 1.2 kV cm-1 were applied for 30 μs. SCNT embryos were cultured under PZM-3 medium to the 1- to 4-cell stage. For embryo transfer, approximately 200 SCNT eggs were transferred into the oviduct of each synchronized recipients. We obtained the extraembryonic (embryo origin) and endometrium (maternal origin) tissues from 3 cloned fetus with that from normal fetus by natural mating at Day 30. Each experiment was repeated at least 3 times and the data were presented as mean ± SE. Values of P < 0.05 were considered statistically significant. Data from real-time RT-PCR are expressed as log (fold change) ± SE. Expression of Bax-α, p53, and caspase-3 mRNA was significantly higher in the SCNT group than in the control group (P < 0.05). Five cytokines were analyzed: Interleukin (IL)4, IL8, tumor necrosis factor-α, and epidermal growth factor were lower in the control group than in the cloned group (P < 0.05). However, the level of MCP1 was higher in the SCNT group (P < 0.05). Numbers of trophoblast cells in the cloned group were also lower than in the control group (P < 0.05). These findings indicate that failure of endometrium and extraembryonic tissues in cloned pregnancies may originate from abnormal embryo-maternal communication that develops during the implantation period. This work received grant support from the Agenda Program (no. 200901FHT010305535), Rural Development Administration, Republic of Korea.