94 NUCLEAR-CYTOPLASMIC INTERACTION AND DEVELOPMENT OF CLONED EMBRYOS

Abstract

Although cloned canine offspring have been produced, the efficiency of nuclear transfer (NT) is still far from expectations. The most important factor interfering with the success of NT is the shortage of canine matured oocytes. In the present study, the nuclear-cytoplasmic interaction and in vitro development of porcine oocytes cloned with canine fetal fibroblasts (cFF) and canine mesenchymal stem cells (cMSC) were assessed by examining the developmental rate, embryonic morphology, total cell number, canine genome expression, and in vivo development following the transfer of iNT embryos into the oviducts of surrogate mother. Isolated cMSC from bone marrow were characterized by early transcription factors (Oct3/4, Sox2, and Nanog) and by differentiation into adipogenic, osteogenic, and neuronal cells under controlled in vitro conditions. Porcine cumulus–oocyte complexes (COC) were matured in vitro as described by Kim YS et al. (2005 Mol. Reprod. Dev. 70, 308–313). Porcine NT embryos were used for the control group. Canine MSC showed their characteristic property of attaching to plastic culture flasks and forming a monolayer of spindle-shaped cells. Adherent cells showed moderate expression of Oct3/4, Sox2, and Nanog protein by immunostaining. Following osteogenic cell induction, cMSC transformed into mineralized nodules by von Kossa staining. Oil red O staining revealed that MSC produced lipid droplets after incubation in adipogenic media. Neuronal-like cells with multipolar, round cell bodies organized into a network-like structure were confirmed by immunostaining of neuronal markers (NFM). The tissue-specific mRNA levels were confirmed after differentiation by RT-PCR (osteogenic cell, Osteocalcin; adipogenic cell, PPAR γ; neuronal cell, NFM). The control group blastocysts (porcine oocytes-pMSC) exhibited significantly (P < 0.05) greater development and total cell numbers of than blastocysts of interspecies groups (porcine oocytes-cFF and cMSC). The canine genome GAPDH was detected in cFF, cMSC, and interspecies NT embryos using PCR, whereas there was no detection in porcine oocytes and the negative control. Most of the embryos arrested at the 8- to 16-cell stage and only 5% of embryos developed to blastocyst stage. The interspecies-NT embryos (average 35 embryos/recipient dog) were surgically transferred into the oviducts of 3 recipient dogs. Two of them showed exterior signs of implantation; i.e. a slight thickening of the uterus and small pea-sized lumps. No pregnancies were detected in the recipients after ovariohysterectomy surgery. These results demonstrate the potential of cMSC isolated from bone marrow to differentiate into mesoderm (osteogenic and adipogenic cell) and ectoderm (neuronal cell)-like cells under specific induction conditions. Although interspecies NT embryos could not implant in the canine uterine wall, the results provided valuable information on canine MSC and interspecies NT.

This work was supported by Grant No. 20070301034040 from Bio-Organ, Republic of Korea.