Development of Interspecies Cloned Monkey Embryos Reconstructed with Bovine Enucleated Oocytes

Abstract

Abstract. This study was carried out to determine whether culture media reconstructed with bovine enucleated oocytes and the expression pattern of Oct-4 could support dedifferentiaton of monkey fibroblasts in interspecies cloned monkey embryos. In this study, monkey and bovine skin fibroblasts were used as donor cells for reconstruction with bovine enucleated oocytes. The reconstructed monkey interspecies somatic cell nuclear transfer (iSCNT) embryos were then cultured under six different culture conditions with modifications of the embryo culture media and normal bovine and monkey specifications. The Oct-4 expression patterns of the embryos were examined at the two-cell to blastocyst stages using immunocytochemistry. The monkey iSCNT embryos showed similar cleavage rates to those of bovine SCNT and bovine parthenogenetic activation (PA). However, the monkey iSCNT embryos were not able to develop beyond the 16-cell stage under any of the culture conditions. In monkey and bovine SCNT embryos, Oct-4 could be detected from the two-cell to blastocyst stage, and in bovine PA embryos, Oct-4 was detectable from the morula to blastocyst stage. These results suggested that bovine ooplasm could support dedifferentiation of monkey somatic cell nuclei but could not support embryo development to either the compact morula or blastocyst stage. In conclusion, we found that the culture conditions that tend to enhance monkey iSCNT embryo development and the expression pattern of Oct-4 in cloned embryos (monkey iSCNT and bovine SCNT) are different than in bovine PA embryos. Key words: Bovine oocyte, Embryo, Interspecies cloning, Monkey, Oct-4 transcription factor (J. Reprod. Dev. 54: [306][307][308][309][310][311][312][313] 2008) stablishment of embryonic stem cells (ESCs) from nuclear transferred (NT) non-human primate (NHP) embryos has yet to be perfected [1]. Because the availability of NHPs is limited, the cost of NHP research can be prohibitive. Therefore, an alternative source of recipient cytoplasm that can support the reprogramming of NHP nuclei would effectively remove this constraint and deserves further investigation. Several studies have shown that the ooplasm of the bovine, rabbit, sheep, domestic cat and ovine can support early development of embryos produced by NT using somatic cell nuclei derived from different mammalian species [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. Recent successes in live offspring born from clones of gaur [6,9], mouflon [10] and African wildcat [14] have demonstrated that interspecies somatic cell nuclear transfer (iSCNT) can be used to preserve endangered species. Among the different choices of oocytes, the bovine oocyte is one of the most popular recipient cytoplasts for iSCNT because a large number of oocytes can be retrieved from ovaries which can be easily obtained from an abattoir. Most important, the in vitro culture system for bovine embryos is well established. Several research reports have demonstrated that the bovine oocyte is a good candidate for use as the recipie...

The development and expression of pluripotency genes in embryos derived from nuclear transfer and in vitro fertilization

“…developmental ability of intra-species cloned embryos was significantly higher than that of inter-species cloned embryos (Murakami et al, 2005;Li et al, 2006;Zhao et al, 2006;Lorthongpanich, et al, 2008).…”

The Effects of Manipulation Medium, Culture System and Recipient Cytoplast on In Vitro Development of Intraspecies and Intergeneric Felid Embryos

“…However, the total cell numbers of those blastocyst embryos were so low compared with the intraspecies cloned domestic cat and bovine blastocysts that we labeled them as blastocyst-like embryos. It was of interest that the same kind of morphology has also been reported in intergeneric cloned monkey embryos when reconstructed using bovine cytoplasts [18]. Furthermore, considering the fact that Yin et al [2] succeeded in producing cloned domestic kittens by transferring the cloned domestic cat embryos with morphology and total cell numbers similar to those in our study into the oviduct of the recipients, this same in vivo environment after embryo transfer might have supported the development of embryos through implantation till the birth of kittens [2].…”

“…While African wildcat, sand cat and DC are the same genus (Felis), MC belongs to a different genus (Pardofelis), and it can be suggested that the taxonomic distance between the donor and recipient species might be too far, resulting in the DCo not being able to support the development of the NT embryos [40]. Although the use of oocytes from a different genus as recipient cytoplasts in intergeneric NT seems to be efficient for a few mitotic divisions of the resultant cloned embryos, this combination of donor and recipient cells might not lead to the complete nuclear-cytoplasmic events needed for development of cloned embryos, since heteroplasmy or mitochondrial incompatibilities might occur at the time of genomic activation [14,18,26,38,39]. Furthermore, our recent study showed that MCf + DCo intergeneric SCNT resulted in abnormal transcriptional levels of Oct-4, DNMTs, HAT1 and HDAC1 genes, which might be involved in the low developmental potential of these reconstructed embryos [41].…”

“…Although the remaining population of the MC is not known, the International Union for the Conservation of Nature and Natural Resources (IUCN) classifies the MC as intermediate and the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) has listed the MC as endangered in Appendix I. Since endangered species have a limited supply of recipient oocytes, the oocyte cytoplasm of domestic species, such as the bovine, sheep, rabbit, DC and dog, has been used for dedifferentiation of the somatic cell nucleus from the rat, pig, monkey, sheep [11], gaur [12], buffalo [13], giant panda [14], banteng [15], wolf [16], goat [17] monkey [18], human [19,20] and several felid species [6][7][8][9][10]21]. Gomez et al [10] suggested that DC oocytes can be used for wildcat preservation by interspecies or intergeneric NT techniques.…”

Two-staged nuclear transfer can enhance the developmental ability of goat–sheep interspecies nuclear transfer embryos in vitro

“…The success of producing cloned embryos (Jiang et al 2005;Murakami et al 2005;Li et al 2006;Lorthongpanich et al 2008;Sha et al 2009;Sugawara et al 2009), even offspring (Lanza et al 2000;Loi et al 2001) by interspecies somatic cell nuclear transfer (iSCNT) indicated that the technique of iSCNT can be adopted to increase the population size of endangered mammals or even to restore extinct species. Even now, compared with intraspecies SCNT, the efficiency of iSCNT was even lower.…”