Heterospecific Nuclear‐transferred Embryos Derived from Equine Fibroblast Cells and Enucleated Bovine Oocytes

Abstract

This study was conducted to reconstruct heterogeneous embryos using equine skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplast for investigating the reprogramming of equine somatic cell nuclear in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Adult horse skin fibroblast cells serum-starved were used as donor somatic cells. Bovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electofusion. The fused eggs were activated by inomycin with 2 mm/ml 6-dimethylaminopurine (6-DMAP). The activated reconstructed embryos were co-cultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum (FCS) for 168 h. The results showed that the first completed cleavage of xenonuclear transfer equine embryos occurred between 30 and 48 h following activation. 52% of the injected oocytes were successfully fused, 72% of the fused eggs underwent the first egg cleavage and 17% of the heterospecific nuclear-transferred zygotes developed to 4- or 8-cell embryo stages. This study demonstrated that the reconstructed embryos have undergone the first embryonic division and the reprogramming of equine fibroblast nuclei can be initiated in bovine-enucleated oocytes.

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

“…Alternatively, not every species has been able to develop to the blastocyst stage when reconstructed with bovine oocytes; it has been reported that cloned mouse [38], equine [39] and domestic cat [22] embryos derived from fibroblasts arrested their development at the eight-cell stage, which is similar to this study. In addition, rabbit oocytes were able to the dedifferentiate somatic cell nuclei of several species, especially cloned intergeneric domestic cats [26].…”

“…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].…”

Development of Interspecies Cloned Monkey Embryos Reconstructed with Bovine Enucleated Oocytes

“…They found that only 17% (8/48) of the equine iSCNT embryos could develop to the four-to eight-cell stage, and none of them could develop to the blastocyst stage, while 15% (10/68) of the bovine PA embryos (control group) reached the blastocyst stage. These reports [11,12,37,40,41] suggest that the iSCNT The cell numbers of the monkey iSCNT embryos from each treatment were significantly different from those of the bovine PA and bovine SCNT embryos (P≤0.01); however, the bovine SCNT and bovine PA embryos (Tr 5 and Tr 6) were not significantly different (P≥0.05).…”

“…In our study, the monkey iSCNT embryos could not develop beyond the eight-cell stage, which was consistent with previous reports concerning iSCNT mouse [37,41], dog [42] and camel [11] embryos using bovine [37,41,42] and ovine [11] enucleated oocytes as the recipient cytoplasm. Zhou et al [12] have investigated reprogramming of the equine somatic cell nucleus after injection into bovine enucleated oocyte cytoplasm and the developmental potential of the reconstructed embryos. They found that only 17% (8/48) of the equine iSCNT embryos could develop to the four-to eight-cell stage, and none of them could develop to the blastocyst stage, while 15% (10/68) of the bovine PA embryos (control group) reached the blastocyst stage.…”

“…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.…”