Abstract
Somatic cell nuclear transfer (SCNT) is a useful technology; however, its efficiency is low. In this study, we investigated the effects of cytoplasmic transfer into enucleated oocytes on the developmental competence and quality of cloned preimplantation bovine embryos via terminal deoxynucleotidyl transferase dUTP nick-end labeling, quantitative reverse transcription PCR, and immunocytochemistry. We used cytoplasm injection cloning technology (CICT), a new technique via which the cytoplasmic volume of an enucleated oocyte could be restored by injecting ∼30% of the cytoplasm of a donor oocyte. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly higher (p < 0.05) in the CICT group than in the SCNT group (28.9 ± 0.8% vs. 20.2 ± 1.3%, respectively). Furthermore, the total cell number per day 8 blastocyst was significantly higher in the CICT group than in the SCNT group (176.2 ± 6.5 vs. 119.3 ± 7.7, p < 0.05). Moreover, CICT increased mitochondrial activity, as detected using MitoTracker® Green. The mRNA levels of DNA methyltransferase 1 and DNA methyltransferase 3a were significantly lower (p < 0.05) in the CICT group than in the SCNT group. The mRNA level of DNA methyltransferase 3b was lower in the CICT group than in the SCNT group; however, this difference was not significant (p > 0.05). Taken together, these data suggest that CICT improves the in vitro developmental competence and quality of cloned bovine embryos.
Highlights
Reconstructed embryos can be generated by injecting a highly differentiated somatic cell nucleus into an enucleated oocyte
We investigated the effects of cytoplasmic transfer into enucleated oocytes on the developmental competence and quality of cloned preimplantation bovine embryos via terminal deoxynucleotidyl transferase dUTP nick-end labeling, quantitative reverse transcription PCR, and immunocytochemistry
We examined the effects of cytoplasmic transfer on cleavage of cloned preimplantation bovine embryos at day 2 and their blastocyst developmental competence at day 8
Summary
Reconstructed embryos can be generated by injecting a highly differentiated somatic cell nucleus into an enucleated oocyte. More than 20 mammalian species have been successfully cloned via somatic cell nuclear transfer (SCNT), this technique is inefficient (Su et al, 2015). The low efficiency of SCNT is likely due to incomplete reprogramming of the donor nucleus, and epigenetic defects are thought to underlie most of the developmental problems of these embryos (Ng and Gurdon, 2005; Vajta and Gjerris, 2006). Successful cloning is considered to be dependent on reprogramming of differentiated somatic cells into a totipotent embryonic-like state (Dean et al, 2001; Latham, 2005; Tian, 2004).
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