Abstract
Artificial activation of oocytes is an important step for successful parthenogenesis and somatic cell nuclear transfer (SCNT). Here, we investigated the initiation of DNA synthesis and in vivo development of canine PA embryos and cloned embryos produced by treatment with 1.9 mM 6-dimethylaminopurine (6-DMAP) for different lengths of time. For experiments, oocytes for parthenogenesis and SCNT oocytes were cultured for 4 min in 10 μM calcium ionophore, and then divided into 2 groups: (1) culture for 2 h in 6-DMAP (DMAP-2h group); (2) culture for 4 h in DMAP (DMAP-4h group). DNA synthesis was clearly detected in all parthenogenetic (PA) embryos and cloned embryos incorporated BrdU 4 h after activation in DMAP-2h and DMAP-4h groups. In vivo development of canine parthenogenetic fetuses was observed after embryo transfer and the implantation rates of PA embryos in DMAP-2h were 34%, which was significantly higher than those in DMAP-4h (6.5%, p < 0.05). However, in SCNT, there was no significant difference in pregnancy rate (DMAP-2h: 41.6% vs. DMAP-4h: 33.3%) and implantation rates (DMAP-2h: 4.94% vs. DMAP-4h: 3.19%) between DMAP-2h and DMAP-4h. In conclusion, the use of DMAP-2h for canine oocyte activation may be ideal for the in vivo development of PA zygotes, but it was not more effective in in vivo development of canine reconstructed SCNT oocytes. The present study demonstrated that DMAP-2h treatment on activation of canine parthenogenesis and SCNT could effectively induce the onset of DNA synthesis during the first cell cycle.
Highlights
Successful somatic cell nuclear transfer (SCNT) using adult somatic cells has been reported in various mammals [1,2,3,4] and has been a useful tool for producing disease models or for bio-resource purposes
In the DMAP-2h group, DNA synthesis was initiated in all PA zygotes at 2 hpa (Table 1)
In the DMAP-4h group, DNA synthesis in 90% of PA zygotes started at 2 h after activation (Table 1)
Summary
Successful somatic cell nuclear transfer (SCNT) using adult somatic cells has been reported in various mammals [1,2,3,4] and has been a useful tool for producing disease models or for bio-resource purposes This technique is affected by a variety of factors such as oocyte status, donor cell cycle, oocyte activation, and reprogramming. In a previous study which the producing intergeneric canine cloned embryos using bovine recipient oocytes, the various duration of 6-DMAP exposure affected cleavage rate on in vitro development, but not on blastocyst formation [16]. The present study investigated the effect on DNA synthesis initiation in canine parthenogenetic zygotes and reconstructed SCNT oocytes after exposure to 6-DMAP for different durations and the association between DNA synthesis initiation and embryonic development in vivo
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