Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.
Interspecies somatic cell nuclear transfer (iSCNT) could be a useful method for embryo research of wildlife animals or endangered species. Because it is hard to obtain the oocytes or embryos of wildlife animals, its embryo research is not progressing well. Therefore, iSCNT is one of the alternative ways for wildlife animal embryo research and conservation of their genetic source. Until now, iSCNT has been applied to conservation of wildlife animals including guar, mouflon, banteng and African wildcat. The domestic pig oocytes have been used for iSCNT of other species such as tiger, sheep and dog and they successfully developed to the blastocyst stage. According to this concept, we performed wild-captured Korean raccoon (Nyctereutes procyonoides koreensis) iSCNT using porcine oocytes matured in vitro. Raccoon fibroblasts from ear skin samples of male raccoon were used as donor cells in 3 to 5 passages. The donor cells were cultured in DMEM supplemented with 15% FBS. Enucleated porcine oocytes were fused with raccoon fibroblasts by electrofusion. The iSCNT embryos were cultured in PZM-3 at 39°C for 7 days in an atmosphere of 5% CO2 and 5% O2. A total of 158 iSCNT embryos were cultured. More than 77% of the raccoon somatic cells successfully fused with the porcine oocytes; 68.5% of the iSCNT raccoon embryos developed to the 2- to 8-cell stage at Day 2 (1-cell: 9.7%, 2-cell: 14.4%, 4-cell: 34.1%, 6-cell: 12.7%, 8-cell: 7.3%, fragmented: 21.8%). This is similar to porcine SCNT results that 62.5% of the SCNT porcine embryos developed (1-cell: 8.0%, 2-cell: 4.2%, 4-cell: 23.6%, 6-cell: 13.6%, 8-cell: 23.8%, fragmented: 26.8%). But no embryos were further developed to blastocyst stage at Day 7 in iSCNT. In fragmentation evaluation in iSCNT embryos using by Hoechst stain at Day 2, two-cell stage embryos and four-cell stage embryos showed the normal numbers of nucleus. However, 6-cell stage embryos showed 4 to 5 nuclei and 8-cell stage embryos also showed 5 to 6 nuclei. Almost iSCNT embryos showed the developmental block at 4-cell stage embryos. This result was probably caused by an incomplete reprogrammed raccoon cell after iSCNT. Therefore, we treated with trichostatin A (TSA), a histone deacetylase inhibitor that has been used to enhance nuclear reprogramming following SCNT. Ninety-seven iSCNT raccoon-pig embryos were treated with 5 nM TSA during 15 h before being cultured in PZM-3. The TSA-treated iSCNT embryos showed similar developmental status to non-treated embryos (1-cell: 13.5%, 2-cell: 12.5%, 4-cell: 35.0%, 6-cell: 10.1%, 8-cell: 6.3%, fragmented: 22.5%). No embryos were further developed to blastocyst stage at day 7. Our results showed that 4-cell stage embryos of raccoon-porcine iSCNT embryos may be produced by iSCNT methods, but they were unable to support complete reprogramming of raccoon-porcine iSCNT embryos. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. 007133022011), Rural Development Administration, Republic of Korea.
Artificial activation is one of the essential factors for successful development of embryos derived from parthenogenetic activation or somatic cell nuclear transfer (SCNT). This study was carried out to confirm whether the short-term treatment of 6-dimethylaminopurine (6-DMAP) or demecolcine would improve developmental competence of porcine parthenogenetic activated embryos and find out the optimal activation protocol. Ovaries were collected from gilts at a local slaughterhouse and cumulus–oocyte complexes were cultured for 44 h in the tissue culture medium-199 with 10% of porcine follicular fluid. Matured oocytes were chemically activated with 10 min treatment of 0.2 mm thimerosal (Thi) followed by 30 min treatment of 8 mm dithiothrethiol (DTT). Additional treatments of 6-DMAP (2 mm) and/or demecolcine (0.4 μg mL–1) for a short term (40 min) or a long term (3 h) were conducted. Activated embryos were cultured in PZM-3. Cleavage rate, blastocyst (BL) formation rate and total cell number of BL were assessed at Day 2, 7 of culture, respectively. All data were subjected to one-way ANOVA followed by Tukey’s test using Prism version 4.0 (Graphpad Software, San Diego, CA, USA) to determine differences among experimental groups. To begin with, we evaluated that effect of short-term treatment of 6-DMAP or demecolcine. Short-term treatment of 6-DMAP improved developmental competence of activated embryos compared to long-term treatment (47.0 ± 1.0% v. 50.5 ± 1.5% for cleavage rate, 5 ± 3% v. 3 ± 1% for BL formation rate, 70 ± 13 v. 37 ± 6 for total cell numbers). Moreover, short-tem treatment of demecolcine also inhibited second polar body extrusion effectively compared with nontreated control (14 ± 2.1 v. 8.3 ± 4.1%). We confirmed that short-term treatment of 6-DMAP or demecolcine is valid. To optimize activation procedure with short-term treatment, development of embryos derived from each treatment were compared among nontreated control (C), 6-DMAP only treatment (6O), demecolcine only (DO) treatment, and 6-DMAP + demecolcine treatment (6 + D) groups. Cleavage rates of 4 groups were similar (53.5 ± 3.0, 62.1 ± 2.9, 53.6 ± 3.0 and 57.0 ± 3.0%, respectively). However, 6 + D group shows significantly greater BL formation rate (18.0 ± 2.5%) compared with C, 6O, or DO groups (9.4 ± 1.9, 14.0 ± 2.3 and 4.3 ± 1.3%, respectively). Total cell number of BL also increased in 6 + D group (52.8 ± 13.0) compared with C, 6O, or DO groups (47 ± 21.5, 31.5 ± 4.22 and 45.2 ± 13.7, respectively). In conclusion, we elucidated that short-term treatment of 6-DMAP or demecolcine improves developmental competence of embryos compared with long-term treatment. We recommend a combination of Thi/DTT with short-term treatment of both 6-DMAP and demecolcine for optimal activation procedure of porcine embryos. This study was supported by BK21, KOSEF (#M10625030005-07N250300510), HANWHA L&C Corp, and Gyeonggido Veterinary Service.
Recently, great focus has been on the rescue of endangered animals through somatic cell nuclear transfer (SCNT). Because it is difficult to obtain the oocytes of endangered species, interspecies SCNT (iSCNT) methods have been attempted. Numerous iSCNT embryos have shown unsuccessful development due to aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). In particular, aberrant EGA may cause the arrest of nucleoli formation and developmental block in embryos. According to this concept, we performed raccoon iSCNT using porcine oocytes and analyzed iSCNT embryo development pattern and formation of nucleoli. Enucleated porcine oocytes were fused with raccoon fibroblasts by electrofusion. Cleavage and blastocyst formation were evaluated under a stereomicroscope at 48 and 168 h post-activation (hpa), respectively. To confirm the formation of nucleoli, which can be detected by C23 antibody labeling in many mammalian species, C23 immunocytochemistry was performed at 48 and 72 hpa. A total of 158 iSCNT embryos were cultured; 68.5% of the raccoon iSCNT embryos were cleaved at 48 hpa (1-cell stage: 9.7%; 2-cell stage: 14.4%; 4-cell stage: 34.1%; 6-cell stage: 12.7%; 8-cell stage: 7.3%; fragmented: 21.8%). But, the embryos seen as 5- to 8-cell stage did not have the same number of nuclei as their blastomere number. When raccoon iSCNT embryos were stained by Hoechst 33342, 5- to 8-blastomere raccoon iSCNT embryos had only 4 nuclei. The raccoon iSCNT embryos did not develop past the 4-cell stage and failed to form blastocysts. In the control group, 65.2% of pig SCNT embryos were cleaved at 48 hpa (1-cell stage: 8.0%; 2-cell stage: 4.2%; 4-cell stage: 23.6%; 6-cell stage: 13.6%; 8-cell stage: 23.8%; fragmented: 26.8%), and 10.0% of pig SCNT embryos developed to blastocysts. In raccoon iSCNT embryos, raccoon nuclei failed to form nucleoli at 48 and 72 hpa. By contrast, pig SCNT embryos showed 18.8 and 87.9% nucleoli formation at 48 and 72 hpa. Our results demonstrate that 4-cell-stage embryos of raccoon-porcine hybrid embryos may be produced by SCNT methods. The pig oocytes partly supported the remodeling and reprogramming of the raccoon somatic cell nuclei, but they were unable to support nucleoli formation. Moreover, aberrant nucleoli formation caused the unsuccessful development of raccoon SCNT embryos to the blastocyst stage. This work was supported by a grant from the Next Generation BioGreen 21 program (no. PJ008121012011), Rural Development Administration, Republic of Korea.
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