Reconstructed Somatic Cell Nuclear Transfer Embryos Research Articles

The Effects of an In Vitro Oocyte Maturation System and Chlorogenic Acid Supplementation during Embryo Culture on the Development of Porcine Cloned Embryos Derived from Native Vietnamese Ban Pigs

The aim of this study was to improve the production efficiency of Vietnamese native Ban pig embryos using somatic cell nuclear transfer (SCNT). Fibroblast cells from Ban pigs were injected into the enucleated cytoplasts of crossbred gilts, and the reconstructed embryos were subsequently cultured. In the first experiment, cytoplasts were isolated from oocytes matured in either a defined porcine oocyte medium (POM) or in TCM199 medium supplemented with porcine follicular fluid. Both media were supplemented with gonadotropic hormones, either for the first 22 h of in vitro maturation (IVM) or for the entire 44 h of IVM. In the second experiment, the reconstructed SCNT embryos were cultured with or without 50 μM chlorogenic acid (CGA). Furthermore, this study examined parthenogenetic embryos. The IVM medium and duration of hormone treatment did not affect embryo development. CGA supplementation to the culture medium significantly increased blastocyst formation rates in parthenogenetic embryos but not in SCNT embryos. However, CGA supplementation significantly reduced the apoptotic index in blastocysts regardless of embryo source. In conclusion, the IVM method did not affect SCNT embryo production, while CGA supplementation during embryo culture improved the quality of SCNT embryos in indigenous pig breeds.

Manipulating the Epigenome in Nuclear Transfer Cloning: Where, When and How.

The nucleus of a differentiated cell can be reprogrammed to a totipotent state by exposure to the cytoplasm of an enucleated oocyte, and the reconstructed nuclear transfer embryo can give rise to an entire organism. Somatic cell nuclear transfer (SCNT) has important implications in animal biotechnology and provides a unique model for studying epigenetic barriers to successful nuclear reprogramming and for testing novel concepts to overcome them. While initial strategies aimed at modulating the global DNA methylation level and states of various histone protein modifications, recent studies use evidence-based approaches to influence specific epigenetic mechanisms in a targeted manner. In this review, we describe—based on the growing number of reports published during recent decades—in detail where, when, and how manipulations of the epigenome of donor cells and reconstructed SCNT embryos can be performed to optimize the process of molecular reprogramming and the outcome of nuclear transfer cloning.

Activation treatment of recipient oocytes affects the subsequent development and ploidy of bovine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos.

We evaluated the potential effect of anisomycin, an antibiotic produced by Streptomyces griseolus, on the parthenogenetic activation of bovine oocytes and reconstructed somatic-cell nuclear transfer (SCNT) embryos. A higher cleavage and blastocyst rate were achieved with anisomycin (70.3% and 27.8%) and 6-dimethylaminopurine (DMAP) (73.3% and 30.2%), relative to oocytes parthenogenetically activated with cycloheximide (CHX) (54.1% and 20.2%). In reconstructed SCNT embryos, a greater proportion of embryos reached the blastocyst stage after anisomycin (32.2%) compared to DMAP (22.3%) and CHX (23.5%) treatment. Furthermore, the quality of embryos-assessed by the total number of cells and the inner cell mass-to-total-cell ratio-was higher with anisomycin (166.2 ± 6.9 and 26.9 ± 1.9) compared to DMAP (135.0 ± 8.7 and 39.4 ± 3.5) and CHX (149.1 ± 8.4 and 36.3 ± 2.5), while a lower percentage of chromosomal abnormalities was observed with anisomycin compared to DMAP and CHX treatments, both in parthenotes (though not significant) and in SCNT embryos (P < 0.05). Therefore, anisomycin can enhance the in vitro developmental potential in parthenotes and reconstructed SCNT embryos, specifically improving the quality of SCNT embryos and decreasing the abnormal ploidy of parthenotes and SCNT embryos compared to the traditional protocols of chemical activation with DMAP and CHX. These results may have important implications for the success of reproductive technologies, including SCNT and intracytoplasmic sperm injection, in different mammalian species.

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum.

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.

Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos

SummarySuccessful mammalian cloning employing somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II-arrested (MII) oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing pluripotency in somatic cell nuclei1-3. However, these poorly defined maternal factors presumably decline sharply after fertilization since cytoplasm of pronuclear stage zygotes is reportedly inactive4, 5. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase (M-phase) can also support derivation of embryonic stem cells (ESCs) following SCNT6-8, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in M-phase but not in interphase cytoplasm are “trapped” inside the nucleus during interphase and effectively removed during enucleation9.Here, we investigated the presence of reprogramming activity in the interphase cytoplasm of 2-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated M-phase and interphase zygotes and 2-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Then, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ESC, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ESCs capable of contributing to traditional germline and tetraploid chimeras. In addition, direct transfer of cloned embryos, reconstructed with ESC nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to utilize interphase cytoplasm in SCNT could impact efforts to generate autologous human ESCs for regenerative applications since donated or discarded embryos are more accessible than unfertilized, MII oocytes.

Promotion of human early embryonic development and blastocyst outgrowth in vitro using autocrine/paracrine growth factors.

Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6–8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner’s criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility treatment and future derivation of patient-specific embryonic stem cells.

Trichostatin A Improves Histone Acetylation in Bovine Somatic Cell Nuclear Transfer Early Embryos

Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming following somatic cell nuclear transfer (SCNT), and may underlie the observed reduced viability of cloned embryos. In the present study, we tested the effects of the histone deacetylase inhibitor (HDACi), trichostatin A (TSA), on development and histone acetylation of cloned bovine preimplantation embryos. Our results indicated that treating activated reconstructed SCNT embryos with 50 nM TSA for 13 h produced eight-cell embryos with levels of acetylation of histone H4 at lysine 5 (AcH4K5) similar to fertilized counterparts and significantly greater than in control NT embryos (p < 0.005). Further, TSA treatment resulted in SCNT embryos with preimplantation developmental potential similar to fertilized counterparts, as no difference was observed in cleavage and blastocyst rates or in blastocyst total cell number (p > 0.05). Measurement of eight selected developmentally important genes in single blastocysts showed a similar expression profile among the three treatment groups, with the exception of Nanog, Cdx2, and DNMT3b, whose expression levels were higher in TSA-treated NT than in in vitro fertilized (IVF) embryos. Data presented herein demonstrate that TSA can improve at least one epigenetic mark in early cloned bovine embryos. However, evaluation of development to full-term is necessary to ascertain whether this effect reflects a true increase in developmental potential.

Goat MII Ooplasts Support Preimplantation Development of Embryos Cloned from Other Species

The preimplantation development competences of somatic cell nuclear transfer (SCNT) embryos reconstructed with enucleated goat ( Capra hircus) Metaphase II (MII) oocytes matured in vivo and whole cells derived from adult fibroblasts of several mammalian species (goat, boer goat, bovine, tahr, panda) and human patient were evaluated. The experimental results indicated that these reconstructed SCNT embryos could complete preimplantation development to form blastocysts. The fusion rate and blastocyst rate of intra-species SCNT embryos ( Capra hircus as control) were 78.67 (557/708); 56.29% (264/469), that of sub-species or inter-species SCNT embryos were: boer goat 78.18% (541/692); 33.90% (40/118), bovine 70.53% (146/207); 22.52% (25/111), tahr 53.51% (61/114); 5.26% (3/570), panda 79.82% (1159/1452); 8.35% (75/898) and human 68.76% (317/461); 5.41% (16/296), respectively. It is concluded that (1) there are no relationships between fusion rate and relativeness of the recipient cytoplasm to nucleus donor cells, (2) cytoplast of the goat MII oocyte can support the preimplantation development of SCNT embryos reconstructed with nucleus from other species, (3) the blastocyst rate of close relative inter-species SCNT embryos is higher than that of distant relative inter-species SCNT embryos.

Birth of viable female dogs produced by somatic cell nuclear transfer

Since the only viable cloned offspring born in dogs was a male, the purpose of the present study was to produce female puppies by somatic cell nuclear transfer (SCNT). Adult ear fibroblasts from a 2-month-old female Afghan hound were isolated and used as donor cells. In vivo-matured canine oocytes surgically collected (approximately 72 h after ovulation) from the oviducts of 23 donors were used for SCNT. After removal of the cumulus cells, oocytes were enucleated, microinjected, fused with a donor cell, and activated. A total of 167 reconstructed SCNT embryos were surgically transferred (Day 0) into the oviducts of 12 recipient bitches (average 13.9 embryos/recipient, range 6–22) with spontaneous, synchronous estrous cycles. Three pregnancies were detected by ultrasonography on Day 23, maintained to term, and three healthy female puppies (520, 460, and 520 g), were delivered by Caesarean section on Day 60. These puppies were phenotypically and genotypically identical to the cell donor. In conclusion, we have provided the first demonstration that female dogs can be produced by nuclear transfer of ear fibroblasts into enucleated canine oocytes.

53 CELL CYCLE SYNCHRONIZATION OF DONOR CELLS AT G1 PHASE AND DEVELOPMENTAL ABILITY OF NUCLEAR TRANSFER EMBRYOS IN MINIATURE PIGS

The cell cycle of donor cells is one of the essential factors for the success of somatic cell nuclear transfer (SCNT), and G0/G1-phase cells have been widely used as donor cells. However, cells synchronized at the G0/G1-phase also have a population of cells with cell cycles other than G0/G1-phase, and we cannot precisely know the cell cycle of donor cells being used for SCNT. In this experiment, we reconstructed SCNT embryos from donor cells that were synchronized at the G1-phase or at the G0/G1-phase and compared their developmental ability in two different culture media. Immature oocytes were isolated from ovaries collected from domestic gilts at a local slaughterhouse and were co-cultured with follicle shells for in vitro maturation (Hoshino et al. 2003 Theriogenology 59, 260). Donor cells were collected from fibroblast cells of miniature Potbelly pigs. Cells synchronized at the G1-phase were prepared shortly after dividing M-phase cells that had been synchronized using 2-methoxyestradiol, as described by Urakawa et al. (2004 Theriogenology 62, 714–728). The G0/G1 cells were also prepared from a fully confluent culture of cells. Donor cells were fused with enucleated oocytes and simultaneously activated by two electric pulses. Reconstructed embryos were cultured in two different media [Whitten and Biggers medium supplemented with 0.5 mg/mL hyaluronic acid sodium salt (WM) and porcine zygote medium-3 (PZM-3, Yoshioka et al. 2002 Biol. Reprod. 66, 112–119)] under 5% CO2 in air. Cleavage rate and development rate to the blastocyst stage were assessed after 48 and 168 hr of culture, respectively. The results are summarized in Table 1. Developmental rate to the blastocyst stage of SCNT embryos reconstructed from G1 cells and cultured in PZM-3 (40%) was significantly higher than that of embryos cultured in WM (25%). The SCNT embryos of the G1 cells showed significantly lower cleavage rate (51%) than that of the G0/G1 cells (69%). However, the developmental rates to the blastocyst stage per cleaved embryo in WM were significantly higher in G1 cells (50%) compared with G0/G1 cells (32%). In addition, the total cell number of the SCNT blastocysts was comparable between the cultures in WM (58 ± 4) and PZM-3 (46 ± 5), although the ratio of inner cell mass cells to the total cell number was significantly higher in PZM-3 (32%) compared with WM (14%). These results suggest that PZM-3 may fit with the culture of SCNT embryos, and that the G1 synchronized cells could be stably reprogrammed for early embryonic development in SCNT embryos and be useful as donor cells for analyzing the processes of nuclear reprogramming. Table 1. Development of nuclear transfer embryos reconstructed from G1 or G0/G1 cells in different culture media