Somatic Nuclear Transfer Embryos Research Articles

Involvement of METTL3-mediated m6A methylation in the early development of porcine cloned embryos

METTL3-mediated N6-methyladenosine (m6A) modification is critical for gametogenesis and early embryonic development. However, the function of METTL3-mediated m6A modification in the early development of somatic nuclear transfer embryos (SCNT) remains unclear. Here, we found that METTL3 mRNA and protein levels exhibit dynamic changes during the early development of porcine SCNT embryos. The levels of METTL3 mRNA and protein in SCNT embryos at specific developmental stages differ from those in parthenogenetic activation (PA) counterparts. SiRNA injection effectively reduced the levels of METTL3 mRNA and protein in 4-cell embryos and blastocysts. METTL3 knockdown significantly reduced the cleavage and blastocyst rates of SCNT embryos. METTL3 knockdown significantly reduced the number of total cells and trophectoderm (TE) cells in the resulting blastocysts and perturbed cell lineage allocation. In addition, METTL3 knockdown reduced the levels of m6A modification in 4-cell embryos and blastocysts. Importantly, METTL3 knockdown decreased the expression levels of CDX2, GATA3, NANOG and YAP, and increased the expression levels of SOX2 and OCT4. Taken together, these results demonstrate that METTL3-mediated m6A modification regulates early development and lineage differentiation of porcine SCNT embryos.

109. AGGREGATING CLONED WITH IN VITRO FERTILISED EMBRYOS RESULTS IN CHIMAERAS AND IMPROVED FETAL SUVIVAL IN CATTLE

Cloning cattle by somatic cell nuclear transfer (NT) results in low survival and high frequencies of abnormal placentation and fetal development. We postulate that such anomalies may be overcome by complementing NT embryos with in vitro fertilised (IVF) embryos to form chimaeras. The gender and germline composition of chimaeras can be experimentally manipulated. Using embryological methods, we aim to produce chimaeric fetuses that are functionally male and produce sperm derived from the somatic NT embryo. Provided sufficient contribution from the IVF embryo, such chimeras should develop more normally than clones. At the 12- to 16-cell stage, individual male NT embryos were aggregated with female IVF embryos derived from X-sorted sperm. Following aggregation, there were no significant difference in blastocyst development between NT/IVF aggregates and disaggregated and re-aggregated IVF and NT controls (86/183 = 47% v. 77/233 = 33% v. 47/109 = 43%, respectively). Suitable quality embryos were transferred individually into synchronised recipient animals. Pregnancy establishment at Day (D) 35 was not significantly different between aggregate, IVF and NT groups (18/57 = 32% v. 11/45 = 24% v. 6/31 = 19%, respectively). Whilst there was no difference in survival between aggregates and IVF controls to ~D100, aggregates survived significantly better than NT controls (16% v. 18% v. 0%; respectively; P < 0.05). In the aggregate group, 7/8 fetuses recovered were phenotypically male. Using RT-PCR, expression of the female-specific mRNA for Xist was detected in 4/5 liver samples, indicating chimaerism. Despite improved survival to ~D100 compared to NT, 3/7 fetuses in the aggregate group still displayed evidence of abnormalities, such as fetal overgrowth. Further studies will explore alternative aggregation strategies and germline transmission of the NT-derived genome in chimaeras.

Aggregating Embryonic but Not Somatic Nuclear Transfer Embryos Increases Cloning Efficiency in Cattle1

Our objectives were to compare the cellular and molecular effects of aggregating bovine embryonic vs. somatic cell nuclear transfer (ECNT vs. SCNT) embryos and to determine whether aggregation can improve cattle cloning efficiency. We reconstructed cloned embryos from: 1) morula-derived blastomeres, 2) six adult male ear skin fibroblast lines, 3) one fetal female lung fibroblast line (BFF), and 4) two transgenic clonal strains derived from BFF. Embryos were cultured either singularly (1X) or as aggregates of three (3X). In vitro-fertilized (IVF) 1X and 3X embryos served as controls. After aggregation, the in vitro development of ECNT but not that of SCNT or IVF embryos was strongly compromised. The inner cell mass (ICM), total cell (TC) numbers, and ICM:TC ratios significantly increased for all the aggregates. The relative concentration of the key embryonic transcript POU5F1 (or OCT4) did not correlate with these increases, remaining unchanged in the ECNT and IVF aggregates and decreasing significantly in the SCNT aggregates. Overall, the IVF and 3X ECNT but not the 1X ECNT embryos had significantly higher relative POU5F1 levels than the SCNT embryos. High POU5F1 levels correlated with high in vivo survival, while no such correlation was noted for the ICM:TC ratios. Development to weaning was more than doubled in the ECNT aggregates (10/51 or 20% vs. 7/85 or 8% for 3X vs. 1X, respectively; P < 0.05). In contrast, the SCNT and IVF controls showed no improvement in survival. These data reveal striking biological differences between embryonic and somatic clones in response to aggregation.

Developmental rate and ploidy of embryos produced by nuclear transfer with different activation treatments in cattle

Bovine oocyte activation is one of the essential elements that determine the success of nuclear transfer and the subsequent development of cloned embryos. Three methods for oocyte activation, including 5 μM ionomycin (5 min, Group 1) alone, ionomycin + 1.9 mM 6-dimethylaminopurine (DMAP, 3 h, Group 2), and ionomycin + 10 μg/ml cycloheximide (CHX, 3 h, Group 3) were compared for the development of embryos produced by somatic nuclear transfer (SCNT) to parthenotes and IVF counterparts. At 19-h post-activation/insemination (hpa/hpi), 27.5% of oocytes in Group 2 cleaved and this rate was greater ( P < 0.05) than other groups (Group 1, 2.1%; Group 3, 3.0%). None of the oocytes in the IVF control group cleaved at 19–22 hpi. At 24 hpa, the rates of cleavage of oocytes in Group 2 (52.1%) were greater ( P < 0.05) than those in Groups 1 and 3 (7 and 38.3%, respectively). Only six oocytes (3.3%) in the IVF control group cleaved at 24 hpi. The overall cleavage rates of oocytes in Group 2 (85.5%) at 48 hpa were greater ( P < 0.05) than other treatments, but it did not show any difference when compared with the IVF control group (75.0%). The development rate to two-cell stage embryos of Group 2 was consistently greater at all observation points followed by Groups 3 and 1. Similar results were obtained in SCNT embryos, but the rates of cleavage at 48 hpi and blastocyst development in Group 2 (68.4 and 16.3%, respectively) did not differ from Group 3 (63.0 and 13.1%, respectively). The chromosomal composition in the parthenotes and SCNT embryos differed ( P < 0.05) among treatments. In Groups 1 and 3, greater percentages of haploid parthenotes (86 and 71%, respectively) were observed. In contrast, 84% of parthenotes in Group 2 had abnormal ploidy (44% polyploid and 40% mixoploid). In the case of SCNT embryos, Groups 1 and 3 had greater percentages of diploid chromosomal sets (77 and 70%, respectively), whereas 54% in Group 2 were polyploid or mixoploid. These results indicate that DMAP treatment after ionomycin greatly increases the developmental rates of parthenotes, but did not differ in blastocyst development compare with CHX treatment. However, DMAP treatment increased the time-dependent cleavage rate to two-cell stage embryos. Further, it greatly enhanced the incidence of chromosomal abnormalities in parthenotes and SCNT embryos. Hence, it is concluded that CHX combined with ionomycin is more desirable than DMAP for oocyte activation during nuclear transfer in cattle.

Duration of In Vitro Maturation of Recipient Oocytes Affects Blastocyst Development of Cloned Porcine Embryos

This study investigated the effects of different incubation periods for oocyte maturation and contact inhibition of donor cells as well as different osmolarities for storage of recipient oocytes on fusion rates, cleavage rates, and blastocyst yields of porcine somatic nuclear transfer (SCNT) derived embryos. In addition, the in vivo developmental potential of cloned embryos derived from the most promising SCNT protocol was tested by transfer to recipient gilts. Storage of in vitro-matured oocytes for 7.5 h in calcium-free TL-HEPES medium at 295 or 320 mOsmol prior to activation yielded significantly (p < 0.05) higher parthenogenetic blastocyst rates compared to storage in TL-HEPES with an osmolarity of 270 mOsmol (24.4 +/- 3.0% and 26.2 +/- 4.3% vs. 18.3 +/- 6.4%, respectively, mean +/- SD) and improved the visibility of the polar body. Electrical fusion of fibroblasts to enucleated oocytes matured for 38, 40, or 42 h resulted in similar fusion and cleavage rates (74.8-84.4%). However, nuclear transfer with oocytes matured for 40 h in vitro yielded significantly higher (p < 0.05) development to the blastocyst stage after 7 days of culture (14.7 +/- 1.7%) than with oocytes matured for 38 h (9.5 +/- 2.1%) or 42 h (5.1 +/- 2.1%). Contact inhibition for 24, 48, or 72 h significantly (p < 0.05) increased the proportion of cells at G0/G1 compared with cycling fibroblasts. However, duration of contact inhibition of the donor cells for either 24, 48, or 72 h had no effect on blastocyst rates of SCNT embryos. Four gilts received an average of 150 SCNT embryos (range 138-161) reconstructed with oocytes matured for 40 h; two of these became pregnant; one of them went to term and farrowed four piglets on day 115 of pregnancy. Microsatellite analysis confirmed that the clones were genetically identical with the donor cells. These results show that changes of the in vitro maturation protocol may affect in vitro development of reconstructed porcine embryos, while duration of the contact inhibition period plays a minor role for the success of porcine SCNT. The effects on in vivo development are yet to be determined.

116 ACTIVE METHYLATION AND ACETYLATION OF HISTONE H3-K9 IN MOUSE EMBRYO WITH DIFFERENT PROPORTIONS OF MATERNAL AND PATERNAL GENOME

Epigenetic modification of parental genomes plays a prominent role in regulating genome expression in the early development of embryos. In general, histone H3 of the paternal genome is demethylated at lysine 9 (H3-K9) during the first and second mitotic divisions in fertilized embryos, while the maternal genome is methylated. We investigated the effects of maternal genomes (Mgen) and paternal genomes (Pgen) on H3-K9 methylation and acetylation during the early development of murine embryos. Histone H3-K9 methylation and acetylation were detected by anti-trimethyl histone H3-K9 and anti-triacetyl histone H3-K9 antibodies. The following embryos were used in this study: (1) intracytoplasmic sperm injection (ICSI) embryos (50% Mgen, 50% Pgen); (2) parthenogenetic diploid embryos (100% Mgen, 0% Pgen); (3) somatic nuclear transfer embryos (50% Mgen, 50% Pgen from previous generation); (4) androgenetic diploid embryos (0% Mgen, 100% Pgen); and (5) haploidized somatic nucleus and sperm embryo (about 25% Mgen, about 75% Pgen). Each experiment was repeated five times to obtain more than 120 embryos per group. Our results show that: (1) in the ICSI embryo, histone H3 methylation occurs in Mgen but not in Pgen at the first and second mitotic divisions; (2) in the parthenogenetic embryo, histone H3 methylation occurs in both nuclei at the first and second mitotic divisions; (3) in the somatic nuclear transfer embryo, histone H3 is methylated in all of the nuclei at the first and second mitotic divisions; (4) in the androgenetic embryo, methylated H3-K9 is detected weakly in the heterochromatin enclosed around the nucleolus of the pronuclei of the one-cell embryo, and methylated in the entire nuclei of the two-cell embryo; and (5) in the haploidized somatic and sperm embryo, the pattern of histone H3-L9 methylation resembles that of the ICSI embryo. While histone H3-K9 acetylation occurs in both paternal and maternal genomes during interphase, even when the nuclear membrane is completely degraded and the chromosome is condensed, it disappears rapidly when the chromosome enters the real metaphase, and reappears at the early stage of pronuclear formation in all types of embryo. These results suggest that the absence of maternal genomes results in histone H3-K9 methylation in the paternal genomes during the first and second mitotic divisions of embryos in mice. In addition, histone H3-K9 acetylation is independent of the presence or absence of maternal or paternal genomes during pre-implantation development in mice. This study was supported by grants-in-aid for Creative Scientific Research (13GS0008) and a project for the realization of regenerative medicine (the research field for the technical development of stem cell manipulation) to T.W. from MEXT, Japan.

Effects of different nuclear recipients on developmental potential of mouse somatic nuclear transfer embryos

In order to investigate the effects of different kinds of nuclear recipients from Kunming (KM) mouse on developmental potential of somatic nuclear transfer embryos, the enucleated M II oocytes, enucleated zygotes and 2-cell blastomere were used to produce cloned mouse embryos. Using fibroblast deriving from C57/BL6 ear tissue as nuclear donor, we produced cloned embryos by transferring the fibroblast nuclei into enucleated KM mouse oocytes (single nuclear transfer, SNT), transferring pronuclei from the SNT embryos into enucleated KM zygotes (nuclear into zygote, NZ), and 2-cell blastomere nuclei from SNT embryos into enucleated KM mouse oocytes (nuclear into oocytes, NO); tetraploid embryos (tetraploid embryos, TE) were obtained by fusing two blastomeres, one is from the SNT cloned embryos, and the other from normal 2-cell KM mouse embryos. In group SNT, the cloned embryos could not develop beyond 8-cell stage and the rate of 8-cell stage is only 0.3%; in group NO, the reconstructed embryos could develop to morula stage, the rate of 8-cell stage was significantly greater than that of SNT group (P < 0.05); in group NZ, the development rate was further improved, and the reconstructed embryos could develop into blastocyst stage, the rate of blastocyst was 1.9%; in group TE, as high as 62.3% of the reconstructed embryos could develop into blastocyst. Results suggested that different nuclear recipients could significantly affect the developmental potential of cloned mouse embryos; KM M II oocyte cytoplasm was not so effective as zygotes to reprogram the mouse somatic cell nuclei; serial nuclear transfer could improve the developmental potential of cloned mouse embryos.

Effects of different nuclear recipients on developmental poten-tial of mouse somatic nuclear transfer embryos

Effects of different nuclear recipients on developmental poten-tial of mouse somatic nuclear transfer embryos