Enhancing somatic nuclear reprogramming by Oct4 gain-of-function in cloned mouse embryos

Cloned mouse embryo development to blastocyst stage correlates positively with the expression level of Oct4 (Pou5f1) at the morula stage, as reported previously by our laboratory. However, whether this correlation is based on a cause-effect relationship has remained unclear. To address this question, we artificially increased the level of Oct4 prior and subsequent to somatic cell nuclear transfer, by microinjection of Oct4 mRNA into ooplasts and by transgenic Oct4 induction at the morula stage, respectively. We observed higher developmental rates of cloned embryos to blastocyst when higher levels of Oct4 were superimposed with the initial reprogramming events; whereas increasing Oct4 at later stages of preimplantation development did not have a significant effect on developmental rates. Our results show that supplemental Oct4 facilitates oocyte-mediated reprogramming only during the first cleavages, implying that the higher Oct4 level observed in developmentally competent cloned morulae is a readout of reprogramming events that successfully took place earlier.     

Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei

The majority of cloned animals derived by nuclear transfer from somatic cell nuclei develop to the blastocyst stage but die after implantation. Mouse embryos that lack an Oct4 gene, which plays an essential role in control of developmental pluripotency, develop to the blastocyst stage and also die after implantation, because they lack pluripotent embryonic cells. Based on this similarity, we posited that cloned embryos derived from differentiated cell nuclei fail to establish a population of truly pluripotent embryonic cells because of faulty reactivation of key embryonic genes such as Oct4. To explore this hypothesis, we used an in silico approach to identify a set of Oct4-related genes whose developmental expression pattern is similar to that of Oct4. When expression of Oct4 and 10 Oct4-related genes was analyzed in individual cumulus cell-derived cloned blastocysts, only 62% correctly expressed all tested genes. In contrast to this incomplete reactivation of Oct4-related genes in somatic clones, ES cell-derived cloned blastocysts and normal control embryos expressed these genes normally. Notably, the contrast between expression patterns of the Oct4-related genes correlated with efficiency of embryonic development of somatic and ES cell-derived cloned blastocysts to term. These observations suggest that failure to reactivate the full spectrum of these Oct4-related genes may contribute to embryonic lethality in somatic-cell clones.     

Embryonic development and gene expression of porcine SCNT embryos treated with sodium butyrate

Incomplete epigenetic modification is one of important reasons of inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). It may also underlie the observed reduced viability of cloned embryos. Sodium butyrate (NaBu) is a natural histone deacetylase inhibitor that is produced in the intestine. In the current study, we evaluated the effects of NaBu on preimplantation development, histone acetylation, and gene expression in porcine SCNT embryos. Our results showed that the blastocyst rate (24.88 ± 2.09) of cloned embryos treated with 1.0 mM NaBu for 12 hr after activation was significantly higher (P < 0.05) than that of untreated cloned embryos (13.15 ± 3.07). In addition, treated embryos displayed a global acetylated histone H3 at lysine 14 profile similar to that of in vitro fertilized (IVF) embryos during preimplantation development. Lower levels of Oct4 and Bcl-2, but higher levels of Hdac1, in SCNT embryos at the two-cell and blastocyst stages were observed, compared with those in the IVF counterparts. The four-cell embryos showed no differences in the levels of these genes among IVF embryos or SCNT embryos treated with or without NaBu; however, the levels of Dnmt3b were significantly different. NaBu-treated SCNT embryos showed similar levels of Oct4, Bcl-2, and Dnmt3b as in IVF blastocysts. These results indicated that NaBu treatment in SCNT embryos alters their histone acetylation pattern to provide beneficial effects on in vitro developmental competence and gene expression.     

Nuclear transfer in cattle using in vivo-derived vs. in vitro-produced donor embryos: Effect of developmental stage

To determine the best developmental stage of donor embryos for yielding the highest number of clones per embryo, we compared the efficiencies of nuclear transfer when using blastomeres from morulae or morulae at cavitation, or when using inner-cell-mass cells of blastocysts as nuclear donors. This comparison was done both on in vivo-derived and in vitro-produced donor embryos. In experiment 1, with in vivo-derived donor embryos, nuclei from morulae at cavitation supported the development of nuclear transfer embryos to the blastocyst stage (36%) at a rate similar to that of nuclei from morulae (27%), blastomeres from morulae at cavitation being superior (P < 0.05) to inner-cell-mass cells from blastocysts (21%). The number of blastocysts per donor embryo was significantly (P < 0.05) higher when using nuclei from morulae at cavitation (15.7 ± 4.1) rather than nuclei from morulae (9.8 ± 5.5) or blastocysts (6.3 ± 3.3). With in vitro-produced donor embryos (experiment 2), nuclei from morulae yielded slightly more blastocysts (32%) than nuclei from morulae at cavitation (29%), both stages being superior to nuclei from blastocysts (15% development to the blastocyst stage). Morulae at cavitation yielded a higher number of cloned blastocysts per donor embryo (11.5 ± 5.9) than did morulae (9.3 ± 3.2) and blastocysts (3.3 ± 1.4). Transfer of cloned embryos originating from in vivo-derived morulae, morulae at cavitation, and blastocysts resulted in four pregnancies (10%), three pregnancies (7%), and one (17%) pregnancy on day 45. The corresponding numbers of calves born were 3 (4%), 3 (7%), and 0, respectively. After transfer of blastocysts derived from in vitro nuclear donor morulae (n = 16) and morulae at cavitation (n = 7), two (20%) and two (50%) recipients, respectively, were pregnant on day 45. However, transfer of seven cloned embryos from in vitro donor blastocysts to three recipients did not result in a pregnancy. Using in vitro-produced donor embryos, calves were only obtained from morula-stage donors (13%). Our results indicate that the developmental stage of donor embryos affects the efficiency of nuclear transfer, with morulae at cavitation yielding a high number of cloned blastocysts. © 1996 Wiley-Liss, Inc.     

Somatic cell-like features of cloned mouse embryos prepared with cultured myoblast nuclei

Cloning by somatic cell nuclear transfer requires silencing of the donor cell gene expression program and the initiation of the embryonic gene expression program (nuclear reprogramming). Failure to silence the donor cell program could lead to altered embryonic phenotypes. Cloned mouse embryos produced using myoblast nuclei fail to thrive in standard embryo culture media but flourish in somatic cell culture media favored by the donor myoblasts themselves, forming blastocysts at a significant rate, with robust morphologies, high total cell number, and a normal allocation of cells to the inner cell mass in most embryos. Myoblast cloned embryos continue expressing the GLUT4 glucose transporter, which is typically expressed in muscle but not in preimplantation stage embryos. Myoblast clones also exhibit precocious enrichment of GLUT1 at the cell surface. Both myoblast and cumulus cell cloned embryos exhibit enhanced rates of glucose uptake. These observations indicate that silencing of the donor cell genome during cloning either is incomplete or occurs progressively over the course of preimplantation development. As a result, cloned embryos initially exhibit many somatic cell-like characteristics. Tetraploid constructs, which possess a transplanted somatic cell genome plus the oocyte-derived chromosomes, exhibit a more embryonic-like pattern of gene expression and culture preference. We conclude that preimplantation stage cloned embryos have profoundly altered characteristics that are donor cell type specific and that exposure of cloned embryos to standard embryo culture conditions may lead to disruptions in basic homeostasis and inhibition of a range of essential processes including further nuclear reprogramming, contributing to cloned embryo demise.     

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as well as embryo loss during development may occur even in cloned embryos reconstructed with nuclei from preimplantation-stage embryos, and these abnormalities are not specific to somatic cloning.     

Analysis of nuclear reprogramming in cloned miniature pig embryos by expression of Oct-4 and Oct-4 related genes

Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P<0.05) in cloned hatched blastocysts derived from fibroblasts and many of fibroblast-derived clones failed to reactivate at least one of the tested genes, while most of the germ cell clones and control embryos correctly expressed these genes. In conclusion, our results suggest that the reprogramming of fibroblast-derived cloned embryos is highly aberrant and this improper reprogramming could be one reason of the early lethality and post-implantation anomalies of somatic cell-derived clones.     

Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos

Full-term development has now been achieved in several mammalian species by transfer of somatic nuclei into enucleated oocytes [1, 2]. Although a high proportion of such reconstructed embryos can evolve until the blastocyst stage, only a few percent develop into live offspring, which often exhibit developmental abnormalities [3, 4]. Regulatory epigenetic markers such as DNA methylation are imposed on embryonic cells as normal development proceeds, creating differentiated cell states. Cloned embryos require the erasure of their somatic epigenetic markers so as to regain a totipotent state [5]. Here we report on differences in the dynamics of chromosome methylation between cloned and normal bovine embryos before implantation. We show that cloned embryos fail to reproduce distinguishable parental-chromosome methylation patterns after fusion and maintain their somatic pattern during subsequent stages, mainly by a highly reduced efficiency of the passive demethylation process. Surprisingly, chromosomes appear constantly undermethylated on euchromatin in morulae and blastocysts, while centromeric heterochromatin remains more methylated than that of normal embryos. We propose that the abnormal time-dependent methylation events spanning the preimplantation development of clones may significantly interfere with the epigenetic reprogramming, contributing to the high incidence of physiological anomalies occurring later during pregnancy or after clone birth.     

Pluripotency deficit in clones overcome by clone-clone aggregation: epigenetic complementation?

Abnormal gene expression patterns in somatic cell clones and their attrition in utero are commonly considered a consequence of errors in nuclear reprogramming. We observe that mouse clone blastocysts have less than half the normal cell number, and that higher cell number correlates with correct expression of Oct4, a gene essential for peri-implantation development and embryonic pluripotency. To increase the cell number, we aggregated genetically identical clones at the 4-cell stage. Clone-clone aggregates did not form more blastocysts, but the majority expressed Oct4 normally and had higher rates of fetal and postnatal development. Fertilized blastocysts with low cell numbers, induced by removal of two blastomeres at the 4-cell stage, did not exhibit abnormal Oct4 expression, indicating that improved gene expression and post-implantation development of clone-clone aggregates is not a consequence of increased cell number. Rather, we propose that complementation of non-cell-autonomous defects of genetically identical, but epigenetically different, embryos results in improved gene expression in clone-clone aggregates.     

Aberrant profile of gene expression in cloned mouse embryos derived from donor cumulus nuclei

Somatic cell nuclear transfer has successfully been used to clone several mammalian species including the mouse, albeit with extremely low efficiency. This study investigated gene expression in cloned mouse embryos derived from cumulus cell donor nuclei, in comparison with in vivo fertilized mouse embryos, at progressive developmental stages. Enucleation was carried out by the conventional puncture method rather than by the piezo-actuated technique, whereas nuclear transfer was achieved by direct cumulus nuclear injection. Embryonic development was monitored from chemically induced activation on day 0 until the blastocyst stage on day 4. Poor developmental competence of cloned embryos was observed, which was confirmed by lower cell counts in cloned blastocysts, compared with the in vivo fertilized controls. Subsequently, real-time polymerase chain reaction was used to analyze and compare embryonic gene expression at the 2-cell, 4-cell, and blastocyst stages, between the experimental and control groups. The results showed reduced expression of the candidate genes in cloned 2-cell stage embryos, as manifested by poor developmental competence, compared with expression in the in vivo fertilized controls. Cloned 4-cell embryos and blastocysts, which had overcome the developmental block at the 2-cell stage, also showed up-regulated and down-regulated expression of several genes, strongly suggesting incomplete nuclear reprogramming. We have therefore demonstrated that aberrant embryonic gene expression is associated with low developmental competence of cloned mouse embryos. To improve the efficiency of somatic cell nuclear transfer, strategies to rectify aberrant gene expression in cloned embryos should be investigated.This project was funded mainly by the National University of Singapore (grant number: R-174-000-065-112/303).