Somatic Cell Nuclear Transfer Fetuses Research Articles

Mouse somatic cell nuclear transfer: What has changed and remained unchanged in 25 years.

Somatic cell nuclear transfer (SCNT) is the only reproductive technology used to produce individuals from somatic cells by transferring them to enucleated oocytes. Although more than 25 years have passed since the first mammalian SCNT was reported in sheep, problems such as low birth rates and morphological abnormalities have persisted and limited its practical applications. The mouse is the ideal laboratory animal to unveil these questions due to its established reproductive technologies and extensive knowledge base of its genome and various strains. We investigated the causes of incomplete reprogramming after nuclear transfer of donor somatic cells and found that the loss of imprint in some placenta-specific imprinted genes could induce non-random SCNT abnormalities. By ameliorating aberrantly expressed imprinted genes, we succeeded in increasing the low birth rate and improving morphological abnormalities observed in SCNT fetuses. Furthermore, we sought appropriate mouse strains and cell types as nuclear donors to increase their developmental efficiencies and expand their applications in various fields. Peripheral blood cells are useful as ethical and economical cell species because they can be collected easily, even though SCNT embryos derived from hematopoietic cells show poor developmental abilities after reconstruction. Additionally, it is possible to obtain mice that are reactive to specific antigens of interest by using lymphocytes. Although there are still many limitations to the practical use of SCNT, its utilization is steadily expanding.

Mitochondrial DNA Depletion in Granulosa Cell Derived Nuclear Transfer Tissues.

Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from production of cloned farm animals to derivation of patient-matched stem cells or production of humanized animal organs for xenotransplantation. However, effects of aberrant epigenetic reprogramming on gene expression compromise cell and organ phenotype, resulting in low success rate of SCNT. Standard SCNT procedures include enucleation of recipient oocytes before the nuclear donor cell is introduced. Enucleation removes not only the spindle apparatus and chromosomes of the oocyte but also the perinuclear, mitochondria rich, ooplasm. Here, we use a Bos taurus SCNT model with in vitro fertilized (IVF) and in vivo conceived controls to demonstrate a ∼50% reduction in mitochondrial DNA (mtDNA) in the liver and skeletal muscle, but not the brain, of SCNT fetuses at day 80 of gestation. In the muscle, we also observed significantly reduced transcript abundances of mtDNA-encoded subunits of the respiratory chain. Importantly, mtDNA content and mtDNA transcript abundances correlate with hepatomegaly and muscle hypertrophy of SCNT fetuses. Expression of selected nuclear-encoded genes pivotal for mtDNA replication was similar to controls, arguing against an indirect epigenetic nuclear reprogramming effect on mtDNA amount. We conclude that mtDNA depletion is a major signature of perturbations after SCNT. We further propose that mitochondrial perturbation in interaction with incomplete nuclear reprogramming drives abnormal epigenetic features and correlated phenotypes, a concept supported by previously reported effects of mtDNA depletion on the epigenome and the pleiotropic phenotypic effects of mtDNA depletion in humans. This provides a novel perspective on the reprogramming process and opens new avenues to improve SCNT protocols for healthy embryo and tissue development.

Identification of amniotic fluid metabolomic and placental transcriptomic changes associated with abnormal development of cloned pig fetuses.

Piglets cloned by somatic cell nuclear transfer (SCNT) show a high incidence of malformations and a high death rate during the perinatal period. To investigate the underlying mechanisms for abnormal development of cloned pig fetuses, we compared body weight, amniotic fluid (AF) metabolome, and placental transcriptome between SCNT- and artificial insemination (AI)-derived pig fetuses. Results showed that the body weight of SCNT pig fetuses was significantly lower than that of AI pig fetuses. The identified differential metabolites between the two groups of AF were mainly involved in bile acids and steroid hormones. The levels of all detected bile acids in SCNT AF were significantly higher than those in AI AF. The increase in the AF bile acid levels in SCNT fetuses was linked with the downregulation of placental bile acid transporter expression and the abnormal development of placental folds (PFs), both of which negatively affected the transfer of bile acids from AF across the placenta into the mother's circulation. Alteration in the AF steroid hormone levels in cloned fetuses was associated with decreased expression of enzymes responsible for steroid hormone biosynthesis in the placenta. In conclusion, cloned pig fetuses undergo abnormal intrauterine development associated with alteration of bile acid and steroid hormone levels in AF, which may be due to the poor development of PFs and the erroneous expression of bile acid transporters and enzymes responsible for steroid hormone biosynthesis in the placentas.

Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering.

The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP). 2A-tdTomato sequence was inserted to replace the stop codon of the porcine Oct4 gene by homogenous recombination (HR). Thus, the fluorescence can accurately show the activation of endogenous Oct4. Porcine fetal fibroblast (PFF) lines with knock-in (KI) of the tdTomato gene in the downstream of endogenous Oct4 promoter were achieved using the CRISPR/CAS9 system. Transgenic PFFs were used as donor cells for somatic cell nuclear transfer (SCNT). Strong RFP expression was detected in the blastocysts and genital ridges of SCNT fetuses but not in other tissues. Two viable transgenic piglets were also produced by SCNT. Reprogramming of fibroblasts from the fetuses and piglets by another round of SCNT resulted in tdTomato reactivation in reconstructed blastocysts. Result indicated that a KI porcine reporter system to monitor the pluripotent status of cells was successfully developed.

Establishment of a rabbit Oct4 promoter-based EGFP reporter system.

Rabbits are commonly used as laboratory animal models to investigate human diseases and phylogenetic development. However, pluripotent stem cells that contribute to germline transmission have yet to be established in rabbits. The transcription factor Oct4, also known as Pou5f1, is considered essential for the maintenance of the pluripotency of stem cells. Hence, pluripotent cells can be identified by monitoring Oct4 expression using a well-established Oct4 promoter-based reporter system. This study developed a rabbit Oct4 promoter-based enhanced green fluorescent protein (EGFP) reporter system by transfecting pROP2-EGFP into rabbit fetal fibroblasts (RFFs). The transgenic RFFs were used as donor cells for somatic cell nuclear transfer (SCNT). The EGFP expression was detected in the blastocysts and genital ridges of SCNT fetuses. Fibroblasts and neural stem cells (NSCs) were derived from the SCNT fetuses. EGFP was also reactivated in blastocysts after the second SCNT, and induced pluripotent stem cells (iPSCs) were obtained after reprogramming using Yamanaka's factors. The results above indicated that a rabbit reporter system used to monitor the differentiating status of cells was successfully developed.

13 ULTRASOUND EVALUATION OF FETAL AND PLACENTAL DEVELOPMENT IN SOMATIC CELL NUCLEAR TRANSFER AND ARTIFICIAL INSEMINATION BOVINE PREGNANCIES

Since the first success in sheep, the production of viable cloned offspring by somatic cell nuclear transfer (SCNT) is still doomed by a high incidence of pregnancy failure. In cattle, most gestation losses initially take place during the peri-implantation period. Then, abnormal placentation associated with fetal overgrowth (abnormal offspring syndrome) occurs in late pregnancy. As a consequence, IETS recommendations include regular ultrasound evaluation of SCNT pregnancies, with qualitative ultrasound evaluation of the placentomes, fetal movements and fluids being proposed. The objective of this study was to evaluate the use of new, quantitative parameters for the assessment of feto-placental development in AI and SCNT bovine pregnancies. Twenty-two heifers of 4 different breeds were used as SCNT recipients and 11 Holstein heifers were used as AI controls (C). All SCNT fetuses were produced as previously published in the laboratory, using the same fibroblast donor. Animals were scanned every 2 weeks from Day 150 using a Voluson-i (GE Medical Systems) with a transabdominal multifrequency probe (2.2 to 6.5 MHz), allowing automatic 3D volume and Doppler acquisition. Each time, 7 placentomes were scored from 0 to 3 according to echogenicity, general appearance and degree of oedema and their 2D surface was measured. Fetal intercostal width (ICW) was measured in a coronal view just behind the heart. Doppler velocimetry indices, pulsatility index (PI) and resistance index (RI) of one of the two fetal umbilical arteries were obtained and the diameters of the umbilical vessels were measured (UD). The 3D power Doppler was performed to obtain vascular index (VI), flow index (FI) and vascular flow index (VFI). Data were analysed by ANOVA with GraphPad Prism®. So far, 27 heifers have either delivered (n = 10 SCNT and 7 C) or pregnancies have been terminated because of abnormal offspring syndrome (n = 10 SCNT). One thousand five hundred and 197 placentomes have been analysed for 2D and 3D analyses, respectively. The mean placentome score is significantly higher (less normal) in SCNT compared with C (P < 0.0001) but does not vary according to gestational age. The placentome surface is also significantly larger in SCNT vs C (P < 0.0001) but also in SCNT that did not go to term vs those that were alive at birth (P < 0.002). Fetal ICW and UD are consistently larger in SCNT vs C (P < 0.0001) at all stages of pregnancy and there is a significant correlation (r2 = 0.81) between ICW at 15 days before term and birth weight. The PI and RI were not different between SCNT and C. Intra-operator reproducibility of 3D analyses was very high (intra-class correlation coefficient: 80 to 95% for a 95% confidence interval). There was no significant difference for VI, FI, or VFI between SCNT and C. Ongoing work taking into account pregnancy outcomes indicates that placentome scores are useful indicators of pregnancy outcome in SCNT pregnancies. In contrast, the abnormal vascularization observed by histology in SCNT placentomes does not appear to be accompanied by abnormal placental blood flow when analysed using quantitative 3D Doppler. This project received financial support from ANR (ref. PCS-09-GENM-022).

22 VASCULAR ENDOTHELIAL GROWTH FACTOR mRNA EXPRESSION IN FETAL MEMBRANES DURING EARLY PREGNANCY AS A BIOMARKER FOR DEVELOPMENT OF HYDROPS PLACENTA LATER IN PREGNANCY IN BOVINE EMBRYOS CREATED THROUGH SOMATIC CELL NUCLEAR TRANSFER (SCNT)

Pregnancies obtained via somatic cell nuclear transfer (SCNT) show an increased incidence of pregnancy complications, including poor placental vascularization, hydrops placenta, and abnormal placental morphology. In a preliminary study of hydrops placentas from SCNT clones v. control cow placentas close to term, we observed a thickening of the endometrial epithelium, which appeared pseudostratified; in addition, the subepithelial endometrial vascular bed consisted of large, rare arterioles with thick basement membranes compared with the normal capillary bed observed in control animals. The fetal membranes of hydrops placenta from bovine SCNT clones also exhibited macroscopic morphological differences, which included edema, increased membrane thickness, and a gelatinous appearance. All of these morphological differences were similar to those we have reported for sheep SCNT clones (Palmieri et al. 2007 Placenta 28, 577–584). In the search for biomarkers that may indicate abnormal placental development early in pregnancy, placental tissues were collected on day 40 from pregnancies established after transfer of embryos created through SCNT (n = 17) or IVF (n = 6). Nuclear donor cells used to obtain SCNT embryos were those we have previously found to be highly susceptible to hydrops (from 5 to 19%), whereas the incidence of hydrops in IVF and controls in our laboratories did not exceed 0.5%. A sample of fetal membranes was fixed in 10% buffered formalin for histological analysis; another sample was snap-frozen in liquid nitrogen for analysis of mRNA levels for vascular endothelial growth factor (VEGF) and its receptors (KDR and FLT) by quantitative, real-time RT-PCR. Histological analysis of cell proliferation rate was performed using immunohistochemical localization of proliferating cell nuclear antigen and image analysis software (Image-Pro Plus). There were no differences (P = 0.39) in the rate of cell proliferation of the trophoblast in SCNT v. IVF (29.4 ± 4.9% v. 22.1 ± 3%). However, VEGF mRNA expression was less (P < 0.04) in the fetal membranes of SCNT than IVF on day 40 of pregnancy (0.30 ± 0.04 v. 0.53 ± 0.12, normalized to α-actin mRNA levels). The mRNA expression for KDR and FLT were similar in SCNT and IVF. These data suggest that the lower expression of VEGF, a major placental angiogenic factor, in fetal membranes of early pregnant SCNT fetuses may be an early biomarker for the development of hydrops placenta later in pregnancy. NDAES Proj. ND01727.

Quantitative Monitoring of Pluripotency Gene Activation after Somatic Cloning in Cattle1

The development of somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA, we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses of whole-mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos, even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, whereas later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent but was activated by a second round of SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses, providing an attractive model for systematic investigation of epigenetic reprogramming in large mammals.

Why is the fetal allograft not rejected?1

In viviparous species, the conceptus must be protected from a potentially hostile maternal immune system. The major histocompatibility complex (MHC) is a genetic region that encodes MHC class I and class II proteins, which present peptide antigens to T lymphocytes and induce graft rejection. The MHC, class II proteins are only expressed on professional, antigen-presenting cells. However, classical, MHC class I proteins are expressed on all nucleated somatic cells. Protection of the conceptus from immune-mediated rejection involves downregulation of classical MHC class I antigen expression on trophoblast cells, which form the external epithelial layer of the placenta, and maintenance of an immunologically favorable immunosuppressive environment in the uterus. Normally, bovine trophoblast cells do not express MHC class I antigens before d 120 of pregnancy. However, during the last third of gestation, trophoblast cells in the inter-placentomal and arcade regions of the placenta express classical, MHC class I proteins, which could potentially induce fetal rejection, as well as nonclassical, MHC class I proteins. A human, nonclassical, MHC class I antigen, human leukocyte antigen G, is an important immunoregulatory factor required for the maintenance of pregnancy. In cattle, MHC class I expression during the last third of pregnancy has no adverse effects and probably contributes to placental separation at parturition. However, somatic cell nuclear transfer (SCNT) conceptuses, the majority of which are aborted between d 30 and 90 of pregnancy, had trophoblast cell expression of MHC class I antigens before d 34 of pregnancy. In conjunction with increased trophoblast MHC class I expression, SCNT pregnancies exhibited a marked increase in the number of stromal lymphocytes in the uteri of surrogate dams. A retrospective study found that SCNT pregnancies established using MHC class I-homozygous cell lines, in which the immunological barrier is greatly reduced, had significantly improved fetal survival from d 28 to term (51% survival for MHC-homozygous and 5% for MHC-heterozygous SCNT fetuses). Consequently, it appears that the high rate of fetal mortality in SCNT pregnancies is due, at least in part, to inappropriate expression of trophoblast, MHC class I antigens resulting in immune-mediated placental rejection. This suggests that appropriate regulation of MHC class I genes is critical for immunological acceptance of an allogeneic conceptus.

54 COMPOSITION OF ALLANTOIC FLUID IN CATTLE PREGNANT WITH AI-, IVP-, OR NUCLEAR TRANSFER-GENERATED EMBRYOS

Abnormal placentation, pregnancy failure, and hydroallantois are associated with somatic cell nuclear transfer (SCNT) in cattle. Identification of diagnostic markers for abnormal placentation in early gestation would permit therapeutic intervention. Ultrasonography and transvaginal sampling of amniotic and/or allantoic (fetal) fluid enables regular monitoring of fetal health. We report on the composition of serial samples of fetal fluid from individual cows between Days 70–130 of gestation and the potential of steroid and electrolyte composition as an early diagnostic marker for the subsequent occurrence of hydroallantois in SCNT pregnancies in cattle. On Day 70, pregnancy rates were 50% and 60% for cows or heifers implanted with single in vitro-fertilized (IVP, 20/40) or SCNT (25/42) embryos, respectively, and 67% for pregnancies generated by artificial insemination (AI, 12/18). Resulting fetuses were either clones (SCNT) or offspring (IVP/AI) of a donor Holstein bull. Fetal fluids, sampled using ultrasound-guided transvaginal puncture, were collected on Days 70, 100, and 130 of gestation (n = 12 and 139 for amniotic and allantoic samples, respectively). Placental and fetal morphological data were collected following slaughter between Days 135–163 of gestation (n = 14, 20, and 10 for SCNT, IVP, and AI groups, respectively). Fetal fluids were analyzed for progesterone, estrone sulphate, sodium, chloride, potassium, creatinine, urea, calcium, magnesium and phosphate. Pregnancy outcomes for the SCNT group were retrospectively classified as: Fail 100 (pregnancies failing between Days 70–99; n = 6); Fail 130 (failing between Days 100–129; n = 5); Hydrops (greater than 10 L combined amniotic and allantoic fluid at postmortem between Days 135–163; n = 8) and SCNT Pregnant 150 (pregnant between Days 135–163; n = 6). IVP and AI pregnancies were classified as IVP or AI Pregnant 150. Fluid composition was analyzed by ANOVA on log-transformed data. On Day 70, allantoic progesterone and estrone sulphate concentrations were significantly higher (P < 0.05) for the SCNT cows compared to the IVP/AI Pregnant 150 cows. On Day 70, allantoic potassium, chloride, creatinine, and urea concentrations were significantly higher (P < 0.05) for the SCNT Hydrops cows compared to the IVP/AI Pregnant 150 cows. In addition, Day 70 allantoic creatinine and urea concentrations were significantly higher (P < 0.05) for the SCNT Hydrops cows compared to other SCNT groups. By Day 100, allantoic chloride, creatinine, and urea concentrations in SCNT Hydrops cows were significantly lower (P < 0.05) than in IVP/AI Pregnant 150 groups. We conclude that elevated Day 70 allantoic urea and creatinine concentrations are potential early diagnostic markers predicting hydroallantois in recipient cattle carrying SCNT fetuses. Further investigation of these markers in other somatic donor cell lines used for nuclear transfer is warranted to determine their general utility.

Mitochondria and the success of somatic cell nuclear transfer cloning: from nuclear - mitochondrial interactions to mitochondrial complementation and mitochondrial DNA recombination

The overall success of somatic cell nuclear transfer (SCNT) cloning is rather unsatisfactory, both in terms of efficacy and from an animal health and welfare point of view. Most research activities have concentrated on epigenetic reprogramming problems as one major cause of SCNT failure. The present review addresses the limited success of mammalian SCNT from yet another viewpoint, the mitochondrial perspective. Mitochondria have a broad range of critical functions in cellular energy supply, cell signalling and programmed cell death and, thus, affect embryonic and fetal development, suggesting that inadequate or perturbed mitochondrial functions may adversely affect SCNT success. A survey of perinatal clinical data from human subjects with deficient mitochondrial respiratory chain activity has revealed a plethora of phenotypes that have striking similarities with abnormalities commonly encountered in SCNT fetuses and offspring. We discuss the limited experimental data on nuclear-mitochondrial interaction effects in SCNT and explore the potential effects in the context of new findings about the biology of mitochondria. These include mitochondrial fusion/fission, mitochondrial complementation and mitochondrial DNA recombination, processes that are likely to be affected by and impact on SCNT cloning. Furthermore, we indicate pathways that could link epigenetic reprogramming and mitochondria effects in SCNT and address questions and perspectives for future research.

Tissue-specific elevated genomic cytosine methylation levels are associated with an overgrowth phenotype of bovine fetuses derived by in vitro techniques.

Epigenetic perturbations are assumed to be responsible for abnormalities observed in fetuses and offspring derived by in vitro techniques. We have designed an experiment with bovine Day 80 fetuses generated by somatic cell nuclear transfer (SCNT), in vitro fertilization (IVF), and artificial insemination (AI) to determine the relationship between fetal phenotype and genome-wide 5-methylcytosine (5mC) content. When compared with AI controls, SCNT and IVF fetuses displayed significantly increased body weight (61% and 28%), liver weight (100% and 36%), and thorax circumference (20% and 11%). A reduced crown-rump length:thorax circumference ratio (1.175 +/- 0.017 in SCNT and 1.292 +/- 0.018 in IVF vs. 1.390 +/- 0.018 in AI, P < 0.001 and P < 0.002) was the external hallmark of this disproportionate overgrowth phenotype. The SCNT fetuses showed significant hypermethylation of liver DNA in comparison with AI controls (3.46% +/- 0.08% vs. 3.17% +/- 0.09% 5mC, P < 0.03), and the cytosine methylation levels for IVF fetuses (3.34% +/- 0.09%) were, as observed for phenotypic parameters, intermediate to the other groups. Regressions of fetal body and liver weight and thorax circumference on 5mC content of liver DNA were positive (P < 0.073-0.079). Furthermore, a significant negative regression (P < 0.021) of the crown-rump length:thorax circumference ratio on liver 5mC was observed. The 5mC content of placental cotyledon DNA was 46% lower than in liver DNA (P < 0.0001) but did not differ among groups. These data are in striking contrast with the recently reported hypomethylation of DNA from SCNT fetuses and indicate that hypermethylation of fetal tissue, but not placenta, is linked to the overgrowth phenotype in bovine SCNT and IVF fetuses.

Heteroplasmy in bovine fetuses produced by intra- and inter-subspecific somatic cell nuclear transfer: neutral segregation of nuclear donor mitochondrial DNA in various tissues and evidence for recipient cow mitochondria in fetal blood.

Varying degrees of mitochondrial DNA (mtDNA) heteroplasmy have been observed in nuclear transfer embryos, fetuses, and offspring, but the mechanisms leading to this condition are unknown. We have generated a clone of 12 bovine somatic cell nuclear transfer fetuses, using nuclear donor cells, recipient oocytes, and recipient heifers with defined mtDNA genotypes, to study nuclear-mitochondrial interactions and the origins of mtDNA heteroplasmy. Embryos were reconstructed from granulosa cells with Bos taurus mtDNA type A and recipient oocytes collected from three different maternal lineages with B. taurus mtDNA type B, B. taurus mtDNA type C, or B. indicus mtDNA. Sequence differences in the control region (CR) of B. taurus mtDNAs ranged from 6 to 11 nucleotides and differences between B. taurus and B. indicus CRs from 45 to 50 nucleotides. Fetuses were recovered from recipient heifers with B. taurus mtDNA type B on Day 80 after nuclear transfer (eight B. taurus A/B, two B. taurus A/C, and two B. taurus A/B. indicus). Agarose gel analysis of the CR by polymerase chain reaction-based restriction fragment length polymorphism failed to detect nuclear donor mtDNA in 11 investigated tissues of 10 viable fetuses and in DNA samples of two fetuses in resorption (one B. taurus A/B and one B. taurus A/C). A more sensitive analysis of 1801 plasmid clones with CR inserts derived from tissues of a B. taurus A/B. indicus fetus detected no or very low levels of heteroplasmy (0.5-0.7%). However, the analyses detected considerable amounts ( approximately 2.5% and 5%) of recipient heifer mtDNA in blood samples from two fetuses. Our data do not suggest a replicative advantage of somatic nuclear donor cell mtDNA in bovine transmitochondrial clones produced with oocytes from domestic forms of the same or a different aurochs (B. primigenius) subspecies. Detection of mtDNA from the recipient animal in the circulation of two fetuses points to leakage of the placental barrier, mimicking heteroplasmy.