Nuclear Transfer Arrest Embryos Show Massive Dysregulation of Genes Involved in Transcription Pathways.

Somatic cell nuclear transfer (SCNT) technology can reprogram terminally differentiated cell nuclei into a totipotent state. However, the underlying molecular barriers of SCNT embryo development remain incompletely elucidated. Here, we observed that transcription-related pathways were incompletely activated in nuclear transfer arrest (NTA) embryos compared to normal SCNT embryos and in vivo fertilized (WT) embryos, which hinders the development of SCNT embryos. We further revealed the transcription pathway associated gene regulatory networks (GRNs) and found the aberrant transcription pathways can lead to the massive dysregulation of genes in NTA embryos. The predicted target genes of transcription pathways contain a series of crucial factors in WT embryos, which play an important role in catabolic process, pluripotency regulation, epigenetic modification and signal transduction. In NTA embryos, however, these genes were varying degrees of inhibition and show a defect in synergy. Overall, our research found that the incomplete activation of transcription pathways is another potential molecular barrier for SCNT embryos besides the incomplete reprogramming of epigenetic modifications, broadening the understanding of molecular mechanism of SCNT embryonic development.

Demetylation of the sex-determining region Y gene promoter and incidence of disorder of sex development in cloned dog males.

Canine cloning is occasionally accompanied by abnormal sexual development. Some male donor cells produce cloned pups with female external genitalia and complete male gonadal dysgenesis, which is classified as an XY disorder of sex development (XY DSD). In this study, we examine the potential of 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to reduce the phenotypic abnormality XY DSD in somatic cell nuclear transfer (SCNT)- derived pups. We used a 9-year-old normal male German Shepherd dog as a cell donor. Donor cells were treated with 10 nM 5-aza-dC for 4 days before being used for SCNT. At the same stage of cell development, significantly lower levels of DNA methylation of the sex-determining region Y (SRY) promoter was observed in the treated donor cells compared to that in the untreated cells (95.2% versus 53.3% on day 4 for the control and treated groups, respectively). No significant differences were observed in the control or treatment groups concerning fusion rate, pregnancy rate (30 days or entire period), the number of pups, or the incidence of XY DSD. However, more XY DSD dogs were observed in the control group (31.25%) than in the treatment group (14.29%). Hypermethylation of the SRY promoter was observed in the XY DSD cloned pups in both the treatment (84.8%) and control groups (91.1 ± 1.4%) compared to the methylation level in the phenotypically normal male pups of the treatment (23.2 ± 20.9%) and control groups (39.1 ± 20.1%). These results suggest that 5-aza-dC treatment of donor cells can reduce the methylation level of the SRY promoter in donor cells, and thus, 5-aza-dC is advantageous for reducing the incidence of XY DSD in canine cloning.

Mitochondrial Genetic Drift after Nuclear Transfer in Oocytes.

Mitochondria are energy-producing intracellular organelles containing their own genetic material in the form of mitochondrial DNA (mtDNA), which codes for proteins and RNAs essential for mitochondrial function. Some mtDNA mutations can cause mitochondria-related diseases. Mitochondrial diseases are a heterogeneous group of inherited disorders with no cure, in which mutated mtDNA is passed from mothers to offspring via maternal egg cytoplasm. Mitochondrial replacement (MR) is a genome transfer technology in which mtDNA carrying disease-related mutations is replaced by presumably disease-free mtDNA. This therapy aims at preventing the transmission of known disease-causing mitochondria to the next generation. Here, a proof of concept for the specific removal or editing of mtDNA disease-related mutations by genome editing is introduced. Although the amount of mtDNA carryover introduced into human oocytes during nuclear transfer is low, the safety of mtDNA heteroplasmy remains a concern. This is particularly true regarding donor-recipient mtDNA mismatch (mtDNA-mtDNA), mtDNA-nuclear DNA (nDNA) mismatch caused by mixing recipient nDNA with donor mtDNA, and mtDNA replicative segregation. These conditions can lead to mtDNA genetic drift and reversion to the original genotype. In this review, we address the current state of knowledge regarding nuclear transplantation for preventing the inheritance of mitochondrial diseases.

The pathophysiological changes associated with neonatal death of cloned pigs.

Cloned pigs generated by the somatic cell transfer nuclear (SCNT) technique are highly valuable for agriculture, biomedicine, and life sciences. However, the neonatal mortality rate of cloned pigs is very high. The reasons causing the massive loss of cloned pigs during their neonatal ages are unclear. In the present study, we found that the neonatal death of cloned pigs was associated with aberrant purine metabolism, impaired renal morphology and function, and decreased hepatic Hprt1 expression. The downregulation of Hprt1, a key purine metabolism regulation gene, in the liver was responsible for the elevation of an important purine metabolite, uric acid, in the serum, causing abnormalities in kidney morphology and function and leading to death of neonatal cloned pigs. This study provided insights into the pathophysiological mechanisms underlying the neonatal death of clone pigs, and results will help improve their survival rate.

Functional consequences of mitochondrial mismatch in reconstituted embryos and offspring.

Animal cloning technology has been developed to produce progenies genetically identical to a given donor cell. However, in nuclear transfer protocols, the recipient oocytes contribute a heritable mitochondrial genomic (mtDNA) background to the progeny. Additionally, a small amount of donor cell-derived mitochondria accompanies the transferred nucleus in the process; hence, the mtDNAs of two origins are mixed in the cytoplasm (heteroplasmy) of the reconstituted oocyte. Herein, I would like to introduce some of our previous results concerning five key considerations associated with animal cloning, including: mtDNA heteroplasmy in somatic cell nuclear transferred (SCNT) animals, the variation in the transmission of mtDNA heteroplasmy to subsequent generations SCNT cows and pigs, the influence of mtDNA sequence differences on mitochondrial proteins in SCNT cows and pigs, the effects of the introduction of mitochondria derived from somatic cells into recipient oocytes on embryonic development, and alterations of mtDNA heteroplasmy in inter/intraspecies nuclear transfer embryos.

Somatic cell nuclear transfer: failures, successes and the challenges ahead.

Somatic cell nuclear transfer (SCNT) has a broad spectrum of potential applications, including rescue of endangered species, production of transgenic animals, drug production, and regenerative medicine. Unfortunately, the efficiency of SCNT is still disappointingly low. Many factors affecting cloning procedures have been described in several previous reviews; here we review the most effective improvements in SCNT, with a special emphasis on the effect of mitochondrial defects on SCNT embryo/ foetus development, an issue never touched upon before.

Aberrant imprinting in mouse trophoblast stem cells established from somatic cell nuclear transfer-derived embryos.

Although phenotypic abnormalities frequently appear in the placenta following somatic cell nuclear transfer (SCNT), mouse trophoblast stem cells (TSCs) established from SCNT embryos reportedly show no distinct abnormalities compared with those derived from normal fertilization. In this study, we reexamined SCNT-TSCs to identify their imprinting statuses. Placenta-specific maternally imprinted genes (Gab1, Slc38a4, and Sfmbt2) consistently showed biallelic expression in SCNT-TSCs, suggesting their loss of imprinting (LOI). The LOI of Gab1 was associated with decreased DNA methylation, and that of Sfmbt2 was associated with decreased DNA methylation and histone H3K27 trimethylation. The maternal allele of the intergenic differentially methylated region (IG-DMR) was aberrantly hypermethylated following SCNT, even though this region was prone to demethylation in TSCs when established in a serum-free chemically defined medium. These findings indicate that the development of cloned embryos is associated with imprinting abnormalities specifically in the trophoblast lineage from its initial stage, which may affect subsequent placental development.

Blastocyst Formation Rate and Transgene Expression are Associated with Gene Insertion into Safe and Non-Safe Harbors in the Cattle Genome.

Integration target site is the most important factor in successful production of transgenic animals. However, stable expression of transgene without disturbing the function of the host genome depends on promoter methylation, transgene copy number and transcriptional activity in integration regions. Recently, new genome-editing tools have made much progress, however little attention has been paid to the identification of genomic safe harbors. The aim of the present study was to evaluate the effect of insertion site, promoter and copy number of transgene on the production of embryos from cattle fibroblast cells following somatic cell nuclear transfer (SCNT). So, three donor vectors were constructed with EGFP gene under control of different promoters. Each vector was integrated into safe and non-safe harbors in the genome using phiC31 integrase. Transgenic clones with a single copy of each vector were isolated. Each clone was analyzed to find site and frequency of integration, expression level and promoter methylation before SCNT, as well as transgene expression level and blastocyst formation rate after SCNT. The data obtained demonstrated that BF5, as a safe harbor, not only showed a stable expression, but also the rate of in vitro-produced embryos from BF5-clones are similar to that of non-transfected cells.

Deregulated Expression of Mitochondrial Proteins Mfn2 and Bcnl3L in Placentae from Sheep Somatic Cell Nuclear Transfer (SCNT) Conceptuses.

In various animal species, the main cause of pregnancy loss in conceptuses obtained by somatic cell nuclear transfer (SCNT) are placental abnormalities. Most abnormalities described in SCNT pregnancies (such as placentomegaly, reduced vascularisation, hypoplasia of trophoblastic epithelium) suggest that placental cell degeneration may be triggered by mitochondrial failure. We hypothesized that placental abnormalities of clones obtained by SCNT are related to mitochondrial dysfunction. To test this, early SCNT and control (CTR, from pregnancies obtained by in vitro fertilization) placentae were collected from pregnant ewes (at day 20 and 22 of gestation) and subjected to morphological, mRNA and protein analysis. Here, we demonstrated swollen and fragmented mitochondria and low expression of mitofusin 2 (Mfn2), the protein which plays a crucial role in mitochondrial functionality, in SCNT early placentae. Furthermore, reduced expression of the Bcnl3L/Nix protein, which plays a crucial role in selective elimination of damaged mitochondria, was observed and reflected by the accumulation of numerous damaged mitochondria in SCNT placental cells. Likely, this accumulation of damaged organelles led to uncontrolled apoptosis in SCNT placentae, as demonstrated by the high number of apoptotic bodies, fragmented cytoplasm, condensed chromatin, lack of integrity of the nuclear membrane and the perturbed mRNA expression of apoptotic genes (BCL2 and BAX). In conclusion, our data indicate that deregulated expression of Mfn2 and Bcnl3L is responsible for placental abnormalities in SCNT conceptuses. Our results suggest that some nuclear genes, that are involved in the regulation of mitochondrial function, do not work well and consequently this influence the function of mitochondria.

Sharpening the cutting edge: additional considerations for the UK debates on embryonic interventions for mitochondrial diseases.

In October 2015 the UK enacted legislation to permit the clinical use of two cutting edge germline-altering, IVF-based embryonic techniques: pronuclear transfer and maternal spindle transfer (PNT and MST). The aim is to use these techniques to prevent the maternal transmission of serious mitochondrial diseases. Major claims have been made about the quality of the debates that preceded this legislation and the significance of those debates for UK decision-making on other biotechnologies, as well as for other countries considering similar legislation. In this article we conduct a systematic analysis of those UK debates and suggest that claims about their quality are over-stated. We identify, and analyse in detail, ten areas where greater clarity, depth and nuance would have produced sharper understandings of the contributions, limitations and wider social impacts of these mitochondrial interventions. We explore the implications of these additional considerations for (i) the protection of all parties involved, should the techniques transfer to clinical applications; (ii) the legitimacy of focussing on short-term gains for individuals over public health considerations, and (iii) the maintenance and improvement of public trust in medical biotechnologies. We conclude that a more measured evaluation of the content and quality of the UK debates is important and timely: such a critique provides a clearer understanding of the possible, but specific, contributions of these interventions, both in the UK and elsewhere; also, these additional insights can now inform the emerging processes of implementation, regulation and practice of mitochondrial interventions.

Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XY(DSD) gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERß, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XY(DSD) in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination.

Aberrant DNA methylation reprogramming in bovine SCNT preimplantation embryos.

DNA methylation reprogramming plays important roles in mammalian embryogenesis. Mammalian somatic cell nuclear transfer (SCNT) embryos with reprogramming defects fail to develop. Thus, we compared DNA methylation reprogramming in preimplantation embryos from bovine SCNT and in vitro fertilization (IVF) and analyzed the influence of vitamin C (VC) on the reprogramming of DNA methylation. The results showed that global DNA methylation followed a typical pattern of demethylation and remethylation in IVF preimplantation embryos; however, the global genome remained hypermethylated in SCNT preimplantation embryos. Compared with the IVF group, locus DNA methylation reprogramming showed three patterns in the SCNT group. First, some pluripotency genes (POU5F1 and NANOG) and repeated elements (satellite I and α-satellite) showed insufficient demethylation and hypermethylation in the SCNT group. Second, a differentially methylated region (DMR) of an imprint control region (ICR) in H19 exhibited excessive demethylation and hypomethylation. Third, some pluripotency genes (CDX2 and SOX2) were hypomethylated in both the IVF and SCNT groups. Additionally, VC improved the DNA methylation reprogramming of satellite I, α-satellite and H19 but not that of POU5F1 and NANOG in SCNT preimplantation embryos. These results indicate that DNA methylation reprogramming was aberrant and that VC influenced DNA methylation reprogramming in SCNT embryos in a locus-specific manner.

Placental abnormalities in equine pregnancies generated by SCNT from one donor horse.

Placental changes associated with SCNT have been described in several species, but little information is available in this area in the horse. We evaluated the ultrasonographic, gross, and histopathological characteristics of placentas from three successful and five unsuccessful equine SCNT pregnancies, established using cells from a single donor horse. Starting at approximately 6-month gestation, the pregnancies were monitored periodically using transrectal (TR) and transabdominal (TA) ultrasonography (US) to examine the placentas, fetal fluids, and fetuses. Of the five mares that aborted, one mare did so suddenly without any abnormal signs detected by US and four had enlarged umbilical vessels visible on TA-US before abortion. Placental edema (TR-US) and intravascular thrombi in the umbilical cords were seen (TA-US) in two of these four mares; one mare aborted shortly after acute placental separation was identified on TA-US. In three mares that delivered live foals, TA-US showed engorged allantoic vessels and enlarged umbilical vessels. Two of these mares had placental thickening visible on TR-US, interpreted as a sign of placentitis, that subsided after aggressive medical treatment. Seven of the eight placentas were submitted for gross and histopathological examinations after delivery. All placentas had some degree of edema, abnormally engorged allantoic vessels, and enlarged umbilical vessels. Placentitis, large allantoic vesicles, cystic pouches in the fetal part of the cord, and hemorrhages and thrombi in the umbilical vessels were detected only in placentas from mares that aborted. Equine pregnancies resulting from SCNT may be associated with placental pathologies that can be detected using ultrasonography. However, interpreting their severity is difficult. Although placental abnormalities have been observed in SCNT pregnancies in other species, to the best of our knowledge, placentitis has not been previously reported and may be an important complication of equine SCNT pregnancies, leading to pregnancy loss.

Faithful expression of imprinted genes in donor cells of SCNT cloned pigs.

To understand if the genomic imprinting status of the donor cells is altered during the process of SCNT (somatic cell nuclear transfer), cloned pigs were produced by SCNT using PEF (porcine embryonic fibroblast) and P-PEF (parthenogenetic-PEF) cells as donors. Then, the gene expression and methylation patterns of H19, IGF2, NNAT and MEST were compared between PEF vs. C-PEF (cloned-PEF), P-PEF vs. CP-PEF (cloned-P-PEF), respectively. Taken together, the results revealed that there was no significant difference in the expression of imprinted genes and conserved genomic imprints between the donor and cloned cells.

Vascular alterations underlie developmental problems manifested in cloned cattle before or after birth.

Although assisted reproductive techniques are commonly applied in humans and animals, they are frequently associated with major developmental deficits and reduced viability. To explore abnormalities associated with cloning or nuclear transfer (NT) as the most invasive of these methods, we used a bovine model to characterize abnormalities. Detailed necropsy examinations were done on 13 calves that died soon after birth; in addition, we included data from embryos and fetuses (produced by NT) that terminated prematurely. Bovine clones that survived until the neonatal period differed quantitatively and qualitatively from in-vivo-derived cattle. Although alterations affected a variety of organs (e.g. heart, lung and liver), there was a clear association with abberant vascular developmental during the early intrauterine phase. Therefore, we concluded that vascular problems were key alterations induced by cloning (presumably via epigenetic modifications).

Assessment of nuclear transfer techniques to prevent the transmission of heritable mitochondrial disorders without compromising embryonic development competence in mice.

To evaluate and compare mitochondrial DNA (mtDNA) carry-over and embryonic development potential between different nuclear transfer techniques we performed germinal vesicle nuclear transfer (GV NT), metaphase-II spindle-chromosome-complex (MII-SCC) transfer and pronuclear transfer (PNT) in mice. No detectable mtDNA carry-over was seen in most of the reconstructed oocytes and embryos. No significant differences were seen in mtDNA carry-over rate between GV NT (n=20), MII-SCC transfer (0.29 ± 0.63; n=21) and PNT (0.29 ± 0.75; n=25). Blastocyst formation was not compromised after either PNT (88%; n=18) or MII-SCC transfer (86%; n=27). Further analysis of blastomeres from cleaving embryos (n=8) demonstrated undetectable mtDNA carry-over in all but one blastomere. We show that NT in the germ line is potent to prevent transmission of heritable mtDNA disorders with the applicability for patients attempting reproduction.

Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle.

The use of Assisted Reproductive Technologies (ARTs) in modern cattle breeding is an important tool for improving the production of dairy and beef cattle. A frequently employed ART in the cattle industry is in vitro production of embryos. However, bovine in vitro produced embryos differ greatly from their in vivo produced counterparts in many facets, including developmental competence. The lower developmental capacity of these embryos could be due to the stress to which the gametes and/or embryos are exposed during in vitro embryo production, specifically ovarian hormonal stimulation, follicular aspiration, oocyte in vitro maturation in hormone supplemented medium, sperm handling, gamete cryopreservation, and culture of embryos. The negative effects of some ARTs on embryo development could, at least partially, be explained by disruption of the physiological epigenetic profile of the gametes and/or embryos. Here, we review the current literature with regard to the putative link between ARTs used in bovine reproduction and epigenetic disorders and changes in the expression profile of embryonic genes. Information on the relationship between reproductive biotechnologies and epigenetic disorders and aberrant gene expression in bovine embryos is limited and novel approaches are needed to explore ways in which ARTs can be improved to avoid epigenetic disorders.

Post-death cloning of endangered Jeju black cattle (Korean native cattle): fertility and serum chemistry in a cloned bull and cow and their offspring.

To preserve Jeju black cattle (JBC; endangered native Korean cattle), a pair of cattle, namely a post-death cloned JBC bull and cow, were produced by somatic cell nuclear transfer (SCNT) in a previous study. In the present study, we examined the in vitro fertilization and reproductive potentials of these post-death cloned animals. Sperm motility, in vitro fertilization and developmental capacity were examined in a post-death cloned bull (Heuk Oll Dolee) and an extinct nuclear donor bull (BK94-13). We assessed reproductive ability in another post-death cloned cow (Heuk Woo Sunee) using cloned sperm for artificial insemination (AI). There were no differences in sperm motility or developmental potential of in vitro fertilized embryos between the post-death cloned bull and its extinct nuclear donor bull; however, the embryo development ratio was slightly higher in the cloned sperm group than in the nuclear donor sperm group. After one attempt at AI, the post-death cloned JBC cow became pregnant, and gestation proceeded normally until day 287. From this post-death cloned sire and dam, a JBC male calf (Heuk Woo Dolee) was delivered naturally (weight, 25 kg). The genetic paternity/maternity of the cloned JBC bull and cow with regard to their offspring was confirmed using International Society for Animal Genetics standard microsatellite markers. Presently, Heuk Woo Dolee is 5 months of age and growing normally. In addition, there were no significant differences in blood chemistry among the post-death cloned JBC bull, the cow, their offspring and cattle bred by AI. This is the first report showing that a pair of cattle, namely, a post-death cloned JBC bull and cow, had normal fertility. Therefore, SCNT can be used effectively to increase the population of endangered JBC.

Clinical study report on milk production in the offspring of a somatic cell cloned Holstein cow.

This study examined two female offspring of a somatic cell cloned Holstein cow that had reproduction problems and milk production performance issues. The two offspring heifers, which showed healthy appearances and normal reproductive characteristics, calved on two separate occasions. The mean milk yields of the heifers in the first lactation period were 9,037 kg and 7,228 kg. The relative mean milk yields of these cows were 111.2% and 88.9%, respectively, when compared with that of the control group. No particular clinical abnormalities were revealed in milk yields and milk composition rate [e.g., fat, protein and solids-not-fat (SNF)], and reproductive characteristics of the offspring of the somatic cell cloned Holstein cow suggested that the cloned offspring had normal milk production.

Nuclear donor cell lines considerably influence cloning efficiency and the incidence of large offspring syndrome in bovine somatic cell nuclear transfer.

Total five ear skin fibroblast lines (named F1, F2, F3, F4 and F5) from different newborn Holstein cows have been used as nuclear donor cells for producing cloned cows by somatic cell nuclear transfer (SCNT). The effects of these cell lines on both in vitro and in vivo developmental rates of cloned embryos, post-natal survivability and incidence of large offspring syndrome (LOS) were examined in this study. We found that the different cell lines possessed the same capacity to support pre-implantation development of cloned embryos, the cleavage and blastocyst formation rates ranged from 80.2 ± 0.9 to 84.5 ± 2.5% and 28.5 ± 0.9 to 33.3 ± 1.4%, respectively. However, their capacities to support the in vivo development of SCNT embryos showed significant differences (p < 0.05). The pregnancy rates at 90 and 240 day were significantly lower in groups F2 (4.9% and 3.3%) and F3 (5.4% and 5.4%) compared to groups F1 (23.3% and 16.3%), F4 (25.7% and 18.6%) and F5 (25.9% and 19.8%) (p < 0.05). The cloning efficiency was significantly higher in group F5 than those in group F1, F2, F3 and F4 (9.3% vs 4.1%, 1.2%, 2.0% and 5.0%, respectively, p < 0.05). Moreover, large offspring syndrome (LOS) incidence in group F5 was significantly lower than those in other groups (p < 0.05). All cloned offspring from cell line F1, F2, F3 and F4 showed LOS and gestation length delay, while all cloned offspring from F5 showed normal birthweight and gestation length. We concluded that the nuclear donor cell lines have significant impact on the in vivo development of cloned embryos and the incidence of LOS in cloned calves.