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.

Efficient generation of cloned cats with altered coat colour by editing of the KIT gene.

Coat colour largely determines the market demand for several cat breeds. The KIT proto-oncogene (KIT) gene is a key gene controlling melanoblast differentiation and melanogenesis. KIT mutations usually cause varied changes in coat colour in mammalian species. In this study, we used a pair of single-guide RNAs (sgRNAs) to delete exon 17 of KIT in somatic cells isolated from two different Chinese Li Hua feline foetuses. Edited cells were used as donor nuclei for somatic cell nuclear transfer (SCNT) to generate cloned embryos presenting an average cleavage rate exceeding 85%, and an average blastocyst formation rate exceeding 9.5%. 131 cloned embryos were transplanted into four surrogates, and all surrogates carried their pregnancies to term, and delivered 4.58% (6/131) alive cloned kittens, with 1.53% (2/131) being KIT-edited heterozygotes (KITD17/+). The KITD17/+ cats presented an obvious darkness reduction in the mackerel tabby coat. Immunohistochemical analysis (IHC) of skin tissues indicated impaired proliferation and differentiation of melanoblasts caused by the lack of exon17 in feline KIT. To our knowledge, this is the first report on coat colour modification of cats through gene editing. The findings could facilitate further understanding of the regulatory role of KIT on feline coat colour and provide a basis for the breeding of cats with commercially desired coat colour.

Cloned Foal Born from Postmortem-Obtained Ear Sample Refrigerated for 5 Days Before Fibroblast Isolation and Decontamination of the Infected Monolayer Culture.

A 6-year-old mare, a valuable polo horse, died of complications following postcolic surgery. To preserve its genetics, ear skin samples were collected immediately after death and stored in an equine embryo transfer medium at 4°C for 5 days. After trypsin digestion, monolayer fibroblast cultures were established, but signs of massive bacterial infection were found in all of them. As an ultimate attempt for rescue, rigorously and repeatedly washed cells were individually cultured in all wells of four 96-well dishes. New monolayers were established from the few wells without contamination and used for somatic cell nuclear transfer. Four of the six Day 7 blastocysts derived from 14 reconstructed zygotes were transferred in four naturally cycling mares on Day 5 after ovulation. The embryo transfers resulted in 2 pregnancies, one from a fresh and one from a vitrified blastocyst. The vitrified embryo transfer resulted in a healthy offspring, now 21 months old, genetically and phenotypically identical to the somatic cell donor animal.

Cloning for the Twenty-First Century and Its Place in Endangered Species Conservation.

Cloning as it relates to the animal kingdom generally refers to the production of genetically identical individuals. Because cloning is increasingly the subject of renewed attention as a tool for rescuing endangered or extinct species, it seems timely to dissect the role of the numerous reproductive techniques encompassed by this term in animal species conservation. Although cloning is typically associated with somatic cell nuclear transfer, the recent advent of additional techniques that allow genome replication without genetic recombination demands that the use of induced pluripotent stem cells to generate gametes or embryos, as well as older methods such as embryo splitting, all be included in this discussion. Additionally, the phenomenon of natural cloning (e.g., a subset of fish, birds, invertebrates, and reptilian species that reproduce via parthenogenesis) must also be pointed out. Beyond the biology of these techniques are practical considerations and the ethics of using cloning and associated procedures in endangered or extinct species. All of these must be examined in concert to determine whether cloning has a place in species conservation. Therefore, we synthesize progress in cloning and associated techniques and dissect the practical and ethical aspects of these methods as they pertain to endangered species conservation.

Effects of extracellular vesicles derived from steroids-primed oviductal epithelial cells on porcine in vitro embryonic development.

Extracellular vesicles (EVs) play an active role in regulating different physiological events, however, endocrine control of EVs cargo contents remain poorly understood. In this study, we aimed to isolate EVs from the porcine oviductal epithelial cells (POECs) that were primed with steroid hormones including estradiol (E2) and progesterone (P4), mimicking the in vivo conditions of the reproductive cycle and studied their effects on in vitro produced embryonic development. For this purpose, POECs were treated either with 0 concentration (control) or two different combinations of E2 and P4 including 50 pg/mL E2 + 0.5 ng/mL P4 (group H1), and 10 pg/mL E2 + 35 ng/mL P4 (group H2). Embryos were prepared after in vitro maturation either by parthenogenetic activation or somatic cell nuclear transfer (SCNT) technique. Treating parthenogenetic embryo with EVs, led a significantly higher rate of the blastocyst formation in the group supplemented with each EVs, compared to the control group. In addition, TUNEL assay and gene expression level analysis revealed that apoptosis was significantly reduced in the H2 EVs group. Furthermore, EVs from hormone-primed POECs improved the formation rate of porcine SCNT embryos compared to the control group. While in each EVs supplemented group (control EVs, H1 EVs, H2 EVs), the expression of cell reprogramming-related genes in cloned embryos showed a tendency of increase, the effect was stronger in H1 EVs and H2 EVs. In conclusion, EVs derived from POECs cultured in hormonal conditions simulating the in vivo environment had a positive effect on porcine blastocysts formation, which will likely facilitate in the production of cloned embryos.

Blastocyst Cell Number and Allocation Affect the Developmental Potential and Transcriptome of Bovine Somatic Cell Nuclear Transfer Embryos.

Cloning cattle using somatic cell nuclear transfer (SCNT) is inefficient. Although the rate of development of SCNT embryos in vitro is similar to that of fertilized embryos, most fail to develop into healthy calves. In this study, we aimed to identify developmentally competent embryos according to blastocyst cell composition and perform transcriptome analysis of single embryos. Transgenic SCNT embryos expressing nuclear-localized HcRed gene at day 7 of development were imaged by confocal microscopy for cell counting and individually transferred to recipient heifers. Pregnancy rates were determined by ultrasonography. Embryos capable of establishing pregnancy by day 35 had an average of 117 ± 6 total cells, whereas embryos with an average of 128 ± 5 cells did not establish pregnancy (P < 0.05). A lesser average number of 41 ± 3 cells in the inner cell mass (ICM) also resulted in pregnancies (<0.05) than a greater number of 48 ± 2 cells in the ICM. Single embryos were then subjected to RNA sequencing for transcriptome analysis. Using weighted gene coexpression network analysis, we identified clusters of genes in which gene expression correlated with the number of total cells or ICM cells. Gene ontology analysis of these clusters revealed enriched biological processes in coenzyme metabolic process, intracellular signaling cascade, and glucose catabolic process, among others. We concluded that SCNT embryos with fewer total and ICM cell numbers resulted in greater pregnancy establishment rates and that these differences are reflected in the transcriptome of such embryos.

Cytokine supplemented maturation medium improved development to term following somatic cell nuclear transfer (SCNT) in cattle.

CONTEXT: In vitro maturation is an important process in the production of embryos. It has been shown that three cytokines, fibroblast growth factor 2, leukemia inhibitory factor and insulin-like growth factor 1 (FLI), increased efficiency of in vitro maturation, somatic cell nuclear transfer (SCNT) blastocyst production, and in vivo development of genetically engineered piglets. AIMS: Assess effects of FLI on oocyte maturation, quality of oocytes, and embryo development in bovine in vitro fertilisation (IVF) and SCNT. KEY RESULTS: Cytokine supplementation resulted in significant increases in maturation rates and decreased levels of reactive oxygen species. Oocytes matured in FLI had increased blastocyst rates when used in IVF (35.6%vs 27.3%, P <0.05) and SCNT (40.6%vs 25.7%, P <0.05). SCNT blastocysts contained significantly more inner cell mass and trophectodermal cells when compared to the control group. Importantly, SCNT embryos derived from oocytes matured in FLI medium resulted in a four-fold increase in full-term development compared to control medium (23.3%vs 5.3%, P <0.05). Relative mRNA expression analysis of 37 genes associated with embryonic and fetal development revealed one gene had differential transcript abundance in metaphase II oocytes, nine genes at the 8-cell stage, 10 genes at the blastocyst stage in IVF embryos and four genes at the blastocyst stage in SCNT embryos. CONCLUSIONS: Cytokine supplementation increased efficiency of in vitro production of IVF and SCNT embryos and in vivo development of SCNT embryos to term. IMPLICATIONS: Cytokine supplementation is beneficial to embryo culture systems, which may shed light on requirements of early embryo development.

Cattle Cloning by Somatic Cell Nuclear Transfer.

Cloning by somatic cell Nuclear Transfer (SCNT) is a powerful technology capable of reprograming terminally differentiated cells to totipotency for generating whole animals or pluripotent stem cells for use in cell therapy, drug screening, and other biotechnological applications. However, the broad usage of SCNT remains limited due to its high cost and low efficiency in obtaining live and healthy offspring. In this chapter, we first briefly discuss the epigenetic constraints responsible for the low efficiency of SCNT and current attempts to overcome them. We then describe our bovine SCNT protocol for delivering live cloned calves and addressing basic questions about nuclear reprogramming. Other research groups can benefit from our basic protocol and build up on it to improve SCNT in the future. Strategies to correct or mitigate epigenetic errors (e.g., correcting imprinting loci, overexpression of demethylases, chromatin-modifying drugs) can integrate the protocol described here.

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.

Evaluation of porcine urine-derived cells as nuclei donor for somatic cell nuclear transfer.

BACKGROUND: Somatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency. OBJECTIVES: This study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs. METHODS: The viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer. RESULTS: Most sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption. CONCLUSIONS: Sow urine-derived cells could be used to produce SCNT embryos.

Vitamin C enhances porcine cloned embryo development and improves the derivation of embryonic stem-like cells.

Porcine cloning through somatic cell nuclear transfer (SCNT) has been widely used in biotechnology for generating animal disease models and genetically modified animals for xenotransplantation. Vitamin C is a multifunctional factor that reacts with several enzymes. In this study, we used porcine oocytes to investigate the effects of different concentrations of vitamin C on in vitro maturation (IVM), in vitro culture (IVC), and the derivation of nuclear transfer embryonic stem-like cells (NT-ESCs). We demonstrated that vitamin C promoted the cleavage and blastocyst rate of genetically modified cloned porcine embryos and improved the derivation of NT-ESCs. Vitamin C integrated into IVM and IVC enhanced cleavage and blastocyst formation (P < 0.05) in SCNT embryos. Glutathione level was increased, and reactive oxygen species levels were decreased (P < 0.05) due to vitamin C treatment. Vitamin C decreased the gene expression of apoptosis (BAX) and increased the expression of genes associated with nuclear reprogramming (NANOG, POU5F1, SOX2, c-Myc, Klf4, and TEAD4), antioxidation (SOD1), anti-apoptotic (Bcl2), and trophectoderm (CDX2). Moreover, vitamin C improved the attachment, derivation, and passaging of NT-ESCs, while the control group showed no outgrowths beyond the primary culture. In conclusion, supplementation of vitamin C at a dose of 50 µg/ml to the IVM and IVC culture media was appropriate to improve the outcomes of porcine IVM and IVC and for the derivation of NT-ESCs as a model to study the pre- and post-implantation embryonic development in cloned transgenic embryos. Therefore, we recommend the inclusion of vitamin C as a supplementary factor to IVM and IVC to improve porcine in vitro embryonic development.

Follistatin supplementation induces changes in CDX2 CpG methylation and improves in vitro development of bovine SCNT preimplantation embryos.

Caudal Type Homeobox 2 (CDX2) is a key regulator of trophectoderm formation and maintenance in preimplantation embryos. We previously demonstrated that supplementation of exogenous follistatin, during in vitro culture of bovine IVF embryos, upregulates CDX2 expression, possibly, via alteration of the methylation status of CDX2 gene. Here, we further investigated the effects of exogenous follistatin supplementation on developmental competence and CDX2 methylation in bovine somatic cell nuclear transfer (SCNT) embryos. SCNT embryos were cultured with or without follistatin for 72h, then transferred into follistatin free media until d7 when blastocysts were collected and subjected to CDX2 gene expression and DNA methylation analysis for CDX2 regulatory regions by bisulfite sequencing. Follistatin supplementation significantly increased both blastocyst development as well as blastocyst CDX2 mRNA expression on d7. Three different CpG rich fragments within the CDX2 regulatory elements; proximal promoter (fragment P1, -1644 to -1180; P2, -305 to +126) and intron 1 (fragment I, + 3030 to + 3710) were identified and selected for bisulfite sequencing analysis. This analysis showed that follistatin treatment induced differential methylation (DM) at specific CpG sites within the analyzed fragments. Follistatin treatment elicited hypomethylation at six CpG sites at positions -1374, -279, -163, -23, +122 and +3558 and hypermethylation at two CpG sites at positions -243 and +20 in promoter region and first intron of CDX2 gene. Motif analysis using MatInspector revealed that differentially methylated CpG sites are putative binding sites for key transcription factors (TFs) known to regulate Cdx2 expression in mouse embryos and embryonic stem cells including OCT1, AP2F, KLF and P53, or TFs that have indirect link to CDX2 regulation including HAND and NRSF. Collectively, results of the present study together with our previous findings in IVF embryos support the hypothesis that alteration of CDX2 methylation is one of the epigenetic mechanisms by which follistatin may regulates CDX2 expression in preimplantation bovine embryos.

25th ANNIVERSARY OF CLONING BY SOMATIC CELL NUCLEAR TRANSFER: Generation of genetically engineered livestock using somatic cell nuclear transfer.

Genetic engineering (GE) of livestock initially has been accomplished primarily using pronuclear microinjection into zygotes (1985-1996). The applications of the technology were limited due to low integration efficiency, aberrant transgene expression resulting from random integration and the presence of genetic mosaicism in transgenic founder animals. Despite enormous efforts to established embryonic stem cells (ESCs) for domestic species, the ESC GE technology does not exist for livestock. Development of somatic cell nuclear transfer (SCNT) has bypassed the need in livestock ESCs and revolutionized the field of livestock transgenesis by offering the first cell-based platform for precise genetic manipulation in farm animals. For nearly two decades since the birth of Dolly (1996-2013), SCNT was the only method used for the generation of knockout and knockin livestock. Arrival of CRISPRS/Cas9 system, a new generation of gene-editing technology, gave us an ability to introduce precise genome modifications easily and efficiently. This technological advancement accelerated production of GE livestock by SCNT and reinstated zygote micromanipulation as an important GE approach. The primary advantage of the SCNT technology is the ability to confirm in vitro that the desired genetic modification is present in the somatic cells prior to animal production. The edited cells could also be tested for potential off-target mutations. Additionally, this method eliminates the risk of genetic mosaicism frequently observed following zygote micromanipulation. Despite its low efficiency, SCNT is a well-established procedure in numerous laboratories around the world and will continue to play an important role in the GE livestock field.

25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER: Current applications of SCNT in advanced breeding and genome editing in livestock.

SCNT (somatic cell nuclear transfer) has complemented the toolbox of ARTs offering yet another technique to reproduce animals in an unprecedented way. Despite remarkable achievements, SCNT suffers low efficiency, high pregnancy losses and higher than normal stillbirth rates that makes it an expensive technique to reproduce animals. Moreover, due to welfare issues associated with gestation and the newborn offspring, it is banned in some countries. It has become evident that these problems are of epigenetic nature associated with incomplete genome reprogramming, observed more frequently in ruminants and less often and of minor degree in pigs and horses. Genome editing is enormously benefiting from SCNT to turn genome edited cells into animals, even if zygote microinjection of CRISPR/Cas9 will become an alternative route in some occasions. SCNT will also be a route to reprogram somatic cell to pluripotency since bona fide iPSC in livestock are missing while embryonic stem cells have been now established. This opens the way to other technologies like the development of artificial gametes or interspecies nuclear transfer. To strengthen its commercial applications, SCNT will face three major challenges, that is, intellectual property (extremely unclear in genome editing), regulatory approval by the relevant authorities of the resuting potential products and finally, acceptance by the public who will eventually decide with its behavior the life or the death of the technology.

Establishment of bovine expanded potential stem cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.

Effect of Dickkopf-1 and colony stimulating factor-2 on the developmental competence, quality, gene expression and live birth rate of buffalo (Bubalus bubalis) embryos produced by hand-made cloning.

A functional canonical WNT signaling pathway exists in preimplantation embryos and inhibits embryonic development. Recent studies suggest that this pathway is over-expressed in nuclear transferred (NT), compared to IVF embryos. The present study investigated the effects of Dickkopf-1 (DKK1), an inhibitor of canonical WNT signaling pathway and colony stimulating factor-2 (CSF2), an embryokine, on the developmental competence, quality, gene expression and live birth rate of NT buffalo embryos produced by Hand-made cloning (HMC). Following supplementation of the in vitro culture medium on day 5 with DKK1 (100 ng/mL), CSF2 (10 ng/mL), DKK1+CSF2 or no supplementation (control), the blastocyst rate was higher (P < 0.05) with DKK1 and DKK1+CSF2 (42.6 ± 1.4% and 46.6 ± 0.9%, respectively) than with CSF2 or controls (40.6 ± 1.3% and 39.0 ± 1.3%, respectively). The apoptotic index of the blastocysts was lower (P < 0.05) for DKK1, CSF2 and DKK1+CSF2 groups (3.44 ± 0.14, 3.39 ± 0.11 and 3.11 ± 0.22, respectively) compared to controls (6.64 ± 0.25), and was similar to that of the IVF blastocysts (3.67 ± 0.18). Although the total cell number was similar for the DKK1, CSF2, DKK1+CSF2 and control groups (200.4 ± 3.05, 196.4 ± 3.73, 204.7 ± 3.71 and 205 ± 4.03, respectively), the inner cell mass:trophectoderm cell number ratio of DKK1, CSF2 and DKK1+CSF2 groups (0.21 ± 0.01, 0.17 ± 0.01 and 0.22 ± 0.02, respectively) was higher (P < 0.05) than controls (0.13 ± 0.01) and was similar to that of IVF blastocysts (0.19 ± 0.01). Treatment with DKK1 or CSF2 or both increased (P < 0.05) the expression level of OCT4, NANOG,SOX2, GATA6, BCL2, PTEN, P53, FGF4, GLUT1 and IFN-τ, and decreased that of C-MYC, CDX2, CASPASE, DNMT3a, TCF7 and LEF1 in blastocysts, compared to controls. Transfer of DKK1-treated embryos to 13 recipients resulted in 4 pregnancies (30.8%; 2 live births, one abortion and one currently at 9 months of pregnancy) whereas, transfer of DKK1+CSF2-treated embryos to 16 recipients, resulted in 4 pregnancies (25.0%), all of which resulted in live births. No pregnancy was obtained after transfer of control and CSF-treated embryos to 12 and 16 recipients, respectively. These results suggest that DKK1 treatment of NT embryos increases the blastocyst, conception and live birth rate, and improves their quality whereas, CSF2 treatment, does not affect the blastocyst, conception and live birth rate despite improvement in embryo quality.

Assessing the effect of interspecies oocyte nucleolar material dosage on embryonic development.

Sequence differences are considered to be the basic cause of developmental failure in interspecies embryos when more distant species are combined. However, other phenomena, such as insufficient or excessive quantity of specific cellular factors, might also influence the outcome. These effects are usually not considered. One of the organelles shown to contain different amount of proteins is the oocyte nucleolus-like body. Here we show that upon interspecies transfer, a single porcine nucleolus-like body is unable to support the development of a mouse parthenogenetic embryo derived from an enucleolated oocyte. However, when the amount of the porcine nucleolar material is increased to equalize the amount of mouse nucleolar material by transferring two nucleolus-like bodies, mouse embryos are able to pass the developmental block elicited by enucleolation. These embryos progress to the blastocyst stage at rates comparable to controls. Thus, using the model of an interspecies nucleolus-like body transplantation between mouse and pig oocytes, we show that an inadequate amount of nucleolar factors, rather than the species origin, affects the development. In a wider context of interspecies nuclear transfer schemes, the observed incompatibility between more distant species might not stem simply from sequence differences but also from improper dosage of key cellular factors.

Effect of season on the in-vitro maturation and developmental competence of buffalo oocytes after somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) is a valuable technology tool with various uses in transgenic animals, regenerative medicine, and stem cell research. However, the efficiency of SCNT embryos appears to have poor developmental competency. Environmental issues may adversely affect SCNT embryos in buffalo. Thereafter, the present study aimed to explore the effect of season on the maturation of buffalo oocytes and subsequent developmental capability after parthenogenetic activation and SCNT in buffalo. Buffalo oocytes (n = 6353) were collected from local slaughterhouse at various seasons; spring (March-April), summer (May-August), autumn (September-November), and winter (December-January). A significant increase (p < 0.05) was recorded in the maturation rate (57.07%) at autumn compared with spring, summer, and winter (50.46, 50.93, and 50.66%, respectively). No significant differences were recorded in the fusion and the cleavage rates among all seasons. Blastocyst development rate was higher (p < 0.05) in autumn and winter (16.52 ± 8.45% and 15.98 ± 7.17%, respectively) than in spring and summer (9.47 ± 6.71% and 10.84 ± 6.58%, respectively) seasons. It could be concluded that the season had a significant effect on oocyte development competence which can be used for SCNT in buffalo.

Asiatic acid supplementation during the in vitro culture period improves early embryonic development of porcine embryos produced by parthenogenetic activation, somatic cell nuclear transfer and in vitro fertilization.

Asiatic acid is a pentacyclic triterpene enriched in the medicinal herb Centella asiatica, and it has been suggested to possess free radical scavenging and anti-apoptotic properties. The purpose of the current study was to explore the effects of asiatic acid on porcine early-stage embryonic development and the potential mechanisms for any observed effects. The results showed that 10 µM asiatic acid supplementation during the in vitro culture period dramatically improved developmental competence in porcine embryos derived from parthenogenetic activation (PA), somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). Further analysis revealed that asiatic acid attenuated H2O2-induced intracellular reactive oxygen species (ROS) generation. Notably, asiatic acid not only enhanced intracellular GSH levels but also attenuated mitochondrial dysfunction. Gene expression analysis revealed that asiatic acid upregulated expression of the antioxidant-related gene Sod-1 and the blastocyst formation related gene Cox-2, while downregulating expression of the apoptosis-related gene Caspase-9 in SCNT blastocysts. These results suggest that asiatic acid exerts beneficial effects on early embryonic development in porcine embryos and that asiatic acid may be useful for improving the in vitro production of porcine embryos.

Overexpression of MBD3 Improves Reprogramming of Cloned Pig Embryos.

Methyl-CpG-binding domain protein 3 (MBD3) is a core component of the nucleosome remodeling and deacetylase (NuRD) complex, which is crucial for pluripotent stem cell differentiation and embryonic development. MBD3 was shown to play important roles in transcription factor-induced somatic cell reprogramming. Expression level of MBD3 was demonstrated to be higher in somatic cell nuclear transfer-generated cloned pig embryos than in fertilization-derived porcine embryos. However, the functions of MBD3 in nuclear transfer-mediated somatic cell reprogramming are unknown. In this study, MBD3 was overexpressed in cloned pig embryos, and the effects of MBD3 overexpression on gene transcription, DNA methylation, and in vitro developmental competence of cloned pig embryos were analyzed. Results indicated that overexpression of MBD3 in cloned pig embryos not only increased blastocyst rate and number of cells per blastocyst but also upregulated mRNA expression levels and decreased the DNA methylation of NANOG, OCT4, and LINE1 genes to the levels close to those in in vivo fertilization-produced pig embryos. These findings suggest that overexpression of MBD3 improves reprogramming of cloned pig embryos.