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Currently, artificial oocyte activation has attracted wide attention in assisted reproduction due to extensive range of applications, particularly in somatic cell nuclear transfer and deriving pluripotent stem cell lines and it is the unique model to determine the role of paternal genome. Numbers of artificial activating agents have been used extensively to induce the oocytes activation; however, embryos developmental competency of artificially activated oocytes is still very low. In the present study, we determined the functional impact of strontium chloride supplementation with gold nanoparticles (AuNPs) in artificial oocytes activation and subsequent embryonic development. Oocytes were activated artificially in the culture medium containing 250 nM AuNPs with constant concentration of strontium chloride 10.00 mM. We found that adding 250 nM AuNPs with constant concentration of strontium chloride (10.00 mM for 3 hr) in culture medium improves the proportion of embryos reaching to the morula and blastocyst stages from 61.00% and 42.00% (controls) to 75.00% and 58.00% (250 nM AuNPs), respectively. In addition, foster mothers receiving AuNPs-treated embryos showed more implantation percentage and pregnancy rate relative to females received control embryos. Finally, embryos treated with 250 nM AuNPs concentration showed no toxic effect in term of blastocyst development. Collectively, our findings suggest the potential role of AuNPs in early embryonic development for mouse oocytes activated artificially and provide new insights in the field of animal biotechnology and assisted reproduction in humans. [ABSTRACT FROM AUTHOR]

Administration of mesenchymal stem cells (MSC) into the wound seems a promising therapy in the management of hard-to-heal wounds in diabetic patients. We still do not have algorithms that would precisely define appropriate use of this therapy. However, the latest research results indicate multidirectional effects of MSC therapy (improvement of blood supply and tissue granulation within the wound, formation of superficial skin layers), as well as differences in the treatment efficacy between transplantation of cells obtained from a healthy person (allogeneic graft) and transplantation of cells obtained from the diabetic host (autologous graft). Various modifications of MSC therapy lead to more rapid wound healing, and this therapy may be a breakthrough in the treatment of chronic wounds in diabetic patients. in this article, we reviewed the up-to-date knowledge on the use of MSC in the treatment of hard-to-heal wounds in diabetes. [ABSTRACT FROM AUTHOR]

• Assembly gaps were studied for heat transfer of heat pipe solid-core reactor. • Thermal contact resistance is highly related to the size of initial assembly gap. • The reactor startup was analyzed considering the gap effects. • Attention should be paid on the assembly gap while designing the reactor. The solid-core nuclear reactor cooled by the heat pipe has received extensive attention in recent years. It is mainly applied in the space reactor power system which can supply ample energy for the demand of the surface power on extraterrestrial planets. Based on this technology, a number of reactor designs have been proposed since 1990 s. Unlike liquid-cooled reactors, the heat transfer in a solid-core reactor has to go through the assembly gaps which could cause the considerable thermal contact resistance. The thermal contact resistance might limit the heat transfer and change with the expansion of the fuel pin in the core during the startup process. A numerical simulation was conducted to calculate the unsteady startup process of the core and analyze the influence of the thermal contact resistance caused by the assembly gaps. The results showed that the size of the initial assembly gaps might change the way of the heat transfer in the core and determine the temperature difference between the core and the heat pipe. [ABSTRACT FROM AUTHOR]

The dark sleeper, Odontobutis obscura (O. obscura), is a commercially important species of freshwater sleeper native to East Asia. However, its molecular biology system is unexplored, including the interferon (IFN) signaling pathway, which is crucial to the antiviral response. In this study, we characterised the IFN regulation pattern of dark sleeper interferon regulatory factor 3 (OdIRF3), supplementing evidence of the conservation of this classical pathway in fish. First, the open reading frame (ORF) of OdIRF3 was cloned from the liver tissue by Rapid amplification of cDNA ends (RACE). Amino acid sequence analysis suggested that OdIRF3 is homologous with other fish IRF3 and that the N-terminal DNA-binding domain (DBD) and the C-terminal IRF-association domain (IAD) are conserved. Then, the cellular distribution demonstrated that OdIRF3 is located in the cytoplasm region and transfers into the nuclear region under stimulation. For the function identification, OdIRF3 activated several types of IFN promoters and induced downstream interferon stimulated genes (ISGs) expression. Finally, the overexpression of OdIRF3 significantly decreased viral proliferation. Taken together, these data systematically characterised the sequence, cellular location, and function in IFN expression of OdIRF3, shedding light on the molecular biology mechanism of the dark sleeper. • OdIRF3 promotes fish IFNs and ISGs' transcription. • OdIRF3 locates at cytoplasm region and transfers into nuclear region by stimulation. • Overexpression of OdIRF3 suppresses SVCV replication. [ABSTRACT FROM AUTHOR]

The intercellular communication mediated by extracellular vesicles (EVs) has gained international interest during the last decade. Interfering with the mechanisms regulating this cellular process might find application particularly in oncology where cancer cell‐derived EVs play a role in tumour microenvironment transformation. Although several mechanisms were ascribed to explain the internalization of EVs, little is our knowledge about the fate of their cargos, which are crucial to mediate their function. We recently demonstrated a new intracellular pathway in which a fraction of endocytosed EV‐associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into the nucleoplasmic reticulum. Silencing tetraspanin CD9 both in EVs and recipient cells strongly decreased the endocytosis of EVs and abolished the nuclear transfer of their cargos. Here, we investigated whether monovalent Fab fragments derived from 5H9 anti‐CD9 monoclonal antibody (referred hereafter as CD9 Fab) interfered with these cellular processes. To monitor the intracellular transport of proteins, we used fluorescent EVs containing CD9‐green fluorescent protein fusion protein and various melanoma cell lines and bone marrow‐derived mesenchymal stromal cells as recipient cells. Interestingly, CD9 Fab considerably reduced EV uptake and the nuclear transfer of their proteins in all examined cells. In contrast, the divalent CD9 antibody stimulated both events. By impeding intercellular communication in the tumour microenvironment, CD9 Fab‐mediated inhibition of EV uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti‐melanoma therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: Production of transgenic animals is still a low-efficiency biotechnology, and simple alternatives should be used to improve the rate of transgenic bovine production by nuclear transfer. One such alternative is selecting the appropriate donor cell type and transfection method. Objective: To investigate the effect of cell type (fetal or adult fibroblasts, and cumulus cells), and gene transfer method (lipofection and lentiviral transduction) on the incorporation, expression, and fluorescence intensity of transgene on bovine cells analyzed by flow cytometry. Methods: Fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) were transfected using lipofection, or transduced using lentiviral particles produced with Green Fluorescent Protein (GFP) expressing plasmids, and analyzed by flow cytometry. Results: Lentiviral transduction resulted in higher transgene expression rates for all cell types (FF: 88.8 ± 0.98; AF: 91.6 ± 2.96; CC: 60.7% ± 14.7) compared to lipofection (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65; CC: 3.9% ± 1.97). Cumulus cells showed lower transgene expression rates than the other cell types. Regarding fluorescence intensity, there was no significant difference between lipofection and lentiviral transduction; in both treatments, higher fluorescence intensity was obtained when adult cells were used instead of fetal cells. Conclusion: Gene transfer efficiency varies according to cell type, and gene transfer method, with lentiviral transduction achieving higher transgene expression rate, and adult fibroblasts showing better transgene expression. [ABSTRACT FROM AUTHOR]

Substantial rates of fetal loss plague all in vitro procedures involving embryo manipulations, including human-assisted reproduction, and are especially problematic for mammalian cloning where over 90% of reconstructed nuclear transfer embryos are typically lost during pregnancy. However, the epigenetic mechanism of these pregnancy failures has not been well described. Here we performed methylome and transcriptome analyses of pig induced pluripotent stem cells and associated cloned embryos, and revealed that aberrant silencing of imprinted genes, in particular the retrotransposon-derived RTL1 gene, is the principal epigenetic cause of pregnancy failure. Remarkably, restoration of RTL1 expression in pig induced pluripotent stem cells rescued fetal loss. Furthermore, in other mammals, including humans, low RTL1 levels appear to be the main epigenetic cause of pregnancy failure. [ABSTRACT FROM AUTHOR]

Abstract The immune regulatory properties of IL-33 have indicated that this cytokine has the capacity to target several immune cells under a variety of immunological responses, including overt inflammation and tolerance. Due to its versatile mechanistics, we sought to investigate the role of IL-33 on mesenchymal stem cells (MSC), a population of cells with recognizable modulatory functions. Our data indicates that IL-33 does not affect the expression of classical MSC markers such as CD29, CD44 and CD73, or the lack of CD45, CD11b and CD117. Also, we found that IL-33 greatly induces iNOS expression and stimulates the secretion of TGF-β and IL-6. Next, we decided to test IFN-γ/IL-33-treated MSC using a skin transplantation model. Our data indicate that allogeneic skin-grafted animals treated with IFN-γ/IL-33-modulated MSC reject as controls. Complementing this finding, we observed that ex vivo re-stimulated draining lymph nodes (dLN) cells from these mice secrete lower amounts of IFN-γ and a slightly higher amount of IL-17. Beside a reduction in CD4+ and CD8+ T cells number, we preliminarily found an increment in the frequencies of CD4+Foxp3+IL-17+ T cells. Altogether, our data propose that IL-33 and IFN-γ modulate MSC phenotype and function, most likely targeting Th1/Th17 axis. [ABSTRACT FROM AUTHOR]

Background/Aims: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells' affects SCNT embryos development and the crosstalk between epigenetic signals. Results: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos. [ABSTRACT FROM AUTHOR]

Highlights • Handmade cloning (HMC), an economical way to multiply elite buffalo. • More than 15 buffalo have been cloned using HMC. • Frozen semen-derived somatic cells were used to preserve high genetic merit bulls. • Cloned buffalo have produced progenies indicating that buffalo clones are fertile. • HMC is a valued reproductive technique in buffalo. Abstract The first birth of a cloned animal produced through the Handmade cloning (HMC) technique was reported more than 15 years ago in cattle. This method of somatic cell nuclear transfer (SCNT) has subsequently been evolving as a much simpler alternative to the classical micromanipulator-based SCNT. Several farm animal species such as cattle, buffalo, pigs, sheep, and goats have been successfully cloned using HMC. In buffalo, HMC technique is now well established, and several births of cloned calves have been reported by us. Several factors such as source of somatic cells, quality of recipient oocytes, cell cycle stage prior to SCNT, electrofusion and culture conditions, and epigenetic status of somatic cells, have been optimized leading to the production of good quality cloned embryos. The preservation through cloning of proven breeding bulls that have died by producing live offspring using somatic cells isolated from frozen semen as donor cells and birth of a cloned calf from urine-derived cells are impressive examples of the success of HMC in buffalo. In conclusion, HMC is a valued reproductive technique in buffalo that offers the opportunity to make multiple copies of highly valuable animals, particularly proven breeding bulls. In this review, there is a discussion of the advancement of the HMC technique in buffalo and factors responsible for the efficient production of cloned embryos. [ABSTRACT FROM AUTHOR]

Abstract: A wide variety of assisted reproductive techniques (ARTs) are available to aid in managing aspects of equine reproduction. Embryo recovery and transfer can be used to obtain more than one foal per mare per year, and to obtain foals from mares that cannot carry a foal to term. Oocyte recovery and either transfer to the oviduct of an inseminated recipient mare (oocyte transfer), or intracytoplasmic sperm injection (ICSI) and embryo culture can be used to obtain foals from mares with some types of subfertility, such as problems of the tubular tract. ICSI can be used to obtain foals when sperm number or quality is low. Because of its ease of use for the mare owner and efficiency, oocyte recovery and ICSI is being used in some cases for management of normally fertile mares and stallions. Oocytes can be recovered from live mares by the referring veterinarian, and shipped overnight to a laboratory for ICSI, without any decrease in oocyte or embryo viability. In case of unexpected death of a mare, ovaries or oocytes can be transported to the ICSI laboratory for production of embryos. Embryos produced both in vitro and in vivo can be biopsied to determine their genetic makeup before they are transferred. Equine embryos can be vitrified successfully; collapse of the blastocoele cavity allows efficient vitrification of expanded blastocysts. In contrast, cryopreservation of unfertilized equine oocytes still has low success. Genetics of valuable animals can be preserved via nuclear transfer (cloning) and several commercial companies offer this service clinically. [ABSTRACT FROM AUTHOR]

Background: Mitochondrial replacement, a form of nuclear transfer, has been proposed as a germline therapy to prevent the transmission of mitochondrial diseases. Mitochondrial replacement therapy has been licensed for clinical application in the UK, and already carried out in other countries, but little is known about negative or unintended effects on the health of offspring born using this technique.Objective and Rationale: Studies in invertebrate models have used techniques that achieve mitochondrial replacement to create offspring with novel combinations of mitochondrial and nuclear genotype. These have demonstrated that the creation of novel mitochondrial-nuclear interactions can lead to alterations in offspring characteristics, such as development rates, fertility and longevity. However, it is currently unclear whether such interactions could similarly affect the outcomes of vertebrate biomedical studies, which have sought to assess the efficacy of the replacement therapy.Search Methods: This systematic review addresses whether the effects of mitochondrial replacement on offspring characteristics differ in magnitude between biological (conducted on invertebrate models, with an ecological or evolutionary focus) and biomedical studies (conducted on vertebrate models, with a clinical focus). Studies were selected based on a key-word search in 'Web of Science', complemented by backward searches of reviews on the topic of mitochondrial-nuclear (mito-nuclear) interactions. In total, 43 of the resulting 116 publications identified in the search contained reliable data to estimate effect sizes of mitochondrial replacement. We found no evidence of publication bias when examining effect-size estimates across sample sizes.Outcomes: Mitochondrial replacement consistently altered the phenotype, with significant effects at several levels of organismal performance and health, including gene expression, anatomy, metabolism and life-history. Biomedical and biological studies, while differing in the methods used to achieve mitochondrial replacement, showed only marginally significant differences in effect-size estimates (-0.233 [CI: -0.495 to -0.011]), with larger effect-size estimates in biomedical studies (0.697 [CI: 0.450-0.956]) than biological studies (0.462 [CI: 0.287-0.688]). Humans showed stronger effects than other species. Effects of mitochondrial replacement were also stronger in species with a higher basal metabolic rate. Based on our results, we conducted the first formal risk analysis of mitochondrial replacement, and conservatively estimate negative effects in at least one in every 130 resulting offspring born to the therapy.Wider Implications: Our findings suggest that mitochondrial replacement may routinely affect offspring characteristics across a wide array of animal species, and that such effects are likely to extend to humans. Studies in invertebrate models have confirmed mito-nuclear interactions as the underpinning cause of organismal effects following mitochondrial replacement. This therefore suggests that mito-nuclear interactions are also likely to be contributing to effects seen in biomedical studies, on vertebrate models, whose effect sizes exceeded those of biological studies. Our results advocate the use of safeguards that could offset any negative effects (defining any unintended effect as being negative) mediated by mito-nuclear interactions following mitochondrial replacement in humans, such as mitochondrial genetic matching between donor and recipient. Our results also suggest that further research into the molecular nature of mito-nuclear interactions would be beneficial in refining the clinical application of mitochondrial replacement, and in establishing what degree of variation between donor and patient mitochondrial DNA haplotypes is acceptable to ensure 'haplotype matching'. [ABSTRACT FROM AUTHOR]

The environmental polycyclic aromatic hydrocarbons (PAH) and dioxins are carcinogens and their adverse effects have been largely attributed to the activation of AhR. Hesperetin is a flavonone found abundantly in citrus fruits and has been shown to be a biologically active agent. In the present study, the effect of hesperetin on the nuclear translocation of AhR and the downstream gene expression was investigated in MCF-7 cells. Confocal microscopy indicated that 7, 12-dimethylbenz[α]anthracene (DMBA) or 2,3,7,8-tetrachlorodibenzo- p- dioxin (TCDD) -induced nuclear translocation of AhR was deterred by hesperetin treatment. The reduced nuclear translocation could also be observed in Western analysis. Reporter-gene assay further illustrated that the induced XRE transactivation was weakened by the treatment of hesperetin. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay demonstrated that the gene expressions of CYP1A1 , 1A2 , and 1B1 followed the same pattern of AhR translocation. These results suggested that hesperetin counteracted AhR transactivation and suppressed the downstream gene expression. [ABSTRACT FROM AUTHOR]

In 2016, the New Scientist announced the birth and good health of the world’s first baby conceived using spindle nuclear transfer (SNT). The story was immediately circulated worldwide. In this article, we analyze 39 articles published within the first 48 hours of the announcement, in the Mexican, British, and U.S. press. These articles constitute the initial press reactions to the announcement, and as such, they offer a narrative ground on which SNT could thereafter be discussed. We argue that as a media event, the articles performed the task of rendering SNT, a “cultural novelty,” as culturally and technologically feasible. [ABSTRACT FROM AUTHOR]

The successful production of animals and embryonic stem cells using somatic cell nuclear transfer (SCNT) has demonstrated the unmatched nuclear reprogramming capacity of the oocyte and helped prove the degree of plasticity of differentiated cells. The introduction of transcription factors to generate induced pluripotent stem cells (iPSCs) displaced SCNT and, due to its ease of implementation, became the method of choice for cell reprogramming. Nonetheless, iPSC derivation remains inefficient and stochastic. This review article focuses on using the oocyte as a source of reprogramming factors, comparing the SCNT and iPSC mechanisms for remodeling chromatin and acquiring pluripotency. [ABSTRACT FROM AUTHOR]

Purpose: Human oocytes are arguably one of the most important cell types in humans, yet they are one of the least investigated cells. Because oocytes are limited in number, the use of high-quality oocytes is almost entirely in reproduction. Furthermore, regulatory hurdles for research on gametes and regulations on funding related to research on gametes present significant obstacles to research and the advancement of reproductive treatments. Here we report the outcomes of the largest compensated oocyte donation program for research in the USA to date, and probably worldwide.Methods: Women who participated in oocyte donation for research between 2008 and 2017 were contacted in a phone interview and completed a standardized questionnaire.Results: Of 114 participants, 98 oocyte donors completed donation, donating 1787 mature MII oocytes and a total of 86 skin biopsies. Complication rate, including minor complications, of oocyte donation was 8/98, or 8.1%, for which two involved follow-up. Fifty-seven donors answered questions about their experience. Participants were incentivized primarily by money and a desire to help others and reported an overall favorable experience. Most, but not all, human subjects recalled that they had donated for research, and approximately half recalled that their oocytes were being used specifically for stem cell research.Conclusions: Compensated oocyte donation provides a reliable path to obtaining high-quality oocytes for research and is reviewed favorably by oocyte donors. The continuation of programs that offer compensation for oocyte donation is invaluable to continued progress and advancements in stem cell research and human embryology, and for the advancement of novel reproductive treatments. [ABSTRACT FROM AUTHOR]

Mitochondrial/nuclear transfer (M/NT) to avoid the transmission of serious mitochondrial disease raises complex and challenging ethical, legal and social issues (ELSI). In February 2015, the United Kingdom became the first country in the world to legalize M/NT, making the heated debate surrounding this technology even more relevant. This critical interpretive review identified 95 relevant papers discussing the ELSI of M/NT, including original research articles, governmentcommissioned reports, editorials, letters to editors and research news. The review presents and synthesizes the arguments present in the literature in relation to the most commonly raised themes: terminology; identity, relationships and parenthood; potential harm; reproductive autonomy; available alternatives; consent; impact on specific interest groups; resources; "slippery slope"; creation, use and destruction of human embryos; and beneficence. The review concludes by identifying those ELSI that are specific to M/NT and by calling for follow-up longitudinal clinical and psychosocial research in order to equip future ELSI debate with empirical evidence. [ABSTRACT FROM AUTHOR]

Abstract: We investigated whether high‐quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis‐shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis‐shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis‐shapened aged oocytes. In an attempt to find out why high‐quality oocytes maintain a round shape whereas poorer oocytes become mis‐shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis‐shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high‐quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis‐shapened oocytes. [ABSTRACT FROM AUTHOR]

Summary: This paper assesses whether cloning horses is ethical by reviewing ethical arguments against cloning of nonequine species and determining whether they apply to horses, analysing ethical arguments about horse cloning which do not apply to noncompetitive species and considering the ethical dilemmas faced by veterinarians involved in horse cloning. The author concludes that concerns about the health and welfare of cloned horses render the technique ethically problematic and that the onus is on those providing commercial equine cloning services to collate data and provide a stronger evidence base for ethical decision‐making. [ABSTRACT FROM AUTHOR]

Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein (pGFAP) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreERl'/LCMV-EGFPLoxF and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreERn) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that Cre£P7-mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreER/EGFPIj, xP recombination system via SCNT. [ABSTRACT FROM AUTHOR]

Buffalo (Bubalus bubalis) is a major source of milk, meat, and draught power in many developing countries in Asia. Animal cloning holds a lot of potential for fast multiplication of elite buffaloes and conservation of their valuable germplasm. Although the progress of buffalo cloning has been slow in comparison to cattle or pig, several breakthroughs were reported in buffalo cloning such as the production of cloned calves from somatic cells isolated from over one-decade old frozen–thawed semen or from urine-derived cells. Since the initiation of buffalo cloning, several approaches have been tried to refine nuclear transfer protocols. This has resulted in increasing the blastocyst production rate and improving their quality leading to an increase in live birth rate. In this review, we discuss current developments in buffalo cloning, its challenges, and the future roadmap. [ABSTRACT FROM AUTHOR]

Abstract: Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs) technology are two classical reprogramming methods. Donor cell types can affect the reprogramming results in the above two methods. We here used porcine embryonic fibroblasts (PEFs) and adult porcine ear skin fibroblasts (APEFs) and adipose‐derived stem cells (ADSCs) as donor cells for SCNT and source cells for iPSCs to study their in vitro developmental capability and colony‐formation efficiency, respectively. For SCNT, fusion and cleavage rate has no significant difference among PEFs, ADSCs and APEFs. The rate and total cell number of blastocysts in the APEF group were significant lower than that in PEFs and ADSCs. For transcription factor‐mediated reprogramming, the reprogramming efficiency of ADSCs were significantly higher than PEFs and APEFs and there is no significant difference between PEFs and APEFs. Furthermore, PEFs, APEFs and ADSCs can be used to generate iPSCs. Fianlly, somatic cloned pigs could still be successfully generated from APEFs, suggesting terminally differentiated aging adult somatic cells could be reprogrammed into a totipotent state. Considering the easy availability of animal tissue and the costs of establishing cell lines, aging porcine ear fibroblasts can support nuclear transfer‐mediated and transcription factor‐based reprogramming. [ABSTRACT FROM AUTHOR]

Intergenomic gene transfer (IGT) is continuous in the evolutionary history of plants. In this field, most studies concentrate on a few related species. Here, we look at IGT from a broader evolutionary perspective, using 24 plants. We discover many IGT events by assessing the data from nuclear, mitochondrial and chloroplast genomes. Thus, we summarize the two roles of the mitochondrion: a source and a pool. That is, the mitochondrion gives massive sequences and integrates nuclear transposons and chloroplast tRNA genes. Though the directions are opposite, lots of likenesses emerge. First, mitochondrial gene transfer is pervasive in all 24 plants. Second, gene transfer is a single event of certain shared ancestors during evolutionary divergence. Third, sequence features of homologies vary for different purposes in the donor and recipient genomes. Finally, small repeats (or micro-homologies) contribute to gene transfer by mediating recombination in the recipient genome. [ABSTRACT FROM AUTHOR]

Pancreatic islet transplantation is a promising treatment for type 1 diabetes (T1D). Interleukin-35 (IL-35) is a recently discovered cytokine that exhibits potent immunosuppressive functions. However, the role of IL-35 in islet transplant rejection remains to be elucidated. In this study, we isolated islet cells of BALB/c mouse and purified CD4+ T cell subsets of a C57BL/6 mouse. The model for islet transplantation was established in vitro by co-culture of the islet cells and CD4+ T cells. IL-35 (20 ng/ml) was administered every other day. Following co-culture, the islet function and Treg/Th17 ratio were analyzed on days 1, 3, and 5. Furthermore, the Th17/Treg ratio was modulated (1:0–2), and the function of islet cells as well as proliferation of Th17 cells were analyzed. T cell sorting was performed using the magnetic bead sorting method; Treg and Th17 count using flow cytometry; cell proliferation detection using the carboxyfluorescein diacetate succinimidyl ester (CFSE) method, and islet function test using the sugar stimulation test. Results showed that Th17 counts increased in the co-culture system. However, after administration of IL-35, the number of Treg cells increased significantly compared to that in the control group (50.7% of total CD4+ T cells on day 5 in IL-35 group vs. 9.5% in control group) whereas the proliferation rate of Th17 cells was significantly inhibited (0.3% in IL-35 group vs. 7.2% in control group on day 5). Reducing the Th17/Treg ratio significantly improved the function of transplanted islets. Treg inhibited Th17 proliferation and IL-35 enhanced this inhibitory effect. IL-35 mitigates the function of murine transplanted islet cells via regulation of the Treg/Th17 ratio. This might serve as a potential therapeutic strategy for in-vivo islet transplant rejection and T1D. [ABSTRACT FROM AUTHOR]

Embryonic and placental development is highly orchestrated by epigenetic processes. Disruptions in normal placental development, commonly observed in pregnancies produced by nuclear transfer, are associated with abnormal gene expression and altered epigenetic regulation of imprinted and vital placental genes. The objective of this study was to evaluate expression and epigenetic regulation of the imprinted gene TSSC4 in cotyledonary and intercotyledonary tissues from day 60 pregnancies produced by embryo transfer (ET), in vitro fertilization (IVF) and nuclear transfer (NT) in cattle. TSSC4 expression was reduced by 30% in cotyledons at 60 days of gestation in the NT group. The proximal promoter region of TSSC4 showed an increase in the permissive histone mark (H3K4me2) and a reduction in the inhibitory histone mark (H3K9me2) in the cotyledons produced by NT, in relation to cotyledons produced by embryo transfer. Interestingly, H3K9me2 was also significantly reduced in cotyledons produced by IVF, compared to the ET controls. DNA methylation, in CpG-rich regions located at the proximal promoter region and the coding region of TSSC4 did not differ. These results suggest that the reduction in TSSC4 expression, observed following NT, can not be explained by the histone changes investigated in the proximal promoter region of the gene, or by changes in methylation in three regions evaluated. Also, a decrease in the levels of H3K9 dimethylation in IVF samples, indicate that in vitro culturing could corroborate with the alterations seen in the NT group. [ABSTRACT FROM AUTHOR]

Epigenetic abnormalities in cloned animals are caused by incomplete reprogramming of the donor nucleus during the nuclear transfer step (first reprogramming). However, during the second reprogramming step that occurs only in the germline cells, epigenetic errors not corrected during the first step are repaired. Consequently, epigenetic abnormalities in the somatic cells of cloned animals should be erased in their spermatozoa or oocytes. This is supported by the fact that offspring from cloned animals do not exhibit defects at birth or during postnatal development. To test this hypothesis in cloned cattle, we compared the DNA methylation level of two imprinted genes ( H19 and PEG3) and three non-imprinted genes ( XIST, OCT4 and NANOG) and two repetitive elements (Satellite I and Satellite II) in blood and sperm DNAs from cloned and non-cloned bulls. We found no differences between cloned and non-cloned bulls. We also analyzed the DNA methylation levels of four repetitive elements (Satellite I, Satellite II, Alpha-satellite and Art2) in oocytes recovered from cloned and non-cloned cows. Again, no significant differences were observed between clones and non-clones. These results suggested that imprinted and non-imprinted genes and repetitive elements were properly reprogramed during gametogenesis in cloned cattle; therefore, they contributed to the soundness of cloned cattle offspring. [ABSTRACT FROM AUTHOR]

Objective: We recently demonstrated spatial regionalization of maternal transcripts and proteins within unfertilized ovine oocyte. Here, we investigated the likelihood of oocyte polarity for the first time in bovine. Materials and Methods: In this experimental study, in vitro matured bovine oocytes were used for manual bisection [into oocyte halve that were near-to (HNS) and far-from (FS) spindle] or trisection [into MII-spindle (S), the spindle-side half (NS), and the distal half unassociated with the spindle (FS)]. Prepared pools of oocyte substructures were used for comparative quantitative real-time polymerase chain reaction (RT-qPCR). To map the possible preferential sperm entry point (SEP), the spatial relationship between SEP and MII-spindle was measured 5 hours post-fertilization. Results: The proportional amount of maternal mRNA in S oocyte fragment was estimated to be 6 to 11-fold higher than NS and FS counterparts. The relative abundances of Nanog, Oct4, Fgf4 and Tead4 were significantly higher in HNS oocyte fragment compared t0 FS. The relative abundances of Ctnb, Carm1, Rex1, Sox2 and Cdx2 were comparable between HNS and NS oocyte fragments. FS oocyte fragment possessed significantly higher transcripts of Gata4 compared to HNS. The distribution of certain transcripts related to pluripotency and lineage commitment were different depending upon the region of the oocyte; either enriched at S (Tead4, Nanog, Ctnb and Sox2), NS (Oct4), or FS (Gata6). The SEP in almost (90%) fertilized oocytes was located in MII-hemisphere. Conclusion: The observation of spatial restriction of mRNAs and SEP within MII-oocyte may indicate that the principal forces of oocyte polarity are evolutionary conserved. This may in turn highlight the need for refinements in the methodology of intracytoplasmic sperm injection (where a sperm is injected far from the MII-spindle) and somatic cell nuclear transfer (where a major amount of regulative mRNAs that are associated with MIIspindle is removed during enucleation). [ABSTRACT FROM AUTHOR]

Nuclear transfer procedures have been recently applied for clinical and research targets as a novel assisted reproductive technique and were used for increasing the oocyte activity during its growth and maturation. In this review, we summarized the nuclear transfer technique for germinal vesicle stage oocytes to reconstruct the maturation of them. Our study covered publications between 1966 and August 2017. In result utilized germinal vesicle transfer techniques, fusion, and fertilization survival rate on five different mammalian species are discussed, regarding their potential clinical application. It seems that with a study on this method, there is real hope for effective treatments of old oocytes or oocytes containing mitochondrial problems in the near future. [ABSTRACT FROM AUTHOR]

During the past decade the need for Artificial Reproductive Techniques in felids has greatly increased. Mostly, this is a result of growing expectations that these techniques may be applied in conservation biology and thereby contribute to saving wild felids from extinction. In this article we describe three most common methods of obtaining embryos in vitro in the domestic cat and its wild relatives: classic in vitro fertilisation, in vitro fertilisation by intracytoplasmic sperm injection and somatic cell nuclear transfer. Each of the methods provides a cleavage rate of around 50% and approx. 20% of embryos develop to the blastocyst stage. After the transfer of embryos produced by these methods, scientists obtained living offspring of the domestic cat, as well as several wild cats: the tiger, serval, fishing cat, caracal, ocelot, wild cat, sand cat, black-footed cat and the oncilla. These successes, in spite of the low efficiency of the discussed methods, are promising and suggest that biotechniques of reproduction will be valuable tools in the protection of wild species. Somatic cell nuclear transfer will allow to sustain the narrow gene pool in the critically endangered felids. For these reasons it is necessary to conduct further research on the optimization of artificial reproduction techniques in cats [ABSTRACT FROM AUTHOR]

Background: The use of nuclear transfer (NT) has been proposed as a novel reproductive treatment to overcome the transmission of maternally-inherited mitochondrial DNA (mtDNA) mutations. Pathogenic mutations in mtDNA can cause a wide-spectrum of life-limiting disorders, collectively known as mtDNA disease, for which there are currently few effective treatments and no known cures. The many unique features of mtDNA make genetic counselling challenging for women harbouring pathogenic mtDNA mutations but reproductive options that involve medical intervention are available that will minimize the risk of mtDNA disease in their offspring. This includes PGD, which is currently offered as a clinical treatment but will not be suitable for all. The potential for NT to reduce transmission of mtDNA mutations has been demonstrated in both animal and human models, and has recently been clinically applied not only to prevent mtDNA disease but also for some infertility cases. In this review, we will interrogate the different NT techniques, including a discussion on the available safety and efficacy data of these technologies for mtDNA disease prevention. In addition, we appraise the evidence for the translational use of NT technologies in infertility.Objective and Rationale: We propose to review the current scientific evidence regarding the clinical use of NT to prevent mitochondrial disease.Search Methods: The scientific literature was investigated by searching PubMed database until Jan 2017. Relevant documents from Human Fertilisation and Embryology Authority as well as reports from both the scientific and popular media were also implemented. The above searches were based on the following key words: 'mitochondria', 'mitochondrial DNA'; 'mitochondrial DNA disease', 'fertility'; 'preimplantation genetic diagnosis', 'nuclear transfer', 'mitochondrial replacement' and 'mitochondrial donation'.Outcomes: While NT techniques have been shown to effectively reduce the transmission of heteroplasmic mtDNA variants in animal models, and increasing evidence supports their use to prevent the transmission of human mtDNA disease, the need for robust, long-term evaluation is still warranted. Moreover, prenatal screening would still be strongly advocated in combination with the use of these IVF-based technologies. Scientific evidence to support the use of NT and other novel reproductive techniques for infertility is currently lacking.Wider Implications: It is mandatory that any new ART treatments are first adequately assessed in both animal and human models before the cautious implementation of these new therapeutic approaches is clinically undertaken. There is growing evidence to suggest that the translation of these innovative technologies into clinical practice should be cautiously adopted only in highly selected patients. Indeed, given the limited safety and efficacy data, close monitoring of any offspring remains paramount. [ABSTRACT FROM AUTHOR]

Significant progress in the field of biotechnology has allowed for the use of cloning in animals which is being used: to improve genetic makeup, to rescue endangered species, in tissue engineering and to increase farm animal population. Unfortunately, cloning has been met with failure due to a variety of reasons namely early and late abortions, compromised immune systems, circulatory and respiratory problems and a high rate of fetal death. The reasons of these problems are unknown, but may research groups are attempting to understand the underlying molecular and cellular mechanisms involved in cloning efficiency. Atypical epigenetic re-programming appears to be the primary cause of ineffective cloning. Understanding molecular mechanisms involving key regulatory proteins is pivotal in the success of animal cloning. This review shows the current paradigm involving animal cloning efficiency, and also further elucidates applications to improve animal cloning efficiency. [ABSTRACT FROM AUTHOR]

A time-lapse monitoring system has predictive value for selecting good-quality embryos with the highest implantation potential. Using this new tool, we investigated the developmental potential and developmental kinetics of bovine parthenogenetic (PA) and two types of somatic cell nuclear transfer (NT) embryos. Bovine non-transgenic ear cells (bECs) or transgenic cells (bTGCs) were used as donor cells. The cleavage and blastocyst development rates did not significantly differ among the PA, NT-bEC, and NT-bTGC groups, and first cleavage occurred an average of 19.3 hours ( n = 70), 21.6 hours ( n = 60), and 21.3 hours ( n = 62) after activation, respectively (20.4 hours [ n = 192] for all embryos). When embryos were classified into early cleaving (≤20 hours) and late cleaving (>20 hours) groups, the blastocyst formation rate was much higher in the early cleaving groups (PA, 46%; NT-bEC, 50%; NT-bTGC, 39%) than in the late cleaving groups (PA, 18%; NT-bEC, 23%; NT-bTGC, 28%), while the percentage of embryos whose development was blocked between the two- and eight-cell stages was increased in the late cleaving groups. The percentage of embryos classified as early cleaving with a normal morphology was twofold higher in the PA group (20.0%, n = 14) than in the NT-bTGC group (9.7%, n = 6). The timing of each developmental stage varied widely; the timing of first cleavage varied from 7.6 hours in the PA group to 34.5 hours in the NT-bEC group and the timing of expanded/hatching blastocyst appearance varied from 141.6 hours in the PA group to 196.3 hours in the NT-bTGC group, differences of 26.9 and 54.7 hours, respectively (PA>NT-bEC>NT-bTGC). These results demonstrate that time-lapse monitoring provides novel data regarding individual embryo developmental kinetics and helps to predict developmental potential for improved bovine NT embryo selection based on early cleavage (≤20 hours) and normal morphology. [ABSTRACT FROM AUTHOR]

The present study investigated the effects of IVM in hypotonic medium containing reduced (61.6 mM) NaCl compared with isotonic medium containing 108.0mM NaCl (designated L and N respectively) on oocyte maturation and embryonic development in pigs. IVM culture was divided into four periods at 11-h intervals. Oocytes cultured in N for 33 h and then in L for 11 h of IVM (N-N-N-L) showed significantly improved (P,0.05) nuclear maturation of oocytes (75.4-79.0% vs 60.2-85.8%) and blastocyst formation (61.5-66.1% vs 45.2-67.5%) after parthenogenesis (PA) compared with other treatments (L-L-L-L, L-L-L-N, L-L-N-L, N-N-L-L, N-N-L-N, L-L-N-L, L-N-N-L and N-L-N-L). Oocytes matured in L-L-L-L and N-N-N-L had an increased (P,0.05) perivitelline space (11.0-12.5 vs 5.5 μm) and intraoocyte reduced glutathione (GSH) content (1.39-1.41 vs 1.00 pixels per oocyte) relative to oocytes matured in N-N-N-N. Somatic cell nuclear transfer (SCNT) embryos derived from the N-N-N-L treatment had significantly (P,0.05) higher blastocyst formation (53.5%) than embryos derived from Medium-199 (37.4%) and N-N-N-N (41.8%) treatments. Overall, the results demonstrate that maturation of pig oocytes in hypotonic medium with reduced NaCl during the last 11 h of IVM increases the developmental competence of oocytes after PA and SCNT by improving the cytoplasmic microenvironment, including an increased GSH content in IVM oocytes. [ABSTRACT FROM AUTHOR]

Wnt signaling is critical in embryonic development and post-embryonic tissue homeostasis. The aim of the present study was to evaluate the expression levels of canonical Wnt signaling genes in porcine somatic cell nuclear transfer (SCNT) embryos. Quantitative polymerase chain reaction analysis was performed in porcine SCNT embryos, and the results indicated that the temporal expression patterns of canonical signaling genes were similar between in vivo and SCNT embryos from the 2-cell to the blastocyst stage. In addition, aberrant expression in a small number of Wnt signaling genes in SCNT embryos was identified. IWP2, an inhibitor of Wnt processing, was applied to the culture of SCNT embryos. The Wnt signaling pathway in the SCNT blastocysts may be inactivated via IWP2 treatment, reflecting the low expression levels of c-Myc and peroxisome proliferator-activated receptor d. Furthermore, blastocyst hatching was damaged by IWP2 treatment. These findings indicate that the canonical Wnt signaling pathway is important for SCNT embryo development. [ABSTRACT FROM AUTHOR]

Objective: Investigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT). Methods: Porcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3) in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR. Results: Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1, Sox2, and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid. Conclusion: Ascorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus. [ABSTRACT FROM AUTHOR]

Incomplete epigenetic reprogramming of donor cell nuclei is one of the main contributors to the low efficiency of somatic cell nuclear transfer (SCNT). To improve the success of SCNT, somatic cell DNA methylation levels must be reduced to those levels found in totipotent embryonic cells. Recent studies have demonstrated that miR-148a can affect DNA methylation via DNMT1 modulation in various cancers. Therefore, the focus of this study was to examine the influence of miR-148a on DNA methylation in donor cells and in SCNT embryo development. Thus, a stable cell line overexpressing miR-148a was established and used to produce SCNT embryos. Upon examination, DNMT1 was found to be a miR-148a target in porcine fetal fibroblasts (PFF). Furthermore, miR-148a overexpression in PFFs significantly decreased DNMT1 expression and global DNA methylation levels (P < 0.05). Moreover, miRNA-148a expression levels in SCNT embryos were significantly lower at the 2-cell and 4-cell stages when compared to IVF and parthenogenetic embryos. The group overexpressing miRNA-148a also showed a significant increase in blastocyst formation and total cell numbers (P < 0.05). Additionally, miR-148a overexpression altered the immunofluorescence signal of 5-mC and H3K9ac, and enhanced pluripotent gene (Oct4 and Nanog) expression levels during embryo development. These results indicate that miR-148a overexpression enhances the developmental potential of SCNT embryos and modifies epigenetic status. [ABSTRACT FROM AUTHOR]

Background: The protein kinase C (PKC) family comprises central regulators of multiple signal transduction processes and is involved in the progression of many cancers. Nuclear factor Kappa-B (NF-κB) is constitutively expressed in cancer tissues and stimulates the transcription of various tumor-related genes. The present study aims to investigate the clinical significance of PKCα and NF-κB p65 in bladder cancer tissues and the mechanism underlying PKCα induction of bladder cancer cell apoptotic resistance through stimulation of p65 nuclear translocation.Methods: Expression of PKCα and NF-κB subunit p65 was detected in seven bladder cancer cell lines by western blot and in 30 bladder cancer tissue specimens by immunostaining. Immunofluorescence was performed to evaluate p65 nuclear translocation induced by Phorbol 12-myristate 13-acetate (PMA). PKCα/β selective inhibitor Gö6976, PKC pan-inhibitor sotrastaurin, and the PKC siRNA were employed to conduct PKC inhibition/knockdown in bladder cancer cells. Luciferase reporter assays were performed to measure the activity of NF-κB. Flow cytometry and TUNEL analysis were used to assess cell apoptosis.Results: Expression of PKCα and NF-κB was found to positively correlate with tumor progression in 30 tumor tissue specimens. Furthermore, a Pearson's correlation coefficient analysis revealed a positive correlation between PKCα and NF-κB expression. Among the PKC inhibitors, the PKCα/β selective inhibitor Gö6976 yielded the most significant block of PKCα and NF-κB activation by PMA. Knockdown of NF-κB p65 remarkably induced cell apoptosis, but PMA restored p65 expression and significantly suppressed cell apoptosis that was otherwise induced by the p65 knockdown alone.Conclusion: Our study showed that PKCα modulated cell resistance to apoptosis by stimulating NF-κB activation and thus promoted the tumorigenesis of bladder cancer. [ABSTRACT FROM AUTHOR]

Cloning procedures often interfere with conceptus growth and life ex utero, in a set of symptoms known as abnormal offspring syndrome (AOS). The aim of the present study was to compare the developmental pattern of in vivoderived (IVD), IVF-derived and handmade cloning-derived (NT-HMC) Day 225 bovine concepti using established procedures. Pregnancy diagnosis was performed on Day 30 following blastocyst transfer on Day 7. Conceptus morphometry was assessed by ultrasonography on Day 51, and on Day 225 pregnant cows were killed for morphological examination of concepti. Pregnancy outcome was similar between groups, with greater pregnancy losses in the first trimester (70.6%) and smaller fetuses on Day 51 in the NT-HMC group than in the IVD (14.3%) and IVF (20.0%) groups. However, NT-HMC-derived concepti were twofold larger on Day 225 of gestation than controls. A higher frequency (63.5%) of placentomes larger than the largest in the IVD group was observed in the NT-HMC group, which may be relevant to placental function. Conceptus traits in the IVF group were similar to the IVD controls, with only slight changes in placentome types. Morphological changes in cloned concepti likely affected placental function and metabolism, disrupting the placental constraining mechanism on fetal growth in mid- to late pregnancy. [ABSTRACT FROM AUTHOR]

For reprogramming a somatic nucleus during mammalian cloning, metaphase of the second meiotic division (MII) oocytes has been widely used as recipient cytoplasm. High activity of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) is believed to accelerate the remodeling and/or reprogramming of a somatic nucleus introduced into the ooplasm by somatic cell nuclear transfer. We demonstrated previously that the first step in nuclear reprogramming is not directly regulated by MPF and MAPK because activated oocytes in which MPF activity is diminished and MAPK activity is maintained can develop to the blastocyst stage after receiving an M phase somatic nucleus in bovine cloning. In this study, our aim was to test whether MAPK activity is necessary for the first step in nuclear reprogramming and/or chromatin remodeling (phosphorylation of histone H3 at Ser3, trimethylation of histone H3 at Lys 9, and acetylation of histone H3 at Lys14) in bovine somatic cloning. We found that it was not necessary, and neither was MPF activity. [ABSTRACT FROM AUTHOR]

Background: Embryo splitting or twinning has been widely used in veterinary medicine over 20 years to generate monozygotic twins with desirable genetic characteristics. The first human embryo splitting, reported in 1993, triggered fierce ethical debate on human embryo cloning. Since Dolly the sheep was born in 1997, the international community has acknowledged the complexity of the moral arguments related to this research and has expressed concerns about the potential for reproductive cloning in humans. A number of countries have formulated bans either through laws, decrees or official statements. However, in general, these laws specifically define cloning as an embryo that is generated via nuclear transfer (NT) and do not mention embryo splitting. Only the UK includes under cloning both embryo splitting and NT in the same legislation. On the contrary, the Ethics Committee of the American Society for Reproductive Medicine does not have a major ethical objection to transferring two or more artificially created embryos with the same genome with the aim of producing a single pregnancy, stating that 'since embryo splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable'.Objective and Rationale: Embryo splitting has been introduced successfully to the veterinary medicine several decades ago and today is a part of standard practice. We present here an overview of embryo splitting experiments in humans and non-human primates and discuss the potential of this technology in assisted reproduction and research.Search Methods: A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies on embryo splitting in humans and non-human primates. 'Embryo splitting' and 'embryo twinning' were used as the keywords, alone or in combination with other search phrases relevant to the topics of biology of preimplantation embryos.Outcomes: A very limited number of studies have been conducted in humans and non-human primates. The published material, especially the studies with human embryos, is controversial. Some reports suggest that twinning technology will find clinical use in reproductive medicine in the future, whereas others conclude the opposite that human twin embryos created in vitro are unsuitable not only for clinical, but also for research, purposes.Wider Implications: The blastomere biopsy technique of embryo splitting seems to be unsuitable for either clinical or research purposes; however, embryo bisection, a preferable method of cloning in veterinary medicine, has not yet been tested on human embryos. [ABSTRACT FROM AUTHOR]

Abnormal placental development is frequent in nuclear transfer (NT) pregnancies and is likely to be associated with altered epigenetic reprogramming. In the present study, fetal and placental measurements were taken on Day 60 of gestation in cows with pregnancies produced by AI, IVF and NT. Placentas were collected and subjected to histological evaluation, the expression of genes important in trophoblast differentiation and expression of the placental imprinted gene pleckstrin homology-like domain, family A, member 2 (PHLDA2), as well as chromatin immunoprecipitation (ChIP) for histone marks within the promoter of PHLDA2. Fewer binucleated cells were observed in NT cotyledons, followed by IVF and AI cotyledons (P<0.05). Expression of heart and neural crest derivatives expressed 1 (HAND1), placental lactogen (PL), pregnancy-associated glycoprotein 9 (PAG-9) and PHLDA2 was elevated in NT cotyledons compared with AI cotyledons. Expression of PHLDA2 was higher in IVF than AI samples (P<0.05). ChIP revealed an increase in the permissive mark dimethylation of lysine 4 on histone H3 (H3K4me2), surprisingly associated with the silent allele of PHLDA2, and a decrease in the inhibitory mark H3K9me2 in NT samples. Thus, genes critical for placental development were altered in NT placentas, including an imprinted gene. Allele-specific changes in the permissive histone mark in the PHLDA2 promoter indicate misregulation of imprinting in clones. Abnormal trophoblast differentiation could have resulted in lower numbers of binucleated cells following NT. These results suggest that the altered expression of imprinted genes associated with NT are also caused by changes in histone modifications. [ABSTRACT FROM AUTHOR]

Nuclear transfer techniques (a.k.a. mitochondrial replacement therapies) are currently under development to provide a route to eliminating particular instances of mitochondrial disease from the germline. Before these kinds of techniques are implemented clinically it is of primary concern that their safety and efficacy is established. In a recent paper, Hyslop et al. (Nature 534:383-386, 2016. doi:10.1038/nature18303) utilized a specific version of pronuclear transfer to investigate the consequences for gene expression in the developing embryo, which may indicate whether or not developmental pathways have been perturbed. However, the study was only able to include a small number of blastocysts within each treatment group, although a larger number of single cell expression profiles from each blastocyst were acquired. Using simulated datasets we show that the size and experimental design of this study cannot provide conclusive evidence that expression profiles of manipulated or control samples are indistinguishable from one another due to low power. These simulations also illustrate why visual inspections of principle component analyses used in the study cannot replace statistical modeling of treatment effects. [ABSTRACT FROM AUTHOR]