Search

Simple Summary: Currently, not enough is known about the effect of histone modification on the epigenetic reprogramming of somatic cells, and the lack of basic study limits the development of somatic cell nuclear transfer technology. The aim of this study was to explore the influence of lysine crotonylation, a newly discovered histone post-translational modification, on the reprogramming of somatic cells from Cashmere goats. The results showed that the crotonylation level was increased in somatic cells with sodium crotonate treatment. At the same time, the treatment of somatic cells improved the cloned embryo cleavage rate. In conclusion, an increasing crotonylation level could promote the reprogramming of somatic cells and cloned embryo development. This finding provides an important reference for future improvements in the efficiency of in vitro Cashmere goat somatic cell nuclear transfer embryo production. Failure in the epigenetic reprogramming of somatic cells is considered the main reason for lower cloned embryo development efficiency. Lysine crotonylation (Kcr) occupies an important position in epigenetic modification, while its effects on somatic cell reprogramming have not been reported. In this study, we detected the influence of sodium crotonate (NaCr) on the Kcr levels in three types of somatic cells (muscle-derived satellite cells, MDSCs; fetal fibroblast cells, FFCs; and ear tip fibroblast cells, EFCs). The three types of somatic cells were treated with NaCr for cloned embryo construction, and the cleavage rates and Kcr, H3K9cr, and H3K18cr levels in the cloned embryos were analyzed. The results showed that the abnormal levels of Kcr, H3K9cr, and H3K18cr were corrected in the treatment groups. Although there was no significant difference in the cloned embryo cleavage rate in the FFC treatment group, the cleavage rates of the cloned embryos in the MDSCs and EFCs treatment groups were increased. These findings demonstrated that the Kcr level was increased with NaCr treatment in somatic cells from Cashmere goat, which contributed to proper reprogramming. The reprogramming of somatic cells can be promoted and cloned embryo development can be improved through the treatment of somatic cells with NaCr. [ABSTRACT FROM AUTHOR]

This study investigated the effect of variation in the number of somaticcell-cloned embryos and their developmental stage at transfer on pregnancy, as well as the influence of the estrus status of recipient pigs on in vivo development of cloned porcine embryos after embryo transfer. For somatic cell nuclear transfer (SCNT), fibroblast cells were obtained from a male porcine fetus. Recipient oocytes were collected from prepubertal gilts at a local abattoir and then cultured. After SCNT, reconstructed embryos of different numbers and developmental stages were transferred into recipient pigs. The developmental stage of the cloned embryos and the number of transferred embryos per surrogate showed no significant differences in terms of the resulting cloning efficiency. However, the pregnancy rate improved gradually as the number of transferred cloned embryos was increased from 100- 150 or 151-200 to 201-300 per recipient. In pre-, peri-, and post-ovulation stages, pregnancy rates of 28.6%, 41.8%, and 67.6% and 16, 52, and 74 offspring were recorded, respectively. The number of cloned embryos and estrus status of the recipient pig at the time of transfer of the cloned embryo affect the efficiency of pig production; therefore, these variables should be particularly considered in order to increase the efficiency of somatic cell pig cloning.

Spider silk, which has remarkable characteristics, has wide application prospects in many fields. Many researchers have explored potential methods for directly producing spider silk proteins and spidroins with mechanical properties or obtaining recombinant spider silk fibers by genetic engineering methods. However, there are still some shortcomings with these methods, such as inability to simulate the fibrosis process of spider silk. In this study, a high glycine/tyrosine protein gene (HGT) promoter originate from sheep was first cloned by PCR. The HGT promoter was ligated into pcDNA3.1 and pcDNA3.1-HGT was obtained. After linking with the synthesized and polymerized gene 4S, a eukaryotic expression vector pcDNA3.1-HGT-4S was constructed using a series of molecular methods. Sheep fibroblasts transfected with the linearized plasmid using a liposome-mediated method were screened with G418 and a transgenic cell line was established. Cells from the transgenic line were used as nuclear donors to construct embryos with somatic cell nuclear transfer (SCNT). Reconstructed embryos derived from transgenic cells were able to develop in vitro successfully. PCR was carried out and results demonstrated that the synthetic spidroin gene 4S had integrated into the embryo genome. In summary, we explored a method and successfully obtained artificial synthetic spidroin gene transgenic sheep cloned embryos with a hair follicle specific promoter by SCNT. Further research is necessary on transgenic sheep with synthetic spidroin genes expressed in hair follicles. [ABSTRACT FROM AUTHOR]

Summary: The low efficiency of somatic cell nuclear transfer (SCNT) greatly limits its application. Compared with the fertilized embryo, cloned embryos display abnormal epigenetic modification and other inferior developmental properties. In this study, small RNAs were isolated, and miR-34c and miR-125b were quantified by real-time PCR; results showed that these micro-RNAs were highly expressed in sperm. The test sample was divided into three groups: one was the fertilized group, one was the SCNT control group (NT-C group), and the third group consisted of SCNT embryos injected with sperm-borne small RNA (NT-T group). The level of tri-methylation of lysine 9 on histone H3 (H3K9me3) at the 8-cell stage was determined by immunofluorescence staining, and the cleavage ratio, blastocyst ratio, apoptotic cell index of the blastocyst and total cell number of blastocysts in each group were analyzed. Results showed that the H3K9me3 level was significantly higher in the NT-C group than in the fertilized group and the NT-T group. The apoptosis index of blastocysts in the NT-C group was significantly higher than that in the fertilized group and the NT-T group. The total cell number of SCNT embryos was significantly lower than that of fertilized embryos, and injecting sperm-borne small RNAs could significantly increase the total cell number of SCNT blastocysts. Our study not only demonstrates that sperm-borne small RNAs have an important role in embryo development, but also provides a new strategy for improving the efficiency of SCNT in rabbit. [ABSTRACT FROM AUTHOR]

Failure in the epigenetic reprogramming of somatic cells is considered the main reason for lower cloned embryo development efficiency. Lysine crotonylation (Kcr) occupies an important position in epigenetic modification, while its effects on somatic cell reprogramming have not been reported. In this study, we detected the influence of sodium crotonate (NaCr) on the Kcr levels in three types of somatic cells (muscle-derived satellite cells, MDSCs; fetal fibroblast cells, FFCs; and ear tip fibroblast cells, EFCs). The three types of somatic cells were treated with NaCr for cloned embryo construction, and the cleavage rates and Kcr, H3K9cr, and H3K18cr levels in the cloned embryos were analyzed. The results showed that the abnormal levels of Kcr, H3K9cr, and H3K18cr were corrected in the treatment groups. Although there was no significant difference in the cloned embryo cleavage rate in the FFC treatment group, the cleavage rates of the cloned embryos in the MDSCs and EFCs treatment groups were increased. These findings demonstrated that the Kcr level was increased with NaCr treatment in somatic cells from Cashmere goat, which contributed to proper reprogramming. The reprogramming of somatic cells can be promoted and cloned embryo development can be improved through the treatment of somatic cells with NaCr.

The term ‘clone’ in animal biotechnology refers to an organism derived from non-sexual reproduction, which is both a direct offspring and a genetic copy of the parent organism. To date, the pig appears to be the most interesting object in cloning research. Somatic cell nuclear transfer in pigs has a wide range of potential applications in various fields of human scientific and economic activities. However, the efficiency of producing cloned embryos in swine is still lower than that of other livestock species, in particular horses and cattle. Somatic cell nuclear transfer is a technically complex multi-stage technology, at each stage of which the pig oocytes, which are more susceptible to changes of surrounding conditions, are affected by various factors (mechanical, physical, chemical). At the stage of oocyte maturation, changes in the cell ultrastructures of the ooplasm occur, which play an important role in the subsequent nuclear reprogramming of the transferred donor cell. Before transfer to the oocyte donor somatic cells are synchronized in the G0/G1 stage of the cell cycle to ensure the normal ploidy of the cloned embryo. When removing the nucleus of pig oocytes maturated in vitro, it is necessary to pay attention to the problem of preserving the viability of cells, which were devoid of their own nuclear material. To perform the reconstruction, a somatic cell is placed, using micro-tools, in the perivitelline space, where the first polar body was previously located, or in the cytoplasm of an enucleated oocyte. The method of manual cloning involves the removal of the oocyte nucleus with subsequent fusion with the donor cell without the use of micromanipulation techniques. The increased sensitivity of oocytes to the environmental conditions causes special requirements for the choice of the system for in vitro culture of cloned pig embryos. In this work, we have reviewed the modern methods used for the production of cloned embryos and identified the technological issues that prevent improving the efficiency of somatic cloning of pigs.

The objective of this study was to investigate the survival and development of porcine cloned embryos vitrified by Cryotop carrier at the zygote, 2- and 4-cell stages. The quality of resultant blastocysts was evaluated according to their total cell number, apoptotic cell rate, reactive oxygen species (ROS) production, glutathione (GSH) content and mRNA expression levels of genes related to embryonic development. The survival rates of zygotes, 2- and 4-cell embryos after vitrification did not differ from those of their fresh counterparts. Vitrification still resulted in significantly decreased blastocyst formation rates of these early-stage embryos. Moreover, the total cells, apoptotic rate, ROS and GSH levels in resultant blastocysts were unaffected by vitrification. The mRNA expression levels of PCNA, CPT1, POU5F1 and DNMT3B in the blastocysts derived from vitrified early-stage embryos were significantly higher than those in the fresh blastocysts, but there was no change in expression of CDX2 and DNMT3A genes. In conclusion, our data demonstrate that the early-stage porcine cloned embryos including zygotes, 2- and 4-cells can be successfully vitrified, with respectable blastocyst yield and quality. • Vitrification had no effect on the viability of porcine cloned embryos at the zygote, 2- and 4-cell stages. • Respectable blastocyst yields were obtained from vitrified early-stage porcine cloned embryos. • The resultant blastocysts exhibited a good quality [ABSTRACT FROM AUTHOR]

In this study, the effect of trichostatin A (TSA)-mediated epigenomic modulation of nuclear donor cells on the in vitro developmental potential of caprine somatic cell cloned embryos was examined. The enucleated ex vivo-matured oocytes were subzonally injected with adult ear skin-derived fibroblast cells exposed or not exposed to TSA (at a concentration of 50 nM). The experiment was designed on the basis of three different approaches to TSA-dependent modulation of donor cell-descended genome: before being used for somatic cell nuclear transfer/SCNT (Group I); immediately after activation of nuclear-transferred (NT) oocytes (Group II); or combined treatment both before being used for SCNT and after activation of NT oocytes (Group III). In the control Group IV, donor cell nuclei have not been treated with TSA at any stage of the experimental design. In TSA-treated Groups I and II and untreated Group IV, cleavage activities of cloned embryos were at the similar levels (80.6%, 79.8% and 77.1%, respectively). But, significant difference was observed between Groups III and IV (85.3 vs. 77.1%). Moreover, in the experimental Groups I and III, the percentages of cloned embryos that reached the blastocyst stages remarkably increased as compared to those noticed in the control Group IV (31.2% vs. 36.7% vs. 18.9%, respectively). In turn, among embryos assigned to Group II, blastocyst formation rate was only slightly higher than that in the control Group IV, but the differences were not statistically significant (25.8% vs. 18.9%). To sum up, TSA-based epigenomic modulation of somatic cell-inherited nuclear genome gave rise to increased competences of caprine cloned embryos to complete their development to blastocyst stages. In particular, sequential TSA-mediated modulation of both nuclear donor cells and activated NT oocytes led to improvement in the blastocyst yields of cloned goat embryos, which can result from enhanced donor cell nuclear reprogrammability. [ABSTRACT FROM AUTHOR]

Copyright of Xinjiang Agricultural Sciences is the property of Xinjiang Agricultural Sciences Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

There is a clinical demand for efficient cryopreservation of cloned camel embryos with considerable logistic and economic advantage. Vitrification of in vivo derived embryos has been reported in camels, but there is no study on vitrification of cloned embryos. Moreover, whether characteristic differences between cloned and in vivo derived embryos imply different vitrification requirement is unresolved. Here, we compared survival, re-expansion and pregnancy rates of cloned embryos vitrified using two commercial vitrification kits (Cryotec and Kitazato), developed basically for human embryos, and a vitrification protocol developed for in vivo camel embryos (CVP). Cloned embryos responded dynamically to vitrification-warming steps in commercial kits, with a flat shrinkage in the final vitrification solution and a quick re-expansion to the original volume immediately after transferring to the isotonic warming solution. Contrarily, full shrinkage was not observed in CVP method, and majority of embryos were still collapsed post-warming. The immediate re-expansion was highly associated and predictive of higher survival and total cell number, and also better redox state of embryos vitrified by Cryotec and Kitazato kits compared to CVP method. Importantly, while 30% blastomere loss, verified by differential dye exclusion test, was tolerated in vitrified embryos, >50% blastomeres loss in non-expanded blastocysts implied the minimal essential cell survival rate for blastocoelic cavity re-expansion in vitrified cloned camel blastocysts, irrespective of vitrification method. A protocol-based exposure of embryos to cryoprotectants indicated that cryoprotectant toxicity, per se , may not be involved in lower cryosurvival of embryos in CVP vs. Cryotec and Kitazato. The initial pregnancy rates were numerically higher in Cryotec and Kitazato frozen transfers compared to fresh transfer (56.3, 60 and 33.3%, respectively), and importantly, a higher percentage of established pregnancies in vitrified groups passed the critical 3 months period of early embryonic loss compared to sibling fresh clone pregnancies (50, 40, and 10%, respectively). Results confirmed the suitability of Cryotec and Kitazato kits for vitrification of cloned camel embryos and that vitrification may improve pregnancy outcome by weeding out poor competent embryos. • A 30% blastomere loss may be tolerated in vitrified cloned camel embryos. • A >50% blastomere loss may not be tolerated in vitrified cloned camel embryos. • Immediate re-expansion is predictive of survival of vitrified cloned camel embryos. • Vitrification solutions developed for in vivo embryos may not be appropriate for in vitro embryos. • Vitrification improves the pregnancy outcome of cloned camel embryos. [ABSTRACT FROM AUTHOR]

The present study sought to examine whether trichostatin A (TSA)‐assisted epigenetic transformation of porcine bone marrow (BM)‐derived mesenchymal stem cells (BM‐MSCs) affects the transcriptional activities of pluripotency‐related genes (Oct4, Nanog, c‐Myc, Sox2 and Rex1), multipotent stemness‐related gene (Nestin) and anti‐apoptotic/anti‐senescence‐related gene (Survivin). Epigenetically transformed or non‐transformed BM‐MSCs that had been transcriptionally profiled by qRT‐PCR and had been analysed for different stages of apoptosis progression provided a source of nuclear donor cells for the in vitro production of cloned pig embryos. TSA‐mediated epigenomic modulation has been found to enhance the multipotency extent, stemness and intracellular anti‐ageing properties of porcine BM‐MSCs. This has been confirmed by the relative abundances for Nanog, c‐Myc Rex1, Sox2 and Survivin mRNAs in TSA‐exposed BM‐MSCs that turned out to be significantly higher than those of TSA‐unexposed BM‐MSCs. Additionally, TSA‐assisted epigenomic modulation of BM‐MSCs did not impact the caspase‐8 activity, Bax protein expression and the incidence of TUNEL‐positive cells. In conclusion, the considerably elevated quantitative profiles of Sox2, Rex1, c‐Myc, Nanog and Survivin mRNA transcripts seem to trigger improved reprogrammability of TSA‐treated BM‐MSC nuclei in cloned pig embryos that thereby displayed remarkably increased blastocyst formation rates as compared to those noticed for embryos derived from TSA‐untreated BM‐MSCs. [ABSTRACT FROM AUTHOR]

In this study, we first investigated the effects of 3-methyladenine (3-MA), an autophagy inhibitor, and the inducer – rapamycin (RAPA) on the incidence of programmed cell death (PCD) symptoms during in vitro development of porcine somatic cell nuclear transfer (SCNT)-derived embryos. The expression of autophagy inhibitor mTOR protein was decreased in porcine SCNT blastocysts treated with 3MA. The abundance of the autophagy marker LC3 increased in blastocysts following RAPA treatment. Exposure of porcine SCNT-derived embryos to 3-MA suppressed their developmental abilities to reach the blastocyst stage. No significant difference in the expression pattern of PCD-related proteins was found between non-transfected dermal cell and transfected dermal cell groups. Additionally, the pattern of PCD in SCNT-derived blastocysts generated using SC and TSC was not significantly different, and in terms of porcine SCNT-derived embryo development rates and total blastocyst cell numbers, there was no significant difference between non-transfected cells and transfected cells. In conclusion, regulation of autophagy affected the development of porcine SCNT embryos. Regardless of the type of nuclear donor cells (transfected or non-transfected dermal cells) used for SCNT, there was no difference in the developmental potential and quantitative profiles of autophagy/apoptosis biomarkers between porcine transgenic and non-transgenic cloned embryos. These results led us to conclude that PCD is important for controlling porcine SCNT-derived embryo development, and that transfected dermal cells can be utilized as a source of nuclear donors for the production of transgenic cloned progeny in pigs. [ABSTRACT FROM AUTHOR]

Abstract: Pre‐implantation embryo metabolism demonstrates distinctive characteristics associated with the development potential of embryos. We aim to determine if metabolic differences correlate with embryo morphology. In this study, gas chromatography – mass spectroscopy (GC‐MS)‐based metabolomics was used to assess the culture media of goat cloned embryos collected from high‐quality (HQ) and low‐quality (LQ) groups based on morphology. Expression levels of amino acid transport genes were further examined by quantitative real‐time PCR. Results showed that the HQ group presented higher percentages of blastocysts compared with the LQ counterparts (P < 0.05). Metabolic differences were also present between HQ and LQ groups. The culture media of the HQ group showed lower levels of valin, lysine, glutamine, mannose and acetol, and higher levels of glucose, phytosphingosine and phosphate than those of the LQ group. Additionally, expression levels of amino acid transport genes SLC1A5 and SLC3A2 were significantly lower in the HQ group than the LQ group (P < 0.05, respectively). To our knowledge, this is the first report which uses GC‐MS to detect metabolic differences in goat cloned embryo culture media. The biochemical profiles may help to select the most in vitro viable embryos. [ABSTRACT FROM AUTHOR]

The objectives of the present study were to establish a porcine neural stem cell (NSC) line and to determine if these NSCs could be used to produce cloned pigs. NSCs were isolated from the brains of three embryonic day 30 fetal pigs and were induced to differentiatein vitro. NSCs and the differentiated cells were harvested for analysis of markers by immunostaining and reverse-transcription polymerase chain reaction (RT-PCR). The NSCs at passage 10 were used for nuclear transfer, and the cloned embryos at the two-cell stage were transferred into the oviducts of surrogate mothers. The results showed that three NSC lines (2 male and 1 female) were successfully established. All NSCs at passage 17 continued to express nestin and Sox2. NSCs could differentiate into neurons (TUBB3+), astrocytes (GFAP+), and oligodendrocytes (O4+). After NSC nuclear transfer, 2020 two-cell stage embryos formed. After embryo transfer, 6 of 10 surrogates were pregnant, and 40 piglets (18 males and 22 females) were born. Twenty-two of these piglets reached sexual maturity and were found to be fertile. The other piglets died within 45 days post-partum. In conclusion, 3 porcine NSC lines capable of self-renewal and differentiation were established, and the cloned embryos derived from these cells could develop to term. Thus, NSCs could be efficient alternative nuclear donors for pig cloning. [ABSTRACT FROM AUTHOR]

The study was undertaken to examine whether various strategies, including contact inhibition and serum starvation, that were used for artificial synchronization of mitotic cycle of porcine fibroblast cell lines affect differently the distribution of cell cycle stage frequencies and the occurrence of apoptotic cell death in the analysed cell samples. In vitro cultured (contact-inhibited or serumstarved) somatic cells were subjected to flow cytometric diagnostics of mitotic cycle together with the detection of late-apoptotic cell fractions with hypodiploid number of nuclear DNA molecules. Moreover, impact of the methods applied to synchronize the cell division cycle of different types of nuclear donor fibroblast cells (adult cutaneous and foetal fibroblasts) on the preimplantation developmental outcomes of cloned pig embryos was investigated. The developmental capabilities of nuclear-transferred (NT) embryos that were reconstituted with contact-inhibited or serum-depleted adult cutaneous fibroblast cells to reach the morula and blastocyst stages remained at the levels of 169/278 (60.8%) and 76/278 (27.3%) or 121/265 (45.7%) and 46/265 (17.4%), respectively. The proportions of NT embryos originating from contact-inhibited or serum-deprived foetal fibroblast cells that completed their development to the morula and blastocyst stages were 223/296 (75.3%) and 108/296 (36.5%) or 165/261 (63.2%) and 67/261 (25.7%), respectively. In conclusion, the flow cytometric analysis of cultured porcine adult cutaneous and foetal fibroblast cells revealed the high efficiency of the artificial synchronization of mitotic cycle at the G0/G1 stages as a consequence of applying the methods of either contact inhibition or serum deprivation. For both types of fibroblast cells used to reconstruct the enucleated oocytes, the strategies that were utilized to synchronize the cell division cycle of nuclear donor cells considerably influenced the in vitro developmental abilities of NT pig embryos. Developmental competencies to reach the morula/blastocyst stages for cloned embryos that had been reconstructed with contact-inhibited or serum-starved foetal fibroblast cell nuclei were significantly higher than those for embryos that had been reconstructed with contact-inhibited or serum-starved adult cutaneous fibroblast cell nuclei. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2022 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

The study was carried out in order to evolve, adopt and optimize the new system for preparation of nuclear recipient cells at different stages preceding the somatic cell nuclear transfer (SCNT) in pigs, including in vitro maturation (IVM) of oocytes. The system was applied to facilitate and accelerate the epigenomic reprogrammability for gene expression of donor cell nuclei that had been transplanted into cytoplasmic microenvironment of recipient oocytes and subsequently underwent the dedifferentiating and re-establishing the totipotent epigenetically conditioned status of their transcriptional activity during the preimplantation development of cloned embryos. The use of trichostatin A (TSA)-mediated epigenetic modulation of in vitro-maturing porcine nuclear recipient oocytes that had been pre-treated with R-roscovitine (R-RSCV) resulted in significantly increased blastocyst formation rate among the cloned embryos compared to the R-RSCV- and TSA-unexposed group (almost 44% vs. 26%). [ABSTRACT FROM AUTHOR]

Abstract: The present study was undertaken to investigate the preimplantation developmental competence of cloned pig embryos that were derived from fibroblast cell nuclei by different methods for the activation of reconstructed oocytes. In subgroups IA and IB, nuclear-transferred (NT) oocytes derived from either adult cutaneous or fetal fibroblast cells that had been classified as nonapoptotic by intra vitam analysis for programmed cell death using the YO-PRO-1 DNA fluorochrome underwent sequential physical (i.e., electrical) and chemical activation (SE-CA). This novel method of SE-CA, which was developed and optimized in our laboratory, involves treatment of reconstituted oocytes with direct current pulses and subsequent exposure to 7.5 μM calcium ionomycin, followed by incubation with 30 μM R-roscovitine (R-RSCV), 0.7 mM 6-dimethylaminopurine and 3.5 μg/mL cycloheximide. In subgroups IIA and IIB, NT oocytes were subjected to the standard method of simultaneous fusion and activation mediated by direct current pulses. The proportion of cloned embryos in subgroup IA that reached the morula and blastocyst stages was 145/248 (58.5%) and 78/248 (31.5%), respectively. The proportions of cloned embryos in subgroup IB that reached the morula and blastocyst stages were 186/264 (70.5%) and 112/264 (42.4%), respectively. In turn, subgroup IIA yielded proportions at the morula and blastocyst stages of 110/234 (47.0%) and 49/234 (20.9%), respectively. Subgroup IIB yielded proportions at the morula and blastocyst stages of 144/243 (59.3%) and 74/243 (30.5%), respectively. In summary, the SE-CA of NT oocytes reconstructed from either type of nonapoptotic/nonnecrotic (i.e., YO-PRO-1-negative) fibroblast cell resulted in porcine cloned embryos with considerably better in vitro developmental outcomes than those of cloned embryos generated using the simultaneous fusion and activation approach. To our knowledge, this is the first report of the successful stimulation of porcine NT oocytes using electric pulses followed by an additional activation with a higher dose (1.5 times) of calcium ionomycin and subsequent exposure to a combination of 30 μM R-RSCV and lower concentrations (by 3 times) of 6-dimethylaminopurine and cycloheximide. Moreover, we report here the first use of R-RSCV, a novel meiosis-promoting factor-related p34cdc2 kinase inhibitor, in the oocyte activation protocol for the somatic cell cloning of pigs. [Copyright &y& Elsevier]

The present study compared developmental potential, telomerase activity and transcript levels of X-linked genes (HPRT, MECP2, RPS4X, SLC25A6, XIAP, XIST and ZFX) in bovine somatic cell nuclear transfer (SCNT) embryos reconstructed with cells derived from a freemartin (female with a male co-twin) or from normal female cattle (control). The rates of cleavage, development to blastocyst and hatched blastocyst stage, and the mean numbers of total and inner cell mass cells in the freemartin SCNT embryos were not significantly different from those of control SCNT embryos (p > 0.05). The levels of telomerase activity analyzed by RQ-TRAP in the freemartin SCNT embryos were also similar to those of the normal SCNT embryos. Transcript levels of HPRT, MECP2, RPS4X and XIAP, measured by quantitative real-time RT-PCR, were not significantly different between the control and freemartin SCNT embryos (p > 0.05). However, the transcript levels of SLC25A6, XIST and ZFX were significantly decreased in the freemartin SCNT embryos compared to control SCNT embryos (p < 0.05). Transfer of 71 freemartin SCNT embryos to 22 recipient cows resulted in 4 (18%) pregnancies, which were lost between days 28 and 90 of gestation. Taken together, the present study demonstrates that the transcript levels of several X-linked genes, especially XIST, showed an aberrant pattern in the freemartin SCNT embryos, suggesting aberrant X inactivation in freemartin clones which may affect embryo survival. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: The birth rate of cloned animals following somatic cell nuclear transfer (SCNT) is very low and the surviving animals have various developmental defects. We compared the morphology and transcriptional profile of extraembryonic tissue from three 26-d old SCNT pig fetuses with that from control fetuses. Transcriptional profiling using long-oligonucleotide microarray technology revealed 34 genes that were differentially expressed between the three groups. The differential expression of several genes involved in translational regulation was confirmed by real-time quantitative PCR and Western blot analysis. Interestingly, the expression of a translational inhibition-related gene encoding a eukaryotic translation initiation factor 4E-binding protein was significantly elevated in the SCNT samples. We concluded that the low birth rate of cloned animals could be related to abnormal expression of translational regulators in extraembryonic tissue during early pregnancy. [Copyright &y& Elsevier]

Abstract: Limited success of somatic cell nuclear transfer is attributed to incomplete reprogramming of transferred nuclei. The objective was to determine if 5-aza-2′-deoxycytidine (5-aza-dC) and trichostatin A (TSA) promoted reprogramming and improved development. Relative to untreated controls, treatment of donor cells, cloned embryos, and continuous treatment of both donor cells and cloned embryos with a combination of 0.01μM 5-aza-dC and 0.05μM TSA significantly increased the blastocyst rate (11.9% vs 31.7%, 12.4% vs 25.6%, and 13.3% vs 38.4%, respectively) and total cell number (73.2 vs 91.1, 75.2 vs 93.7, and 74.6 vs 96.7). Moreover, blastocyst rate and inner cell mass (ICM) cell number of embryos continuously exposed to both reagents were significantly higher than that of a TSA-treated group (38.4% vs 23.9% and 27.4 vs 18.2). The DNA methylation level of 2-cell embryos was decreased significantly, whereas the histone acetylation level increased dramatically after donor cell treatment and continuous treatment with both reagents. However, these epigenetic features of cloned blastocysts were not significantly different than the untreated control group. Following embryo treatment, DNA methylation and histone acetylation levels of cloned blastocysts were unchanged, except for the group given 0.5μM TSA (acetylation level was significantly increased, but development potential was reduced). In conclusion, development of cloned bovine embryos was enhanced by 5-aza-dC and TSA; furthermore, the combination was more effective than either one alone. [Copyright &y& Elsevier]

In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1-7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2-7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

• Low concentration 5-Aza-C improved maturation of pig oocytes. • 5-Aza-C treated oocytes had improved ability to develop in vitro after IVF or PA. • Enucleated 5-Aza-C treated oocytes had better reprogramming ability upon SCNT. • Enucleated 5-Aza-C treated oocytes may be a better cytoplast for SCNT. Treatment of donor cells and/or cloned embryos with cytidine analogues, having an Aza group at its 5th carbon (5-Aza), such as 5-Azacytidine (5-Aza-C) or 5-Aza-2′-deoxycytidine (5-Aza-dC) improves the in vitro development of cloned embryos produced by somatic cell nuclear transfer (SCNT). In vitro maturation (IVM) of immature pig oocytes treated with 5-Aza-C not only results in greater (P < 0.05) meiotic maturation to the MII stage but also enhances the capacity of 5-Aza-C treated oocytes for early embryonic development after parthenogenetic activation (PA), in vitro fertilization (IVF) or SCNT in a dose-dependent manner (0–10 μM). Cloned embryos generated from 5-Aza-C (0.01 μM) treated oocytes had an increased capacity to develop to the blastocyst stage (14.1 ± 1.5% compared with 9.6 ± 1.8%), greater probability of hatching (61.8 ± 1.5% compared with 45.0 ± 3.9%) and contained a greater number of cells per blastocyst (38.5 ± 4.4 compared with 30.5 ± 3.4) than those produced from non-treated control oocytes (P < 0.05). Data from the present study indicate that treatment of oocytes with 5-Aza-C may be an important approach to enhance the meiotic maturation and subsequent in vitro development of pig embryos. Future studies should be conducted to determine the underlying mechanism of improved early embryonic development of 5-Aza-C treated oocytes. [ABSTRACT FROM AUTHOR]

Imprinting disorder during somatic cell nuclear transfer usually leads to the abnormality of cloned animals and low cloning efficiency. However, little is known about the role of donor cell imprinting in the development of cloned embryos. Here, we demonstrated that the imprinting (H19/Igf2) in porcine fetus fibroblasts derived from the morphologically abnormal cloned fetuses (the abnormal imprinting group) was more hypomethylated, and accordingly, significantly higher H19 transcription and lower Igf2 expression occurred in comparison with those in fibroblasts derived from morphologically normal cloned fetuses (the normal imprinting group) or donor fetus fibroblasts (the control group). When these fibroblasts were used as donor cells, the abnormal imprinting group displayed an even lower imprinting methylation level, in correspondence to the significantly downregulated expression of Dnmt1, Dnmt3a and Zfp57, and a markedly reduced blastocyst rate, while the normal imprinting group took on the similar patterns of imprinting, gene expression and embryo development to the control group. When 5-aza-dC was applied to reduce the fibroblasts imprinting methylation level in the normal imprinting group, cloned embryos displayed the more severely impaired imprinting and significantly lower blastocyst rate. While the upregulated H19 transcription in the abnormal imprinting group was knocked down, the imprinting statuses were partly rescued, and the cleavage and blastocyst rates significantly increased in cloned embryos. In all, donor cell imprinting disorder reduced the developmental efficiency of cloned embryos. This work provides a new insight into understanding the molecular mechanism of donor cells regulating the cloned embryo development., Competing Interests: CONFLICTS OF INTEREST The authors declare that no conflicting financial interests exist.

A novel method termed the biological transcomplementary activation (B-TCA) has been recently utilized for the stimulation of porcine oocytes reconstituted by somatic cell nuclear transfer (SCNT). The use of cytosolic components originating from fertilized (FE) rabbit zygotes as the stimuli for the B-TCA of SCNT-derived pig oocytes appeared to be a highly efficient strategy applied to promote the in vitro development of cloned embryos, leading to a significant improvement in the blastocyst yield (43.6%) compared to the yields achieved using the standard protocol of simultaneous fusion and electrical activation (SF-EA; [31.3%]) or the protocol of delayed electrical activation (D-EA) independent of extracellular Ca(2+) ions (0%). The FE rabbit zygote cytoplast-mediated B-TCA resulted in the increased blastocyst formation rate of porcine cloned embryos as compared to the B-TCA triggered by either cytoplasts isolated from pig parthenogenotes (PAs; [27.8%]) or rabbit PA-descended cytoplasts (0%). A considerably lower percentage of blastocysts containing apoptotic and/or necrotic (annexin V-eGFP-positive) cells were obtained from the SCNT-derived oocytes stimulated by the FE rabbit zygote cytoplast-based B-TCA (22.2%) compared to those stimulated using the SF-EA protocol (35.1%). In contrast to the B-TCA induced by FE rabbit zygote cytoplasts, apoptosis/necrosis incidence decreased totally among the cloned pig blastocysts that developed from reconstituted oocytes undergoing the porcine PA cytoplast-evoked B-TCA. In conclusion, the FE rabbit zygote cytoplast-mediated B-TCA turned out to be a relatively effective strategy for the in vitro production of porcine blastocyst clones of higher quality compared to those created using the standard SF-EA approach., (Copyright © 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)