Development Of Somatic Cell Nuclear Transfer Embryos Research Articles

Optimization of donor cell production for somatic cell nuclear transfer in the critically endangered Vietnamese Ỉ pig

We aimed to establish efficient donor cells to produce piglets by somatic cell nuclear transfer (SCNT) of the endangered Vietnamese Ỉ pig. In Experiment 1, we assessed the effects of cell passages on the in vitro development of SCNT embryos. Cells with five and six passages showed significantly cleaved and blastocyst formation rates (86.72 and 86.64; 35.68 and 35.51, respectively, P < 0.05). The highest average total cell number per blastocyst was observed in groups of cells with five and six passages (50.45 and 50.18, respectively). Experiment 2 was performed to assess the sex of donor cells on the subsequent development of SCNT embryos. There was no significant difference in the cleaved and blastocyst formation rates, and the average total cell between female and male groups (86.51 % vs 86.94 % and 35.31 % vs 35.08 %, 50.29 % vs 50.67 %, respectively, P > 0.05). Experiment 3 was performed to assess the effect of cell lines on the development of SCNT embryos. Our results showed no significant difference in the success rate of fibroblast nuclear transfer into recipient oocytes, the cleaved and blastocyst formation rates, and the average total cell number per blastocyst among the cell lines 6004, 9154, 9155, 9156 and 9157 (P > 0.05). Experiment 4 was performed to assess the ability of SCNT embryos to induce pregnancy and to develop term. SCNT embryos were produced from Ỉ fibroblast cells established based on the results of Experiments 1, 2 and 3. Transfer of blastocyst stage embryos into 19 recipients (100–120 embryos in each) resulted in 14 pregnancies, in which 8 pregnant females terminated on Day 22–42 and 6 others produced 20 cloned piglets from donor cells of a female pig but 5 piglets died before birth and 15 healthy cloned piglets. However, 3 out of 15 healthy piglets died of unknown causes within 24h of birth and 3 out of 15 healthy piglets died at 3–5 days of age due to diarrhoea, 9 out of 15 healthy piglets are now 3 months of age. Finally, we established a protocol for the donor cell production which enabled the production of the endangered Ỉ pig embryos by SCNT and maximized blastocyst production rate by more than 35 % and pregnant rate after the transfer of cloned Ỉ pig embryos to recipients at 73.68 % for the first time in Vietnam.

A retrospective analysis of sheep generated by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) is one of the primary methods for production of genetically engineered sheep, which allows for gene editing or transgene introduction in somatic cells. The use of SCNT eliminates the risk of genetic mosaicism in embryos and animals that is commonly observed after zygote micromanipulations. This retrospective analysis of SCNT in sheep performed at Utah State University, spanning from 2016 to 2021, examined parameters that may impact pregnancy and full-term development, including donor oocytes (donor age), donor cell lines, SCNT parameters (time of oocyte activation following SCNT, number of transferred embryos, in vitro maturation and culture conditions), and recipients (surgical number and ovulatory status), as well as factors that may correlate with large offspring syndrome or abnormal offspring syndrome (LOS/AOS) in the fetuses and lambs. Our findings indicated that compared to prepubertal oocytes, the SCNT embryos produced from adult sheep oocytes had comparable in vitro maturation rates, pregnancy and full-term development rates, as well as SCNT efficiency. In addition, earlier activation time of SCNT embryos (e.g. 24–26 h post maturation) was correlated to the early pregnancy loss rate, full-term rate, and SCNT efficiency. Compared to our standard serum-containing medium, commercial serum-free culture medium showed a positive correlation with the full-term development of sheep SCNT embryos. Transferring 15–30 embryos per recipient resulted in consistently good pregnancy rates. Surgical numbers and ovulatory status (having at least one follicle between 6 and 12 mm in size or a corpus hemorrhagicum (CH)) of recipients did not affect pregnancy and full-term development rates. In summary, this retrospective analysis identified parameters for improving pregnancy and full-term development of SCNT embryos in sheep.

Comparative transcriptome profile of embryos at different developmental stages derived from somatic cell nuclear transfer (SCNT) and in-vitro fertilization (IVF) in riverine buffalo (Bubalus bubalis).

Somatic cell nuclear transfer (SCNT) is a very important reproductive technology with many diverse applications, such as fast multiplication of elite animals, the production of transgenic animals and embryonic stem (ES) cells. However, low cloning efficiency, a low live birth rate and the abnormally high incidence of abnormalities in the offspring born are attributed to incomplete or aberrant nuclear reprogramming. In SCNT embryos, the aberrant expression pattern of the genes throughout embryonic development is responsible for the incomplete nuclear reprogramming. The present study was carried out to identify the differential gene expression (DEGs) profile and molecular pathways of the SCNT and IVF embryos at different developmental stages (2 cell, 8 cell and blastocyst stages). In the present study, 1164 (2 cell), 1004 (8 cell) and 530 (blastocyst stage) DEGs were identified in the SCNT embryos as compared to IVF embryos. In addition, several genes such as ZEB1, GDF1, HSF5, PDE3B, VIM, TNNC, HSD3B1, TAGLN, ITGA4 and AGMAT were affecting the development of SCNT embryos as compared to IVF embryos. Further, Gene Ontology (GO) and molecular pathways analysis suggested, SCNT embryos exhibit variations compared to their IVF counterparts and affected the development of embryos throughout the different developmental stages. Apart from this, q-PCR analysis of the GDF1, TMEM114, and IGSF22 genes were utilized to validate the RNA-seq data. These findings contribute valuable insights about the different genes and molecular pathways underlying SCNT embryo development and offer crucial information for improving SCNT efficiency.

Reduction of H3K9 methylation by G9a inhibitors improves the development of mouse SCNT embryos

Removal of somatic histone H3 lysine 9 trimethylation (H3K9me3) from the embryonic genome can improve the efficiency of mammalian cloning using somatic cell nuclear transfer (SCNT). However, this strategy involves the injection of histone demethylase mRNA into embryos, which is limiting because of its invasive and labor-consuming nature. Here, we report that treatment with an inhibitor of G9a (G9ai), the major histone methyltransferase that introduces H3K9me1/2 in mammals, greatly improved the development of mouse SCNT embryos. Intriguingly, G9ai caused an immediate reduction of H3K9me1/2, a secondary loss of H3K9me3 in SCNT embryos, and increased the birth rate of cloned pups about 5-fold (up to 3.9%). G9ai combined with the histone deacetylase inhibitor trichostatin A further improved this rate to 14.5%. Mechanistically, G9ai and TSA synergistically enhanced H3K9me3 demethylation and boosted zygotic genome activation. Thus, we established an easy, highly effective SCNT protocol that would enhance future cloning research and applications.

Treatment of Donor Cells with Oxidative Phosphorylation Inhibitor CPI Enhances Porcine Cloned Embryo Development.

Somatic cell nuclear transfer (SCNT) technology holds great promise for livestock industry, life science and human biomedicine. However, the development and application of this technology is limited by the low developmental potential of SCNT embryos. The developmental competence of cloned embryos is influenced by the energy metabolic status of donor cells. The purpose of this study was to investigate the effects of CPI, an oxidative phosphorylation inhibitor, on the energy metabolism pathways of pig fibroblasts and the development of subsequent SCNT embryos. The results showed that treatment of porcine fibroblasts with CPI changed the cellular energy metabolic pathways from oxidative phosphorylation to glycolysis and enhanced the developmental ability of subsequent SCNT embryos. The present study establishes a simple, new way to improve pig cloning efficiency, helping to promote the development and application of pig SCNT technology.

DNA methylation regulates RNA m6A modification through transcription factor SP1 during the development of porcine somatic cell nuclear transfer embryos.

Epigenetic modifications play critical roles during somatic cell nuclear transfer (SCNT) embryo development. Whether RNA N6-methyladenosine (m6A) affects the developmental competency of SCNT embryos remains unclear. Here, we showed that porcine bone marrow mesenchymal stem cells (pBMSCs) presented higher RNA m6A levels than those of porcine embryonic fibroblasts (pEFs). SCNT embryos derived from pBMSCs had higher RNA m6A levels, cleavage, and blastocyst rates than those from pEFs. Compared with pEFs, the promoter region of METTL14 presented a hypomethylation status in pBMSCs. Mechanistically, DNA methylation regulated METTL14 expression by affecting the accessibility of transcription factor SP1 binding, highlighting the role of the DNA methylation/SP1/METTL14 pathway in donor cells. Inhibiting the DNA methylation level in donor cells increased the RNA m6A level and improved the development efficiency of SCNT embryos. Overexpression of METTL14 significantly increased the RNA m6A level in donor cells and the development efficiency of SCNT embryos, whereas knockdown of METTL14 suggested the opposite result. Moreover, we revealed that RNA m6A-regulated TOP2B mRNA stability, translation level, and DNA damage during SCNT embryo development. Collectively, our results highlight the crosstalk between RNA m6A and DNA methylation, and the crucial role of RNA m6A during nuclear reprogramming in SCNT embryo development.

Unreprogrammed H3K9me3 prevents minor zygotic genome activation and lineage commitment in SCNT embryos

Somatic cell nuclear transfer (SCNT) can be used to reprogram differentiated somatic cells to a totipotent state but has poor efficiency in supporting full-term development. H3K9me3 is considered to be an epigenetic barrier to zygotic genomic activation in 2-cell SCNT embryos. However, the mechanism underlying the failure of H3K9me3 reprogramming during SCNT embryo development remains elusive. Here, we perform genome-wide profiling of H3K9me3 in cumulus cell-derived SCNT embryos. We find redundant H3K9me3 marks are closely related to defective minor zygotic genome activation. Moreover, SCNT blastocysts show severely indistinct lineage-specific H3K9me3 deposition. We identify MAX and MCRS1 as potential H3K9me3-related transcription factors and are essential for early embryogenesis. Overexpression of Max and Mcrs1 significantly benefits SCNT embryo development. Notably, MCRS1 partially rescues lineage-specific H3K9me3 allocation, and further improves the efficiency of full-term development. Importantly, our data confirm the conservation of deficient H3K9me3 differentiation in Sertoli cell-derived SCNT embryos, which may be regulated by alternative mechanisms.

Inhibition of Wnt activity improves peri-implantation development of somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) can reprogram differentiated somatic cells into totipotency. Although pre-implantation development of SCNT embryos has greatly improved, most SCNT blastocysts are still arrested at the peri-implantation stage, and the underlying mechanism remains elusive. Here, we develop a 3D in vitro culture system for SCNT peri-implantation embryos and discover that persistent Wnt signals block the naïve-to-primed pluripotency transition of epiblasts with aberrant H3K27me3 occupancy, which in turn leads to defects in epiblast transformation events and subsequent implantation failure. Strikingly, manipulating Wnt signals can attenuate the pluripotency transition and H3K27me3 deposition defects in epiblasts and achieve up to a 9-fold increase in cloning efficiency. Finally, single-cell RNA-seq analysis reveals that Wnt inhibition markedly enhances the lineage developmental trajectories of SCNT blastocysts during peri-implantation development. Overall, these findings reveal diminished potentials of SCNT blastocysts for lineage specification and validate a critical peri-implantation barrier for SCNT embryos.

Blastocyst cell number and allocation affect the developmental potential and transcriptome of bovine Somatic Cell Nuclear Transfer embryos.

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

Farrerol directly activates the deubiqutinase UCHL3 to promote DNA repair and reprogramming when mediated by somatic cell nuclear transfer

Farrerol, a natural flavanone, promotes homologous recombination (HR) repair to improve genome-editing efficiency, but the specific protein that farrerol directly targets to regulate HR repair and the underlying molecular mechanisms have not been determined. Here, we find that the deubiquitinase UCHL3 is the direct target of farrerol. Mechanistically, farrerol enhanced the deubiquitinase activity of UCHL3 to promote RAD51 deubiquitination, thereby improving HR repair. Importantly, we find that embryos of somatic cell nuclear transfer (SCNT) exhibited defective HR repair, increased genomic instability and aneuploidy, and that the farrerol treatment post nuclear transfer enhances HR repair, restores transcriptional and epigenetic network, and promotes SCNT embryo development. Ablating UCHL3 significantly attenuates farrerol-mediated stimulation in HR and SCNT embryo development. In summary, we identify farrerol as an activator of the deubiquitinase UCHL3, highlighted the importance of HR and epigenetic changes in SCNT reprogramming and provide a feasible method to promote SCNT efficiency.

Cloning horses by somatic cell nuclear transfer: Effects of oocyte source on development to foaling

The cloning of horses is a commercial reality, yet the availability of oocytes for cloned embryo production remains a major limitation. Immature oocytes collected from abattoir-sourced ovaries or from live mares by ovum pick-up (OPU) have both been used to generate cloned foals. However, the reported cloning efficiencies are difficult to compare due to the different somatic cell nuclear transfer (SCNT) techniques and conditions used. The objective of this retrospective study was to compare the in vitro and in vivo development of equine SCNT embryos produced using oocytes recovered from abattoir-sourced ovaries and from live mares by OPU. A total of 1,128 oocytes were obtained, of which 668 were abattoir-derived and 460 were OPU-derived. The methods used for in vitro maturation and SCNT were identical for both oocyte groups, and the embryos were cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham medium supplemented with 10% fetal calf serum. Embryo development in vitro was assessed, and Day 7 blastocysts were transferred to recipient mares. The embryos were transferred fresh when possible, and a cohort of vitrified-thawed OPU-derived blastocysts was also transferred. Pregnancy outcomes were recorded at Days 14, 42 and 90 of gestation and at foaling. The rates of cleavage (68.7 ± 3.9% vs 62.4 ± 4.7%) and development to the blastocyst stage (34.6 ± 3.3% vs 25.6 ± 2.0%) were superior for OPU-derived embryos compared with abattoir-derived embryos (P < 0.05). Following transfer of Day 7 blastocysts to a total of 77 recipient mares, the pregnancy rates at Days 14 and 42 of gestation were 37.7% and 27.3%, respectively. Beyond Day 42, the percentages of recipient mares that still had a viable conceptus at Day 90 (84.6% vs 37.5%) and gave birth to a healthy foal (61.5% vs 12.5%) were greater for the OPU group compared with the abattoir group (P < 0.05). Surprisingly, more favourable pregnancy outcomes were achieved when blastocysts were vitrified for later transfer, probably because the uterine receptivity of the recipient mares was more ideal. A total of 12 cloned foals were born, 9 of which were viable. Given the differences observed between the two oocyte groups, the use of OPU-harvested oocytes for generating cloned foals is clearly advantageous. Continued research is essential to better understand the oocyte deficiencies and increase the efficiency of equine cloning.

Effects of alpha lipoic acid treatment during in vitro maturation on the development of porcine somatic cell nuclear transfer embryos.

Oxidative stress influences the embryo production efficiency in vitro. We investigated the effects of alpha lipoic acid (ALA) treatment during the in vitro maturation (IVM) period on the porcine somatic cell nuclear transfer (SCNT) embryo production. After IVM, maturation rates of the 12.5- and 25-μM ALA-treated groups were not significantly different from those of the 0-μM ALA-treated group. Compared to those in the 0-μM ALA-treated group, the reactive oxygen species and glutathione levels were significantly decreased and increased, respectively, in the cytoplasm of matured oocytes in the 12.5-50-μM ALA-treated groups. Apoptosis rate in cumulus cells after IVM was significantly lower in the 12.5-50-μM ALA-treated groups than in the 0-μM ALA-treated group. Blastocyst formation rate was significantly higher in parthenogenetic oocytes treated with 12.5-μM ALA than in the 0-, 25-, and 50-μM ALA-treated groups. Similarly, in SCNT embryos, the 12.5-μM ALA-treated group showed a significantly higher blastocyst formation rate than the 0-μM ALA-treated group. Apoptosis rate in SCNT blastocysts was significantly decreased by 12.5-μM ALA treatment. The results showed that treatment with 12.5-μM ALA during IVM improves porcine SCNT embryo development and partial quality.

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

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

Simultaneous Inhibition of Histone Deacetylases and RNA Synthesis Enables Totipotency Reprogramming in Pig SCNT Embryos.

Combining somatic cell nuclear transfer (SCNT) with genome editing technologies has emerged as a powerful platform for the creation of unique swine lineages for agricultural and biomedical applications. However, successful application of this research platform is still hampered by the low efficiency of these technologies, particularly in attaining complete cell reprogramming for the production of cloned pigs. Treating SCNT embryos with histone deacetylase inhibitors (HDACis), such as Scriptaid, has been routinely used to facilitate chromatin reprogramming after nuclear transfer. While increasing histone acetylation leads to a more relaxed chromatin configuration that facilitates the access of reprogramming factors and DNA repair machinery, it may also promote the expression of genes that are unnecessary or detrimental for normal embryo development. In this study, we evaluated the impact of inhibiting both histone deacetylases and RNA synthesis on pre- and post-implantation development of pig SCNT embryos. Our findings revealed that transcription can be inhibited for up to 40 h of development in porcine embryos, produced either by activation, fertilization or SCNT, without detrimentally affecting their capacity to form a blastocyst and their average number of cells at this developmental stage. Importantly, inhibiting RNA synthesis during HDACi treatment resulted in SCNT blastocysts with a greater number of cells and more abundant transcripts for genes related to embryo genome activation on days 2, 3 and 4 of development, compared to SCNT embryos that were treated with HDACi only. In addition, concomitant inhibition of histone deacetylases and RNA synthesis promoted the full reprograming of somatic cells, as evidenced by the normal fetal and full-term development of SCNT embryos. This combined treatment may improve the efficiency of the genome-editing + SCNT platform in swine, which should be further tested by transferring more SCNT embryos and evaluating the health and growth performance of the cloned pigs.

Reprogramming barriers in bovine cells nuclear transfer revealed by single-cell RNA-seq analysis.

Many progresses have recently been achieved in animal somatic cell nuclear transfer (SCNT). However, embryos derived from SCNT rarely result in live births. Single‐cell RNA sequencing (scRNA‐seq) can be used to investigate the development details of SCNT embryos. Here, bovine fibroblasts and three factors bovine iPSCs (3F biPSCs) were used as donors for bovine nuclear transfer, and the single blastomere transcriptome was analysed by scRNA‐seq. Compared to in vitro fertilization (IVF) embryos, SCNT embryos exhibited many defects. Abnormally expressed genes were found at each stage of embryos, which enriched in metabolism, and epigenetic modification. The DEGs of the adjacent stage in SCNT embryos did not follow the temporal expression pattern similar to that of IVF embryos. Particularly, SCNT 8‐cell stage embryos showed failures in some gene activation, including ZSCAN4, and defects in protein association networks which cored as POLR2K, GRO1, and ANKRD1. Some important signalling pathways also showed incomplete activation at SCNT zygote to morula stage. Interestingly, 3F biPSCNT embryos exhibited more dysregulated genes than SCNT embryos at zygote and 2‐cell stage, including genes in KDM family. Pseudotime analysis of 3F biPSCNT embryos showed the different developmental fate from SCNT and IVF embryos. These findings suggested partial reprogrammed 3F biPS cells as donors for bovine nuclear transfer hindered the reprogramming of nuclear transfer embryos. Our studies revealed the abnormal gene expression and pathway activation of SCNT embryos, which could increase our understanding of the development of SCNT embryos and give hints to improve the efficiency of nuclear transfer.

Oocytes obtained by ovum pick-up from live mares as an alternative to abattoir-derived oocytes for the development of equine embryos produced by somatic cell nuclear transfer

The production of equine embryos by somatic cell nuclear transfer (SCNT) is limited by the availability of immature oocytes. The main source of oocytes for SCNT comes from ovaries obtained from slaughter houses. However, in some countries there is no horse abattoirs or their number is limited which precludes the use of SCNT for the production of equine cloned embryos. Ovum pick-up (OPU) is a reproductive assisted technique which is growing rapidly in the equine industry which allows obtaining oocytes from live mares. OPU is mainly used in combination to ICSI (intracytoplasmic sperm injection) for the production of in vitro embryos. Oocytes collected from live recipient mares by OPU could be an alternative for SCNT in countries in which there are limited number of equine slaughter houses. However, it is unknown the effect of the oocytes’ source on the development of equine cloned embryos. The aim of this study was to compare the development of equine SCNT embryos produced using oocytes either retrieved by OPU or recovered from abattoir-sourced ovaries. For the study, a total of 1128 oocytes were used, of which 663 were from abattoir-sourced ovaries and 495 were from live mares programmed for OPU. The harvested ovaries were transported to the lab within 1 h for processing. The methods used for in vitro maturation (IVM), SCNT andembryo culture in vitro were identical for both sources of oocytes. The rates of maturation, cell fusion, cleavage and blastocyst formation at Day 7 were evaluated. Transferrable grade blastocysts were transferred to recipient mares 4 to 5 days after ovulation and pregnancy was diagnosed at Days 14 and 42 by ultrasound. The maturation rate of OPU-derived oocytes was lower than that of abattoir-derived oocytes (50.3 ± 2.7% vs . 61.9 ± 3.4%; P < 0.05). However, the rates of cell fusion (90.7 ± 2.6% vs. 81.9 ± 5.2%), cleavage (68.8 ± 3.9% vs . 61.9 ± 5.0%) and blastocyst formation (34.3 ± 2.8% vs. 25.4 ± 2.1%) were greater (P < 0.05) for OPU-derived embryos compared with abattoir-derived embryos. While similar proportions of OPU- and abattoir-derived blastocysts initiated pregnancy at Day 14, a greater proportion of OPU-derived blastocysts developed to Day 42 of gestation (13 of 50 embryos) compared with abattoir-derived blastocysts (3 of 27 embryos; P < 0.05). The results show that abattoir-sourced oocytes subjected to IVM have the potential to support the development of SCNT embryos that can initiate pregnancy. However, given the observed difference in subsequent in vivo development, the use of OPU-derived oocytes for equine SCNT embryo production is recommended. Further research is needed to improve the IVM of abattoir-sourced oocytes

Double Sperm Cloning: Could Improve the Efficiency of Animal Cloning

Somatic cell nuclear transfer (SCNT) is an assisted reproductive technology that produces an individual from a single somatic cell nucleus and an enucleated oocyte. SCNT has presented an extensive application in the multiplication of superior germplasm, production of transgenic animals, the rescue of endangered animals, and therapeutic cloning. Despite that, the competence of SCNT remains very low due to poor reprogramming of somatic cells and epigenetic modifications. The somatic cells used in SCNT as donor nuclei lack some important components which normally present in sperm cells that are transmitted during fertilization. Sperm-derived factors play an important role in nucleus reprogramming during SCNT and embryonic development. Loss of such factors in somatic cells is reflected as an imperative reason for the abnormal development of SCNT embryos. To improve the efficiency of somatic cells’ nuclear reprogramming during SCNT, double sperm cloning (DSC) could be an alternative approach to produce clone embryos/animals. Here, we focus on the recent advances of SCNT and their bottleneck and explored the possibility of cloning efficiency using sperm as donor nuclei.

Effect of ACY-1215 on cytoskeletal remodeling and histone acetylation in bovine somatic cell nuclear transfer embryos

The latest studies indicated that in addition to alterations in abnormal chromosome epigenetic modifications, the abnormal cytoskeletal changes are also an important cause for the developmental failure of somatic cell nuclear transfer (SCNT) embryos. In the present study, the effects of ACY-1215, a specific inhibitor of HDAC6, on the acetylation of α-tubulin, histone epigenetic modification, spindle formation and embryonic development of early bovine SCNT embryos were studied. The results showed that acetylation of α-tubulin, H3K9, and H4K16 was significantly lower in SCNT embryos than in vitro fertilization (IVF) embryos. After ACY-1215 treatment, the acetylation level of α-tubulin, H3K9, and H4K16 of SCNT embryos was closer to that of IVF embryos. ACY-1215 treatment reduced spindle abnormalities, delayed the time of first cleavage of embryos, increased the total cell number and trophectoderm cells numbers, and reduced apoptosis in SCNT blastocysts. ACY-1215 regulated the process of embryonic epigenetic modification and cytoskeletal protein acetylation, corrected abnormal development of SCNT embryos, and improved SCNT embryonic development potential.

Interleukin 17D Enhances the Developmental Competence of Cloned Pig Embryos by Inhibiting Apoptosis and Promoting Embryonic Genome Activation.

Simple SummaryThe cloning technique is important for animal husbandry and biomedicine because it can be used to clone superior breeding livestock and produce multipurpose genetically modified animals. However, the success rate of cloning currently is very low due to the low developmental efficiency of cloned embryos, which limits the application of cloning. The low developmental competence is related to the excessive cell death in cloned embryos. Interleukin 17D (IL17D) is required for the normal development of mouse embryos by inhibiting cell death. This study aimed to investigate whether IL17D can improve cloned pig embryo development by inhibiting cell death. Addition of IL17D protein to culture medium decreased the cell death level and improved the developmental ability of cloned pig embryos. IL17D treatment enhanced cloned pig embryo development by regulating cell death-associated gene pathways and promoting genome-wide gene expression, which is probably via up-regulating the expression of a gene called GADD45B. This study provided a new approach to improve the pig cloning efficiency by adding IL17D protein to the culture medium of cloned pig embryos.Cloned animals generated by the somatic cell nuclear transfer (SCNT) approach are valuable for the farm animal industry and biomedical science. Nevertheless, the extremely low developmental efficiency of cloned embryos hinders the application of SCNT. Low developmental competence is related to the higher apoptosis level in cloned embryos than in fertilization-derived counterparts. Interleukin 17D (IL17D) expression is up-regulated during early mouse embryo development and is required for normal development of mouse embryos by inhibiting apoptosis. This study aimed to investigate whether IL17D plays roles in regulating pig SCNT embryo development. Supplementation of IL17D to culture medium improved the developmental competence and decreased the cell apoptosis level in cloned porcine embryos. The transcriptome data indicated that IL17D activated apoptosis-associated pathways and promoted global gene expression at embryonic genome activation (EGA) stage in treated pig SCNT embryos. Treating pig SCNT embryos with IL17D up-regulated expression of GADD45B, which is functional in inhibiting apoptosis and promoting EGA. Overexpression of GADD45B enhanced the developmental efficiency of cloned pig embryos. These results suggested that IL17D treatment enhanced the developmental ability of cloned pig embryos by suppressing apoptosis and promoting EGA, which was related to the up-regulation of GADD45B expression. This study demonstrated the roles of IL17D in early development of porcine SCNT embryos and provided a new approach to improve the developmental efficiency of cloned porcine embryos.

Antioxidant Activity and Anti-Apoptotic Effect of the Small Molecule Procyanidin B1 in Early Mouse Embryonic Development Produced by Somatic Cell Nuclear Transfer.

As an antioxidant, procyanidin B1(PB1) can improve the development of somatic cell nuclear transfer (SCNT) embryos; PB1 reduces the level of oxidative stress (OS) during the in vitro development of SCNT embryos by decreasing the level of reactive oxygen species (ROS) and increasing the level of glutathione (GSH) and mitochondrial membrane potential (MMP). Metabolite hydrogen peroxide (H2O2) produces OS. Catalase (CAT) can degrade hydrogen peroxide so that it produces less toxic water (H2O) and oxygen (O2) in order to reduce the harm caused by H2O2. Therefore, we tested the CAT level in the in vitro development of SCNT embryos; it was found that PB1 can increase the expression of CAT, indicating that PB1 can offset the harm caused by oxidative stress by increasing the level of CAT. Moreover, if H2O2 accumulates excessively, it produces radical-(HO-) through Fe2+/3+ and damage to DNA. The damage caused to the DNA is mainly repaired by the protein encoded by the DNA damage repair gene. Therefore, we tested the expression of the DNA damage repair gene, OGG1. It was found that PB1 can increase the expression of OGG1 and increase the expression of protein. Through the above test, we proved that PB1 can improve the repairability of DNA damage. DNA damage can lead to cell apoptosis; therefore, we also tested the level of apoptosis of blastocysts, and we found that PB1 reduced the level of apoptosis. In summary, our results show that PB1 reduces the accumulation of H2O2 by decreasing the level of OS during the in vitro development of SCNT embryos and improves the repairability of DNA damage to reduce cell apoptosis. Our results have important significance for the improvement of the development of SCNT embryos in vitro and provide important reference significance for diseases that can be treated using SCNT technology.