Efficiency Of Somatic Cell Nuclear Transfer Research Articles

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.

Chromatin accessibility memory of donor cells disrupts bovine somatic cell nuclear transfer blastocysts development.

The post-transfer developmental capacity of bovine somatic cell nuclear transfer (SCNT) blastocysts is reduced, implying that abnormalities in gene expression regulation are present at blastocyst stage. Chromatin accessibility, as an indicator for transcriptional regulatory elements mediating gene transcription activity, has heretofore been largely unexplored in SCNT embryos, especially at blastocyst stage. In the present study, single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) of in vivo and SCNT blastocysts were conducted to segregate lineages and demonstrate the aberrant chromatin accessibility of transcription factors (TFs) related to inner cell mass (ICM) development in SCNT blastocysts. Pseudotime analysis of lineage segregation further reflected dysregulated chromatin accessibility dynamics of TFs in the ICM of SCNT blastocysts compared to their in vivo counterparts. ATAC- and ChIP-seq results of SCNT donor cells revealed that the aberrant chromatin accessibility in the ICM of SCNT blastocysts was due to the persistence of chromatin accessibility memory at corresponding loci in the donor cells, with strong enrichment of trimethylation of histone H3 at lysine 4 (H3K4me3) at these loci. Correction of the aberrant chromatin accessibility through demethylation of H3K4me3 by KDM5B diminished the expression of related genes (e.g., BCL11B) and significantly improved the ICM proliferation in SCNT blastocysts. This effect was confirmed by knocking down BCL11B in SCNT embryos to down-regulate p21 and alleviate the inhibition of ICM proliferation. These findings expand our understanding of the chromatin accessibility abnormalities in SCNT blastocysts and BCL11B may be a potential target to improve SCNT efficiency.

A long non-coding RNA essential for early embryonic development improves SCNT efficiency in goats.

Although abundant long non-coding RNAs (lncRNAs) have been found to be highly expressed in early embryos, the functions and mechanisms of most lncRNAs in regulating embryonic development remain unclear. This study was conducted to identify the key lncRNAs during embryonic genome activation (EGA) for promoting embryonic development after somatic cell nuclear transfer (SCNT) in goats. We screened and characterized lncRNAs from transcriptome data of in vitro-fertilized, 2-cell (IVF-2c) and 8-cell embryos (IVF-8c) and 8-cell SCNT embryos (SCNT-8c). We obtained 12 differentially expressed lncRNAs that were highly expressed in IVF-8c embryos compared to IVF-2c, and less expressed in SCNT-8c embryos. After target gene prediction, expression verification, and functional deletion experiments, we found that the expression level of lncRNA3720 affected the early embryonic development in goats. We cloned full length lncRNA3720 and over-expressed it in goat fetal fibroblasts (GFFs). We identified histone variants by analyzing the transcriptome data from both GFFs and embryos. Gene annotation of the gene library and literature search revealed that histone variants may have important roles in early embryo development, so we selected them as the potential target genes for lncRNA3720. Lastly, we compensated for the low expression of lncRNA3720 in SCNT embryos by microinjection, and showed that the development rate and quality of SCNT embryos was significantly improved. We speculate that lncRNA3720 is a key promoter of embryonic development in goats by interacting with histone variants.

Methylation Genome-Wide Profiling in Lowly and Highly Efficient Somatic Cell Nuclear Transfer in Pigs

Swine is a common model organism for biomedical research. Epigenetic reprogramming in somatic cell nuclear transfer (SCNT) embryos does not fully recapitulate the natural DNA demethylation events at fertilisation. This study aimed to conduct genome-wide methylation profiling to detect differentially methylated regions (DMRs) responsible for epigenetic differences in stem cells that displayed high and low efficiency of SCNT and to elucidate the low efficiency of cloning rate in pigs. Adipose tissue mesenchymal stem cells (AMSC)s lines were isolated from adipose tissue of adult male pigs (n = 20; high-efficiency cells = 10; and low-efficiency cells = 10). Reduced representation bisulfite sequencing (RRBS) was performed on an Illumina HiSeq1500. Paired-end reads were filtered to remove the adapter contamination, and low-quality reads using TrimGalore! Filtered reads were mapped to the reference genome using Bismark. MethylKit was used to identify differentially methylated regions (DMRs) (bases and tiles), showing statistically significant differential methylation between high and low-efficiency AMSCs. Hierarchical cluster analysis according to methylation patterns clearly defined groups with low and high cloning efficiency. We report 3704 bases with statistically significant differences in methylation and 10062 tiles with statistically significant differences in methylation. Most differentially methylated sites are intergenic 62%, 31% are intronic, 4% are in exons, and 4% in promoters. Moreover, 37% of differentially methylated sites are located in known CpG islands (CGIs), and 4% in CpG island shores (CGSs).

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.

Somatic Cell Nuclear Transfer Using Freeze-Dried Protaminized Donor Nuclei.

Somatic cell nuclear transfer (SCNT) is the only nuclear reprogramming method that allows rewinding an adult nucleus into a totipotent state. As such, it offers excellent opportunities for the multiplication of elite genotypes or endangered animals, whose number have shrunk to below the threshold of safe existence. Disappointingly, SCNT efficiency is still low. Hence, it would be wise to store somatic cells from threatened animals in biobanks. We were the first to show that freeze-dried cells allow generating blastocysts upon SCNT. Only a few papers have been published on the topic since then, and viable offspring have not been produced. On the other hand, lyophilization of mammalian spermatozoa has made considerable progress, partially due to the physical stability that protamines provide to the genome. In our previous work, we have demonstrated that a somatic cell could be made more amenable to the oocyte reprogramming by the exogenous expression of human Protamine 1. Given that the protamine also provides natural protection against dehydration stress, we have combined the cell protaminization and lyophilization protocols. This chapter comprehensively describes the protocol for somatic cell protaminization, lyophilization, and its application in SCNT. We are confident that our protocol will be relevant for establishing somatic cells stocks amenable to reprogramming at low cost.

Cattle Cloning by Somatic Cell Nuclear Transfer.

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

2-Cell-like Cells: An Avenue for Improving SCNT Efficiency.

After fertilization, the zygote genome undergoes dramatic structural reorganization to ensure the establishment of totipotency, and then the totipotent potential of the zygote or 2-cell-stage embryo progressively declines. However, cellular potency is not always a one-way street. Specifically, a small number of embryonic stem cells (ESCs) occasionally overcome epigenetic barriers and transiently convert to a totipotent status. Despite the significant potential of the somatic cell nuclear transfer (SCNT) technique, the establishment of totipotency is often deficient in cloned embryos. Because of this phenomenon, the question arises as to whether strategies attempting to induce 2-cell-like cells (2CLCs) can provide practical applications, such as reprogramming of somatic cell nuclei. Inspired by strategies that convert ESCs into 2CLCs, we hypothesized that there will be a similar pathway by which cloned embryos can establish totipotent status after SCNT. In this review, we provide a snapshot of the practical strategies utilized to induce 2CLCs during investigations of the development of cloned embryos. The 2CLCs have similar transcriptome and chromatin features to that of 2-cell-stage embryos, and we propose that 2CLCs, already a valuable in vitro model for dissecting totipotency, will provide new opportunities to improve SCNT efficiency.

Recapitulating early human development with 8C-like cells.

In human embryos, major zygotic genome activation (ZGA) initiates at the eight-cell (8C) stage. Abnormal ZGA leads to developmental defects and even contributes to the failure of human blastocyst formation or implantation. An invitro cell model mimicking human 8C blastomeres would be invaluable to understanding the mechanisms regulating key biological events during early human development. Using the non-canonical promoter of LEUTX that putatively regulates human ZGA, we developed an 8C::mCherry reporter, which specifically marks the 8C state, to isolate rare 8C-like cells (8CLCs) from human preimplantation epiblast-like stem cells. The 8CLCs express a panel of human ZGA genes and have a unique transcriptome resembling that of the human 8C embryo. Using the 8C::mCherry reporter, we further optimize the chemical-based culture condition to increase and maintain the 8CLC population. Functionally, 8CLCs can self-organize to form blastocyst-like structures. The discovery and maintenance of 8CLCs provide an opportunity to recapitulate early human development.

Modification of alternative splicing in bovine somatic cell nuclear transfer embryos using engineered CRISPR-Cas13d.

Animal cloning can be achieved by somatic cell nuclear transfer (SCNT), but the resulting live birth rate is relatively low. We previously improved the efficiency of bovine SCNT by exogenous melatonin treatment or by overexpression of lysine-specific demethylase 4D (KDM4D) and 4E (KDM4E). In this study, we revealed abundant alternative splicing (AS) transitions during fertilization and embryonic genome activation, and demonstrated abnormal AS in bovine SCNT embryos compared with in vitro fertilized embryos. We used the CRISPR-Cas13d RNA-targeting system to target cis-elements of ABI2 and ZNF106 pre-mRNA to modify AS, thus reducing the ratio of abnormal-isoform SCNT embryos by nearly 50% and achieving a high survival rate (11%-19%). These results indicate that this system may provide an efficient method for bovine cloning, while also paving the way for further improvements in the efficiency of SCNT.

Current Progress and Prospects in Rabbit Cloning.

Somatic cell nuclear transfer (SCNT) shows great value in the generation of transgenic animals, protection of endangered animals, and stem cell therapy. The combination of SCNT and gene editing has produced a variety of genetically modified animals for life science and medical research. Rabbits have unique advantages as transgenic bioreactors and human disease models; however, the low SCNT efficiency severely impedes the application of this technology. The difficulty in SCNT may be attributable to the abnormal reprogramming of somatic cells in rabbits. This review focuses on the abnormal reprogramming of cloned mammalian embryos and evaluates the progress and prospects of rabbit somatic cell cloning.

A diverse English keyword search reveals the value of scriptaid treatment for porcine embryo development following somatic cell nuclear transfer.

Incomplete epigenetic reprogramming of histone deacetylation (HDAC) is one of the main reasons for the low efficiency of somatic cell nuclear transfer (SCNT). Scriptaid is a synthetic HDAC inhibitor (HDACi) that may improve the efficiency of porcine SCNT. This study aimed to determine whether scriptaid increases the number of blastocyst cells or the cleavage rate. We conducted a meta-analysis of the pertinent literature published over the past decade. A total of 73 relevant papers were retrieved using a diverse English keyword search, and 11 articles were used for the meta-analysis. Scriptaid was positively correlated with blastocyst rate but had no effect on cleavage rate or blastocyst cell number. A subgroup analysis of blastocyst cell number showed that the staining method was the source of the heterogeneity. In SCNT embryos, scriptaid treatment after activation can promote embryonic development, but there may be adverse effects on early development. HDACi research should focus on SCNT birth efficiency.

Effect of Embryo Aggregation on In Vitro Development of Adipose-Derived Mesenchymal Stem Cell-Derived Bovine Clones.

Somatic cell nuclear transfer (SCNT) is a method with unique ability to reprogram the epigenome of a fully differentiated cell. However, its efficiency remains extremely low. In this work, we assessed and combined two simple strategies to improve the SCNT efficiency in the bovine. These are the use of less-differentiated donor cells to facilitate nuclear reprogramming and the embryo aggregation (EA) strategy that is thought to compensate for aberrant epigenome reprogramming. We carefully assessed the optimal time of EA by using in vitro-fertilized (IVF) embryos and evaluated whether the use of adipose-derived mesenchymal stem cells (ASCs) as donor for SCNT together with EA improves the blastocyst rates and quality. Based on our results, we determined that the EA improves the preimplantation embryo development per well of IVF and SCNT embryos. We also demonstrated that day 0 (D0) is the optimal aggregation time that leads to a single blastocyst with uniform distribution of the original blastomeres. This was confirmed in bovine IVF embryos and then, the optimal condition was translated to SCNT embryos. Notably, the relative expression of the trophectoderm (TE) marker KRT18 was significantly different between aggregated and nonaggregated ASC-derived embryos. In the bovine, no effect of the donor cell is observed on the developmental rate, or the embryo quality. Therefore, no synergistic effect of the use of both strategies is observed. Our results suggest that EA at D0 is a simple and accessible strategy that improves the blastocyst rate per well in bovine SCNT and IVF embryos and influence the expression of a TE-related marker. The aggregation of two ASC-derived embryos seems to positively affect the embryo quality, which may improve the postimplantation development.

Improving porcine SCNT efficiency by selecting donor cells size

ABSTRACT Considerable advancements have recently been achieved in porcine somatic cell nuclear transfer (SCNT), but the efficiency remains low. Donor cell size might play an important role in SCNT, but its effects in pigs remain unclear. This study aimed to evaluate the efficiency of porcine SCNT by selecting donor cells of suitable size. Porcine fetal fibroblasts (PFFs) were divided into three groups, group S (small, d ≤ 13 μm), group M (medium, 13 μm<d ≤ 18 μm), and group L (large, d > 18 μm), and their biological characteristics were analyzed. Next, SCNT was performed using PFFs of different sizes to evaluate the developmental potential of reconstructed embryos. The data showed that PFFs in groups S, M and L accounted for 17.5%, 47.7% and 34.8% of cells, respectively. Morphologically, cells in group S exhibited clear and regular cell membranes and nuclei, whereas cells in groups M and L displayed varying degrees of cell membrane protuberance, karyo-pyknosis, autophagy and mitochondrial abnormalities. In addition, the growth status and proliferation capabilities of cells in group S were significantly better than those of group M and group L. The percentage of cells at G0/G1 in group S and M were significantly greater than group L. The senescence rate of group S was lower than group M and group L. The apoptosis rate of group S was significantly lower than that of group L but comparable to that of group M . The cleavage rate of group S was also significantly greater than that of group M but comparable to that of group L . The blastocyst rate of group S was significantly greater than that of group M and group L. The blastocyst cell numbers of group S were also significantly greater than those of group M and group L. These findings suggested that small PFFs with a diameter of less than 13 μm are more suitable donor cells for SCNT in pigs. Abbreviations: DMEM: Dulbecco’s modified Eagle’s medium; FBS: Fetal bovine serum; PBS: Phosphate buffer saline; PFFs: Porcine fetal fibroblast cells; SCNT: Somatic cell nuclear transfer.

Functions and Regulation of Endogenous Retrovirus Elements during Zygotic Genome Activation: Implications for Improving Somatic Cell Nuclear Transfer Efficiency.

Endogenous retroviruses (ERVs), previously viewed as deleterious relics of ancestral retrovirus infections, are silenced in the vast majority of cells to minimize the risk of retrotransposition. Counterintuitively, bursts of ERV transcription usually occur during maternal-to-zygotic transition (MZT) in preimplantation embryos; this is regarded as a major landmark event in the zygotic genome activation (ZGA) process, indicating that ERVs play an active part in ZGA. Evolutionarily, the interaction between ERVs and hosts is mutually beneficial. The endogenization of retrovirus sequences rewires the gene regulatory network during ZGA, and ERV repression may lower germline fitness. Unfortunately, owing to various limitations of somatic cell nuclear transfer (SCNT) technology, both developmental arrest and ZGA abnormalities occur in a high percentage of cloned embryos, accompanied by ERV silencing, which may be caused by the activation failure of upstream ERV inducers. In this review, we discuss the functions and regulation of ERVs during the ZGA process and the feasibility of temporal control over ERVs in cloned embryos via exogenous double homeobox (DUX). We hypothesize that further accurate characterization of the ERV-rewired gene regulatory network during ZGA may provide a novel perspective on the development of preimplantation embryos.

Improved development of mouse somatic cell nuclear transfer embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors†

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.

Extranuclear Inheritance of Mitochondrial Genome and Epigenetic Reprogrammability of Chromosomal Telomeres in Somatic Cell Cloning of Mammals.

The effectiveness of somatic cell nuclear transfer (SCNT) in mammals seems to be still characterized by the disappointingly low rates of cloned embryos, fetuses, and progeny generated. These rates are measured in relation to the numbers of nuclear-transferred oocytes and can vary depending on the technique applied to the reconstruction of enucleated oocytes. The SCNT efficiency is also largely affected by the capability of donor nuclei to be epigenetically reprogrammed in a cytoplasm of reconstructed oocytes. The epigenetic reprogrammability of donor nuclei in SCNT-derived embryos appears to be biased, to a great extent, by the extranuclear (cytoplasmic) inheritance of mitochondrial DNA (mtDNA) fractions originating from donor cells. A high frequency of mtDNA heteroplasmy occurrence can lead to disturbances in the intergenomic crosstalk between mitochondrial and nuclear compartments during the early embryogenesis of SCNT-derived embryos. These disturbances can give rise to incorrect and incomplete epigenetic reprogramming of donor nuclei in mammalian cloned embryos. The dwindling reprogrammability of donor nuclei in the blastomeres of SCNT-derived embryos can also be impacted by impaired epigenetic rearrangements within terminal ends of donor cell-descended chromosomes (i.e., telomeres). Therefore, dysfunctions in epigenetic reprogramming of donor nuclei can contribute to the enhanced attrition of telomeres. This accelerates the processes of epigenomic aging and replicative senescence in the cells forming various tissues and organs of cloned fetuses and progeny. For all the above-mentioned reasons, the current paper aims to overview the state of the art in not only molecular mechanisms underlying intergenomic communication between nuclear and mtDNA molecules in cloned embryos but also intrinsic determinants affecting unfaithful epigenetic reprogrammability of telomeres. The latter is related to their abrasion within somatic cell-inherited chromosomes.

Sperm-borne small RNAs improve the developmental competence of pre-implantation cloned embryos in rabbit.

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.

25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER: Scientific and technological approaches to improve SCNT efficiency in farm animals and pets.

The birth of Dolly through somatic cell nuclear transfer (SCNT) was a major scientific breakthrough of the last century. Yet, while significant progress has been achieved across the technics required to reconstruct and in vitro culture nuclear transfer embryos, SCNT outcomes in terms of offspring production rates are still limited. Here, we provide a snapshot of the practical application of SCNT in farm animals and pets. Moreover, we suggest a path to improve SCNT through alternative strategies inspired by the physiological reprogramming in male and female gametes in preparation for the totipotency required after fertilization. Almost all papers on SCNT focused on nuclear reprogramming in the somatic cells after nuclear transfer. We believe that this is misleading, and even if it works sometimes, it does so in an uncontrolled way. Physiologically, the oocyte cytoplasm deploys nuclear reprogramming machinery specifically designed to address the male chromosome, the maternal alleles are prepared for totipotency earlier, during oocyte nuclear maturation. Significant advances have been made in remodeling somatic nuclei in vitro through the expression of protamines, thanks to a plethora of data available on spermatozoa epigenetic modifications. Missing are the data on large-scale nuclear reprogramming of the oocyte chromosomes. The main message our article conveys is that the next generation nuclear reprogramming strategies should be guided by insights from in-depth studies on epigenetic modifications in the gametes in preparation for fertilization.

Sperm-borne miR-202 targets SEPT7 and regulates first cleavage of bovine embryos via cytoskeletal remodeling.

In mammals, sperm-borne regulators can be transferred to oocytes during fertilization and have different effects on the formation of pronuclei, the first cleavage of zygotes, the development of preimplantation embryos and even the metabolism of individuals after birth. The regulatory role of sperm microRNAs (miRNAs) in the development of bovine preimplantation embryos has not been reported in detail. By constructing and screening miRNA expression libraries, we found that miR-202 was highly enriched in bovine sperm. As a target gene of miR-202, co-injection of SEPT7 siRNA can partially reverse the accelerated first cleavage of bovine embryos caused by miR-202 inhibitor. In addition, both a miR-202 mimic and SEPT7 siRNA delayed the first cleavage of somatic cell nuclear transfer (SCNT) embryos, suggesting that miR-202-SEPT7 mediates the delay of first cleavage of bovine embryos. By further exploring the relationship between miR-202/SEPT7, HDAC6 and acetylated α-tubulin during embryonic development, we investigated how sperm-borne miR-202 regulates the first cleavage process of bovine embryos by SEPT7 and demonstrate the potential of sperm-borne miRNAs to improve the efficiency of SCNT.