High Expression of ZFP42 Improves Early Development of Pig Embryos Produced by Handmade Cloning.

Handmade Cloning (HMC) is a pivotal technique for cloning pig embryos. Despite its significance, the low efficiency of this method hampers its widespread application. Although numerous factors and signaling pathways influencing embryo development have been studied, the mechanisms underlying low developmental capacity and insufficient reprogramming of cloned embryos remain elusive. In the present study, we sought to elucidate key regulatory factors involved in the development of pig HMC embryos by comparing and analyzing the gene expression profiles of HMC embryos with those of naturally fertilized (NF) embryos at the 4-cell, 8-cell, and 16-cell stages. The results showed that ZFP42 expression is markedly higher in NF embryos than in cloned counterparts. Subsequent experiments involving the injection of ZFP42 messenger RNA (mRNA) into HMC embryos showed that ZFP42 could enhance the blastocyst formation rate, upregulate pluripotent genes and metabolic pathways. This highlights the potential of ZFP42 as a critical factor in improving the development of pig HMC embryos.

Analysis of production efficiency of cloned transgenic Yucatan miniature pigs according to recipient breeds with embryo transfer conditions.

The purpose of this study was to compare the efficiency of the production of cloned transgenic Yucatan miniature pigs (YMPs) using two recipient breeds, i.e., YMPs and domestic pigs (DPs), under various embryo transfer conditions. We initially assessed the in vitro developmental competence of embryos obtained via somatic cell nuclear transfer (SCNT) from three different transgenic donor cells. No difference was observed among the three groups regarding developmental competence. Furthermore, the cloning efficiency remained consistent among the three groups after the transfer of the SCNT embryos to each surrogate mother. Subsequently, to compare the efficiency of the production of cloned transgenic YMPs between the two recipient breeds using varying parameters, including ovulation status (preovulation and postovulation), duration of in vitro culture (IVC) (incubated within 24 h and 24-48 h), and the number of transferred SCNT embryos (less than and more than 300), we assessed the pregnancy rates, delivery rates, mean offspring counts, and cloning efficiency. Regarding the ovulation status, YMPs exhibited higher pregnancy rates, delivery rates, and cloning efficiency compared with DPs in both statuses. Moreover, the pregnancy rates, delivery rates, and cloning efficiency were affected by the ovulation status in DPs, but not in YMPs. The comparison of IVC duration between groups revealed that YMPs had higher pregnancy rates vs. DPs in both conditions. SCNT embryos cultured for 24-48 h in YMPs yielded higher delivery rates and cloning efficiency compared with those cultured for less than 24 h in DPs. Finally, the analysis based on the number of transferred SCNT embryos showed that both the pregnancy and delivery rates were higher in YMPs vs. DPs. However, the highest average number of offspring was obtained when more than 300 SCNT embryos were transferred into DPs, whereas the cloning efficiency was higher in YMPs vs. DPs. Our results suggest that YMPs are more suitable recipients than are DPs under various conditions for the production of cloned transgenic YMPs.

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

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

Reprogramming mechanism dissection and trophoblast replacement application in monkey somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) successfully clones cynomolgus monkeys, but the efficiency remains low due to a limited understanding of the reprogramming mechanism. Notably, no rhesus monkey has been cloned through SCNT so far. Our study conducts a comparative analysis of multi-omics datasets, comparing embryos resulting from intracytoplasmic sperm injection (ICSI) with those from SCNT. Our findings reveal a widespread decrease in DNA methylation and the loss of imprinting in maternally imprinted genes within SCNT monkey blastocysts. This loss of imprinting persists in SCNT embryos cultured in-vitro until E17 and in full-term SCNT placentas. Additionally, histological examination of SCNT placentas shows noticeable hyperplasia and calcification. To address these defects, we develop a trophoblast replacement method, ultimately leading to the successful cloning of a healthy male rhesus monkey. These discoveries provide valuable insights into the reprogramming mechanism of monkey SCNT and introduce a promising strategy for primate cloning.

Overexpression of BRG1 improves early development of porcine somatic cell nuclear transfer embryos.

The epigenetic modification levels of donor cells directly affect the developmental potential of somatic cell nuclear transfer (SCNT) embryos. BRG1, as an epigenetic modifying enzyme, has not yet been studied in donor cells and SCNT embryos. In this study, BRG1 was overexpressed in porcine fetal fibroblasts (PFFs), its effect on chromatin openness and gene transcription was examined, subsequently, the development potential of porcine SCNT embryos was investigated. The results showed that compared with the control group, the percentage of G1 phase cells was significantly increased (32.3 % ± 0.87 vs 25.7 % ± 0.81, P < 0.05) in the experimental group. The qRT-PCR results showed that the expression of H3K9me3-related genes was significantly decreased (P < 0.05), HAT1 was significantly increased (P < 0.05). Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) results revealed that SMARCA4、NANOG、SOX2、MAP2K6 and HIF1A loci had more open chromatin peaks in the experimental group. The RNA-seq results showed that the upregulated genes were mainly enriched in PI3K/AKT and WNT signaling pathways, and the downregulated genes were largely focused on disease development. Interestingly, the developmental rate of porcine SCNT embryos was improved (27.33 % ± 1.40 vs 17.83 % ± 2.02, P < 0.05), the expression of zygotic gene activation-related genes in 4-cell embryos, and embryonic development-related genes in blastocysts was significantly upregulated in the experimental group (P < 0.05). These results suggest that overexpression of BRG1 in donor cells is benefit for the developmental potential of porcine SCNT embryos.

Overcoming the H4K20me3 epigenetic barrier improves somatic cell nuclear transfer reprogramming efficiency in mice.

Epigenetic reprogramming during fertilization and somatic cell nuclear transfer (NT) is required for cell plasticity and competent development. Here, we characterize the epigenetic modification pattern of H4K20me3, a repressive histone signature in heterochromatin, during fertilization and NT reprogramming. Importantly, the dynamic H4K20me3 signature identified during preimplantation development in fertilized embryos differed from NT and parthenogenetic activation (PA) embryos. In fertilized embryos, only maternal pronuclei carried the canonical H4K20me3 peripheral nucleolar ring-like signature. H4K20me3 disappeared at the 2-cell stage and reappeared in fertilized embryos at the 8-cell stage and in NT and PA embryos at the 4-cell stage. H4K20me3 intensity in 4-cell, 8-cell, and morula stages of fertilized embryos was significantly lower than in NT and PA embryos, suggesting aberrant regulation of H4K20me3 in PA and NT embryos. Indeed, RNA expression of the H4K20 methyltransferase Suv4-20h2 in 4-cell fertilized embryos was significantly lower than NT embryos. Knockdown of Suv4-20h2 in NT embryos rescued the H4K20me3 pattern similar to fertilized embryos. Compared to control NT embryos, knockdown of Suv4-20h2 in NT embryos improved blastocyst development ratios (11.1% vs. 30.5%) and full-term cloning efficiencies (0.8% vs. 5.9%). Upregulation of reprogramming factors, including Kdm4b, Kdm4d, Kdm6a, and Kdm6b, as well as ZGA-related factors, including Dux, Zscan4, and Hmgpi, was observed with Suv4-20h2 knockdown in NT embryos. Collectively, these are the first findings to demonstrate that H4K20me3 is an epigenetic barrier of NT reprogramming and begin to unravel the epigenetic mechanisms of H4K20 trimethylation in cell plasticity during natural reproduction and NT reprogramming in mice.

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

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

Effect of additional cytoplasm injection on the cloned bovine embryo organelle distribution and stress mitigation.

Although somatic cell nuclear transfer (SCNT) is a critical component of animal cloning, this approach has several issues. We previously introduced the cytoplasm injection cloning technology (CICT), which significantly improves the quality and quantity of cloned embryos. This study examined the residual status of fused cytoplasmic organelles, such as the endoplasmic reticulum (ER) and lysosomes, in the CICT group during early embryo development. We found that extra-cytoplasmic organelles stained using the ER-Tracker™ Green dye and LysoTracker™ Deep Red probe were fused and dispersed throughout the recipient oocyte and were still visible in day 8 blastocysts. We screened for ER stress, autophagy, and apoptosis-related genes to elucidate the association between the added organelles and improved embryo quality in CICT-cloned embryos. We found that CHOP, ATF4, ATG5, ATG7, and LC3 genes showed non-significantly up- or downregulated expression between CICT- and in vitro fertilization (IVF)-derived embryos but showed significantly (p < 0.05) upregulated expression in SCNT-cloned embryos. Surprisingly, a non-significant difference in the expression of some genes, such as ATF6 and caspase-3, was observed between the CICT- and SCNT-cloned embryos. Our findings imply that compared to conventional SCNT cloning, CICT-derived cloned embryos with additional cytoplasm have much higher organelle activity, lower autophagy, lower rates of apoptosis, and higher embryo development rates.

Human Cloning: Biology, Ethics, and Social Implications.

Abstract: This scholarly article delves into the multifaceted domains of human cloning, encompassing its biological underpinnings, ethical dimensions, and broader societal implications. The exposition commences with a succinct historical and contextual overview of human cloning, segueing into an in-depth exploration of its biological intri-cacies. Central to this biological scrutiny is a comprehensive analysis of somatic cell nuclear transfer (SCNT) and its assorted iterations. The accomplishments and discoveries in cloning technology, such as successful animal cloning operations and advances in the efficiency and viability of cloned embryos, are reviewed. Future improvements, such as reprogramming procedures and gene editing technology, are also discussed. The discourse extends to ethical quandaries intrinsic to human cloning, entailing an extensive contemplation of values such as human dignity, autonomy, and safety. Furthermore, the ramifications of human cloning on a societal plane are subjected to scrutiny, with a dedicated emphasis on ramifications encompassing personal identity, kinship connections, and the fundamental notion of maternity. Culminating the analysis is a reiteration of the imperative to develop and govern human cloning technology judiciously and conscientiously. Finally, it discusses several ethical and practical issues, such as safety concerns, the possibility of exploitation, and the erosion of human dignity, and emphasizes the significance of carefully considering these issues.

Ian Wilmut (1944-2023).

Embryologist who cloned Dolly the sheep.

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.

Genetic clues to reprogramming power and formation of mouse oocyte.

Oocyte features the unique capacity to reprogram not only sperm but also somatic nuclei to totipotency, yet the scarcity of oocytes has hindered the exploration and application of their reprogramming ability. In the meanwhile, the formation of oocytes, which involves extensive intracellular alterations and interactions, has also attracted tremendous interest. This review discusses developmental principles and regulatory mechanisms associated with ooplasm reprogramming and oocyte formation from a genetic perspective, with knowledge derived from mouse models. We also discuss future directions, especially to address the lack of insight into the regulatory networks that shape the identity of female germ cells or drive transitions in their developmental programs.

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.

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.

Astaxanthin enhances the development of bovine cloned embryos by inhibiting apoptosis and improving DNA methylation reprogramming of pluripotency genes.

Low cloning efficiency limits the wide application of somatic cell nuclear transfer technology. Apoptosis and incomplete DNA methylation reprogramming of pluripotency genes are considered as the main causes for low cloning efficiency. Astaxanthin (AST), a powerfully antioxidative and antiapoptotic carotenoid, is recently shown to improve the development of early embryos, however, the potential role of AST during the development of cloned embryos remains unclear. This study displayed that treating cloned embryos with AST significantly increased the blastocyst rate and total blastocyst cell number in a concentration dependent manner, and also alleviated the damage of H2O2 to the development of cloned embryos. In addition, compared with the control group, AST significantly reduced the apoptotic cell number and rate in cloned blastocysts, and the significantly upregulated expression of anti-apoptotic gene Bcl2l1 and antioxidative genes (Sod1 and Gpx4) and downregulated transcription of pro-apoptotic genes (Bax, P53 and Caspase3) were observed in the AST group. Moreover, AST treatment facilitated DNA demethylation of pluripotency genes (Pou5f1, Nanog and Sox2), in accompany with the improved transcription levels of DNA methylation reprogramming genes (Tet1, Tet3, Dnmt1, Dnmt3a and Dnmt3b) in cloned embryos, and then, the significantly upregulated expression levels of embryo development related genes including Pou5f1, Nanog, Sox2 and Cdx2 were observed in comparison with the control group. In conclusion, these results revealed that astaxanthin enhanced the developmental potential of bovine cloned embryos by inhibiting apoptosis and improving DNA methylation reprogramming of pluripotency genes, and provided a promising approach to improve cloning efficiency.

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

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

A long non-coding RNA essential for early embryonic development improves somatic cell nuclear transfer somatic cell nuclear transfer efficiency in goats.

In brief: Early embryonic development in goats is a complex and an important process. This study identified a novel long non-coding RNA (lncRNA), lncRNA3720, that appears to affect early embryonic development in goats through histone variants. Abstract: Although abundant 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, two-cell (IVF-2c) and eight-cell embryos (IVF-8c) and eight-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 the 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 were significantly improved. We speculate that lncRNA3720 is a key promoter of embryonic development in goats by interacting with histone variants.

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

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

Epigenetic Iinsights into the Senescence of Porcine Fetal Fibroblasts induced by Passaging.

Epigenetic status of fetal fibroblasts (FFs) is one of the crucial factors accounted for the success of somatic cell nuclear transfer and gene editing, which might inevitably be affected by passaging. But few systematic studies have been performed on the epigenetic status of passaged aging cells. Therefore, FFs from large white pig were in vitro passaged to the 5, 10, and 15 (F5, F10, and F15) passages in the present study to investigate the potential alteration of epigenetic status. Results indicated the senescence of FFs occurs with the passaging, as assessed by the weakened growth rate, increased ß-gal expression, and so on. For the epigenetic status of FFs, the higher level both of DNA methylation and H3K4me1, H3K4me2, H3K4me3 was observed at F10, but the lowest level was observed at F15. However, the fluorescence intensity of m6A was significantly higher in F15, but lower (p < 0.05) in F10, and the related mRNA expression in F15 was significantly higher than F5. Further, RNA-Seq indicated a considerable difference in the expression pattern of F5, F10, and F15 FFs. Among differentially expressed genes, not only the genes involved in cell senescence were changed, but also the upregulated expression of Dnmt1, Dnmt3b, Tet1 and dysregulated expression of histone methyltransferases-related genes were detected in F10 FFs. In addition, most genes related to m6A such as METTL3, YTHDF2, and YTHDC1 were significantly different in F5, F10, and F15 FFs. In conclusion, the epigenetic status of FFs was affected by being passaged from F5 to F15.

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

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