Embryo Aggregation Research Articles

Tetraploid embryo aggregation produces high-quality blastocysts with an increased trophectoderm in pigs.

Tetraploid complementation is an ideal method for demonstrating the differentiation potential of pluripotent stem cells. In this study, we selected the most efficient tetraploid production method for porcine embryos and investigated whether tetraploid blastomere aggregation could enhance the quality of tetraploid embryos. Three methods were investigated to produce tetraploid embryos: First, tetraploid embryos were produced using electro-fusion of two-cell stage parthenogenetic blastomere (FUTP). Second, somatic cell was injected into the mature oocyte and fused to produce tetraploid embryos. Third, oocytes were matured with Cytochalasin B (CB) for the late 22h of in vitro maturation to inhibit the first polar body (PB1). Following that, non-PB1 oocytes were treated with CB for 4h after parthenogenetic activation. There was no significant difference in the blastocyst development rate and tetraploid production rate of the embryos produced through the three methods. However, FUTP-derived blastocysts had a significantly lower percentage of apoptotic cells compared to other methods. The developmental competence of embryos, expression of trophectoderm cell marker genes, and distribution of YAP1 protein were investigated in tetraploid embryos produced using the FUTP method. The FUTP method most effectively prevented apoptosis during porcine tetraploid embryo formation. Tetraploid aggregation-derived blastocysts have a high proportion of trophectoderm with increased expression of the CDX2 mRNA and high YAP1 intensity. High-quality blastocysts derived from a tetraploid embryo aggregation can serve as suitable source material for testing the differentiation potential of pluripotent stem cells for blastocyst complementation in pigs.

Developmental competence of chimeric porcine embryos through the aggregation of parthenogenetic embryos and somatic cell nuclear transfer embryos

The efficiency of somatic cell nuclear transfer (NT) in pigs is low and requires enhancement. We identified the most efficient method for zona pellucida (ZP) removal and blastomere aggregation in pigs and investigated whether the aggregation of NT and parthenogenetic activation (PA) of blastomeres could reduce embryonic apoptosis and improve the quality of NT-derived embryos by investigating. Embryonic developmental competence after ZP removal using acid Tyrode's solution or protease (pronase E). The embryonic developmental potential of NT-derived blastomeres was also investigated using well-of-the-well or phytohemagglutinin-L. We analyzed apoptosis in aggregate-derived blastocysts. The aggregation rate of protease-treated embryos was lower than that of Tyrode’s solution-treated embryos (69.2% vs. 88.3%). No significant difference was observed between phytohemagglutinin-L and well-of-the-well (35.7%–38.5%). However, 2P1N showed a higher number of blastocysts compared to 3N (73.8% vs. 24.3%) and an increased blastocyst diameter compared to the control and 1P2N (274 μm vs. 230–234 μm). In blastomeres aggregated using phytohemagglutinin-L, the apoptotic cell ratio was significantly higher in 1P2N and 3N than in 3P (5.91%–6.46% vs. 2.94%, respectively). Our results indicate that aggregation of one NT embryo with two PA embryos improved the rate of blastocysts with increased blastocyst diameter.

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.

Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres.

Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the species to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. For this purpose, we compared in vitro development of synchronic (Sync) or asynchronic (Async) and asynchronic with a tetraploid (Async4n) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA fragmentation. Additionally, the developmental rates of Async4n aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. Async aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by Async4n aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by Async4n aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of two-cell-fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos.

Controlled Cytoplast Arrest and Morula Aggregation Enhance Development, Cryoresilience, and In Vivo Survival of Cloned Sheep Embryos.

Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphase-arrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.

16 Embryo aggregation and adipose-derived mesenchymal donor cells in bovine somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) is a powerful tool, but its efficiency remains low. The use of less differentiated donor cells or the embryo aggregation (EA) strategy improves the SCNT rates in several species. It remains unexplored whether the combined use of both strategies results in a synergistic effect that improves SCNT efficiency in bovine. To evaluate that, we assessed the optimal time of EA using IVF embryos (aim 1) and we evaluated whether the use of adipose-derived mesenchymal stem cells (ASC) as donor for SCNT together with EA improves the blastocyst rates and quality (aim 2). For aim 1, cumulus–oocyte complexes (COCs) were collected from slaughterhouse ovaries, invitro matured (TCM-199), fertilized (16×106 spermatozoa mL−1 for 5h) and cultured (synthetic oviductal fluid media in a humidified gas mixture at 39°C). After IVF, the zona pellucida was enzymatically removed and zona-free (ZF) embryos were cultured individually (1X) or 2 embryos placed together within a microwell (2X) (Day 0, n=70). This procedure was performed at Days 3, 4, 5, 6, or 7 (n=76, 78, 94, 96, 90, respectively) and blastocyst rate was assessed at Day 8. Contribution of both embryos to the 2X blastocyst was confirmed by staining Day 0 IVF embryos either with green or red Mitotracker (ThermoFisher Scientific) before EA. For aim 2, fibroblast (FB) and ASC cells were isolated from the skin and subcutaneous adipose tissue of the same adult animal, respectively. Cloned embryos were produced by ZF enucleation, fusion of one ASC or FB cell, and activation with 5μM ionomycin/6-(dimethylamino)purine (6DMAP). After activation, cloned embryos were aggregated (FB2X or ASC2X) or individually cultured (FB1X or ASC1X). Blastocyst rates were recorded at Day 7 of invitro culture. Three biological replicates were evaluated for each aim. Embryo developmental differences were determined using Fisher’s exact test. Relative expression of OCT4, SOX2, and KRT18 was measured by RTqPCR at the blastocyst stage and analysed by Kruskal–Wallis statistical test. Regarding aim 1, no differences for developmental rates were found for Day 0, 3, 4, and 5 groups (57%, 60%, 61.5%, 61%), but the blastocyst rate was only improved in Day 0 and Day 3 relative to their respective 1X controls (Day 0 2X 54.2% vs. Day 0 1X 25.5% and Day 3 2X 52.6% vs. Day 3 1X 25.3%). No aggregation occurred in Day 6 and Day 7 groups. All blastomeres were homogeneously distributed in the 2X blastocyst. Regarding aim 2, no effect of the donor cell was observed on the blastocyst rate (FB1X 26.8%, n=82; ASC1X 21.7%, n=198; FB2X 39.7%, n=126; ASC2X 33%, n=204), whereas EA improved the blastocyst rate of ASC-derived embryos (ASC1X 21.7% vs. ASC2X 33%). Overall, no synergistic effect of the use of both strategies was observed. Relative expression of KRT18 was significantly different between ASC1X and ASC2X embryos. Although OCT4 and SOX2 expression did not differ between groups, EA tended to bring the values closer to that of an IVF embryo. No effect of the donor cell was observed on the embryo relative expression. Our results suggest that EA at Day 0 improves the blastocyst rate in bovine SCNT and IVF embryos. EA of 2 ASC-derived embryos seemed to normalise the embryo quality and may improve post-implantation development.

Chromatin remodeler INO80 mediates trophectoderm permeability barrier to modulate morula-to-blastocyst transition.

Inositol requiring mutant 80 (INO80) is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells. However, the roles and mechanisms of INO80 in porcine pre-implantation embryonic development remain largely unknown. Here, we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development. The INO80 protein is highly expressed in the nuclei during morula-to-blastocyst transition. Functional studies revealed that RNA interference (RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm. Mechanistically, single-embryo RNA sequencing revealed that INO80 regulates multiple genes, which are important for lineage specification, tight junction assembly, and fluid accumulation. Consistent with the altered expression of key genes required for tight junction assembly, a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts. Importantly, aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium. Taken together, these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification, tight junction assembly, and fluid accumulation.

15 Blastocysts altered CDX2 and SOX2 gene expression and pregnancy failure after embryo transfer in yak heterospecific somatic cell nuclear transfer

Heterospecific cloning is a tool for the genetic rescue of endangered animals. Our objective was to evaluate the effects of heterospecific yak (Bos grunniens) cloned embryo aggregation on the expression levels of NANOG, OCT4, CDX2, and SOX2 genes, and to compare with IVF, parthenogenetic zona-free (P-ZF), and homospecific bovine cloned embryos (BB1x). Oocytes were recovered from the ovaries of slaughtered cows and invitro matured for 22h. The zona pellucida was removed by protease treatment and then mature oocytes were enucleated by micromanipulation. Enucleated oocytes were placed in phytohemagglutinin to induce adherence with a somatic donor cell followed by electrofusion (with two 30-µs pulses of 1.2 kV/cm, 0.1s apart). Two hours after fusion, reconstructed embryos were activated using ionomycin followed by 6-(dimethylamino)purine (6-DMAP) treatment for 3h and cultured in synthetic oviductal fluid (SOF) medium for 7 days. The experimental groups were IVF, P-ZF, BB1x, heterospecific yak-bovine cloned embryos (1 embryo per microwell, YB1x), and heterospecific yak-bovine cloned embryos aggregated (2 embryos per microwell, YB2x). In all experimental groups, cleavage and blastocyst rates were assessed 7 days after activation. In addition, 5 blastocysts were pooled for each biological replicate, and pluripotency-specific genes (NANOG, SOX2, CDX2, and OCT4) were analysed by quantitative PCR. Data were analysed by the ΔΔCT method using the geometric mean of ACTB (actin) and GAPDH as internal standard followed by one-way ANOVA. Cleavages rates were significantly lower in the YB1x group compared with the other groups. Moreover, blastocyst rates in YB2x (31.34%, n=67) were significantly higher than in YB1x (13.86%, n=101) and BB1x (13.33%, n=45) groups, but there were no significant differences compared with the IVF (43.82%, n=89) and P-ZF (25%, n=68) groups. In contrast, although no significant differences were observed among groups in the expression of NANOG and OCT4 genes, the expression of CDX2 was lower in YB2x and YB1x blastocysts compared with the BB1X, P-ZF, and IVF (control) groups. In addition, a decrease in SOX2 gene expression was observed in the YB2x and YB1x blastocysts compared with the BB1X group. Blastocysts from YB1x (n=5) and YB2x (n=18) groups were transferred to recipient cows (n=23) on Day 7. Forty days after embryo transfer, presence of uterine fluid was detected by ultrasound in 3 recipient cows (from YB2x), suggesting embryo loss. In concordance with our previous reports, yak heterospecific SCNT blastocysts showed underexpression of CDX2 and SOX2 compared with the overexpression observed for these genes in bovine homospecific SCNT blastocysts. Thus, yak heterospecific SCNT blastocysts may have compromised developmental competence associated with altered expression of CDX2 and SOX2 that cannot be rescued by the aggregation of 2 reconstructed embryos.

Embryo aggregation allows the production of kodkod (Leopardus guigna) blastocysts after interspecific SCNT

The kodkod (Leopardus guigna) is a small felid endemic of Chile and is considered a vulnerable species. Domestic cat oocytes have been successfully used as recipient cytoplast to reprogram somatic cells from different felids by interspecific somatic cell nuclear transfer (iSCNT). The developmental competence of felid embryos generated by iSCNT can be improved by the aggregation method using a zona-free culture system. The objective of this research was to evaluate the developmental competence of kodkod embryos generated by iSCNT using domestic cat oocytes and the aggregation method. For this purpose, five experimental group were done: (1) cat embryos generated by IVF, (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by iSCNT (K1x) and (5) aggregated kodkod embryos generated by iSCNT (K2x). Cleavage, morulae and blastocyst rates were estimated. The blastocyst diameter was evaluated. The gene expression level of pluripotency (OCT4, SOX2 and NANOG) and differentiation markers (CDX2 and GATA6) was analyzed in blastocysts. Morulae rate was higher in the IVF group and when cloned embryos were cultured in aggregates (IVF: 68.2%, Ca2x: 58.0% and K2x: 62.4%) compared to individually cultured kodkod embryos (K1x: 37.0%) (P < 0.05). Embryo aggregation increased blastocysts formation in the Ca2x group (30.9%) to a similar rate compared to the IVF group (44.5%) (P > 0.05). No blastocysts were generated in the K1x group, whereas blastocysts formation was obtained in K2x group (5.9%). The diameter of blastocysts from the K2x group (172.8 μm) was significantly lower than blastocysts from the Ca2x group (P < 0.05). The relative expression of OCT4 was lower in blastocysts from Ca1x than in blastocysts from IVF (P < 0.05). Furthermore, CDX2 expression was lower in blastocysts from Ca2x than in blastocysts from Ca1x and IVF groups (P < 0.05). In kodkod embryos, only one blastocyst from the K2x group expressed OCT4. No expression of SOX2, NANOG, CDX2 and GATA6 was detected in kodkod blastocysts. In conclusion, after iSCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. The aggregation method increases the morulae rate of kodkod cloned embryos and allows blastocysts formation. However, kodkod blastocysts have a poor morphological quality and a lacking expression of pluripotency and differentiation markers, probably caused by an incomplete nuclear reprogramming.

Effects of PXD101 and Embryo Aggregation on the In Vitro Development of Mouse Parthenogenetic Embryos.

To improve the isolation efficiency of parthenogenetic embryonic stem cells (pESCs) in mice, it is necessary to optimize the method to increase in vitro developmental competence of mice parthenogenetic blastocysts. Therefore, this study aims to investigate an optimal method for the production of mouse parthenogenetic blastocysts and isolation of pESC colonies by comparing the effects of two methods: (1) the treatment of histone deacetylase inhibitor PXD101 before, during, or after parthenogenetic activation; (2) parthenogenetic embryo aggregation; and (3) their combination treatment. The results suggest that application of PXD101 treatment and embryo aggregation could both improve the development of mouse parthenogenetic blastocysts (50 nM PXD101 treated 4 hours during activation and further 4 hours after activation: 40.0% vs. 20.0%; p < 0.05; two-cell embryo aggregation: 38.3% vs. 20.0%; p < 0.05) and also enhance the isolation rate of pESC colonies (PXD101: 33.3% vs. 11.8%; p < 0.05; two-cell embryo aggregation: 36.4% vs. 11.8%; p < 0.05). The combination of their treatments had the higher rate of parthenogenetic blastocyst development (41.7%) and significantly higher rate of pESC colony isolation from parthenogenetic blastocysts (45.0%); therefore, we concluded that the combination of these two methods (50 nM PXD101 treated for 8 hours and then aggregated at two-cell stage with 0.25% pronase for 10 minutes in our self-made concave) is considered the optimal way for the in vitro development of parthenogenetic blastocysts and subsequent pESC colony isolation in mice, opening new opportunities for application of this combination method to improve the parthenogenetic embryo development in other species.

Embryo aggregation regulates in vitro stress conditions to promote developmental competence in pigs.

Embryo aggregation is a useful method to produce blastocysts with high developmental competence to generate more offspring in various mammals, but the underlying mechanism(s) regarding the beneficial effects are largely unknown. In this study, we investigated the effects of embryo aggregation using 4-cell stage embryos in in vitro developmental competence and the relationship of stress conditions in porcine early embryogenesis. We conducted aggregation using the well of the well system and confirmed that aggregation using two or three embryos was useful for obtaining blastocysts. Aggregated embryos significantly improved developmental competence, including blastocyst formation rate, blastomere number, ICM/TE ratio, and cellular survival rate, compared to non-aggregated embryos. Investigation into the relationship between embryo aggregation and stress conditions revealed that mitochondrial function increased, and oxidative and endoplasmic reticulum (ER)-stress decreased compared to 1X (non-aggregated embryos) blastocysts. In addition, 3X (three-embryo aggregated) blastocysts increased the expression of pluripotency, anti-apoptosis, and implantation related genes, and decreased expression of pro-apoptosis related genes. Therefore, these findings indicate that embryo aggregation regulates in vitro stress conditions to increase developmental competence and contributes to the in vitro production of high-quality embryos and the large-scale production of transgenic and chimeric pigs.

20 Aggregation of yak heterospecific somatic cell nuclear transfer embryos improves cloning efficiency

Cloning endangered species has the limitation that generally the number of available oocytes is limited. Reprogramming the nuclei heterospecifically using an enucleated oocyte from a different species is an alternative. Aggregation of SCNT (somatic cell nuclear transfer) embryos from the same specie results in improved embryo development. However, after aggregation of heterospecific SCNT embryos from different genera, no effects were observed (Moro et al. 2015 Reproduction 50, 1-10). The objective of this study was to evaluate the influence of aggregation of yak (Bos grunniens) embryos produced by heterospecific SCNT using enucleated oocytes from an animal from the same genus Bos taurus. As control homospecific SCNT of Bos taurus, parthenogenic zone-free embryos and IVF embryos were used. Cumulus-oocyte complexes were recovered from bovine slaughterhouse ovaries by follicular aspiration. The cumulus-oocyte complexes were matured in tissue culture medium 199 containing 10% fetal bovine serum, 10μgmL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine, and 2% antibiotic-antimycotic for 22h, at 6.5% CO2 in humidified air and 38.5°C. After denudation, mature oocytes were stripped of the zona pellucida using a protease and then enucleated by micromanipulation. Staining was performed with Hoechst 33342 to observe MII. Enucleated oocytes were placed in phytohemagglutinin to induce adherence with the donor cell followed by electrofusion. All reconstituted embryos were activated using ionomcine. This was followed by a treatment with 6-dimethylaminopurine for 3h. Zona-free reconstituted cloned embryos were cultured in the wells of the well system, placing one (1×) or two (2×) per microwell, in synthetic oviductal fluid medium. The experimental groups were parthenogenic zone free; IVF; reconstituted embryos bull fibroblast-enucleated oocyte from cow (BC1×); reconstituted embryos yak fibroblast-enucleated oocyte from cow (YC1×); and reconstituted embryos aggregated yak fibroblast-enucleated oocyte from cow (YC2×). In all experimental groups, cleavage of at least one embryo in the wells and blastocyst formation at Day 7 were assessed. The effect of cloned embryo aggregation on blastocyst rates was analysed using Fisher exact tests (GraphPad Prisma 8), and results are shown on Table 1. Results demonstrated that aggregation of two SCNT heterospecific embryos increased the blastocyst formation rate of yak (P&amp;lt;0.05). In conclusion aggregation in yak heterospecific SCNT embryos from species of the same genus (Bos) can improve development to blastocyst. Table 1.Aggregation of yak heterospecific somatic cell nuclear transfer embryos Experimental group1 No. of embryos No. of embryos-wells2 Cleavage (%) Blastocyst (%) PZF 68 68 66 (97.06%)a 17 (25.00%)acd IVF 89 - 81 (91.01%)ab 39 (43.82%)b BC1× 45 45 41 (91.11%)b 6 (13.33%)cd YC1× 101 101 77 (76.24%)c 14 (13.86%)c YC2× 134 67 61 (91.04%)ab 21 (31.34%)ab a-dDifferent superscripts in the same column indicate significant difference (Fisher's exact test, P&amp;lt;0.05). 1PZF, parthenogenetic zone free; IFV, IVF fecundation; BC1×, clone of bovine; YC1×, clone of yak-bovine; YC2×, clone of yak-bovine added. 2Wells used with embryos.

97 Loss of aggregation capacity of bovine invitro-produced embryos and blastocyst-derived trophoblasts from Day 6 of development

Embryo aggregation consists of placing more than one zona-free (ZF) embryo in contact during development to obtain a unique structure. It has been reported in different species that aggregated cloned embryos show certain benefits compared with nonaggregated embryos. One way to obtain these benefits in IVF embryos would be to generate a transient chimera by the introduction of trophoblastic cells. Bovine trophoblastic cells can be obtained by embryo bisection of blastocysts, cutting asymmetrically to use trophoblasts (Tr) for aggregation and leaving aside the portion that contains the inner cell mass (ICM). Taking all this into account, the objectives of this work are to study the aggregation of Tr at different days of development and to determine the appropriate time of aggregation. To this aim, cumulus-oocyte complexes (COCs) collected from slaughterhouse ovaries were matured in tissue culture medium 199 containing 10% fetal bovine serum, 10µgmL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine and 2% antibiotic-antimycotic for 24h, at 6.5% CO2 in humidified air and 38.5°C. We performed IVF with 16×106 spermatozoa per mL for 5h. Afterwards, presumptive zygotes were cultured in synthetic oviductal fluid (SOF) for 7 days in a humidified atmosphere at 38.5°C, 5% O2, 5% CO2, and 90% N2. In Experiment 1, embryo bisection of Day 7 blastocysts was performed manually under stereoscopic observation with a microblade to obtain Tr. These were aggregated, with the bisected part containing the ICM (n=22) or with ZF embryos of Days 4 (n=23), 5(n=25), or 6 (n=22) and blastocysts (n=25), and placed in microwells in a 100-μL SOF drop covered by mineral oil (Gambini et al. 2012 Biol. Reprod. 87, 15; https://doi.org/10.1095/biolreprod.112.098855). In Experiment 2, ZF synchronous whole embryos were aggregated in microwells at different developmental days: Day 3 (n=18), 4 (n=18), 5 (n=47), 6 (n=48), and 7 (n=45). In both experiments, aggregation was assessed at Day 8. In Experiment 1, no aggregation was observed between the Tr and the embryos or the bisected ICM. Experiment 2 showed embryo aggregation on Days 3 (55%), 4 (27%), and 5 (61%), whereas on Days 6 and 7 no aggregation was observed. According to these results, we can conclude that, in our culture conditions, Tr obtained by blastocyst bisection have no capacity for aggregation. Day 6 and 7 whole ZF embryos also do not aggregate. As a general conclusion, there is a period from Days 0-5 of the invitro development of bovine embryos in which aggregation is possible. Aggregation of blastocyst-derived Tr to cloned or high-value IVF embryos, aiming for quality improvement, is not an effective strategy.

Effects of fusion-activation interval and embryo aggregation on in vitro and in vivo development of bovine cloned embryos

Nuclear reprogramming in somatic cell cloning is one of the key factors for proper development, with variations in the protocol appearing to improve cloning efficiency. This study aimed to determine the effects of two fusion-activation intervals and the aggregation of bovine cloned embryos on subsequent in vitro and in vivo development. Zygotes produced by handmade cloning were exposed to two fusion-activation intervals (2 h or 4 h), and then cultured in microwells either individually (1 × 100%) or after aggregation of two structures (2 × 100%). Zona-intact oocytes and zona-free oocytes and hemi-oocytes were used as parthenote controls under the same fusion-activation intervals. Day-7 cloned blastocysts were transferred to synchronous recipients. Cleavage (Day 2), blastocyst (Day 7) and pregnancy (Day 30) rates were compared by the χ2 test (P < .05). Extending fusion-activation interval from 2 to 4 h reduced cleavage (91.0 vs. 74.4%) but not blastocyst (34.8 vs. 42.0%) rates. On a microwell basis, cloned embryo aggregation (2 × 100%) increased cleavage (91.5% vs. 74.4%) and blastocyst (46.0% vs. 31.3%) rates compared to controls (1 × 100%), but did not improve the overall embryo production efficiency on Day 7 (23.0% vs. 31.3%), on a per reconstructed embryo basis, respectively. Treatments had no effects on in vitro developmental kinetics, embryo quality, and in vivo development. In summary, the fusion-activation interval and/or the aggregation of cloned bovine embryos did not affect cloning efficiency based on the in vitro development to the blastocyst stage and on pregnancy outcome.

17 Effect of the zona-free aggregation on the developmental competence of kodkod (Leopardus guigna) embryos generated by interspecies somatic cell nuclear transfer

The kodkod is considered a vulnerable species by the International Union for Conservation of Nature. Phylogenetically, the kodkod is classified in the Leopardus genus, which has only 36 chromosome pairs compared with the domestic cat, which has 38. The proposed hypothesis was that domestic cat oocytes are capable of reprogramming somatic cells from kodkod after interspecies somatic cell NT (SCNT), allowing the in vitro embryo development up to blastocyst stage. Five experimental groups were made based on the technology and culture system: (1) cat embryos generated by IVF (IVF), (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by interspecies SCNT (K1x), and (5) aggregated kodkod embryos generated by interspecies SCNT (K2x). Interspecies SCNT was performed using a zona-free method. Reconstructed embryos were activated by 2 electrical pulses of 140 kV cm−1 for 40 µs and then incubated for 5h in 10μg mL−1 of cycloheximide and 5μg mL−1 of cytochalasin B. Embryos were cultured in SOF media using the well of the well system in a 5% O2, 5% CO2, and 90% N2 atmosphere at 38.5°C for 8 days. The morulae and blastocysts rates were estimated, and diameter of cloned blastocysts was measured. The relative expression of OCT4, SOX2, and NANOG was evaluated in blastocysts by RT-qPCR using the standard curve method; SDHA was used for normalization. The Kruskal-Wallis test was used to evaluate the developmental parameters and gene expression. The t-test was used to evaluate blastocyst diameter. Statistical differences were considered at P&amp;lt;0.05. The number of replicates was IVF=10, Ca1x=8, Ca2x=6, K1x=3, and K2x=8. The morulae rate was lower when clone embryos were cultured individually (IVF=97/153, 63.4%; Ca2x=28/51, 54.9%; K2x=63/110, 57.3%; Ca1x=48/126, 38.1%; K1x=22/87, 25.3%; P&amp;lt;0.05). In the domestic cat, blastocysts rate was higher in IVF (58/153, 37.9%) and Ca2x (28/51, 29.4%) groups than in the Ca1x group (21/126, 16.7%; P&amp;lt;0.05). No blastocysts were generated in the K1x group (0/87), whereas 5.5% of blastocysts were obtained from the K2x (6/110; 5.5%); this was not statistically different compared with the K1x group (P&amp;gt;0.05). No differences were found in blastocyst diameter between the Ca1x (220.4µm) and Ca2x (251.2µm) groups (P&amp;gt;0.05). However, the diameter of the blastocysts from the K2x group (172.8µm) tended to be lower than that of the blastocysts from the Ca2x group (P=0.05). Regarding gene expression, only 1 of the 6 kodkod blastocysts expressed OCT4, and none expressed SOX2 and NANOG. On the other hand, the relative expression of OCT4 tended to decrease in blastocysts from the Ca1x and Ca2x groups compared with the IVF group (P=0.09), but no differences were found in the expression of SOX2 and NANOG among groups (P&amp;gt;0.05). In conclusion, after interspecies SCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. However, the embryo aggregation did not significantly improve the blastocyst rate and gene expression.

Contribution of rat embryonic stem cells to xenogeneic chimeras in blastocyst or 8-cell embryo injection and aggregation.

The ultimate goal of regenerative medicine is the transplantation of a target organ generated by the patient's own cells. Recently, a method of organ generation using pluripotent stem cells (PSCs) and blastocyst complementation was reported. This approach is based on chimeric animal generation using an early embryo and PSCs, and the contribution of PSCs to the target organ is key to the method's success. However, the contribution rate of PSCs in target organs generated by different chimeric animal generation methods remains unknown. In this study, we used 8-cell embryo aggregation, 8-cell embryo injection, and blastocyst injection to generate interspecies chimeric mice using rat embryonic stem (ES) cells and then investigated the differences in the contribution rate of the rat ES cells. The rate of chimeric mouse generation was the highest using blastocyst injection, followed in order by 8-cell embryo injection and 8-cell embryo aggregation. However, the contribution rate of rat ES cells was the highest in chimeric neonates generated by 8-cell embryo injection, and the difference was statistically significant in the liver. Live functionality was confirmed by analyzing the expression of rat hepatocyte-derived drug-metabolizing enzyme. Collectively, these findings indicate that the 8-cell embryo injection method is the most suitable for generation of PSC-derived organs via chimeric animal generation, particularly for the liver.

Novel histone deacetylase inhibitors and embryo aggregation enhance cloned embryo development and ES cell derivation in pigs.

The histone deacetylase inhibitor (HDACi) has been investigated for treating cancers and many other diseases as well as enhancing the reprogramming efficiency in cloned embryos for decades. In the present study, we investigated the effects of two novel HDAC inhibitors, i.e., HDACi-14 and -79, at the concentrations of 0, 1, 2, or 4 μM on the development of embryos cloned by the oocyte bisection cloning technique (OBCT). Blastocyst rates for the reconstructed embryos reached 60% in the 2 μM HDACi-14-treated groups, which was higher (P < 0.05) compared to the untreated group (36.9%). Similarly, HDACi-79 treatment at 2 and 4 μM also conferred higher (P < 0.05) blastocyst rates than that of the untreated group (79.4, 74.2, and 50.0%, respectively). Both HDACi-14 and -79 treatments had no beneficial effect on total cell numbers and apoptotic indices of cloned embryos (P > 0.05). Histone acetylation profile by both HDACi-14 (2 μM) and -79 (2 μM) treatments demonstrated a drastic increase (P < 0.05) mainly in two-cell stage embryos when compared to the control group. After seeding on the feeder cells, the aggregated cloned blastocysts produced by the HDACi-79 treatment showed a significant increase of primary outgrowths compared to the control group (60.0% vs. 42.9%; P < 0.05). Finally, the cloned embryo-derived ES cell lines from aggregated cloned embryos produced from the HDACi-79-treated, HDACi-14-treated and control groups were established (5, 3, and 2 lines, respectively). In conclusion, the novel histone deacetylation inhibitors improve blastocyst formation and potentially increase the derivation efficiency of ES cell lines from the cloned porcine embryos produced in vitro. Depending on the purposes, some fine-tuning may be required to maximize its beneficial effects of these newly synthesized chemicals.

Strategies for improvement of cloning by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) is a powerful tool that is being applied in a variety of fields as diverse as the cloning and production of transgenic animals, rescue of endangered species and regenerative medicine. However, cloning efficiency is still very low and SCNT embryos generally show poor developmental competency and many abnormalities. The low efficiency is probably due to incomplete reprogramming of the donor nucleus and most of the developmental problems are thought to be caused by epigenetic defects. Applications of SCNT will, therefore, depend on improvements in the efficiency of production of healthy clones. This review has summarised the progress and strategies that have been used to make improvements in various animal species, especially over the period 2010–2017, including strategies based on histone modification, embryo aggregation and mitochondrial function. There has been considerable investiagation into the mechanisms that underpin each strategy, helping us better understand the nature of genomic reprogramming and nucleus–cytoplasm interactions.

Effects of Embryo Aggregation and PXD101 on the In Vitro Development of Mouse Somatic Cell Nuclear Transfer Embryos.

To improve the cloning efficiency of somatic cell nuclear transfer (SCNT) and to establish nuclear transfer embryonic stem cells (NT-ESCs) reliably, it is necessary to produce high-quality blastocysts derived from mice SCNT embryos. Therefore, the present study aims to investigate an optimal method for mouse SCNT embryo production and NT-ESCs derivation by comparing the effects of two methods: the treatment of histone deacetylase inhibitor PXD101 after SCNT, embryo aggregation and their combination treatment. The results suggest that embryo aggregation at four-cell stage and 50 nM PXD101 treated for 10 hours during and after activation could improve both mouse SCNT embryos' development (PXD101: 40.0% vs. 18.5%; p < 0.05; aggregation: 40.2% vs. 18.5%; p < 0.05) and also enhance the isolation rate of NT-ESCs (PXD101: 38.2% vs. 12.5%; p < 0.05; aggregation: 39.0% vs. 12.5%; p < 0.05). The combination of their treatments had a higher development rate (43.6%) and significantly higher NT-ESCs isolation rate (54.7%), therefore, we concluded that the combination of these two methods (50 nM PXD101 treated for 10 hours after SCNT and then aggregated at four-cell stage) is considered as the optimal way for the in vitro development of SCNT embryo and subsequent NT-ESCs isolation in mice, providing a new approach for the practical improvement of mouse cloning techniques and opening new opportunities to improve cloning efficiencies in other species.

Mouse↔rat aggregation chimaeras can develop to adulthood

In order to examine interactions between cells originating from different species during embryonic development we constructed interspecific mouse↔rat chimaeras by aggregation of 8-cell embryos. Embryos of both species expressed different fluorescent markers (eGFP and DsRed), which enabled us to follow the fate of both components from the moment of aggregation until adulthood. We revealed that in majority of embryos the blastocyst cavity appeared inside the group of rat cells, while the mouse component was allocated to the deeper layer of the inner cell mass and to the polar trophectoderm. However, due to rearrangement of all cells and selective elimination of rat cells, shortly before implantation all primary lineages became chimaeric. Moreover, despite the fact that rat cells were always present in the mural trophectoderm, majority of mouse↔rat chimaeric blastocysts implanted in mouse uterus, and out of those 46% developed into foetuses and pups, half of which were chimaeric. In contrast to mural trophectoderm, polar trophectoderm derivatives, i.e. the placentae of all chimaeras were exclusively of mouse origin. This strongly suggests that the successful postimplantation development of chimaeras is enabled by gradual elimination of xenogeneic cells from the nascent placenta. The size of chimaeric newborns was within the limits of control mouse neonates. The rat component located preferentially in the anterior part of the body, where it contributed mainly to the neural tube. Our observations indicate that although chimaeric animals were able to reach adulthood, high contribution of rat cells tended to diminish their viability.