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SummarySomatic cell nuclear transfer (NT) is associated with aberrant changes in epigenetic reprogramming that impede the development of embryos, particularly during zygotic genome activation. Here, we characterized epigenetic patterns of H3K4me3, H3K9me3, and H3K27me3 in mouse NT embryos up to the second cell cycle (i.e. four-celled stage) during zygotic genome activation. In vivo fertilized and parthenogenetically activated (PA) embryos served as controls. In fertilized embryos, maternal and paternal pronuclei exhibited asymmetric H3K4me3, H3K9me3, and H3K27me3 modifications, with the paternal pronucleus showing delayed epigenetic modifications. Higher levels of H3K4me3 and H3K9me3 were observed in NT and PA embryos than in fertilized embryos. However, NT embryos exhibited a lower level of H3K27me3 than PA and fertilized embryos from pronuclear stage 3 to the four-celled stage. Our finding that NT embryos exhibited aberrant H3K4me3, H3K9me3, and H3K27me3 modifications in comparison with fertilized embryos during early zygotic genome activation help to unravel the epigenetic mechanisms of methylation changes in early NT reprogramming and provide an insight into the role of histone H3 in the regulation of cell plasticity during natural reproduction and somatic cell NT.

We injected mouse zygotes with combinations of Cas9 protein, Cas9 mRNA, and two gRNAs targeting a single exon of type I interferon receptor (IFNAR1) to determine the gene targeting efficiencies. Cas9 protein produced on-target mutations more efficiently than Cas9 mRNA when each was used with a single gRNA, regardless of which gRNA was used. When Cas9 mRNA and Cas9 protein were co-injected, the on-target efficiency could reach 97.0% when both gRNAs were used, which was higher than when either gRNA was used alone (61.3% and 75.5%, respectively; P

Blastocysts hatch from the zona pellucida (ZP) to enable implantation into the uterine endometrial epithelium, but little is known regarding the effect of hatching sites on pregnancy outcomes. Murine hatching embryos were categorized into five groups based on initial trophectoderm projection (TEP)/ZP position corresponding to the inner cell mass center. In blastocysts (3.5 dpc) post-12 hours in vitro culture, TEP rates of A-site (44.4%) and B-site (38.6%) embryos were higher than those of C-site (12.5%) and D-site (3.1%) embryos, while the O-site (1.4%) was the lowest (P < .05). Post-ET A-site (55.6%) and B-site (65.6%) birth rates were higher than those of C-site embryos (21.3%) and controls (P < .05). Furthermore, live birth rate of B-site embryos remained higher than C-site embryos (68.8% vs 31.3%; P < .05) when both were transferred into the same recipients. Different TEP site blastocysts exhibited different implantation competences: the implantation rate of C-site embryos was lower than that of both A- and B-site groups (67.7% vs 84.3% and 83.2%, respectively; P < .05) at 2 days post-ET. C-site embryos also had a distinctly higher ratio of developmental defects (47.5%) than A- and B-site embryos (22.5% and 14.6%, respectively), with implantation failure mainly associated with poor birth rate, a finding corroborated by differential gene expression analysis such as LIF, LIFR, and S100a9. Surprisingly, acidified Tyrode's solution (AAH)-treated B-site blastocysts had a significantly increased birth rate (77.1%) than C-site (55.3%) and controls (43.4%). Site specificity and differential gene expression during embryo hatching can be applied in ART screening. More importantly, assisted hatching by AAH is effective and feasible for improving pregnancy and term development, particularly at the B-site, for humans and in animal husbandry.

Methyl-CpG-binding domain 3 (Mbd3) is a core repressor complex component. Although Mbd3 is required for the pluripotency of embryonic stem cells (ES), the role of Mbd3 in mouse ES (mES) cell apoptosis remains undefined. In this study naïve-state mES were derived and maintained in the presence of a selective protein kinase C pathway inhibitor (PKCi; Gӧ6983) to study the function of Mbd3 during mES apoptosis. Mbd3 overexpression in mES decreased the total cell number and viability, and it also dramatically increased the rate of apoptosis. Further investigation of Mbd3 overexpression revealed a 3-fold increase in the proapoptotic/prosurvival protein ratio (Bax/Bcl-2) and elevated RNA expression levels of apoptosis-related genes, including Bim, Trail, Fasl, and caspase 3, with reduced Bcl-2 RNA expression levels. Removal of PKCi from the mES cell culture resulted in upregulated Mbd3 expression and apoptosis, similar to the effects of Mbd3 overexpression. Furthermore, specific knockdown of endogenous Mbd3 partially rescued the mES apoptosis induced by the removal of PKCi, thus increasing the total cell number and viability while decreasing the rate of apoptosis. Additionally, Bax, Bim, Trail, and caspase 3 RNA expression levels were partially reduced, and that of Bcl-2 was partially increased. Our findings support Mbd3 as a pivotal regulator of apoptosis in mES.

Goat oocyte in vitro maturation is associated with a variable efficiency of embryo development after in vitro fertilization (IVF). Here, we developed a novel maturation procedure to evaluate the cellular effect of cysteamine (Cys), leukemia inhibitory factor (LIF) and Y27632 on oocyte in vitro maturation in native Chinese Yangtze river white goats. Oocytes were collected by slicing ovary tissues and matured for 24 h in vitro prior to IVF. Presumptive fertilized oocytes were cultured in embryo media for 8 days. Maturation rates were similar in gonadotropin basal maturation medium and the same medium supplemented with Cys, LIF, or Y27362 (41.0-48.0%; P 0.05). However, when two substances were co-supplemented into the medium, the maturation rate was higher in the Cys+LIF group than in the LIF+Y27362 and Cys+Y27362 groups (60.0% vs. 43.1% and 25.8%, respectively; P 0.05). Co-supplementation of all three substances into the medium achieved the highest maturation rate (67.5%; P 0.05). Compared with oocytes in gonadotropin basal maturation medium, those in medium supplemented with Cys showed increased fertilization (56.1% vs. 72.1%), cleavage (36.7% vs. 44.8%), and blastocyst development (1.7% vs. 4.2%), respectively (P 0.05). Cys+LIF supplementation further improved fertilization (81.6%), cleavage (54.9%), and blastocyst development (6%; P 0.05). Furthermore, combined supplementation of all three substances resulted in the best fertilization (84.9%), cleavage (70.7%), and blastocyst development (10.3%; P 0.05). Resultant IVF blastocysts possessed an average cell number as high as 276 ± 45 per embryo. This is the first study to report increased efficiency of caprine oocyte maturation by combined Cys, LIF, and Y27632 supplementation into basal maturation medium, leading to improved fertilization and embryo development in vitro post-IVF.

The guinea pig is an excellent but underused animal model due to its reproductive biology, which poses difficulties in inducing superovulation, embryo manipulation

The study was designed to determine the impact of magnesium (Mg2+) on bovine embryo development. We found that two commercially available sources of bovine serum albumin (BSA) and fetal bovine serum (FBS) contained different amounts of Mg2+ residue: 4 ppm in ICPbio BSA, 114 ppm in Sigma BSA, and 44 ppm in FBS. When CR1 was used as basal medium, PVA and ICPbio BSA produced the lowest blastocyst yield (2.2–2.3%), whereas Sigma BSA increased blastocyst yield to 18.9% (P 0.05). We next found that increasing concentrations of MgCl2 in the culture medium (ICPbio BSA) elevated blastocyst rate from 2.6% (0 mM), 38.4% (0.35 mM) to 50.2% (1.4 mM; P 0.05). However, blastocyst rate was reduced to 31.4% (4.2 mM) and 29.4% (5.6 mM) when MgCl2 supplement was increased (P 0.05). In embryo transfer experiments, higher rates of pregnancy (54.3 vs. 41.5%) and calving (44.3 vs. 32.5%) were achieved in the CR1-Mg2+-supplemented BSA group compared with the FBS group with co-culture, respectively (P

The aim of this study was to determine whether vitrification is an effective method when used for Japanese Black Cattle (Wagyu) in vivo-derived embryos, collected following a superovulation treatment and embryo transfer (MOET) programme. In vivo-derived morula and blastocysts collected on day 7 after artificial insemination, were vitrified using a modified droplet vitrification (MDV) procedure and subsequently warmed for transfer (ET) into synchronized recipients. Fresh embryos, and embryos cryopreserved using a standardized slow freezing procedure (direct thaw/direct transfer, DT) served as ET controls. Two different follicle-stimulating hormone (FSH) sources, Folltropin(®) Canada (FSH BAH, 24 donors) and a brand prepared by the Chinese Academy of Science (FSH CAS, 16 donors), were compared in a series of superovulation outcomes following well-established FSH administration protocols. Following data analysis, the total number of ovulations recorded at the time of embryo flushing (10.5 vs 8.5; p = 0.28) and the total number of transferable embryos (6.2 vs 5.1; p = 0.52) were similar between the two FSH sources. ET for MDV (39.7%, n = 78), DT (35.2%, n = 71) and fresh controls (47.1%, n = 34) resulted in similar pregnancy rates (p > 0.05). When MDV was used, a higher pregnancy rate (42.6%) resulted from the transfer of vitrified morulae, when compared to the DT counterparts (24.3%), (p = 0.05). Transfer of vitrified morulae resulted also in higher pregnancy rate, when compared to the transfer of vitrified blastocysts (42.6% vs. 29.4%; p < 0.05). Transfer of DT blastocysts resulted in higher pregnancy rate than morulae, similarly cryopreserved (47.1% vs. 24.3%, p < 0.05). In conclusion, MDV is an effective alternative methodology for cryopreservation of in vivo-derived embryos. This study gives also indication that, compared to vitrified blastocysts, MDV of morula stage embryos results in higher pregnancy rates following warming and transfer into synchronized recipients.

Epigenetic modification and expression of key pluripotent factors are critical for development, cell fate determination, and differentiation in early embryos. In this study, we systematically examined the dynamic patterns of histone modifications (H3K4me3 and H3K27me3) and Nanog expression during the development of preimplantation rabbit embryos. Rabbit oocytes, 1-, 2-, 4-, 8-, and 16-cell embryos, morulae, and blastocysts were collected at specific time points following superovulation and assessed for nuclear H3K4me3, H3K27me3, and Nanog expression by immunofluorescence microscopy. The frequency of H3K4me3-positive nuclear staining was highest in oocytes through 4-cell embryos (100%), decreased in 8-cell (97.2%) and 16-cell (94.4%) embryos (P > 0.05), declined dramatically in morulae (86.7%) (1- through 8-cell embryos vs morulae, P < 0.05), and was the lowest in blastocysts (76.2%) (P < 0.05). Nuclear staining of H3K27me3 was negative in oocytes and embryos through the 16-cell stage but was positive in 25.9% of morulae and 34.2% of blastocyst (P < 0.05). Similarly, rabbit oocytes and embryos through the 16-cell stage did not express Nanog, but Nanog was expressed in 24.9% of morulae and 36.5% of blastocysts (P < 0.05). The observed decrease in H3K4me3 and increase in H3K27me3 as development progressed in preimplantation rabbit embryos, together with late Nanog expression, indicates a correlation of these factors with early embryonic cell fate determination and differentiation. Our study provides a specific and dynamic profile of histone modifications and gene expression that will be important for the derivation of rabbit embryonic stem cells and improving rabbit cloning by somatic cell nuclear transfer.

Exogenous genes can be site-specifically integrated into the genomic DNA of animals by homologous recombination, generating transgenic animals. These animals have a clear hereditary background, although position effects of the exogenous genes and potential functional disruption of host genes can be caused by the genetic inserts. Therefore, the generation of mammary gland bioreactors via gene-targeting methods is a great asset for producing recombinant proteins in milk. Here, we describe a method of generating gene-targeted goats with the human alpha-lactalbumin gene (hα-LA) integrated into the beta-lactoglobulin gene (BLG) locus. The milk from these goats will be less allergenic and will be enriched with components of human milk protein.

Exogenous genes can be site-specifically integrated into the genomic DNA of animals by homologous recombination, generating transgenic animals. These animals have a clear hereditary background, although position effects of the exogenous genes and potential functional disruption of host genes can be caused by the genetic inserts. Therefore, the generation of mammary gland bioreactors via gene-targeting methods is a great asset for producing recombinant proteins in milk. Here, we describe a method of generating gene-targeted goats with the human alpha-lactalbumin gene (hα-LA) integrated into the beta-lactoglobulin gene (BLG) locus. The milk from these goats will be less allergenic and will be enriched with components of human milk protein.

Hair follicle stem cells (HFSCs) are an important source for skin tissue engineering studies and clinical applications. Here, we describe a differential enrichment approach to derive HFSCs from hair follicles of vibrissae and ear skin using the Rho-associated protein kinase (ROCK) inhibitor Y-27632. In the presence of Y-27632, primary cultured hair follicle cells grew in clustered colonies surrounded by keratinocyte-like cells and simultaneously expressed three HFSC markers: CD34, K15, and ITGB1. HFSCs cultured in medium containing Y-27632 were presented at a stable ratio of 30.7%, 34.1%, and 32.9% after passages 5, 10, and 15, respectively. By contrast, in medium containing epidermal growth factor, clustered HFSC colonies disappeared after 6 passages and lacked HFSC marker expression. After withdrawal of Y-27632 from the medium, HFSCs rapidly differentiated into keratinocyte-like cells. Furthermore, HFSCs derived with Y-27632 formed spherical clusters in collagen matrix

The rabbit is a useful animal model for regenerative medicine. We previously developed pluripotent rabbit embryonic stem cell (rbESC) lines using fresh embryos. We also successfully cryopreserved rabbit embryos by vitrification. In the present work, we combined these two technologies to derive rbESCs using vitrified–thawed (V/T) embryos. We demonstrate that V/T blastocysts (BLs) can be used to derive pluripotent rbESCs with efficiencies comparable to those using fresh BLs. These ESCs are undistinguishable from the ones derived from fresh embryos. We tested the developmental capacity of rbESCs derived from V/T embryos by BL injection experiments and produced chimeric kits. Our work adds cryopreservation to the toolbox of rabbit stem cell research and applications and will greatly expand the available research materials for regenerative medicine in a clinically relevant animal model.

The aim of this study was to assess modified droplet vitrification (MDV) for the cryopreservation of early developmental mouse embryos. Mouse embryos were equilibrated in holding solution for 3 min followed by immersion in vitrification solution for 30-45 s, and then three embryos per 3-μL vitrification droplet were directly dropped into liquid nitrogen. Vitrified embryos were warmed to examine their developmental potential both in vitro and in vivo. The results demonstrated that MDV vitrified and warmed embryos had a survival rate of 98.1-99.6% (P0.05); however, blastocyst development post warming and culture in vitro demonstrated that vitrified 4-celled, 8-celled, 16-celled, morulae, and blastocyst embryos had significant higher developmental potentials (94.7-99.5%) than those from zygotes (9.2%) and 2-celled embryos (85.7%) (P0.05). Compared to CryoLoop and CryoTech vitrification, MDV showed similar results with regards to rates of survival, blastocyst development, but with the higher hatching rate (76.1% vs. 64.0-67.3%) (P0.05). Cryopreservation by MDV resulted in a similar blastocyst developmental potential in 4-celled and 16 celled embryos from ICR (94.7-99.5%), C57BL/6J (94.7-96.4%), and their crossbred F1 strain (97.9-98.9%) (P0.05). After embryo transfer of vitrified ICR embryos from 4-celled, 16-celled, morulae and blastocyst stage, 40.7-43.7% of the embryos developed into live offspring (P0.05), but MDV vitrification resulted in the highest birth rate (43.8%) compared to CryoLoop (38.3%) and CryoTech (35.4%) (P0.05), when 4-celled mouse embryos were used for vitrification. Our study clearly demonstrated that MDV is the most efficient vitrification to cryopreserve embryos at least 4-celled and advanced stages, which can be used to preserve important mouse genomes from different strains and different developmental stages.

We investigated the effects of 5′-end truncated CRISPR RNA-guided Cas9 nuclease (tru-RGN, 17/18 nucleotides) on genome editing capability in NIH/3T3 cells, and its efficiencies on generating Factor VII (FVII) gene-knockout (KO) mice. In cultured cells, RGNs on-target editing activity had been varied when gRNAs was truncated, higher at Site Two (tF7–2 vs. F7–2, 49.5 vs. 30.1%) while lower in other two sites (Site One, tF7–1 vs.F7–1, 12.1 vs. 23.6%; Site Three, tF7–3 vs.F7–3, 7.7 vs 10.9%) (P FVII KO mice were generated with higher efficiency at Site Two (80.1 vs. 35.8%) and Site One (55.0 vs 3.7%) (P 0.05) when compared with standard RGN controls. Knockout FVII mice demonstrated a delayed prothrombin time and decreased plasma FVII expression. Our study first demonstrates that truncated gRNAs to 18 complementary nucleotides and Cas9 nucleases, can effectively generate FVII gene KO mice with a significantly higher efficiency in a site-dependent manner. In addition, the off-target frequency was much lower in KO mice than in cell lines via RGN expression vector-mediated genome editing.

The Japanese Black cattle (Wagyu) plays a significant part in the Japanese beef industry because it is numerically the largest breed group and comprises ~93% of the national purebred beef cow herd. Japanese Black cattle are genetically predisposed to intense marbling and to a high percentage of unsaturated fat resulting in a meat characterised by both high quality and price on the market. Like other breeds, genetic improvement and production traits in Wagyu can be fostered by the implementation of traditional reproductive strategies such as multiple-ovulation embryo transfer. A multiple-ovulation embryo transfer case study was performed in a leading production centre in Hainan island, South China. Donors (n = 40) were split into 2 groups receiving either a total dose of 400 mg of FSH Folltropin (FSH USA; Folltropin Bioniche Inc., USA; n = 24), or 10 mg of an FSH formulation produced from the Institute of Zoology of the Chinese Academy of Sciences (FSH CHN; n = 16). In both cases, dosages were equally distributed over a 4-day administration schedule. Both donors and recipients (198 heifers) were synchronized for fixed-time embryo transfer (ET) and AI, respectively, by adopting the Ovsynch protocol. Such protocol consists of GnRH administration at Day 0, followed by prostaglandin administration at Day 7 and a second administration of GnRH at Day 9. Artificial insemination was performed on donor animals at 12 and 24 h from the last GnRH administration, whereas for recipient synchronization receiving fresh embryos, the second GnRH administration was given at the time of second AI on donors, and ET was performed 7 days following the first AI. Final synchronization at the time of ET, judged by the ultrasonic presence of a functional corpus luteum, was 53% (105/198). The following parameters for FSH USA and FSH CHN were found to be not significantly different (Student's t; mean ± s.e.): i) ovulations (10.5 ± 1.2 v. 8.5 ± 1.2; P = 0.2); ii) embryos (6.3 ± 1.2 v. 5.1 ± 1.1; P = 0.5), and iii) embryos from ovulated donors (6.8 ± 1.3 v. 5.8 ± 1.1; P = 0.6). Recovered embryos from the 2 groups were also not different: i) degenerated embryos (1.0 ± 0.4 v. 0.8 ± 0.2; P = 0.6); ii) morula (4.0 ± 0.8 v. 2.6 ± 0.5; P = 0.1), and iii) early blastocysts (4.0 ± 1.2 v. 3.3 ± 0.5; P = 0.6). Blastocysts were recovered only from donors treated with FSH USA. Out of 233 recovered embryos, 34 were transferred as fresh and 71 as frozen/thawed. Pregnancy rate at 60 days following ET for fresh and frozen/thawed embryos was 47.1 and 35.2%, respectively (P = 0.2). Within frozen embryos, pregnancy rates derived from transferred morulas and blastocysts were 25 and 45.5% (P = 0.07). When considering the two sources of hormones, overall pregnancy rates were similar between the two groups (28/71, 39.4% v. 13/34, 38.2%; P = 0.9). Finally, pregnancy rates from the transfer of fresh embryos (9/21, 42.8% v. 7/13, 53.8%; P = 0.6) and frozen/thawed embryos (19/50, 38% v. 6/21, 23.8%; P = 0.6) were also not different. In conclusion, all parameters in this study did not differ between the 2 sources of FSH; however, a lower incidence of degenerated embryos and a higher pregnancy rate following transfer of frozen/thawed embryos occurred when FSH USA was used in donor animals.