• Journal of Embryo Transfer
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• v.15 no.2
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• pp.175-180
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• 2000

The possible use of micromanipulative biopsy and PCR of the biopsied embryonic cells was tested to produce sexed bovine embryos in practical terms. By micromanipulation and PCR techniques, higher survival rate and accurate sexing of demi-embryos were btained. Bovine oocytes matured and fertilized in vitro were co-cultured with bovine oviductal epithelial cell (BOEC) monolayer in USU-6 medium supplemented with 15% FBS, and the embryos of 37% (327/885) were developed to blastocysts. Among 111 blastocysts produced by invitro, only 7 (6.3%) embryos were found unable to determine their sex, probably due to the loss of cells, since no PCR product was found from those cells. All the remaining 104 (93.7%) demi-embryos survived micromanipulation and demonstrated male-specific product or bovine-specific product alone suggesting that correct sexing of the sample. Forty-three point one percent(25/58) of manipulated and cryopreserved demi-embryos after thawing were survived. Final verification of the sexed embryos is necessary to make sure the same sex in fetus and newborn calf upon embryo transfer. The established sexing method on a large number of bovine embryos from previous and this study suggests that this a could be used practically in the field.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.121-129
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• 2022

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is an N6-methyladenosine (m⁶A) RNA modification regulator and a key determinant of prem-RNA processing, mRNA metabolism and transportation in cells. Currently, m⁶A reader proteins such as hnRNPA2/B1 and YTHDF2 has functional roles in mice embryo. However, the role of hnRNPA2/B1 in porcine embryogenic development are unclear. Here, we investigated the developmental competence and mRNA expression levels in porcine parthenogenetic embryos after hnRNPA2/B1 knock-down. HhnRNPA2/B1 was localized in the nucleus during subsequent embryonic development since zygote stage. After hnRNPA2/B1 knock-down using double stranded RNA injection, blastocyst formation rate decreased than that in the control group. Moreover, hnRNPA2/B1 knock-down embryos show developmental delay after compaction. In blastocyste stage, total cell number was decreased. Interestingly, gene expression patterns revealed that transcription of Pou5f1, Sox2, TRFP2C, Cdx2 and PARD6B decreased without changing the junction protein, ZO1, OCLN, and CDH1. Thus, hnRNPA2/B1 is necessary for porcine early embryo development by regulating gene expression through epigenetic RNA modification.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.693-700
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• 2007

The present study was conducted to examine the effect of different levels of essential and nonessential amino acid in NCSU-23 medium on the in vitro-produced porcine embryo as it develops from the zygote to the blastocyst stage. Four experiments were performed, each with a completely randomized design involving 5 to 8 replications of treatments. In order to know the effect of nonessential amino acids in NCSU-23 medium, 0, 5, 10 and $20{\mu}/ml$ MEM were supplemented there to, (Exp. 1) and the medium was supplemented with same level of essential amino acids (Exp. 2). The combined effect of nonessential (0, 5, 10 and $20{\mu}/ml$ MEM) and essential amino acids (0, 5, 10 and $10{\mu}/ml$ MEM) in NCSU-23 medium (Exp. 3), first 72 h with non-essential amino acids (at 0, 5, 10 and $20{\mu}/ml$ MEM), and last 4 d with essential amino acids with the same level as NEAA (Exp. 4) were examined. The embryo development was monitored and the quality of blastocysts was evaluated by counting the number of total cells and determining the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells. When Eagle's nonessential amino acids (MEM) added to NCSU-23 medium, it significantly increased the likelihood of development to the 2- to 4-cell stage and subsequent blastocyst development. Supplementation of different levels of essential amino acids in the NCSU-23 medium decreased cleavage rate, rate of morula and blastocyst development and the number of ICMs. In the case of the combined effect of essential and nonessential amino acids, better and significant results were found for blastocysts, hatching blastocysts and for ICM numbers which were also dose dependent. With respect to the biphasic effect of nonessential and essential amino acids, nonessential amino acids increased cleavage whereas essential amino acids increased the total cell number. Neither the nonessential nor the essential group of amino acids, on their own, affected blastocyst cell number or the differentiation of cells in the blastocyst. In conclusion, this study determined the role of nonessential and essential amino acids in the culture of the porcine embryo and showed that the embryo requires different levels of amino acids as it develops from the zygote to the blastocyst stage.

• Journal of Embryo Transfer
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• v.29 no.4
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• pp.345-350
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• 2014

We investigate the effect of L-glutathione (GSH), an antioxidant, treatment during the somatic cell nuclear transfer (SCNT) procedures on the in vitro development and DNA methylation status of bovine SCNT embryos. Bovine in vitro matured (IVM) oocytes were enucleated and electrofused with a donor cell, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine. The recipient oocytes or reconstituted oocytes were treated with $50{\mu}M$ GSH during these SCNT procedures from enucleation to activation treatment. The SCNT embryos were cultured for 7 days to evaluate the in vitro development, apoptosis and DNA methylation in blastocysts. The apoptosis was measured by TUNEL assay and caspase-3 activity assay. Methylated DNA of SCNT embryos at the blastocyst stages was detected using a 5-methylcytidine (5-MeC) antibody. The developmental rate to the blastocyst stage was significantly higher (P<0.05) in GSH treatment group ($32.5{\pm}1.2%$, 78/235) than that of non-treated control SCNT embryos ($22.3{\pm}1.8%$, 50/224). TUNEL assay revealed that the numbers of apoptotic cells in GSH treatment group ($2.3{\pm}0.4%$) were significantly lower (P<0.05) than that of control ($3.8{\pm}0.6%$). Relative caspase-3 activity of GSH treated group was $0.8{\pm}0.06$ fold compared to that of control. DNA methylation status of blastocysts in GSH treatment group ($13.1{\pm}0.5$, pixels/embryo) was significantly lower (P<0.05) than that of control ($17.4{\pm}0.9$, pixels/embryo). These results suggest that antioxidant GSH treatment during SCNT procedures can improve the embryonic development and reduce the apoptosis and DNA methylation level of bovine SCNT embryos, which may enhance the nuclear reprogramming of bovine SCNT embryos.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.95-104
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• 2017

This study was conducted to establish the optimal chemical post-activation conditions in porcine embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) using 4 different chemical compositions (cytochalasin B (CB), cyclohexamide (CHX), demecolcine (DC), 6-dimethylaminopurine (DMAP). Porcine embryos were produced by PA and SCNT and then, cultured for post-activation with CB ($7.5{\mu}g/mL$), CB ($7.5{\mu}g/mL$) + CHX ($10{\mu}g/mL$), CB ($7.5{\mu}g/mL$) +DC ($0.4{\mu}g/mL$), and CB ($7.5{\mu}g/mL$) + DMAP (2 mM). In PA embryonic development, cleavage rates have been significantly higher in CB group (94.7%) and CB+DMAP group (94.1%) than that of CB+CHX and CB+DC group (88.1 and 84.3%, respectively). There have been no significant differences in blastocyst formation rates among the four groups. In cell number of blastocyst was shown in CB group (42.3%) significantly higher than CB+CHX and CB+DC group (40.6 and 40.6%, respectively). In SCNT embryonic development, CB+DMAP group (89.7%) significant differences were found on embryo cleavage rates when compared with other three groups. Blastocyst formation rates in CB+DMAP group (26.9%) were significantly higher when compared with CB, CB+CHX, and CB+DC groups (25.5, 20.2, and 22.1%, respectively). In blastocyst cell number, CB+DMAP group (41.4%) was found higher significant difference compared with other three groups. Additionally, we have investigated survivin expression in early development stages of porcine SCNT embryos for more confirmation. Our results establish that CB group and CB+DMAP group for 4 h during post-activation improves pre-implantation improvement of PA and SCNT embryos.

• Reproductive and Developmental Biology
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• v.33 no.1
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• pp.35-40
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• 2009

The objectives of this study was to evaluate the efficiency of the bacteria eliminated sperm by percoll gradient method on sperm quality and embryo cleavage in vitro in pig. The semen of miniature pig collected by gloved-hand method pre-warmed ($37^{\circ}C$) in thermos bottle, and separated by 65% percoll. Analysis of sperm ability was estimated by examining viability, capacitation and acrosome reaction using chlortetracycline (CTC) and the abnormality. Also, fertility of sperm was monitored with cleavage rate of embryo after IVF using separated and un-separated sperm by percoll. The result, viability of separated sperm was significantly(p<0.05) higher($83.6{\pm}$2.0 vs $59.0{\pm}4.4%$) than un-separated sperm. The results of CTC analysis showed the percentage of F- and B-patterned separated sperm was higher in separated that than un-separated sperm. On the contrary, the percentage of AR-patterned form unseparaed sperm was significantly(p<0.05) higher($13.6{\pm}0.8$ vs $8.1{\pm}0.6%$) than separated sperm. Also, abnormality of un-separated sperm was significantly(p<0.05) higher($2.2{\pm}0.4$ vs $16.8{\pm}2.8%$) than separated sperm. However, the cleavage rates of embryo using separated sperm by percoll and un-separated sperm had not significantly difference on 2 cell stage(9.25 vs 11.88%), 4 cell stage(26.76 vs 24.51%) and >4 cell stage(63.99 vs 63.61%) at 48h of IVF. Therefore, the sperm separated by percoll method showed improvement in sperm quality than un-separated sperm in miniature pig.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.101-108
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• 2002

Human Prourokinase (proUK) offers potential as a novel agent with improved fibrin specificity and, as such, may offer advantages as an attractive alternative to urokinase that is associated with clinical benefits in patients with acute peripheral arterial occlusion. For production of transgenic cow as human proUK bioreacotor, we conducted this study to establish efficient production system for bovine transgenic embryos by somatic cell nuclear transfer (NT) using human prourokinase gene transfected donor cell. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human prourokinase target gene into a pcDNA3 plasmid. Cumulus cells were used as donor cell and transfected with the expression plasmid using the Fugene 6 as a carrier. To increase the efficiency for the production of transgenic NT, development rates were compared between non-transfected and transfected cell in experiment 1, and in experiment 2, development rates were compared according to level of GFP expression in donor cells. In experiment 1, development rates of non-transgenic NT embryos were significantly higher than transgenic NT embryos (43.3 vs. 28.4％). In experiment 2, there were no significant differences in fusion rates (85.4 vs. 78.9％) and cleavage rates (78.7 vs. 84.4％) between low and high expressed cells. However, development rates to blastocyst were higher in low expressed cells (17.0 vs. 33.3％), and GFP expression rates in blastocyst were higher in high expressed cells (75.0 vs. 43.3％), significantly.

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.639-645
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• 1997

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplanted embryos. The oocytes collected from slaughterhouse ovaries were matured 24h in TCM199+10% FBS and exposed to $39^{\circ}C$ or room temperature to allow cytoplasmic maturation and gain activation competence. Donor embryos were treated for 12h with $10{\mu}g/ml$ nocodazole or $0.05{\mu}g/ml$ demicolcine to synchronize the cell cycle stage at 26h after the onset of culture. The blastomeres and recipient oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. In the treatment of oocyte activation and cell cycle regulation of donor nuclei, the room temperature exposure and nocodazole treatment group had significant effect on the developmental rates to morula/blastocyst(21.7% vs 12.1~16.7%), but had no significant effect on the fusion rates between donor blastomeres and recipient oocytes. The developmental rates of bovine nuclear transplanted embryos appeared to be higher significantly in mTALP medium under 5% $O_2$ condition and in TCM199 with bovine oviduct epithelial cell under 20% $O_2$ condition(22.2%) than other groups. In embryo transfer of nuclear transplanted embryos, there were no significant differences in calving rates between the use of excellent and good grade donor embryos.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.183-187
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• 2009

In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro fertilization (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22h post insemination were treated at different concentration of actinomycin D (0, 5, 50 and 500 ng/ml in NCSU medium). Treated embryos were incubated at $39^{\circ}C$ in 5% $CO_2$, 5% $O_2$ for 8h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2days and BL development rate at 7days after in vitro culture. A significantly lower rate of cleavage was found in the 500 ng/ml group compared to others (500 ng/ml vs. 0, 5, 50 ng/ml; 27.8 % vs. 50.0%, 41.2%, 35.9%), and BL formation rate in 500 ng/ml was lower than that of others (500 ng/ml vs. 0, 5, 50 ng/ml; 8.0% vs. 12.6%, 11.2%, 12.6%). In experiment 2, to evaluate apoptotic cells, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng/ml actinomycin D). A high number of BL derived control group contained at least one apoptotic cell. Actinomycin D treated BLs responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and increase in the incidence of apoptotic cell death. In 500 ng/ml group, the incidence of apoptosis increased at 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is useful tool for the apoptosis study of porcine preimplantation embryos.

• BMB Reports
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• v.52 no.6
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• pp.403-408
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• 2019

Dorsoventral patterning of body axis in vertebrate embryo is tightly controlled by a complex regulatory network of transcription factors. Ventx1.1 is known as a transcriptional repressor to inhibit dorsal mesoderm formation and neural differentiation in Xenopus. In an attempt to identify, using chromatin immunoprecipitation (ChIP)-Seq, genome-wide binding pattern of Ventx1.1 in Xenopus gastrulae, we observed that Ventx1.1 associates with its own 5'-flanking sequence. In this study, we present evidence that Ventx1.1 binds a cis-acting Ventx1.1 response element (VRE) in its own promoter, leading to repression of its own transcription. Site-directed mutagenesis of the VRE in the Ventx1.1 promoter significantly abrogated this inhibitory autoregulation of Ventx1.1 transcription. Notably, Ventx1.1 and Xcad2, an activator of Ventx1.1 transcription, competitively co-occupied the VRE in the Ventx1.1 promoter. In support of this, mutation of the VRE down-regulated basal and Xcad2-induced levels of Ventx1.1 promoter activity. In addition, overexpression of Ventx1.1 prevented Xcad2 from binding to the Ventx1.1 promoter, and vice versa. Taken together, these results suggest that Ventx1.1 negatively regulates its own transcription in competition with Xcad2, thereby fine-tuning its own expression levels during dorsoventral patterning of Xenopus early embryo.