• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.207-214
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• 1996

To examine the developmental capacity of manipulated embryos after ultrarapid refreezing and thawing, mouse embryos were biopsied at 4-cell stage, frozen twice at 4-cell and morula stages, respectively, and then transferred to rec-ipients. Single blastomeres were biopsied from 4-cell embryos by a modified aspiration method. Biopsied 4-cell embryos were equilibrated into freezing medium at room temperature for 2.5 min, loaded into 40 $\mu$I of freezing medium in 0.25 ml plastic straw and then directly immersed into liqiud nitrogen. Freezing medium for 4-cell embryos consisted of 4.0 M ethylene glycol and O.25 M sucrose in dPBS supplemented with 6 mg/lm BSA. Morulae were frozen into freezing medium containing 5.0 M glycerol instead of ethylene glycol. Thawing was conducted by agitating each straw in 3TC water for 20 sec. The c content of each straw was expelled into 0.5 ml of dilution medium, which consisted of 0.25 M sucrose and 3 mg/ml BSA in dPBS. The thawed embryos were rehydrated in dilution medium for 10 min, washed 3 times with dPBS and then cultured in M16 medium at 37$^{\circ}C$, 5% CO$_2$ in air. Blastocysts that developed from frozen or refrozne biopsied embryos were transferred to recipients on Day 3 of pseudopregnancy, respectively. In vitro and in vivo developmental rates of the biopsied and intact 4~cell embryos after freezing and thawing were 78 (10l/130) and 25% (10/40), and 91 (114/125) and 30% (12/40), respectively. Although the rates of in vitro development of biopsied and intact embryos to blastocyst stage were significantly different after freezing and thawing (P

• Korean Journal of Animal Reproduction
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• v.11 no.3
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• pp.155-160
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• 1987

This study was carried out to determine the effects of cryoprotectants, freezing and thawing rates on the survival of 8-cell mouse embryos. The female ICR mice were induced to superovulated by intraperitoneal injections of 5 i.u. PMSG and 5 i.u. HCG given 48h apart and then were paired with males of the same strain. They were killed and embryos were flushed from the oviducts and uteri on 3 days after injection of HCG. Embryos were flushed with modified Dulbecco's phosphate buffered saline and equilibrated with 1.5M-dimethyl sulfoxide (DMSO) or 1.5M-glycerol by 3-step procedure. The freezing rates of the embryos were 1$^{\circ}C$/min from room temperature to -5$^{\circ}C$ and the embryos were seeded at -5$^{\circ}C$. After being held for 3 min at the seeding temperature, the rates were 0.3$^{\circ}C$/min from -5$^{\circ}C$ to -35$^{\circ}C$. From -35$^{\circ}C$ to -7$0^{\circ}C$, the rates were divided into 0.1$^{\circ}C$/min, 1$^{\circ}C$/min and 1$0^{\circ}C$/min, respectively. After being held for 5 min at -7$0^{\circ}C$, the embryos were plunged directly into liquid nitrogen. The embryos were thawed at 4$^{\circ}C$/min and 12$^{\circ}C$/min from -196$^{\circ}C$ to 37$^{\circ}C$, and for 2 min in 37$^{\circ}C$ water bath, respectively. The average number of ovulation points and embryos recovered were 42.7 and 34 appearing 79.5% recovery rate. Eight cell embryos in the embryos recovered were 26.3. The survival rates of embryos according to the freezing rates in the presence of 1.5M-DMSO were 73.5~80.6% at 0.1$^{\circ}C$/min, 75.0~79.5% at 1$^{\circ}C$/min and 52.8~54.7% at 1$0^{\circ}C$/min, but in the presence of 1.5M-glycerol were 62.9~67.6% at 0.1$^{\circ}C$/min, 61.4~68.3% at 1$^{\circ}C$/min and 25.5~30.2% at 1$0^{\circ}C$/min. The survival rates of embryos were not affected by the thawing rates.

• Animal Bioscience
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• v.36 no.8
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• pp.1180-1189
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• 2023

Objective: Discovering the mechanism of cell specification is important to manipulate cellular lineages. To obtain lineage-specific cell lines, the target lineage needs to be promoted, and counterpart lineages should be suppressed. Embryos in the early blastocyst stage possess two different cell populations, the inner cell mass (ICM) and trophectoderm. Then, cells in the ICM segregate into epiblasts (Epi) and primitive endoderm (PrE). PrE cells in embryos show specific expression of platelet-derived growth factor (PDGF) and its receptor, PDGF receptor A (PDGFRA). In this study, we suppressed PDGF signaling using two methods (CRISPR/Cas9 injection and inhibitor treatment) to provide insight into the segregation of embryonic lineages. Methods: CRISPR/Cas9 RNAs were injected into parthenogenetically activated and in vitro fertilized embryos. The PDGF receptor inhibitor AG1296 was treated at 0, 5, 10, and 20 µM concentration. The developmental competence of the embryos and the number of cells expressing marker proteins (SOX2 for ICM and SOX17 for PrE) were measured after the treatments. The expression levels of the marker genes with the inhibitor were examined during embryo development. Results: Microinjection targeting the PDGF receptor (PDGFR) A reduced the number of SOX17-positive cell populations in a subset of day 7 blastocysts (n = 9/12). However, microinjection accompanied diminution of Epi cells in the blastocyst. The PDGF receptor inhibitor AG1296 (5 µM) suppressed SOX17-positive cells without reducing SOX2-positive cells in both parthenogenetic activated and in vitro fertilized embryos. Within the transcriptional target of PDGF signaling, the inhibitor significantly upregulated the Txnip gene in embryos. Conclusion: We identified that PDGF signaling is important to sustain the PrE population in porcine blastocysts. Additionally, treatment with inhibitors was a better method to suppress PrE cells than CRISPR/Cas9 microinjection of anti-PDGF receptor α gene, because microinjection suppressed number of Epi cells. The PDGF receptor might control the number of PrE cells by repressing the proapoptotic gene Txnip. Our results can help to isolate Epi-specific cell lines from blastocysts.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.1
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• pp.9-20
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• 1999

The genetic defects in human gametes and embryos can cause adverse effects on overall reproductive events. Biopsy of embryos for preimplantation genetic diagnosis (PGD) offers a new possibility of having children free of the genetic disease. In addition, advanced embryo culture method may enhance the effectiveness of embryo biopsy for the practical application of PGD. This experimental study was undertaken to evaluate the effects of coculture on the development in vitro of biopsied mouse embryos as a preclinical model for PGD of human embryos. Embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BLfemale/CBAmale). Using micromanipulation, 1, 2, 3 or 4 blastomeres of 8-cell stage embryos were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acidic Tyrode's solution (ATS). After biopsy of blastomeres, embryos were cultured in vitro for 110 hours in Ham's F-10 supplemented with 0.4% BSA or cocultured on the monolayer of Vero cells in the same medium. The frequence of blastocyst formation were recorded, and the embryos beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. There was no significant difference in the blastocyst formation between the zona intact control group and the zona drilling (ZD) only, or biopsied groups. The hatching rate of all the treatment groups except 4/8 group was significantly higher than that of control group. In all the treatment groups, there was a significant reduction in the mean cell number of embryos beyond blastocyst stage ($50.2{\pm}14.0$ in control group vs. $41.2{\pm}7.9$ in ZD, $39.3{\pm}8.8$ in 7/8, $29.7{\pm}6.4$ in 6/8, $25.1{\pm}5.7$ in 5/8, and $22.1{\pm}4.3$ in 4/8 groups, p<0.05). When the same treatments were followed by coculture with Vero cells, a similar pattern was seen in the blastocyst formation and the hatching rate. However, in all the treatment groups, there was a significant increase in the mean cell number of embryos beyond blastocyst stage with coculture, compared with the parallel groups without coculture. In the cleavage rate of biopsied blastomeres cultured for 110 hours after IVF, there was no significant difference between coculture and non-coculture groups (87.2% vs. 78.7%). However, the mean cell number of embryos developed from the biopsied blastomeres was significantly higher in coculture group ($11.5{\pm}4.7\;vs.\;5.9{\pm}1.9$, p<0.05). In conclusion, biopsy of mouse embryos after ZD with ATS is a safe and highly efficient method for PGD, and coculture with Vero cells showed a positive effect on the development in vitro of biopsied mouse embryos and blastomeres as a preclinical model for PGD of human embryos.

• Journal of Plant Biotechnology
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• v.30 no.2
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• pp.167-172
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• 2003

Immature zygotic embryos of Eleutherococcus senticosus seeds matured rapidly within one month when the seeds comprising zygotic embryos were pieced to small size and cultured on 1/2 MS medium. Frequency of somatic embryos formation was declined rapidly when the zygotic embryos germinated and grew to plantlets. Embryogenic cells were induced by consecutive subculture of somatic embryos on MS medium with 1.0mg/L2,4-D. After heart-shaped somatic embryos were induced by suspension culture, these embryos were plated onto petri dish to support maturation of embryos. Germination of embryos occurred on medium with 5mg/L GA$_3$and transferred to culture bowl to stimulate the further growth. Frequency of soil survival of plantlets was influenced by soil mixture (perlite and peatmoss). The suitable combination of perlite and peatmoss was 1:5, and the soil survival rate was 78% after 4 months. The soil transferred plantlets were over-wintered in field condition after defoliation. New year sprouting of plants was achieved successfully and they grew to adult plants. These results indicate that the systematic procecure of plant production in E. senticosus for micro propagation.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.20-20
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• 2001

It has been reported that cloning cattle is inefficient. One of the problems was placental abnormality, finally resulting in fetal mortality after transfer of nuclear transfer (NT) bovine embryos. This study was focused on the allocations of embryonic cells to the inner cell mass (ICM) or to the trophectoderm(TE) in NT bovine blastocysts. Somatic cells were derived from a Day 45 fetus of gestation, individually transferred into enucleated oocytes and developed to the blastocyst stage in vitro. Differential staining was used to assess the qualify of blastocysts derived from NT, IVF and in vivo. Development rate of NT embryos to blastocysts (25.0%, 41/164) was similar to that of IVF embryos (28.7%, 49/171). The total cell number of NT blastocysts (101.3$\pm$45.9) was not different compared with that of IVF embryos (107.9$\pm$34.2, P>0.05), but was lower than in vivo embryos (122.5$\pm$21.6, P<0.05). Ratio of ICM/total cells was higher in NT embryos (51.6$\pm$ 18.6%) than in IVF and in vivo embryos (42.3$\pm$ 15.3% and 34.9$\pm$8.9%, respectively) (P<0.05). Most IVF (56.8%, 25/44) and in vivo blastocysts(80.8%, 21/26) was distributed in the proportion of ICM/total cells ranging from 20 to 40% group. However, most NT blastocysts was biased in the 40-60%(34.1%, 15/44) and >60% (31.8%, 14/44) groups. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocation of NT embryos to the ICM cells.

• Korean Journal of Plant Tissue Culture
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• v.26 no.4
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• pp.289-291
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• 1999

Hypocotyl explants from 7 days old seedlings of one $F_1$ hybrid cultivar and two pure lines of cucumber formed embryogenic calli at frequencies of up to 8% when cultured on Murashige and Skoog medium (MS) supplemented with 1 mg/L 2,4-D for 3 weeks. Embryogenic calli gave rise to somatic embryos. When slices of somatic embryos were cultured on the same medium for 4 weeks, they formed embryogenic calli. Embryogenic cell suspension cultures were established with embryogenic calli in MS liquid medium with 1 mg/L 2,4-D. Embryogenic potential of cell suspension cultures was maintained by subculturing every seven days. When the level of 2,4-D in the medium was lowered to 0.2 mg/L by diluting with liquid MS basal medium, embryogenic cell suspension cultures underwent development into numerous somatic embryos. When plated onto MS basal medium, over 95% of somatic embryos developed into plantlets. Plantlets were transplanted to potting soil and grown to maturity.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.232-239
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• 2019

In general, cloned pigs have been produced using the somatic cell nuclear transfer (SCNT) technique with various types of somatic cells; however, the SCNT technique has disadvantages not only in its low efficiency but also in the development of abnormal clones. This study aimed to compare early embryonic development and quality of SCNT embryos with those of induced pluripotent stem cells (iPSCs) NT embryos (iPSC-NTs). Ear fibroblast cells were used as donor cells and iPSCs were generated from these cells by lentiviral transduction with human six factors (Oct4, Sox2, c-Myc, Nanog, Klf4 and Lin28). Blastocyst formation rate in iPSC-NT (23/258, 8.9%) was significantly lower than that in SCNT (46/175, 26.3%; p < 0.05). Total cell number in blastocysts was similar between two groups, but blastocysts in iPSC-NT had a lower number of apoptotic cells than in SCNT (2.0 ± 0.6 vs. 9.8 ± 2.9, p < 0.05). Quantitative PCR data showed that apoptosis-related genes (bax, caspase-3, and caspase-9) were highly expressed in SCNT than iPSC-NT (p < 0.05). Although an early development rate was low in iPSC-NT, the quality of cloned embryos from porcine iPSC was higher than that of embryos from somatic cells. Therefore, porcine iPSCs could be used as a preferable cell source to create a clone or transgenic animals by using the NT technique.

• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.229-238
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• 1997

Chromosome condensation and swelling of the donor nucleus have been known as the early morphological indicators of chromatin remodelling after injection of a foreign nucleus into an enucleated recipient cytoplasm. The effects of non-preactivation and electrical preactivation of recipient cytoplasm, prior to fusing a donor nucleus, on the profile of nuclear remodelling in the nuclear transplant rabbit embryos were evaluated. The embryos of 16-cell stage were collected and synchronized to G1 phase of 32-cell stage. The recipient cytoplasms were obtained by removing the first polar body and chromosome mass by non-disruptive microsurgical procedure. The separated G1 phase blastomeres of 32-cell stage were injected into non-preactivated recipient cytoplasms. Otherwise, the enucleated recipient cytoplasms were preactivated by electrical stimulation and the separated G1 phase blastomeres of 32-cell stage were injected. After culture until 20h post-hCG injection, the nuclear transplant oocytes were electrofused by electrical stimulation. The nuclei of nuclear transplant embryos fused into non-preactivated and/or preactivated recipient cytoplasm were stained by Hoechst 33342 at 0, 1.5, 2, 4, 6, 8, 10 hrs post-fusion and were observed under an fluorescence microscopy. Accurate measurements of nuclear diameter were revealed with an ocular micrometer at 200$\times$. Upon blastomere fusion into non-preactivated recipient cytoplasm, a prematurely chromosome condensation at 1.5 hrs post-fusion and nuclear swelling at 8 hrs post-fusion were occurred as 91.6% and 86.1%, respectively. But the nuclei of nuclear transplant embryos fused into preactivated recipient cytoplasm, as o, pp.sed to non-preactivated recipient cytoplasm, were not occurred chromosome condensation and extensive nuclear swelling. Nuclear diameter fused into non-preactivated and preactivated recipient cytoplasm at hrs post-fusion was 30.2$\pm$0.74 and 15.2$\pm$1.32${\mu}{\textrm}{m}$, respectively. These results indicated that onset of unclear condensation and swelling which was associated with oocytes activation were critical steps in the process of chromatin swelling. Futhermore, complete reprogramming seemed only possible after remodelling of the donor nucleus by chromosome condensation and nuclear swelling.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.147-153
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• 2001

Objective: The present study was performed to investigate the efficiency of partial laser assisted hatching (p-LAH; lased 1/2 ZP width from ZP edge) on hatching of mouse blastocysts. Methods: We used non-contact $1.48{\mu}m$ diode laser (MTM, Switzland) to create a precise hole on zona pellucida. 2-cell embryos were collected from the mouse (ICR) oviduct at 48 hours after hCG administration. Collected 2-cell embryos were cultured in the P-1 medium supplemented with 0.4% BSA. For experiments, embryos at 8-cell stage were used after $20{\sim}22$ hours in culture. After conventional (c-LAH) or partial laser assisted hatching, the embryos were further cultured in P-1 medium supplemented with 0.4% BSA for 3 days. To compare efficiency of complete and partial laser assisted hatching, hatching rate, hatching time and blastocyst diameter and zona pellucida thickness at hatching time were investigated. Embryos were examined every 12 hours. Blastocyst diameter and zona pellucida thickness at hatching time were measured with an ocular micrometer. Results: Hatching rates of p-LAH group (84.2%) was significantly higher than that of control group (39.3%), but there was no difference between the p-LAH (84.2%) and c-LAH (91.2%). p-LAH group was hatched 12 hours earlier than control group, but hatched 12 hours later than c-LAH group. The diameter of blastocyst at hatching time of p-LAH group ($113.1{\pm}6.4{\mu}m$) was smaller than that of control group ($122.2{\pm}5.0{\mu}m$), but larger than that of c-LAH group ($102.2{\pm}2.7{\mu}m$). Zona pellucida thickness at hatching time of p-LAH group ($6.4{\pm}0.9{\mu}m$) was thicker than that of control group ($4.5{\pm}1.5{\mu}m$), but thinner than that of c-LAH group ($10.0{\pm}0.8{\mu}m$). Conclusion: These results suggest that p-LAH may maintains the cell arrangement of early embryos to ensure successful development and prevent precocious hatching of blastocyst when compare to c-LAH and conventional (acidic tyrode) AH. Thus, p-LAH may provide a valuable and effective AH technique for human ART program.