• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.315-323
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• 1994

Mammalian oviductal epithelial cells have been known to improve in vitro fertilization and embryonic development. Recently, co-cultured human embryos with the epithelial cells in human genital tract has been reported to improve the pregnancy rate. The purpose of the study was to investigate the effects of the epithelial cells of human genital tract on the development of mouse early embryos and human fertilized oocytes. The epithelial cells of human genital tract were collected from the fallopian tubes which were obtained during hysterectomy in fertile women and from the endometrium during endometrium biopsy. Collected human ampullary cells(HACs) and endometrial cells(HECs) were cultured for 10 days to establish primary monolayer. Second passaged HACs and HECs were obtained by trypsinization were cryopreserved in PBS with 1.5 M DMSO for later use. To investigate the effect when co-cultured with HACs and HECs, we tried to apply strict quality control on mouse embryo, from two cell to blastocyst prior to human trial. The results of quality control were as follows; In Group I (Ham's F10 with 10% FCS), Group IT (co-cultured with HACs) and Group ill (co-cultured with HECs), developmental rates to blastocyst were 63.3%(253/400), 76.0%(304/ 400),74.0%(296/400), respectively. Hatching rates were 36.8%(147/400), 41.80/0(167/400), 38.0%(152/400), respectively(p<0.05). To perform the human IVF, cryopreserved HACs were thawed at 37$^{\circ}C$ waterbath, seeded on the well dish and cultured for 48 hI'S. The pronuclear stage embryos were transferred to the seeded well dish. After 24 hRS, co-cultured embryos were examined and transferred to patient's uterus. The results of human IVF when co-cultured with HACs were that fertilization and developmental rates were 61.8% (256/414), 95.3% (244/256) as compared with 57.2% (279/488) and 94.6%(264/279) in Ham's F10 supplemented with 10% FCS(control). However, 62.9% (161/256) of co-cultured human embryos showed good embryos(no or slight fragmentation) as compared with 53.8 % (150/279) in control(p < 0.05). Pregnancy rate was 40.0% (12/30) when co-cultured with HACs whereas 30.6%(11/36) in control. In conclusions, co-culture system using HACs and HECs improved the developmental and hatching rates of mouse embryo. Also, in human IVF system when co-cultured with HACs, it improved both the quality of human embryos and the pregnancy rate.

• Current Research on Agriculture and Life Sciences
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• v.6
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• pp.145-156
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• 1988

This study was conducted to investigate the effect of embryo stage on bisection rates of embryos, on development of separated demi-embryos and on subsequent development to full term following transfer of demi-embryos to recipients. The results obtained in this study are summarized as follows. The 2-, 4-, 8-cell and morula embryos were mostly obtained at 36-42, 48-54, 62-66 and 72-78 hours after injection of hCG, respectively, and the average number of embryos recovered per head was 17.0. The bisection rate of 8-cell embryos was 83.6%, which was significantly higher than that of morula embryos, 65.5%. But the development of morula demi-embryos to blastocyst after in-vitro culture was better than that of 8-cell demi-embryos and the rate was 76.5% and 60.9%, respectively. When the 2, 10 and 15 cultured demi-embryos or intact embryos were transferred to each recipient mouse, respectively. the highest pregnancy rate was obtained when 15 embryos were transferred. And the overall offspring production rate of intact embryos was higher than that of demi-embryos.

• KSBB Journal
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• v.10 no.1
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• pp.15-22
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• 1995

The embryos(6-8mm) isolated from seeds of Diospyros kaki were cultured on Murashlge-Skoog(MS), Woody Plant Medium(WPM), Campbell Durzen(CD), Lictvay's Medium(LM), Kao-Michaluk(KM), Nitsch, White, Heller, Wolter-Skoog(WS) media. The results showed that MS and WPM media were most suitable to the development of embryos into plantlets with length of $5.4{\pm}1.2 cm$ and 5-6 leaves. However, when LM and KM media were used, the addition of 1 to $2{\mu} moles/\ell GA_3$ was required for the germination of the embryos. Superoxide dismutase (SOD) activities, one of the changing factors in leaves according to physiological status displayed to be exceptionally significant in the leaves of plantlets germinated from seeds in potting sand soil contrary to those of cultured embryos specially around germination period.

• Korean Journal of Plant Resources
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• v.19 no.3
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• pp.427-431
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• 2006

In vitro high-frequency plant regeneration of Muscari comosum var. plumosum through somatic embryogenesis was obtained via two developmental pathways: direct embryos and multiple shoots regenerated from embryogenic callus. Flower bud with pedicel, receptacle, petal and ovary wall, floral stalk and leaf as explants were cultured in MS medium supplemented with various plant growth regulators. Embryos formed directly from pedicel, receptacle and floral stalk. Depending on explant sources, the optimal medium was MS medium supplemented with 0.2 mg/L IBA and 0.3 mg/L BA, 3.0 mg/L IBA and 3.0 mg/L BA, and MS-free medium for pedicel, receptacle, and floral stalk, respectively. Multiple shoots regenerated from embryogenic cal]i which was initiated from petal, ovary and leaf were observed in MS medium with different concentrations and combinations of hormone. The most suitable medium for each type of explant was 3.0 mg/L IBA and 3.0 mg/L BA(petal and ovary) and 5.0 mg/L IBA and 5.0 mg/L BA (leaf) Furthermore, the combination of 0.1 mg/L 2,4-D and 1.0 mg/L BA was also good for all sources of explants not only for direct embryo formation, but also, for embryogenic callus induction.

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.307-317
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• 1998

This study was to investigate the vitrification method for cryopreservation technique of bovine in vitro fertilization(IVF) embryos. The morphological appearance and viability following vitrification of IVF bovine blastocysts and expanded blastocysts were examined. Embryos obtained 6, 7, 8 or 9 days after IVF were vitrified in both 40% ethylene glycoI(EG) plus 0.3M trehalose and 20% polyvinylpyrrolidone(PVP) in DPBS(ETP, Exp. 1) and ETP solution added 0.375M dextrose (ETPD, Exp. 2). The viability of Days 6, 7, 8 and 9 vitrified /thawed embryos at 24∼48 h culture after thawing was 11.9%, 19.8%, 23.4% and 15.3% in ETP(Exp. 1), and 34.6%, 54.5%, 37.9% and 13.0% in ETPD(Exp. 2), respectively. The viability of vitrified embryos produced from the culture days after IVF did not differ in Exp. 1, but significantly differ in Exp. 2(P＜0.05). The viability of blastocysts and expanded blastocysts significantly differed(P＜0.05) in 15.2% and 23.3%(Exp. 1), and 25.0% and 45.8%(Exp. 2). The result of Exp. 1 was similar to that of Exp. 2 on the viability of embryo according to developmental stages, but ETP solution plus sugar(dextrose) was increased the viability of vitrified embryos. In Experiment 3, The viability of vitrified embryos was not different between 12% and 20% PVP concentrations in ETP solution according to culture days or developmental stages. To investigate the effect of addition of sugar, two type of carbohydrates and a mixture of cryoprotectants for vitrification on the survival of bovine IVF embryos, bovine Days 7 to 9 blastocysts and expanded blastocysts were cryopreserved in either 20% glycerol plus 20% EG, 0.375M sucrose and 0.375M dextrose (GESD, Exp. 4) or 20% glycerol plus 20% EG, 0.3M trehalose and 20% PVP(GETP, Exp. 5) in DPBS. Survival rates of Day 7, 8 and 9 embryos at 24∼48h culture after thawing were 71.4%, 94.6% and 40.5% in GESD, and 59.5%, 81.5% and 62.5% in GETP, respectively. Hatching rates of Day 7, 8 and 9 embryos after thawing were 28.6%, 35.1% and 16.2% in GESD, and 27.0%, 33.3% and 18.8% in GETP, respectively. These results indicates that a mixture of cryoprotectants(glycerol and EG) and addition of sugar can improve the survival rates of the bovine IVF embryos(Day 7 or 8) vitrified, and the expanded blastocyst embryos are more suitable for vitrification than early blastocysts stage.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.281-287
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• 2013

A major barrier to progress in pig to primate organ transplantation or cell therapy is the presence of terminal ${\alpha}$-1,3-galactosyl epitopes on the surface of pig cells. Therefore, the purpose of this experiment was to establish and cha- racterize mesenchymal stromal/stem cells (MSCs) derived from ${\alpha}$-1,3-galactosyltransferase (GalT) knock out (GalT KO) pig to confirm their potential for cell therapy. Bone marrow (BM)-MSCs from GalT KO pig of 1 month old were isolated by Ficoll-Paque PLUS gradient and cultured with A-DMEM + 10% FBS on plastic dishes in 5% $CO_2$ incubator at 38.5. GalT KO BM-MSCs were analyzed for the expression of CD markers ($CD45^-$, $29^+$, $90^+$ and $105^+$) and in vitro differentiation ability (adiopogenesis and osteogenesis). Further, cell proliferation capacity and cell aging of GalT KO BM-MSCs were compared to Wild BM-MSCs by BrdU incorporation assay (Roche, Germany) using ELISA at intervals of two days for 7 days. Finally, the cell size was also evaluated in GalT KO and Wild BM-MSCs. Statistical analysis was performed by T-test (P<0.05). GalT KO BM-MSCs showed fibroblast-like cell morphology on plastic culture dish at passage 1 and exhibited $CD45^-$, $29^+$, $90^+$ and $105^+$ expression profile. Follow in ginduction in StemPro adipogenesis and osteogenesis media for 3 weeks, GalT KO BM-MSCs were differentiated into adipocytes, as demonstrated by Oilred Ostaining of lipid vacuoles and osteocytes, as confirmed by Alizarinred Sstaining of mineral dispositions, respectively. BrdU incorporation assay showed a significant decrease in cell proliferation capacity of GalT KO BM-MSCs compared to Wild BM-MSCs from 3 day, when they were seeded at $1{\times}10^3$ cells/well in 96-well plate. Passage 3 GalT KO and Wild BM-MSCs at 80% confluence in culture dish were allowed to form single cells to calculate cell size. The results showed that GalT KO BM-MSCs($15.0{\pm}0.4{\mu}m$) had a little larger cell size than Wild BM-MSCs ($13.5{\pm}0.3{\mu}m$). From the above findings, it is summarized that GalT KO BM-MSCs possessed similar biological properties with Wild BM-MSCs, but exhibited a weak cell proliferation ability and resistance to cell aging. Therefore, GalT KO BM-MSCs might form a good source for cell therapy after due consideration to low proliferation potency in vitro.

• Korean Journal of Animal Reproduction
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• v.24 no.1
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• pp.97-108
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• 2000

The objectives of this study were performed to increase the efficiency of the culture conditions of embryos produced in vitro, and to assess the developmental potential after transfer of those embryos into recipients. The mean number of folliclular oocytes recovered from an ovary was 10.7. The rates of maturation and fertilization in Grade I oocytes were significantly (P＜0.05) higher than Graden and III. Developmental rate into blastocyst in the culture group of TCM-199 with BOEC were significantly higher (P＜0.05) than the groups of TCM-199 and conditioned medium (24.7% vs. 12.4% and 18.2%). The survivability of post-thawed blastocysts equilibrated for 3 min in EFS solution was significantly (P＜0.05) lower than l0 for 1 and 2 min (32.1% vs. 82.9% and 73.3%). Significantly higher (P＜0.05) survival rate in blastocysts was seen after freezing than in morulae stage embryos. Out of all 105 recipients, 49 (46.7%) were confirmed in pregnant. On pregnancy of cattle, 48 calves were born from 40 recipients. The ratio of twin and single calves was 30.5% (32/40 and 7.6% (8/40), respectively. However, the others composed of abnormal, as judging as 6 (12.2%) for abortion and 3 (6.1%) for stillbirth during the pregnant period.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.83-87
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• 2004

The objective of this study was performed to establish the in vitro culture system of porcine in vitro maturation and in vitro fertilization(IVM/IVF) embryo. These studies was to determine the effects of melatonin, nitric oxide donor(SNP), and the combination effects of SNP and melatonin in porcine IVM/IVF embryos. In routine porcine IVM/IVF procedure, oocytes were cultured for 40∼44h incubation, and the zygotes were cultured for 40∼44h in NCSU 23 medium. Then 2 to 8 cell embryos were removed cumulus cell and were allotted randomly to NCSU 23 containing different concentration of melatonin, SNP and SNP plus melatonin in 5% $O_2$, 5% $CO_2$ and 90% $N_2$ at 38.5$^{\circ}C$. Cell numbers of blastocyst were also counted using double fluorescence stain method. In NCSU 23 medium treated with melatonin 0, 1, 5 and 10 nM, the developmental rate of morula plus blastocysts were 33.3%, 39.1%, 33.3% and 27.9%, respectivly. This result show that the developmental rate of morula and blascytocys treated with 1 nM melatonin was higher than in any other groups(P<0.05). The developmental rates of morula plus blastocysts were 41.9% in 0 uM SNP, 25.6% in 50 uM and 28.4% in 100 uM, respectively. The developmental rate of morula plus blastocysts were decreased treated with SNP in NCSU 23. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM, SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), the developmental rates beyond morula stage of porcine embryos were 31.3%, 34.1%, 39.5%, 29.4% and 39.5%, respectively. The addition of SNP 50 uM plus maltonin 1 nM, developmental rates of blastocyst was higher rate than in any other groups. Cell numbers of blastocyst in NCSU 23 treated with melatonin 0, 1, 5 and 10 nM were 41.0, 42.6, 39.6 and 33.0, respectively. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM , SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), cell numbers of developed blastocyst were 36.3, 34.6, 39.0, 39.9 and 39.0, respectively. These result show that the cell numbers of blastocyst treated with 0, 1 and 5 nM melatonin were higher than in 10 nM group(P<0.05), but cell numbers of blatocyst produced by SNP plus melatonin were not significantly difference in all experimental groups.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.119-126
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• 2004

The present study was carried out to examine the effect of four different media (NCSU (North Carolina State University)-23, PZM (Porcine Zygotes Medium)-3, PZM-4 and TCM (Tissue Culture Medium)-l99) and two oxygen concentrations (39 , 5% $O_2$, 5% $CO_2$ and 90% $N_2$, 5% $CO_2$ in air) on in vitro production of porcine IVM/IVF embryos. The results were summarized as follows: The rates of GVBD and nuclear maturations were not significantly different (p>0.05) for 44 hours of culture with four media in two oxygen concentrations. The rates of polyspermy, penetrated sperm(s) and male and female prouclei formation were not significantly different (p>0.05). among four media in two oxygen concentrations. The cleavage rates were not significantly different (p>0.05) among four media in two oxygen concentrations. At day 7 under gas atmosphere of 5% $O_2$, 5% $CO_2$ and 90% $N_2$, the blastocyst formation was significantly higher (p<0.05) in PZM-3 (19.9$\pm$2.4) than other media. Also, NCSU-23 medium gave high rate of blastocyst formation at day 7 under gas atmosphere of 5% $CO_2$ in air (p<0.05). Based on the result of differential staining of porcine blastocyst at dat 7, inner cell mass cell and total cell numbers were not significantly different (p>0.05) among four media in two oxygen concentrations. However, the observed total cell number was higher in PZM-3 medium (36.8$\pm$6.5) than other madia. In conclusion, these results suggested that in vitro production of porcine embryos in PZM-3 medium under a gas atmosphere of 5% $O_2$, 5% $CO_2$ and 90% $N_2$ was effective on the blastocyst formation rate and total blastocyst cell number.

• Korean Journal of Animal Reproduction
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• v.15 no.1
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• pp.49-57
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• 1991

This study was carried out to investigate the effect of equilibration time, precooling and straw loading method on the post-thaw survival rates of mouse embryos cryopreserved by vitrification method. The effect of the vitrification procedure on the damage of the embryos was also examined by the straining of nuclei using Hoechst 33342. The results obtained were summarized as follows; 1. The equilibration in Medium-1 for 10 minutes was considered to be the optimum equilibration time. Embryos equilibrated in Medium-1 for 10 minutes(81.0%) showed significantly(P<0.05) higher survival rates than those equilibrated for 5 minutes(40.0%) or 15 minutes(74.1%). 2. The survival rate of embryos cryopreserved using the double Medium-2 column(81.0%) was significantly(P<0.01) higher than that using the single Medium-2 column, whish was considered to be due to the double Medium-2 column method being more reliable for preventing contamination of diluent solution of 1M sucrose. 3. The survival rate of compacted morula stage embryos cryopreserved with the precooled and non-precooled Medium-2 was not significantly(P<0.05) different. 4. The number of blastomeres of embryos at late blastocyst stage after culture of mouse morulae for 24 to 28hours was significantly(P<0.05) decreased by freezing embryos using vitrification(53.3${\pm}$1.6 vs 41.4${\pm}$1.5), which was considered to be due to the damage of embryos during vitrification and the delay of embryo development by handling in vitro. 5. The vitrification procedure is considered to be a very simple and efficient method for cryopreservation of embryos at early developmental stage.