• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.73-83
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• 1990

These studies were carried out to find the proper conditions for in vitro maturation and fertilization of bovine follicular oocytes and culture methods capable of further developing early embryos. For these objectives, the cleavage rate of oocytes matured and fertilized in vitro was investigated under medium supplemented with hormones and estrous cow serum and season of oocytes collection as well as different cumulus cell stage before insemination. Finally, 2~8 cell embryos were cultured in in vitro and in vitro culture system to investigate developmental capacity into morula. 1. Cleavage rate of oocytes matured in vitro was 27%(20/73) for A(LH＋FSH＋estradil-17$\beta$＋10% FBS), 38%(27/71) for B(LH＋10% ECS) and 27%(15/56) for C(10% ECS), respectively. Supplement B showed more higher rate and 4~8 cell embryos were also obtained much more in this group(67%, 18/27). In vitro maturation rate of follicular oocytes cultured in TCM 199 supplemented withLH and 10% ECS was 88%(75/85). 2. Cleavage rate(15%, 10/65) of oocytes collected in summer was significantly lower than in fall(47%, 42/89). 3. Cleavage rate(15%, 10/65) of oocytes collected in summer was significantly lower than in fall(47%, 42/89). 3. Cleavage rate(15%, 10/65) of oocytes with partially removed cumulus cell before insemination was more higher than that(44%, 27/62) of oocytes with intact cumulus cell. 4. The frequency of development from early cleaved embryos into morula was 6%(4/65), 12%(4/33) for co-culture of cumulus cell monolayer and bovine oviduct epithelial cell monolayer, respectively and 25%(6/25) in ligated rabbit oviduct.

• Korean Journal of Animal Reproduction
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• v.8 no.1
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• pp.22-28
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• 1984

This experiment was carried out to investigate effects of DMSO or ethylene glycol as a cryopotectant and of freezing methods on survival rate of forozen-thawed rat 2-cell embryos by morphological observation. 2-cell embryos were recovered from oviducts of Sprague Dawley females mated with males of same strain on day 2 of pregnancy after inducing superovulation by intrapertioneal injection of PMSG and HCG. In slow freezing and thawing groups, embryos were frozen to -79$^{\circ}C$ or -196$^{\circ}C$ in a glass test tube containing 0.2ml PBI with 1.5M DMSO or 1.2M ethylene glycol at a rate of 0.3-1.0C/min. and thawed slowly. When samples were frozen to -79$^{\circ}C$, higher survival rate was obtained in the medium containing DMSO (43.9%) than ethylene glycol (41%). And similar result was obtained (32.5% in DMSO vs. 31.4% in ethylene glycol) when samples were frozen. In rapid freezing and thawing groups, embryos were frozen to -79 or -196$^{\circ}C$ in a glass test tube containing 0.2ml of PBI with 1.5M DMSO or 1.2M ethylene glycol by rapid cooling, and thawed rapidly. When samples were frozen to -79$^{\circ}C$, 1.5M DMSO (13.2%) was more effective than 1.2M ethylene glycol (6.1%). When the storage temperature was -196$^{\circ}C$, survival rates were 9.8% in 1.5M and 5.4% in 1.2M ethylene glycol.

• Journal of Embryo Transfer
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• v.13 no.1
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• pp.11-19
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• 1998

This study was to determine the effect of ionomycin and 6-dimethylaminopurine (6-DMAP) and/or elcetrical stimulation on the oocyte activation and production of rabbit nuclear transplant embryos. The oocytes were collected from the oviduct of superovulated rabbits at 14 h post hCG injection and cultured in TCM-199 containing 10% FBS until 19 h post hCG injection. To determine the optimum concentration and exposure time of 6-DMAP, some oocytes were activated with 5 $\mu$M ionomycin for 5 min and then in 2.0 mM 6-DMAP for 0.5 to 3.0 h, or in 1.0 to 3.0 mM 6-DMAP for 2.0 h. Other control oocytes were stimulated electrically(3X, 1.25 kV/cm, 60 $\mu$sec) in 0.3 M mannitol solution supplemented with 100 $\mu$M CaCl$_2$ and MgCl$_2$. The nuclear donor embryos of 8-cell stage were synchronized to G$_1$ phase of 16-cell stage, and the recipient cytoplasms were obtained from removal of the first polar body and a portion of membrane bound cytoplasm of the oocytes collected at 15 h post hCG injection. A separated blastomere was injected into the perivitelline space of the enucleated oocytes. The oocytes injected with nucleus were cultured until 19 h post hCG and then electrofused and activated by electrical stimulation with or without ionomycin and 6-DMAP. These nuclear transplant embryos were cultured in TCM-199 containing 10% FBS in 39˚C, 5% CO2 incubator for 120 h. For the oncytes activated parthenogenetically with electrical stimulation with or with-out ionomycin and the various concentration of exposure time of 6-DMAP, the highest cleavage(92.3%) and development to blastocyst stage(41.0%) were resulted from the oocytes activated by ionomycin and 2.0 mM 6-DMAP for 2.0 h, which were found to be significantly(P<0.05) higher than the cleavage(45.2%) and developement to blastocyst stage(14.3%) from the oocytes activated with electrical stimulation. The significantly(P<0.05) more oocytes(71.4%) developed to 4 cell stage at 24 h post activation by ionomycin and 6-DMAP than those by electrical stimulation(18.9%). For the nuclear transplant embryos, the cleavage rate was similarly high in oocyte activation by electrical stimulation with(79.4%) or without ionomycin and 6-DMAP(70.5%). However, the embryo development to blastocyst stage was significantly(P<0.05) higher in oocyte activation by electrical stimulation with ionomycin and 6-DMAP(44.4%) than by electrical stimulation only(25.0%). The significantly(P<0.05) more nuclear transplant embryos(45.6%) developed to 4 cell stage at 18 h post activation by electrical stimulation with ionomycin and 6-DMAP than those by electrical stimulation only(10.6%). These results indicated that the supplemental oocyte activation by ionomycin and 6-DMAP with electrical stimulation enhanced and accelerated the preimplanted in vitro development of the rabbit nuclear transplant embryos.

• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.209-215
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• 1992

These studies were carried out to overcome 2-cell block and in vitro development to blastocysts in vitro fertilization of mouse embryos. The unfertilized ova were obtained by superovulation in ICR mice of 4 to 6 weeks old. Tyrode's 280 solution was used as basal media, and the pH range of media examined was designed from 6.5 to 7.5 with 0.2 interval and the range of osmolality from 250 to 370 mOsm with 20 interval, and the period of sperm preincubation examined was 30, 60, 120, and 180 minutes. The ova developed to 2-cell embryos after 26hrs of incubation with preincubated sperm were evaluatated as in vitro fertilized ones. The results obtained were summarized as follows: 1. The optimal ranges of pH and osmolality of culture media and of sperm preincubation time for in vitro development of in vitro fertilized ova to blastocyst were pH 7.1 to 7.3, 250 to 350 mosmol and 60 to 180 min, respectively. 2. With the media of pH 7.1, 310 mOsm and sperm preincubation period of 120min in another experiment of large sample size, the in vitro fertilized ova was found 66.5% and the in vitro development of in vitro fertilized ova to blastocyst was found 35.8%. From the above results it was concluded that the optimal conditions of pH and osmolality of the media for mouse IVF and embryo culture, and the period of sperm preincubation might be 7.1, 310 mOam and 120min, respectively.

• Development and Reproduction
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• v.18 no.3
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• pp.153-160
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• 2014

Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second mid-preimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.1
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• pp.25-33
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• 2006

Objective: The aim of this study was to investigate whether embryonic stem (ES) cells can be established from isolated blastomeres of mouse embryos. Methods: Blastomeres were separated from mouse (C57Bl/6J) 2- or 4-cell embryos. Isolated blastomeres or whole 4-cell embryos were co-cultured with mitosis-arrested STO feeder cells in DMEM supplemented with recombinant murine leukemia inhibitory factor and ES-qualified fetal bovine serum. After the tentative ES cell lines were maintained from isolated blastomeres or whole embryos, some of them were frozen and the others were sub-cultured continually. Characteristics of tentative ES cell lines as were evaluated for specific genes expressions with immunocytochemistry and RT-PCR. Results: One ES cell line (3.0%) was established from isolated blastomere of 2-cell embryo and one cell line (4.0%) from isolated two blastomeres of 4-cell embryo. And five cell lines (16.7%) were established from whole 4-cell embryos. Both cell lines from isolated blastomere and whole embryo expressed mouse ES cell specific markers such as SSEA-1, Oct-4 and alkaline phosphatase. Marker genes of three germ layers were expressed from embryoid bodies of both cell lines. Conclusion: This study suggests that mouse ES cells could be established from isolated blastomeres, although the efficiency is lower than whole embryos. This animal model could be applied to establishment of autologous human ES cells from biopsied blastomeres of preimplantation embryos in human IVF-ET program.

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.127-132
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• 2014

This study was carried out to investigate the effects of tissue inhibitor of matalloproteinase-1 (TIMP-1), Activin A and Heparin binding epidermal growth factor (HB-EGF) on in vitro production of bovine embryos. In experiment 1, presumptive zygotes were cultured in the medium supplemented with TIMP-1 ($0.5{\mu}g/ml$), Activin A (100 ng/ml), or HB-EGF (100 ng/ml) at $39^{\circ}C$ in a humidified atmosphere of 5% (v/v) $CO_2$, 5% (v/v) $O_2$ and 90% (v/v) $N_2$. In experiment 2, TIMP-1 + HB-EGF or Activin A + HB-EGF combinations were supplemented in the culture medium. The developmental rate to blastocysts, hatching rate and total cell numbers of the blastocysts were evaluated in both experiments. The embryos cultured in medium without growth factor supplementation was used as control group. In experiment 1, the embryos cultured in medium supplemented with TIMP-1 and Activin A showed significantly higher developmental rate to blastocysts than those cultured with HB-EGF and control (36.9%, 34.1%, 21.2% and 23.1%, respectively) (P<0.0001). However, the hatching rate of blastocyst was significantly higher in embryos with HB-EGF than those with TIMP-1, Actvin A and Control groups (84.4%, 58.8%, 51.4% and 49.3%, respectively) (P<0.001). Total cell number per blastocyst was also significantly higher in embryos with HB-EGF group ($174.3{\pm}2.5$) than those with TIMP-1, Activin A (149.7 and 150.0, respectively) (P<0.05) and Control (119.0) (P<0.001). In experiment 2, embryos cultured with combined treatment of Activin A and HB-EGF resulted in significantly higher rates of blastocysts formation (48.0%), hatching rate (89.7%) and total cell number in blastocyst ($182.3{\pm}2.1$) than those with TIMP-1 and HB-EGF combination group (32.0%, P<0.001; 76.6%, P<0.05; $165.7{\pm}4.2$, P<0.001, respectively). Our data demonstrate that in vitro production of bovine embryos could be improved by combined supplementation of Activin A and HB-EGF in culture medium.

• Journal of Veterinary Science
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• v.23 no.2
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• pp.40.1-40.13
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• 2022

Background: Somatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency. Objectives: This study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs. Methods: The viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer. Results: Most sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption. Conclusions: Sow urine-derived cells could be used to produce SCNT embryos.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.249-255
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• 2014

Programmed cell death or apoptosis is associated with changes in $K^+$ concentration in many cell types. Recent studies have demonstrated that two-pore domain $K^+$ ($K_{2P}$) channels are involved in mouse embryonic development and apoptotic volume decrease of mammalian cells. In cerebellar granule neurons that normally undergo apoptosis during the early developmental stage, TASK-1 and TASK-3, members of $K_{2P}$ channels, were found to be critical for cell death. This study was performed to identify the role of $K^+$ channels in the $H_2O_2$-induced or cryo-induced cell death of mouse and bovine embryos. Mouse and bovine two-cell stage embryos (2-cells) exposed to $H_2O_2$ for 4 h suffered from apoptosis. The 2-cells showed positive TUNEL staining. Treatment with high concentration of KCl (25mM) inhibited $H_2O_2$-induced apoptosis of 2-cells by 19%. Cryo-induced death in bovine blastocysts showed positive TUNEL staining only in the cells near the plasma membrane. Cryoprotectant supplemented with 25 mM KCl reduced apoptosis slightly compared to cryoprotectant supplemented with 5 mM KCl. However, the combination of antioxidants (${\beta}$-mercaptoethanol) with 25 mM KCl significantly decreased the rate of $H_2O_2$-induced and cryo-induced apoptosis compared to treatments with only antioxidants or 25 mM KCl. These results show that blockage of $K^+$ channel efflux for a short-time reduces $H_2O_2$- and cryo-induced apoptosis in mouse and bovine embryos. Our findings suggest that apoptosis in mouse and bovine embryos might be controlled by modulation of $K^+$ channels which are highly expressed in a given cell type.

• Korean Journal of Animal Reproduction
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• v.10 no.1
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• pp.42-48
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• 1986

These experiments were carried out to develop new techniques identifying XX-bearing embryos prior to implantation by immunological method. Antiserum to histocompatibility-Y(H-Y) antigen was prepared in adult SD(sprague-dawley) female rat by repeated immunization of newbone testis supernatant from males of the same strain. ELISA test was used to identify the H-Y antibody of antiserum. Total 124 mouse embryos (8-cell stage) were treated with H-Y antiserum and complement in BSA free Ho, pp. and Pitt's medium and cultured under the gas phase of 5% CO2 in air at 37$^{\circ}C$ for 24 to 48 hrs. The morphological characteristics of embryos treated were observed under the phase-contrast micro scope. The results obtained in these experiments were summarized as follows: 1. Optimal Density of H-Y antibody were a, pp.ared to be 0.27-0.47 by ELISA test. 2. Of total 124 embryos treated with H-Y antiserum and complement 69(55.6%) embryos developed to blastocyst and 55(44.4%) destroyed or arrested.