• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.111-118
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• 1997

Intracytoplasmic sperm injection (ICSI) recently has been utilized widely as the most successful technique to overcome the unfertilization problem in cases of severe male infertility in couples who could not be treated by conventional IVF. Recently, indications of ICSI have been extended further and more fertilized oocytes become available. Thus, it is necessary to examine the efficiency of freezing the surplus embryos obtained from ICSI. We compared the survival rate and the future outcome of cryopreserved embryos obtained either after conventional IVF or ICSI during the same period. After ICSI or IVF, five best-quality embryos from each patient were transferred in the stimulation cycle and the surplus pronuclear (PN) stage oocytes or multicellular embryos were cryopreserved by slow freezing protocol with 1,2-propanediol (PROH) as a cryoprotectant. A total of 792 embryos from ICSI trial were thawed and 65.2% (516/792) survived. The survival rates of PN stage oocyte, multicellular embryo and PN + multicellular embryo were 63.5%, 68.2%, 64.0%, respectively. After 111 transfers, 34 pregnancies were achieved, corresponding to a clinical pregnancy rate of 30.6% per transfers. We thawed 1033 embryos from IVF trials and 57.5% (594/1033) survived. In IVF cycle, the survival rates of PN stage oocyte, multicellular embryo and PN + multicellular embryo were 58.2%, 65.2%, 40.2%, respectively. Thirty eight clinical pregnancies were established after 134 transfers, corresponding to a pregnancy rate of 28.4% per transfer. The cleavage rate of thawed PN stage oocytes from ICSI trial (61.3%) was significantly higher than those from conventional IVF (53.4%). The developmental rates of good embryo (${\geqq}$ grade II) in thawed PN stage oocytes obtained from conventional IVF and ICSI were 63% and 65%, respectively. We concluded that PN stage oocytes, multicellular embryos resulting from ICSI procedure can be successfully frozen/thawed with reasonable clinical pregnancy rates comparable to those of IVF.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.209-217
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• 1995

The present study was carried out to develop a cloning technology of mouse embryos by nuclear transplantation with electrofusion and to produce cloned offsprings by transfer of reconstituted embryos. A single nucleus from two- and eight-cell embryos was transplanted into the enucleated two-cell embryos by rnicromanipulation. The fusion of nucleus with recipient cytoplasm and the subsequent development of reconstituted embryos in vitro as well as in vivo to term were examined to determine the optimal electrofusion parameters for nuclear transplantation in mouse embryos. The successful enucleation of donor embryos was 84.9 and 83.3% in two- and eight-cell stage, respectively, and the successful injection of nucleus from two- and eight-cell donor embryos into the perivitelline space of enucleated two-cell embryos were 85.1 and 84.7%, respectively. No significant differences were found in enucleation or injection rate between the cell stages of donor embryos. When the blastomeres of intact two-cell mouse embryos were electrofused in 0.3 M mannitol medium(100 $\mu$sec., 3 pulses), the fusion rate was similarly 93.2, 92.2 and 92.0% in 1.0, 1.5 and 2.0 kV /crn, respectively, but in vitro development to blastocyst of the fused two-cell embryos was significantly(P<0.05) lower in 2.0 kV/cm (63.4%) than in 1.0 kV/cm (91.7%) or 1.5 kV/cm (82.4%). The development in vitro to eight-cell stage of the reconstituted embryos with nucleus from two-cell stage(45.5%) was significantly(P<0.05) higher than that from eight-cell stage blastomeres (16.7%). The number of blastomeres of the intact embryos at blastocyst stage was 50i0.6 and 55$\pm$2.4 in in vitro and in vivo cultured mouse embryos, respectively, but significantly(P<0.05) decreased to 35$\pm$0.7 in nuclear transplanted blastocyst embryos. The conception rate of mice following embryo transfer was 32.1% in the reconstituted two-cell embryos using two-cell donor nuclei, which was comparable to the fresh two-cell embryos(40.6%). However, the rate of development in vivo to term following embryo transfer of the reconstituted two-cell embryos using two-cell donor nuclei (23.5%) was significantly(P<0.05) lower compared with the percentage of two-cell fresh embryos(31.5%).

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.85-93
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• 1997

These experiments were carried out to examine the development capacity of sexed and then bisected embryo from 8-cell to morula stage. Antisera to histocompatibility-Y(H-Y) antigen were prepared in inbred SD female rat by repeated immunization of spleen cell or testis supernatant from males of same strain. Male and female embryos were separated by delaying development of embryos against H-Y antibody. After sexing, rabbit embryos were bisected and aggregated. The results obtained from the these experiments were summuerized as follows: 1. When mouse and rabbit 8-, 16-cell and morular embryos were culature in H-Y antiserum, the ratio of embryo which has developed to hatching blastocyst was 53.4, 46.3 and 57.4% in mouse embryos, and 49.0, 52.0 and 61.0% in rabbit embryo, respectively. The ratio of mouse and rabbit embryos developed to hatching blastocyst showed nearly natural sex rate(50%), except rabbit mourla showed a little higher ratio(61.0%) as compared to natural sex ratio. 2. When rabbit demi-embryos from 8-, 16-cell embryo and morula were cultured, the percentage of demi-embryos was 70, 68 and 58% without zona pellucida removed, and 62, 69 and 51% with zona pellucida. The rate of aggregation was higher in 8- and 16-cell demi-embryos than in morula demi-embryo. 4. When sexed-demi-embryo was aggregated with another demi-embryo with demi-embryo with same sex, the rate of embryo developed to blastocyst was 60, 50 and 25%, respectively. Eight-cell demi-embryo showed highest rate. In conclusion, it showed that H-Y antiserum which was made by rat spleen cell enabled sexing rabbit embryos. And when rabbit sexed 8-, 16-cell and morula demi-embryo were aggregated, they were developed to eu-blastocyst which suggested the potential of sexed embryo manipulation.

• Korean Journal of Animal Reproduction
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• v.19 no.2
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• pp.89-93
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• 1995

In this study was demonstrate that retrovirus-mediated gene transfer is one of the promising alternatives to the conventional pronuclear DNA microinjection approach, especially in transferring the exogenous genes into the boving embryos. By co-culturing of zona of zona-free one-cell stage embryos with the retrovirus-producing cells for 24 hours followed by 6 days of culture in virus-free medium, we could get morulae and blastocysts expressing the E. coli LacZ genes which were transferred by our retrovirus vector. The results obtained in this study are summarized as follows : 1. Addition of 5$\mu\textrm{g}$/ml of polybrene in the embryo and virus-producing cell co-culture medium did not affect development of zona-free one-cell embryo. 2. Compared with the intact embryos removal of zona at one-cell stage before co-culturing with the virus-producing cells for one day caused only slight decrease of embryo develpment. 3. Co-culture of 625 zona-free one-cell stage embryos with the virus-producing cells resulted in 65(10.4%) morulae or blastocysts, and 12.3%(8/65) of the morulae or blastocysts were E. coli LacZ positive.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.113-121
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• 2005

This study was conducted to investigate the effects of different hexoses (glucose, mannose, galactose and fructose) on in vitro development of parthenogenetic embryos in the pigs. When the parthnogenetic embryos were cultured in medium with concentrations of 5mM glucose or 1mM galactose, the rates of embyos developed to morula and blastocyst stages were significantly higher than those in another culture conditions (P<0.05). However, high concentration of galactose inhibited development to morula and blastocyst stages. Addition of hexoses at early stage of porcine parthenogenetic embryos were effective for in vitro development. Especially, the embryos cultured in medium with glucose at early stage were effective for development to 2-cell $(72\%)$ and blastocyst $(19\%)$ stages compared with embryo cultured without glucose. From the present results, it is suggested that development of porcine parthenogenetic embryos can improve in medium with 5mM glucose. The concentration of 1mM galactose was also effective for development of porcine parthenogenetic embryos. It also show that parthenogenetic embryos cultured with glucose at early stage can improve in vitro development.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.93-97
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• 1999

The development potential of bovine somatic cells was evaluated using nuclear transfer. A single donor cell derived from fetus of HanWoo(Korean Native Cattle) was selected and deposited into perivitelline space of each enucleated oocyte before electrical fusion and activation. Nuclei of donor cells starved for 7 days (37%) tended to support the development of reconstitute embryo the blastocyst stage better than those of donor cells starved 3, 14 and 30 days. The cleavage rate was significantly lower(P<0.05) in reconstitute embryos derived from large size donor cells(51.2%), than those from small medium size donor cells(76.6 and 73.5, respectively). The developmental rate to blastocyst of reconstructed embryos from medium size donor cells was higher than those from small and medium size donor cells. This study demonstrates that an appropriate culture period for induction into quiescent stage and the size of donor cells effect on the efficiency of nuclear transfer using cultured bovine cells.

• Animal cells and systems
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• v.5 no.1
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• pp.85-90
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• 2001

Yolk platelets, one of the main food stores in the embryonic development, are composed of proteins. However, little is known about the identity of proteins utilized at certain stages of embryogenesis. In this study, we followed the fates of embryonic storage proteins by using an anti-polyubiquitin monoclonal antibody (mAB) as a probe. The mAb recognized the major storage proteins of Drosophila, Xenopus and chicken eggs. In the Drosophila embryo, the mAb-reactive 45-kDa protein was not used until stage 11 but was used up at stage 16 when the embryo completed segmentation. In the Xenopus embryo, the mAb-reactive 111 kDa protein was mostly utilized between stages 42 and 45 implying that the protein might be an energy source used just prior to feeding on food. By N-terminal sequencing the storage protein of Xenopus embryo was identified as a lipovitellin 1. This study confirms that storage proteins are used almost simultaneously at certain stages of embryogenesis and that vitellogenin 1 is the last storage protein in Xenopus embryogenesis.

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.35-42
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• 1998

This experiment was carried out to improve in vitro development of rabbit one-cell embryos to the blastocyst stage. One-cell rabbit embryos were collected at 19\ulcorner20hr after superovulation induction and incubated at 39\ulcorner in 5% CO2 for 72hr. In order to find optimum conditions in medium that affects the rabbit embryo's development in vitro, RDH medium which mixed with RPMI1640, DMEM and Ham's F10 was compared with the previously reported mediums (Ham's F10 and RD) for embryo development and cell numbers. Three additives (BSA, taurine and glucose) were tested for the development of rabbit one-cell embryos in vitro. When the embryos were cultured in RDH medium, their development was markedly promoted as compared with Ham's F-10 or RD alone. Glucose exhibited no significant effects on embryo development and cell numbers. BSA a, pp.ared to promote transition from morula to blastocyst stage and taurine increased cell numbers of cultured embryos markedly regardless of medium. BSA and taurine together in RDH medium showed the additive effects on embryos development and cell number.

• Journal of Ginseng Research
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• v.16 no.1
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• pp.37-43
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• 1992

Structural changes of the endosperm of Panax ginseng C.A. Meyer from fertilization to germination were investigated by light microscope. The endosperm of the ginseng seed is cellular type. Since endosperm cells adjacent embryo continuously breakdown and disappear with the elongation of embryo, the real of endosperm is gradually decreased. As the anatropous ovules of immature seed with green seed coat developes more and more, ovary cells adjacent ovary cavity become abundant by the periclinal division, their size is decreased, hypotrophy of cell wall discern, and they are gradually differentiated in seed coat. Though embryo responds strongly to basic dye at the stage of completion of endosperm formation, tissue of endosperm responds to acidic dye positively Cell wall of embryo and endosperm are composed of primary cell wall not lignified. Endosperm cells adjacent embryo begin to breakdown in the endosperm tissue of indehiscent seed before the beginning of the after-ripening. Dehiscent seed of which seed coat is opened through after-ripening represent the form as a seedling in the result of embryo developments with the formation of organs; radicle, cotyledon, plumule. Umbilifom layer represents strong positive response to the toluidine blue and the basic function. Umbiliform layer that endosperm cells breakdown and disappear is observed clearly at the periphery of the embryo cotylemon, while slightly at the periphery of the radicle.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.221-227
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• 1994

This study was conducted to examine the condition of in vitro culture system and the viability after embryo transfer of in vitro matured-in vitro fertilized (IVM-IVF) bovine embryos. The in vitro development to the blastocyst stage was enhanced by supplying bovine serum albumin(BSA) to co-culture medium with bovine oviduct epithelial tissue(BOET) compared with that in medium supplemented with fetal bovine serum(FBS) (41.2% vs. 26. 3%, P<0.05). After transfer of IVM-IVF blastocysts into the uterine horn of recipient females (Aberdeen Angus), one was pregnant to term and produced a head of male Korean native calf. These results confirm that the in vitro development of IVM-IVF bovine embryos is affected with different protein source in co-culture with BOET, and IVM-IVF embryos can develop to term after in vitro culture and embryo transfer.