• The Korean Journal of Zoology
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• v.18 no.2
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• pp.87-101
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• 1975

Electron microscopic studies on the ultrastructure of the mouse oocyte were made to investigate the inhibition of germinal vesicle breakdown by dibutyryl cAMP. The nuclear membrane of the dibutyryl cAMP-treated oocyte is characterized by a decreased degree of folding, maintains the normal double membrane structure, and shows an increased occurrence of the nuclear pore. It is suggested that these may be related to the suppression of the maturation of oocytes at the germinal vesicle. Mitochondria in the control cell were shown to be spread evenly throughout the cytoplasm and structurally underdeveloped or transitionary having little cristae development. On the contrary, mitochondria in the treated oocyte were found to be localized mainly around the nucleus and to show a greater extent of cristae development. The oocyte treated with dibutyryl cAMP appears to have fewer and structurally simpler lysosomes as compared to the control. The Golgi complex in the control oocyte exhibits the typical granular and lamellar structure, whereas that in the treated cell is poorly developed. Many multivesicular bodies, tonofilaments, and free ribosomes were observed in the control as well as in treated cells. The microvilli become structurally irregular, and a development of the perivitelline space is apparent in the treated oocyte. It is concluded that there is no basic difference in the ultrastructure between the oocytes treated with dibutyryl cAMP for 24 hours in the medium and those collected directly from the follicle. However, the finding that dibutyryl cAMP induces a development of more pores along the nuclear membrane strongly suggests the possibility that this compound inhibits the maturation of oocytes by influencing the permeability of the nuclear membrane.

• Korean Journal of Ichthyology
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• v.11 no.1
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• pp.86-93
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• 1999

Artificial fertilization of flounder, Limanda herzensteini caught at Pohang brook was performed in April 13, 1995 and hatched larvae were reared for 25 days to describe the development of eggs and the morphogenesis of larvae. The fertilized egg was buoyant, colorless and spherical in shape, measuring 0.86~0.96 mm in diameter (mean: 0.90 mm), The egg has no oil globule, and its perivitelline space was narrow. Hatching in the indoor tank with $16.0^{\circ}C$ in mean water temperature started from the 64 hours after fertilization. The newly hatched larva, measuring about 2.80 mm in total length, had 10~11+25~27=35~38 myomeres. The anus is open at about the anterior point of the body. The 3 to 4 days old larva was 3.90 mm in total length. In this larva the eyes were black and the mouth was open. The yolk was consumed in 8 to 9 days. In 20 to 22 days after hatching, the larvae grew 6.1 mm in total length. The eyes were located symmetry on both sides of head.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.119-127
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• 2018

The purpose of this study is to examined the electrofusion and activation conditions for the production of porcine somatic cell nuclear transfer (SCNT) embryos. In this study, immature oocytes were cultured in TCM-199 with and without hormones for 22 hours. Skin fibroblasts cells of porcine were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion was performed with two different pulses that each one pulse (DC) of 1.1 kV/cm or 1.5 kV/cm for $30{\mu}sec$. After fusion subsequent activation were divided into three groups; non-treatment (control) and treatment with 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B for 4 hours. Transferred embryos were cultured in PZM-3 (Porcine Zygote Medium-3) in $5%\;CO_2$ and 95% air at $39^{\circ}C$ for 7 day. Apoptosis-related genes (Caspase-3, BCL-2, mTOR, and MMP-2) were analyzed by immunofluorescence staining. There was no significant difference between two different electrofusion stimuli in the cleavage rate; $64.9{\pm}4.8%$ in 1.1 kV/cm and $62.7{\pm}4.0%$ in 1.5 kV/cm. However, blastocyst formation rate (%) was significantly different among three different activation groups (no treatment, 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B) combined with electrofusion of 1.1 kV/cm. The blastocyst formation rate was $12.6{\pm}2.5$, $20.0{\pm}5.0$, and $34.9{\pm}4.3%$ in control, 2 mM 6-DMAP, and $7.5{\mu}g/ml$ cytochalasin B, respectively. Immunofluorescence data showed that expression levels of caspase-3 in SCNT embryos undeveloped to blastocyst stage were higher than those in the blastocyst stage embryos. Expression levels of Bcl-2 in blastocyst stage embryos were higher than those in the arrested SCNT embryos. These results showed that the combination of an electric pulse (1.1 kV/cm for $30{\mu}sec$) and $7.5{\mu}g/ml$ cytochalasin B treatment was effective for production of the porcine SCNT embryos.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.22-22
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• 2001

To produce reconstituted rabbit embryos with fetal fibroblasts, the present study was evaluated the efficiencies of the different fusion and activation conditions as assessments of subsequent development and chromosome in the embryos. New Zealand White rabbits were used throughout the study. Fetal fibroblasts collected from 22-d of fetuses were cultured in DMEM ＋ 10% FBS in 5% $CO_2$ in air. The culture was maintained for 10 passages. In every passage half of cell suspension were kept In frozen. From rabbits treated with FSH in 30% PVP solution and hCG, oocytes were surgically collected from oviducts at 14 h post-hCG injection and stripped off their cumulus cells by re-pipetting in a 300 IU hyaluronidase solution. Oocytes with an extruded first polar body and dense cytoplasm were enucleated by micromanipulation in Ham's F-10 medium＋7.5 g/$m\ell$ cytochalasin B. Euncleation was confirmed under a fluorescence microscope after staining with 5 g/$m\ell$ bisbenzimide for 2 min. Each enucleated oocyte was injected with a fetal fibroblast into a perivitelline space. Reconstructed eggs were compared fusion rates either at 2.0 ㎸/cm or 1.6 ㎸/cm(60 sec, double pulses). After fusion, all eggs were activated with the combination of 5 M ionomycin (5 min) and 10 g/$m\ell$ cycloheximide (CHX, 3h), and cultured in CRlaa medium and transferred into TCM199＋10% FBS on day 3. Although there was not significantly differ in fusion rate between treatments (60%, 2.0 ㎸/cm vs. 79.4%, 1.6 ㎸/cm), none of them in the eggs fused with 2.0 ㎸/cm developed to blastocyst. In comparison of development and chromosome status between different activation treatments (Group 1; 5 M ionomycin/10 g/$m\ell$ CHX, Group 2; 5 M ionomycin/5 g/$m\ell$ CHX ＋ 2 mM DMAP after fusion with 1.6 ㎸/cm), there were not differ in cleavage and development rates (67.3% and 28.9% in Group 1; 67% and 33% in Group 2). All out of 8 embryos evaluated in Group 1 appeared a normal diploid chromosome sets and mean number of cells (Mean SEM) on day 4.5 of culture was 141.5 23.15 (n=8). It can be concluded that the use of cycloheximide has not happened in chromosome abnormalities, and fetal fibroblasts can be used for cloning in rabbit.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.169-175
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• 2013

Recently, the transgenic animal production technique is very important for the production of bio-parmaceutical as animal bio-reactor system. However, the absence of survival evaluation in vitro produced transgenic embryos has been a problem of the low productivity of transgenic animal because of absent of pre-estimate of pregnancy after transgenic embryos transferred into recipient. Therefore, this study is conducted to improve efficiency of transgenic cattle production by improving the non-surgical embryo transfer (ET) method. Transgenic bovine embryos were produced by injection of feline immunodeficiency virus enhanced green fluorescent protein (FIV-EGFP) lentiviral vector into perivitelline space of in vitro matured MII stage oocytes, and then in vitro fertilization (IVF) was occured. Normal IVF and EGFP expressing blastocysts were transferred into recipients. Results indicated that 2 expanded blastocysts (34.7%) transferred group showed significantly (P<0.05) higher pregnancy rate than 1 expanded blastocyst (26.8%) transferred group. In case of parity of recipient, ET to heifer (34.9%) showed significantly (P<0.05) higher pregnancy rate than ET to multiparous recipient (21.2%). However, there are no significant differences of pregnancy rate between natural induced estrus and artificial induced estrus groups. Significantly (P<0.05) higher pregnancy rate was obtained from recipient group which have normal corpus luteum with crown group (34.8%) than normal corpus luteum without crown (13.6%). Additionally, treatment of $100{\mu}g$ Gn-RH injection to recipient group (38.6%) 1 day before ET significantly (P<0.05) increase pregnancy rate than non- Gn-RH injection to recipient group (38.6%). We also transferred 2 EGFP expressing expanded blastocysts to each 19 recipients, 7 recipients were pregnant and finally 5 EGFP transgenic cattle were produced under described ET condition. Therefore, our result suggested that transfer of 2 good-quality expanded blastocysts to $100{\mu}g$ of Gn-RH injected recipient which have normal corpus luteum with crown is feasible to produce transgenic cattle.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.185-191
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• 2013

The purpose of this study is to improve production efficiency of vitrified-thawed transgenic bovine embryos. Transgenic bovine embryos were produced by injection of FIV-GFP lentiviral vector into perivitelline space of in vitro matured MII stage oocytes, and then in vitro fertilization. EGFP-expressing transgenic bovine blastocysts were cultured in serum-containing and serum-free medium. These blsatocysts were vitrified by pull and cut (PNC) container made with 0.25 cm plastic straw. Results indicate that total developmental rates of normal IVF embryo cultured in serum-containing and-free medium into blastocyst were not significantly different (22.3 vs 21.5%) and those of GFP-expressing transgenic bovine embryo into blastocyst showed no significant difference between serum-containing (13.9%) and-free medium (13.1%). However, developmental rate of GFP transgenic embryo was significantly (P<0.05) lower than its of normal IVF embryos. In additional study, we vitrified GFP transgenic normal bovine blastocysts using PNC vitrification method. Survival rate of vitrified-thawed GFP transgenic blastocyst (23.1%) was significantly (P<0.05) lower than its of normal blastocysts (68.9%). Although, survival rate of vitrified-thawed GFP transgenic blastocyst was lower than its of normal blastocyst, our result may suggested that PNC vitrification method is feasible to cryopreserve transgenic embryos. Our next plan will be the production of GFP express transgenic bovine derived from vitrified-thawed embryos using PNC method.

• Development and Reproduction
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• v.13 no.4
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• pp.305-312
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• 2009

Multipotent spermatogonial stem cells (mSSCs), derived from uni-potent SSC, are a type of reprogrammed cells with similar characteristics to embryonic stem cells (ESCs). The aim of this study was to evaluate the potential for transgenesis of mSSC derived from outbred mice and the production of transgenic animal by the mSSC-insertion into embryo. mSSCs, established from outbred mice (ICR strain) in the previous study, were maintained and then transfected with a lenti-viral vector expressing green fluorescent protein (GFP), CS-CDF-CG-PRE. Embryonic stem cells (ESCs) were derived from inbred transgenic mice (C57BL/6-Tg (CAG-EGFP)) and were used as an experimental control. Transfected mSSCs were well proliferated in vitro and maintained their characteristics and normal karyotype. Ten to twelve mSSCs and ESCs were collected and inserted into perivitelline space of 8-cell mouse embryos, and then transferred them into uteri of poster mothers after an additional 2-days of culture. Percentage of mSSC-derived offsprings was 4.8% (47/980) and which was lower than those (11.7% (67/572)) of ESC-derived ones (P<0.05). However, even though different genetic background of mSSC and ESC origin, the production efficiency of coat-colored chimeric offspring in mSSC group was not different when compared it with ESC (6.4% (3/47) vs. 7.5% (5/67)). From these results, we confirmed that mSSC derived from outbred mice has a pluripotency and a potential to produce chimeric embryos or mice when reaggregatation with mSSC is performed.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.175-181
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• 2008

This study was conducted to investigate an effective recipient oocyte and culture system for producing of Hanwoo (Korean native cattle) somatic cell nuclear transfer (SCNT) embryos. Hanwoo ear skin fibroblasts were used as donor cells. In vitro matured Hanwoo or Holstein oocytes were enucleated, and single donor cells were transferred into the perivitelline space of the enucleated oocytes. The couplets were subsequently fused and activated. The reconstructed embryos were cultured in a conventional or sequential culture system. In the former, embryos were cultured in CR2aa medium for eight days; in the latter, embryos were cultured in modified CR2aa-A (mCR2-A) for three days and then further cultured in modified CR2aa-B (mCR2-B) for five days. In the experiment with the recipient oocyte, the rate of embryo development to the blastocyst stage was significantly (p<0.05) higher in Hanwoo recipient oocytes than in Holstein ones (48.8% vs 38.9%). BIastocysts derived from Hanwoo recipient oocytes contained significantly (p<0.05) higher numbers of total cells than those derived from Holstein recipient oocytes ($156.0{\pm}68.2$ vs $134.7{\pm}54.8$). There was no difference in the mean proportion of apoptotic cells in blastocysts between the sources of recipient oocytes. In the experiment with the embryo culture system, the blastocyst rate was somewhat higher in sequential system than in conventional system (50.0% vs 43.5%), though there was no significant difference. The numbers of total ($160.0{\pm}69.0$ vs $156.7{\pm}68.4$) and apoptotic cells ($14.0{\pm}10.4$ vs $11.8{\pm}6.4$) were not different between the culture systems. In conclusion, the present study demonstrated that Hanwoo recipient oocytes and the sequential culture system were more effective in supporting the production of Hanwoo SCNT embryos.

• 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.24 no.1
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• pp.59-67
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• 2000

This study was investigated the developmental potential of bovine embryos following nuclear transfer with bovine fetal fibroblasts (BFF). BFF were isolated from a male 45-day-old-fetus. Non-starved BFF labeled with MitoTracker were transferred into perivitelline space of enucleated oocytes. BFF-oocyte units were fused by electric pulse, and then fused oocytes were activated with calcium ionophore A23187 and subsequently 6-dimethylaminopurine (6-DMAP). The resulting zygotes were placed into CRlaa bovine embryo culture medium. Transfer of the nucleus into enucleated oocyte led to premature chromosome condensation, swelling and pronucleus formation. Remodeled oocytes were developed to the mitotic and 2-cell stage at 18 to 26 h after nuclear transfer. The incidence of in vitro development to the blastocyst stages was 21% of fused oocytes. Mitochondria of BFF eliminated rapidly and were not detected at 8 h after fusion. These results suggest that BFF can be successfully reprogrammed in enucleated bovine oocytes, and that reconstructed embryos can develop to the blastocyst stage.