We investigate the effect of L-glutathione (GSH), an antioxidant, treatment during the somatic cell nuclear transfer (SCNT) procedures on the in vitro development and DNA methylation status of bovine SCNT embryos. Bovine in vitro matured (IVM) oocytes were enucleated and electrofused with a donor cell, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine. The recipient oocytes or reconstituted oocytes were treated with 50 μM GSH during these SCNT procedures from enucleation to activation treatment. The SCNT embryos were cultured for 7 days to evaluate the in vitro development, apoptosis and DNA methylation in blastocysts. The apoptosis was measured by TUNEL assay and caspase-3 activity assay. Methylated DNA of SCNT embryos at the blastocyst stages was detected using a 5-methylcytidine (5-MeC) antibody. The developmental rate to the blastocyst stage was significantly higher (P<0.05) in GSH treatment group (32.5±1.2%, 78/235) than that of non-treated control SCNT embryos (22.3±1.8%, 50/224). TUNEL assay revealed that the numbers of apoptotic cells in GSH treatment group (2.3±0.4%) were significantly lower (P<0.05) than that of control (3.8±0.6%). Relative caspase-3 activity of GSH treated group was 0.8±0.06 fold compared to that of control. DNA methylation status of blastocysts in GSH treatment group (13.1±0.5, pixels/ embryo) was significantly lower (P<0.05) than that of control (17.4±0.9, pixels/embryo). These results suggest that antioxidant GSH treatment during SCNT procedures can improve the embryonic development and reduce the apoptosis and DNA methylation level of bovine SCNT embryos, which may enhance the nuclear reprogramming of bovine SCNT embryos.

The present study was conducted to examine the effect of antioxidant treatment during parthenogenetic activation procedure on the reactive oxygen species (ROS) levels and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by a combination of electric stimulus and 2 mM 6- dimethylaminopurine (6-DAMP) before in vitro culture. During the activation period, oocytes were treated with 50 μM β-mercaptoethanol (β-ME), 100 μM L-ascorbic acid (Vit. C) or 100 μM L-glutathione (GSH). To examine the ROS level, porcine parthenogenetic embryos were stained in 10 μM dichlorohydrofluorescein diacetate (H2DCFDA) dye 20 h after culture, examined under a fluorescence microscope, and the fluorescence intensity (pixels) were analyzed in each embryo. The parthenogenetic embryos were cultured for 6 days to evaluate the in vitro development. The apoptosis was measured by TUNEL assay. The H2O2 levels of parthenogenetic embryos were significantly lower in antioxidant treatment groups (26.9±1.6～29.1±1.3 pixels/embryo, p<0.05) compared to control (33.2±1.7 pixels/embryo). The development rate to the blastocyst stage was increased in antioxidant treatment groups (32.0～32.5%) compared to control (26.9%, p<0.05), although, there was no difference in apoptosis among groups. The result suggests that antioxidant treatment during parthenogenetic activation procedure can inhibit the ROS generation and enhance the in vitro development of porcine parthenogenetic embryos.

This study was conducted to examine the optimal concentration and treatment time of antioxidants for inhibition of the ROS generation in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine oocytes were activated parthenogenetically, during which oocytes were treated with various antioxidants to determine the optimal concentrations and kind of antioxidants. Determined antioxidants were applied to oocytes during in vitro maturation (IVM) and/or SCNT procedures. Finally, antioxidant-treated SCNT embryos were compared with in vitro fertilized (IVF) embryos. H2O2 levels were analyzed in embryos at 20 h of activation, fusion or insemination by staining of embryos in 10 μM 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA) dye, followed by fluorescence microscopy. H2O2 levels of parthenogenetic embryos were significantly lower in 25 μM β- mercaptoethanol (β-ME), 50 μM L-ascorbic acid (Vit. C), and 50 μM L-glutathione (GSH) treatment groups than each control group (24.0±1.5 vs 39.0±1.1, 29.7±1.0 vs 37.0±1.2, and 32.9±0.8 vs 36.3±0.8 pixels/embryo, p<0.05). There were no differences among above concentration of antioxidants in direct comparison (33.6±0.9～35.2±1.1 pixels/embryo). Thus, an antioxidant of 50 μM Vit. C was selected for SCNT. H2O2 levels of bovine SCNT embryos were significantly lower in embryos treated with Vit. C during only SCNT procedure (26.4±1.1 pixels/embryo, p<0.05) than the treatment group during IVM (29.9±1.1 pixels/embryo) and non-treated control (34.3±1.0 pixels/embryo). Moreover, H2O2 level of SCNT embryos treated with Vit. C during SCNT procedure was similar to that of IVF embryos. These results suggest that the antioxidant treatment during SCNT procedures can reduce the ROS generation level of SCNT bovine embryos.

The present study was conducted to examine the generation of reactive oxygen species (ROS) during micromanipulation procedures in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine enucleated oocytes were electrofused with donor cells, activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. Oocytes and embryos were stained in dichlorodihydrofluorescein diacetate or 3'-(p-hydroxyphenyl) fluorescein dye and the H2O2 or ˙OH radical levels were measured. In vitro fertilization (IVF) was performed for controls. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each oocyte and embryo. The H2O2 and ˙OH radical levels of reconstituted oocytes were increased during manipulation (37.2~49.7 and 51.0~55.2 pixels, respectively) as compared to those of mature oocytes (p<0.05). During early in vitro culture, the ROS levels of SCNT embryos were significantly higher than those of IVF embryos (p<0.05). These results suggest that the cellular stress during micromanipulation procedures can generate the ROS in bovine SCNT embryos.

The present study was conducted to examine the reactive oxygen species (ROS) generation levels and subsequent DNA damage in the bovine cultured somatic cells. Bovine ear skin cells were classified by serum starvation, confluence and cycling cells. Cells were stained in 10 μM dichlorohydrofluorescein diacetate (H2DCFDA) or 10 μM hydroxyphenyl fluorescein (HPF) dye to measure the H2O2 or ˙OH radical levels. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each cell. H2O2 and ˙OH radical levels of cultured somatic cells were high in confluence group (7.1±0.7 and 8.4±0.4 pixels/cell, respectively) and significantly low in serum starvation group (4.9±0.4 and 7.0±0.4 pixels/cell, respectively, p<0.05). Comet tail lengths of serum starvation (148.3±5.7 μm) and confluence (151.1±5.0 μm) groups were found to be significantly (p<0.05) increased in comparison to that of cycling group (137.1±7.5 μm). These results suggest that the culture type of donor cells can affect the ROS generation, which leads the DNA fragmentation of the cells.

The present study was conducted to examine the reactive oxygen species (ROS) generation levels in porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by the combination of electric stimulus and 6‐ DMAP before in vitro culture. Porcine oocytes and parthenogenetic embryos were stained in 10 μM dichlorohydrofluorescein diacetate (DCF) or 10 μM hydroxyphenyl fluorescein (HPF) dye each for 30 min at 39℃. The fluorescent emissions from the samples were recoded as JPEG file and the intensity of fluorescence in oocytes and embryos were analyzed. H2O2 and ˙OH radical levels of porcine oocytes were reduced immediately after electric stimulation. However, H2O2 and ˙OH radical levels of parthenogenetic embryos were increased with time elapsed after electric stimulation from 0 h to 3 h and after DMAP culture. During in vitro culture, H2O2 and ˙OH radical levels were gradually increased from the one‐cell stage to the two‐ and four‐cell stages. The result of the present study suggests that the ROS was not increased by electric pulse in porcine embryos. Rather than it seems to be associated with the stage of development and the culture condition.

We attempted to control the maturation promoting factor (MPF) activity and investigated the subsequent reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos. Serum‐starved adult skin fibroblasts were fused to enucleated oocytes treated with 2.5 mM caffeine or 150 μM roscovitine. The MPF activity, nuclear remodeling patterns, chromosome constitutions and development of SCNT embryos were evaluated. Methylated DNA of embryos was detected at various developmental stages. The MPF activity was increased by caffeine treatment or reduced by roscovitine treatment (p<0.05). Blastocyst development was higher in the caffeine‐treated groups (27.6%) than that of the roscovitine‐treated group (8.3%, p<0.05). There was no difference in the apoptotic cell index among the three groups. However, the mean cell number of blastocysts was increased in the caffeine‐treated group (p<0.05). Higher methylation levels were observed in the Day 3 embryos of the roscovitine‐treated group (50.8%), whereas lower methylation levels were noted at Day 5 in the caffeine‐treated group (12.5%, p<0.05). These results reveal that the increase in MPF activity via a caffeine‐treatment creates a more suitable condition for nuclear reprogramming after SCNT.

The aim of this study was to examine the microtubule distributions of somatic cell nuclear transfer (SCNT) and parthenogenetic porcine embryos. Porcine SCNT embryos were produced by fusion of serum-starved fetal fibroblast cells with enucleated oocytes. Reconstituted and mature oocytes were activated by electric pulses combined with 6-dimethlyaminopurine treatment. SCNT and parthenogenetic embryos were cultured in vitro for 6 days. Microtubule assembly of embryos was examined by confocal microscopy 1 hr and 20 hr after fusion or activation, respectively. The proportions of embryos developed to the blastocyst stage were 25.7% and 30.4% in SCNT and parthenogenetic embryos, respectively. The frequency of embryos showing β-tubulins was 81.8% in parthenogenetic embryos, whereas 31.3% in SCNT embryos 1 hr after activation or fusion. The frequency of the embryos underwent normal mitotic phase was low in SCNT embryos (40.6%) compared to that of parthenogenetic ones (59.7%) 20 hr after fusion or activation (p<0.05). The rate of SCNT embryos with an abnormal mitosis pattern is about twice compared to that of parthenogenetic ones. The spindle assembly and its distribution of SCNT embryos in the first mitotic phase were not different from those of parthenogenetic ones. The result shows that although microtubule distribution of porcine SCNT embryos shortly after fusion is different from parthenogenetic embryos, and the frequency of abnormal mitosis 20 hr after fusion or activation is slightly increased in SCNT embryos, microtubule distributions at the first mitotic phase are similar in both SCNT and parthenogenetic embryos.

This study was performed to confirm the microtubule assemblies and methylation patterns of porcine IVF and parthenogenetic embryos. Cumulus-oocyte complexes were collected and matured in vitro for 42 hr. Oocytes were fertilized by prepared fresh sperm or activated parthenogenetically by exposure to electric stimulation and 6-dimethylaminopurine. Porcine IVF and parthenogenetic embryos were cultured in vitro for 6 days. Embryos were stained by immunofluorescence staining method to observe the dynamic of nucleus and microtubules in the first mitotic phase and the methylation patterns in different developmental stages. After then, samples were confirmed and analyzed through a laser-scanning confocal microscope. IVF embryos had a centrosome originated from sperms, which was shown a ɤ-tubulin spot. However, ɤ-tubulin spot was not observed in parthenogenetic embryos. A lower methylation level was observed in IVF embryos compared to parthenogenetic ones at the morula and blastocyst stages. In conclusion, it is considered that microtubule assemblies and genetic regulation mechanism differ between parthenogenetic and IVF embryos.

This study was conducted to investigate the development and gene expression in miniature pig nuclear transfer (mNT) embryos produced under different osmolarity culture conditions. Control group of mNT embryos was cultured in PZM-3 for 6 days. Treatment group of mNT embryos was cultured in modified PZM-3 with NaCl (mPZM-3, 320 mOsmol) for 2 days, and then cultured in PZM-3 (270 mOsmol) for 4 days. Blastocyst formation rate of the treatment group was significantly higher than the control and the apoptosis rate was significantly lower in treatment group. Bax- and caspase-3 mRNA expression were significantly higher in the control than the treatment group. Also, the majority of imprinting genes were expressed aberrantly in in vitro produced mNT blastocysts compared to in vivo derived blastocyst H19 and Xist mRNA expression were significantly lower in the control than the treatment group or in vivo. IGF2 mRNA expression was significantly higher in the control than the treatment group or in vivo. IGF2r mRNA expression was significantly lower in the control. Methylation profiles of individual DNA strands in H19 upstream T-DMR sequences showed a similar methylation status between treatment group and in vivo. These results indicate that the modification of osmolarity in culture medium at early culture stage could provide more beneficial culture environments for mNT embryos.

This study was conducted to examine the effect of several saccharides on the induction of capacitation and acrosome reaction (AR) and to examine the effects of mono and polysaccharides on the penetration activity of boar spermatozoa. Spermatozoa were inseminated in medium with fucose, galactose and mannose as monosaccharide, and fucoidan, galactan and mannan as polysaccharide. The penetration rates were significantly (p<0.05) lower in medium with galactose (40.6%), mannose (38.1%), fucose (41.6%) and fucoidan (36.6%) compared with control (56.7%). The rates of AR were increased (40.7 to 59.8%) by the preincubation periods prolonged from 0 to 4 hr (p<0.05). Similar tendencies were observed in AR when spermatozoa were treated with monosaccharides, but not significantly differ among the groups treated with different time of preincubation with some exception of galactose. When spermatozoa were treated with polysaccharides, the rates of AR were significantly (p<0.05) increased by preincubation time prolonged from 0 to 4 hr with an exception of fucoidan. In conclusion, the present study suggests that penetration rate of spermatozoa is higher in presence of polysaccharides than monosaccharides. Also, it may resume that the comparing to control, the all saccharides (L-fucose, D-galactose, D-mannose, fucoidan, galactan and mannan)-treated groups slightly increase the AR pattern as preincubation time prolonged.

In vitro development of porcine embryo is affected by culture condition. One possible factor is osmolarity of culture medium. This study examined whether high osmolarity of culture medium at the early culture stage improves development of preimplantation porcine in vitro fertilization (IVF) and nuclear transfer (NT) embryos. NT and IVF embryos were divided into three groups and the basic medium was PZM-3 (250～270 mOsmol, control group). The control group of embryos was cultured in PZM-3 for whole culture period. Other two groups of embryos were cultured in a modified PZM-3 with 0.05 M sorbitol or 0.05 M sucrose (300～320 mOsmol, sorbitol or sucrose group) for the first 2 days, and then cultured in PZM-3 for further culture. NT embryos cultured in sucrose group showed a significantly higher developmental rate to the blastocyst stage with a decreased apoptosis rate compared to the sorbitol (p<0.05). For IVF, sucrose group showed a significantly increased the blastocyst formation rate with a decreased apoptosis rate compared to the control (p<0.05). This study represents that the high osmolarity in the early embryo culture stage can enhance the in vitro development of porcine NT and IVF embryos to the blastocyst stage with reduced apoptosis of cells.

본 연구는 물리적 탈핵 방법인 aspiration 방법과 squeezing 방법을 사용해 핵이식된 소 체세포 복제란의 발달능을 조사하였다. 두 방법에 의해 탈핵된 수핵란에 한우 귀피부 세포를 융합하여 체외에서 7～8일간 배양하여 발육능을 검사하였다. 배양 7일째 배반포 발달율은 aspiration 방법 (31.9±13.4%)과 squeezing 방법 (33.6±15.7%) 간에 차이가 없었다. 또한 배양 8일째 배반포 발달율도 squeezing 방법 (35.3±15.1%)과 aspiration 방법 (37.8±10.4%) 간에 차이가 없는 것으로 나타났다. 배양 7일째 배반포의 평균 세포수에 있어 각 처리 간에 차이가 없는 것으로 조사되었으며 (aspiration: 110.3±39.2, squeezing: 103.7±42.8), 세포자연사의 비율에 있어서도 역시 유의적인 차이가 없는 것으로 조사되었다 (aspiration: 2.8±2.6 %, squeezing: 4.3±4.4%). 본 연구의 결과는 물리적 탈핵 방법인 aspiration 방법과 squeezing 방법 모두 소 체세포 복제수정란의 생산을 위한 유용한 방법임을 보여준다.

본 연구는 활성화처리 방법 및 배양 조건이 돼지 단위발생란의 체외발달 및 apoptosis에 미치는 영향을 알아보기 위해 실시되었다. 도축장 유래 난소로부터 채취된 미성숙 난자를 42~44시간 동안 성숙배양한 후 사용하였다. Apoptosis는 TUNEL 방법을 사용하여 조사하였다. 실험 1에서는 성숙배양된 난자들을 electric pulse(1.2 kV/cm for 30μsec 2회, E), E + 6-dimethylaminopurine(6-DMAP) 또는 E + cycloheximide(CH) 방법으로 활성화 처리하여 PZM-3를 이용하여 5% CO2, 38.5℃에서 배양하였다. 실험 2에서는 전기자극을 이용하여 활성화처리된 난자들을 각각 PZM-3 또는 NCSU-23 배양액 내에서 배양하였다. 각 배양액 내의 난자들은 각각 20% O2 조건으로 나뉘어 배양하였다. E + 6-DMAP(36.5%) 또는 E + CH 구(32.5%)에서 E 구(27.7%)보다 유의적으로 높은 배반포 형성율을 보였다(P<0.05). 처리별 apoptosis 발생율은 각각 5.3%(E), 7.7%(6-DMAP) 및 7.1%(CH)였다. 실험 2에서는 PZM-3 구의 배반포 형성율이 NCSU-23 구에 비하여 산소분압조건과 관계없이 다소 높았다(28.2{sim}29.7% vs. 22.6~24.4%). PZM-3 및 20% O2 조건하에서 유의적으로 낮은 apoptosis 발생 비율을 나타냈다(9.2%, P<0.05). 그러므로 돼지 단위발생란을 chemical agent를 이용한 추가 활성화처리 후 PZM-3, 20% O2, 조건으로 배양하면 더 나은 배반포 발생율을 얻을 수 있다고 생각된다.

핵이식 방법을 이용하여 성공적인 복제를 이루기 위해서 인위적인 활성화 처리는 필수적인 요소이다. 본 연구는 전기자극에 의해 활성화된 난자를 chemical agent를 이용하여 추가적인 활성화 처리를 하였을 때 돼지 단위발생란의 발달에 미치는 영향을 알아보고자 수행되었다. 체외에서 40~44시간 동안 배양된 난자를 전기자극(E)으로 활성화 처리한 후 Thimerasol + Dithiothreitol(Thi+DTT), 6-Dimethylaminopurine(6-DMAP) 및 Cycloheximide(CH)를 사용하여 추가 활성화 처리를 하였다. 활성화 방법(E, E+Thi+DTT, E+6-DMAP 및 E+CH)에 따른 단위발생란의 배반포까지의 발달율을 조사한 결과, chemical agent에 의해 추가 활성화된 단위발생란이 전기자극만으로 처리된 구의 단위발생란보다 유의적으로 높은 발달율을 보였다(21.5~28.1% vs. 18.0%, P<0.05). 특히, E+Thi+DTT를 이용하였을 때 발달율이 유의적으로 높게 나타났다(28.1%, P<0.05). 활성화 처리별 전핵 형성율을 조사한 결과, chemical agent에 의해 추가 활성화 처리된 구에서 하나의 극체(1PN) 형성률은 처리별로 차이를 보이지 않았으나(59.9~64.7%), 2PN 형성율은 추가 활성화 처리구에서 전기자극만을 사용하였을 때보다 유의적으로 높게 나타났다(7.2~9.7% vs. 4.3%, P<0.05). 이상의 결과를 살펴볼 때, 전기자극 후 chemical agent를 이용한 추가 활성화는 단위발생란의 배반포까지의 발달능력을 증가시키는 것으로 생각된다.