Phenylalanine ammonia-lyase (PAL)는 L-phenylalanine을 trans-cinnamic acid 와 ammonia로 변환시키는 효소로 식물에서 조직의 분화, 발달, 생장, 색소 등의 이차대사물질인 phenylpropanoids의 생합성 경로에 관여하는 것 으로 알려져 있다. 버섯에서도 이 효소가 존재함이 알려져 있지만 아직 그 기능과 역할이 잘 알려져 있지 않 다. 또한 자실체 발달과 관련하여 이 효소의 활성과 유전자 발현에 대한 정보도 매우 부재한 실정이다. 식용 버섯인 양송이 (Agaricus bisporus)에는 갓의 색이 갈색인 품종과 백색인 품종이 존재한다. 이에 따라 본 연구 에서는 국내에서 육종된 색이 다른 두 양송이 품종인 갈색의 ‘호감’ 품종과 백색의 ‘새한’ 품종을 대상으로 PAL 효소와 그 유전자가 자실체 발달 단계에서 발현되는 양상을 조사하였다. PAL 유전자 발현은 real-time RT-PCR 방법으로 조사하였고 PAL 효소 활성은 L-phenylalanine을 기질로 하여 분광광도계 방법으로 측정하 였다. PAL 유전자는 갈색의 ‘호감’ 품종은 자실체가 발달함에 따라 갓에서의 PAL1 과 PAL2 gene 모두 발현 이 감소하였다. 갓의 표피만을 대상으로 PAL 발현을 조사하였을 때 PAL1 과 PAL2 gene 모두 발현이 높았 다. PAL 효소 활성 측정 결과, 갈색과 백색 품종 모두 자실체의 발달이 진행됨에 따라 갓에서의 PAL 효소 활성이 감소하였다. PAL 효소 활성은 갓 표피에서 높게 측정되었다. 이러한 결과는 PAL 효소 활성과 PAL 유전자 발현 양상이 일치함을 보여주었다. 흥미롭게도 갈색품종과 백색품종 간 PAL 활성을 비교하였을 때 PAL 활성은 백색품종에 비해 갈색품종에서 조사된 모든 자실체 발달 단계에서 높았다.

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. However, there was no standard criterion to measure the oxygen consumption of embryos. Here, we measured oxygen consumption of bovine embryos at various developmental stages was measured using a scanning electrochemical microscopy (SECM). We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell-stage to morula-stage), indicating that oxygen consumption reflects the cell number ( versus , p<0.05). In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos ( versus , p<0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro-derived bovine blastocyst-stage embryos (p>0.05). In the frozen-thawed blastocyst-stage embryos, live embryos showed significantly higher oxygen consumption than dead embryos ( versus , p<0.05). These results indicate that the measuring oxygen consumption by SECM can be used to evaluate bovine embryo quality.

In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro fertilization (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22h post insemination were treated at different concentration of actinomycin D (0, 5, 50 and 500 ng/ml in NCSU medium). Treated embryos were incubated at in 5% , 5% for 8h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2days and BL development rate at 7days after in vitro culture. A significantly lower rate of cleavage was found in the 500 ng/ml group compared to others (500 ng/ml vs. 0, 5, 50 ng/ml; 27.8 % vs. 50.0%, 41.2%, 35.9%), and BL formation rate in 500 ng/ml was lower than that of others (500 ng/ml vs. 0, 5, 50 ng/ml; 8.0% vs. 12.6%, 11.2%, 12.6%). In experiment 2, to evaluate apoptotic cells, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng/ml actinomycin D). A high number of BL derived control group contained at least one apoptotic cell. Actinomycin D treated BLs responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and increase in the incidence of apoptotic cell death. In 500 ng/ml group, the incidence of apoptosis increased at 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is useful tool for the apoptosis study of porcine preimplantation embryos.

본 연구는 OPS 기법에 의한 돼지 수정란의 동결-융해 시 수정란의 발달 단계와 superoxide dismutase (SOD)가 수정란의 생존능력에 미치는 영향을 검토하였다. 돼지 체외수정 배반포는 OPS방법에 의해 동결 후 융해하여 0~10unitsml의 SOD 존재 하에 48시간 체외배양하였다. 동결-융해 후 형태학적으로 정상적인 수정란의 비율은 초기, 중기 및 확장배반포간에 유의적인 차이는 인정되지 않았다(30.8~38.6%). 그러나 발육단계가 높을수록 형태적으로 정상인 수정란의 비율이 높은 경향을 나타냈다. 수정란의 융해 후 48시간 추가 배양했을 때, 발육이 진행된 수정란은 후기배반포기에 동결한 수정란이 38.7%로 유의적으로 높았으며(P<0.05), 1 unit/ml의 SOD를 첨가한 경우 비교적 높은 생존율을 나타내었다. 본 연구의 결과로부터, 수정란의 OPS방법에 의한 동결-융해 후 생존성의 향상을 위해서는 후기배반포기 단계에 동결하는 것이 유리하며, SOD의 첨가는 수정란의 손상을 어느 정도 방지할 수 있을 것으로 사료된다.

The present experirnents on cryopreservation were carried out to investigate effect of solution toxicity, equilibration time and cell stages on the post-thaw survival of mouse morulae and blastocyst embryos cryopreserved by vitrification in EFS solution. The mouse embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen and thawed rapidly. After the mouse morulae embryos were exposed to EFS solution for 2 and 5 ruin. at room temperature and then they were washed in 0.5 M sucrose solution and basal mediurn(D-PBS + 10% FCS), they were cultured to examined cryoprotectant toxicity induced injury during exposure, most of embryos developed to expanded blastocysts(100 and 90.0%). However, when the exposure time was extended to 10 and 20 min, these development rates dropped dramatically in 10 ruin. (75.0%) and 20 ruin. (4.5%), respectively. When the compacted morulae were vitrified in EFS solution after equilibration for 2 and 5 min, the embryos have developed to normal blastocyst following thawing, washing and culture processes was 89.3 and 89.6%. However, when the exposure time was expanded to 10 ruin, this survival rate dropped to 68.8%. When the blastocyst were vitrified in EFS solution after equilibration for 2, 5 and 10 minutes, the survival rate of embryos which developed to normal blastocyst following thawing and culture processing were 58.5, 46.7 and 22.4%, respectively. The optimal time of equilibration of mouse morula and blastocysts in EFS solution seemed o be 2 and 5 ruin.

This study evaluated the influence of cell stage of donor nucleus on nuclear injection, electrofusion and in vitro development in the rabbit to improve the efficiency of nuclear transplantation in the rabbit. The embryos of 8-, 16- and 32-cell stage were collected from the mated does by flushing viducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FGS) at 44, 54 and 60 hours after hCG injection. The blastorneres separated from these embryos were used as donor nucleus. The ovulated oocytes collected at 14 hours after hCG injection were used as recipient cytoplasm following removing the nucleus and the first polar body. The separated blastomeres were injected into the enucleated oocytes by micromanipulation and were electrofused in 0.28 M mannitol solution at 1.5 kV /cm, 60 sec for three times. The fused oocytes were cocultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FGS for 72~120 hours at 39 in a 5% incubator. The cultured nuclear transplant embryos were stained with Hoechst 33342 solution and the number of cells were counted by fluorescence microscopy. The successful injection rate of 8-, 16- and 32-cell-stageblastomeres into enucleated oocytes was 86.7, 91.0 and 93.9%, respectively. The electrofusion rate of 8-, 16- and 32-cell-stage blastomeres with enucleated oocytes was 93.3,89.3 and 79.0%, respectively. Development of blastomeres to blastocyst was similar with 8-,16- and 32-cell-stage donor nuclei(26.2, 25.8 and 26.6%, respectively, P<0.05). The mean number of cell cycle per day during in vitro culture in nuclear transplant embryos which received 8-, 16- and 32-cell- stage nuclei was 1.87, 1.81 and 1.43, respectively.

The present experiments on cryopreservation were designed to examine the effects of solution toxicity, equilibration time and cell stages on the post-thaw survival of bovine IVF embryos. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with 35 g /ml FSH, 10 g /ml LH, 1 g /ml estradiol-17 and granulosa cells at 39 under 5% in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro(IVC) with bovine oviductal epithelial cells for 7 to 9 days. The bovine IVF embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen, and thawed rapidly. 1. after the bovine blastocysts were exposed to EFS solution for 2 min. at room temperature and then they were washed in 0.5 M sucrose solution and TCM-199, they were cultured to examined cryoprotectant induced injury during exposure, Most of the embryos(95.0%) developed to reexpanded blastocoels. However, when the exposure time was extended to 5 and 10 min, these development rates dropped dramatically in 5 min. (69.5%) and 10 min. (47.4%), respectively, 2. When the bovine IVF embryos were vitrified in EFS solution after the equilibration for 1 and 2 min. exposure, The embryos to have reexpanded blastocoels following thawing, washing and culture processes were found to he 82.6 and 73.9%, respectively. However, when the exposure time was extended to 3 min, this survival rate dropped to 18.2%. The optimal time for equilibration of bovine IVF blastocysts in EFS solution seemed to he 1~2 min. 3. When the bovine IVF embryos were equilibrated for 1 min. the significantly (P<0. 05) higher post-thaw survival rates were obtained from the embryos of blastocyst stage(81.3%) than morulae stage(5. 1%). The optimal cell stage for viterification with EFS solution proven to he blastocyst stage in bovine IVF embryos. 4. The number of blastomeres of blastocyst stage was examined with nuclear staining with Hoechst 33342 during 7 to 9 days post-insemination. The cell counts of frozen bovine IVF embryos were found significantly(P7.5 and those of the fresh embryos 76.67. 1, which were cultured in the sarne period and conditions as frozen embryos.

The post-thaw survival of mouse embryos of the various developmental stages was determined after cryopreservation by vitrification in a solution containing ethylene glycol, Ficoll and sucrose (EFS). All the embryos were equilibrated for 2 minutes just prior to freezing. The number of blastomeres during in vitro development was counted by nuclei higher rates of post-thaw survival were obtained from the embryos of 2-cell(92.2%), 8-cell(77.2%) or morula stage(90.0%) than those of blastocyst stage(62.7%). The number of blastomeres per embryo following in vitro culture for 24 hours was significantly(P<0.05) smaller as 66.0f22.3 in vitrified and thawed morulae than fresh morulae(91.712.2).

브로콜리 74 계통 화기 구조를 통한 소포자 배 발생을 살펴본 결과 18개 계통에서 소포자 유래 배 발생이 확인되었다. 특히 08-8-8 계통과 08-8-33 계통에서 petri dish 당 20개 정도의 소포자 유래 배 발생이 확인되었다. 높은 소포자 유래 배 발생 계통인 08-8-8 계통과 소포자 유래 배 발생이 되지 않는 08-8-10 계통을 대상으로 화뢰의 크기에 따른 소포자 발달 단계 및 밀도를 조사하였다. 소포자 유래 배 발생 효율이 높은 08-8-8 계통과 소포자 유래 배 발생 효율이 낮은 08-8-10 계통 사이에 화뢰의 구조적 차이는 나타나지 않았다. 그러나 화뢰의 크기가 M 사이즈인 경우에만 주두의 길이가 화엽의 길이보다 더 길었다. 화뢰의 크기와 소포자 수와는 정의 상관을 나타내었으나 S 사이즈의 화뢰인 경우에는 소포자를 관찰할 수 없었다. 화뢰 당 소포자 밀도는 08-8-10 계통보다 08-8-8 계통에서 높게 나타났으며, 화뢰 M 사이즈의 1핵성 후기 소포자 비율은 08-8-8 계통에서는 91.3%, 08-8-10 계통에서는 45.7%로 나타났다.