해산송사리, Oryzias dancena 2배체와 유도 3배체 각 조직에서의 세포주기를 비교, 분석하였다. G1, S, G2+M기의 분포 빈도에서 꼬리지느러미인 경우 2배체에서는 85.8%, 7.6% 및 6.9%이었으며 유도 3배체에서는 91.2%, 3.6% 및 5.2%이었다. 간 조직인 경우 2배체에서는 78.4%, 10.6%, 및 11.0%이었으며 유도 3배체는 86.2%, 5.9% 및 7.9%이었다. 아가미 조직인 경우 2배체에서는 79.3%, 9.4% 및 11.3%이었으며 유도 3배체는 85.7%, 5.4% 및 8.9%이었다. 2배체와 유도 3배체에서 조직 간 세포 주기 빈도에서 유의한 차이가 있었다 (p<0.05). Mitosis (체세포분열)은 유도 3배체에 비해 2배체가 더욱 활성이 있었으며, 이러한 체세포분열은 2배체와 유도 3배체 모두에서 꼬리지느러미 조직 보다는 간 조직과 아가미 조직에서 더욱 왕성하였다.

The aim of this study was to compare the appearance rate of vaginal cytology during estrous cycle in small pet bitches. A characteristic features of vaginal cytology during the estrous cycle were the high proportion of superficial cell in proestrus, anuclear cell in estrus, small intermediate cell in diestrus, and small intermediate cell in anestrus, respectively. There were no statistically significant differences of appearance rate of vaginal cytology among small pet bitches in the each phase of estrous cycle. These results indicated that the vaginal cytology was useful method for estimating estrous cycle and optimal breeding time in small pet bitches.

The aim of this study was to evaluate the changes of protein patterns in granulosa cells and corpus luteum in ovaries during the estrus cycle in cows. The estrus cycle was devided into five steps of follicular, ovulatory, early-luteal, mid-luteal and late-luteal phases. In results, 61 spots of total 85 spots were repeated on follicular phase and 51 spots of total 114 spots were repeated on ovulatory phase. The 40 spots of total 129 spots were repeated on early-luteal phase and 49 spots of total 104 spots were repeated on mid-luteal phase. Also 41 spots of total 60 spots were repeated on late-luteal phase. On the other hands, the 16 spots were indicated difference in follicular phase and ovulation phase had a difference 10 spots. It was showed difference No. 103 spot in ovulation phase, No. 135 spot in early-luteal phase and No. 175 and 176 spots in mid-luteal phase. Also, the 11 spots were expressed specifically in mid-luteal phase and No. 178 and 179 spots were difference of expression in late-luteal phase. We confirmed that there were 7 spots for ovulation, 4 spots for luteinization and 2 spots for luteolysis. Spot No. 89～93 in ovulation phase were transferrin, and spot No.94～98 were HSP60. Spot No. 103 was Dusty PK, spot No. 135 was OGDC- E2, and spot No. 175 and 176 were Rab GDI beta from luteinization. Spot No. 178 and 179 in luteolysis were vimentin. This results suggest that will be help to basic data about infertility.

The trace element nutrient selenium discharges its well-known nutritional anti-tumor activity. Converging data from epidemiological, ecological and clinical studies have shown that selenium can decrease the risk for some types of human cancers, especially those of the prostate, lung, and colon. Mechanistic studies have indicated that selenium has many desirable attributes of chemoprevention targeting cancer cells through DNA single strand breaks, the induction of reactive oxygen species. However, there is no reports about the relationship between methylseleninic acid (MSeA), one of methylselenol metabolites and cell cycle arrest in LNCaP human prostate cancer cells. Our data showed that MSeA arrested G1/S pahse of cell cycle arrest and inhibited DNA synthesis in LNCaP cells and those cellular events by MSeA were due to the induction of p27 protein which is a well-known cyclin-dependent kinase inhibitor. Taken together, cell cycle arrest occurred by MSeA may contribute to the growth-inhibition of prostate cancer cells.

Vaginal cytology with behavioral observation was performed in 34 estrous cycles in 16 Miniature Schnauzer dogs to evaluate the its usefulness. The mean duration of proestrus and estrus in Miniature Schnauzer based on behavioral observation and vaginal cytology was (Mean S.D., range: ) and days for proestrus, and and days for estrus, respectively. The duration of each phase of the estrous cycle was not significantly different based on between behavioral observation and vaginal cytology. The gestational length from the first day of male acceptance was days, days from the first male refusal, and days from the onset of cytologic diestrus, respectively. Vaginal cytology during the estrous cycle were significantly characteristic of large intermediate cells in proestrus, anuclear cells in estrus, small intermediate cells in diestrus, and parabasal cells and small intermediate cells in anestrus (p<0.001), respectively. Cornification index (CI) by vaginal cytology was higher in proestrus () and estrus (), then it was decreased in diestrus () and anestrus ().

Shit-tzu견에서 발정 주기, 교배 적기 및 배란 시기를 정확하게 추정하기 위한 질 세포 검사를 확립하기 위하여 Shih-tzu 견 12두를 대상으로 발정 주기동안 질 세포 검사 및 estradiol-와 progesterone 농도를 측정하여 다음과 같은 결과를 얻었다. 발정주기별 질 세포상의 특징적인 변화로서 발정 전기에는 무핵 세포와 적혈구, 발정기에는 표층 세포와 무핵 세포 및 적혈구가 주종을 이루는 세포였으며 발정 휴지기에는 소형 중간 세포와

비글개 6두에서 11회(임신견 7두, 비 임신견 4두)의 발정 주기 및 임신 기간 동안 질 상피 세포 검사 및 혈장 progesterone과 estradiol- 농도를 측정하여 질 상피 세포상과 번식 호르몬의 관계를 조사하고 배란 및 교배 적기 판정을 위한 기초 자료를 제공하고자 본 실험을 실시하였다. 임신 예와 비 임신 예에 있어서 발정 전기, 발정기 및 발정 휴지기의 기간은 각각 및 그리고 및 일이었다. 임선 예에 비해 비 임신견의 발정 휴지기가 길

It has been reported that light-emitting diodes(LED) can be used in the treatment of oral diseases. Although bio-stimulatory effects of LED irradiation such as promotes stimulation of wound healing have been well known, there are few reports about molecular mechanism associated with cell cycle by LED irradiation. The purpose of present study was to examine the molecular event in cell cycle of LED irradiation on primary human gingival fibroblast(hGF) in vitro. The source of light for irradiation was a continuous-wave LED emitting at a wavelength of 635nm, and manufactured that energy density was 5mW/cm2 on sample surface. The hGF were irradiated for 1 hour at 37℃ in 5% CO2 humidified chamber. Experimental samples were acquired at 0 (right after irradiation), 8 and 24 hour after irradiation. To investigate the molecular mechanisms associated with cell cycle, growth phase was determined by flow cytometry and mRNA expression of cyclin A, cyclin B, cyclin D1, cyclin E, cdc2, PCNA, p18, p27, p21, and p53 were determined by real time RT-PCR. Flow cytometric analysis demonstrated the percentage of cells in the G1 and S phase were decreased, but the G2 phase increased, which showed cells irradiated by LED were transitioned from S to G2 phase. For mRNA expression, cyclin B, cdc2, PCNA and p53 were increased at 0 hour after irradiation, and most of cell cycle molecules were increased at 8 hour after irradiation. At 24 hour after irradiation, cyclin A, cyclin E, PCNA and p18 were increased. Taken together, LED irradiation induced proliferation of hGF cells through transition from S to G2 phase.

본 연구는 demecolcine 처리에 의한 탈핵과 수핵란 세포질의 세포 주기가 소 체세포 핵이식란의 발육에 미치는 영향을 검토하였다. 체외에서 16~20시간 성숙배양된 난자를 극체 방출 유무 및 MI, MII기 난자로 구분하여 0.4㎕/mL demecolcine으로 40분간 처리 후 염색체 부위가 돌출된 난자는 탈핵 후 핵이식에 공시하였다. 소의 귀 피부 세포를 탈핵란에 이식하여 전기융합과 활성화 처리(Ca-ionophore+DMAP)를 거쳐 체외 배양하였다. Demecolcine처리 후 86.2%의 난자가 염색체 부위의 돌출을 보여 이 중 98.8%가 탈핵에 성공하였다. Demecolcine은 핵이식란의 발육에 영향을 주지 않았다. 제1극체 방출란 유래 핵이식란의 배반포 발육율은 극체 미방출란 유래 핵이식란에 비하여 유의적으로 높았다(18.2% vs.4.6%, P<0.05). 한편, MI 난자 유래 핵이식란의 분할율 및 배반포 발육율은(69.4%와 5.9%) MII 난자 유래 핵이식란에 비하여 유의적으로 낮았다(96.7%와 23.9%, P<0.05). 본 연구의 결과는 demecolcine 처리가 소 난자의 탈핵에 매우 효과적이며 MII기 난자가 MI기 난자에 비하여 수핵란 세포질로 더 적절하나 극체 미방출란 및 MI기 난자도 비록 제한적이기는 하지만 핵이식란의 배반포 발육을 지원할 수 있음을 보여준다.

본 연구에서는 한우 태아의 시기별로 35일령, 50일령, 70일령 및 90일령의 fetal fibroblast cell line을 생산하였고, bovine-specific primer와 Y chromosome-specific primer를 이용하여 PCR에 의해 성을 판별하여 각각 암수 2 line의 한우 fetal fibroblast cell line을 확립하였다. 이들 cell line을 계대배양하여 passage number가 10 이상에서 염색체 분석을 실시하였는데 모두에서 80%이상의 세포가 60개의 정상 염색체수의 나타내어 계대배양이 karyotype에 영향을 미치지 않는 것으로 나타났다. Serum starvation과 confluent 배양 방법을 이용하여 Go 상태로 유도되었는지 확인하기 위해 PCNA antibody를 이용하여 Western blotting 분석을 실시하였는데 PCNA 발현이 현저히 감소되는 것을 확인할 수 있었고, 다시 정상 medium으로 환원시켰을 때 세포분열이 재개되어 Go상태로 유도되었음을 확인할 수 있었다. 또한 serum stravation 방법이 conflent한 배양방법보다 PCNA 발현양이 적은 것으로 나타나 좀더 효율적인 Go 상태 세포 주기 조절방법으로 판명되었다.

To improve the efficiency of production of cloned embryos and animals by nuclear transplantation in the rabbit, the effect of cell cycle of donor nuclei and type of recipient cytoplasm on the in vitro developmental potential and production efficiency of offspring was determined. The embryos of 16-cell stage were collected from the mated does at 48h post-hCG injection and they were synchronized to G phase of 32-cell stage. The oocytes collected at 14h post-hCG injection were freed from cumulus cells and then enucleated. One group of the enucleated cytoplasms was activated by electrical stimulation prior to injection of donor nucleus, and the other group was not pre-activated. The separated Gphase blastomeres of 32-cell stage embryos were injected into the perivitelline space of recipient cytoplasms. After culture for 20h post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation and the fused nuclear transplant embryos were co-cultured for 120h and the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye and their blastomeres were counted. Some of the nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The electrofusion rate was similar between the types of donor nuclei and recipient cytoplasms used. However, the nuclear transplant embryos using G phase donor nuclei were developed to blastocyst at higher rate(60.3%) than those using S phase ones(24.7%). Also, when non-preactivated oocytes were used as recipient cytplasms, the develop-mental rates of nuclear transplant embryos to blastocysts were significantly(P< 0.05) higher(57.1%) than those using preactivated ones(20.8%). The cell counts of nuclear transplant embryos developed to blastosyst stage were increased signficantly(P<0.05) more in the non-preactivated recipient cytoplasm(163.7 cells), as compared whit the preactivated recipient cytoplasm(85.4 cells), A total of 49 nuclear transplant embryos were tranferrid into 5 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer. these results showed that the blastomeres of G1 phase and non-preactivated oocytes might be utillzed efficiently as donor nuclei and recipient cytoplasms in the nuclear transplant procedure, thought the offspring production remained still low.

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplant embryos. The oocytes collected from slaughterhouse ovaries were matured for 24 h and then enucleated and cultured to allow cytoplasmic maturation and gain activation competence. And then the donor embryos were treated for 12 h with 10 g /ml nocodazole and 7.5 g /ml cytochalasin B to synchronize the cell cycle stage at 26 h after the onset of culture. The blastomeres were transferred into the perivitelline space of the enucleated nocytes and blastomeres and oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. The age of the recipient(30 vs 40 h) had no significant effect on the fusion rates(82.4 vs 82.1%) and the developmental rates to morula /blastocyst(9.8 vs 11.0%). Effect of Nocodazole treatment on the donor cell cyle synchronization to improve the developmental rates of bovine nuclear transplant embryos was significantly higher than control group(21.4 vs 10.1%, p<0.05). Significant differences were in the percentage of fusion rates(72.9,77.1vs 61.9%) in three types of fusion medium(PBS(+), mannitol and sucrose, p<0.01). The developmental rates of bovine nuclear transplant embryos appeared to be highest in mSOF medium under 5% 0 condition, but no significant differences were found when compared with TCM199-BOEC and mSOF under two different oxygen ratio(5 and 20%).

large scale production of cloned embryos requires the technology of multiple generation nuclear transplantation(NT) using NT embryos as the subsequent donor nuclei. The purposes of this study were producing the second generation cloned rabbit embryos, and also to determine the electrofusion rate and in vitro developmental potential comparatively in the cloned embryos of the first and second NT generation. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gi /S transition of 32-cell stage. The first generation NT embryos which were developed to 8-cell were synchronized in Gi /S transition phase of the following 16-cell stage and used as donor nuclei for second generation Synchronization of the cell cycle of blastomeres was induced, first, using an inhibitor of microtuble polymerization, colcemid for 10 hours to arrest blastomeres in M phase, and secondly, using a DNA synthesis inhibitor, aphidicolin for 1.5 to 2 hours to arrest them in Gi /S transition boundary. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 14 hours after hCG injection. The separated donor blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 sec at 1.25 kV /cm in 0.28 M rnannitol solution The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39 in a 5% incubator. Following in vitro culture of the first and second generation cloned embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The results obtained were summarized as follows: 1. The electrofusion rate was found to be similar as 79.4 and 91.5% in the first and second generation NT rabbit embryos, respectively. 2. The in vitro developmental potential to blastocyst stage of the second generation NT embryos (23.3%) was found significantly(p<0.05) lower, compared with that of the first generation NT embryos (56.8%). 3. The mean blastomeres counts of embryos developed to blastosyst stage following in vitro culture for 120 hours and also their daily cell cycles during the culture period were decreased significantly (p<0.05) to 104.3 cells and 1.33 cylces in the second NT generation, compoared with 210.4 cells and 1.54 cycles in the first NT generation, respectively.