Programmed cell death (PCD) is thought as a well-controlled process by which unwanted cells are selectively eliminated. During the last decade many researches have elucidated molecules and their interactions involved in cell death by using largely in vitro induction of cell death or survival signals in a more defined manner, While these critical information and novel findings provide us with clearer understanding of mechanisms underlying cell death, it does by no means explain how PCD occurs and which cells or tissues are affected during normal embryonic development in vivo. In this study, we used zebrafish to examine whether the PCD is occurring selectively or randomly in developing embryos by whole mount in situ TUNEL analysis with specific markers for neural cells. The result revealed that the degree and distribution of TUNEL staining varied considerably throughout gastrulation stage, and there was also a number of TUNEL-negative embryos. Most of TUNEL-positive cells were scattered randomly throughout the blastoderm. During the gastrulation stage about 75 % of the embryos analyzed exhibited more than 5 TUNEL-positive cells. As the dorsal epiblast begins to thicken rather abruptly near the end of gastrulation, TUNEL-positive cells were mainly located along the dorsal side. Although there were some variations in TUNEL staining during segmentation and pharyngeal stages, TUNEL staining continued to be localized to the central nervous system, and was also detected in the sensory organs, trigeminal ganglions, and the primary sensory neurons. High levels of the cell death in developing brain between 20-somite and prim-6 stages are thought to play a role in the morphogenesis and organization of the brain. At prim-16 stage, cell death is considerably reduced in the brain region. Dying cells are mainly localized to the prospective brain region where ectodermal cells are about to initiate neurogenesis. As development progressed, high levels and more reproducible patterns of cell death were observed in the developing nervous system. Intensive TUNEL staining was restricted to the trigeminal ganglions, the primary sensory neurons, and sensory organs, such as olfactory pits and otic vesicles. Thus, PCD patterning in zebrafish embryos occurs randomly at early stages and becomes restricted to certain region of the embryos. The spatio-temporal pattern of PCD during the early embryonic development in zebrafish will provide basic information for further studies to elucidate genes involved in. regulation of PCD largely unknown in vivo during vertebrate embryogenesis.

This experiment was carried out to evaluate the effect of early mouse embryonic development in vitro by co-culture with bovine and porcine oviductal epithelial cells (BOEC and POEC). The 2-cell embryos were collected from the oviducts of the superovulated and mated cultured in D-PBS /15% FCS at 48 hours after hCG injection. The in vitro developmental rate of blastocyst formation in the embryos were examined under the fllowing treatments; 1) TCM 199 added 15% HCS, 2) Ham's F-10 added 15% HCS, 3) MediCult IVF medium, 4) TCM 199 added 15% HCS + BOEC, 5) TCM 199 added 15% HCS + POEC, 6) Ham's F40 added 15% HCS + BOEC, 7) Ham's F-10 added 15% HCS + POEC,8) MediCult IVF medium + BOEC, 9) MediCult IVF medium + POEC. For a comparative study of in vitro development for 96 hours after hCG injection, were cultured with oviductal epithelial cell and media only. The obtained results were 2-cell embryos developed to the blastocyst stage in TCM 199, Ham's F-10 and MediCult IVF medium at the rates of 84.4,83.2 and 81.6%. respectively. The higher developmental rates(91~97%) of blastocyst formation was appeared when the embryos were co-cultured with a monolayer of bovine or porcine oviductal epithelial cells in TCM 199 or Ham's F-10 and MediCult IVF media. No significant difference in developmental rates was shown between bovine and porcine oviductal epithelial cells but significant difference in co-culture system in comparison between media only system and co-cultures. In conclusions, oviductal epithelial cells, BOEC and POEC, when co-culture with mouse early embryos improved the rates of development, blastocyst and hatching. Therefore, it is suggested that co-culture system using oviductal epithelial cells improve early embryonic developtnent in mouse.

담배나방(Heliothis assulta Guenee)의 배자 발생을 알아보기 위하여 실내에서 우화시킨 성충의 난(卵)을 이용하여 온도에 따른 배자발생기간을 조사하였으며, 에서의 조직분화 및 기관형성과정을 관찰하여 다음과 같은 결과를 얻었다. 1. 난(卵)은 적도직경 0.53mm, 높이 0.56mm인 구형이며, 표면에는 정방형(正方形)의 요철(凹凸)무늬가 있고, 난(卵)의 정부(頂部)에는 1개의 micropyle이 있었다. 2. 담배나방의 배자발생기간은 20, 25, 30, 에서 각각 147, 81, 61, 67시간이 있으며 에서의 배자기 치사율이 크게 증가하였다. 3. 난(卵)의 외형(外形)은 발생이 진행됨에 따라 색깔 및 무늬의 변화가 뚜렷하였으며 발생시간에따라 특징적이었다. 4. 에서는 배자발생이 시작된 후 14시간이 경과하면 기관형성이 일어나며, 44시간 경과 후에는 소화기관 형성이 완성되고, 80시간 후에 부화하였다. 5. 발생 초기에 난(卵)의 장축을 따라 형성된 배자는 발생시작 24시간 경과 후에는 회전하고 , 이어서 배의 장축을 중심으로 회전하며, 머리부분이 micropyle을 향하여 부화시까지 고정되었다.

Early life stage mortality in fish is one of the problems faced by loach aquaculture. However, our understanding of immune system in early life stage fish is still incomplete, and the information available is restricted to a few fish species. In the present work, we investigated the expression of immune-related transcripts in loach during early development. In fishes, recombination-activating gene 1 (RAG-1) and sacsin (SACS) have been considered as immunological function. In this study, the expression of the both genes was assessed throughout the early developmental stages of loach using real-time PCR method. maRAG-1 mRNA was first detected in 0 dph, observed the increased mostly until 40 dph. Significant expression of maRAG-1 was detected in 0 to 40 dph. These patterns of expression may suggest that the loach start to develop its function after hatching. On the other hand, maSACS was detected in unfertilized oocyte to molura stages and 0 to 40 dph. maSACS mRNA transcripts were detected in unfertilized oocytes, suggesting that they are maternally transferred.

To overcome the risk of the ovarian hyperstimulation syndrome (OHSS) in patients have polycystic ovarian syndrome (PCOS) and to prepare emergency fertility preservation in patients undergoing anticancer treatment, several researchers have reported IVM of oocytes retrieved from ovaries exposed by only hCG priming. However, the maturation rate and the developmental potential of embryos from IVM oocytes are significantly lower than those of oocytes matured in vivo. Here, we investigated the optimal time point for immature oocyte collection at post hCG only injection for in vitro maturation, in vitro fertilization and blastocyst formation. Immature GV oocytes were collected from 25 days old B6D2F1 female mouse at 12 hr, 14 hr, 16 hr or 24 hr post hCG injection. Oocytes were collected from antral or late secondary follicle by puncturing with 26 G needle. Collected oocytes were cultured in G2 medium with 10% FBS, FSH, estradiol, and hCG for 16 hr in vitro and subjected in vitro fertilization and further embryonic development. To examine follicular maturation, we estimated the numbers of primordial, primary, secondary follicle and antral follicle on ovaries of each time point post hCG. To confirm the optimal time point post hCG injection for collecting immature oocytes, we recovered the oocytes from each time point. There is no difference in the number of oocytes per mice. Oocytes collected at 14 hr post hCG injection were shown higher maturation rate to MII stage and blastocyst formation compare to other three groups (p<0.01). However, there is no difference in the maturation rate on the other three groups. Also, apoptotic signal with TUNEL assay or anti-PARP staining was not change in ovaries from all experimental groups. Granulosa cell proliferation test with anti Ki-67 or anti AMH was not show any difference. According to these results, there are no significant differences in four different time points at 12 hr, 14 hr, 16 hr or 24 hr of collection of immature oocytes in hCG primed mouse. However, oocytes from 14 hr post hCG injection showed higher percentages of maturation rate, in vitro fertilization rate, blastocyst formation.

Cryopreservation has become a powerful method of the assisted reproduction technology and supports fertility preservation of cancer and other indication patients. After controlled ovarian hyperstimulation, surplus oocytes and embryos were recommended to store using cryopreservation. Recently, vitrification is replaced with traditional slow freezing protocol, because of improved survival rates and clinical outcomes. Vitrification requires a high concentration of CPAs that may induce significant osmotic and metabolic damage to cells including oocytes even in a short exposure of a few minutes. Generally, MPF plays a crucial role in the cell cycle regulation and maintaining the meiotic arrest of oocytes. In fact, it has been observed to decline in MII ovine oocytes after vitrification and would be suggested that one of the main causes of low fertilization rate and developmental competence derived from cryoinjury during vitrification. Therefore, the aim of this study was to evaluate the effect of caffeine treatment on the activity of MPF, MAPK level in vitrified/warmed mouse mature eggs. Caffeine, Phosphataseinhibitor, may maintain active form of MPF. We evaluated their survival after warming procedure, fertilization, cleavage, and developmental rates. Ovulated MII eggs were retrieved from 6 weeks old B6D2F1 female mouse at 14hr post hCG injection. Collected MII eggs were maintained in HTF medium containing 10% KSR with or without caffeine for 1hr. Eggs were vitrified in 7.5%EG +7.5%DMSO equilibrium solution, 15%EG + 15%DMSO + 0.5M sucrose vitrification solution with or without caffeine. Also warming solution contained sucrose (0.5M, 0.25M, 0.125M, and 0M) with or without caffeine. After warming, eggs were cultured in HTF medium with or without caffeine for 2 hr then fertilized with epididymal sperm in vitro and cultured in KSOM for 5 days to analyze embryonic development. Survival rates were similar in all experimental groups. However, fertilization rate was higher in with caffeine group compare to without caffeine significantly (80% vs. 85%, p<0.05). 2-cell and blastocyst formation were increased in caffeine group (p<0.05). MPF activity and MAP kinase activity were recovered in with caffeine group after vitrification/warming process. In conclusion, Caffeine may maintain MPF and MAPK level in vitrified/warmed MII eggs, and enhance fertilization and further embryonic development.

We have launched an investigation for Embryonic Development, Larvae and Juvenile Morphology, of Buenos aires tetra in order to build basic data of Characidae and fish seeding production. We brought 50 couples of Characidae from Bizidduck aquarium in Yeosu-si, Jeollanamdo, from Korea on March of 2015. We put them in the tetragonal glass aquarium (50×50×30 cm). Breeding water temperature was 27.5~28.5°C (mean 28.0±0.05°C) and being maintained. The shape of fertilized egg was round shape, and it was adhesive demersal egg. The egg size was 0.63~0.91 mm (mean 0.74±0.07 mm, n=20). After getting fertilized egg, the developmental stage was gastrula stage, and embryo covered almost two-thirds of Yolk. Incubation was happened after 16 hours 13 minutes from gastrula stage, and the tail of juvenile came out first with tearing egg capsule. Immediately after the incubation, prelarvae had 3.78~3.88 mm length (mean 3.84±0.04 mm, n=5), and it had no mouth and anus yet. 34 days after hatching from the incubation, juvenile had 8.63~13.1 mm (mean 10.9±1.66 mm), and it had similar silver-colored body shape with its mother.

The early growth response protein 1 (Egr-1) is a widely reported zinc finger protein and a well known transcription factor encoded by the Egr-1 gene, which plays key roles in many aspects of vertebrate embryogenesis and in adult vertebrates. The Egr-1 expression is important in the formation of the gill vascular system in flounders, which develops during the post-hatching phase and is essential for survival during the juvenile period. However, the complete details of Egr-1 expression during embryo development in olive flounder are not available. We assessed the expression patterns of Egr-1 during the early development of olive flounders by using reverse transcription polymerase chain reaction (RT-PCR) analysis. Microscopic observations showed that gill filament formation corresponded with the Egr-1 expression. Thus, we showed that Egr-1 plays a vital role in angiogenesis in the gill filaments during embryogenesis. Further, Egr-1 expression was found to be strong at 5 days after hatching (DAH), in the development of the gill vascular system, and this strong expression level was maintained throughout all the development stages. Our findings have important implications with respect to the biological role of Egr-1 and evolution of the first respiratory blood vessels in the gills of olive flounder. Further studies are required to elucidate the Egr-1-mediated stress response and to decipher the functional role of Egr-1 in developmental stages.

이 연구는 2006년 1월부터 3월까지 전남 여수시 거문도 해역에서 어획된 찰가자미의 어미로부터 실험실 내 자연 산란을 유도하여 난발생 및 자치어 형태 발달을 관찰하였다. 수정란은 무색 투명한 분리부성란이고, 유구는 없었으며, 난경은 1.640.03 mm(n=50)였다. 부화에 소요되는 시간은 수온 (평균 )에서 수정 후 168시간 40분이었으며, 부화 직후의 평균 전장은 4.050.18 mm(n=20)였다. 부화 직후 자어는 막지느러미가 발달하였고,

찰가자미 Microstomus achne의 어미로부터 수정란을 받아 수온이 난 발생에 미치는 영향을 연구하였다. 찰가자미의 난발생 속도와 부화율에 미치는 수온의 영향을 조사한 결과, 난발생이 개시되는 생물학적 영도는 평균 였다. 수정에서 부화에 이르기까지 평균 소요시간은 에서 86.5~296.67시간이 소요되었으며, 수온이 높을수록 각 발생단계에 이르는 시간이 짧아지는 경향을 나타내었다. 부화율은 에서 95.8~97.0%였으며, 와 에서는 각각 86.9