코발트는 자연적으로 암석, 토양 물 등에서 발견되는 요소일 뿐만 아니라 산업화에 의해 생성되는 유해물질 중의 하나이다. 환경에서는 코발트 (II)와 코발트 (III)의 두 가지 산화물로 존재하며, 연안 해역으로 유입 시 먹이연쇄를 통해 해양생태계 전반에 영향을 미칠 수 있다. 따라서 본 연구는 유용 수산생물로써 조간대 암반 지역에 서식하는 말똥성게(Hemicentrotus pulcherrimus)를 이용하여 cobalt (Co (II); 10,

Artificial insemination and embryo transfer is one of the most important factors affecting to the production of fawn from deer nuclear transfer in the field of deer farms. This study* was conducted to establish the production technology of nuclear transfered embryo in deer. For estrus synchronization or superovulation tretments in flower deer and elk, each 10 does were inserted into the vagina for 14 days with CIDR (Pfizer New Zealand Ltd., NZ) for elk and Ring-CIDR (Bioculture Co., Ltd., Korea) for flower deer, and then those inserted devices were removed. The estrus synchronization of each 6 does were induced by the intramuscular injection of PGF2α (25 mg/head) and PG600 (hCG 200IU + PMSG 400IU, Intevet, Holland). Then, the superovulation of each 4 does of flower deer and elk was induced by additional injection of FSH (200 mg/ head) twice with an interval of 24 hours , respectively. Follicular oocytes were collected from each 2 does superovulated after 48 hours since the injection of PG600 and FSH. In the meantime, the ovarian response and the number of the collected ovarian follicles were investigated with the surgical operations. As a result, the average number of the collected ovarian follicles were 8.5 and 9.0 in flower deer and elk, respectively. The ovarian follicles collected from each two does were cultured in vitro for 48 hours with m-DMEM medium, and then the cell fusion was carried out after the nuclear transfer by the antler cell. As a result, 5 out of 18 ovarian follicles collected from 2 elk does were reached on the MII stage, but there was no generation resulting from the nuclear transferred embryos by the antler cell after enucleation. In 2 flower does, 7 out of 17 ovarian follicles were reached to the MII stage, but one of them was developed to parthenogenetic embryo as well despite a case of fusion from the nuclear transferred embryo. Embryos were collected in a surgical way on the 7th day after artificial insemination, numbers of average embryos collected were 2.5 and 3.0 in each 2 flower deer and elk does superovulated, respectively. The collected two embryos were transplanted to each 2 does synchronized. As a result, a head of fawn was produced from only one elk doe, where as a head of fawn were delivered from one out of 4 elk does artificial inseminated. Given these findings, we consider that more or less of problems might have occurred in vitro culture system of ovarian follicles in the production of nuclear transfered deer embryos. In addition, the greatest reason why both the aetificial insemination and embryo transfer failed was considered attributable to stress due to anesthesia and catching.

Cloning or somatic cell nuclear transfer (SCNT) using adult somatic cell to derive cloned embryos is a promising new technology with potential applications in both agriculture and regenerative medicine. Mammalian embryos derived by nuclear transfer are capable of development to the blastocyst stage with a relatively high efficiency of 30～ 50%. However, in full-time development, usually only 2% of NT embryos can result in live births due to abnormalities in placenta formation. In SCNT embryos, the donor cell nucleus is epigenetically reprogrammed by oocyte cytoplasm during development. Incomplete reprogramming of the donor cell genome is considered a major reason for low cloning efficiency. Aberrant epigenetic modifications include DNA methylation, histone modification and X-chromosome-inactivation. Due to a lack of basic knowledge regarding the embryos following nuclear transfer, the success rate of cloning is low. Therefore, elucidation of the molecular mechanism of SCNT embryo development will be of great value for further research. MicroRNAs (microRNA) are single-strand RNA molecules of about 19 23 nucleotides in length, which regulate gene expression by imperfect base pairing with target mRNA, subsequently guiding mRNA cleavage or translational repression. Since the first discovery and functional annotation in 1993 of the small RNA, lin-4 and let-7, which are involved in developmental timing and gene regulation during C. elegans larval development, microRNAs have received scientific attention. Now hundreds of microRNAs have been identified in various multicellular organisms, and many microRNAs have been shown to be evolutionarily conserved. The roles proposed for this novel class of tiny RNA molecules are diverse. They are likely to be involved in developmental timing, differentiation, cell proliferation, signaling pathways, apoptosis, metabolism, heterochromatin formation, genome rearrangement, brain development and carcinogenesis. Currently (2006- present) we are working to determine the role of microRNAs on the epigenetic regulation of fertilized and cloned embryo development. The general hypothesis of our research is that genetic and epigenetic factors regulate the development of preimplantation mammalian embryos, and aberrant modulations in cloned embryos are causes of abnormal development and low success rate of cloned embryos.

The vast majority of embryo generated by Assisted Reproductive Technologies (ART) do not result in a live offspring and a multiple birth is the single biggest health risk associated with human fertility treatment, and the used of frozen embryos increased for medical or personal reasons. However, practical and ethical reasons might hamper study of human embryos. Therefore, animal models are necessary to elucidate the molecular and morphological changes during development. In the serial experiments, we employed mouse embryos and a Cdx-inducible ES cell system that transdifferentiates into TS cells. We found aberrant gene expression profiles including apoptosis associated (Bcl2), lineage formation related genes (Cdx-2, Tcfap2c, Oct4, and Nanog), and/or mitochondrial DNA replication related genes (mt-cox-1, mt-cox-2, Polg, Polg2, Tfam) in mouse embryos that showed developmentally retardation between morula to blastocyst transition or post implantation development after embryo transfer to surrogate mothers, compared to control embryos. To determine direct interaction between knockdown genes via siRNA approach and putative down-stream genes involved in blastocyst formation and further development, we carried out qPCR and Chip assay in either mouse embryos or the ES cells. qPCR and Chip assay results showed target gene directly bound to promoter regions of down-regulated genes in TS cells. In conclusion, we suggested that an increased understanding of epigenetic regulation of early embryonic development through animal models may ultimately lead to better methodologies for selecting more competent embryos and and/or protocols for augmenting embryos viability.

The composition of culture media is a key element in the process of in vitro embryo production. With the development of defined culture media, many components that are present in trace amounts in follicular fluid and serum have been excluded from the in vitro embryo production system. Among these are hormones, which have important regulatory roles in growth, metabolism and differentiation and are known to be present in follicular fluid, serum and the female reproductive tract. We have investigated the effects of supplementation of in vitro maturation and/or culture medium with testosterone (T), androstenedione (A4) and thyroid hormones (TH) on bovine in vitro embryo production and the mechanisms of action of TH in developing embryos. Our results show that testosterone, but not androstenedione or thyroid hormone supplementation increased cleavage rates. None of the treatments significantly altered the sex ratio. Addition of thyroid hormones, T3 and T4, to the in vitro culture media resulted in a significant increase in the rate of development to the blastocyst stage. In addition, blastocysts from the T3/T4 treated groups had higher cell number and lower rates of apoptosis. We have confirmed the expression of mRNAs for both Thyroid hormone receptor α and β (TR α and β) in cumulus‐oocyte‐complexes, oocytes, and in both treated and control blastocyst. Quantitatively, the expression of TR mRNA was higher in the treated embryos but the difference was not statistically significant. TR proteins were detectable in blastocysts of both groups with a difference in the distribution pattern. TH treated embryos had peri‐nuclear concentration of TR while in control embryos it was homogenously distributed in the cytoplasm. Preliminary studies of inhibition of TR α and β by siRNA knockdown by micro injection at the zygote stage show a drastic reduction in development suggesting that TH play an essential role in embryo development. They appear to mediate this effect by their receptors, TR α and β. Overall the results show that the presence of hormones in maturation and the culture medium can alter the outcome of in vitro embryo production and highlight the significance of biological components missing from in defined embryo culture media.

In this study, we examined the effectiveness of in vitro fertilization of porcine immature oocytes on the embryo development of blastocysts or hatched blastocysts and the number of cells according to the in vitro fertilization conditions. In the in vitro fertilization of in vitro matured porcine oocytes, there were no significant differences between treatment groups regarding fertilization rate, blastocyst rate, and embryo development of hatched blastocysts according to the storage periods of liquid sperm of 24, 48, and 72 hours. The embryo development rate of hatched blastocysts after the fertilization according to different spermatozoa concentrations (, , and cells/ml) showed the highest rate in the group with a spermatozoa concentration of cells/ml; in particular, this rate was significantly higher than that in the cells/ml group (p<0.05). The total number of blastocysts cells as well as trophectoderms (TE) that developed in each treatment group were also significantly higher in the cells/ml group than in any other groups (p<0.05). In contrast, the embryo development rate of blastocysts according to different co-incubation periods of sperm and oocyte (1, 3, and 6 hr) was high in the 6-hour group; in particular, the rate was significantly higher than that of the I-hour group (p<0.05). Furthermore, the total number of oocytes cells and TEs that developed was significantly higher in the 6-hour group than any other group (p<0.05). In this study, the most effective treatment conditions for porcine embryo development and high cell number were found to be as follows: a sperm storage period of less than 72 hours, a spermatozoa concentration of cells/ml, and a 6-hour co-incubation period for sperm and ooocyte.

산업화에 의해 발생되어진 여러 가지 오염물질 가운데 중금속인 니켈, 코발트, 수은은 연안 해역으로 유입 시 극히 미량일지라도 생체 내 축적되어 해양생물의 생리 및 발생에 장애를 일으키는 것으로 알려져 있으며 먹이연쇄 (food chain)를 통해 생물확대 (biomagnification)됨으로써, 해양생태계 전반에 영향을 미칠 수 있다. 본 연구는 유용 수산생물로써 조간대 암반 지역에 서식하는 말똥성게 (Hemicentrotus pulcherrimus)를 이용하여 니켈, 코발트, 수은의 생태 위해성을 조사하고자 한다. 성숙한 말똥성게 (H. pulcherrimus)에 0.5 M KCl를 주입하여 방란 및 방정을 유도한 후, 수집한 정자 및 난자를 니켈 (10, 25, 50, 100, 500 ppb), 코발트 (10, 100, 500, 1000, 2500 ppb) 그리고 수은 (10, 25, 50, 100, 500 ppb) 농도에서 10분간 인공수정 한 후, 수정 및 배아발생을 관찰하였다. 실험결과 수정률은 노출 된 각각의 니켈과 코발트 농도에 유의적인 영향을 받지 않았다. 수은은 농도가 증가할수록 농도 의존적으 로 감소하는 경향을 나타내었다. 배아 발생률은 니켈, 코발트 및 수은 농도가 증가 할수록 농도 의존적으 로 유의적인 감소를 나타내었다. 말똥성게 (H. pulcherrimus)의 배아 발생률에 대한 니켈, 코발트 및 수은 의 영향을 독성치로 나타냈을 때, 무영향농도 (NOEC)는 10 ppb로 모두 같은 값으로 나타났고, 최소영향 농도 (LOEC)는 각각 25 ppb, 10 ppb, 10 ppb로 나타났으며, 반수영향농도 (EC50)는 각각 34.19 ppb, 71.84 ppb, 144.66 ppb로 나타났다. 본 연구 결과, H. pulcherrimus의 배아 발생률을 이용한 생태독성 평가시, 니켈>코발트>수은 순으로 독성이 강한 것으로 나타났으며, 이들 중금속이 연안해역에 유입되어 10 ppb를 초과할 경우 연안 생태계 내에 서식하는 생물의 재생산에 유해한 영향을 미칠 것으로 판단된다.

페놀류는 석유정제, 섬유산업, 도로포장 및 광산산업 등의 폐수에 다량 함유되어있는 내분비계교란 및 독성 물질로서 연안해역으로 유입 시 해양 서식 생물의 생식세포 및 배아 발생 등에 영향을 미치 는 것으로 알려져 있다. 성게는 수정 및 배아 발생과 같은 초기생활사 연구에 중요한 생물 종으로서 유해물질 위해성 평가에 유용하게 이용되고 있다. 본 연구는 우리나라 조간대 암반 지역에 주로 서식 하는 말똥성게 (Hemicentrotus pulcherrimus)를 이용하여 페놀이 말똥성게 (H. pulcherrimus)의 생식 세포 및 배아 발생에 어떤 영향을 미치는지에 대하여 관찰하였다. 성숙한 말똥성게 (H. pulcherrimus) 에 0.5 M KCl를 주입하여 방란 및 방정을 유도한 후, 수집한 정자 및 난자를 페놀 농도 (10, 100, 500, 1000, 5000, 10000 ppb)에서 10분간 인공수정 한 후, 수정 및 배아발생을 관찰하였다. 수정률 은 페놀 농도에 유의적인 영향을 받지 않았으나 배아 발생은 페놀 농도가 높을수록 정상적으로 발생 하는 개체가 감소하는 농도 의존성을 나타냈다. Metallothionein mRNA 유전자는 페놀 농도가 증가할 수록 발현이 높게 나타났으며 Nodal mRNA 유전자는 유의적인 차이가 나타나지 않았다. 말똥성게 (H. pulcherrimus) 배아 발생에 미치는 페놀의 영향을 독성치로 나타낼 때, 무영향농도 (NOEC)는 10 ppb, 최소영향농도 (LOEC)는 100 ppb를 나타냈으며 반수영향농도 (EC50)는 1565.86 ppb를 나타내 었다. 본 연구 결과로서, 연안 해역에 유입되는 페놀이 무영향농도 (NOEC) 10 ppb를 초과할 경우, 성게를 비롯한 조간대 암반에 서식하는 생물종의 생식 및 발생에 유해한 영향을 미칠 것으로 판단된 다.

This study was to investigate the effect of flavonoid treatment on in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos, and their pregnancy and delivery rate after embryo transfer into recipient. In experiment 1, to optimize the flavonoid concentration, parthenogenetic day 2 (≥ 2-cell) embryos were cultured in 0 (control), 1, 10 and 20 μM flavonoid for 6 days. In the results, in vitro development rate was the highest in 10 μM flavonoid group (57.1%) among treatment groups (control, 49.5%; 1 μM, 54.2%; 20 μM, 37.5%), and numbers of total and ICM cells were significantly (p<0.05) higher in 10 μM flavonoid group than other groups. We found that 10 μM flavonoid treatment can significantly (p<0.05) decrease the apoptotic index and derive high expression of anti-oxidant, anti-apoptotic, cell growth and development marker genes such as Mn-SOD, Survivin, Bax inhibitor, Glut-5, In-tau, compared to control group. In experiment 2, to produce the cloned Jeju Black Cattle, beef quality index grade 1 bull somatic cells were transferred into enucleated bovine MII oocytes and reconstructed embryos were cultured in 10 μM flavonoid added medium. When the in vitro produced day 7 or 8 SCNT blastocysts were transferred into a number of recipients, 10 μM flavonoid treatment group presented higher pregnancy rate (10.2%, 6/59) than control group (5.9%, 2/34). Total three cloned Jeju Black calves were born. Also, two cloned calves in 10 μM flavonoid group were born and both were all healthy at present, while the one cloned calf born in control group was dead one month after birth. In addition, when the result of short tandem repeat marker analysis of each cloned calf was investigated, microsatellite loci of 11 numbers matched genotype between donor cell and cloned calf tissue. These results demonstrated that the flavonoid addition in culture medium may have beneficial effects on in vitro and in vivo developmental capacity of SCNT embryos and pregnancy rate.