Placenta is the main nutrition source for the fetus during pregnancy. Thus, it has a pivotal function in the pregnant process. Many functions of the placenta have been elucidated. An abnormal placenta is associated with a high rate of pregnancy failure in somatic cloned bovine. Differentially expressed genes (DEGs) were examined in a comparison between normal and cloned bovine placenta using annealing control primer (ACP)-based GeneFishing PCR. Using 120 ACPs, nearly 80 genes were identified and the fragments of 42 DEGs were sequenced. 38 of these genes were known genes and four were unknown. To determine the DEGs result, six target clones expressing on one-side of a normal and a clone placenta were selected. Through an analysis of the target genes using the real-time PCR, the expressing pattern was found to be somewhat different from the DEGs. Additionally, several genes appeared with the same expression pattern. Taken together, this suggests that the target genes would be essential for research into what influences the placental formative mechanisms during fetal development.

This study was conducted to compare the expression pattern of the specific factors associated with pregnancy and angiogenesis during early pregnancy in Hanwoo. Synchronized female Hanwoo (4～6 year‐old) were inseminated artificially. After 10 weeks after artificial insemination (AI), the pregnancy was tested by rectal palpation method. Three pregnant and non‐pregnant Hanwoo were used in this experiment, respectively. The plasma progesterone level was measured by ELISA. Western blot analysis was performed to detect the expression of pregnancy associated glycoprotein (PAG) or angiogenic factors (VEGF, B‐FGF, ANP‐1, and TIE‐2). The plasma P4 level was increase gradually in pregnant group and maintained high level. The concentration of PAG was significantly higher from 5th weeks in pregnant group compared to that of non‐pregnant group (p<0.05). The concentrations of the VEGF (p<0.05), B‐FGF (p<0.05), and ANP‐1 (p<0.05) were significantly increased from 6th or 7th week after AI in pregnant group, respectively. And the intensity of TIE‐2, ANP‐1 receptor, was well matched with ANP‐1 (p<0.05). Taken together, it can be postulated that the blood vessels connected with fetus and dam were formed dramatically around 40 days after AI, because the expression levels of the angiogenic factors were increased significantly from this time in pregnant Hanwoo.

This study was conducted to investigate an effective recipient oocyte and culture system for producing of Hanwoo (Korean native cattle) somatic cell nuclear transfer (SCNT) embryos. Hanwoo ear skin fibroblasts were used as donor cells. In vitro matured Hanwoo or Holstein oocytes were enucleated, and single donor cells were transferred into the perivitelline space of the enucleated oocytes. The couplets were subsequently fused and activated. The reconstructed embryos were cultured in a conventional or sequential culture system. In the former, embryos were cultured in CR2aa medium for eight days; in the latter, embryos were cultured in modified CR2aa-A (mCR2-A) for three days and then further cultured in modified CR2aa-B (mCR2-B) for five days. In the experiment with the recipient oocyte, the rate of embryo development to the blastocyst stage was significantly (p<0.05) higher in Hanwoo recipient oocytes than in Holstein ones (48.8% vs 38.9%). BIastocysts derived from Hanwoo recipient oocytes contained significantly (p<0.05) higher numbers of total cells than those derived from Holstein recipient oocytes (156.0+-68.2 vs 134.7+-54.8)). There was no difference in the mean proportion of apoptotic cells in blastocysts between the sources of recipient oocytes. In the experiment with the embryo culture system, the blastocyst rate was somewhat higher in sequential system than in conventional system (50.0% vs 43.5%), though there was no significant difference. The numbers of total (160.0+-69.0 vs 156.7+-68.4) and apoptotic cells (14.0+-10.4 vs 11.8+-6.4)) were not different between the culture systems. In conclusion, the present study demonstrated that Hanwoo recipient oocytes and the sequential culture system were more effective in supporting the production of Hanwoo SCNT embryos.

본 연구는 체세포 복제 한우 송아지의 생산에서 생시체중과 생존성과의 관계에 대하여 살펴보고자 실시하였다. 293두의 한우 대리모에 580개의 복제란을 이식하였다. 복제란의 임신율은 이식 후 50일까지 72.3%로 높았으나, 이후 급격하게 감소하였다. 평균 임신 기간은 복제 송아지에서 287일(279~295일)이었으며, 인공수정 송아지도 287일(255~293일)로 각각 나타났다. 복제 송아지의 생시체중(30.3kg)은 인공수정 송아지(23.7kg)에 비하여 유의하게 높은 것으로 나타났다(p<0.05). 자연분만(n=17, 29.9kg)과 제왕절개(n=14, 32.3kg)로 태어난 복제 송아지의 생시체중은 차이가 없었다. 하지만, 생후 175일 이전에 사망한 복제 송아지(n=18, 32.8kg)의 생시체중이 175일 이상 생존한 복제 송아지(n=11, 28.3kg)보다 유의적으로 높게 나타났다(p<0.05). 흥미로운 점은 생시체중이 15kg 이하(n=5) 또는 35kg 이상(n=9)인 복제 송아지들은 모두 생후 175일 이전에 폐사하였다. 생후 175일 이전에 폐사한 복제 송아지들(n=20)의 사망 원인은 미성숙 개체 2두(10.0%), 폐와 간 이상 2두(10.0%), 폐의 원인 4두(20.0%), 기형 4두(20.0%), 출생 후 급사(sudden death syndrome) 4두(20.0%) 및 기타 원인불명이 4두(20.0%) 등으로 분류되었다. 이상의 결과를 종합하여보면, 복제 송아지의 정상 생시체중이 6개월 이상을 생존하는데 가장 중요한 요소들 중의 한 가지임을 확인하였다.

DNA 메틸화는 조직특이적인 유전자 조절에 관여하고, 정상적인 배 발달에 필수적이다. POU5F1은 octamer-binding transcription factor 4 (Oct-4)를 encode하며, 초기 분화에 중요한 전사인자이다. 본 실험에서 소의 Oct-4가 조직특이적이고 발달의존적인 epigenetic 표지 인지를 검토하고자, 착상 전 수정란에서 Oct-4 전사산물과 상류 promoter 영역의 CpGs의 메틸화를 조사하였다. Oct-4 전사산물은 정자 그리고 2-cell에서 8-cell 수정란까지 낮은 수준으로 존재하지만, 상실배와 배반포에서 높게 검출되었다. 이러한 결과는 배 발달 과정의 상실배 단계에서 Oct-4의 de novo 발현이 시작됨을 의미한다. Oct-4 상류 promoter 영역에는 메틸화 가변 영역 (tissue-dependent differentially methylated region, T-DMR)이 존재한다. Oct-4 메틸화 가변 영역의 메틸화 상태는 정자, 성체 체조직과 난자에서 서로 다르고, 수정란으로부터 배반포 단계까지 변화하였는데, 이는 착상 전 초기 배 발달 과정에 active 메틸화와 탈메틸화가 일어남을 의미한다. 이상의 결과, Oct-4 유전자 상류 promoter 영역은 DNA 메틸화의 타깃이고, 그 메틸화 상태는 소 수정란 발달 동안에 다양하게 변화한다.

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.

There are replete numbers of reports which have apparently shown that established patterns of methylation are critical for normal mammalian development. Here, we report expression of the DNA methyltransferases (Dnmts) family during mouse early development. Transcription of Dnmt1o occurs in one-cell and morula stage embryos, whereas Dnmt1s transcripts were detectable in all cells and tissues examined during the study. Dnmt3a1 transcript was detected in all cells and Dnmt3a2 transcript was particularly detected in the oocyte and 1-cell stages. Low level Dnmt3b1 transcripts were expressed ubiquitously in oocyte, 1-cell, and preimplantation embryos except 2～4 cell stages. Dnmt3b3 transcripts were only detected in E7.5 embryo and ovary. Furthermore, Dnmt3l transcripts were detectable in all cells and tissues examined. Unlike Dnmt1, both Dnmt3a and Dnmt3b proteins existed in the nucleus of preimplantation embryos till the morula stage. These Results suggest that differences Dnmts expression level exist and genomic DNA methylation patterns may be determined partly through differential expression of Dnmts during early development.

Di-n-butyl phthalate (DBP), diisononyl phthalate (DINP) and di-(2-ethylhexyl) adipate (DEHA) are ubiquitously distributed chemicals that are widely used as plasticizers and also found at low levels in foods. The aims of this study were to determine whether perinatal exposure to DBP, DINP and DEHA could alter normal patterns of neonatal development. Dams were provided with pulverized soy-free diet containing 20, 200, 2,000 and 10,000 ppm of DBP, 40, 400, 4,000 and 20,000 ppm of DINP, or 480, 2,400 and 12,000 ppm of DEHA from gestational day 15 to postnatal day 21. Exposure to the high doses of DBP, DINP and DEHA during gestational period significantly decreased food consumption and body weight gain of dams. These chemicals reduced neonatal body weight as well as that of the after maturation. Also, exposure to DINP of all the doses used and the higher doses (2,400 and 12,000 ppm) of DEHA decreased AGD at PND 1 in male neonates, though that to DBP did not affect AGD in males. In female neonates, an increase in AGD was observed in DBP- and DINP-exposed animals at the highest doses. Moreover, these chemicals affected survival rate of pups at PND 5, and delayed onset of eye opening in all chemical-exposed groups at PND 17. These results suggest that perinatal exposure to these chemicals may affect the normal development and / or growth of offspring.

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.