Spermatogonial stem cells(SSCs) only are responsible for the generation of progeny and for the transmission of genetic information to the next generation in male. Other in vitro studies have cultured SSCs for proliferation, differentiation, and genetic modification in mouse and rat. Currently, information regarding in vitro culture of porcine Germline Stem Cell(GSC) such as gonocyte or SSC is limited and is in need of further studies. Therefore, in this study, we report development of a successful culture system for gonocytes of neonatal porcine testes. Testis cells were extracted from 10～14-day-old pigs. These cells were harvested using enzymatic digestion, and the harvested cells were purified with combination of percoll, laminin, and gelatin selection techniques. The most effective culture system of porcine gonocytes was established through trial experiments which made a comparison between different feeder cells, medium, serum concentrations, temperatures, and O2 tensions. Taken together, the optimal condition was established using C166 or Mouse Embryonic Fibroblast(MEF) feeder cell, Rat Serum Free Medium(RSFM), 0% serum concentration, 37℃ temperature, and O2 20% tension. Although we discovered the optimal culture condition for proliferation of porcine gonocytes, the gonocyte colonies ceased to expand after one month. These results suggest inadequate acquirement of ingredients essential for long term culture of porcine GSCs. Consequently, further study should be conducted to establish a successful long-term culture system for porcine GSCs by introducing various growth factors or nutrients.

본 연구는 한우 체세포를 이용하여 생산된 복제란을 한우 대리모에 이식하여 임신이 확인된 개체에서 임신 기간 중 주요 호르몬의 발현 특성을 인공수정으로 임신된 대리모와 비교 분석하고자 실시하였다. 한우 섬유아세포를 이용하여 생산된 체세포 복제란을 자연발정으로 동기화된 한우 대리모에 이식하여 임신이 확인된 개체를 공시하였으며(n=8), 대조군으로는 인공수정으로 임신된 대리모을 사용하였다(n=5). 발정 관찰 후 60일경에 직장검사로 임신을 확인하였다. 주요 스테로이드 호르몬인 progesterone(P4)와 estradiol-l7 (E2) 농도는 방사선동위원소 면역분석시험(RIA) 방법을 이용하였으며, 혈중 cortisol 농도는 ELISA 방법으로 측정하였다. 인공수정한 대리모의 경우 E2 농도가 분만 시기에 급격하게 증가하였으나, P4 농도는 분만 시기에 급격하게 감소하는 경향을 나타내었다. 이에 반해 복제란 이식우의 혈장 P4 농도는 분만 50일 전부터 분만 10일전까지는 대조군과 유사하게 유지되었으나, 분만예정일에는 전혀 떨어지지 않고 높은 수준으로 유지되었다. 한편, 복제란 이식우에서 분만 때까지 정상적으로 임신이 유지된 대리모들의 경우는 임신 기간 동안 cortisol 농도는 임신 후반기까지 낮게 유지되며 별다른 변화를 나타내지 않았다. 반면에 유산이 일어난 개체의 경우에는 임신 100일경에 cortisol의 농도가 급격하게 증가하는 것을 확인하였다. 이상의 결과를 종합하여 보면, 복제란 이식우의 경우 분만예정일 전 후에 일어나는 급격한 호르몬의 변화가 일어나지 않음을 확인할 수 있었으며, 이러한 현상은 복제란 이식우의 분만 지연과 밀접한 관계가 있는 것으로 사료된다.

This study was conducted to investigate the development and gene expression in miniature pig nuclear transfer (mNT) embryos produced under different osmolarity culture conditions. Control group of mNT embryos was cultured in PZM-3 for 6 days. Treatment group of mNT embryos was cultured in modified PZM-3 with NaCl (mPZM-3, 320 mOsmol) for 2 days, and then cultured in PZM-3 (270 mOsmol) for 4 days. Blastocyst formation rate of the treatment group was significantly higher than the control and the apoptosis rate was significantly lower in treatment group. Bax- and caspase-3 mRNA expression were significantly higher in the control than the treatment group. Also, the majority of imprinting genes were expressed aberrantly in in vitro produced mNT blastocysts compared to in vivo derived blastocyst H19 and Xist mRNA expression were significantly lower in the control than the treatment group or in vivo. IGF2 mRNA expression was significantly higher in the control than the treatment group or in vivo. IGF2r mRNA expression was significantly lower in the control. Methylation profiles of individual DNA strands in H19 upstream T-DMR sequences showed a similar methylation status between treatment group and in vivo. These results indicate that the modification of osmolarity in culture medium at early culture stage could provide more beneficial culture environments for mNT embryos.

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.

The aim of this study was to examine more effective cryoprotectant for the cryopreservation of mouse preantral follicles. Enzymetically isolated preantral follicles from 12-day-old mice were cryopreserved by a slow freezing protocol with 1.5 M propanediol (PROH), dimethyl sulphoxide (DMSO) or glycerol (GLY) and then grown and matured in vitro for 11 days after thawing. The survival of preantral follicles immediately after freezing and thawing was not different among the PROH (68.2%), DMSO (72.4%) and GLY (72.1%). After grown and matured in vitro, the rates of survival and metaphase II oocytes were 54.9% and 36.6% for PROH which was significantly higher rates (p<0.05) compared with the rates obtained from DMSO (16.9% and 9.0%) and GLY (16.3% and 7.5%). The diameter of metaphase II oocytes from pre antral follicles frozen in PROH (67.4+-1.8 um) was significantly (p<0.05) smaller than that of the fresh preantral follicles (69.1+-2.3 um). The results from the present study revealed that PROH is more suitable cryoprotectant for the cryopreservation of mouse preantral follicles.

This study investigated the developmental ability and gene expression of somatic cell nuclear transfer embryos using ear skin fibroblast cells derived from miniature pig. When miniature pig (m) and landrace pig (p) were used as donor cells, there were no differences in cleavage (79.2 vs. 78.2%) and blastocyst rates (27.4 vs. 29.7%). However, mNT blastocysts showed significantly higher apoptosis rate than that of pNT blastocysts (6.1 vs. 1.7%) (p<0.05). The number of nuclei in pNT blastosysts was significantly higher than that of mNT (35.8 vs. 29.3) (p<0.05). Blastocysts were analyzed using Realtime RT-PCR to determine the expression of Bax-, Bcl-xl, H19, IGF2, IGF2r and Xist. Bax- was higher in mNT blastocyst than pNT blastocyst (p<0.05). There was no difference in Bcl-xl between two NT groups. Bax-/Bcl-xl was, however, significantly higher in mNT blastocyst compared to pNT. The expression of imprinting genes were aberrant in blastocysts derived from NT compared to in vivo blastocysts. H19 and IGF2r were significantly lower in mNT blastocysts (p<0.05). The expression of IGF2 and Xist was similar in two NT groups. However, imprinting genes were expressed aberrantly in mNT compared to pNT blastocysts. The present results suggest that the NT between donor cells derived from miniature pig and recipient oocytes derived from crossbred pig might affect reprogramming of donor cell, resulting in high apoptosis and aberrant expression patterns of imprinting genes.

Surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS) is one of the recently developed proteomic technologies which is based on capturing proteins and peptides by chemically modified surfaces and highly sensitive for the analysis of complex biological samples. In the present study, to gain insights into oocyte maturation and early embryo development, SELDI-TOF-MS was used to find the protein candidates that are specifically or prominently expressed in porcine oocytes at the in vitro matured metaphase II (MIIl) and germinal vesicle (GV) stages. By selected CM10 chip, 16 candidates were found to be up-regulated in GV stage oocytes compared with in MII stage oocytes, their molecular weights were 8,180 (2 candidates), 10,226 (5 candidates), 15,767 (5 candidates) and 16,770 (4 candidates) Da respectively. And the expression of 29 candidates were higher in MII than in GV stage oocytes, their molecular weight were 10,832 (3 candidates), 17,743 (8 candidates), 20,122 (3 candidates), 22,131 (3 candidates), 24,857 (7 candidates) and 33,507 (5 candidates) Da, respectively. The expression of selected 13 candidates (0.2 and 1.0 % error tolerances) were analyzed using real time RT-PCR. The proteins that differentially regulated during oocyte in vitro maturation in the pigs may be potential biomarkers of oocyte maturation and quality.

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.

본 연구는 체세포 복제란 이식우의 분만에 있어서 혈중 스테로이드호르몬, TGF-β1 농도와 분만지연의 상관 관계를 살펴보고자 실시하였다. 대조군으로는 인공수정(AI)을 통하여 임신한 암소(cow)들을 사용하였다(AI-R). 모든 AI-R들은 자연분만(n=5, 임신 284+-0.71일)을 하였다. 분만징후를 보이지 않는 체세포 복제란 이식우(n=5, SCNT-R)들은 분만 예정일보다 10일 정도 지난 임신 292일째에 제왕절개(Caesarean section, C-sec)를 실시하여 분만하였다. 혈액 및 태반 샘플을 분만 전.후에 채취하여 형태 및 중량 등을 측정하였다. 혈장호르몬인 Progesterone(P4)와 Estradiol-17β (E2) 농도는 방사선동위원소 면역 분석 시험(RIA) 방법을 이용하여 측정하였다. 혈장 및 태반분엽의 TGF-β1 농도는 ELISA 방법으로 측정하였다. SCNT-R에서 회수한 태반의 무게는 AI-R의 것과 비교하여 유의적으로 무거웠다(p<0.05). 분만 직전 SCNT-R들의 혈장 내 P4 농도는 AI-R들의 그것과 비교하여 유의하게 높았다(p<0.01). 하지만 SCNT-R들의 혈장 내 E2 농도는 AI-R과 비교하여 상대적으로 낮게 나타났다(p<0.01). 한편, 분만 전.후 SCNT-R들에서 혈장 또는 태반분엽의 TGF-β1 단백질 발현 수준은 AI-R들과 비교하여 각각 유의적으로 높은 수준을 유지하였다(p<0.01). 이상의 결과를 종합하여 보면, 분만 시 P4 및 E2의 이상 발현과 높은 수준의 혈장 및 태반 내 TGF-β1 단백질은 체세포 복제태아의 분만지연을 야기하는 중요한 요인들 중의 하나일 것이라 사료된다.

This study was conducted to examine the mRNA expression of apoptosis-related and imprinted genes and methylation pattern of the differentially methylated region (DMR) of H19 gene in day 35 of SCNT pig fetuses. The day 35 of natural mating (control) or cloned (clone) pig fetuses were recovered from uterus. Endometrium from dam and liver from fetus were obtained, respectively. mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. The Bcl-2 mRNA expression in clone was significantly lower than that of control (p<0.05). The mRNA expression of H19 gene in both endometrium and liver was significantly higher in clone than that of control, respectively (p<0.05). The level of IGF-2 mRNA in liver of clone was significantly lower than that of control (p<0.05), whereas the mRNA expression of IGF2-R gene in liver of clone was significantly higher than that of control (p<0.05). The DMR of H19 was lower methylation pattern in clone than that of control. These results suggest that the aberrant mRNA expression of apoptosis-related and imprinted genes and the lower DMR methylation pattern of imprinted gene may be closely related to the inadequate fetal development of cloned fetus.

본 연구는 체세포를 이용하여 생산된 복제 한우 수소의 번식능력을 검토하기 위해 실시하였다. 복제 한우 수소(C-38 및 C-39) 또는 일반 한우 종모우로부터 정액을 채취하여 정자의 수 및 동결 전후의 생존성 등을 살펴보았으며, 정자의 운동성 등은 computer assisted sperm analysis(CASA)를 이용하여 측정하였다. 또한, 이들의 수정 능력을 확인하기 위하여 체외수정과 인공수정을 각각 실시하였다. 정액 성상에서는 복제 수소들과 일반 종모우 간에 정액의 양, 정자의 농도 및 동결융해 후의 생존성 등에서 차이가 나타나지 않았다. CASA를 이용한 분석에서 운동성, 곡선 운동 속도(VCL), 직선 운동 속도(VSL) 및 평균 진행 속도(VAP) 등은 복제 수소의 정액이 일반 종모우의 정액에 비하여 유의적으로 높았다(p<0.05). 체외수정에 따른 수정란의 분화율 및 배반포로의 발달율은 복제 수소와 일반 종모우 간에 차이가 나타나지 않았다. 복제소 정액(C-38)을 이용하여 인공수정을 한 5두의 체세포 복제 대리모에서 암수 각각 한 두씩의 건강한 복제 후대 송아지 2두를 생산하였다. 이상의 결과를 종합하여 보면, 실험에 공시된 복제 수소 개체 간의 차이가 나타나기는 하였지만, 복제 수소는 정액 성상과 정자의 운동성 등에서 일반 종모우와 차이가 없었으며. 또한 인공수정을 통해 송아지를 생산함으로써 정상적인 번식능력이 있음을 확인하였다.