Earticle

NOD/SCID 마우스는 선천성 면역결핍을 지닌 마우스로서 이종 세포 및 조직 이식을 위한 실험동물로서 가장 많이 활용되고 있다. 본 연구는 돼지의 골수조직에서 채취한 조혈줄기세포를 면역결핍마우스의 정맥 주입을 통하여 생체 내 주입을 실시한 결과, 마우스의 조혈조직에서 대단히 높은 돼지 T 면역세포의 증식이 관찰되었다. 유세포 분석기를 이용해 돼지 골수 조혈세포 생체 이식 6주의 마우스에서의 돼지 T 면역세포의 증식과 분화 특성을 분석한 결과, 마우스 조혈조직인 골수(5.4±1.9%), 비장(15.4±7.3%), 간(21.3±1.4%), 림프절(33.5±32.8%)에서 돼지 조혈줄기세포 유래 T 세포의 증식과 분화가 관찰되었고, 돼지 helper T 세포와 cytotoxic T 세포의 발달도 확인되었다. 또한 조직 면역염색을 통하여 마우스의 비장조직에 이식한 돼지 면역세포의 증식을 관찰하였다. 본 연구는 NOD/SCID 마우스를 이용해 돼지 조혈줄기세포로부터 T 면역세포로의 분화 및 발달과정을 생체 내에서 분석할 수 있는 유용한 동물모델로서 이용할 수 있음을 보여준다.

The nonobese diabetic / severe combined immune deficiency (NOD/SCID) has been used for determination of proliferation and differentiation of hematopoietic stem cells as xenotransplantation animal model. In this study, we transplanted porcine hematopoietic cells from bone marrow into NOD/SCID mice via intravenous injection to confirm the activity of differentiation and proliferation for porcine hematopoietic cells in vivo. Interestingly, we observed the results of high efficiency with pig T lymphocytes in hematopoietic organs, liver, spleen, lymph node, and bone marrow in NOD/SCID mice. The porcine CD3+ T cells were detected with 5.4±1.9% in bone marrow, 15.4±7.3% in spleen, 21.3±1.4% in liver, and 33.5±32.8% in lymph node of NOD-SCID mice at 6 weeks after transplantation. Furthermore, immunohistochemical analysis showed the high engraftment of porcine T lymphocytes in spleen of NOD/SCID mice. Our data suggest that NOD/SCID mice are excellent animal model to determinate the generation and function of pig T lymphocytes.

The present study investigated the effects of follicle stimulating hormone (FSH) and human chorionic gonadotrophin (hCG) on the nuclear maturation of canine oocytes. Oocytes were recovered from mongrel female ovaries in various reproductive states; follicular, luteal or anestrous stage. Oocytes were cultured in serum-free tissue culture medium (TCM)-199 supplemented with various concentrations of FSH (Exp. 1: 0, 0.5, 1.0 or 10 IU) or hCG (Exp. 2: 0, 0.5, 1.0 or 10 IU) or both (Exp. 3: 1 IU FSH + 1 IU hCG) for 72 hr to determine the effective concentration of these hormones, and to examine their combined effect. After maturation culture, oocytes were denuded in PBS containing 0.1% (w/v) hyaluronidase by gentle pipetting. The denuded oocytes were stained with 1.9 μM. Hoechst 33342 in glycerol and the nuclear state of oocytes was evaluated under UV light. More (p<0.05) oocytes matured to MII stage when follicular stage oocytes were supplemented with 1 IU FSH (6.2%) compared with the control, 0.1 or 10.0 IU FSH (0 to 1.2%). Significantly higher (p<0.05) maturation rate to MII stage was observed in follicular stage oocytes supplemented with 1.0 IU hCG (7.2%) compared with the control or other hCG supplemented groups (0 to 1.5%). However, the combination of FSH and hCG did not improve the nuclear maturation rate of canine oocyte (2.4 %) compared with FSH (6.2%) and hCG alone (7.2%). In conclusion, FSH or hCG alone significantly increased the maturation of canine oocytes to MII stage.

가축의 번식과 수요를 조절할 수 있는 생물학적 자극 통제 수단으로 새로운 돼지 페르몬성 냄새 물질를 탐색하고자, 기질 분자로서 2-cyclohexyloxytetrahydrofurane (A), 2-phenoxytetrahydrofurane (B) 유도체들의 설명인자인 물리화학 파라미터와 돼지 페로몬의 수용체 (pOBP)에 대한 결합 친화력 상수(p[Od.]50) 간의 2D-QSAR 모델을 유도하고 검토하였다. 2D-QSAR 모델은 결합 친화력 상수를 약 96.4% 설명하는 매우 양호한 모델(r2=0.964)로서 분자내 치환기의 소수성(SL) 상수에 관한 적정값이 (SL)opt.=1.418일 때 가장 높은 결합 친화력을 나타냄을 알았다. 그러므로 분자내 치환기의 소수성 인자가 결합 친화력 상수에 가장 큰 영향을 미치는 중요한 요소이었다.

To search of a new porcine pheromonal odorant for biostimulation control system technologies to offer a potentially useful and practical way to improve reproductive efficiency in livestock species, the two dimensional quantitative structure-activity relationship (QSAR) models between physicochemical parameters as descriptors of 2-cyclohexyloxytetrahydrofurane (A), 2-phenoxytetrahydrofurane (B) analogues and binding affinity constants (p [Od.]50) for porcine odorant-binding protein (pOBP) as receptor of pig pheromones were derived and disscused. The statistical quality of the optimized 2D-QSAR model is good (r2=0.964) and accounts for 96.4% of the variance in the binding affinity constants. It was found that the binding affinity constants were dependent upon the optimal value, (SL)opt.=1.418 of substituent lipole (SL) in molecules. Therefore, the SL constant was very important factor for binding affinity.

During early embryo development, Oct-4 is an important transcription factor for the early differentiation. The present study was first examined methylation status in distal enhancer and promoter region of Oct-4 during mouse pre-implantation embryo development. In oocyte and sperm, high methylation was observed in both distal and proximal of promoter in Oct-4. Following fertilization, relatively high methylation level remained until 8-cell stage embryos, but decreased at the morula and blastocyst stage. Specific gene knock down of Oct-4 by siRNA injection into zygote induced higher methylation rates of both distal and proximal region ofpromoter of Oct-4. These results suggest a functional link between the DNA methylation status of distal and promoter region in the Oct-4 gene and the gene sequence-specific transcriptional silencing by exogenous siRNA injection during mouse pre- implantation embryos.

본 연구는 소 체외수정란과 체세포 복제란의 초자화 동결 및 융해 후 생존능을 검토하였다. 배반포로 발육된 체외수정란 및 체세포 복제란을 초자화 동결법에 의해 동결하였다가 융해하여 생존율 및 배양 후 부화율을 검사하였다. 체외수정란 배반포를 초자화 동결 융해한 결과, 확장배반포가 배반포기 난자에 비하여 생존율(82.1%, 96/117)과 부화율(64.1%, 75/117)에서 모두 유의적으로 높았다(p<0.05). 핵이식 배반포 복제란을 초자화 동결 융해한 경우도 체외수정란과 비슷한 경향을 보여 확장 및 부화배의 생존율과 부화율이 각각 81.1%(30/37)와 78.3%(29/37)로, 배반포(각각 71.8 및 53.8%)에 비하여 다소 높게 나타났다. 본 연구의 결과는 초자화 동결 방법에 의해서 소 체외수정란과 체세포 복제란을 성공적으로 동결할 수 있으며, 특히 후기 배반포기 단계에서 초자화 동결 시 높은 생존율과 부화배 형성율을 얻을 수 있음을 보여준다.

This study was conducted to examine the development of in vitro fertilized (IVF) and nuclear transfer (NT) embryos following vitrification. IVF and NT embryos developed to the blastocyst stage were equilibrated by 3 steps, vitrified and thawed, and their survival and hatching rates were examined. In IVF embryos, higher survival (82.1%, 96/117) and hatching rates (64.1%, 75/117) were obtained respectively after thawing and culture in expanded blastocysts compared to blastocysts (p<0.05). High survival and hatching rates were also obtained by vitrification of NT blastocysts, especially in expanded and hatching blastocysts (81.1 and 78.3%, respectively). The result of this study shows that IVF and NT blastocysts, especially late stage blastocysts, are successfully cryopreserved by vitrification.

본 연구는 인간 난관액 또는 자궁액 내에 존재하는 에너지원이 생쥐 2-세포기 배의 체외 발달에 미치는 영향을 조사하기 위하여 실시하였다. ICR 암 생쥐에 5 IU hCG 주사 후 46～50시간에 2-세포기 배를 회수하였다. 회수된 배는 3가지 배양 조건 [대조군: 0 mM, Group A: glucose(G) 0.5 mM + pyruvate(P) 0.32 mM + lactate(L) 10.5 mM, Group B: G 3.15 mM + P 0.1 mM + L 5.83 mM]에서 72시간 배양하였다. 배양 24 시간에 상실배 출현율은 group A (72.3%)와 group B (56.6%)가 대조군(34.9%)보다 유의하게 높았다(p<0.05). 그러나 48시간에 배반포기 배 출현율은 대조군(51.8%)이 group A (39.8%)와 group B (28.9%)보다 유의하게 (p<0.05) 높았다. 72시간에 투명대 부착 (ZiB, 41.0～51.8%), 투명대 탈출 (ZeB, 18.1～32.5%) 및 총 배반포기 배 출현율 (68.7～73.5%)은 실험군 간에 통계적인 차이가 없었다. 배반포기 배의 평균 세포수와 ICM 세포수는 group A (70.8, 13.4)와 group B (64.4, 11.8)가 대조군 (53.1, 5.7)보다 유의하게(p<0.05) 많았고, 통계적인 유의차는 없었으나 group A가 group B보다 많은 경향이었다. 총 세포수에 대한 ICM 비율은 group A(22.9%)와 group B(23.7%)가 대조군(14.2%)보다 유의하게(p<0.05) 높았다. 영양배엽(TE) 세포수(34.1～45.1)는 실험군 간에 통계적인 차이가 없었다. ICM에 대한 TE 비율(ICM:TE ratio)은 대조군(1:6.0)이 group A(1:3.4)나 group B(1:3.4)보다 유의하게(p<0.05) 높았다. 생쥐 2-세포기 배를 배양하여 72시간까지의 배 발달율을 살펴보면 배양액에 에너지원을 첨가하는 것이 효과적이었으며, 자궁액 농도보다는 난관액 농도로 에너지원을 조절했을 때 배 발생 능력이 높은 경향을 보였다.

This study was conducted to examine the effects of energy substrates in different concentration of carbohydrates in the human oviduct and uterus on the in vitro development of mouse 2-cell embryos. Two-cell embryos were collected from ICR female mice at 46～50 hr after 5 IU hCG injection, and cultured in three different media [Control: 0 mM, Group A: glucose (G) 0.5 mM + pyruvate (P) 0.32 mM + lactate (L) 10.5 mM, Group B: G 3.15 mM + P 0.1 mM + L 5.83 mM] for 72 hr. Rates of morula formation of group A (72.3%) and B (56.6%) were significantly higher (p<0.05) than that of control (34.9%) at 24 hr. However, blastocyst rate was significantly higher (p<0.05) in control (51.8%) than group A (39.8%) and B (28.9%) at 48 hr. At 72 hr, no differences were found in the number of zona-intact, zona-escape and total blastocysts among groups. Mean and ICM cell numbers were significantly higher (p<0.05) in group A (78.0, 13.4) and B (64.4, 11.8) than control (53.1, 5.7), respectively. The percent of ICM were significantly higher (p<0.05) in group A (22.9%) and B (23.7%) than control (14.2%). No differences were found in the TE cell numbers (34.1~45.1). The ICM:TE ratio was significantly higher (p<0.05) in control (1:6.0) than group A (1:3.4) and B (1:3.4). This study shows that energy substrates added to culture media especially, the oviductal level of carbohydrates increase the developmental capacity of 2-cell mouse embryos.

본 연구는 인공수정 기법을 이용한 칡소의 증식효율을 검토하고자 칡소 종모우의 정액을 채취하여 종모우별 정액성상, 동결융해 후 정액의 특성, 수태율 평가를 위한 체외수정 및 인공수정 후 수태율과 송아지의 모색 분포 등에 대하여 검토하였다. 정액은 호반모를 가진 수컷 6두에서 채취하였으며, 동결 융해 후 체외수정을 실시하였다. 총 18두의 칡소 및 일반 한우 암컷에 인공수정 후 수태율을 확인하였고, 생산된 40두의 송아지를 대상으로 모색을 분석하였다. 칡소 정액의 동결 전 일반 성상은 개체 간에 차이가 없었으며, 동결 후의 생존율과 활력에서도 개체별 차이가 없었다. 체외수정에 의한 수정능 분석 결과, 수정율에는 차이가 없었으나 배반포 발육율이 일반 한우에 비하여 칡소 정액 체외수정 시 25.9%로 유의하게 높았다(p<0.05). 인공수정 후 임신율에는 일반 한우와 칡소 간에 차이가 없었다. 칡소 정액의 인공수정 후 태어난 송아지의 모색 표현형은 40두의 송아지 중 호반모(Tiger)가 17두(42.5%)로, 황흑모, 흑황모, 흑모 및 황모에 비하여 높았으나 전체적으로 낮은 발현율을 나타내었다. 또한 호반모 암컷에 인공수정한 경우에도 55%의 송아지만 호반모로 발현되었다. 이상의 결과는 인공수정에 의한 칡소의 증식이 가능하나 호반모 칡소의 발현 빈도를 높이기 위한 연구가 필요할 것으로 사료된다.

This study was conducted to examine the characteristics of fresh and frozen semen, proliferating efficiency by AI, and the coat color of offsprings in Korean Native Striped Cattle (Bos namadicus Falconer, Chikso). Semen were collected from 6 heads of tiger-coated male Chikso. In vitro fertilization (IVF) was conducted with frozen-thawed semen and in vitro matured Korean native brown cattle (general Hanwoo)) oocytes. Total 18 heads of Hanwoo and Chikso were inseminated using Chikso semen. Coat colors of total 40 offsprings produced by AI were evaluated. The characteristics of the fresh and frozen-thawed Chikso semen did not differ among individuals. In vitro fertilization rate of Chikso semen was not different from that of general Hanwoo semen. However, developmental rate to the blastocyst stage of IVF embryos was higher in Chikso semen (25.9%) than in general Hanwoo semen (p<0.05). There was no difference in conception rate after AI between Chikso and general Hanwoo. The coat colors of offsprings varied, only 42.5% (17/40 heads) of offsprings had tiger coat color. Futhermore, only 55% of offsprings produced from the tiger-coated recipients had tiger coat color. This result shows that proliferation of Chikso by AI is possible, but further research approaches may be needed to enhance the productivity of tiger-coated Chikso.

Astaxanthin is a kind of carotenoid compounds, having a antioxidant and anti-inflammatory activities. The antioxidative mechanism by which carotenoid scavenge free radicals has been clearly elucidated, but has not tried for the development of mammalian preimplantation embryo. This study was conducted to investigate the antioxidative effect of astaxanthin on in vitro development of porcine in vitro fertilized embryos. Porcine embryos derived from in vitro fertilization (IVF) were cultured in 5% CO2 in air at 38.5℃ in PZM-3 medium supplemented with different dosages of astaxanthin (0, 1, 5 and 10 M) and taurine (0, 1, 2.5 and 5 mM) as a positive control, and execute to compare the effects of various antioxidants such as taurine, melatonin and asculatin on in vitro development. The proportions of embryos developed to the blastocyst stage were increased when 1 and 5 M of astaxanthin (26.6 and 23.4%, respectively) and 1 and 2.5 mM taurine (25.8 and 26.4%, respectively) were supplemented, compared to controls (p<0.05). Also, various antioxidant-treated groups were significantly higher rates of blastocysts (astaxanthin, 27.4%; taurine, 29.1%; melatonin, 26.8%; aesculetin, 27.9%, respectively ) than control (18.8%). There was no difference in mean cell number of blastocysts between antioxidants and control. This result indicates that astaxanthin has an antioxidant feature when porcine IVF embryos were cultured in vitro.

Aging causes thymus involution, and genes in thymus play an important role in the development of the immune system. In this study, we compared genes expressed in thymus of neonatal and peripubertal rats using annealing control primers (ACPs)-based GeneFishing polymerase chain reaction (PCR) and semiquantitative reverse transcription (RT)-PCR. We identified 10 differentially expressed genes (DEGs) with 20 ACPs. Of 10 DEGs, bystin-like, collagen type V alpha 1 (COL5A1), and T-cell receptor beta-chain segment 2 (TCRB2) that are related to immune-function were detected in rat thymus. Bystin-like and TCRB2 were up-regulated, while COL5A1 was down-regulated in peripubertal thymus. Semiquantitative RT-PCR confirmed postnatal changes in expression of bystin-like, COL5A1, and TCRB2. These results suggest that bystin-like, COL5A1, and TCRB2 could regulate immune function controlled in thymus as age increases.