현재 한우에서는 영양대사물질 수준에 대한 연구가 미흡한 수준이다. 따라서 본 연구는 한우 번식우에 있어서 임신과 비임신 상태에 있어서 혈액내 주요 영양대사물질을 분석하였다. 한우 번식우 54두를 방목을 하였을 경우에는 혈액내 주요대사물질 6개 항목(AST, ALT, Cholesterol, Glucose, BUN, NEFA)을 분석하였다. 사육방법과 관련된 NEFA수준 조사에서 방목우 임신우 평균은 221±105 uEq/L이고, 사사 사육 임신우 평균 240±121 uEq/L로 나타났으나 유의적인 차이는 없었다. 콜레스테롤 수준은 사사 사육시 임신우 113.33±17.03 mg/dL이었고, 비임신우는 137.25±21.77 mg/dL로 비임신우에서 유의적으로(p<0.05) 높은 결과를 보였다. 사사 사육시 NEFA 수준(임신우 204±119 uEq/L과 비임신우 286±215 uEq/L)도 콜레스테롤 수준과 동일한 경향을 보였다. 분만 전ㆍ후 AST 수치는 각각 105.53±24 IU/L와 81.59±12.55 IU/L으로 분석되어 분만 후에서 유의적으로(p<0.05) 감소한 결과를 보였다. NEFA 수준에서는 분만 전 122±52 uEq/L, 분만 후 497±239 uEq/L 로서 분만 후에서 유의적으로(p<0.05) 높은 결과를 보였다. 이러한 NEFA 수준의 증가는 분만 후 번식우가 필요로 하는 영양대사물질이 증가하였기 때문인 것으로 사료된다. 이러한 결과를 종합하면 한우 번식우의 적정한 영양수준 관리는 생산성을 향상 시킬 수 있을 것으로 사료되며 한우 번식우에 대한 영양수준 연구는 추가적으로 연구가 이루어져야 할 것으로 사료된다.

In the present study, we examined the effect of different lecithin concentrations on spermatozoa characteristics after freeze-thawing. Hanwoo semen was collected from one bull and divided into five groups (tris-citric acid semen extender with 0, 0.1, 0.25, and 0.5% lecithin groups as well as a 20% egg yolk group). Semen extender with 20% egg yolk was used as control treatment. After the freeze-thawing of semen, spermatozoa motility, motility parameters, viability, acrosomal membrane integrity, mitochondrial membrane potential, and plasma membrane integrity were examined. In experiment 1, the effect of different lecithin concentrations on spermatozoa motility and associated parameters was examined. The 0.1% lecithin-treated spermatozoa showed greater fast progressive motility (%) in addition to higher VCL (μm/s), VSL (μm/s), and VAP (μm/s) when compared to other lecithin concentration groups and controls. In experiment 2, the effect of different lecithin concentrations on spermatozoa viability was examined. The 0.1% and 0.25% lecithin addition groups (55.4±7.3 and 51.7±11.2%) exhibited similar viability compared to the control group (54.1±12.6%). In experiment 3, the effects of different lecithin concentrations on viability, acrosomal membrane integrity, and mitochondrial membrane integrity of spermatozoa were examined. The percentage of live spermatozoa with an intact acrosome and high mitochondrial membrane potential in the 0.1% lecithin group was not significantly different compared to the control group (31.2±13.3 vs. 30.5±10.9%). In experiment 4, the effect of different lecithin concentrations on the plasma membrane integrity of spermatozoa was examined. The percentage of spermatozoa with a normal plasma membrane was similar between the 0.1% lecithin and control groups (31.2±13.3 vs. 30.5±10.9%). In conclusion, we suggest that semen extender supplemented with 0.1% lecithin can replace 20% egg yolk without reducing spermatozoa quality.

Understanding the behavior of transgenes introduced into oocyte or embryos is essential for evaluating the methodologies for transgenic animal production. To date, many studies have reported the production of transgenic pig embryos with, however, low efficiency in environment of blastocyst production. The aim of present study was to determine the expression and duration of transgene transferred by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of GFP and then for the transmission of the transgene. Briefly, fresh spermatozoa were bound to exogenous DNA after treatment by Triton X-100 and Lipofectin. When ICSI-MGT was performed using sperm heads with tails removed, the yield of blastocyst (25.3%), treated with Lipofectin (18.8%) and Triton X-100 (19.2%) were observed. Treatments of Lipofectin or Triton X-100 did not further improve the rates of blastocysts. Moreover, the apoptosis rates of embryos were obtained from the control and LIpofectin groups (8.7%, 9.7%, respectively), but were significantly higher in the Triton X-100 group (13.0%). Our results demonstrated that ICSI-MGT caused minimal damage to oocytes that could develop to full term. Moreover, the embryos derived by ICSI-MGT have shown prolonged exogenous DNA expression during preimplantation stage in vivo. However, more efforts will be required to improve the procedures of both sperm treatments cause of high frequency of mosaicisms.

Oct4 and Nanog are transcription factors involved in pluripotency of stem cells. In general, Oct4 is up-regulated by Nanog. In previous results, however, Oct4 was differentially regulated by various doses of Nanog in P19 cells. High dose Nanog down-regulated the Oct4 expression. This negative feedback event was performed by DNMT and HDAC through the inhibitor assays (5-AZA-cytidine and trichostatin A). To identify the precise recruited sites for DNMT and HDAC, ChIP assay was performed in differential doses of Nanog. As a result, HDAC1, HDAC2, DNMT3A and Nanog were recruited to CR2, CR3, CR1, and CR4 upon high dose Nanog, respectively. Next, to investigate the differentiation potency of the cells upon high dose Nanog, RT-PCR with specific markers for three germ layers was performed. However, there was no increase for three germ layers in high dose Nanog treated cells except E-cadherin expression. E-cadherin is a specific marker for epithelial cells. Taken together, high dose Nanog induces Oct4 down-regulation and results in differentiating embryonic carcinoma cells to epithelial cell type. These results will be helpful for study on regulation of pluripotency-related genes in embryonic stem cells. * This study was supported by 2012 Post Doctoral Fellowship Program of National Institute of Animal Science, Rural Development Administration, Republic of Korea. This work received grant support from the Agenda Program (No.PJ007577), Rural Development Administration, Republic of Korea.

Phosphoprotein Enriched in Astrocytes (PEA15) is a 15kD-sized intracellular signaling protein, highly expressed in astrocytes and constitutively expressed in peripheral tissues. Recently it was found that PEA15 expression was elevated in patients suffering type 2 diabetes and suggested to be involved in the syndrome of insulin resistance. To investigate the functional role of PEA15 for the control of blood glucose level, we produced a transgenic pig over-expressing mouse PEA15 (mPEA15). As a model animal, pig has many advantages. They have a higher fecundity and a short generation time and are physiologically similar to human. Using the transgenic pig, we carried out a series of experiments to establish a link between PEA15 expression and the insulin resistance. Our results suggested that, compared with control pig, mPEA15 pig has, (1) a higher blood resistin level, (2) a lower cell membrane-embeded GLUT4 level, and (3) a lower glucose clearing ability based on oral glucose tolerance test (OGTT). When our results combined, it can be concluded that mPEA15 over-expressing pig has many symptoms of insulin resistance and these pigs will become a useful disease model to investigate diabetes mellitus in the near future.

Oct4 and Nanog are well-known transcription factors related with self renewal of embryonic stem cell. In low-dose of Nanog, transcription of oct4 is increased; however, oct4 is down-regulated upon high-dose of Nanog. There is a negative feedback loop between oct4 and Nanog. To identify this regulation, we generated 4 nested sets for mouse oct4 promoter. Luciferase activities of oct4 were declined upon high-dose Nanog in all constructs. The declined effects of oct4 upon high-dose Nanog were moderated with DNMT and HDAC inhibitors (5-AZA-cytidine and trichostatin A) in 3 constructs (1867, 1346, 754). But, one construct (2179) was only sensitive to TSA. Taken together, these effects were also represented in semi-quantitative RT-PCR and Western blotting data. These data suggest that negative regulation of oct4 gene upon high-dose Nanog would be accomplished by DNMT and HDAC. Further, it will be studied whether these constraining molecules bind to CR1-4 region of oct4 promoter upon low- and high-dose of Nanog.

The overexpression of Phosphoprotein Enriched in Astrocytes (PEA15) gene is commonly found in human diabetic patients. The overexpression of this gene in skeletal muscle and fat tissues have been reported to cause insulin resistance, thereby impairing insulin stimulated glucose uptake. We introduced a gene of mouse PEA15 (mPEA15) and enhanced green fluorescent protein (EGFP) into fertilized one cell pig zygotes using microinjection, and produced a piglet that showed overexpression of mPEA15 in the muscle tissues and expression of EGFP in the ear tissues and hooves. RT-PCR RFLP, southern blot and FISH analysis showed that the tissues carried the transgene. Real-time RT-PCR and western blots demonstrated that PEA15 gene was overexpressed in the various tissues and muscle tissues, respectively. These facts suggest that expression vector system is normally expressed in the trnasgenic (TG) pigs. To use as animal diseases model for type 2 diabetes, further study is necessary to confirm whether diabetes occur in these TG pigs, especially insulin resistance.