This study examined the effects of tamoxifen at different concentrations and treatment periods on proliferation of a human cervical cancer cell line, HeLa. As in the previous studies, Estrogen did not have an effect on the cellular proliferation at low concentrations but significantly reduced proliferation at high concentration (5 ug/ml) based on MTT assay. Treatment with tamoxifen had similar effects. It did not have any significant effect on the HeLa cell proliferation at low concentrations, but significantly reduced proliferation at high concentration (10 uM). In addition, combined treatment with both estrogen and tamoxifen did not alter the inhibitory effects of either estrogen or tamoxifen on cellular proliferation. The inhibitory effects by tamoxifen on HeLa cell proliferation did not differ among different treatment periods. This suggests that tamoxifen may exert anti- proliferative effects at high concentration but does not have synergistic or antagonistic effects against estrogen on HeLa cell proliferation.

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells; however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to locate Ski protein in the rat ovary during luteinizationto predict the possible role of Ski. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rats, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of the corpus luteum (CL). Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski)was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggests that its expression is regulated post-transcriptionally.

For reconstituting genetic resource(Korean Native Chicken: KNC) with grem-line chimeric chicken made with cryopreserved biastdermal cells, the experiments were carried out to optimize cryopreservating conditions. Stage X biastdemal cells were collected from KNC embryos and dissociated. Cells were susupended in medium containing cyopretectant and fetal bovine serum(FBS), and distributed into plastic ampules. Cell susupensions were seeded to induce ice formation at — 7℃ to — 35℃ at in the experiments, the effect of modification of dissociation way, concentration of FBS and cell density on the vaibility of frezen-thawed cells were investigated by trypan blue exclusion. Then change the way of cell dissociation from pipetting to short time vortexing, viability of frozen- thawed cell tended to be increaced from 29 % to 52 %. Increase concentraition of FBS in frozen medium from 20 % to 80 % made viability of thawed cell from 28 % to 35 %. The viability of thawed cells were 33.9% frozen at 2 embryos/ 0.5 ml, and 43.6 % frozen at 20embryos/0.5 ml. Furthermore, combination of three modifications make big improvement. The viability of frozen-thawed cell was 60 % for combinated method, and 41 % for general method. This result means the advance to practical cryoreservation of blastdermal cell of the KNC(Ogolgye breed).

Sloan-Kettering virus gene product of a cellular pro-oncogene c-Ski is an unique nuclear pro-onco protein and belongs to the Ski/Sno proto-oncogene family. Ski plays multiple roles in a variety of cell types, it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. Ski protein is implicated in proliferation/differentiation in a variety of cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of this study was, by means of immunohistochemical methods, to locate Ski protein in the rat ovaries during ovulation and corpora lutea(CL) formation to predict the possible involvement of Ski in luteinization. In addition, to examine whether the initiation of luteinization with luteinizing hormone(LH) directly regulates expression of Ski in the luteinized granulosa and luteal cells after ovulation by in vivo models. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rat, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone(LH) surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of corpus luteum(CL). These results indicate that Ski is profoundly expressed in the luteinized granulosa cells and luteal cells of CL during luteinization, and suggest that Ski may play a role in luteinization of granulosa cells.

Ski protein is a nuclear transcription factor that does not bind DNA directly. Due to its unique binding properties with multiple factors, Ski could perform various roles in the regulation of both cellular proliferation and differentiation. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells; however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to examine whether the initiation of luteinization with luteinizing hormone (LH) directly regulates expression of Ski in the luteinized granulosa and luteal cells after ovulation by in vitro models. RT-PCR and real time PCR analysis respectively revealed that LH had no effect on c-Ski mRNA expression in the cultured granulosa cells regardless of LH treatment. Though Ski protein isabsent in granulosa cells of preovulatory follicle, its mRNA(c-Ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated post-transcriptionally. Moreover, expression of mRNA of Arkadia, an E3 ubiquitin ligases, in luteinizing granulosa cells in vivo was assessed by real time-PCR. The levels of Arkadia mRNA expression were unchanged during follicular growth and post ovulatory luteinization. These findings suggest that Ski protein level may be regulated during luteinization at translational and/or post-translational level but not by Arkadia.

MAC-T cells, bovine mammary epithelial cell line, have been utilized to investigate bovine lactation system. A lactogenic phenotype of the cell is generally induced by combination of dexamethasone, insulin and prolactin (PRL). Effect of vitamin A derivative retinoic acid (RA), well reported as an inducer for differentiation in many cells, to MAC-T cell has not been studied. The objective of this study was to confirm effect of differentiation potential by RA treatment in MAC-T cells and to test effect of combination of RA and PRL treatment. In RA or PRL treatment groups, both has induced morphological change to secrete milk of MAC-T cells. Combination of RA and PRL treatment group has presented noticeable lactogenic phenotype among the all group. This phenotype observed at four days after treatment and showed critical morphological change that was rouphly spherical structure at eight days. RA alone treatment showed slightly inhibition of proliferation in the MAC-T cells, but co-treatment with PRL was improved the cell growth more than control group. MTT assay result and Bcl-xL/Bax ratio of mRNA abundance also was entirely consistent with earlier one. RA-induced differentiation of MAC-T cells has increased αs1-casein, αs2-casein and β-casein mRNA expression compared to PRL treatment group. Expression of αs1-casein, αs2-casein and β-casein genes represented the maximum value in the combination of RA and PRL treatment group at four days. The value of each casein gene expression was 4-, 5.5- and 5.9-fold, respectively, as compared with PRL alone treatment in the MAC-T cells. Protein level of β-casein releasing to the medium also induced the highest level at four days. These results provide evidence that RA can induce the differentiation of MAC-T cells and have synergetic effect with PRL.

The reactive oxygen species (ROS) generated during the somatic cell transfer nuclear (SCNT) procedures may cause the mitochondrial dysfunction and DNA damage, which may result in restricts the reprogramming of SCNT embryos and play a key direct role in apoptosis. The present study was conducted to investigate the effect of antioxidant treatment during the SCNT procedures on the inhibition of mitochondria and DNA damages in bovine SCNT embryos. The reconstituted oocytes were treated with antioxidants of 25 μM β-mercaptoethanol (β-ME) or 50 μM vitamin C (Vit. C) during the SCNT procedures. In vitro fertilization (IVF) was performed for controls. Mitochondrial morphology and membrane potential (ΔΨ) were evaluated by staining the embryos with MitoTracker Red or JC-1. Apoptosis was analyzed by Caspase-3 activity assay and TUNEL assay, and DNA fragmentation was measured by comet assay at the zygote stage. Mitochondrial morphology of non-treated SCNT embryos was diffused within cytoplasm without forming clumps, while the IVF embryos and antioxidant treated SCNT embryos were formed clumps. The ΔΨ of β-ME (1.3±0.1, red/green) and Vit. C-treated (1.4±0.2, red/green) SCNT embryos were significantly higher (p<0.05) than that of non-treated SCNT embryos (0.9±0.1, red/ green), which similar to that of IVF embryos (1.3±0.1, red/green). Caspase-3 activity was not difference among the groups. TUNEL assay also revealed that little apoptosis was occurred in SCNT embryos as well as IVF embryos regardless of antioxidant treatment. Comet tail lengths of β-ME and Vit. C-treated SCNT embryos (337.8±23.5 μm and 318.7 ±27.0 μm, respectively) were shorter than that of non-treated SCNT embryos (397.4± 21.4 μm) and similar to IVF embryos (323.3±10.6 μm). These results suggest that antioxidant treatment during SCNT procedures can inhibit the mitochondrial and DNA damages of bovine SCNT embryos.

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.

Recent advances in stem cell biology have shown that terminally differentiated somatic cells can be directly converted to the different types of somatic cells such as neurons and cardiomyocytes with defined sets of transcription factors without going through a pluripotent state. Recently, it was demonstrated that the hepatocyte-specific transcription factors, Hnf4α plus Foxa1, Foxa2 or Foxa3 could erase somatic memory and reset hepatocyte program on the differentiated somatic genome. Here, we show that Foxa3 together with Hnf4α could efficiently reprogram fibroblasts into hepatocytes. However, the direct conversion into hepatocytes is not observed with Hnf4α plus Foxa1. After two weeks of retroviral transduction of Hnf4α and Foxa3, we observed epithelial colonies emerged from starting fibroblasts and were able to establish stable hepatocyte cell lines, namely induced hepatocytes (iHep cells). The iHep cells closely resemble primary hepatocytes in a number of characteristics such as their polygonal shapes, the hepatic gene expression patterns and the presence of E-cadherin signals as determined by immunocytochemistry. In addition, iHep cells show the storage of glycogen as revealed by Periodic acid-Schiff (PAS) staining, indicating that iHep cells are functionally similar to their in vivo counterparts. Taken together, our findings suggest that the combination of hepatic transcription factors, Hnf4α with Foxa3 but not Foxa1 could induce hepatocyte fate on the differentiated somatic cells.

INTRODUCTION In rodent, molecular markers of spermatogonia, spermatocyte, spermatid and sperm have been identified. It has been reported that DBA, PGP 9.5 and NanpG can be the markers for spermatogonia in pig. For further understanding the spermatogenesis of the pig on morphological and molecular level, we report identification of testicular cells in neonatal and pubertal pig testis, and a putative marker for spermatogonia and spermatid in pig testis. MATERIALS AND METHODS Neonatal (3 day) and pubertal testis (150 day) was cut and fixed in Bouin’s solution for immunohistochemistry (IHC). Gonocytes were isolated from neonatal testis for immunocytochemistry (ICC). Based on references (Phillips et al., 2010), thirteen antibodies (VASA, Oct4, NanoG, PGP 9.5, DAZL, SCF, GFR-alpha 1, PLZF, c-kit, integrin-beta1, Thy1, Sohlh1 and CD9) were used for IHC and ICC. Paraffin section was performed for IHC. Gonocytes were attached to the APS-coated slides for ICC. HRP-conjugated and florescent-labeled secondary antibody was used for IHC and ICC, respectively. RESULTS In histological analysis, spermatogonia and Sertoli cells, which are enclosed by seminiferous tubule and connective tissue, were observed in neonatal testis. However, complete spermiogenesis, including spermatocyte, spermatid and spermatozoa, was not observed in neonatal testis. Spermatocyte, spermatid and elongated spermatid were observed in pubertal testis but matured spermatozoa were not observed. As a result, two antibodies (PGP 9.5 and GFR-alpha1) of thirteen antibodies were available for IHC and ICC. As reported in other studies, PGP 9.5 protein was detected in spermatogonia of ne-onatal in IHC. In addition, it was observed in spermatogonia of pubertal testis. GFR- alpha1 protein was detected in spermatogonia of neonatal testis and spermatid of pubertal testis. In ICC, PGP 9.5 protein was detected in gonocytes as reported in other studies. GFR-alpha1 was also observed in gonocyte isolated from pig testis. In this study, we found that 1) only spermatogonia exist in neonatal pig testis (3 day), 2) GFR-alpha1 is a new marker for spermatogenesis in pig testis.

소의 정장은 정관, 정소상체와 부생식선으로부터의 분비물로 구성된다. 정자는 정소에서 생성되어 정소상체에서 성숙하고, 사출되는 과정에서 부생식선으로 분비되는 다양한 성분 들 과의 접촉 한다. 자연교배시 정장은 사출된 정자와 같이 암컷의 생식기도에 도달하여 생리 학적 및 면역학적인 영향을 미치는 것으로 보고되었다. 정장성분의 분석에 관한 연구들은 정자의 수정능 획득 기작을 파악하기 위한 목적과 프로테옴(단백질체학)의 연구기법들을 활 용하여 수정율에 증가에 관련된 marker들을 발견하려는 목적에서 수행되었으며 궁극적으 로 종모우의 수정율을 증가시키기 위한 것이다. 정자의 수정능 획득시 정자의 세포막에는 지 질 구성의 변화가 일어나는데, 정자의 세포막에서 콜레스테롤이 배출되기 때문인 것으로 보고 되어 왔다. 배출되는 콜레스테롤은 정장 단백질이나 난관세포로 이행된다. 본 연구는 수정 능 획득과 콜레스테롤의 배출 기작에 관련된 정장 단백질들을 한우와 칡소의 정장에서 동 정하여 수정률향상을 위한 marker들로서 활용할 수 있는지를 판단하기 위한 목적으로 수행 되었다. 정장단백질 중에서 정자의 콜레스테롤 배출과 관련되어 수정능획득에 영향을 미치 는 정장단백질들로서 BSP-30 및 spermadhesin Z13 등이 보고되었었다. 본 연구에서는 한 우 4 두와 (3 두: 한우시험장, 1 두: 전라북도 축산시험장)와 칡소 3 두 (2 두: 전라북도 축 산시험장, 1 두: 유전자원시험장)에서 정액을 채취하고, 원심분리하여 정장을 분리하였다. 정장은 단백질 정량 후 SDS-PAGE 방법으로 1차원 전기영동을 실시한 후 분리된 단백질들 을 비교하였고, 특이 단백질 밴드를 동정하였다. 그 이후 2차원 전기영동으로 정장 단백질 을 분리하고 PDQuest 프로그램으로 spot들을 분석하였으며, 특이 단백질들을 동정하였다. 1차원 전기영동으로 분리 후 동정된 소의 정장 단백질은 plasma serine protease inhibitor 였고, 2차원 전기영동으로 분리 후 동정된 소의 정장 단백질들은 Pdc-109, BSP-30, seminal plasma protein A3, spermadhesin Z13 및 spermadhesin-1(aSFP)이었다. 일부 동정된 단백질들은 발현된 양과 위치의 차이가 존재하였다. 이들 단백질 중에서 Pdc-109, BSP-30, seminal plasma protein A3는 수정에 긍정적인 역할을 하고, spermadhesin Z13와 spermadhesin- 1(aSFP)는 수정에 부정적인 역할을 하는 것으로 보고되었었다. 본 연구결과를 기 반으로 이들 정장 단백질의 발현양상과 유전자들의 변이를 조사하고, 그 결과를 토대로 정 장 단백질의 작용기작을 조절하면 소의 수정률 향상에 기여할 것으로 기대된다. * 연구는 농촌진흥청 차세대 바이오그린21사업(과제번호: PJ008047)의 지원에 의해 이루어 진 것임.

The main purpose of this study is to estimate the effect of adding Tea-N-Tris to the freezing buffer for miniaturepig sperm. In particular, we attempted to identify the association between the MMPs expression and the survival and viability of sperms. Prior to freezing, sperms in LEY without Tea-N-Tris showed 40.3±2.8% viability and 60.3±1.3% acrosome intact rate at 4℃. After freezing, sperms stored in LEY (lactose+Egg yolk) with Tea-N-Tris (=TLE) showed the highest viability (57.4±1.8%) and acrosome intact rate (65.6±4.6%). In accordance with this, DNA fragmentation was the highest among sperms frozen in LEY while the lowest fragmentation was observed among sperms frozen in TLE. When these sperms were used for in vitro fertilization (IVF), the LEY group showed lower rate of blastocyst development, although the difference was not statistically significant. Meanwhile the rate of blastocyst development appeared similar when sperms from TLE and TFGE (Tea-N-Tris+Fructose+ Glucose+Egg yolk) group were used for IVF. We observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of MMP-9 and MMP-2 was the highest in sperms frozen in LEY, Meanwhile, sperms from the TFGE and TLE group showed lower level of MMP-9 and MMP-2 expression in the order of TLE being the lowest. Together, these results indicate that adding Tea-N-Tris to the sperm freezing buffer would not only suppress MMPs expression but also minimize DNA fragmentation, providing a mean to improve the success rate in the in vitro manipulation of miniaturepig sperms. * This work was supported by BioGreen 21 Program (No. PJ008029). Rural Development Administation, Republic of Korea.

칡한우는 한우의 품종의 하나로 FAO에 등록되어 있고, 1,700여두가 사육되고 있는 것으 로 추정하고 있다. 그러나 사육두수가 적어 멸종위험품종으로 분류되고 있고, 특히 번식 가 능한 수소의 숫자가 매우 적어 근친의 위험에 노출되어 있다. 칡소에 대한 연구는 유전적 특성(이 등, 2002), 정액동결(조 등, 2011), 모색분포(박 등, 2007) 등으로 아주 제한적으로 보고되고 있어서 증식에 필요한 정액 동결관련 연구가 필요할 것으로 생각된다. 따라서 본 연구에서는 칡한우 수소의 활용도를 높이기 위하여 생후 15개월령 단계에서 2-3일 간격으 로 2회 채취하여 동결한 후 거세·비육하여 도체성적을 평가하였다. 정액채취는 인공질(FHK, Japan), 동결희석제는 Triladyl(Minitube, Germany) 희석제를 이 용하였다. 정자의 motility는 CASA system(Minitube, Germany)상에서 Total motile (TM)과 Progressive motile (PM) 비율을 측정하였다. 15개월령 칡소와 30개월령 칡소의 신선 및 동결융해 정액의 활력을 조사한 결과, 15개월 령 수소의 정액량이 평균 2.3 ml이었고, 30개월 수소의 정액량은 평균 5.0 ml로서 유의하게 높았다(p<0.05). 채정 직후의 신선정자의 TM 비율이 15개월 및 30개월 수소가 각각 평균 93.7% 및 88.3%, PM 비율은 각각 97.0% 및 88.3%로서 PM 비율은 15개월령이 유의하게 높았다(p<0.05). 한편 동결융해 정자의 TM 비율은 각각 평균 56.0% 및 58.0%였고, PM 비 율은 각각 평균 64.0% 및 70.7%로서 차이가 없었다. 칡소 15개월 정액채취가 완료된 후 거세 및 비육하여 출하한 결과와 한우 거세우의 도축 성적을 비교한 결과, 육량은 A등급이 칡한우는 35.7%, 한우는 27.5%였고, B등급은 각각 42.9% 및 52.75였다. 육질등급은 1++등급이 각각 35.7% 및 15.0%, 1+등급이 각각 35.7% 및 30.8%, 1등급은 각각 14.3% 및 32.0% 그리고 2등급이 각각 14.3% 및 20.1%였다. 본 연구를 통하여 칡한우 수소 생후 15개월령에서 정액채취, 동결 및 활용이 가능한 것 으로 판단되며, 향후 칡한우 고유 유전자원의 확보 및 증식에 활용도가 높을 것으로 기대된 다.

닭 정액의 주요성분인 glutamine은 정장의 주요 성분으로 정자의 생리 활성에 매우 중요 한 것으로 알려져 있으며 조류 정액 희석액의 주요성분으로 이용되어져 왔다. 그러나 glutamine은 액상의 배양액에서 변성이 일어날 수 있어 세포배양에는 alanyl glutamine을 이용하 여 장기 세포배양에 이용되고 있다. 본 연구에서는, alanyl glutamine을 이용하여 개발된 희 석제를 이용하고 닭 정액의 냉장보존에 이용하였으며, 닭 정자의 장기 생존성에 미치는 영 향을 조사하였다. 국립축산과학원 가축유전자원시험장에서 사양관리중인 오골계 수탉 또 는 레그혼에서 복부 마사지법으로 채취된 정액을 이용하였다. 채취한 정액은 96 mM L- Glutamine 또는 Alanyl Glutamine을 함유하는 희석액에 각각 1:8의 부피비율로 상온에서 희석하 여 상업용 냉장고와 4℃로 조절된 BOD 배양기에 보존을 실시하였다. 냉장되는 온도의 저 하 속도를 낮추기 위하여 상온에서 희석하는 온도와 동일한 온도의 증류수 300 ml을 비이 커에 담고 물위에 1.5 ml E-tube에 담아서 파리필름으로 밀봉하였다. 희석된 정자의 활력은 상업용 냉장고에 보존할 경우, 4일차까지 75.6%의 활력정자가 유지 되었으나 5일 이후에는 급격히 생존성이 저하되었으며 6일째에는 활력 정자의 수가 18%로 낮게 나타났다. 한편, BOD 배양기에서 보존된 동일한 정자는 9일까지 약 73%의 활력정자가 유지되었으며, 10일 이후에는 활력 정자의 수가 급격히 감소되는 현상을 관찰하였으며, 15일째에는 약 20%의 정자 활력이 유지되는 현상을 관찰하였다. 이러한 정자의 활성 유지는 정자의 미토콘드리 아 에 필요한 글루타민 에너지 공급원을 안정적으로 공급하는 기능에 의하여 유지 되는것으로 추정되며 특히 hyper activated sperm의 비율을 줄여주어 정자군의 노화를 방지할 수 있는 효과가 있는 것으로 추정되었다. 그러므로 조류 정액을 냉장 보존할 때, 알라닐글루타민을 희석액의 주요 성분으로 이용될 수 있음을 보여주고 있다.

Spermatogonial stem cells (SSC) undergo self-renewal division and generate spermatogenesis to produce mature spermatozoa. Very recently in some species, include rodent and farm animals, SSC has been isolated and cultured in vitro. In this study, we analysed SSC marker of both 5 days old and pubertal pig testis by histological method. In 5day pig testis, prior to set of spermatogenic differentiation, the seminiferous tubules contain a relatively large number of SSCs than in pubertal pig testis. Then putative pig SSCs were successfully isolated from 5 day pig testis, and cultured long term using CD34 positive cell culture media contained GDNF, bFGF, LIF and EGF. The SSC colonies were appeared at 3 days after cells were seed. The SSC colonies were alkaline phosphatase positive and strongly expressed PGP 9.5, PLZF and DBA, but not expressed GATA4 and LH receptors. The SSC colonies were stably proliferated in GDNF, bFGF, LIF and EGF contained CD34 positive cell culture media up to 60 days. This study will be facilitated to study of in vitro and ex vivo spermatogenesis and of production of transgenic pigs using sperm vector.

Hematopoietic stem cells (HSCs) are the self‐renewing, multipotent progenitors that give rise to all types of mature blood cells. The hallmark properties of HSCs are the ability to balance self‐renewal versus differentiation cell fate decisions to provide sufficient primitive cells to sustain haematopoiesis, while generating more mature cells with specialized capacities. In the present experiment, we optimized the techniques for isolation and identification of hematopoietic stem cells from cow peripheral blood. The objective of this study was to optimize the more accurate methodology for separation of mononuclear cells (MNCs) from peripheral blood and identification of HSCs by using a specific cell surface marker i.e. CD34. A total 10 peripheral blood samples were collected from Holstein dairy cows from jugular vein. We used Ficoll 400 in different concentrations from 1 to 12% and Ficollpaque Plus (1.077 g/ml) at different centrifugation speed and time. After Giemsa staining, we found more than 98% recovery of monocytes with Ficollpaque Plus (1.077 g/ml). It was demonstrated that Ficollpaque Plus (1.077 g/ml) and centrifugation at 400xg for 30 min is the best method for separation of MNCs from bovine peripheral blood. Separated MNCs were immediately subjected for magnetic activated cell sorting (MACS) by using CD34 microbead kit. HSCs (CD34+ cells) recovery was 0.307% of peripheral blood. Peripheral blood MNCs and CD34+ cells were morphologically characterized by Giemsa staining. CD34+ cells were also confirmed by immunochemistry using FITC conjugated CD34 antibodies. HSCs were also confirmed by progenitor assay including burst forming unit‐erythroid (BFU‐E), colony forming cells‐ granulocyte (CFC‐G), colony forming cells‐ macrophage (CFC‐M), colony forming cells‐ granulocyte macrophage (CFU‐GM) and colony forming cells‐ granulocyte erythroid macrophage monocyte (CFCGEMM) on Methocult 4435.

Accumulating evidence suggests that chemotherapy can cause long‐term detrimental effects and alter the biology of the recipient environment. Therefore, a subsequent report claimed that the transplantation of female germline stem cells (FGSCs) into the ovaries of recipient mice that were pretreated with a high dose of busulfan and cyclophosphamide (B/C) resulted in the successful production of offspring. Therefore, this study was conducted to further clarify the impact of female germ cell transplantation on female ovaries after B/C treatment. RT‐PCR analysis showed that the period of germ cell depletion coincide with decreased Figla, Lhx8, Nobox, Kit, and Sox3 gene expression in the B/Ctreated ovary. However, depletion of female germ cells is mediated by a Fas/FasL‐, Kit/ Kitl‐, TNF‐, p53‐ and autophagy‐ independent pathway. Also, histological analysis is similar to that of Nobox null‐derived ovaries, indicating that follicle death after B/C treatment might be caused by down‐regulating of Nobox pathway. When female mice during 15 weeks after B/C treatment were checked for reproductive activity, B/C treated mice did not produce their pups. In addition, when 3×106 GFP positive primordial follicles were injected into B/C treated female mouse ovaries, donor follicle were not able to colonize into the ovaries of recipients. In conclusion, these data from a preclinical mouse model strongly suggested that female ovary until 15 weeks after B/C treatment could not support environment for maturing of exogenous FGSCs.

Althogh Spermatogonial stem cells (SSCs) are widely studied in mice, study of pig SSCs is not sufficient for the isolation, long-term culture, and characterization. To identify the effect of growth factor in cultured pig SSC, newly generated pSSCs like cell from neonatal 5days porcine testis were cultured and investigated for the pSSCs like cell formation. Glial derived neurotrophic gactor (GDNF), fibroblast growth factor (FGF), leukemia inhibitory factor (LIF), and epidermal growth factor (EGF) were applied to culture media to identify the pSSC like cell growth and stem cell formation. The criteria for the determining of stem cell characters, morphology, number of colonies, putative stem cell marker were analysed by microspic, polymerase chain reaction (PCR) and immunocytochemistry (ICC) methods. Most of the pSSCs like cells were formed approximately 100 μm size with sphere shape. Most of the feeder cells were highly dependent on FGF that feeder cells were not stably attached on plate without FGF and colony formation of pSSC was not observed consequently. Immunocyto chemistry data revealed that this cells expressed the ubiquitin-C-terminal hydrolase 1 (UCHL-1, PGP9.5) and Dolichos Biflorus Agglutinin (DBA) in addition of 20 ng/ml EGF, 10 ng/ml FGF, 10 ng/ml GDNF, 10 U3/ml LIF. In addition, Alkaline Phosphatase ()was positive in all period of culture. This study suggest that various growth factorsinp SSC culture system is important to regulate and maintain the pSSC. In conculsion, although the precise role of growth factor in pSSC proliferation need to be clarified, combination of growth factor might be critical in order to derivation and proliferation of neonatal pSSCs and spermatogenesis.

Since embryonic stem cells (ESCs) were first established from explant cultures of in vivo day 3.5 mouse embryos, the establishment of ESCs from species such as primates and rat has been developed. However, this success relies on the development of culture medium suitable for human and rat cells, which has different requirements from the murine ESC. In general, the establishment of ESC from pig and cow is of great interest both the agricultural perspective and for biomedical application. Large animal models, particularly pig, are likely to provide models of human genetic diseases and transplantation research where rodent models are inappropriate. However, establishment of ESCs establishment from pigs has remained an elusive goal. In the present study, we focused on signaling transduction regulation in pig epiblast stem cells (pEpiSCs). Pig epiblasts were isolated from early tubular stage embryos collected in vivo day 10.5～12 after insemination. Epiblasts were separated from trophoblast and underlying primitive endoderm using 21G needles and fine forceps. Epiblasts were cultured on mitomycin C (10 μl/ml) treated mouse embryonic feeder cells in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% minimal essential medium (MEM) nonessential amino acids, 1% penicillin/ streptomycin, 1% glutamine, 0.007% β-mercaptoethanol, 5 ng/ml bFGF and 1 ng/ml LIF. After plating rapid differentiation of isolated epiblasts to extraembryonic cell types was visualized in most cultures but stem cells were enclosed by these differentiated cells. We have established seven pig epiblast stem cells lines (pEpiSC1-7) from Days 10.5–12 pig embryos. pEpiSC expressed the pluripotent markers including OCT4, NANOG, SOX2 and NODAL at 3-5 passage. In addition, the modification of culture condition by the inclusion of particular protein kinase inhibitor such as Akt inhibitor, PD0325091(PD), delyed rapid differentiation of pEpiSCs. These results showed that stemness of pEpiSCs can be maintained by regulation of signaling pathway. * This work was partly supported by a grant from the NPR (2011-0013703) and the Next-Generation BioGreen 21 Program (No. PJ008209), Rural Development Administration, Republic of Korea.

Genomic reprogramming factors in the GV cytoplasm improved cloning efficiency in mice through the pre‐exposure of somatic cell nuclei to a GV cytoplasmic extract prior to nuclear transfer. In this study, a pig GV oocyte extract (pGV extract) was developed. Treatment of pig fibroblasts with the pGV extract promoted colony formation after 2–3 weeks in culture, concomitant with the expression of stem cell markers (Oct‐4, Rex1, Nanog, Sox2) and repression of differentiated cell markers (CKAP2, NPR3 ). Using fibroblasts transfected with human Oct‐4 promoter‐driven enhanced green fluorescent protein (Oct4‐EGFP), pGV extract treatment induced the reactivation of the Oct‐4 promoter in Oct4 ‐ EGFP cells by 10 days post‐treatment. These transgenic donor cells were injected into 8‐cell embryos. Oct‐4 promoter activity was subsequently detected in most ICM cells of the host blastocyst. Interestingly, reconstructed embryos with pGV extract‐treated Oct4‐ EGFP fibroblast nuclei showed prolonged expression of Oct4 in the ICM of embryos. Additionally, the pGV extract promoted somatic cell reprogramming and cloned embryo development when assessed by measuring histone H3‐K9 hypomethylation, the expression of Oct4 and Nanog in blastocysts, and the production of increased numbers of high‐ quality blastocysts. Under specific culture conditions, pGV extract‐treated fibroblast cells differentiated into neuronal, pancreas, cardiac, and endothelial lineages that were confirmed by antibodies against specific marker proteins. These data provide evidence for the generation of stem‐like cells from differentiated somatic cells by treatment with GV oocyte extracts in pig.