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This study examined factors affecting survival rate of epididymal sperm from Korean native black goat using 0.25 mL straw. The sperm was cryopreserved by the height above 5 cm or 14 cm from the surface of liquid nitrogen (LN2) for 10 min and then plunged into the LN2. The motility of sperm after thawing was estimated using CASA system and movement analysis. The motile sperm rates after thawing at 14 cm freezing height was significantly higher than at 5 cm (85.3% vs. 73.0%). Also the hyper- activation rates showed that the more actively moving sperm counts appeared at 14 cm height experiment (55.3% vs. 47.6%). This phenomenon was also observed in the dilution of electro-ejaculted semen. So, these results show that testis originated sperm could be used for artificial insemination and fertility evaluation in vivo is needed to evaluate their contribution to animal genetic resource banks.

가금 산업에 있어서 닭 정액의 희석은 저온 저장과 동결 보존에 있어 매우 중요한 과 정이며 농가 간 정액의 교류를 통한 산업적 이용성이 높은 연구 영역으로 판단된다. 그 럼에도 불구하고 닭 정액의 희석에 관한 연구는 미진한 편이며, 정액의 희석 비율 또한 4~8배에 머물고 있어 소와 같은 포유류에 비하여 많이 낮은 편이다. 본 연구에서는 닭 정액의 희석 비율을 높이기 위하여 fatty acid free BSA (FAF-BSA) 0.1%을 첨가한 BPSE 희석액을 이용하여 레그혼의 신선 정액을 10배 희석하였다. 희석된 정액을 17℃에 서 6시간 저온 보존한 후, 인공수정을 실시하였다. 수정 후 10일 동안 회수한 수정란을 37.8℃ 배양기에서 발육시켰으며 발생 5일자 수정란에서 수정률을 조사하고 발생 19일에 하란을 실시여 21일에 수정란의 부화율을 조사하였다. 대조군으로 BSA를 0.1%가 함유된 BPSE를 이용하고 동일하게 보존하여 인공수정을 실시하였다. FAF-BSA를 활용한 실험군 에서 수정률이 유의적 수준으로 증가하였으며(53.2% vs. 38.3%, p<0.05), 부화율 또한 증가하였다(90.9% vs. 78.33%, p<0.05). 이러한 결과는 FAF-BAS를 활용하여 닭 정액을 희석하고 저온 저장할 때 그 이용성과 효율성이 증가한다는 것을 보여주고 있으며 닭 정 액이 필요한 농가와 종계의 정액을 이용하고자 하는 농가에서 그 활용성이 높을 것으로 판단된다.

The Jeju Black Cattle (JBC) are a type of traditional Korean native cattle with a characteristic black fur that covers the entire body. Jeju black cattle are smaller than other breeds and their disposition is very calm. The breed’s qualities and genetic uniqueness helped it to be named as a protected, state-designated national monument in 2013. However, they are endangered crisis. Therefore, in vitro fertilization (IVF) technology is needed to preserve these endangered animals and to further improve their traits. In this context, semen analysis is the most commonly used procedure to evaluate male fertility potential in humans and animals. This study was performed on 3 Jeju Black Cattle (JBC) Bulls belonging to Jeju Special Self-Governing Province Promotion Institute. [JBC A, B, C grade]. The freezing medium (20% egg yolk plus 20% triladyl) to a final concentration of 100×106 sperm cells/mL. For sperm cooling, the 0.5 mL plastic straws filled with diluted samples were kept in refrigerator at 4°C for 2 h. They were placed 3 or 7 cm over liquid nitrogen (LN2) vapor for 10 or 30 min and then directly plunged into LN2 for storage. Thawing was done by transferring the frozen straws into water bath at 37°C for 30 sec for analysis. The sperm motility in each group was assessed using the Sperm Analysis Imaging System (SAIS Plus; Medical Supply Co, Ltd., Korea). The sperm vitality in each group was assessed using the eosin-nigrosin stain. The sperm morphology in each group was assessed using the diff-quik kit. The motility was significantly lower in JBC-C frozen-thawing sperm group (35∼65%) than in JBC-C fresh sperm group (98.5%) (p<0.05). The vitality of fresh sperm group (45∼61%) was similar to that of frozen-thawed sperm group (30∼65%). The morphology of fresh sperm group (72∼89%) was similar to that of frozen-thawed sperm group (83∼93%). These results demonstrate that there were similar in sperm quality (vitality and motility) between fresh sperm and frozen-thawed sperm.

The Jeju Black Cattle (JBC) are a type of traditional Korean native cattle with a characteristic black fur that covers the entire body. The breed’s qualities and genetic uniqueness helped it to be named as a protected, state-designated national monument in 2013. However, they are endangered crisis. Therefore, in vitro fertilization (IVF) technology is needed to preserve these endangered animals and to further improve their traits. In this study, we compared the sperm fertility and developmental potential of IVF embryos using Hanwoo sperm supplied by Korea Animal Improvement Association and JBC sperm made by Jeju Special Self-Governing Province Livestock bull semen Promotion Institute. [Hanwoo A, B (KPN) and JBC A, B grade]. Sperm was inseminated in 44 μL IVF drop contained 10 oocytes with sperm concentration of 1×106 cells/mL, and then 2 μL heparin and 2 μL PHE (20 μM penicillamine, 10 μM hypotaurine, 2 μM epinephrine) were added. At 2 days after IVF, the in vitro fertilization rate was significantly higher in Hanwoo-B sperm group (82.3±1.1) than in JBC-A sperm group (64.6±2.3) (p<0.05). The highest percentage of blastocyst formation was produced in Hanwoo-B sperm group (43.0±3.9) among four treatment sperm groups. The trophectoderm (TE) cell number was higher in Hanwoo-A sperm group (122±7.8) than in JBC-A sperm group (73±12.6). The inner cell mass (ICM) cell number of Hanwoo-A sperm group (46±2.5) was similar to that of JBC-A sperm group (46±4.0). Thus, the total cell number of day 8 blastocyst was higher in Hanwoo-A sperm group (168±8.9) than in JBC-A sperm group (119±15.9). These results demonstrate that there were different in sperm quality (viability and motility) between Hanwoo sperm (60∼70%) and JBC sperm (50∼60%), and their in vitro development capacity was also presented as difference. We need to embryo transfer study for field trial.

Mammalian testes are maintained at a lower temperature than the abdominal region for healthy spermatogenesis. Germ cell apoptosis has been described in heat-damaged testes by cryptorchidism, but the mechanism is not yet fully understood. To elucidate the cause of germ-cell death by cryptorchidism, we surgically induced cryptorchidism in dog testes and performed histological and molecular analyses. Histological data showed that the seminiferous tubules of cryptorchid testes and epididymis contained significantly fewer germ cells. Total RNA sequencing was performed to screen overexpressed genes in cryptorchid dog testes. Clusterin RNA was significantly higher (approximately 12.8-fold) in cryptorchid testes than in normal testes. In addition, cleaved caspase-3 and -8 were detected at higher levels in cryptorchid dog testes. Real time RT-PCR and western blotting analysis showed that the expression of clusterin was higher in cryptorchid dog testes. Furthermore, clusterin was detected in extracellular regions of cryptorchid dog testes during 4 weeks after surgery. Thus, we demonstrated germ-cell specific apoptosis and expression of clusterin in extracellular regions of cryptorchid dog testes. This result will facilitate further study of spermatogenesis and the specific mechanisms by which cryptorchidism causes male infertility.

2018년 제2차 말산업 육성법(2017∼2021) 발표를 하는 등 최근 말 산업의 부가가치는 매우 높은 복합산업으로 각광받고 있으며, 말 산업 규모는 2016년 기준 약 4,120억원, 산업체 수는 약 2,278개소에 이르며, 승마인구는 47,471명, 체험 승마 인구는 890,951명 으로 모든 말 산업 분야 규모가 매년 증가하고 있는 추세이다. 본 연구에서는 제주산마 의 액상정액 및 동결정액의 성상 개선을 위하여 펜톡시필린 수준을 설정하여 정자의 운 동성, 생존율, 정자막 온전성을 평가하였다. 액상정액에 펜톡시필린 2 mM 처리구에서 48 시간 이후 정자 운동성이 높은 경향을 나타내었다. 정액의 동결-융해 후 30분이 경과하였 을 때 정자 성상 분석에서는 펜톡시필린 4mM(T1) 처리구와 8 mM (T2)처리구에서 Progressive Motility(PM)가 53.25±2.87, 50.28±2.14로 40.09±5.15와 41.27±2.82인 대조구 와 16 mM(T3)에 유의적으로 높게 나타났으며 (p<0.05), Progressive Fast Motility(PFM) 도 펜톡시필린 4 mM(T1) 처리구와 8 mM(T2) 처리구가 각각 22.44±1.62와 22.74±3.07로 대조구와 16 mM(T3) 처리구의 3.47±1.48와 14.66±3.68보다 유의적으로 높게 나타났다 (p<0.05). 융해 후 1시간이 경과하였을 때 정자 성상분석에서는 총 운동성(Total Motility) 에서 T1과 T2가 68.96±1.64와 67.90±6.72로 53.48±4.84와 58.14±2.65인 대조구와 T3에 비해 유의적으로 높은 운동성을 보였다(p<0.05).

Spermatogonial stem cells (SSCs) can produce sperms transferring genetic information into the next generation in seminiferous tubule of testes. Accordingly, an efficient isolation technique of SSCs with extremely low numbers from testes is required for successful downstream researches related to maintenance, differentiation and cryopreservation of SSCs. To date, a variety of isolation techniques in a variety of species have been used for retrieving SSCs from testicular tissue. However, comparison of their efficiency has been not revealed clearly. Accordingly, among isolation methods described previously, we tried to elucidate a technique showing the best isolation efficiency in the retrieval of SSCs from testes derived from mice. For these, SSCs were isolated from mouse testis as follows: differential plating (DP), EpCAM, Thy1 or GFRα1 antibody-based magnetic-activating cell sorting (MACS) post-DP, EpCAM, Thy1 or GFR α1 antibody-based MACS, EpCAM, Thy1 or GFRα1 antibody-based MACS post-MACS based on GFRα1 antibody (double MACS for positive selection), and CD34 or α-SMA antibody-based MACS post-MACS based on GFRα1 antibody (double MACS for negative selection). Subsequently, SSCs isolated from each method were stained by alkaline phosphatase (AP) staining and percentage of AP positive SSCs was compared to find an optimal method among isolation method candidates. As the results, SSCs isolated by DP for 8 h showed numerically the highest percentage of AP positive SSCs. In case of MACS post-DP, SSCs isolated by MACS based on Thy1 antibody post-DP (MACSThy1 post-DP) revealed numerically higher percentage of AP positive cells than those on EpCAM and GFRα1 antibodies post-DP. Moreover, numerically the highest percentage of AP positive SSCs was detected when SSCs were isolated from mouse testis by MACS based on GFRα1 (MACSGFRα1) compared to EpCAM and Thy1 antibodies. In case of double MACS for positive selection, the usage of EpCAM antibody in the second MACS post-GFRα1 antibody-based MACS (double MACSGFRα1/EpCAM) showed numerically the highest percentage of AP positive SSCs compared to Thy1 and GFRα1 antibodies. On the other hands, after GFRα1 antibody-based MACS sorting, the usage of α-SMA antibody in the second MACS (double MACSGFRα1/α-SMA) for negative selection showed numerically higher percentage of AP positive SSCs than CD34 antibody. The subsequent comparison of isolation efficiency derived from each method demonstrated that MACSGFRα1 and double MACSGFRα1/EpCAM resulted in significantly the best isolation efficiency. Accordingly, we could elucidate that MACSGFRα1 and double MACSGFRα1/EpCAM were techniques sorting effectively SSCs from mouse testes.

Aberrant expressions of Meg3 hamper the developmental potential of induced pluripotent stem cells (iPSCs) not only the birth of a chimera but also the generation of fully derived iPSC-derived clones by tetraploid complementation. This study was conducted to examine the effect of vitamin C (VC) on the quality of iPSCs in pig. Porcine iPSCs were generated from ear fibroblast cells using the SeV vector expressing 4 human factors (POU5F1, SOX2, C-MYC, and KLF4) with or without VC. Colonies emerged at Day 14 of transduction. Colony number of VC group was higher than that of control. Next, clonal lines of iPSCs were separately sub-cultured from every single colony under 2i (CHIR99021 and PD0325901)-LIF and MEF feeder culture condition and the expression of Meg3 and pluripotency-related genes (Pou5f1 and Lin28) were examined in each iPSC line. The expression level of these genes differed among iPSC lines. Porcine iPSCs that maintained self-renewal showed a high level of Meg3 as well as pluripotency-related genes expression. Whereas, porcine iPSCs with a low level of Meg3 could not maintain the typical self-renewal morphology over 10 passages. Our results suggest that the quality of porcine iPSCs could be improved by VC treatment, and the expression status of Meg3 may be closely related to the developmental potential of iPSCs in pigs.

To establish induced pluripotent stem cell (iPSC) by reprogramming cells that is differentiated types, specific transcription factors are needed. Some researchers examined whether chemical molecules or proteins can replace transcription factors to alter the de fined cell fate. Previously, we confirmed that the treatment of bone morphogenetic protein 4 (BMP4) could intermediately reprogram the mouse skin fibroblasts (MSFs) to stem-like cells. In this study, to reprogram the cell more efficiently and definitely, we investigated the synergic effects of BMP4 and 4 kinds of small molecules, RG108, 5-azacytidine, Na-butyrate and PD173074, that regulates some signal transduction pathway. The ventral skin epidermis of an adult mouse were used to confirm the synergic reprogramming activity of small molecules. After the cells were cultured with BMP4, they were formed sphere-like organoid bodies (OBs) with BMP4 and each small molecules for 5 days. The activity of alkaline phosphatase was the highest in BMP4+RG108 group. Also, all the small molecule treated groups expressed high Oct-4 level than BMP4 treated group. To confirm the stem cell potency of these cells, we induced their differentiation into cardiomyocytes. The cells exhibited similar mRNA expression of cardiac mesoderm markers such as connexin 40 and GATA binding protein 4, and the cardiomyocyte marker troponin T among groups. However, the expression of one of the cardiac mesoderm marker gene, Nk2 transcription factor-related locus 5, was significantly decreased in BMP4+RG108 group. These results suggest that treatment of some small molecules may enhance the BMP4-mediated somatic stem cell reprogramming.

Canine induced pluripotent stem cells (ciPSCs) have great potential for veterinary regenerative medicine. To generate integration-free iPSCs from aged dog’s fibroblasts, canine adult fibroblasts (CAFs) were reprogrammed using an RNA-based strategy involving the non-integrating, self-replicating Venezuelan equine encephalitis (VEE) virus. Using this RNA system, CAFs were co-transfected with the T7-VEE-OKS-iG RNA replicon plus B18R mRNA for 4 h. Subsequently, the transfection medium was replaced with Advanced-Dulbecco’s modified Eagle’s medium (Advanced-DMEM) containing 10% fetal bovine serum and 200 ng/mL of the B18R protein. One day after the final transfection, cells were grown in stage 1 medium and selected with puromycin (0.5 μg/mL) until Day 10. On Day 11, the stage 1 medium was changed to mouse embryonic fibroblast- conditioned medium containing 20% KnockOut Serum Replacement (KSR) medium. Putative ciPSC colonies first appeared between Days 15 and 25. Interestingly, two distinct types of ciPSC colonies were identified, which showed TRA-1-60 expression or alkaline phosphatase activity. Furthermore, analysis of the gene-expression levels suggested that these putative ciPSCs were in a transition state. Although further studies will be needed, this study will contribute to the development of cell-based therapeutics for aged dogs.

본 연구는 alpha 1,3-galactosyltrasfersase 유전자 기능 제거 돼지(GT KO)의 심장을 이 식 한 후 9일째 사망한 원숭이에서 사망의 원인을 추적하고자 실시하였다. 사망한 심장 의 조직을 병리학적으로 분석한 결과, 초급성 및 급성 거부반응의 징후는 발견되지 않았 고, 대표적인 심장질환의 하나인 심근경색과 심근 섬유증이 심장 전체에 광범위하게 발 견되었다. 이종이식에 의해 변화된 유전자의 발현 양상을 알아보기 위하여 동결 보존된 심장의 심실에서 총 RNA를 추출한 후 new generation sequencing을 실시하였다. 심장이 식에 활용한 돼지와 한 복으로 태어난 돼지의 심장을 이종이식일에 채취하여 대조군으로 사용하였다. 차등발현 유전자 데이터를 DAVID 분석을 실시하여 26개의 유의한 생물학적 경로 관련 유전자에서 발현 변화가 크다는 것을 알 수 있었다. 그 중에서 발현 변화 정 도가 높았던 경로는 심장질환 환자에서 빈번하게 발견되는 비대 심장근육증(hypertrophic cardiomyopathy), Valine, leucine and isoleucine degradation, 확장 심장근육증(Dilated cardiomyopathy), 부정맥유발 우심실 심장근육증(Arrhythmogenic right ventricular cardiomyopathy) 이었다. KEGG를 통한 기능별 분석을 실시하여 심장근육증 관련 SGCA, SGCB, SGCD, SGCG, MYH6, MYH7, MYBPC3, and TNNI3, DMD 유전자의 유의적인 발현 감소를 발견하였다. 심장근육증의 대표적인 증상인 심근섬유증 진단 마커로 활용되 는 α-SMA, COL1A1, COL1A2, POSTN, VIM P4HA1, P4HA3, THY1, NPPA, NPPB 유전 자의 발현이 유의적으로 증가하였고, MYH6의 발현은 유의적으로 감소하는 것을 quantitative PCR 방법으로 검증하였다. 또한 심근 경색 관련 matricellular 단백질 유전자인 THBS1, TN-C, SPARC, POSTN의 유의적인 발현 증가를 quantitative PCR 방법과 면역 조직염색 방법으로 검증하였다. 이러한 결과로 GT KO 돼지의 심장을 이식받은 원숭이가 9일째 사망한 원인은 거부반응이 아닌 병리 및 생리적 이상에서 발생하는 심근경색 및 심근 섬유증에 의한 심장 근육의 이상으로 판단된다.

Unlike allograft, xenotransplantation has problems such as hyperacute rejection (HAR), acute rejection and chronic rejection. HAR was rapid humoral immune responses against the pig carbohydrate Galα1,3-Gal epitope by human complement regulatory reaction, but HAR was almost overcome through knockout of α1,3-galactosyltransferase gene. The acute rejection is appeared within a few days to week caused by ischemia, diffuse intravascular coagulation and lack of cellular infiltrate. In this study, we bred the transgenic pigs containing US11 and hDAF (CD55) gene, evaluate transgene transmission ratio and efficiency of Reproduction rate in natural breeding. In addition, performed Cytotoxicity experiment about human NK cells and complement against each piglet ear fibroblast that have gene expression of US11, hDAF or both hDAF and US11. In breeding evaluate, sow has normal litter size, and piglets sex rate was followed 1:1. Transmission ratio of transgenes was indicated that wild type was 10%, US11 gene only was 20%, hDAF gene only was 20%, and both US11 gene and hDAF gene was 50% in 10 piglets. In cytotoxicity assay, US11 gene showed that effect to reduce cytotoxicity rate from human NK cell, and hDAF gene was significantly reduce the cytotoxicity ratio from complement. Besides, piglet cells that has both US11 gene and hDAF gene has effect to reduce cytotoxicity of NK cell and complement. In conclusion, our study showed that US11 and hDAF genes has effective to reduced Xenotransplantation rejection, and natural breeding was efficient to product transgenic pig.

In human organ transplantation area, the biggest problem is the shortage of donor organs. To solve this problem, xenotransplantation, such as pig-to-human, can be a solution. Despite the physiological and anatomical similarities in pig to human, the immune- rejection still remain a major barricade. The alpha-1,3-galactosyltransferase (GT) gene is responsible for synthesis of xenoreactive antigen galactose-1,3-galactose and many researchers have afforded in elimination of the xenoreactive antigen and producing genetically modified pigs for xenotransplantation. Despite of the effort, the problems in immune rejection are not entirely eliminated yet. Here, we constructed a vector expressing multiple human genes, hCD55, hCD39, hTFPI, and hC1 inhibitor, to reduce immune rejection and GT targeting vector. The two vectors were co-transfected into porcine ear fibroblast (PEF), and neomycin was used as a selective marker to isolate cells inserted the two vectors. We confirmed the TG cell lines by PCR and western blot analysis. Using established cell lines, we will produce the Knock-in pigs by SCNT. Also we expect that the cloned pigs carrying those multiple immune-modulation genes will overcome the current obstacles in xenotransplantation.