In vitro production of mammalian embryos has been achieved with the oocytes derived from middle-size follicles (MF, mainly 3-6 mm in diameter) in many species including domestic animals. In the ovaries, however, there are more small-size follicles with less than 3 mm in diameter (SF). If we can develop an efficient system to produce embryos in vitro from the oocytes from SF. In this presentation, I would like to review about embryo production in vitro from the oocytes derived from SF. As well as the diameter of oocytes, the number of cumulus cells surrounding the oocyte derived from SF is significantly smaller those of oocytes from MF. The comparative analysis in electrophoresis about secretions of cumulus-oocyte complexes derived from SF and MF demonstrated a significant difference in the proteins with a molecular weight. Proteins secreted from cumulus cells, vascular endothelial growth factor (VEGF), are 34- to 42-kDa proteins, including seven family members. The molecular weight of VEGF was similar with the secretion we observed. Supplementation of medium for in vitro maturation with VEGF significantly improved the oocytes competence not only to complete the meiosis in vitro but also to develop to the blastocyst stage following parthenogenetical activation. Removing cumulus cells 20 h after the start of culture for in vitro maturation also significantly improved the competence of oocytes derived from SF to achieve the meiosis. A combination of these new techniques may improve more the meiotic and developmental competences.

The Y chromosome is a type of sex chromosome existing primarily in male mammalian species. The Y chromosome passes through the male gamete and determines male sex in humans, non-human primates, and other mammals. The mammalian Y chromosome varies from the X chromosome and the rest of the chromosomes primarily by size and its male sex-determining/spermatogenesis function. In the Y chromosome, male sex-determining function is exclusively located on the short arm, while the spermatogenesis function is distributed widely on the short and long arm. Deletions or mutations particularly in the male-specific region of Y chromosome (MSY) may cause male infertility. During the last few decades, researchers put forth an enormous effort to discover Y chromosome specific genes, and their encoded RNAs and proteins in humans, primates, and rodents. As a result, most of the genes and encoded proteins responsible for male-sex determination, testis development, and spermatogenesis have been discovered in humans, however not well established in non-human primates and rodents. Also, there might be a percent of proteins missing in human Y chromosome. The aim of this study is to annotate the proteins that encoded on the Y chromosome of humans, chimpanzee, and mouse using extensive bioinformatics tools. The human (annotation release 107), chimpanzee (annotation release 103), and mouse (annotation release 105) proteins were first retrieved from the National Center for Biotechnology Information (NCBI) eukaryotic genome annotation resource database. Then, the annotated human proteins (66 proteins) were compared with the core databases of human proteome project such as neXtProt, PeptideAtlas, and the Human Protein Atlas. The X-homologous of human Y chromosome-encoded proteins were searched using the NCBI Protein BLAST program. The cellular pathways and protein-protein interactions involving human Y chromosome-encoded proteins were searched using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping database, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, and the Pathway Studio software. Finally, the human Y chromosome-encoded protein homologs/orthologs in chimpanzee and mouse were analyzed using the NCBI bl2seq program. This analysis resulted a significant number of homologous/orthologous proteins between human, chimpanzee and mouse. Our findings provide the scientific community with updated information on the Y chromosome-encoded proteins in humans, chimpanzee, and mouse.

Actin nucleation factors, which promote the formation of new actin filaments, have emerged in the last decade as key regulatory factors controlling asymmetric division in mammalian oocytes. Actin nucleators such as formin-2, spire, and the ARP2/3 complex have been found to be important regulators of actin remodeling during oocyte maturation. We found that actin nucleation promoting factor called WASP homolog-associated protein with actin, membranes and microtubules (WHAMM) play crucial roles in mouse oocyte maturation by generation of ER-associated actin filaments during meiotic spindle migrations. We also investigate regulatory mechanism of actin nucleator spire and discovered the novel roles of Zinc in regulating spire localization and cytoplasmic actin mesh formation. Another class of actin-binding proteins including cofilin, tropomyosin, capping proteins and tropomodulin, are thought to control actin cytoskeleton dynamics at various steps of oocyte maturation. The heterodimeric actin-capping protein (CP) binds to the fast-growing (barbed) ends of actin filaments and plays essential roles in various actin-mediated cellular processes. When CP is knockdowned or inhibitory component was overexpressed, asymmetric divisions of oocytes have been compromised. It turns out that knockdown or inhibition of CP deplete cytoplasmic actin mesh level, which have been known to be essential for maintain cytoplasmic actin mesh. Another actin binding proteins, tropomodulin 3 (Tmod3), binds to the slow-growing end of actin filaments and knockdown or expression deletion mutant of Tmod3 also decrease actin mesh level in maturing oocyte and it severely ablated asymmetric division of oocyte. Finally, tropomyosin 3, actin filament binding proteins protect actin filament from depolymerization, is also important to maintain cortex integrity in maturing oocyte, therefore showed the importance maintenance of actin filaments during oocyte maturation. Taken together, our study on various actin nucleator and actin binding study showed the importance of actin dynamics in mammalian oocyte maturation and early embryonic developments.

Kisspeptin-10 (KP-10) has been reported to act as a tumor metastasis suppressor via its receptor, G protein-coupled receptor 54 (GRP54). The KP-10/GPR54/BMPs signaling pathway plays an important role in embryonic kidney development. However, its function in osteoblast differentiation is unknown. The aim of this study was to confirm the molecular mechanism for the action of KP-10 on osteoblast differentiation. Expression of the Bone morphogenetic protein-2 (BMP2) and osteogenic genes were determined by RT-PCR and real-time PCR analysis in C3H10T1/2 cells. Transient transfection assays were performed to confirm the effects of KP-10 on BMP2-Luc activity. BMP2 and phospho-Smad1/5/9 protein levels were determined by Western blot analysis. Alkaline phosphatase (ALP) staining experiment was performed to evaluate ALP activity. To further confirm the effect of KP-10-induced GPR54, we used GPR54 Knock out (KO) C3H10T1/2 cells. KP-10 significantly increased osteogenic gene such as Runx2, ALP and Dlx5 in C3H10T1/2 cells. The ALP staining levels were also increased by KP-10. Interestingly, BMP2 mRNA, protein expression and promoter activity were also increased by KP-10. However, KP-10-induced BMP2 expressions were not increased in GPR54 KO cells. These results suggest that KP-10 increases BMP2 expression through GPR54. Next, Western blot analysis shown Smad1/5/9 phosphorylation were enhanced in a time-dependent manner by KP-10 treatment. It is well known that BMP2 increased phosphorylation of Smad1/5/9 via BMP2 receptor. In addition, KP-10 increased NFATc4 mRNA levels and NFATc4 overexpression enhance BMP2 mRNA levels. To confirm the KP-10-induced BMP2 action, we used KP-10-treated medium in wild type cells and GPR54 KO cells. The osteogenic genes were not elevated by KP-10-treated medium (GPR54 KO cells) whereas increased expression levels by KP-10 medium (wild type cells). These data indicate that KP-10 induced osteoblast differentiation through NFATc4-mediated BMP2 signaling.

The mammalian yolk sac endoderm is an essential but understudied tissue that patterns and nourishes the embryo. This talk will present the isolation and characterization of several categories of rodent yolk sac endoderm stem cells. Specifically, we have isolated yolk sac endoderm stem cell lines from preimplantation embryos and from post-gastrulation yolk sacs. In both cases, we obtained two versions of stem cells that appear to differ in their degree of lineage maturation. I will discuss the relationship of these various isolates within the same species (rat or mouse), between species (rat vs. mouse), and with previously published isolates. I will then discuss potential applications in developmental biotechnology and toxicology as well as the human relevance of this research.

Early detection of Hanwoo cow’s estrus is an important issue in the management of group-housed livestock. In particular, failure to detect estrus in a timely and accurate way can become a limiting factor in achieving efficient reproductive performance. Although a rich variety of methods has been introduced for the detection of estrus, a more accurate and practical method is still required. The behavior of 50 estrus and 100 non estrus Hanwoo cows was video recorded and analysed in 2 Korean native cattle farm. All 50 estrus cattle (100%) showed the mounting or mounted behavior, but only 5 of 100 non estrus cattle (5%) showed the mounting or mounted behavior. One hundred and fifty Hanwoo cows with an age between 2 to 5 years that were expected to come into estrus within 3 days were randomly assigned to each compartment for the estrus group. The heated cows were video recorded for about 24 hours until after post estrus. The results showed that Hanwoo cows can be considered on estrus when it stand immobile during mounted by any other cow in more than or equal to 3.15 s and 3.22 s in chest-tail head mount and head-above back mount and that occurs consecutively at least three times within 876.4 s interval. The algorithm was also developed using the thresholds of the mount duration, mount interval and consecutive occurrence number. The Hanwoo cow’s estrus detection system using IR sensor (CEDSIRS) was composed of IR sensors, a controller, a CPU, a mobile, and so on. If total COUNT numbers per hour was above 1 and it was maintained more than 7 hours, we determined that a cow was in estrus and 16 h past the time from the first estrus detection was regarded as optimum AI time. The 55 of 57 estrus cows (96.5 %) were detected to estrus. Only 5 of 57 cows detected to estrus (8.8%) were decided to weak estrus and 2 of them were not detected. The total conception rate was 85.7%. The estimated price of CEDSIRS is 3,000,000 Won. Therefore, CEDSIRS will improve largely the average conception rate and economic benefits of Hanwoo cow farms.

Adipose tissue is the largest energy storage in the body, with the endocrine, paracrine and autocrine function, and they constitute a network regulatory signal, and participate in energy balance and metabolism regulation in adipose tissue. When adipose tissue is excessively accumulated or obesity, inflammatory signaling pathway is activated as the secretion increase of a variety of inflammatory cytokines, then, the body is under the state of chronic inflammation, causing insulin resistance and many metabolic diseases, such as type 2 diabetes, atherosclerosis, cancer and other chronic metabolic disease, and bringing a serious health crisis to humans. And, excessive fat deposition reduces the feed conversion rate, leading to the increase of livestock and poultry production cost and the reduction of meat food quality. Therefore, the regulation of adipogenic differentiation has become an important field in the study of human health and animal production. 1. The source of adipose tissue The formation of adipose tissue is due to the increase of adipose cell number caused by differentiation and the increase of adipose cell volume and adipose accumulation during development. This process is that adipose mescenchymal stem cells (AMSCs) are transformed to preadipocytes in adipogenic environment, and differentiation related specific transcription factors start to express and induce the specific expression of adipose cell genes and terminal differentiation, finally, mature adipose cells are formed after adipose accumulation. Recent studies have suggested that dedifferentiated fat cells (DFATs) may be an important source of adipose tissue. Mature adipose cells can be dedifferentiated to the sub cells (DFATs) with the dividing ability in vitro culture, and DFATs are pluripotent and can be redifferentiated to adipose cells or transdifferentiated to other cell types, such as cartilage cells, bone cells, muscle cells, etc. by induction. This suggests that DFATs are progenitor cells with the stem cell properties, showing the great potential in tissue engineering and regenerative medicine. Research on the mechanism of DFATs redifferentiation and transdifferentiation has an important significance for human health and animal production. 2. Regulation of adipocyte differentiation and transcription The adipocyte differentiation lies in the transcription level regulation, involving the cascade and cooperative effects of multiple transcription factors, among which, the core transcription factor is peroxisome proliferator activated receptors-γ (PPARγ), which specifically expresses in adipose tissue, combines the promoters of downstream genes promoter and induces their expression, such as lipoprotein lipase (LPL), insulin sensitive glucose transporter 4 (GLUT4), fatty acid synthase (FAS), adipose-specific fatty acid binding protein-2 (AP2) and adiponectin, and promotes the differentiation and maturation of adipose cells. 3. Transcription regulation of PPARγ by Kruppel like factors Kruppel like factors (KLFs) are a class of transcription factors with zinc finger structure, which is involved in the regulation of cell proliferation, cell apoptosis, cell differentiation and tumor formation in a variety of animal cell types. Since KLF15 is first proved to have the transcriptional regulation capability of adipose differentiation by Gray, et al. in 2002, other KLFs are also found to be involved in the adipose differentiation regulation. According to the recent studies of KLFs regulation of adipose differentiation, Christopher, et al. in 2009 summarized KLFs transcription regulation network in the PPARγ upstream. This network includes 8 types of KLFs, namely, KLF2-7, KLF11 and KLF15, among which, KLF2, KLF3 and KLF7 are involved in the negative regulation of adipose formation, while KLF4-6, KLF11 and KLF15 positively regulates adipose formation, and they express according to a certain time sequence during adipocyte differentiation. 4. Regulation of adipose differentiation by curcumin Curcumin is a kind of polyphenols extracted from Curcuma longa L.. Curcumin can reduce the mice obesity formation, directly interfere with the preadipocytes differentiation and decrease the adipocyte number and adipose accumulation. Moreover, curcumin plays a role in the early stage of adipocyte differentiation, and inhibit the mitotic proliferation process and the expression levels of PPARγ, C/EBPα, and certain downstream transcription factors. 5. Regulation of AMSCs and DFATs adipogenic differentiation in pigs It is generally believed that pigs are the most suitable animal models for the application of human clinical medicine. Also, pigs are the largest source of human meat food, and one of animals with the most fat content. Therefore, research on the regulation of porcine adipocyte differentiation has an important significance for the establishment of human disease model and the production of low fat and lean meat pigs. This report summarizes the expression patterns of different KLFs and the effect of curcumin on the KLFs and PPARγ expression during the adipogenic differentiation of porcine AMSCs and DFATs in recent years.

Reprogramming is a process in which a developmentally determined cell fate is re-established to another fate by artificial modifications. Reprogramming to pluripotent state has been studied extensively in somatic cell nuclear transfer and induced pluripotent stem cells (iPSCs). Interestingly, recent studies showed that expression of a set of transcription factors also can induce transdifferentiation, also known as direct conversion or direct reprogramming. There are two major approaches in direct reprogramming: one is target cell-specific factor-mediated direct reprogramming and the other is epigenetic flexibility-inducing factor-mediated direct reprogramming. We are interested in generating induced neural cells via direct reprogramming by using pluripotency factors as epigenetic flexibility inducers and understanding the mechanistic basis of the process. We hope that these reprogramming technologies will provide a new paradigm of research in regenerative medicine as well as disease study and drug development.

Introducing lineage-specific transcription factors (TFs) into somatic cells enables the induction of distinct cellular identities without the need to first pass through a pluripotent stem cell (PSC) state. We and others have demonstrated the direct conversion of somatic cells into adult stem cells or progenitor cells, such as angioblast-like progenitor cells, hematopoietic stem cells, and neural stem cells. The process underlying direct conversion is known to be relatively simpler and faster than that of induced pluripotent stem cell (iPSC) generation. Furthermore, directly converted cells have been shown to exhibit therapeutic potential following transplantation into respective disease models without obvious evidence for tumor formation. Thus, TF-mediated direct conversion technology has been considered as an alternative to iPSC technology for patient-specific cell- and tissue-replacement therapies. Here we show our recent findings describing the robust direct conversion of differentiated somatic cells into distinct cellular identities. Furthermore, we also show the recent 3D organoid technology for generating brain tissues from human pluripotent stem cells.

Cryopreservation of miniature pig sperm is essential because of high demand of organ transplant in mass production. However, miniature pig sperm are vulnerable to oxidative stress more than other mammals. Erythritol is a naturally occurring sugar alcohol with powerful antioxidant property. Thus, the aim of our study is to verify if erythritol could reduce lipid peroxide and enhance viability of frozen thawed miniature pig sperm. Ejaculated semen samples were frozen with cryoprotectant subjected to erythritol treatment (0, 10, 100, 500 mM). After frozen thawed, spematozoa viability were examined using the computer assisted sperm analysis (CASA) system. The product of lipid peroxidation, malondialdehyde (MDA) were quantified using spectrophotometer with DPPH and ABTS assays as ROS scavenger markers. Our result showed that erythritol enhanced sperm viability (p<0.05), reduced lipid peroxides significantly (p<0.05), proving the concentration of 100 mM erythritol to be an effective for lowing oxidative damage. Data from our study suggest that erythritol exhibits significant lipid peroxidation scavenging characteristics which may prevent oxidative damage, enhance viability of frozen thawed sperm and thus could be a effective additive as cryoprotectant.

Germ cells originate outside of the fetal gonads and migrate toward the genital ridges through the embryonic tissue. Germ cell is the most important and valuable cell in livestock because germ cell is the only cell type that can transfer the genetic information and content into the next generation. In this study, we established the primordial germ cell (PGC) lines derived from the fetal gonads of 6 day-old-embryonic chicks, and then cryopreserved for long-term storage. First, we determined each chick embryo sex by genomic PCR with DNA extracted from blood. After dissociation of the whole gonads from individual embryos, total gonadal cells were plated into the culture dish and cultured with 20% fetal bovine serum-contained culture media. PGC lines were derived from three different chicken strains; White Leghorn (WL), Korean Oge (KO), and a commercial line, Hyline. There was no significant difference between the efficiencies of the PGC line derivation according to the different chicken strains. Thus, PGC culture and long-term storage system could be applied to maintain the endangered avian species and also produce the offspring through germline chimera production system.

Processes of cryopreservation consists of three steps: dilution with the extender/cooling (Step 1), addition of cryoprotectant (Step 2), and freezing/thawing (Step 3) and spermatozoa are exposed different kind of environment and stress in each step. We categorized sperm samples as good freezablitiy (GF), damaged by cryoprotectant (DCP), and damaged by freezing (DF) and identified characteristics of each group in different step of cryopreservation. In Step 2, DCP was significantly decreased in motility, rapid speed and increased in slow speed. DF was significantly decreased in only motility whereas there were no significant difference between GF and DF and significantly higher than DCP in Step 2. Motility, rapid, medium speed of all group were significantly decreased in Step 3 and GF was significantly higer than other groups. AR pattern of all groups were significantly increased in Step 3 whereas GF was significantly lower than other groups. Additionally AR pattern of DF was significantly increased in Step 2. F pattern of DF and DCP were significantly decreased in Step 3. There no difference of B pattern in whole process. Mitochondrial activity of DCP was significantly decreased in Step 2 and mtichondrial activity of all groups were significantly decreased in Step 3. However mtichondrial activity of GF was higher than other groups. Viability result shows same significant difference with mitochondrial pattern. The present study compared with various sperm parameters in different groups which has different freezability. We defined different two types of group that damaged from different step of cryopreservation. DF and DCP is mainly damaged in Step 3 and Step 2 respectively. The results of current study suggest that various sperm parameters can be used as physical markers in freezability.

Spermatogonial stem cells (SCCs) is foundation for spermatogenesis throughout male adult life because they have ability of self-renewal and differentiation into spermatozoa. Storage of such SSCs is very important to study on male reproduction, which would contribute human male infertility to be treated. However, during cryopreservation, the most cells are damaged by cryoinjury such as apoptosis, necrosis, osmotic stress, oxidative stress and so on. For the reason, in cryopreservation technique, targeting purpose is what cells are stored stably without cryoinjury. The purpose of this study was to develop the cryoprotectant for decrease in cryoinjury of SSCs by using melatonin and necrostatin-1 as additive cryoprotectant. The SSCs with melatonin or necrostatin-1 was frozen for 1 month, and then thawed to evaluate survival, recovery and proliferation rate. The result showed that necrostatin-1 50 mM was significantly greater than DMSO control. Furthermore, we conducted the characterization of cryo-thawed SSCs with necrostatin-1 50 mM to confirm whether the SSCs could maintain the undifferentiated state. As a result, the normal expression of each marker, which is PLZF, GFRa1 and VASA, was observed except for C-kit, meaning that the cells could maintain the undifferentiated state regardless of cryopreservation. Therefore, the result indicates that the cryo-thawed SSCs have ability of proliferation and self-renewal. In conclusion, our finding verifies that cryopreservation of SSC with necrostatin-1 50 mM could be helpful to preserve the SSCs stably, contributing to various studies on male reproduction and infertility treatment

승용말 주요 생산 국가와 협회에서 약 90% 정도가 인공수정으로 망아지를 생산하고 있고, 인공수정에 이용되는 정액은 냉장정액이 90%, 동결정액은 10% 정도 활용되고 있다. 본 연구에 서는 국내 승용말 인공수정 저변 확대에 필요한 씨수말 냉장 정액의 장기보존성 향상을 위하여 냉장 보존용 희석제와 희석 후 보관 형태가 정자의 생존성 및 운동성에 미치는 효과에 대하여 조사하였다. 한국마사회 장수목장에서 관리 중인 씨수말 2두를 이용하였으며, 정액의 희석에는 INRA-96(IMV, France) 및 EZ-mixin(ARS, USA) 희석제를 각각 이용하였고, 냉장(4℃) 상태에 서 96시간까지 보관하면서 정자의 생존율과 운동성을 분석하였다. 보관 형태는 aerobic 및 anaerobic 상태로 하였다. 정자의 운동성은 CASA 시스템을 이용하여 분석하였다. 각각 결과는 mean±SD로 나타냈으며, 통계처리는 t-test를 이용하였고 p<0.05 수준에서 유의성 검정을 하였 다. 승용말 정액 채취 직후 생존율과 운동성은 각각 평균 65.5% - 73.8% 및 40.8% 및 47.8% 였 다. 실험 목적에 따라 INRA96 및 EX-mixin 그리고 aerobic 및 anaerobic 상태로 24, 48, 72 및 96시간 동안 보존 하였다. EZ-mixin 희석제의 aerobic 및 anerobic 상태에서 보존 시간 경과에 따라 생존율은 42.3% - 4.0%, 운동성은 50.4% - 16.0%로 낮아졌다. INRA96희석제에서 보존에 서 생존율은 aerobic 54,3% - 49.9% 및 anaerobic 58.7% - 47.5%로 낮아지는 경향이었으나 유의 차는 없었다(p<0.05). 한편 운동성은 aerobic 34.5% - 37.4% 및 anaerobic 36.6% - 31.9%로 차이 가 없었다. 따라서 각각 희석제에 따른 혐기 및 호기성 상태로의 보존은 생존율과 운동성에도 영향이 없었으나, 희석제의 조성에 따른 생존율과 운동성은 유의한 차이가 인정되었다.

본 연구는 흑염소 동결정액 생산을 위해 활용되는 당의 종류가 동결융해 후 정자 생 존성 및 운동성 등에 미치는 영향을 조사하고자 수행하였다. 공시축의 사양관리는 개체별 별도 사육공간에서 사료 및 음수를 자유 채식케 하였으며 정액채취를 위해 12개월령 이 상의 성성숙(Sexual Maturity)이 완료된 흑염소 5두를 공시하였고 정액채취 빈도는 주 1 회로 하였다. 정액 채취는 보정 후 충전식 전기 자극기(11900, Mini-tube, Germany)를 활용하였고 Probe를 직장(Rectum) 내 삽입 후 숫 흑염소의 부생식선이 위치하는 부위에 밀착시켜 자동 전기자극 모드(0～10V)에서 2～3초당 0.5V씩 전압이 증가되도록 하여 2∼5회간 사출된 정액을 채취하였다. 정액의 탁도, 운무상을 현장에서 확인한 다음 실험실로 이 동 후 위상차 현미경(LABOPHOT-2, NIKON, Japan) 및 Markler-Chamber(Sefi-medical, USA)를 활용하여 원정액의 생존율 및 운동성을 하였다. 동결보존 희석액 조성은 Tris(hydroxymethyl)amino-methane 250mM, Citric-acid monohydrate 80mM, Sugar Supplements 60mM, Penicillin G Streptomycin 1%, Egg-Yolk 20%, Glycerol 7%로 하 였다. 동결정액의 생산은 당의 종류별로 원정액을 1차 희석한 다음 저온실로 이동하여 2차 희석 및 Glycerol 평형을 유도한 다음 0.25ml Straw에 충진시켜 액체질소를 활용한 동결을 실시하였다. 융해 후 정자 특성분석은 위상차 현미경과 컴퓨터정자분석기 (Computer Assisted Sperm Analysis)를 활용하였다. 생존율, 운동성, 선형성, 직진도 등 의 검사를 실시한 결과 생존율은 Glucose, Trehalose, Fructose(80%, 80%, 75%)였고, 운 동성은 Trehalose, Glucose, Fructose(97.8%, 94%, 88%)순 이였으며, 선형성은 Trehalose, Glucose, Fructose(31.4%, 31.9%, 34%)순으로 낮은 경향을 보였다. 직진도는 Glucose, Fructose, Trehalose(51.3%, 53.1%, 53.5%)순으로 조사되었다. 동결보존 희석액에 첨가되 는 당류의 분자량은 동결 과정 중 삼투압조절, 탈수, 원형질막의 보존과 융해 이후 생존 율 운동성 등에 영향을 미치는 측면을 감안한다면 위 연구결과와 같이 당류 별로 각각 상이한 결과를 나타낸 것은 당류가 정자의 생존율 및 운동성 등에 매우 중요한 역할을 하는 것으로 판단된다. 이에 동결보존 희석액내 첨가되는 당류의 선정이 매우 중요하며 더불어 동결보존 시 활용되는 항동해제의 적정농도와 생존율 향상을 위한 흑염소 동결보 존의 표준화가 확립된다면 흑염소 개량과 번식효율 향상으로 농가소득 향상에 기여할 것 으로 사료된다.

본 연구는 칡소의 동결정액 생산 시 항산화제 첨가가 정자 생존성 및 운동성 등에 미 치는 영향을 조사하고자 수행하였다. 공시축의 사양관리는 한우사양관리(국립축산과학원, 2007) 기준에 준하여 사육하였다. 공시축은 정액채취를 위해 승가훈련이 완료된 칡소 5두 를 공시하였고 정액채취 빈도는 주 1회로 하였다. 정액 채취는 암소 보정 후 공시축의 승가를 유도한 다음 인공질에 음경을 삽입하여 채 정 후 원정액 정자가 80∼90% 생존율을 나타내는 것을 동결정액 생산에 활용하였다. 동 결보존 희석액의 기본조성은 Tris(hydroxymethyl)amino-methane 250mM, Citric-acid monohydrate 80mM, Fructose 60mM, Penicillin G Streptomycin 1%, Egg-Yolk 20%, Glycerol 7%에 항산화제인 Ascorbic-acid를 무처리, 0.5mM, 1.0mM, 2.0mM로 농도로 하 여 본 실험에 공시하였다. 동결정액의 생산은 처리군별로 원정액을 1차 희석한 다음 저온 실로 이동하여 2차 희석 및 Glycerol 평형을 유도한 다음 0.5ml Straw에 충진시켜 액체 질소를 활용한 동결을 실시하였다. 융해 후 정자 특성분석은 위상차 현미경과 컴퓨터정자 분석기(Computer Assisted Sperm Analysis)를 활용하여 생존율, 운동성, 선형성, 직진도 등을 검사한 결과 생존율은 2.0mM, 무처리, 1.0mM, 0.5mM(83.3%, 81.6%, 65%, 60%)였 고 운동성은 무처리, 2.0mM, 1.0mM, 0.5mM(97%, 96.5%, 94.4%, 91.3%)순이였고, 선형성 은 2.0mM, 무처리, 1.0mM, 0.5mM(29%, 29.7%, 32.9%, 36.4%)순으로 낮은 경향을 보였 다. 직진도는 2.0mM, 무처리, 1.0mM, 0.5mM(65.6%, 62.5%, 62.2%, 60.4%)순으로 낮은 경향을 보였다. 동결보존 희석액에 항산화제인 Ascorbic-acid 첨가함으로써 정자의 대사 과정에 따라 생성되는 활성산소의 발생 억제 등으로 인하여 동결융해 후 생존율에 영향 을 미치는 것을 감안하여 분석한다면 항산화제의 농도별로 각각 상이한 결과를 나타낸 것은 Ascorbic-acid의 첨가가 정자의 동결 융해 후 생존율 및 운동성 등에 영향을 미쳤 을 것으로 판단된다. 이와 같은 결과를 바탕으로 칡소 동결정액 생산 시 생존율 극대화를 위해 적정 항산화제 및 첨가 농도를 확립하고 이를 활용한다면 가축유전자원 보존·증식 에 기여할 것으로 사료된다.

Multiple interferon tau (IFNT) genes exist in bovine. An antiluteolytic substance secreted by the bovine conceptus and primarily responsible for maternal recognition of pregnancy is bovine trophoblast protein 1 (bIFNT1), one of new type I interferon tau (IFNT) genes. The objectives of this research were to investigate whether multiple, distinct gene encode bIFNT1 and other type I bIFNT gene in the bovine genome or not and to examine the expression of bIFNT1 and other bIFNTc1 mRNAs during conceptus development. The transcription of these genes could be regulated through caudal-related homeobox-2 (CDX2) and ETS2 and/or AP1(JUN) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. The presence of mRNAs encoded by bIFNT1 and type I bIFNTc1 genes were examined quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis of total cellular RNA (tcRNA) extracted from on the days 17, 20 and 22 bovine conceptuses. bIFNT1 was highly expressed on the day 17 and transcripts were gradually and weakly detectable on the days 20 and 22. However, the other bIFNTc1 gene examined transcripts was highly expressed on the day 20 and transcripts were weakly detectable on the days 17 and 22 bovine conceptuses. Furthermore, human choriocarcinoma JEG3 was co-transfected with an -1kb-bIFNT1/c1-Luc constructs and several transcription factor expression plasmids. Compared to each -1kb-bIFNT1/c1-Luc increased when this constructs were co-transfected with, ETS2, AP1(JUN), CREBBP and/or CDX2. Also, bIFNTc1 gene was had higher effect on activity by alone ETS2, and AP1(JUN) expression factors in choriocarcinoma JEG3 cell. However, bIFNT1 gene expression of the upstream region was not idented. These results demonstrate that these genes display differential, tissue-specific expression and developmental regulation during pregnancy.

Interferon tau (IFNT), produced by the mononuclear trophectoderm, signals the process of maternal recognition of pregnancy in ruminants, but its expression in vivo is not well characterized. Objectives of this study were to determine IFNT gene isoforms expressed in the bovine uterus, and to identify differences in promoter sequences of IFNT genes that differ in their expression. Through the RNA-seq analysis of bovine conceptuses on days 17, 20 and 22 (day 0 = day of estrus), the expression of only two IFNT transcripts, IFNT1 and IFNTc1, were found, which were indeed classified into the IFNT gene clade. IFNT mRNAs were highest on day 17, and then decreased on days 20, and 22, which were also supported by the results of quantitative RT-PCR. Bovine ear-derived fibroblast (EF) cells were then cotransfected with luciferase reporter constructs carrying 5‘-upstream (positions -1000 to +51) regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids. CDX2, either alone or with other Ap-1, ETS2 and/or CREBBP transcription factors, was found to increase luciferase activity approximately 10 and 18 fold more than twice of those cotransfected with bIFNT1, c1-Luc construct. Furthermore, The degree of transcriptional activation by a combination of the AP1, ETS2, CREBBP and/or CDX2 expression vectors was similar to that of CDX2 along plasmid. However, expression patterns of these Luc activity differented. Whereas bIFNTc1-Luc showed lowest antivity had than bIFNT1-Luc reports. Although, lowest antivity had of the bIFNTc1 –Luc report, cotransfected with the bIFNTc1-Luc construct and AP1(JUN) or/and ETS2 expression plasmid, Luc activity was enhanced approximately 2 and 4-fold more than the bIFNT1-Luc. Furthermore, along CDX2 expression factor had high effect on activity of bIFNT1-Luc reporter than the c1 gene in EF cells. These results suggest that two forms of IFNT genes are expressed in utero and their transcriptional regulations differ.

The cancer and Parkinson's disease associated protein DJ-1 is multifunctional protein that involves in diverse cellular process. DJ-1 protein has a cellular protective role and promoted cell survival under an oxidative stress. However, the cellular protective mechanism of DJ-1 is not fully understand, and we needs to be further study their functions in novel organisms. In the present study, we investigated the protective role of DJ-1 against induced oxidative stress in canine cell line. On the basis of these experiments, canine DJ-1 overexpressing and null cell lines were established. The stable overexpression and down regulation of DJ-1 efficiency confirmed by the western blot analysis. Subsequently, the DJ-1 gene transfected cell lines and control cells were subjected to induced the oxidative stress, and then cell viability, cell proliferation assay, cellular apoptosis detection analysis (Annexin V and TUNEL assay), intracellular ROS and mitochondrial activity were measured appropriately. The results showed that DJ-1 overexpressed cells were up-regulated cell viability under oxidative stress conditions induced by the rotenone and hydrogen peroxide (H2O2), whereas loss of DJ-1 cells were down-regulated the cell survival activity. Additionally, overexpression of DJ-1 cells increased cell resistance to oxidative stress and inhibited the elevation of cell death and cellular ROS induced apoptosis. Moreover, DJ-1 overexpressed cells was increased mitochondrial functions by using confocal microscopy with MitoTracker staining. On the contrary to this, DJ-1 null cells show defective cellular protection and mitochondria activity against oxidative stress conditions. Our data indicate that canine DJ-1 protein attenuates cellular apoptosis and ROS generation, enhances the cellular survival activity and promote mitochondrial function under the oxidative stress, likewise other mammalian cells. Importantly, DJ-1 overexpression may be an important part of a protective strategy as a sensor for oxidative stress.

Prolonged communication between oocytes and the surrounding somatic cells is one of the unique reproductive physiology in canine. Paracrine Kit ligand (KITL) signaling is a well-known communication between granulosa cells and the oocyte. KITL is a cytokine growth factor secreted by granulosa cells that signals via the c-kit receptor expressed by oocytes. Paracrine factors, including growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), exert their effects by binding with the kinase receptors expressed on the granulosa cells. However, the regulations of GDF9 and BMP15 in the canine KITL expression are currently poorly understood. Therefore, we investigated the effects of GDF9 and BMP15 on the expression of KITL in canine ovarian granulosa cells in vitro. In Annexin V assay recombinant GDF9 and BMP15 did not induce apoptosis in the cultured ovarian granulosa cells. When treated, FSH significantly increased KITL expression, and hCG suppressed its expression. When both FSH and hCG were treated, the expression of KITL was affected by GDF9 and BMP15 in dose and time dependent manner in the luteal granulosa cells. GDF9 (10 ng/mL) significantly decreased KITL expression after12 h. BMP15 (10 ng/mL) significantly also decreased KITL expression after 24 h. Western blot and immunochemistry results indicate that GDF9 activated Smad2/3. After blocking ALK 4/5/7 receptors by SB, GDF9 failed to activate Smad2/3, also BMP15 did not activate Smad1/5/8 after blocking ALK 2/3/6 receptors by DM. So GDF9 exerts its effects via using ALK 4/5/7 receptors to activate SMAD2/3 signaling, and BMP15 binds ALK 2/3/6 receptors to activate SMAD1/5/8 signaling. The expression of KITL was not changed by SB or DM treatment. However, the effect of GDF9 and BMP15, which decreased the expression of KITL, was suppressed by SB or DM treatment. In conclusion, this study provides the first evidence that recombinant GDF9 and BMP15 decrease KITL expression in canine ovarian granulosa cells.