Earticle

In pigs, expression and amounts of biologically active interferon tau (IFNT) and transforming growth factor-β (TGF-β) at the conceptus-maternal interface increase significantly as conceptuses elongate and begin the implantation process. Epidermal growth factor (EGF) acts through EGF receptor (EGFR) present in the blastocyst seems to regulate embryonic production of IFNT. To understand the mechanisms regulating the interaction between the hatched blastocyst and maternal uterine environment, the production of IFNT and TGF-β were investigated. Hatched pig blastocysts (days 7～10) were cultured and exposed to EGF (0, 1, 10 and 100 ng/mL) for 24, 48 and 72 h. Protein concentrations of IFNT in the cultured media were determined by commercial enzyme-linked immunosorbent assay (ELISA) kit. Epidermal growth factor (10 and 100 ng/mL) increased embryonic production of IFNT and TGF-β production by hatched blastocysts. The above results suggest that epidermal growth factor produced by epithelial cell stimulates the production of IFNT by pig trophoblasts. The capacity of conceptus to increase IFNT and TGF-β production in response to EGF stimulation may be important for the establishment of pregnancy in pig.

Fertilized zygotic embryos rapidly cleave and proliferate without cell cycle checkpoint such as G1/S, G2/M check point, and they do not show apoptosis during early embryo cleavage stages. In vivo fertilized mouse embryos were exposed to 14 Gy X-ray irradiation immediately after pronuclear formation and cultured until the blastocyst stage. We found the X-ray irradiated embryo did not develop to blastocyst, however, 1-cell zygotes injected by CDKN1A siRNA overcome the morula arrest and developed to blastocyst. We also examined regulator genes for CDKN1A using qRT-PCR, but the expression levels of genes were not changed. In conclusion, CDKN1A regulate stage specific DNA damage between morula and blastocyst stages.

One of the most important issues in animal embryology is the ability of the oocyte to develop, that is, pluripotency. So, in this experiment, I want to get information about the early development of pluripotency by observing the presence of certain antibodies in the pluripotency. These results are expected to lead to positive results in many experiments including animal breeding and replication. In this experiment, CDX2, OCT4 and E-cadherin, among the many antibodies known to affect pluripotency, were observed for oocytes development in pigs. In mouse, CDX2 was found in late second stage development and OCT4 is known to be expressed specifically in the ICM region in blastocyst. E-cadherin was expressed in the junctional region of cells after the 2-cell stage. Blastocysts appeared in areas with junctions and cell walls. We observed the early stages of oocytes development in pigs based on PA (Parthenogenesis CDX2 and OCT4 were simultaneously imaged using an optical microscope in immunohisto chemical staining and compared to DAPI. E-cadherin was also experiment in the same way. In the development stage of oocyte development, CDX2 was expressed in blastocyst and expressed in nucleus except ICM (inner cell mass) region, and OCT4 was expressed in whole nucleus including ICM region in blastocyst. E-cadherin was expressed in the cell junctions and cell walls in the blastocyst. Our results showed that three antibody is pigs were expressed in pigs, as in mouses, and that they had an effect on pluripotency.

In mammals, cyclin-dependent kinases (CDKs) are involved in regulating both the cell cycle and transcription. Although CDK1 is known to act as the kinase subunit of maturation- promoting factor (MPF), the roles of the other CDKs in mammalian oocyte maturation are not yet understood. Here, we show that inhibition of various CDKs by small molecule inhibitors has different effects on the maturation and transcriptional activity of pig oocytes in vitro. Inhibition of CDK1 did not significantly affect cumulus cell expansion, but its kinase activity was necessary for germinal vesicle breakdown (GVBD). The inhibitions of CDK2, CDK4, or CDK6 had no effect on cumulus expansion or GVBD. The catalytic activity of CDK7 was crucial for GVBD but less important for cumulus expansion, whereas inhibition of CDK9 severely blocked both cumulus cell expansion and GVBD. CDK1, —2, —4, and —6 appeared to be dispensable for nuclear transcription, as their inhibitions did not affect nascent RNA production in oocytes. However, inhibition of CDK7 or CDK9 dramatically decreased the transcriptional activity in oocytes. Finally, we found that the GVBD arrest triggered by CDK9 inhibition was not due to altered MPF activity, but rather the inhibition of transcription. Overall, our results show that CDK7 and CDK9 are important for the nuclear maturation and transcriptional activity of pig oocytes.

SETD2 (SET domain containing protein 2) is known as a histone H3 lysine 36 (H3K- 36)-specific methyl-transferase, and suggesting that it has an important role in gene active transcription in human cells. In the current study, to investigate the dynamic change of SETD2 in pig, we determined the SETD2 expression in porcine fetal fibroblasts, oocytes and preimplantation embryos derived from in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) by immunofluorescence using specific antibodies and laser scanning confocal microscopy. In porcine fetal fibroblasts, SETD2 expression was detected in inter phase, not in M (mitosis) phase. SETD2 signal was observed in non-surrounded nucleolus (NSN) stage oocytes. However, there were no signals were detected in surrounded nucleolus (SN), metaphase I (MI), and metaphase II (MII) stage oocytes. In IVF embryos, SETD2 signal was detectable in sperm, but this signal was lost after fertilization, and then became detectable at 2-cell stage, peaked at the 4-cell stage which is porcine embryonic gene activation time. Similar to the pattern found in IVF embryos, SETD2 signal in PA embryo was not detected at 1-cell stage, but detected at 2-cell stage, and maintained to blastocyst stage. Interestingly, unlike IVF and PA embryos, SETD2 signal could not be lost in 1-cell stage of SCNT embryos, and this signal was detectable in whole SCNT embryonic developmental stage. Overall, these data indicated that the SETD2 may be a mark of embryonic gene activation in porcine preimplantation embryos. Aberrant SETD2 expression occur in 1-cell stage of porcine SCNT embryos may be a factor of inducing low of cloning.

Less than two layers of cumulus cells in cumulus cell-oocyte complexes (COCs) considered morphologically poor in pig. Much to our regret, COCs of more than half included in poor group. If we could take full advantage of all of these poor quality COCs, we could dramatically improve the efficiency of in vitro embryo maturation. During in vitro maturation, some maturation factors are interacted bidirectionally between oocyte and cumulus cells, also interaction occurs between COCs and other COCs. We hypothesized that poor COCs suffer a failure in complete embryo maturation due to their insufficient maturation factor secretion. Therefore, we tried whether it increases the maturity and utilization of poor COCs if we co-culture good COCs and poor COCs. We harvested 6,201 COCs, classified them into grades I～IV. We investigated maturity, glutathione levels, embryo development capacity, blastocyst quality, and cumulus gene expression levels with suitable methods. 6,201 COCs were classified grade I (N=534, 8.6%), grade II (N=1,886, 30.4%), grade III (N=3,109, 50.1%) and grade IV (N=672, 10.9%). Our results showed that oocyte maturation, glutathione levels, embryo development capacity, blastocyst quality, and cumulus gene expression levels of BCL-2 and PCNA were similar to co-cultured good quality groups, all of which were substantially higher than the poor group. Our results suggest that co-culture strategy have considerably increased the utilization of poor COCs without reducing their ability for maturity and development capacity.

Melatonin has antioxidant and scavenger effects in the cellular antioxidant system. It acts by protecting cells from oxidative injury via superoxide-free radicals and hydrogen peroxide (H2O2). Recent studies have shown that treatment with melatonin improves somatic cell nuclear transfer (SCNT) embryo development and increases the success of somatic cell reprogramming. However, the mechanisms for this remain unclear. This research investigated the protective effects and underlying mechanisms of action for melatonin in porcine SCNT embryos under oxidative stress. The results suggested that the developmental competence of porcine SCNT embryos was significantly enhanced after melatonin treatment. In addition, melatonin attenuated the H2O2-induced increase in reactive oxygen species levels, decrease in glutathione levels, and mitochondrial dysfunction. Importantly, melatonin also inhibited phospho-histone γH2A.X expression and comet tail formation, suggesting that it prevents H2O2-induced DNA damage. Meanwhile, the expression of genes involved in homologous recombination (MRE11a, BRAC1, and RAD51) and non-homologous end-joining (PRKDC, XRCC6, and TP53BP1) pathways for the repair of double-stranded breaks was reduced upon melatonin treatment in porcine SCNT embryos on day 5 of development under H2O2-induced oxidative stress. Taken together, these results indicated that melatonin promotes porcine SCNT embryo development by preventing oxidative stress-induced DNA damage via quenching of free radical formation. Our results reveal a previously unrecognized regulatory effect of melatonin in response to oxidative stress and DNA damage. This provides a novel mechanism for the improvements in SCNT embryo development that were reported to be associated with exposure to this hormone.

Zinc (Zn2+) is one of essential factors during mammalian oocyte maturation and fertilization. Previous studies showed that depletion of cellular Zn by metalion chelator impair asymmetric division of oocyte. But the detailed mechanism of these phenomena is unclear. We found that depletions of zinc by cell-permeable heavy metal chelator N,N,N',N'- tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) caused the decrease of cytoplasmic actin mesh level. Spire2-GFP is co-localized with zinc at the cortex and intracellular vesicle. By the treatment of TPEN, number of Spire2-GFP decorated vesicle is drastically decreased, indicating that Zn2+ is essential for the localization of the spire in mouse oocyte. Two putative zinc-binding regions were located in the C-terminal part of Spire2. Mutations of zinc binding site on spire abolish its localization at the intracellular vesicle. Over expression of C-terminal region containing zinc binding site of spire impair oocyte maturations and decrease cytoplasmic actin mesh. Taken together, these results suggest that intracellular zinc is crucial for the proper localizations of spire in the mouse oocyte, and unraveling the novel regulatory mode of actin nucleator spire by Zn2+.

Fatty acid synthase (FASN) is an enzyme that catalyzes the synthesis of long chain fatty acid. During development, FASN plays a role in growth rather than the energy storage pathways. In this study, we hypothesis that knockout of FASN may affect the early embryonic development through induction of the endoplasmic reticulum (ER) stress. The function of FASN was studied using the Crispr/Cas9 technology. We found that FASN knockout induced ER stress by generation of reactive oxygen species (ROS), further resulted in the activation of adaptive unfolded protein response (UPR), caused the splicing of XBP1. In addition, FASN knockout increased ATF4 and CHOP expression, influenced phosphorylation of PERK. At last, Ca2+ was released from the ER and then taken up by the mitochondrial and influenced mitochondrial function, initiated apoptosis. These results demonstrated that FASN knockout induced generation of ROS, which mediated the activation of UPR via the ER and subsequent apoptosis in porcine early embryos.

C-phycocyanin (C-PC) is a biliprotein enriched in blue-green algae that is known to possess antioxidant, antiapoptosis, anti-inflammatory, and radical-scavenging properties in somatic cells. But the protective effect of C-PC on porcine embryo developmental competence in in vitro is little known. In the present study, we investigated the effect of C-PC on the development of porcine early embryos as well as the underlying its mechanisms. Different concentrations of C-CP (1, 2, 5, 8, 10 μg/mL) was added to the porcine zygote medium 5 (PZM-5) during in vitro culture. The results showed that 5 μg /mL C-PC significantly increased blastocyst formation. Blastocyst formation and its quality were significantly increased in 50 μM H2O2 treatment group following 5 μg/mL C-PC addition. C-PC prevented H2O2-induced compromise of mitochondrial membrane potential, release of cytochrome c from the mitochondria and reactive oxygen species generation. Furthermore, apoptosis, DNA damage level and autophagy in the blastocysts were attenuated by supplement of C-PC in H2O2-induced oxidative injury group compared with control. Taken together, these results suggest that C-PC has beneficial effects on the development of porcine parthenotes by attenuating mitochondrial dysfunction and oxidative stress.

Present study, it is demonstrated that MMP-9 serum as well as follicular concentrations are related to a successful IVF resulting in pregnancy. Currently, a limited number of studies have investigated intrafollicular and serum MMPs, with inconsistent conclusions. However, MMPs theoretically could be important players in IVF processes. Moreover, different behaviors of MMP-2 and MMP-9 during the embryo development were confirmed; only MMP-9 has shown a vast difference between follicular and maturation oocyts. The main objective of this study is to examine the mode of MMPs activation during bovine in vitro fertilization. In particular, the relative employment of two major embryo culture medium in embryo development; e.g. ES(non-bovine serum) and CR(bovine serum) was compared. This results found that the level of MMP-2 and MMP-9, which degrades basal membrane, was different in the ES and CR in cultured at each stage of embryo development. In ES cultured system, MMP-9 was highly expressed throughout blastocyst inner cell mass and exhibited strong gelatinase activity. The MMP-9 expression continually increased as the embryo develops from oocyte maturation to fertilization, Meanwhile, the level of MMP-2 remained insignificant throughout these periods. However, decreased MMP-2 and 9 expression on the CR culture system. Interestingly, MMP-9 was actively expressed in ES culture system, inner cell mass is showing levels higher than trophoblast cell. Our result suggests a possibility that different subtype of MMP partakes major functions in the remodeling of inner cell mass in blastocyst. Such differences in MMP expression may impact to the distinct mode of follicular development in the two species.

The feeding strategies are important to improve milk production, milk quality and cost-efficiently. It has been studies that the amount of concentrated fed should depend on the milk yield. However, there is no evidence of difference in colostrum composition based on different levels of feed. Here, we investigate the correlation between feeding levels and colostrum protein. We observed significant changes of colostrum protein between two groups, which were eating the standard diet and high-energy diet. Milk proteins, colostrum, were collected from the day of calving and 3rd day of calving. The change of milk protein was analysis using two-dimensional (2-D) gel electrophoresis and these proteins were identified using PMF analysis. Five protein were identified in milk from the day of calving. The level of αS2-casein precursor and β-casein were higher in the colostrum from high-energy diet group than standard-diet while, IgG3 heavy chain constant region, non-classical MHC class Ⅰ antigen isoform X2 and β-casein A2 variant were higher in colostrum of standard diet group. Twelve proteins were identified in milk from 3rd day of calving. All of these protein highly observed in colostrum from standard diet group than high-energy diet. In addition, Most of this protein known as immune-related factor and colostrum of standard diet group contain many of those factors than high-energy group, although we observed the mature milk yield is a slightly lower in the group of standard diet. On other hand, excess supply of diet can lead the milk production, which has less immune-related factor in colostrum.

Mammary gland development is critically dependent on the interaction between the stromal and the epithelial compartments within the gland. In this study, we established a co-culture of bovine mammary epithelial Mac-T cells and murine preadipocyte 3T3-L1 cells. Mac-T cells were co-cultured with 3T3-L1 cells for four days and production of milk proteins was induced for three days. After seven days of co-culturing, the number of alveolar-like colonies in the presence of 3T3-L1 cells was significantly higher than that in control group. Expression levels of αS1-casein and β-casein mRNAs were significantly increased by co-culturing with 3T3-L1 cells. In addition, casein protein production was significantly higher in the co-culture of Mac-T cells with 3T3-L1 cells than in control Mac-T cells. Substances that induced casein production in Mac-T cells also stimulated adipogenesis in 3T3-L1 cells. We suggest that a co-culture system of bovine mammary epithelial cells and preadipocyte cells is an efficient method for in vitro milk protein production.

The colostrum proteins are an important energy source for newborns and improves their innate immune system. Recently, there are many interest about beneficial factors in colostrum to health and many products using colostrum are attentive into dietary supplements in global industry. The aim of this study was to compare the enriched proteins between the colostrum from the first and the third day after calving using proteomic analysis and to analyze which enriched protein will be useful to industry of dietary supplements. In this study, cows in the experimental group were fed a standard composition of feed for 12 months, after which we collected the colostrum on the first and the third day after calving. By comparison between the first and the third day colostrum, several factors, including beta-lactoglobulin, fibrinogen gamma-B chain, complement C3, zinc-alpha-2 glycoprotein, bP47 protein, beta casein, and alpha-S2 casein were enriched in the third day colostrum, whereas immunoglobulin gamma 1 and beta- casein A2 were enriched in the first day colostrum. The results suggest that the colostrum composition depends on time and the first day colostrum is important to establish the primary specific immune system, whereas the third day colostrum might regulate the non-specific immune system and increase nutrition using casein and the third day colostrum might be useful dietary products for supporting the immune system.

With the global demand for dairy protein for consumption growing annually, there has been increasing activity in the research field of dairy protein synthesis and production. From a manipulation perspective, it is more difficult to use live cattle for laboratory studies on the production of milk as well as of dairy protein such as casein, as compared with using laboratory animals like rodents. Therefore, we aimed to develop a mouse model of bovine mammary alveolar ducts for laboratory-scale studies. We studied the formation of the bovine mammary gland ductal structure by transplanting the MAC-T bovine alveolar cell line into mice. MAC-T cells (1×107) were suspended in Matrigel and injected into the dorsal tissue of 8-week-old male BALB/C nude mice. Histological analysis of tissue dissected from the MAC-T cell-transplanted mice after 6 weeks showed the typical morphology of the tubuloalveolar female gland, as well as glands made up of branching ducts that were surrounded by smooth muscle with small alveoli budding off the ducts. In addition, the epithelial markers CK14 and CK18 were expressed within the duct-like structure. Prolactin was detected in the duct interior in these CK14+ and CK18+ cells but not in the non-transplanted MAC-T cells. These results showed that duct-like tissue had been successfully formed after 6 weeks of transplantation of the CK14+ and CK18+ MAC-T cells into mice dorsal tissue. This mouse model will be a useful tool for further research on the bovine mammary gland.

Premature ovarian failure is an important problem for young women with cancer during chemotherapy. To preserve the fertility of young female cancer patients, it is necessary to find out proper methods and materials against chemotherapy-induced ovarian failure. In the previous study, we reported that melatonin treatment partially prevents the depletion of dormant follicle pool via repressing simultaneous activation of dormant primordial follicles by cisplatin. However, the protective effect of melatonin was partial, but not sufficient. Here, we found a hormone, ghrelin to maximize the protective effect of melatonin on ovarian failure in cisplatin-injected mice. Interestingly, co-administration of melatonin and ghrelin more effectively works against follicle disruption by cisplatin. Simultaneous treatment of melatonin and ghrelin almost restored the number of primordial follicles and corpus-luteum in cisplatin-treated ovaries, compared with the single administration group. We found that melatonin and ghrelin receptors were highly present in the cell membrane of premature oocytes of primordial follicles. In addition, both melatonin and ghrelin treatment decreased the phosphorylation of PTEN and FOXO3a resulting in inhibition of FOXO3a’s location in the nucleus and reduction of p27Kip1 expression in primordial follicles. Nuclear FOXO3a and p27Kip1 expression were critical for keeping dormant status of primordial follicles in cisplatin-treated ovary. In conclusion, these findings suggest that combination of melatonin and ghrelin is able to be used in clinics for fertoprotective adjuvant therapy during cisplatin-mediated chemotherapy in young female cancer patients.

형질전환 동물의 생산기술은 기존동물의 이용효율을 획기적으로 극대화시킬 수 있으며, 특히 돼지는 다른 포유동물에 비해 생리 및 장기 형태가 인간과 가장 유사하여 이종이식 용 형질전환 연구의 매개체로서 이용되고 있다. 그 중 Massachusetts General Hospital (MGH) 미니돼지는 주요 조직적합항원(major histocompatibility complex)을 고정화 하여 이종이식 연구 활용을 위해 개발되었다. 그러나 일반돼지와 번식 생리학적인 특성에서 유사성을 나타내는지에 대해서 아직 연구된 바가 없다. 따라서 본 연구는 MGH 미니돼지 의 발정 특성을 분석하여 일반돼지와 유사한 양상을 보이는지 확인하기 위하여, MGH 미 니돼지로부터 alpha-1,3 Galactocyltransferase (GalT) 유전자 좌위에 membrane cofactor protein(MCP) 유전자가 삽입된 Homozygote (GalT –MCP/-MCP), Heterozygote (GalT –MCP/+) 다 중 형질전환 돼지와 CD73 (ecto-5’-nucleotidase) 유전자가 발현된 돼지의 발정주기를 MGH 일반돼지와 비교하여 분석하였다. 발정주기는 17개월부터 63개월령 사이의 돼지를 대상으로 14개월에 걸쳐 분석하였으며, 매일 정해진 시간에 암컷을 대상으로 외음부의 상태를 육안으로 파악하여 발정 유무를 판단하였다. 그 결과, GalT –MCP/-MCP, GalT –MCP/+ 및 CD73 돼지의 발정 주기는 각각 18.05±8.46, 21.79±12.05, 25.60±13.97일로 MGH 일 반 돼지의 22.17±9.12일과 유의적인 차이는 보이지 않았다. 발정이 지속되는 기간은 각 각 3.18±0.95, 3.06±0.85, 3.13±0.84일로 MGH 일반돼지의 3.15±0.99일과 같이 정상적인 발정 기간이 관찰되었다. 그러나 MGH 돼지의 임신기간은 117±1.3일로 일반적으로 알려 진 돼지의 평균 임신기간 114일과는 차이가 있었다. 결론적으로 MGH 돼지의 임신기간 은 일반돼지보다 길지만, 발정주기와 기간은 큰 차이를 보이지 않았다. 이러한 자료는 MGH 돼지의 안정적인 증식과 계통 조성에 활용될 수 있을 것이라 생각된다.

Porcine litter size is a quantitative trait and its heritability is especially low. So it is necessary to identify porcine reproductive gene and protein. The establishment of pregnancy requires performance of a receptive endometrium. The endometrium secretes a wide array of growth factors, cytokines and proteins. Based on these background, we analyzed the endometrial tissue protein of porcine and would find out biomarker proteins related to porcine litter size. We sorted the two groups according to litter size of porcine: a small litter size group (SLSG) (n=2) and a large litter size group (LLSG) (n=2). The porcine endometrial tissue samples were preprocessed for proteomic analysis. In order to comparison, samples of each 2 mg endometrium protein were separated form pI and molecular weight in the same conditions by applying a pH 3.0～10.0 IPG gels for the first dimension and then 8～16% SDS-PAGE gel for the second dimension. After proteins were visualized by staining with Commassie brilliant blue (CBB), imange analysis was performed with Image Master detect variations in protein spots between large litter size group and small litter size group endometrium. And then differential proteins were identified using MALDI-TOF analysis. The master images of 2-DE gel images obtained from 2 mg samples of large litter size group and small litter size group endometrial proteins at pH 3.0～10.0 revealed more than 400 protein spots in pH 3.0～10.0 range. When we analyzed the levels of expression of proteins that protein spots appeared more than 1.5-fold difference in endometrial tissue from porcine. In comparison of SLSG(small litter size group) with LLSG(large litter size group), a total of 9 protein spots differentially expressed on porcine endometrial tissue 2-DE gels, among which 5 spots were up-regulated proteins as retinol dehydrogenase 16-like isoform 1, Acrosin-binding protein, alpha-N-acetylgalactosaminidase. phosphoglycerate kinase 2, Acrosin-binding protein in LLSG. And 4 spots were up-regulated proteins as phosphoglycerate kinase 2, prenylcysteine oxidase in SLSG.

Plasma membrane contains ion channel, protein and peptide receptor, phospholipid and various organelles that play a role metabolism, ion transport, antigen-antibody reaction, hormone response and physiology of reaction in eukaryotic cells. Especially, viability and motility directly related with plasma membrane proteins in sperm. Therefore, this study was to investigate change of proteins by plasma membrane integrity (PMI) in bull sperm. One hundred fifty ejaculates obtained from twenty three bull were diluted in semen extender. Sperm PMI was measured by SYBR14 and propidium iodide (PI) double staining method and analyzed using flow cytometry. Sperm was classified as more than 60% PMI (High PMI) and less than 40% PMI (Low PMI). Proteins were extracted from sperm of each groups using mammalian protein extraction buffer. The proteins was separated using immobilized pH gradient strip by isoelectric point and electrophoresed using SDS-PAGE. Protein intensity of visualized protein spots were detected using image analyzing program, and protein mass was analyzed. Molecular and biological function of proteins was investigated in universal protein resource based on protein mass and coverage rate. In results, 21 protein spots were repeatedly found in the High and Low PMI groups, and molecular functional analysis demonstrated that the proteins were primarily involved in ATP binding and cytokine activity. Especially, MAP kinase mkk-4, cyclin-dependent kinase 1, and heat shock 70 kDa protein 1-like were upregulated, whereas interferon alph-H, interleukin-4, succinyl-Coa ligase subunit beta, and adenylate kinase isoenzyme 1 were down-regulated in the High PMI. Additionally, biological functions demonstrated that proteins involved in cell proliferation and division were up-regulated in the High PMI group. These results may provide understanding of plasma membrane proteins in bull sperm, and will be used in male reproduction for developing specific bio-markers in the future.

The freezing semen extender of stallions has been used routinely for artificial insemination (AI) of the mares. The INRA96 was often used as a base solution for freezing extender for stallion semen in addition of egg yolk and glycerin. In this study, the possible use of SM, domestically developed semen extender for fresh and cool semen, as a base solution for freezing semen extender was tested. The semen was collected from 3 Thoroughbred stallions (an average age of 6.66±0.19) using CSU artificial vagina. Semen was diluted in SM or INRA96 at 1:2 ratio and centrifuged to remove the supernatant in where seminal plasma was placed. Sperms were resuspended with freezing medium prepared with 2% egg yolk and 2.5% glycerol added to SM or INRA96 and frozen in 0.5 cc straws. For thawing process, straws were immersed in the 37℃ water bath for 30 sec. After thawing, total and progressive motility, membrane integrity, viability and mitochondria membrane potential were measured to compare the freezing efficiency of these base solutions. Statistical analysis was performed using SAS program. Total and progressive motility, membrane integrity, membrane integrity, viability, and mitochondria membrane potential of frozen/thawed sperms with SM based freezing medium were not significantly different with those of sperms frozen/thawed with INRA 96 based freezing medium. This result suggests that SM is as effeiceint as INRA96 as a base solution for freezing medium. However, the membrane integrity (28±6.6), viability (22.8±3.8), and mitochondria membrane potential (24.6±11.8) of frozen/thawed sperms with SM were very low. Also total (32.3±4.2) and progressive motility (1.72± 0.52) of these sperms were not acceptable for commercial use. In conclusion, the efficiency of SM as a base solution for freezing medium of stallion semen is comparable with INR96.

The spermatogonial stem cell (SSC) transplantation technique has been utilized for the production of donor-derived sperms in the recipient animals. Depletion of the endogenous germ cells in the seminiferous tubules is the key technique for successful SSCs transplantation because it provides a place for transplanted donor germ cells settle in the seminiferous tubules. We previously reported that intra-testicular injection of 70% glycerin partially removed endogenous germ cells, but the effect varied among stallions. The main objective of this study was to find out an optimal dose and injection method by which endogenous germ cells of stallions are successfully depleted without disturbing functions of sperm production and libido. Each group of stallions (n=3) were treated with 1) 15 mg of busulfan/kg of BW, all at once (group 1), 2) 15 mg of busulfan/kg of BW, at 5 different times (group 2), or 3) none (control, group 3). The percentages of survival rate in group 1, 2, and 3 were 33 (1/3), 100 (3/3), and 100% (3/3), respectively. The percentage of Sertoli cell only cross section of seminiferous tubules were significantly higher (p<0.05) in group 2 (78.2±7%) compared to group 3 (0%). The averages of testicular weight (g) between group 2 (143.5±8.5 g) and 3 (179±1.0 g) were not significantly different (p<0.05). In group 2, the number of sperm production and motility (total and progressive) decreased after busulfan treatment. However, libido of stallions was not altered after treatment. In conclusion, weekly split IV injection of busulfan can be utilized to deplete endogenous germ cells for the purpose of preparing recipient stallions for spermatogonial stem cell transplantation.

This study examined factors affecting the analysis of motility of chicken semen. The viability of spermatozoa was estimated using varying dilution ratios and supplementation with BSA or fatty acid free (FAF)-BSA as protein sources in semen diluent. Fresh semen was examined after preparing dilutions in BPSE of 1/8, 1/16 and 1/32 at 25℃. The motility of incubated semen at each dilution was observed at 3 min (89.9%, 69.9 % and 53.2%), 30 min (86.7%, 71.4% and 51.7%), 1 h (89.5%, 74.0% and 53.5%) and 3 h (78.5%, 66.5% and 45.7%), respectively. The addition of BSA or FAF-BSA to BPSE diluent significantly increased the viability of semen in 1/32 dilution with results of 53.2% (control), 84.8% (BSA) and 92.9% (FAF-BSA) (p<0.05). This phenomenon was also observed in the dilution of frozen semen, where FAF-BSA treatment increased the viability of thawed semen from 17.6% to 34.0% in a 1/8 dilution (p<0.05). When the protein sources were used in the dilution, the survival rates of diluted chicken semen were also increased with time lapse. These results show that FAF-BSA may act to protect chicken semen and is suitable as a basic component of chicken semen diluent for the method of analyzing rooster semen after freezing.

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 both the sperm fertility and developmental capability of IVF embryos using Hanwoo sperm obtained by Korea Animal Improvement Association and JBC sperm obtained by Jeju Special Self-Governing Province Livestock 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 fertilization rate was higher in Hanwoo sperm group (80.3±5.2) than in JBC sperm group (73.3±5.2). The percentage of morula formation was the higher in JBC sperm B group (43.0) than in the other groups. However, the percentage of blastocyst formation was higher in the Hanwoo B sperm group (42.3±3.9) than in the other sperm groups. The total cell number of blastocyst was the higher in Hanwoo sperm group (168±8.9) than in JBC sperm group (119± 15.9). These results demonstrate that semen characteristics and in vitro development potential are similar for JBC-B and Hanwoo, but the quality of JBC-A is slightly lower. We need to embryo transfer study for field trial.

농촌진흥청 국립축산과학원에는 이종장기 이식을 위한 초급성 거부반응과 보체 활성화 를 억제시킨 alpha 1,3-galactosyltransferase knock out/MCP (GTKO/MCP) 돼지, 바이러 스 저항성 3D8 scFv (3D8) 돼지, Erythropoietin(hEPO) 돼지, htPA 돼지, Factor8와 vWf 돼지와 같이 다양한 형질전환 돼지들을 사육 중에 있다. 형질전환 돼지의 안정적인 번식 과 유지를 위해서는 돼지 정자 활용이 필수이다. 본 연구에서는 국립축산과학원에서 보 유하고 있는 형질전환 돼지의 정자 품질과 관련된 정자 성상을 일반돼지의 정자성상과 비교 평가하였다. 정액은 수압법을 이용하여 GTKO/MCP, hCD73, hEPO, htPA, Factor8, vWf, 3d8, 일반돼지에서 채취를 하였고, 채취된 정액은 Androhep plus를 이용하여 1:1로 희석하였다. 희석된 정자는 Diff qucik stain을 이용하여 형태, 광학현미경(20배)을 이용하 여 전진 운동성, SYBR-14/PI를 이용하여 생존성, FITC/PSA를 이용하여 첨체 온전성을 평가하였다. 본 연구에서 그룹은 Factor8, vWf, htPA, hEPO, 3D8, hCD73, GTKO/MCP 및 일반돼지로 구성하였다. 정액 성상 분석결과, 정자의 형태는 71.2±4.2, 85±6.4, 78.7± 7.7, 86.3±5.4, 74.0±4.0, 96.6±0.6, 72.3±5.3, 93.0±3.0으로 각각 조사되었고, 정자의 운동 성은 80.0±4.0, 85.0±2.8, 75.0±9.5, 85.0±2.2, 85.8±3.2, 77.5±2.5, 70.0±4.4, 90.0±0.0으 로 각각 조사되었으며, 정자의 생존성은 82.4±2.7, 89.9±0.9, 83.9±5.0, 87.4±1.5, 90.0± 1.5, 92.8±1.6, 83.6±3.0, 93.9±0.9로 각각 조사되었다. 마지막으로 정자의 첨체 온전성은 98.6±0.2, 98.6±1.0, 98.7±1.0, 98.1±1.5, 97.2±1.9, 99.5±0.3, 95.1±1.2, 98.1±0.9로 각각 그룹별로 조사되었다. 형질전환 돼지의 성상분석 결과, 일반돼지의 성상 분석결과와 유의 적인 차이는 나타나지 않았다. 본 연구에 공시된 형질전환 돼지에 도입된 외래 유전자는 돼지 정자의 기능적 이상을 나타내지 않는다는 것을 의미하며, 이는 유전자 조작이 돼지 정자의 성상에 영향을 미치지 않는 것으로 결론된다.

Porcine pluripotent stem cells (pPSCs) have provided potentials for agricultural biotechnology and biomedical models. However, authentic pPSCs have not yet established because standards for pPSCs-specific markers and culture conditions are not clear. Therefore, we have generated various types of pPSC lines, porcine epiblast stem cells (pEpiSC type 1 and type 2) derived from in vivo derived epiblasts and porcine induced pluripotent stem cell (piPSC) lines derived from porcine fetal fibroblasts (PFFs) with lentiviral transduction of six reprogramming factors (hOct4, hNanog, hSox2, hc-Myc, hKLF4 and hLin28) or sendai viral transduction of four reprogramming factors (hOct4, hSox2, hc-Myc and hKLF4). As the results, Lenti viral piPSCs and pEpiSCs type 1 have showed AP positive but Sev viral piPSCs and pEpiSCs type 2 have showed AP negative. However, both AP positive and negative pPSCs have expressed pluripotent associated genes (Oct4, Nanog and Sox2). In addition, all of these cell lines have showed in vitro differentiation potentials. An increase of S and G2/M phase in pPSCs was shown when compared to that in the control (PFFs) based on cell cycle analysis. However, surface marker of SSEA-1 was expressed only in pEpiSCs type 2 and Sev viral piPSCs, whereas surface marker of SSEA-4 was expressed only in pEpiSCs type 1 and Lenti viral piPSCs. Additionally, Tra-1-60 and Tra-1-81 were positively expressed only in Lenti viral piPSCs. In conclusion, these results showed that it requires to demonstrate specific markers and culture conditions for pPSCs because criteria for pPSCs are not clear.

Stem cells can be differentiated into numerous cell types and then can be used to research intractable disease and regenerative medicine. Induced pluripotent stem cells (iPSCs) are produced through an artificial manipulation in vitro, and have the advantage of being to customized to patient. In this study, we used the Sendai virus to produce porcine iPSC (p-iPSC) from Jeju Black Pig (JBP) cells. JBP cell was infected for 24 h with Sendai virus (1×104 cells/mL). One week after infection, they were spread onto mouse embryonic fibroblast (MEF) feeder cell layer. When we used JBP #4007 2nd passage cells for iPSC generation, primary colony was appeared between 3～4 weeks. The first and second colony were dissociated by treatment with trypsin and hereafter formed colony were mechanically dissociated by insulin syringe. During culture of the JBP-iPSC, some colonies were stained by alkaline phosphatase (AP) staining kit for confirming expression of the pluripotency marker. The stem cell markers as undifferentiation state, Oct4, Sox2, SSEA-1 and Nanog, were stained using JBP-iPSC at 5 passage. The JBP-iPSC colony have been maintaining in nowaday 14 passages, and the AP and immunostain were expressed. These results demonstrate that p-iPSC can derive from Jeju Black Pig cell using Sendai virus, and it will be used to stem cell research for animal disease model.

Jeju Black Cattle (JBC, endangered native Korean cattle), a species characterized by their pitch-black coat color, live in Jeju Special Self-Governing Province. Pluripotent stem cells (PSCs) derived from farm animals have potential for various applications in transgenic breeding, basic research and agriculture. However, there were a few reports for establishment of bovine induced PSC (iPSC) and their culture conditions were not optimized yet. In this study, JBC bull #5293 (date of birth, October 31, 2013) ear cells were transfected by retroviral vectors that include 5 pluripotent reprogramming factors, hOCT4, hSOX2, hKLF4, hc-MYC and hNANOG. The JBC cells were exposed to retrovirus in suspended state in a fibroblast culture medium (10% FBS+DMEM) for 3 h and then plated on culture dish (3×104 cells/60mm-dish). After three days, infected cells were dissociated and replated to a new feeder-free culture dish or mouse embryonic fibroblast (MEF) feeder cell dish (3×104 cells/60 mm-dish). And these cells were cultured in two different ES media (15% FBS+DMEM; 15% FBS or 20% SR+DMEM/F12; 20% SR) commonly supplemented with 5 ng/mL bFGF and 1,000 U LIF. Produced iPSC-like colony was separated by mechanical dissociation and transferred to a new MEF cell dish. In the result, cell aggregation before colony formation was predominantly appeared in 20% SR group compared with 15% FBS group, while the JBC cell growth was faster in 15% FBS group. In comparison of feeder-dependent or -free culture, differentiation was more frequently appeared in feeder-free condition. However, a iPSC-like colony was recovered in 15% FBS group under feeder-free culture. These iPSC-like colonies were maintained up to 3 passages. When we checked the pluripotent cell marker alkaline phosphatase (AP), the activity was strongly detected in 2nd passage JBC iPS-like cells. Further experiments were needed to find the better stable culture condition able to more passages culture (>15) for establishment of JBC-iPSCs.

Xenotransplantation has been considered as an alternative to moderate shortage of donor organ for transplantation. To achieve successful xenotransplatation, we need to overcome immune rejection. Even though, hyperacute rejection has been overcome by α1,3-galactosyltransferase knockout pig, cellular immune rejection still remains as a subsequent barrier. Interleukin-10 (IL-10) is known as anti-inflammatory and immunomodulatory cytokine which has been shown to limit inflammatory responses by inhibiting macrophage activation in several animal experiments. To study of effect of human IL-10 (hIL-10) on pig-to-human xenotransplantation, we estabilished porcine kidney epithelial cell line (PK15) expressing hIL-10. When the cells were co-cultured with human monocyte-derived macrophages by phorbol myristate acetate activation, the cytotoxicity of macrophages was decreased by hIL-10. Furthermore, there is a decreased production of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-23, and increased anti-inflammatory cytokines like IL-10, but not transforming growth factor beta, in the presence of hIL-10. Also, macrophage polarization toward M2-like phenotype were induced by hIL-10 from transgenic PK15 cells. Finally, we found that hIL-10 expressing from porcine cells induces the macrophage polarization into M2-like macrophages, reducing the cytotoxic effect of human macrophages. Therefore, these findings suggest that hIL-10 transgenic pig seems to be considered as an useful model to overcome xenograft rejection.

In this study, we report successful expression of recombinant human erythropoietin (hEPO) in the egg white of transgenic hens using a feline immunodeficiency virus (FIV)- based lentiviral vector as an exogenous gene deliverer. hEPO is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. FIV vectors permit high levels of transgene expression in quails and chickens. We constructed a FIV vector containing a hEPO cDNA sequence driven by an oviduct-specific ovalbumin promoter, and microinjected into the subgerminal cavity of stage X chick embryos to generate transgenic chicken. Out of 208 injected eggs, 10 chicks were hatched after 21 days of incubation, and one of the G0 hatched chicken expressed the vector-encoded hEPO gene in sperm. Successful germline transmission of the transgene was also confirmed in G1 transgenic chicks produced from crossing G0 transgenic roosters with non-transgenic hens. One rooster was mated to wild-type hens to produce 518 G1 progeny. PCR analysis of blood samples from these progeny revealed that there were four G1 transgenic offspring, corresponding to a 0.77% germline transmission rate. Subsequently, Southern blot analysis of the genomic DNA from four G1 transgenic chickens was carried out to verify the stable genomic integration and copy number of the transgene in the genome. Quantitative analyses of the blood and egg white samples taken from G1 transgenic chickens resulted in 4,810～6,600 IU/mL (40.1～55.0 ㎍/mL) of hEPO in egg white, whereas 18～25 mIU/mL (0.15～0.2 ng/mL) in serum. The biological activity of the recombinant hEPO in egg white was comparable to its commercially available counterpart. We conclude that successful expression of recombinant hEPO in the egg white of transgenic chickens implies an important step towards efficient production of human cytokine from the transgenic animal bioreactor.

The demand for organ transplantation has rapidly increased all over the world during the past decade. Genetically modified pigs provide a solution to the severe shortage of organs available for human transplantation. Porcine α-1,3-galactosyltransferase (GGTA1) gene is generates Gal-T epitopes that trigger hyperacute rejection in pig-to-human transplantation. Since production of GGTA1 knock-out pigs in 2002, non-gal antigens are considered to be the next xenoantigen involved in the rejection phenomenon. Here, we targeted the GGTA1 and CMP-Neu5Ac hydroxylase (CMAH) genes with CRISPR-Cas9 systems resulting in double knock-out pigs that no longer express α-Gal or Neu5Gc. Similar to GGTA1 gene, CMAH is widely expressed on the endothelial cells of many mammals except humans and this epitope is a potential porcine target for the antinon- gal antibody in humans. CMAH is responsible for the expression of Neu5Gc that key non-gal antigen. Additionally, hCD46 controls complement activation and when this gene expressed sufficiently as a transgene protects xenografts against complement-mediated rejection. This is report to describe generation of transgenic pigs that modify GGTA1, CMAH and hCD46. We expect to remove α-gal and Neu5Gc antigens and express hCD46 from pig for reducing human antibody mediated cytotoxicity.