Earticle

It has been passed already more than 20 years since it is thought to be pig-to-human xenotransplantation. However, the main 3 limitations; zoonosis, immunological rejection and ethical problems were still remained for xenotransplantation. The ethical issues are a matter to be resolved in the form of consideration slowly and importantly, also nationally and legally. A lot of scientist of the world are trying to solve the remaining limitations which were immune rejection and zoonosis. As the limitation of xenotransplantation, the zoonosis is a disease caused by viral bacterial parasites that can be transmitted to humans from animal (pig-to-human) at the same time. The best solution of this should be the germfree animal source. In other respects, about the immune rejection, there are 3 steps of immune rejection by the time of occurrence which are hyper-acute rejection, acute cellular/humoral rejection and chronic rejection. An alpha- 1,3-galactosyl transferase(GALT) KO pigs were produced and investigated for overcoming hyper-acute rejection in xenotransplantation by various research groups. After GALT which is thought to be the only factor of hyper-acute rejection from pig-to-human xenotransplantation, there were some unexpected results occurred by increasing nongal epitopes such as CMAH. Furthermore, not only complement regulatory proteins but also thrombomodulin (TBM), tissue factor pathway inhibitor (TFPI) and other coagulation factors were over-expressed in minipig are considered as candidate of modification genes to solve the acute vascular-humoral rejection problem. Other than that, to overcome the acute cellular rejection problem, the cellular immune activator/inhibitors such as CD47, IL-10, CTLA-4, CD80/86(B7) have been considered. Therefore, for successful xenotransplantation, the overexpression of several immune escaping genes on the base of GALT KO pig is considered to be the best solution and is currently on the way.

Programmable nucleases, which include zinc-finger nucleases, transcriptional activatorlike effector nucleases, and RNA-guided engineered nucleases derived from the prokaryotic CRISPR/Cas system, enable targeted genetic modifications in cultured cells, animals, and plants, tools of great value in research, medicine, and biotechnology. Cpf1 is a recently reported effector endonuclease protein of the class 2 CRISPR-Cas system. Cpf1 has several differences from Cas9: cleavage with 5’ overhangs, shorter guide RNA, and a longer distance between the seed sequence and cleavage site, which could provide potential advantages for some cases of genome editing such as nonhomologous end joining-based gene insertion and efficient genome editing using homology- directed repair. However, limited information is available about Cpf1 activity profiles in mammalian cells, precluding its wide use for genome editing. Here, we performed en masse evaluation of guide RNA and Cpf1 activity using synthetic target sequences and deep sequencing. Using this approach, we determined target sequence- dependent activity profiles and protospacer adjacent motif sequences of Cpf1. We found that sequence features of high activity AsCpf1 guide RNAs are distinct from those of SpCas9. Evaluation of activity at mismatched target sequences showed that Cpf1 target sequences can be divided into seed, trunk, and promiscuous regions depending on mismatch tolerability. The Cpf1 characterization profile will greatly facilitate Cpf1-based genome editing.

Infertility is defined as the inability to conceive after 1 year of unprotected intercourse. Approximately 15% of all human couples are recognized as infertile, and about 50 % of these cases are associated with male fertility factors. However, the causes of male infertility are largely unknown. To date, multiple factors have been implicated in reduced sperm count and motility including genito-urinary infections, environmental exposure to hazardous chemicals, anatomic and physical obstructions, hormonal imbalances and immunologic disorders. Spermatogenesis is a complex developmental process regulated by pituitary-testis axis. Another important molecule involved in the spermatogenesis is insulin-like growth factor-1 (IGF-1). IGF-1 affects various reproductive processes and plays an essential role in the onset, progress, and regulation of spermatogenesis. IGFs are produced mainly in the liver in response to growth hormone (GH) from the anterior pituitary. In the male reproductive tract, IGF-1 is secreted by Leydig cells and Sertoli cells in the testis. IGF-1 receptor has been identified on Sertoli cells, Leydig cells, secondary spermatocytes, spermatids, and spermatozoa as well. However, only a few studies have investigated the association of the IGF-1 level and male infertility. To obtain a better understanding of the role of IGF-l in male fertility, we determined the relationship between serum IGF-1 concentration, seminal plasma IGF-1 concentration, and sperm parameter abnormalities. To this end, a total of 79 men were enrolled and were prospectively analyzed with parameters including age, body mass index, smoking status, urological history, and fertility history. Patients were divided into four groups based on their semen parameters: normal sperm (n=31), abnormal sperm motility (athenospermia, n=12), abnormal sperm morphology (teratospermia, n=20), and two or more abnormal parameters (multi, n=16). Patient seminal plasma and serum IGF-1 concentrations were determined. Men with teratospermia had significantly lower levels of serum IGF-1 compared with men with normal sperm morphology (p<0.05). Men with both teratospermia and athenospermia had significantly lower levels of serum IGF-I as well (p<0.01). Seminal plasma IGF-1 levels, however, did not differ significantly between the groups investigated here. Further investigations will be required to determine the exact mechanisms by which growth hormone and IGF-1 affect sperm quality.

Obesity caused by excessive fat accretion in adipose tissue or the whole body is a major health issue in a significant portion of the US population. Economically, the CDC estimates that the US spends $147 billion annually in extra health costs related to obesity. In the poultry industry, decreasing 1% of body fat in the chicken is valued at a total of $30 million per year. The recent advances in bioinformatics and DNA microarray technology allow rapid genetic analysis to take place on a genome-wide scale and have revolutionized the way specific diseases are studied. Our comparative analysis of microarray data across animal species led us to identify several novel adipose tissue-specific genes and demonstrate functions of these genes in adipose development. Because genetics and developmental aspects of adipocyte biology are highly conserved across species, animals including the quail provided excellent models to understand the function of these genes in adipocyte development and lipid metabolism. This approach of studying obesity promises to provide a better understanding of the underlying mechanisms responsible for obesity and to discover new candidate genes and their signaling pathways, leading to the development of more effective therapeutic and nutraceutical interventions.

The initial segment (IS) in rodents is functionally and structurally distinct from other epididymal segments and plays an important role in sperm maturation. The MAPK/ ERK- 1/2 pathway is maintained active in the IS by testicular luminal factors and plays crucial roles in the maintenance and differentiation of the IS epithelium. Tight junctions (TJs) are constituents of the blood-epididymis barrier, which mediates the paracellular transport of ions, solutes, and water and controls epithelial cell differentiation, thereby contributing to the establishment of a unique luminal environment. We examine here the role of the MAPK/ERK1/2 pathway in the regulation of TJ proteins in the IS. Inhibition of mitogen activated protein kinase kinase (MAPKK or MEK1/2) with PD32- 5901, followed by reduction of ERK1/2 phosphorylation (pERK), decreased zonula occludens (ZO)-2 expression and increased ZO-3 expression in TJs but had no effect on ZO-1 expression. In control mice, in addition to being located in TJs, claudin (Cldn)-1, Cldn-3, and Cldn-4 were detected in the basolateral membrane of epithelial cells, with enriched expression of Cldn-1 and Cldn-4 in basal cells. PD325901 reduced the expression of Cldn-1 and Cldn-4 at all locations without affecting Cldn-3. Occludin was undetectable in the IS of control mice, but PD325901 triggered its expression in TJs. No effect was observed for any of the proteins examined in the other epididymal regions. Our results indicate the participation of the MAPK/ERK1/2 pathway in the regulation of cell-cell events that control the formation and maintenance of the blood-epididymis barrier.

Transplantation of male gem cells including spermatogonial stem cells (SSCs) in experimental animal models has been used to study SSC biology and to produce transgenic animals. The species-specific recipient model preparation is important for the characterization of male gem cells and the production of offspring. Here, effects of surgically induced cryptorchidism investigated in dog as a new recipient model for SSC transplantation. Artificially unilateral or bilateral cryptorchidism was induced in ten mature male dogs by surgically returning the testis and epididymis to the abdominal cavity. The testes and epididymis were collected every week after the induction of artificial cryptorchidism (surgery) for one month. To determine the effect of surgical cryptorchidism, the seminiferous tubule diameter was measured and immunohistochemistry using PGP9.5 and GATA4 antibodies was analyzed. The diameters of the seminiferous tubules of abdominal testes were significantly reduced compared to those of the scrotal testes. Immunohistochemistry results showed that PGP9.5 positive undifferentiated spermatogonia were significantly reduced after surgical cryptorchidism induction, but there were no significant changes in GATA-4 positive sertoli cells. To evaluate the testis function recovery rate, orchiopexy was performed on two dogs after 30 days of bilateral cryptorchidism. In the orchiopexy group, functional sperm were detected, and it produced puppy. In this study, optimum experimental conditions and time provided for surgical preparation of a recipient canine model for SSC transplantation. Additionally, this data will contribute to recipient preparation by using surgically induced cryptorchidism in non-rodent species.

Epigenetic change is dynamic during germ cell development. DNA methylation and histone modification are the most important epigenetic process to regulate the gene expressions. They are very close reciprocal relationship on the specific genomic regions called CpG islands (CGI). The CGIs are located on the promoter regions and recruit various epigenetic regulators including, CFP1, KDM2A, KDM2B, TET1 and MLL. They contain a common domain which is the zinc finger CxxC domain. The CxxC domain reads non-methylated CpG and recruits other regulatory elements such as SET1, PRC, COMPASS and SIN3A to modify Histone proteins. CFP1 contains a CxxC domain. CFP1 protein therefore imposes an ability to distinguish its important regulatory element, “non-methylated CpG” from the genome. After binding the CpG, CFP1 recruits SET1 complex, which is involved in the histone H3 lysin 4 (H3K4) methylation. However, the functional consequence of CFP1 in the germ cell development remains unknown. In this study, we demonstrated that CFP1 is critical for the both spermatogenesis and oogenesis using conditional knockout system.

Porcine embryonic stem-like cells (pESLCs) are able to undifferentiated proliferation and self renewal. However, due to the lack of a system that can effectively culture pESLCs, it is difficult to long-term culture of pESLCs. Integrins composed of α and β subunits and αβ combination are molecular receptor acting on the cell surface when cells are bound to extracellular matrix such as fibronectin and collagen. Integrins regulate function of cells through the inside-out and outside-in signaling regarding motility, survival, differentiation and proliferation of cells. However, there is lack of information on integrins regarding to pESLCs. Therefore, the aim of this study was to confirm the presence of integrins in pESLCs through the integrins screening. Fluorescence immuno assay showed that integrin α1 and α2 were not expressed, whereas, integrin α3, α4, α5, α6, α7, α8, α9, αv and β1 were expressed in pESLCs. Also, fluorescence intensity showed that intensity of integrin α5, α7, and α8 was higher than twice compared to other groups (610.58±26.83; 419.69±13.84; 646.09±4.53; p<0.05), but, integrin α6 was lower intensity than other groups (50.55±2.65; p<0.05). To identify of integrin heteromiders subsequently, we experimented the cell attachment assay. Cultures with 10 μg/mL vitronectin and 60 μg/mL tenascin C showed significantly highest rate compared to controls, indicating the presence of integrin heterodimer α9β1 and αvβ1 on the membranes of pESLCs respectively (p<0.05). Therefore, integrin α3, α4, α5, α6 and α7 exist as subunits and integrin α9β1 and αvβ1 exist as heterodimers in pESLCs.

Mammalian spermatogenesis consisted of two major parts that included self-renewal of spermatogonia stem cells (SSCs) and meiotic differentiation. Identification of the stage- specific gene expression in developmental germ cells were critical to understand molecular regulation of spermatogenesis. In neonatal testicular tubules, only cells were early spermatogonia and sertoli cells, and they began to differentiate to spermatozoa by chaing the hormonal regulation at puberty. In mice, several established markers including Oct4, Gfr-α1, Plzf, Utf1, Vasa, Sall4 and Lin28 were known to maintain self-renewal and stemness. Based on these previous studies, the expressions of Vasa, Sall4 and Lin28 were determined in neonatal, pre-pubertal, pubertal and post-pubertal stages of boar germ cells. As expected, the expression of Vasa, Sall4 and Lin28 were identified in PGP9.5 positive cells which is regarded as an undifferentiated spermatogonia, from the birth to pre-pubertal stages. However, expression patterns of these genes were changed after puberty that Vasa was expressed in secondary speramatocytes that were positive to C-kit and Scp3, but negative to Acrosin during the pubertal and post-pubertal stages. Expression of Sall4 was identified in the cells of late meiotic stages that were positive to C-kit, but negative to Scp3. In addition, Lin28 was expressed in acrosin positive, but C-kit and Scp3 negative cells. In the present study, expressions of Vasa, Sall4 and Lin28 were determined during meiotic spermatogenesis. The expression patterns of these genes were identical to mice before puberty, however each gene expressions after puberty were differ. This data indicates that Vasa, Sall4 and Lin28 can be the useful marker to understand spermatogenesis in boar testis.

Mitochondrial functions are required for early embryonic development in pigs. Reactive oxygen species (ROS) can produce from the maintenance of mitochondrial functions during in vitro culture (IVC) of embryos. However, the role of mitochondria specific ROS (mito-ROS) in porcine early embryonic development remains unknown. Therefore, the objective of present study was to confirm the changes in functions, aggregation and dynamics of mitochondria by the regulation of mito-ROS in porcine embryo development. Here, we performed the IVC from classified two groups (G1; high lipid and G2; low lipid contents) at the zygote stage. Blastocyst developmental rate and total nuclei numbers in G2 embryos were lower (p<0.05) than that of G1 embryos. To investigate the superoxide as mitochondrial-target ROS on preimplantation development of G1 and G2 embryos, we performed the MitoSOX staining. Superoxide levels significantly increased (p<0.05) in G2 embryos. And, changes of mitochondrial membrane potential (MMP), ATP levels and expression of mitochondria fission protein were investigated by using JC-1 kit, ATP determination kit and Western blot analysis. MMP and ATP levels were significantly decreased (p<0.05) in G2 embryos compared with G1 embryos. Protein level of mitochondrial fission protein Drp1 was increased (p<0.05) in the cleavage stage of G2 embryos. To confirm the effects of reducing mito-ROS on the porcine embryo development, we treated with the Mito-TEMPO, mitochondria specific ROS scavenger, for G2 embryos. Mito-TEMPO improved preimplantation development to the blastocyst stage and reduced mito-ROS level in the G2 embryos (p<0.05). These results demonstrated that regulation of mito-ROS improves the blastocysts development and their qualities through the control of mitochondrial functions and dynamics of porcine embryos.

It has been established that a shift from the T helper 1 (Th1) cytokine profile to the T helper 2 (Th2) profile during pregnancy contributes to the successful pregnancy. Interleukin-12 (IL12) is a Th1 cytokine composed of IL12A and IL12B and activates the JAK-STAT pathway and promotes Th1 cell development by binding heterodimeric receptors, IL12RB1 and IL12RB2. Interleukin-10 (IL10), a well-known Th2 cytokine, regulates the expression of Th1 cytokines, MHC class II antigens, and co-stimulatory molecules on immune cells via heterotetramer receptor composed of IL10RA and IL10RB. Although the Th1:Th2 cytokine balance for the maintenance of pregnancy is well studied in humans and rodents, expression and function of IL12 and IL10 are not fully understood in pigs. Thus, this study determined expression of IL12, IL10 and their receptors at the maternal-conceptus interface in pigs. Real-time RT-PCR analysis showed levels of IL12A and IL12B mRNAs in the uterine endometrium were high during early pregnancy, but maintained low expression levels thereafter. Levels of IL12RB1 mRNA during pregnancy showed a biphasic pattern with the highest levels on D15 and D60 of pregnancy, while levels of IL12RB2 mRNA did not change during pregnancy. Levels of IL10 mRNA in the uterine endometrium were highest on D15 of pregnancy and decreased toward term pregnancy. Levels of IL10RA mRNA showed highest levels on D30, while levels of IL10RB mRNA were biphasic with highest levels on D12 and D30 of pregnancy. Immunohistochemical analysis showed that IL12 protein was localized specifically to scattered cell types in endometrial stroma during the estrous cycle and early pregnancy. IL10 protein was detected on scattered cells around the blood vessels in the endometrium during early pregnancy, then detected in all endometrial stroma during mid- to late pregnancy. Conceptus and chorioallantoic tissues during pregnancy also expressed IL12, IL10 and their receptors. These results showed that there were changes in IL12 and IL10 expression in the uterine endometrium during pregnancy, suggesting that a shift toward IL10, a Th2 cytokine, over IL12, a Th1 cytokine, occurs at the maternal conceptus interface for the successful establishment of pregnancy in pigs.

The gene targeting efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. However, the efficiency of gene targeting in somatic cells is lower than that in mouse embryonic stem cells. The purpose of this study was to determine whether structure of knock-in vector with different size of homologous arm influence the efficiency of knock-in on the β-casein gene locus in the somatic cells. Three kinds of knock-in vectors were constructed; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5’ arm region and 1.8 kb or 0.64 kb of 3’ arm region, and neomycin resistance gene(neor ) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5’ terminal of endostatin gene and inserted into exon 7 of the β-casein gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. And then, Antibiotics selection was performed 10～12 days using 500 ug/mL G418. After selection, G418-resistance colonies were analyzed by 3'arm, 5'arm and targeted or non-targeted PCR. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3’ arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine β-casein gene in the mammary gland.

Polo-like kinase 1 (Plk1) plays crucial roles in various stages of oocyte maturation. Recently, we demonstrated that synthesized peptidomimetics AB103-8, targeting the polo- box domain (PBD) of Plk1 disrupted oocyte meiotic maturation and meiosis resumption. However, as a peptidic character of AB103-8 appeared to be defective due to poor membrane permeability and protease stability. To overcome the drawbacks, we designed and synthesized a series of pyrrole-based small-molecule inhibitors and screened against porcine oocyte maturation rates. Among the synthesized compounds, the macrocyclic inhibitor compound 4 displayed the highest inhibition potential against the oocyte meiotic maturation and embryos blastocyst formation. Furthermore, the addition of this compound to culture medium efficiently blocked the maturation of porcine and mouse oocytes, indicating that the lead compound could penetrate zona pellucida and cell membrane. We also investigated the Plk1 inhibiting ability of this compound. Treated mouse oocytes confirmed the Plk1 inhibition by showing abnormal spindle formation. Collectively, these results suggest that the novel Pyrrole-based compounds could be used for the basis of developing contraceptive agents.

Germ cells are alternative cell sources for deriving pluripotent stem cells. When cultured with feeder cells and adequate cytokines, migrating primordial germ cells (PGCs) can be reprogrammed into pluripotent stem cells, named embryonic germ cells (EGCs). In this study, we attempted to establish and characterize pig embryonic germ cells from fetal gonads. Consequently, EGC lines were derived from the genital ridges of a porcine dpc 30 fetuses in media containing LIF, FGF2 and SCF. After establishment, this cells were cultured and stabilized in LIF or FGF2 contained media. These cell lines were maintained in both condition over an extended time period and were able to spontaneously differentiate into the three germ layers in vitro. Interestingly, cell lines cultured in LIF or FGF2 expressed different pluripotency markers. While LIF-treated pig EGCs (LIF-pEGCs) expressed only few pluripotent markers including OCT4, SOX2 and NANOG, FGF2-treated pEGCs (FGF2-pEGCs) expressed pluripotency markers such as OCT4, SOX2, NANOG and SSEA4. As a result of molecular analysis, it was verified that LIF- and FGF2-EGCs represented intermediated state of EGCs and complete reprogrammed EGCs respectively, and reprogramming of pig gonadal PGCs is progressed in stepwise manner, from PGCs via intermediated EGCs to EGCs. In conclusion, it is verified that FGF2 signaling have important roles in reprogramming and maintaining pEGCs from fetal gonads.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis in male. Among the testicular germ cells, SSCs are considered as an important population because of their capability to multiply in numbers which is also known as self-renewal, and to differentiate into spermatocytes by subsequent meiotic processes finalized with the production of spermatozoa. As SSCs are present in a very small proportion among the total testis cells, in vitro proliferation of SSCs in undifferentiated state is very important in the field of experiments related to germ cell culture. The objective of the study was to establish an effective feeder cell for murine germ cell development in vitro. We used three types of feeder cells to grow murine germ cells, such as STO, tissue specific feeder layer, and C166 feeder cells and green fluorescence protein (GFP) rat SSCs were used to grow on them. A continuous culture was performed for each feeder groups, but C166 feeder cells found unable to proliferate germ cells even after several weeks. We observed a comparatively better proliferation rate of rat germ cells grown on tissue specific feeder layer where cultured germ cell colony and cell count were higher than STO feeder. Immunocytochemistry of undifferentiated SSC marker showed significantly higher expression for germ cell cultured on tissue specific feeder layer. Similarly, mRNA level of undifferentiated spermatogonia were found higher in case of tissue specific feeder layer and STO, although the expression level of differentiated spermatogonia was similar. Finally, the functional characteristics of SSCs were studied by cultured germ cell transplantation into recipient mice. We used donor rat germ cells cultured on both feeder groups for 4 months, where SSCs from tissue specific feeder layer showed higher functional and stemness activity. Transplantation of SSCs cultured for 8 months from tissue specific feeder layer group were performed to recipient rat and we could produce GFP offspring from those recipients. Thus the experiment conclude that tissue specific cells could be an effective feeder layer for murine SSCs culture based on their survivability and proliferation along with retaining the perfect functional characteristics and stemness properties.

The Hippo signaling pathway is essential for regulating proliferation, differentiation, and apoptosis in mammalian cells. It has been reported that the members of Hippo signaling pathway are highly expressed in mammalian ovaries and uteri. However, the regulatory mechanism of this pathway in the uterus during estrous cycle regulation remains unclear. Serine/Threonine Protein Kinase 4 (STK4) (known as MST1) is a major factor of Hippo signaling pathway. However STK4 in the mouse uterus has not yet been examined. The purpose of our study was to determine the expression of STK4 during the estrous cycle and regulation by estrogen in the mouse uterus. We found that Stk4 mRNA was dynamically expressed in uterine endometrium during the estrous cycle. The expression of Stk4 was significantly increased at the stage of estrus and diestrus among the estrus cycle, whereas its expression was decreased at proestrus and metestrus stages. The localization of Stk4 was highly detected in Luminal epithelial (LE) and Glandular epithelial (GE) in uterine endometrium. In addition, we demonstrated that Estrogen (E2) administration into ovariectomized mice increased the expression of Stk4 in the uterus. The expression of Stk4 by E2 was gradually increased depending on the time up to 6 hours after the treatment and then decreased. Moreover, the treatment the Estrogen receptor inhibitor, ICI 182, 780 (ICI), before E2 injection significantly reduced the alleviation of Stk4 expression by E2. Our results indicate that the Stk4 is expressed in Luminal epithelial (LE) and Glandular epithelial (GE) of the uterus and its expression in regulated by the ovarian hormone, Estrogen. These informations will give us on sights to understand uterine dynamics during the estrous cycle.

Spermatogenesis is the process of proliferation and differentiation of spermatogonial stem cells into adult spermatozoa within seminiferous tubules. Sperm cells undergo extensive epigenetic modifications during spermatogenesis. One of the epigenetic modifications, histone acetylation induces a loose chromatin structure and active transcription which facilitates binding of transcription factors at sperm chromosome. Therefore, histone acetylation is known to play an important role in spermatogenesis. However, factors involved in histone acetylation during spermatogenesis are not well known. We found a factor TLE3(Transducin Like Enhancer of Split 3). The transcriptional co-repressor TLE family is known to regulate histone acetylation by interacting with histone deacetylase( HDAC). We examined the expression level of TLE3 in various mouse tissues. As a result of RT-PCR, TLE3 showed significantly higher expression in testis than that in other tissues. Immunofluorescent and Immunohistochemical analysis revealed that TLE3 protein was located in sertoli cell and spermatid in seminiferous tubules of adult mouse testis. In addition, the differential regulation in TLE3 protein expression site was observed in the testis during postnatal development. We also analyzed the expression pattern of HDAC2, which is known to cooperate with TLE3. Both HDAC2 and TLE3 protein were detected in spermatogonial stem cells at one-weeks-old mouse, however, Their expression site were transferred to sertoli cell at six-weeks-old mouse. aken together, TLE3 may play a role in regulating histone acetylation via interaction with HDAC2 in the testis. Futher studies are needed to look into a relation between TLE3 and histone acetylation during spermatogenesis and postnatal development in testis.

The DJ-1 was found to be the first case that an oncogene also play a role for causative gene of neurodegenerative diseases. The DJ-1 is a multi-functional protein that play important role in tissue and participates in a number of intracellular signaling pathways. Especially, 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 canine. In the present study, we investigated the protective role of DJ-1 against induced oxidative stress, cell viability, cell proliferation assay, cellular apoptosis detection analysis, intracellular ROS were measured appropriately. The results showed that DJ-1 overexpressed cells were up-regulated cell viability under oxidative stress conditions, 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. The data indicate that canine DJ-1 protein enhances the cellular survival activity and promote cell survival activity under the oxidative stress by attenuates cellular apoptosis and ROS generation.

The aim of this study was to evaluate effect of Propolis on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryo-preserved in 11% lactose with 20% egg yolk (LEY) and Triladyl containing propolis (0, 10, 30, and 50 ng/mL) with 0.05% ethanol. Cryo-preserved boar sperms was thawed at 37.5℃ for 45 sec in water-bath. Sperm viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed sperm, which was cryo-preserved using LEY and triladyl, was decreased in 0, 30, and 50 propolis treatment groups. However, there was no significant difference. Acrosome reaction and mitochondrial intact of sperm was not influenced by all of propolis treatment using both of freezing extender. Motility of frozen-thawed sperm with LEY containing 10 ng/mL propolis was significantly higher than in 0, 10, and 50 ng/mL propolis treatment groups (p<0.05), whereas there was no significant difference compared to control. Especially, 0 and 30 ng/mL propolis treatment groups were significantly decreased compared to control (p<0.05). Similar to result in frozen-thawed sperm with LEY, motility of frozen-thawed sperm with triladyl was significantly increased in 10 ng/mL propolis treatment groups compare to control and other propolis treatment groups (p<0.05). In conclusion, 10 ng/mL propolis improves mobility in frozen-thawed boar sperm with LEY and triladyl, whereas 30 and 50 ng/mL propolis reduced motility. But, the addition of propolis during cryopreservation did not influence to the membrane function of frozen-thawed spermatozoa. Therefore, it is necessary to continue the study on the effects of propolis on boar spermatozoa during cryopreservation.

가축유전자원으로서 흑염소의 정액은 후대 생산과 유전적 개량을 위하여 활용될 필요 성이 있으나, 아직까지 많은 연구가 진행되어 있지 않다. 국내 염소 동결정액의 생산과 이를 이용한 인공수정에 관한 국내 연구는 많이 없는 상태로 인공수정과 체외수정에 관 한 자료가 많지 않다. 본 연구에서는 흑염소에서 정소상체 정자와 전기자극으로 채정된 사출 정액의 동결성을 비교 검토하였다. 특히 염소 정액은 난황의 인지질을 분해하는 효 소를 함유하기 때문에 정자의 대사를 촉진하여 정자의 장수성을 낮추는 것으로 알려져 있고, 동결정액의 생산성을 저해하는 것으로 보고되었다. 정액 희석제로 triladyl-egg yolk 와 triladyl-egg yolk plasma를 활용하고, 동결 정액을 제조하였다. 정소상체유래 정자는 6～7개월령의 숫염소를 거세하여 정소를 적출하였으며, 정소상체에 공급하는 혈관을 외 과적으로 제거하고, calcium magnesium free PBS를 이용하여 혈액을 거즈에 흡수시키 고, 메스와 시약스푼을 이용하여 정소상체를 절단하고 압착하여 흐르는 농후정자를 걷어 내어 같은 양의 희석제로 혼합하여 정액을 회수하였다. 전기자극기를 이용하여 농후한 정액을 15 mL 코니컬 튜브에 받았으며, 1회용 비닐 꼬깔을 이용하여 50 mL 코니컬을 절단하여 깔때기를 제조하여 사용하였다. 희석된 정액을 300×106개/mL 농도로 희석하고, 저온 중탕법으로 2시간에 걸쳐 상온에서 5℃로 서서히 냉각하였고, 스티로폼을 활용한 간이 동결법으로 동결을 실시하였으며, 질소 표면 위 5 cm에서 10분간 노출하여 동결스 트로를 제작하였다. 정소상체 유래의 정자는 전기자극에 의하여 채정된 정액보다 융해후 생존성이 75±9.4%로 53.4±6.2%보다 우수하였으며, triladyl-egg yolk plasma를 활용한 정 액은 65.3±7.2% 및 72.4±3.0%로 차이가 존재하지 않았다. 그러므로 염소의 정액은 정소 상체 유래의 정자가 동결성이 더 높은 것으로 판단되며, 이는 정자의 손상이 채정 후 후 처지에 의하여 저하되고 있음을 보여주고 있다.

칡소는 이모색을 가진 한우의 한 갈래로 한우로 등록되고 있으며, 육종을 위한 개체 선 발을 위하여 증식을 실시하고 있다. 이를 위하여 동결 정액 생산이 지자체를 중심으로 이루어지고 있으며, 근친을 막기 위한 정액 교환이 활발하게 이루어져지고 있다. 동결정 액의 생산은 유전자원을 확보하는 가장 기본적인 기술로 그 중요도가 높으며, 인공수정 과 체외수정에 필요한 정액을 안정적으로 공급할 수 있다. 그럼에도 불구하고 칡소의 경 우, 동결 정액의 생존성 및 활력도가 균일하지 못한 것이 현실이며, 이는 개체와 실험자 의 기술적 차이에 의한 것으로 판단된다. 본 연구에서는 칡소 동결정액의 생산 효율성을 높이기 위하여 Cathepsin B inhibitor로 알려져 있는 E64를 5, 50, 100 μM로 조정하여 처 리하여 동결 후 생존성을 조사하였다. 동결 정액을 제조하는 방법은 1단계 동결방법으로 triladyl-egg yolk 희석액을 이용하였으며, 상온에서 신선정액을 50～100×106개/mL 농도로 맞추고, 저온 중탕기법으로 2시간에 걸쳐 상온에서 5℃로 서서히 냉각하였으며, E64가 함유된 2차 희석액으로 1:1로 희석하여 실험군을 준비하였다. 정액의 동결은 스티로폼을 활용한 간이 동결법으로 동결을 실시하였으며, 질소 표면 위 5 cm에서 10분간 노출하고, 액체 질소에 침지하여 동결스트로를 제작하였다. 37℃에서 40초간 융해 후 정자의 생존 성을 조사하였을 때, 정액의 동결성이 우수한 개체에서 대조군은 71.2±2.0%의 생존율을 관찰하였으나, 100 μM E64 처리군은 82±4.3%로 유의적으로 높게 관찰되었다(p<0.05). 5 와 50 μM E64 처리군의 생존율은 77.5±2.0%와 75.0±5.0%로 관찰되어 유의적 차이를 관찰할 수 없었다(p >0.05). 이러한 결과를 근거로 칡소의 동결 보존에 있어서 E64는 동 결정액의 생산성 향상에 도움이 될 것으로 판단되며, 체외수정 및 발생에 관한 영향에 대한 연구결과가 기대되고 있다.

정액을 동결하여 가축유전자원으로 보존하는 것은 국가 유전자원을 확보하는 가장 기 본적인 기술로 중요도가 높으며, 소의 경우 동결정액 생산기술은 상용화가 이루어진 기 술로 인공수정과 체외수정에 가장 많이 이용되는 기술이다. 그럼에도 불구하고, 희소한우 의 경우, 동결 정액의 생존성과 활력도가 균일하지 못한 것이 현실이며, 이는 개체의 차 이에 의한 것으로 판단된다. 본 연구에서는 희소한우의 동결정액의 효율을 높이기 위하 여, 본 연구에서는 비타민 B12의 전구물질인 pyridoxine을 첨가하여 동결과정에서 생존성 을 조사하였다. 동결정액을 제조하는 방법은 1단계 동결방법을 이용하였으며, triladyl 희 석액을 이용하였다. 상온에서 신선정액을 80～120×106개/mL 농도로 맞추고, 저온 중탕법 으로 2시간에 걸쳐 상온에서 5℃로 서서히 냉각하였으며, 동일한 방법으로 pyridoxine이 함유된 2차 희석액으로 1:1로 희석하여, pyridoxine의 최종 농도가 5, 50, 100 및 200 μM 로 조정하여 첨가하지 않은 대조군과 비교 검토하였다. 정액의 동결은 스티로폼을 활용 한 간이 동결법으로 동결을 실시하였으며, 질소 표면 위 5 cm에서 10분간 노출하여 동 결스트로를 제작하였다. 37℃에서 40초간 융해 후 정자의 생존성을 조사하였을 때, 대조 군과 실험군의 유의적 차이가 나타나지 않았으나, computer assisted sperm analysis를 실시하였을 때, 활력도가 우수한 정자의 분포도가 농도 의존적으로 증가하는 것으로 관 찰되었다. 그러므로 유전자원으로 보존되는 정자의 생존율에는 pyridoxine은 큰 효과가 없는 것으로 판단되나, 정자의 장수성과 연관된 활력도를 증진시키는 것으로 판단되며, 그 세포생물학적 기능은 추후 연구를 통하여 구명될 것으로 판단된다.

본 연구는 희소 재래가축으로 지정된 칡소의 생식세포 유전자원 중 동결정액 생산 · 보 존을 위해 활용되는 씨숫소를 대상으로 지용성 비타민제 첨가급여에 따른 동결정액 생산 시 채정량 및 정자 생존성 등에 미치는 영향을 조사하고자 수행하였다. 공시축의 사양관 리는 한우사양관리(국립축산과학원, 2007) 기준에 준하여 사육하였다. 지용성 비타민 Vitamin- A 35,000 IU, Tocopherol acetate 10 IU/일을 최초 정액채취일 30일 전부터 급이 하였다. 공시축은 정액채취를 위해 승가훈련 및 성성숙이 완료된 칡소 5두를 공시하였고, 정액채취 빈도는 월 2회로 하였다. 정액 채취는 암소 보정 후 공시축의 승가를 유도한 다음, 인공질에 음경을 삽입하여 채 정을 실시하였다. 동결정액 생산용 원정액은 현미경 검사결과, 정자의 생존율이 80∼85% 이상 확인되는 것을 활용하였다. 동결정액 생산용 희석액 조성은 Tris amino-methane 250 mM, Citric-acid monohydrate 80 mM, Fructose 60 mM, Penicillin G+Streptomycin 1%, Egg-Yolk 20%, Glycerol 7%로 하였다. 동결정액의 생산은 원정액을 1차 희석한 다음, 저 온실로 이동시켜 5℃로 온도 하강을 실시한 이후, Glycerol이 첨가된 희석액으로 2차 희 석을 실시한 이후 항동해제 평형을 유도하였다. 동결정액의 생산은 액체질소를 활용한 간이동결로 실시하였다. 동결이후 생존율 검사는 38℃에서 15초 이상 융해 후 현미경 검 사로 실시하였으며, 운동성 직진도 등은 컴퓨터정자분석기(Computer Assisted Sperm Analysis) 를 활용하여 조사하였다. 정액채취 결과, 지용성 비타민 처리군에서는 13.5 mL 내외 로 채정되었고, 무처리군은 9.0 mL 내외였다. 원정액의 생존율은 처리군 및 무처리군 모 두 85% 내외였다. 동결융해 이후 생존율을 검사한 결과, 생존율은 처리군 72.0%, 무처리 군 72.5%로 조사되었다. 운동성은 처리군 92.6%, 무처리군 94.8%로 조사되었고, 선형성 은 처리군 31.2%, 무처리군 32%였다. 직진도는 처리군 50%, 무처리군 51.3% 각각 조사 되었다. 이와 같은 결과는 지용성 비타민 급여가 칡소 생식기능에 영향을 미치는 것으로 판단되며, 추가적인 연구를 진행하여 지용성 비타민의 적정 급여 수준을 설정하고, 동결 정액 생산을 위한 표준화기술에 적용한다면 가축 유전자원 보존 · 증식에 기여할 것으로 사료된다.

Recombination activating gene-2 (Rag2) participate in the immune system process through V(D)J recombination of B-cells and T-cells activation process. The deletion of Rag2 gene impaired the immune regulation through complete loss of matured B-cells and T-cells. However, in this research we found that Rag2-ablated mice showed very marginal expression for both B-cells (CD20 and CD79a) and T-cells (CD4 and CD8) markers. These data indicated the possibility of leakiness of B-cells and T-cells at least in a marginal level in the spleen and thymus of Rag2-deficient mice. It also suggested that effectiveness of Rag2 deletion could be regulated based on the strain and age of the concerned mice. Thereafter, we investigated the miRNAs and genes expression profile of Rag2 knockout mice spleen and found that some of the deregulated genes are putative targets of the differentially expressed miRNAs and might be transcriptionally connected. Moreover, the deregulated miRNAs and genes are also actively involved in both B-cells and T-cells activation signaling network. Finally, we concluded that miRNAs along with the related genes are important regulator of B-cells and T-cells signaling cascade, and the Rag2 deletion might impaired B-cells and T-cells development in a miRNAs-dependent manner.

Many diseases, including myocardial infarction, autoimmune disease, viral diseases, neurodegenerative diseases, and cancers, are frequently diagnosed with the aberrant expression of microRNAs (miRNAs) and their allied pathways. This indicates the crucial role of miRNAs in maintaining biological and physiological processes. miR-7641 is a miRNA whose role in disease has not been fully investigated. In the present study, we investigated the expression pattern of miR-7641 and its target genes in different cancer cells, as well as in patients with breast cancer and colorectal cancer. Direct inhibition of miR-7641 using a locked nucleic acid upregulated the expression of its target genes, sensitized cancer cells, and enhanced the efficiency of therapeutic agents such as doxorubicin. In addition, inhibition of miR-7641 boosted doxorubicin-mediated apoptosis of cancer cells via upregulation of apoptotic molecules Caspase 9 (CAS9) and poly ADP ribose polymerase (PARP) and downregulation of anti-apoptotic molecule BCL2. Thus, these findings indicated that miR-7641 is a clinically important therapeutic target for cancers. Inhibition of miR-7641 expression could be an efficient treatment strategy for patients with breast and colorectal cancer. In addition, inhibitors of miR-7641 could be used in combination with other therapeutic agents, such as doxorubicin, to enhance the efficiency of cancer therapy.

Silver nanoparticles (AgNPs) is one of the nanomaterials as most popular and commonly used in consumer products in biomedical devices, antibiotic agent, wound dressings, text tile and also candidate of anti-cancer material. F9 cell, the teratocarcinoma stem cell line has been widely used as a model for the analysis of molecular mechanisms associated with differentiation and retinoic acid (R.A) is well known molecule that induce F9 cell differentiation. There are many study about induce differentiation in stem cells by AgNPs and in anti-cancer therapy, induce cancer stem cell differentiation is very important to killing cancer. In this study, we demonstrated that AgNPs showing toxicity in F9 cells as anti-cancer cell effect and also induce F9 cell differentiation. F9 cells are induce cell toxicity and apoptosis after treated AgNPs and also make F9 cell differentiation like induced by R.A. Signaling pathway analysis showing that gene and protein expression going different and more sensetive to anti cancer drug, cisplatin, after induced differentiation by AgNPs. We found that potential of AgNPs as not only killing cancer but also make cancer stem cell going to differentiation.

Graphene oxide (GO) is a novel material derived from two-dimentional carbon lattice of graphene and has attracted increasing interest in the field of biomedicine as grug delivery, gene delivery, biosensers. Also G.O is well known that low toxicity in cells but effect in germ cell development are poorly understood. Leydig cells and Sertoli cells are somatic cells that surpport germ cells during spermatogenesis. In this study, we found that toxicity to leydig and sertoli cell line induced by G.O following variants size. The small size (about 27 nm) showing more high toxicity and cell damage, apoptosis in those somatic cells compare with large size (about 80 nm). Small size G.O is more easy to upkate to cells and effect to cells. Taken together, our study reveals that G.O showing cell toxicity, even if low, and possibility that interrupt spermatogenesis by damage of leydig and sertoli cells. It is important information that apply small size of G.O as biomeical drug.

Obesity is a risk factor for various diseases, including cardiovascular disease, diabetes, renal disease, hypertension, cancer, and neural disease. Adipose tissue in animals is important for the mobilization of lipids, milk production, deposition of fat in different depots, and muscle and meat production. Understanding the genetic and physiological causes of metabolic disease is a priority in biomedical genome research. In this study, we examined several variables in mice fed a high-fat diet, including serum composition, body weight, total calorie intake, and differentially ex\-pressed genes. Body weight and blood glucose levels were not significantly different between animals fed high-fat and normal diets. However, high-fat diet groups showed reduced calorie and food intakes. Levels of sodium, ionized calcium, glucose, hematocrit, hemoglobin, pH, PCO2, PO2, TCO2+, HCO3+, base excess, and SO2 in the blood were not significantly different between mice fed high-fat and normal diets. Serum potassium concentration, however, was lower in mice a high-fat diet. Differentially expressed genes were also compared between the two groups. The purpose of this study was to discover new genes as a result of annealing control primer (ACP) PCR using 20 random primers. Five down regulated genes were identified and three of others were up-regulated by high-fat diet. Known genes were excluded from this result. In addition, the relationships among candidate genes and high-fat diet should be investigated according to potassium concentration in the blood. In conclusion, mice fed normal and high-fat diets showed no significant difference in body weight, whereas high-fat diet led to changes in blood composition and differential expression of several genes. These findings may provide a better understanding of the mechanisms underlying the association between obesity and metabolic diseases.

Interferon-tau (IFNT) gene was originally recognized as as low molecular weight secretory protein produced by the conceptus during the peri-implantation period in ruminant ungulates. Two isoform interferon tau (IFNT) genes exist in bovine conceptus. An antiluteolytic substance secreted by the bovine conceptus and primarily responsible for maternal recognition of pregnancy is bovine trophoblast protein 1 (bIFNT1), a new type I bovine interferon tau (bIFNTc1) gene. However, studies of new type I bovine interferon- tau (bIFNTc1) gene regulation, an anti-luteolytic factor produced by conceptuses of the ruminant ungulates, have been carried out, but a definitive mechanism for its transcription has not been elucidated. In the present study, Human choriocarcinoma JEG3 cells were co-transfected with an bovine IFNTc1 (–2.2-kb, –631-bp)-luciferase reporters (–2.2-kb, –631-bp-Luc) construct and several transcription factor expression plasmids. bIFNTc1 gene was had very effect on activity by alone ETS2, and/or AP1 (JUN) expression factors in choriocarcinoma JEG3 cell. However, bIFNTc1 (–2.2-kb vs –631-bp)-luciferase reporters, expression level of the upstream region was not identified. The degree of transcriptional activation of the bIFNTc1(–2.2-kb)-luciferase reporter gene was had more effect by ETS2 and/or AP1, expression plasmid. These results indicate that an more ETS2 and/or AP1-binding site may be located between positions –2.2 kb to –631 bp of the bIFNTc1 gene. Also, transcription of the (–631-IFNT1c1)- Luc increased more than 30 times when the potential relationship between AP1 and ETS2, and their association with a co-activator, cAMP-response element binding protein- binding protein (CREBBP). These observation suggest that AP1 and ETS2 are the most probable binding partners for CREBBP in the potentiation of bIFNTc1 gene transcription. These results indicate that new type I bovine interferon tau (bIFNTc1) gene transcription is regulated by AP1 and ETS2, and suggest that AP1 and ETS2 could be a key molecule in determining bIFNTc1 gene transcription by the trophectoderm.

CD26 (DPP IV/Dipeptidyl peptidase IV)는 다양한 기능을 갖는 120 kDa 크기의 당단백 질로서 콩팥, 간, 혈관 표피 세포, 난소 및 림프구 등에서 발현되며 이 단백질은 생체 내 에서 면역계 및 내분비계의 조절에 중요한 역할을 하는 것으로 알려져 있다. 또한, CD26 억제제는 당뇨 질환 치료제로 이용되고 있으며, 본 연구에서는 당뇨 질환 모델 동물을 생산하기 위하여 cd26이 과발현된 돼지 체세포를 확립하였다. 질환 모델 생산에 앞서, CD26 유전자의 기능이 돼지 체세포에서 밝혀진 바가 없어 그 역할을 규명하기 위하여 분석을 실시하였다. 돼지 체세포에서 cd26의 활성이 세포사멸과 연관성이 있는지 조사하 였다. 과발현 세포에서 cd26의 발현을 일반 귀조직 유래 체세포와 비교한 결과 유의적으 로 높은 발현을 확인하였으며, 세포 사멸 관련 유전자인 Bad와 Caspase3의 발현율을 Realtime RT-PCR로 분석한 결과, 과발현 세포에서 유의적으로 높은 발현을 보였고, TUNEL 염색에서도 동일한 결과를 확인하였다. 또한, 미토콘드리아의 기능을 확인코자 JC-1 염색 및 관련 유전자의 발현을 조사하였다. 그 결과, 미토콘드리아의 potential은 대 조구와 비교해 볼 경우 낮은 경향을 나타내었다. Western blotting 분석을 통하여 Caspase3와 Caspase9의 단백질 수준을 확인한 결과, mRNA 수준에서 나타난 바와 같이 과발 현 세포에서 높은 발현을 나타내었다. 따라서, CD26 유전자가 과발현될 경우, 세포 사멸 을 유도하는 것으로 여겨진다.