Proteomic analysis of cloned porcine conceptuses during the implantation period.

Differentially regulated proteins within porcine somatic cell nuclear transfer (SCNT)-derived conceptuses were compared with conceptuses that were derived from natural matings on day 14 of pregnancy. Proteins that were expressed prominently on day 14 were identified in SCNT-derived conceptuses using 2-D PAGE and MALDI-TOF MS. Sixty eight proteins were identified as being differentially regulated in the SCNT-derived conceptuses. Among these, 62 were down-regulated whereas the other six proteins were up-regulated. Glycolytic proteins, such as pyruvate dehydrogenase, malate dehydrogenase and lactate dehydrogenase, were down-regulated in the SCNT-derived conceptuses whereas apoptosis-related genes as annexin V, Hsp60, and lamin A were up-regulated. Thus, apoptosis-related genes are expressed at significantly higher levels in the SCNT-derived conceptuses than in the control conceptuses, whereas metabolism-related genes are significantly reduced.

Heating-mediated purification of active FGF21 and structure-based design of its variant with enhanced potency.

Fibroblast growth factor 21 (FGF21) has pharmaceutical potential against obesity-related metabolic disorders, including non-alcoholic fatty liver disease. Since thermal stability is a desirable factor for therapeutic proteins, we investigated the thermal behavior of human FGF21. FGF21 remained soluble after heating; thus, we examined its temperature-induced structural changes using circular dichroism (CD). FGF21 showed inter-convertible temperature-specific CD spectra. The CD spectrum at 100 °C returned to that at 20 °C when the heated FGF21 solution was cooled. Through loop swapping, the connecting loop between ß10 and ß12 in FGF21 was revealed to be associated with the unique thermal behavior of FGF21. According to surface plasmon resonance (SPR) experiments, in vitro cell-based assays, and model high-fat diet (HFD)-induced obesity studies, heated FGF21 maintained biological activities that were comparable to those of non-heated and commercial FGF21s. Based on sequence comparison and structural analysis, five point-mutations were introduced into FGF21. Compared with the wild type, the heated FGF21 variant displayed improved therapeutic potential in terms of body weight loss, the levels of hepatic triglycerides and lipids, and the degree of vacuolization of liver in HFD-fed mice.

Developmental arrest of scNT-derived fetuses by disruption of the developing endometrial gland as a result of impaired trophoblast migration and invasiveness.

Somatic cell nuclear transfer (scNT)-derived pig placenta tissues of gestational day 30 displayed avascularization and hypovascularization. Most of the cytotrophoblast-like cells of the developing scNT-derived placenta villi were improperly localized or exhibited impaired migration to their targeting loci. Id-2, Met, MMP-9, and MCM-7 were barely detectable in the cytotrophoblast cells of the scNT-derived placenta villi. Active MMP-2 and MMP-9 expression was significantly down-regulated in the scNT-embryo transferred recipient uteri. scNT clones exhibited a hypermethylated pattern within the pig MMP-9 promoter region and the significance of GC box in the regulation of MMP-9 promoter activity. Marked apoptosis was observed in the developing endometrial gland of scNT-embryo transferred recipient uteri. Collectively, our data strongly indicated that early gestational death of scNT clones is caused, at least in part, by disruption of the developing endometrial gland as a result of impaired trophoblast migration and invasiveness due to the down-regulation of active MMP-9 expression.

Induction of Ski Protein Expression upon Luteinization in Rat Granulosa Cells.

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

Structural and functional identification of the uncharacterized metallo-ß-lactamase superfamily protein TW9814 as a phosphodiesterase with unique metal coordination.

Metallo-ß-lactamase (MBL) superfamily proteins have a common αß/ßα sandwich fold and perform a variety of functions through metal-mediated catalysis. However, because of the enormous scale of this superfamily, only a small percentage of the proteins belonging to the superfamily have been annotated structurally or functionally to date. Therefore, much remains unknown about the MBL superfamily proteins. Here, TW9814, a hypothetical MBL superfamily protein, was structurally and functionally investigated. Guided by the crystal structure of dimeric TW9814, it was demonstrated that TW9814 functions as a phosphodiesterase (PDE) in the presence of divalent metal ions such as manganese(II) or nickel(II). A docking model between TW9814 and the substrate bis(p-nitrophenyl)phosphate (bpNPP) showed the importance of the dimerization of TW9814 for its bpNPP-hydrolyzing activity and for the interaction between the enzyme and the substrate. TW9814 showed outstanding catalytic efficiency (kcat/Km) under alkaline conditions compared with other PDEs. The activity of TW9814 appears to be regulated through a disulfide bond, which is a feature that is not present in other MBL superfamily members. This study provides a platform for the functional characterization of other hypothetical proteins of the MBL or other superfamilies.

Folic acid pretreatment and its sustained delivery for chondrogenic differentiation of MSCs.

Dietary uptake of folic acid (FA) improves cartilage regeneration. In this work, we discovered that three days of FA treatment is highly effective for promoting chondrogenic differentiation of tonsil-derived mesenchymal stem cells (TMSCs). In a three-dimensional pellet culture, the levels of typical chondrogenic biomarkers, sulfated glycosaminoglycan, proteoglycan, type II collagen (COL II), SRY box transcription factor 9 (SOX 9), cartilage oligomeric matrix protein (COMP), and aggrecan (ACAN) increased significantly in proportion to FA concentration up to 30 µM. At the mRNA expression level, COL II, SOX 9, COMP, and ACAN increased 3.6-6.0-fold with FA treatment at 30 µM compared with the control system that did not receive FA treatment, and the levels with FA treatment were 1.6-2.5 times greater than those in the kartogenin-treated positive control system. FA treatment did not increase type I collagen α1 (COL I α1), an osteogenic biomarker which is a concern with most chondrogenic promoters. At the high FA concentration of 100 µM, significant decreases in chondrogenic biomarkers were observed, which might be related to DNA methylation. A thermogel system incorporating TMSCs and FA provided sustained release of FA over several days, similar to the FA treatment. The thermogel system confirmed the efficacy of FA in promoting chondrogenic promotion of TMSCs. The increased nuclear translocation of core-binding factor ß subunit (CBFß) and the runt-related transcription factor 1 (RUNX1) expression after FA treatment, together with molecular docking studies, suggest that the chondrogenic enhancement mechanism of FA is mediated by CBFß and RUNX1.

Comparative gene expression analysis of somatic cell nuclear transfer-derived cloned pigs with normal and abnormal umbilical cords.

Gene expression profiling of compromised umbilical cords (CUCs) derived from somatic cell nuclear transfer (scNT) clones was performed to determine why scNT-derived clones often exhibit malformed umbilical arteries. Umbilical cord samples were obtained from 65 scNT piglets, and of these, nine displayed a CUC. Microscopic analyses of the scNT clones with CUCs (scNT-CUCs) revealed complete occlusive thrombi that were not detected in the arteries of scNT clones with normal umbilical cords (scNT-Ns). Moreover, whereas the allantoic ducts of the scNT-Ns contained columnar epithelium, the scNT-CUCs lacked this epithelial layer. Compared to scNT-Ns, the scNT-CUCs exhibited severe histological damage, including tissue swelling and vein and arterial damage with complete occlusive thrombi. To investigate functional abnormality, gene expression profiles were created in duplicate using the Platinum Pig 13K oligonucleotide microarray, which contains 13,610 probes of 70 bp in length and is capable of interrogating 13,297 targets with up to one probe per target. Probe sets were selected according to a 2-fold or greater increase or decrease of gene expression in scNT-CUCs compared to scNT-Ns. Most genes expressed in scNT-Ns were also expressed by scNT-CUCs. However, most genes involved in transcriptional regulation, such as JUN, JUNB, and FOSL2, showed a significant decrease in expression in the scNT-CUCs, which may produce a ripple effect capable of altering the transcriptomes of many other cellular processes, including angiogenesis, antioxidation, and apoptosis. The scNT-CUCs with thrombosis showed extensive apoptosis leading to placental insufficiency and related pathology. Considering that the umbilical cord plays a role in the transportation of metabolites to the fetus, placental insufficiency in scNT-CUCs may be caused by an increase in apoptotic protein expression from scNT-derived umbilical cords with hypoplastic arteries, and our results provide evidence that porcine oligonucleotide microarray analysis is a useful tool for screening scNT-derived abnormalities in pigs.

Overexpression of Jazf1 induces cardiac malformation through the upregulation of pro-apoptotic genes in mice.

The transcription factor Juxtaposed with another zinc finger gene 1 (JAZF1) is a zinc finger protein that binds to the nuclear orphan receptor TR4. Recent evidence indicates that TR4 receptor functions as both a positive and negative regulator of transcription, but the role of JAZF1 in transcriptional mechanisms has not been elucidated. Recently, the incidence rate of congenital heart malformations was reported to be significantly elevated in patients who had neurofibromatosis 1 (NF1) with chromosomal microdeletion syndrome. Furthermore, Joined to JAZF1 (SUZ12) is expressed at high levels in the hearts of adult patients with NF1 microdeletion syndrome. Therefore, we hypothesized that ectopic expression of JAZF1 may lead to cardiac malformations that deleteriously affect the survival of neonates and adults. We sought to elucidate the role of JAZF1 in cardiac development using a Jazf1-overexpressing (Jazf1-Tg) mouse model. In Jazf1-Tg mice, Jazf1 mRNA expression was significantly elevated in the heart. Jazf1-Tg mice also showed cardiac defects, such as high blood pressure, electrocardiogram abnormalities, apoptosis of cardiomyocytes, ventricular non-compaction, and mitochondrial defects. In addition, we found that the expression levels of pro-apoptotic genes were elevated in the hearts of Jazf1-Tg mice. These findings suggest that Jazf1 overexpression may induce heart failure symptoms through the upregulation of pro-apoptotic genes in cardiomyocytes.

Identification of maturation and protein synthesis related proteins from porcine oocytes during in vitro maturation.

BACKGROUND: In vitro maturation (IVM) of mammalian oocytes is divided into the GV (germinal vesicle stage), MI (metaphase I stage) and MII (metaphase II stage) stages, and only fully mature oocytes have acquired the ability to be fertilized and initiate zygotic development. These observations have been mostly based on morphological evaluations, but the molecular events governing these processes are not fully understood.The aim of the present study was to better understand the processes involved in the molecular regulation of IVM using 2-DE analysis followed by mass spectrometry to identify proteins that are differentially expressed during oocyte IVM. RESULT: A total of 16 up-regulated and 12 down-regulated proteins were identified. To investigate the IVM process, we specifically focused on the proteins that were up-regulated during the MII stage when compared with the GV stage, which included PRDX 2, GST, SPSY, myomegalin, PED4D, PRKAB 1, and DTNA. These up-regulated proteins were functionally involved in redox regulation and the cAMP-dependent pathway, which are essential for the intracellular signaling involved in oocyte maturation. Interestingly, the PDE4D and its partner, myomegalin, during the MII stage was consistently confirmed up-regulation by western blot analyses. CONCLUSION: These results could be used to better understand some aspects of the molecular mechanisms underlying porcine oocyte maturation. This study identified some regulatory proteins that may have important roles in the molecular events involved in porcine oocyte maturation, particularly with respect to the regulation of oocyte meiotic resumption, MII arrest and oocyte activation. In addition, this study may have beneficial applications not only to basic science with respect to the improvement of oocyte culture conditions but also to mammalian reproductive biotechnology with potential implications.

Oviduct-specific enhanced green fluorescent protein expression in transgenic chickens.

In this study, we confirmed the ability of the 2-kb promoter fragment of the chicken ovalbumin gene to drive tissue-specific expression of a foreign EGFP gene in chickens. Recombinant lentiviruses containing the EGFP gene were injected into the subgerminal cavity of 539 freshly laid embryos (stage X). Subsequently the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Twenty-four chicks (G0) were hatched and screened for EGFP with PCR. Two chicks were identified as transgenic birds (G1), and these founders were mated with wild-type chickens to generate transgenic progeny. In the generated transgenic hens (G2), EGFP was expressed specifically in the tubular gland of the oviduct. These results show the potential of the chicken ovalbumin promoter for the production of biologically active proteins in egg white.

Crystal structure of AmpC BER and molecular docking lead to the discovery of broad inhibition activities of halisulfates against ß-lactamases.

AmpC BER is an extended-spectrum (ES) class C ß-lactamase with a two-amino-acid insertion in the H10 helix region located at the boundary of the active site compared with its narrow spectrum progenitor. The crystal structure of the wild-type AmpC BER revealed that the insertion widens the active site by restructuring the flexible H10 helix region, which is the structural basis for its ES activity. Besides, two sulfates originated from the crystallization solution were observed in the active site. The presence of sulfate-binding subsites, together with the recognition of ring-structured chemical scaffolds by AmpC BER, led us to perform in silico molecular docking experiments with halisulfates, natural products isolated from marine sponge. Inspired by the snug fit of halisulfates within the active site, we demonstrated that halisulfate 3 and 5 significantly inhibit ES class C ß-lactamases. Especially, halisulfate 5 is comparable to avibactam in terms of inhibition efficiency; it inhibits the nitrocefin-hydrolyzing activity of AmpC BER with a Ki value of 5.87 µM in a competitive manner. Furthermore, halisulfate 5 displayed moderate and weak inhibition activities against class A and class B/D enzymes, respectively. The treatment of ß-lactamase inhibitors (BLIs) in combination with ß-lactam antibiotics is a working strategy to cope with infections by pathogens producing ES ß-lactamases. Considering the emergence and dissemination of enzymes insensitive to clinically-used BLIs, the broad inhibition spectrum and structural difference of halisulfates would be used to develop novel BLIs that can escape the bacterial resistance mechanism mediated by ß-lactamases.

Activation and expression of urokinase-type plasminogen activator are modulated by freezing/thawing process through activation of redox signal pathway in primary porcine endometrial cells.

Plasminogen activators (PAs) play a pivotal role in a variety of uterine physiologies, such as endometrial function, trophoblast invasion, and implantation process, but its alteration in expression or activity during cryopreservation of primary uterine cells has received little attention. In this study, we investigated whether PA expression and activity were modulated in first passage primary porcine uterus endometrial epithelium cells (PUEECs) treated with or without a freezing-thawing procedure. Western blotting and zymographic analysis showed that uPA expression and activity increased significantly in frozen-thawed PUEECs in a passage-dependent manner as compared to freshly prepared control cells. Moreover, intracellular reactive oxygen species (ROS) were increased by freezing-thawing and longer culturing, and were more prominent in frozen-thawed PUEECs than in control cells. However, the increase in both uPA expression and activity was greatly reduced or alleviated by treatment with either ROS scavenger N-acetylcysteine or extracellular signal-regulated kinase (ERK) inhibitor PD98059. These results suggest that ROS/ERK-mediated uPA activation may be an important factor in cryo-damage of primary uterine cells.

Comparative proteomic analysis of malformed umbilical cords from somatic cell nuclear transfer-derived piglets: implications for early postnatal death.

BACKGROUND: Somatic cell nuclear transfer (scNT)-derived piglets have high rates of mortality, including stillbirth and postnatal death. Here, we examined severe malformed umbilical cords (MUC), as well as other organs, from nine scNT-derived term piglets. RESULTS: Microscopic analysis revealed complete occlusive thrombi and the absence of columnar epithelial layers in MUC (scNT-MUC) derived from scNT piglets. scNT-MUC had significantly lower expression levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) and angiogenesis-related genes than umbilical cords of normal scNT piglets (scNT-N) that survived into adulthood. Endothelial cells derived from scNT-MUC migrated and formed tubules more slowly than endothelial cells from control umbilical cords or scNT-N. Proteomic analysis of scNT-MUC revealed significant down-regulation of proteins involved in the prevention of oxidative stress and the regulation of glycolysis and cell motility, while molecules involved in apoptosis were significantly up-regulated. Histomorphometric analysis revealed severe calcification in the kidneys and placenta, peliosis in the liver sinusoidal space, abnormal stromal cell proliferation in the lungs, and tubular degeneration in the kidneys in scNT piglets with MUC. Increased levels of apoptosis were also detected in organs derived from all scNT piglets with MUC. CONCLUSION: These results suggest that MUC contribute to fetal malformations, preterm birth and low birth weight due to underlying molecular defects that result in hypoplastic umbilical arteries and/or placental insufficiency. The results of the current study demonstrate the effects of MUC on fetal growth and organ development in scNT-derived pigs, and provide important insight into the molecular mechanisms underlying angiogenesis during umbilical cord development.

Gangliosides are involved in neural differentiation of human dental pulp-derived stem cells.

Human dental pulp-derived stem cells (hDPSCs) have been considered alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. This study investigated the possible role of gangliosides in the neural differentiation of hDPSCs. When hDPSCs were cultured under neural differentiation conditions, expression of neural cell marker genes such as Nestin, MAP-2, and NeuN was detected. Immunostaining and high-performance thin-layer chromatography analysis showed that an increase in ganglioside biosynthesis was associated with neural differentiation of hDPSCs. Specifically, a significant increase in GD3 and GD1a expression was observed during neural differentiation. To confirm the role of gangliosides in neural differentiation, ganglioside biosynthesis was inhibited in hDPSCs by knockdown of UDP-glucose ceramide glucosyltransferase (Ugcg), which prevented differentiation into neural cells. These results suggest that gangliosides may play a role in the neural differentiation process of hDPSCs.

In vitro and in vivo Inhibitory Activity of NADPH Against the AmpC BER Class C ß-Lactamase.

ß-Lactamase-mediated resistance to ß-lactam antibiotics has been significantly threatening the efficacy of these clinically important antibacterial drugs. Although some ß-lactamase inhibitors are prescribed in combination with ß-lactam antibiotics to overcome this resistance, the emergence of enzymes resistant to current inhibitors necessitates the development of novel ß-lactamase inhibitors. In this study, we evaluated the inhibitory effect of dinucleotides on an extended-spectrum class C ß-lactamase, AmpC BER. Of the dinucleotides tested, NADPH, a cellular metabolite, decreased the nitrocefin-hydrolyzing activity of the enzyme with a Ki value of 103 µM in a non-covalent competitive manner. In addition, the dissociation constant (KD) between AmpC BER and NADPH was measured to be 40 µM. According to our in vitro susceptibility study based on growth curves, NADPH restored the antibacterial activity of ceftazidime against a ceftazidime-resistant Escherichia coli BER strain producing AmpC BER. Remarkably, a single dose of combinatory treatment with NADPH and ceftazidime conferred marked therapeutic efficacy (100% survival rate) in a mouse model infected by the E. coli BER strain although NADPH or ceftazidime alone failed to prevent the lethal bacterial infection. These results may offer the potential of the dinucleotide scaffold for the development of novel ß-lactamase inhibitors.

Cytological endometritis in dairy cows: diagnostic threshold, risk factors, and impact on reproductive performance.

We determined the threshold proportion of polymorphonuclear leukocytes (PMNs) for a diagnosis of cytological endometritis (CEM), the risk factors for this condition, and its impact on reproductive performance in dairy cows. Uterine cytology was performed on 407 Holstein cows 4 weeks postpartum to determine the proportions of endometrial cells and PMNs. A receiver operator characteristics curve was used to determine the threshold above which the PMN proportion affected the likelihood of cows conceiving by 200 days postpartum. The optimal threshold was ≥ 14% PMN (sensitivity, 31.3%; specificity, 81.7%; p ＜ 0.05). The farm identity, retained placenta (odds ratio [OR] = 1.87), and septicemic metritis (OR = 3.07) were risk factors for CEM (p ＜ 0.05). Cows with CEM were less likely to resume cyclicity (OR = 0.58) and to conceive by 200 days postpartum (hazard ratio = 0.58). Cows with CEM tended (p ＜ 0.1) to be less likely to become pregnant after their first insemination (OR = 0.65) and to require a greater number of inseminations per conception (2.3 vs. 2.2). In conclusion, a PMN threshold of 14% defined the presence of CEM at 4 weeks postpartum. The farm, retained placenta, and septicemic metritis were risk factors for CEM, which reduces subsequent reproductive performance.

Effect of two treatment protocols for ketosis on the resolution, postpartum health, milk yield, and reproductive outcomes of dairy cows.

We determined the effect of ketosis treatment with propylene glycol (PG) or PG plus l-carnitine and methionine (Metabolase®, Fatro, Bologna, Italy) on the resolution, postpartum health, milk yield, and reproductive performances of dairy cows. Blood from 475 Holstein cows was collected weekly until 4 weeks after calving to measure blood ß-hydroxybutyrate (BHBA) concentrations. Cows with blood BHBA concentration ≥1.2 mmol/L were diagnosed with ketosis and were enrolled. One hundred and fifty cows diagnosed with ketosis were randomly assigned to three treatment groups (Day 0): (1) PG (300 g, PO) for 3 days (PG group, n = 50), (2) PG (300 g, PO) plus l-carnitine (1.25 g) plus methionine (5 g, IV) for 3 days (PG + CM group, n = 50), and (3) no treatment (control group, n = 50). On Day 3, blood was collected to evaluate whether the ketosis had resolved. Cows in the PG and PG + CM groups with blood BHBA ≥1.2 mmol/L were retreated for an additional 2 days, and then blood BHBA concentration was evaluated on Days 5 and 10. Blood glucose and haptoglobin concentrations, rumen fill score (RFS), and body condition score (BCS) were measured on Days 0, 3, 5, and 10. Postpartum complications, milk yield during the first 2 months, and reproductive outcomes were evaluated. The probability of resolution from ketosis was higher (P < 0.05) in the PG + CM group than in the control group on Days 3, 5, and 10 (odds ratio: 2.6-6.3). Blood BHBA in the PG + CM group was lower (P < 0.05) than that of the control group on Days 3 and 5, whereas blood glucose in the PG + CM group was higher (P < 0.05) than that of the control group on Days 3 and 5. RFS in the PG and PG + CM groups was higher than that of the control group on Day 10 (P < 0.01), while BCS loss from Day 0-10 in the control group was higher than those of the PG and PG + CM groups (P < 0.05). Milk yields on the 30th and 60th days postpartum were higher in the PG + CM group than the control and PG groups (P < 0.05). Postpartum complications and intervals between calving and first postpartum insemination or pregnancy did not differ among the groups (P > 0.05). In conclusion, treatment of dairy cows with PG plus l-carnitine and methionine improved the chances of resolution of ketosis and increased milk yield, while affecting neither the incidence of postpartum complications nor reproductive performance.

Structural and Biochemical Characterization of the Curcumin-Reducing Activity of CurA from Vibrio vulnificus.

Curcumin is a yellow-colored ingredient in dietary spice turmeric ( Curcuma longa Linn). This nontoxic polyphenol has antitumor, anti-inflammatory, apoptotic, and antioxidant activities. The ingested curcumin is reduced to multihydrated forms with more potent therapeutic potentials by the curcumin reductase (CurA) from commensal Escherichia coli. In this study, we demonstrated that Vibrio vulnificus CurA ( VvCurA) with 87% sequence similarity to the E. coli CurA exhibits the curcumin-reducing activity through spectrophotometric detection of NADPH oxidation and high performance liquid chromatographic analysis of curcumin consumption and product generation. Afterward, we determined the crystal structures of VvCurA and the VvCurA/NADPH complex, and made the in silico model of the VvCurA/NADPH/curcumin ternary complex through induced fit docking. Based on structural information, active site residues that play critical roles in catalysis have been identified and characterized by mutational and kinetic studies, leading us to propose the reaction mechanism of CurA.

Hanwoo cattle: origin, domestication, breeding strategies and genomic selection.

Hanwoo (Korean cattle) is the native, taurine type of cattle breed of Korea and its history as a draft animal dates back to 5000 Years. In earlier times Hanwoo was used extensively for farming, transportation. Over the period of time, Hanwoo has changed to be meat type cattle. Full-scale production of Hanwoo as meat-type cattle has occurred since 1960s with the rapid growth of the Korean economy. Hanwoo is one of the most economically important species in Korea as it is a significant source of nutrition to the Korean people. Hanwoo beef is the most cherished food of Korea. One of the main goals of researchers is to increase the meat quality, quantity and taste of the beef. In this review we describe the origin, domestication of Hanwoo cattle and breeding program initiated from 1980's. Moreover the advent of technological advancement had provided us a platform to perform genome wide selection on economic traits and its implementation into traditional breeding programs.

Compensatory proliferation of endogenous chicken primordial germ cells after elimination by busulfan treatment.

INTRODUCTION: Primordial germ cells (PGCs) are the major population of cells in the developing bilateral embryonic gonads. Little is known about the cellular responses of PGCs after treatment with toxic chemicals such as busulfan during embryo development. In this study, we investigated the elimination, restorative ability, and cell cycle status of endogenous chicken PGCs after busulfan treatment. METHODS: Busulfan was emulsified in sesame oil by a dispersion-emulsifying system and injected into the chick blastoderm (embryonic stage X). Subsequently, we conducted flow cytometry analysis to evaluate changes in the PGC population and cell cycle status, and immunohistochemistry to examine the germ cell proliferation. RESULTS: Results of flow cytometry and immunohistochemistry analyses after busulfan treatment showed that the proportion of male PGCs at embryonic day 9 and female PGCs at embryonic day 7 were increased by approximately 60% when compared with embryonic day 5.5. This result suggests the existence of a compensatory mechanism in PGCs in response to the cytotoxic effects of busulfan. Results of cell cycling analysis showed that the germ cells in the G0/G1 phase were significantly decreased, while S/G2/M-phase germ cells were significantly increased in the treatment group compared with the untreated control group in both 9-day-old male and female embryos. In addition, in the proliferation analysis with 5-ethynyl-2'-deoxyuridine (EdU) incorporation, we found that the proportion of EdU-positive cells among VASA homolog-positive cells in the 9-day embryonic gonads of the busulfan-treated group was significantly higher than in the control group. CONCLUSIONS: We conclude that PGCs enter a restoration pathway by promoting their cell cycle after experiencing a cytotoxic effect.