The number of abandoned dogs is increasing with the worsening of the economy and the rising of feed value. It was becoming a serious social problem because of the disease transmission and destruction of natural ecosystems by abandoned dogs been wild animal. In order to solve these problems, companion dogs necessary to secure its own genetic information and to establish the systematic tracking system. Using multiplex-PCR method with 27 microsatellite marker (MS marker) divided 3 set, various alleles occurring to 6 dog breed (Labrador Retriever, German Shepherd, English Springer Spaniel, Belgian Malinois, Jindo Dog, PoongSan Dog) make use of markers to determine allele frequency and heterozygosity. MS marker FH2834 and FH2790 have only two allele and most were found in 13 alleles at FH3381 and FH3399. Average heterozygosity of MS marker is 0.534 and especially, heterozygosity represented the highest value of 0.765 at FH3381. So, it was recognized appropriate allele frequency for individual identification and paternity diagnosis in companion dogs. Using multiplex-PCR method with MS marker, various alleles occurring to dog breed make use of markers to deter mine individual identification and paternity diagnosis, traits associated biomarkers and breed-specific marker for faster, more accurate and ways to reduce the analysis cost. Based on this result, a scientific basis was established to the existing pedigree data by applying genetics additionally. Animal registration system is expected to be conducted nationwide in future. The method expects to very useful this system.
The development of embryos reconstructed by somatic cell nuclear transfer (SCNT) is dependent upon numerous factors. Central to development is the quality and developmental competence of the recipient cytoplast and the type of the donor nucleus. Typically metaphase of the second meiotic division (MII) has become the cytoplast of choice. Production of a cytoplast requires removal of the recipient genetic material, however, it may remove proteins which are essential for development or reduce the levels of cytoplasmic proteins to influence subsequent reprogramming of the donor nucleus. In this study, enucleation at MII did not affect the activities of either MPF or MAPK kinases. Immunocytochemical staining showed that both Cyclin B1 (MPF) and Erk1/2 (MAPK) were associated with the meiotic spindle of AI/TI oocytes with little staining in the cytoplasm, however, at MII association of both proteins with the spindle had reduced and a greater degree of cytoplasmic distribution was observed. The analysis of oocyte proteins removed during enucleation is a difficult approach to the identification of factors which may be depleted in the cytoplast. This is primarily due to the large numbers of aspirated karyoplasts which would be required for the analysis.
The experimental manipulation of protooncogenes and their gene products is a valuable research tool for the study of human neoplasia. In this study, the recently identified human cervical cancer protooncogene (HccR-2) was expressed in transgenic mice under the control of the tetracycline regulatory system. Mice expressing the HccR-2 transgene showed an altered myeloid development characterized by an increased percentage of mature and band-form neutrophils in the peripheral blood, liver and spleen. This phenotype is similar to human chronic neutrophilic leukemia (CNL) in many ways, which is a rare chronic myeloproliferative disorder (CMD) that presents as a sustained leukocytosis of mature neutrophils with a few or no circulating immature granulocytes, an absence of peripheral blood monocytosis, basophilia, or eosinophilia, and an infiltration of neutrophils into the liver, spleen and kidney. Thus, the HccR-2 transgenic mouse model is imperative not only for investigating the biological properties of the HccR-2 protooncogene in vivo, but also for analyzing the mechanisms involved in the progression of CNL.
The experimental manipulation of protooncogenes and their gene products is a valuable research tool for the study of human neoplasia. In this study, the recently identified human cervical cancer protooncogene (HccR-2) was expressed in transgenic mice under the control of the tetracycline regulatory system. The phenotype observed was similar in many respects to human chronic neutrophilic leukemia (CNL). Thus, the HccR-2 transgenic mouse model is important not only for investigating the biological properties of the HccR-2 protooncogene in vivo, but also for analyzing the mechanisms involved in the progression of CNL.
Mitochondria diseases have been reported to involve structural and functional defects of complex I-V. Especially, many of these diseases are known to be related to dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I). The dysfunction of mitochondria complex I is associated with neurodegenerative disorders, such as Parkinson's disease, Huntington's disease, and Leber’s hereditary optic neuropathy (LHON). Mammalian mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) is largest and consists of at least 46 different subunits. In contrast, the NDI1 gene of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. The Saccharomyces cerevisiae NDI1 gene using a recombinant adeno-associated virus vector (rAAV-NDI1) was successfully expressed in AML12 mouse liver hepatocytes and the NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the NDI1 gene failed to survive. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting. Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced cells was not affected by rotenone which is inhibitor of complex I, but was inhibited by antimycin A. Furthermore, these results indicate that Ndi1 can be functionally expressed in the AML12 mouse liver hepatocytes. It is conceivable that the NDI1 gene is powerful tool for gene therapy of mitochondrial diseases caused by complex I deficiency. In the future, we will attempt to functionally express the NDI1 gene in mouse embryonic stem (mES) cell.
Sildenafil citrate (SIL) a phosphodiesterase 5 inhibitor (PDE5I) has been used for long time as a first line oral drug for erectile dysfunction. Though it has beneficial effects on erectile organ it also has some adverse effects in other cells and/or tissues related to reproductive system when exposed to longer duration. The objective of the present study is to evaluate the long term effect of SIL on sperm parameters in Wistar albino rat. The animals are divided into two groups, for group I - rats were treated with saline (vehicle alone) and group - II oral administration of 5 mg/kg b.w. of SIL was administrated orally once in a day for 120 days. At the end of the trial period animals were sacrificed and epididymal sperm were subjected to various analysis. Results showed significant reduction in sperm count, motility, viability and morphologically intact sperm in long term PDE5I exposed animals when compared to control. Acrosomal status and fertility test also showed significant reduction in long term PDE5I exposed animals. The present study clearly indicated that long term SIL has shown to induce alteration in sperm quality and quantity, leading to decline in fertility rate. Indicate that SIL impinge on spermatogenesis as well as epididymal function. Understanding the molecular down-stream events involved in long-term exposure to PDE5 inhibitor can be valuable to supervise on related infertility issues and to suggest corrective measures.
The study aim is to investigate the free radicals scavenging and spermatogenic potentials, as well as to analyze any reproductive toxicity of ethanolic extract of Mucuna prureins (M. pruriens) Linn. in spermatozoa, under different dosages in normal male rat. Normal rats were randomly selected and suspension of the extract was administered orally at the dosages of 150, 200 and 250 mg/kg body weight of the different groups of male rats (n=6) once in a day for 60 days and grouped as group II, III and IV respectively. Saline treated rats served as control -group I. On the 60th day the animals were sacrificed and the epididymal sperm were subjected to various analyses like level of ROS production, LPO, enzymatic and non enzymatic antioxidant, morphology, morphometry, chromosomal integrity and DNA damage. Results showed significant reduction in ROS production and peroxidation and significant increase in both enzymic and non-enzymic antioxidants in all concentration treated groups when compared with control. Results from all the drug treated groups showed good sperm morphology, increased sperm count and motility. There was no DNA damage and showed normal chromosomal integrity even in 250 mg/kg dose. When compared with control all the three extract treated groups showed increased ROS scavenging activity. However, group II (200 mg/kg) showed significant changes in all the parameters. From the present study it was confirmed that the M. pruriens has potential to improve the sperm qualitatively and quantitatively through scavenging the excess ROS with any adverse side effects. These observations suggest that ethanolic seed extract of M. pruriens may serve as anti-oxidant that can exploit to treat the oxidative stress mediated male factor infertility.
Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by etopic expression of transcription factors. iPS cells are indistinguishable from ES cells in terms of morphology and stem cell marker expression. Moreover, mouse iPS cells give rise to chimeric mice that are competent for germline transmission. However, mice derived from iPS cells often develop tumors. Furthermore, the low efficiency of iPS cell generation is a big disadvantage for mechanistic studies. Nonviral plasmid‐based vectors are free of many of the drawbacks that constrain viral vectors. The histone deacetylase inhibitor valproic acid (VPA) has been shown to improve the efficiency of mouse and human iPS cell generation, and vitamin C (Vc) accelerates gene expression changes and establishment of the fully reprogrammed state. The MEK inhibitor PD0325901 (Stemgent) has been shown to increase the efficiency of the reprogramming of human primary fibroblasts into iPS cells. In this report, we described the generation of mouse iPS cells devoid of exogenous DNA by the simple transient transfection of a nonviral vector carrying 2A‐peptide‐linked reprogramming factors. We used VPA, Vc, and the MEK inhibitor PD0325901 to increase the reprogramming efficiency. The reprogrammed somatic cells expressed pluripotency markers and formed EBs.
JAZF1 (Juxtaposed with Another Zinc Finger gene 1) transcription factor are Zn-finger proteins that bind to the nuclear orphan receptor TAK/TR4 (Nakajima et al., 2004). The nuclear orphan receptor TAK1/TR4 functions as a positive as well as a negative regulator of transcription. It was recently reported that congenital cardiovascular malformations are significantly more frequent in Neurofibromatosis 1 (NF1) patients with microdeletion syndrome than in those with classical NF1. JAZF1 was expressed in adult heart of patients with microdeletion syndrome. JAZF1 is highly conserved among various species include zebrafish. We hypothesized that the expression of zebrafish Jazf1 may lead to severe forms of congenital heart disease that allow the survival of newborns and adults. In this study, we created Jazf1 transgenic zebrafish which over-express zebrafish Jazf1 cDNA under control of the CMV promoter. Our results suggested that Jazf1 expression may play an important role in zebrafish cardiac development.
This study investigated the changes of plasminogen activators (PAs) activity, expression and localization of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) during the estrous cycle in pigs. Estrous cycle was sorted into three group by pre-ovulation (Pre-Ov), post-ovulation (Post-Ov) and early to mid-luteal stages (Early to mid-L). Analysis for immunohistochemistry was confirmed by location of tPA and uPA. Porcine uterus tissue was cut into 1 × 1 cm squares, and were incubated in DMEM/F-12 medium for 1 h at 38℃, 5% CO2 for measurement of PA activity. Western blotting was implemented for measurement of PA quantity. In results, the blood vessels and secretory glands were increased in Post-Ov stage than Pre-Ov and Early to mid-L stages. The tPA and uPA was located mainly within lumen of blood vessels and secretory glands. The PA activity in Post-Ov (0.99±0.03) stage were significantly (p<0.01) higher than Pre-Ov stage (0.51±0.03) and Early to mid-L stage (0.21±0.04). Expression of PAs were significantly (p<0.05) higher in Early to mid-L stage than other stages. These results indicate that PAs activity and expression may change in uterus tissue during the estrous cycle in pigs.
These study was carried out to investigate the effects of the supplementation with sodium nitroprusside (SN) and nitric oxide (NO) of canine oocytes on IVM rates. Oocytes were incubated in TCM-199 supplement with at 0.03~0.10 mM SN and 0.3~1.0 mM NO for 48 hrs. Oocytes were transferred to 50 ul drops of maturation medium covered mineral oil and cultured in a CO2 incubator (5% CO2, 95% air, 38℃). The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03, 0.05, 0.07, 0.10 mM SN were 25.9±3.5%, 36.4±3.2%, 33.3±3.5%, 28.8±3.2%, respectively. The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03~0.07 mM SN were significantly increased compare to the control (26.0±2.2%). The in vitro maturation rates of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.3, 0.5, 0.7, 1.0 mM NO were 28.0±4.2%, 36.5± 3.6%, 30.0±3.8%, 19.2±3.5%, respectively. The in vitro maturation rate of oocytes in TCM-199 medium supplemented with 0.3 and 0.5 mM NO were significantly increased compare to the control (26.0±2.2%). The in vitro maturation rates of oocytes cultured for 12~48 hrs in TCM-199 medium supplement with 0.05 mM SN were 26.0±3.2%, 28.0±3.4%, 38.0±3.2%, respectively. The in vitro maturation rate of oocytes cultured for 12~48 hrs in TCM-199 medium supplement with 0.5 mM NO were 22.0±3.0%, 30.0±3.8%, 36.0±4.2%, respectively. These result was significantly increased compare to the control.
In the present study, the effect of cysteine and NT or bisphenol A (BP) on in vitro aturation (IVM) of porcine oocytes were examined. COCs was cultured in NCSU-23 medium supplement with 10% FCS which had previously been covered with mineral oil and equilibrated in a humidified atmosphere of 5% CO2 and 95% air at 38℃. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 0.5~10.0 mM cysteine were 34.0±3.2%, 36.0±3.5%, 48.0±3.8%, 22.0±3.2%, respectively. The IVM rate of oocytes cultured in NCSU-23 medium supplement with 0.5~5.0 mM NT for 48 hrs were 24.0±4.2%, 18.0±4.9%, 8.0±2.2%, respectively. NT affects oocyte in vitro maturation rate in a dose-dependent. This result were significantly lower than the control group. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 1.0 mM NT+5.0 mM cysteine (38.0±4.3%) were significantly higher than that of NT treatment. The IVM rate of oocytes cultured in NCSU-23 medium supplement with 0.05~5.0 mM BP for 48 hrs were 20.0±4.7%, 10.0±5.3%, 6.0±3.2%, respectively. The IVM rate of oocytes cultured in NCSU-23 medium supplement with BP was significantly lower cultured non supplement of BP (44.0±3.5%). BP affects porcine oocyte maturation rate in a dose-dependent manner. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 1.0 mM BP+5.0 mM cysteine (32.0±3.2%) were increased than that of BP treatment.
The objective of this study was to determine the effects of E. coli isolated from porcine semen on sperm viability, motility, and semen pH. Semen samples were prepared using commercial extender, SeminarkPro (Noahbio Tech, Korea) that did not contain antibiotics. And 4 different levels of E. coli were artificially innoculated to semen with following concentrations; 4,000 of sperms with 1 of E. coli (T1), 400 with 1 (T2), 40 with 1 (T3), and 4 with 1 (T4). Semen samples were preserved at 17℃ for 5 days in semen storage box until analyzed by flowcytometer. Aliquots were subjected to measure the sperm viability (Live/Dead® stain), motility (mitochondrial function), and semen acidity (pH) from day 0 (day of semen collection) to day 5. Sperm motility and viability were significantly decreased (p<0.05) on day 0 (4 hrs after preservation at 17℃) in T3 and T4 compared to control groups and were significantly decreased (p<0.05) in all groups from day 3. Sample pH was acidic in T3 (6.90~6.86) and T4 (6.86~6.65) from day 3 to day 5 (p<0.05). On the other hand, sample pH was maintained 7.0~7.1 in control, T1, and T2 during the experimental period. Sperm motility and viability were significantly decreased from day 0 to day 5 compared to control in samples contaminated with E. coli above a value of 40:1 (20×106 sperm cells/ml : 5×105 cfu/ml). Even on day 1 in T4 and on day 3 in T3, semen pH was acidic probably due to the acidification of dead spermatozoa. These results suggest that E. coli contamination has a concentration-dependent detrimental effect on extended porcine semen quality.
The present study was conducted to examine the reactive oxygen species (ROS) generation levels and subsequent DNA damage in the bovine cultured somatic cells. Bovine ear skin cells were classified by serum starvation, confluence and cycling cells. Cells were stained in 10 μM dichlorohydrofluorescein diacetate (H2DCFDA) or 10 μM hydroxyphenyl fluorescein (HPF) dye to measure the H2O2 or ˙OH radical levels. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each cell. H2O2 and ˙OH radical levels of cultured somatic cells were high in confluence group (7.1±0.7 and 8.4±0.4 pixels/cell, respectively) and significantly low in serum starvation group (4.9±0.4 and 7.0±0.4 pixels/cell, respectively, p<0.05). Comet tail lengths of serum starvation (148.3±5.7 μm) and confluence (151.1±5.0 μm) groups were found to be significantly (p<0.05) increased in comparison to that of cycling group (137.1±7.5 μm). These results suggest that the culture type of donor cells can affect the ROS generation, which leads the DNA fragmentation of the cells.
During mammalian fertilization, germ cell-specific hyaluronidases, such as sperm adhesion molecule 1 (SPAM1) and hyaluronoglucosaminidase 5 (Hyal5), are important for the dispersal of the cumulus mass. In this study, we demonstrated that bull Hyal5 is a single copy gene on chromosome 4 that is expressed specifically in the testis. In addition, we expressed recombinant bull SPAM1 and Hyal5 in human embryonic kidney 293T cells and showed that these enzymes possessed hyaluronidase activity. We also demonstrated that a polyclonal antibody against bull sperm hyaluronidase inhibits sperm-egg interactions in an in vitro fertilization (IVF) assay. Our results suggested that bull Hyal5 may have a critical role in bull fertilization.
Aurora A kinase is a mitotic serine/threonine kinase whose proposed functions include the maturation of centrosomes, G2/M transition, alignment of chromosomes at metaphase, and cytokinesis. In this study, we investigated the effect of MLN8237, an aurora A kinase inhibitor, on the postovulatory aging of oocytes based on the frequency of oocyte fragmentation, cdk1 kinase activity, and cyclin B degradation. The fragmentation of ovulated oocytes during prolonged culture was inhibited by treatment with MLN8237 in a concentration-dependent manner. The frequency of fragmented oocytes was significantly lower in oocytes treated with 2 μM MLN8237 (13%) than in control oocytes (64%) after two days of culture. Most of the control (non-fragmented) oocytes (91%) were activated after two days of culture. In comparison, only 22% of the MLN8237-treated oocytes were activated; the rest of the oocytes (78%) were still in metaphase with an abnormal spindle and dispersed chromosomes. Next, cdk1 activity and the level of cyclin B were examined. The level of cyclin B and cdk1 activity in MLN8237-treated oocytes were nearly equal to those in control oocytes. Our results indicate that MLN8237 inhibited the fragmentation of ovulated oocytes during prolonged culture, although it blocked the spontaneous decrease in activity of cdk1 and degradation of cyclin B. This mechanism of inhibition is different from that in oocytes treated with nocodazole, which have high levels of cdk1 activity and cyclin B.
Cellular uptake of nanoparticles for stem cell labeling and tracking is a critical technique for biomedical therapeutic applications. However, current techniques suffer from low intracellular labeling efficiency and cytotoxic effects, which has led to great interest in the development of a new labeling strategy. Using silica-coated nanoparticles conjugated with rhodamine B isothiocyanate (RITC) (SR), we tested the cellular uptake efficiency, biocompatibility, proliferation or differentiation ability with murine bone marrow derived hematopoietic stem/progenitor cells. The bone marrow hematopoietic cells showed efficient uptake with SR with dose or time dependent manner and also provided a higher uptake on hematopoietic stem/progenitor cells. Biocompatibility tests revealed that the SR had no deleterious effects on cell cytotoxicity, proliferation, or multi-differentiation capacities in vitro and in vivo. SR nanoparticles are advantageous over traditional labeling techniques as they possess a high level of cellular internalization without limiting the biofunctionality of the cells. Therefore, SR provides a useful alternative for gene or drug delivery into hematopoietic stem/progenitor cells for basic research and clinical applications.
To avoid hyperacute rejection of xenografts, α1,3-galactosyltransferase knock-out (GalT KO) pigs have been produced. In this study, we examined whether Sia-containing glycoconjugates are important as an immunogenic non-Gal epitope in the pig liver with disruption of α1,3-galactosyltransferase gene. The target cells were then used as donor cells for somatic cell nuclear transfer (scNT). A total of 1,800 scNT embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. Real-time RT-PCR and glycosyltransferase activity showed that α2,3-sialyltransferase (α2,3ST) and α2,6-sialyltransferase (α2,6ST) in the heterozygote GalT KO liver have higher expression levels and activities compared to controls, respectively. According to lectin blotting, sialic acidcontaining glycoconjugate epitopes were also increased due to the decreasing of α-Gal in heterozygote GalT KO liver, whereas GalNAc-containing glycoconjugate epitopes were decreased in heterozygote GalT KO liver compare to the control. Furthermore, the heterozygote GalT KO liver showed a higher Neu5Gc content than control. Taken together, these finding suggested that the deficiency of GalT gene in pigs resulted in increased production of Neu5Gc-bounded epitopes (H-D antigen) due to increase of α2,6-sialyltransferase. Thus, this finding suggested that the deletion of CMAH gene to the GalT KO background is expected to further prolong xenograft survival.
Growth hormone (GH) is obligatory for growth and development. But, there is controversy on the GH effect about reproductive processes of sexual differentiation, pubertal maturation, gonadal steroidogenesis, gametogenesis and ovulation. This study was conducted to investigate the effect of GH on estrus, ovulation and embryo implantation. The results obtained were as follows. GH stimulated to increase estrus rate (p<0.05), pregnancy rate (p<0.05), and total fetus number in mice treated for superovulation. Also, the correlation between GH and steroids, E2 and P4, at peri-estrus stage/ peri-ovulation stage/ peri-implantation stage of the superovulation-induced mice was examined. Consequently, GH co-injected with PMSG especially increased P4 level (p<0.05) at peri-estrus stage of superovulationinduced mice. In conclusion, GH co-treatment in superovulation system boosted the rate of estrus, pregnancy and total fetus by increasing progesterone level at peri-estrus stage of superovulation-induced mice.