This study was carried out to study the viability of oocytes when vitrified at various maturation stages. Bovine cumulus-oocyte complexes were recovered from ovaries at a slaughter and then divided into five groups: control group(unvitrified oocytes), 0 hr. group(composed of oocytes vitrified before the onset of maturation) and 10, 14, and 20 hrs groups(vitrified at 10, 14 and 20 hrs after the onset of maturation, respectively). The oocytes remained vitrified for 24 hrs, and then were thawed in 3$0^{\circ}C$. Survival and cleavage rates were investigated by results of in vitro culture and aceto-orcein staining or FDA test. No difference in the incidence of diploid oocytes was observed among the control, non-vitrified group(3.6%) and oocytes vitrified at 14 hrs(6.7%) or 20 hrs(1.7%). However, more diploid oocytes were detected after vitrification at 0 hr.(26.7%) and 10 hrs(21.7%) post maturation. The survival rate of all vitrified immature oocytes(12.0~38.0%) was low, 48.0% of unvitrified oocytes and oocytes vitrified before maturation or 0~ 10 hrs after the onset of maturation were higher than that of other groups. The overall fertilization and cleavage rates of vitrified immature oocytes (32.3 ~ 64.6% and 4.6 ~ 32.3%) were low, and 55.0% of unvitrified oocytes and the rate of immature oocytes were very higher than that of mature oocytes.
Yang, Seoyeon;Lee, Ji-Yeon;Hur, Ho;Oh, Ji Hoon;Kim, Myoung Hee
BMB Reports
/
v.51
no.9
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pp.450-455
/
2018
Tamoxifen (TAM) is commonly used to treat estrogen receptor (ER)-positive breast cancer. Despite the remarkable benefits, resistance to TAM presents a serious therapeutic challenge. Since several HOX transcription factors have been proposed as strong candidates in the development of resistance to TAM therapy in breast cancer, we generated an in vitro model of acquired TAM resistance using ER-positive MCF7 breast cancer cells (MCF7-TAMR), and analyzed the expression pattern and epigenetic states of HOX genes. HOXB cluster genes were uniquely up-regulated in MCF7-TAMR cells. Survival analysis of in slico data showed the correlation of high expression of HOXB genes with poor response to TAM in ER-positive breast cancer patients treated with TAM. Gain- and loss-of-function experiments showed that the overexpression of multi HOXB genes in MCF7 renders cancer cells more resistant to TAM, whereas the knockdown restores TAM sensitivity. Furthermore, activation of HOXB genes in MCF7-TAMR was associated with histone modifications, particularly the gain of H3K9ac. These findings imply that the activation of HOXB genes mediate the development of TAM resistance, and represent a target for development of new strategies to prevent or reverse TAM resistance.
Since cancer is the leading cause of death worldwide, there is an urgent need to understand the mechanisms underlying cancer progression and the development of cancer inhibitors. Signal transducer and activator of transcription 3 (STAT3) is a major transcription factor that regulates the proliferation and survival of various cancer cells. Here, dual-specificity phosphatase 3 (DUSP3) was identified as a regulator of STAT3 based on an interaction screening performed using the protein tyrosine phosphatase library. DUSP3 interacted with the C-terminal domain of STAT3 and dephosphorylated p-Y705 of STAT3. In vitro dephosphorylation assay revealed that DUSP3 directly dephosphorylated p-STAT3. The suppressive effects of DUSP3 on STAT3 were evaluated by a decreased STAT3-specific promoter activity, which in turn reduced the expression of the downstream target genes of STAT3. In summary, DUSP3 downregulated the transcriptional activity of STAT3 via dephosphorylation at Y705 and also suppressed the migratory activity of cancer cells. This study demonstrated that DUSP3 inhibits interleukin 6 (IL-6)/STAT3 signaling and is expected to regulate cancer development. Novel functions of DUSP3 discovered in IL-6/STAT3 signaling regulation would help expand the understanding of cancer development mechanisms.
Sphingosine-1-phosphate (S1P) has a many function involved proliferation, differentiation and survival of many cells. In this study, to investigate whether S1P improve the developmental competence of porcine embryos, 50 nM of S1P were supplemented during in vitro maturation (with EGF or without EGF) medium and/or in vitro culture (IVC) medium. Addition of S1P was significantly increased the rate of oocytes reaching metaphase II (MII) compared to the control (83.5 vs. 64.1%) in without EGF medium, but not with EGF medium (89.5 vs. 84.6%). When treated with $1{\mu}M$ of N1N-dimethylsphingosine (DMS), a sphingosine kinase inhibitor which is blocked endogenous generation of S1P, the meiotic progression rates to MII stage (without EGF: 45.2 and with EGF: 66.7%) were significantly decreased and degeneration rates (without EGF: 51.2 and with EGF: 30.1%) were increased in both medium compared to control group during IVM periods. Also, the rates of blastocyst formation was significantly increased in the S1P treated group compared to control group (29.0 vs. 19.2%) of EGF supplemented medium, whereas there were no effect in the EGF free medium (9.0 vs. 10.5%). After 12 h IVM, the phosphorylation of ERK1 and ERK2, which is major signaling pathway of MAP kinase, were increased in the S1P group than that of control or DMS group. When supplemented of S1P during IVC, the rates of blastocyst formation and total cell number (30.2% and 40.6) were significantly increased in S1P-treated group compared with control (20.1% and 32.5), DMS (12.3% and 25.1), and S1P plus DMS group (24.7% and 33.6). The percentage of apoptosis nuclei in the S1P group was significantly decreased than other groups. Also, the rates of blastocyst formation (26.7 vs. 14%) and total cell number (42.8 vs. 32.5) were significantly increased in the S1P group than those of control group when S1P added during the entire IVM and IVC periods. Taken together, our results indicate that S1P supplementation in IVM and/or IVC medium affects beneficial effect of meiotic maturation and subsequent developmental competence of porcine embryos.
Histone deacetylation mediated by histone deacetylases (HDACs) has been reported as one of the epigenetic mechanisms associated with tumorigenesis. The poor responsiveness of anticancer drugs found with cholangiocarcinoma (CCA) leads to short survival rate. We aimed to investigate mRNA expression of HDACs class I and II, and the effect of HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA), in CCA in vitro. Expression of HDACs was studied in CCA cell lines (M213, M214 and KKU-100) and an immortal cholangiocyte (MMNK1) by semi-quantitative reverse transcription-PCR. SAHA and VPA, as well as a classical chemotherapeutic drug 5 -fluorouacil (5-FU) were used in this study. Cell proliferation was determined by sulforhodamine assay. $IC_{50}$ and $IC_{20}$ were then analyzed for each agent and cell line. Moreover, synergistic potentional of VPA or SAHA in combination with 5-FU at sub toxic does ($IC_{20}$) of each agent was also evaluated. Statistic difference of HDACs expression or cell proliferation in each experimental condition was analyzed by Student's t-test. The result demonstrated that HDACs were expressed in all studied cell types. Both SAHA and VPA inhibited cell proliferation in a dose-dependent manner. Interestingly, KKU-100 which was less senstitive to classical chemotheraoeutic 5-FU was highly was sensitive to HDAC inhibitors. Simultaneous combination of subtoxic doses of HDAC inhibitors and 5-FU signiicantly inhibited cell proliferation in CCA cell lines compared to single sgent treatment($P{\leq}0.01$), while sequentially combined treatments were less effective. The present study showed inhibitory effects of HDACIs on cell proliferation in CCA cell lines, with synergistic antitumor potential demonstrated by simultaneous combination of VPA or SAHA with 5-FU, suggesting a novel alternative therapeutic strategy in effective treatment of CCA.
Tumor angiogenesis, growth and metastasis are three closely related processes. We therefore investigated the effects of barbigerone on all three in the B16F10 tumor model established in both zebrafish and mouse models, and explored underlying molecular mechanisms. In vitro, barbigerone inhibited B16F10 cell proliferation, survival, migration and invasion and suppressed human umbilical vascular endothelial cell migration, invasion and tube formation in concentration-dependent manners. In the transgenic zebrafish model, treatment with $10{\mu}M$ barbigerone remarkably inhibited angiogenesis and tumor-associated angiogenesis by reducing blood vessel development more than 90%. In vivo, barbigerone significantly suppressed angiogenesis as measured by H and E staining of matrigel plugs and CD31 staining of B16F10 melanoma tumors in C57BL/6 mice. Furthermore, it exhibited highly potent activity at inhibiting tumor growth and metastasis to the lung of B16F10 melanoma cells injected into C57BL/6 mice. Western blotting revealed that barbigerone inhibited phosphorylation of AKT, FAK and MAPK family members, including ERK, JNK, and p38 MAPKs, in B16F10 cells mainly through the MEK3/6/p38 MAPK signaling pathway. These findings suggested for the first time that barbigerone could inhibit tumor-angiogenesis, tumor growth and lung metastasis via downregulation of the MEK3/6/p38 MAPK signaling pathway. The findings support further investigation of barbigerone as a potential anti-cancer drug.
Objective: Using domestic cats as a biomedical research model for fertility preservation, the present study aimed to characterize the influences of ovarian tissue encapsulation in biodegradable hydrogel matrix (fibrinogen/thrombin) on resilience to cryopreservation, and static versus non-static culture systems following ovarian tissue encapsulation and cryopreservation on follicle quality. Methods: In experiment I, ovarian tissues (n=21 animals; 567 ovarian fragments) were assigned to controls or hydrogel encapsulation with 5 or 10 mg/mL fibrinogen (5 or 10 FG). Following cryopreservation (slow freezing or vitrification), follicle viability, morphology, density, and key protein phosphorylation were assessed. In experiment II (based on the findings from experiment I), ovarian tissues (n=10 animals; 270 ovarian fragments) were encapsulated with 10 FG, cryopreserved, and in vitro cultured under static or non-static systems for 7 days followed by similar follicle quality assessments. Results: In experiment I, the combination of 10 FG encapsulation/slow freezing led to greater post-thawed follicle quality than in the control group, as shown by follicle viability (66.9%±2.2% vs. 61.5%±3.1%), normal follicle morphology (62.2% ±2.1% vs. 55.2%±3.5%), and the relative band intensity of vascular endothelial growth factor protein phosphorylation (0.58±0.06 vs. 0.42±0.09). Experiment II demonstrated that hydrogel encapsulation promoted follicle survival and maintenance of follicle development regardless of the culture system when compared to fresh controls. Conclusion: These results provide a better understanding of the role of hydrogel encapsulation and culture systems in ovarian tissue cryopreservation and follicle quality outcomes using an animal model, paving the way for optimized approaches to human fertility preservation.
Proceedings of the Korean Society of Developmental Biology Conference
/
2003.10a
/
pp.80-80
/
2003
In cryopreserved rat embryos, survival rates obtained in vitro are not always consistent with the rates obtained in vivo. To determine the optimal conditions for in vivo development to term, rat embryos at the 4-cell, 8-cell and morula stages were vitrified in EFS40 by a 1-step method and transferred into oviducts or uterine horns of recipients at various times during pseudopregnancy. Vitrified and fresh 4-cell embryos only developed after transfer into oviducts of asynchronous recipients on Day -1 to -2 of synchrony, i.e., at a point in pseudopregnancy that was 1-2 days earlier than the embryos. However, although about half the vitrified embryos transferred into oviducts on Day -1 developed to term, only a minority of embryos transferred at later times did so, whether vitrified (10-34%) or fresh (24-33%), suggesting that this may not be the most suitable stage for cryopreservation. Very few 8-cell embryos, either vitrified or fresh, developed when transferred into oviducts on Day 0 to -0.5. However, when transferred into uterine horns, high proportions of vitrified 8-cell embryos (-63%) developed to term in reasonably synchronous recipients (Day 0 to -0.5) but not in more asynchronous ones (6%; Day-1). A majority of vitrified morulae also developed to term (52-68%) in a wider range of recipients (Day 0 to -1), the greatest success occurring with recipients on Day -0.5. Similar proportions of vitrified and fresh 4-cell embryos, 8-cell embryos and morulae developed to term when there was appropriate synchronization between embryo and recipient. Thus vitrification of preimplantation stage rat embryos does not appear to impair their developmental potential in vivo.
DNA methylation is one of the reasons for poor survival of clone animals. The OCT-4 gene is essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that the 5'-promoter region of Oct-4 gene was a target of DNA methylation and the methylation status was changed variously during embryonic development in bovine. The study conducted to examine the expression and methylation pattern of tissue-dependent differentially methylated region (T-DMR) of Oct-4 gene in bovine somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) blastocysts. The Oct-4 gene expression was evaluated by RT-PCR and fluorescence immunocytochemistry. The methylation pattern of T-DMR was analyzed using restriction mapping and bisulfite sequencing methods. The Oct-4 transcripts were highly expressed in IVF, while they were not expressed in SCNT. The Oct-4 protein was not detected or expressed at very low level in SCNT, the intensity of Oct-4 protein, however, was strong in IVF. On the other hand, the T-DMR of Oct-4 gene was hypermethylated in SCNT compared to that of IVF. These results suggested that expression and the failure of demethylation of Oct-4 gene was closely associated with incomplete development of SCNT embryos.
Mast cells (MCs) have been implicated in the pathogenesis of tissue fibrosis. However, the role of MC in the development of liver fibrosis has not been fully elucidated. Stem cell factor (SCF) is known to recruit MCs to the liver following injury as it induces mast cell proliferation, survival and differentiation from resident tissue precursors. This study examines the interaction between activated hepatic stellate cells (HSCs) and MCs in rat fibrotic liver, and SCF production by HSCs during culture in vitro. Rats were studied 4, 7, 14 and 21 days after bile duct ligation (BDL). Fibrogenesis was assessed by a measurement of collagen stained with sirius red F3B. Activated HSCs and MCs were identified by ${\alpha}$-smooth muscle actin (${\alpha}-SMA$) immunohistochemical and alcian blue staining and measured by a computerized image analysis system. SCF production was determined in rat HSC cultures using Western blotting. Mild fibrotic changes were noted in BDL rat livers as early as 4 days after induction of cholestasis. Significant expansion and organization of fibrous tissue has occurred in day 14 BDL rats which progressed to bridging fibrosis by day 21. In BDL rats, both a large number of activated HSCs and MCs were detected in portal tracts and fibrous septa. Both area of activated HSCs infiltration and density of MCs were significantly higher in all BDL group compared with Shams. In BDL rats, both areas of activated HSCs infiltration and density of MCs were no significant difference between day 4 and 7 and were significantly higher in day 14. However, the areas of activated HSCs infiltration were significantly lesser in day 21 and the densities of MCs were significantly higher in day 21 compared with day14 BDL. In BDL rats, both areas of activated HSCs infiltration and density of MCs were highly correlated with areas of fibrosis. Western blotting showed that SCF protein was consistently produced in activated HSCs by culture on plastic and freshly isolated HSCs expressed relatively little 30kD SCF compared to late primary culture activated HSCs (day 14) and passaged HSCs. These results suggest that HSCs activated in vitro produce SCF, and may play an important role in recruiting mast cells to the liver during injury and fibrosis.
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