• Journal of Embryo Transfer
• /
• v.12 no.3
• /
• pp.293-300
• /
• 1997

Supplementation of maturation medium with additional granulosa cells has beneficial effect on in vitro maturation of bovine follicular oocytes and their subsequent cleavage and development in vitro. However, maturation system using granulosa cells have some disadvantages that collection of granulosa cells is cumbersome and metabolic activity of the cells is variable according to ovarian cycle or follicular size. We hypothesized that bovine immsture oocytes matured without granulosa cell coculture can fertilize and develop normally if the medium volume per oocyte is reduced during in vitro maturation. Immature oocytes were matured for 24 hours in a TCM199 containing 10% fetal calf serum, anterior pitultary hormone (0.02 AU /ml Antrinⓡ) and estradiol with or without granulosa cells in vitro. In Group 1, 35 to 40 oocytes were matured in a well of 4-well plastic dish containing 500 $\mu$l of maturation medium and granulosa cells, and 9 to 10 oocytes were matured in a 50-$\mu$l drop of maturation medium without granulosa cells in Group 2. After maturation, oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Inseminated oocytes were cultured in a microdrop (30 $\mu$l) of a synthetic oviduct fluld medium (SOFM) containing BSA, Minimum Essential Medium essential and non-essential amino acids for 9 days. As a preliminary experiment, we investigated the beneficial effect of granulosa cells during maturation on subsequent cleavage and development using the same type of culturedishes (4-well dish). Granulosa cells could not increase embryo cleavage after fertilization but significantly improved (p<0.05) embryo development to expanding blastocyst (Table1 and 2). In Group 1, 68 and 80% of inseminated oocytes have cleaved at 30 hours and 2 days after IVF, respectively, which is similar (p>0.05) to the result of Group 2 (69% at 30 hours and 78% at 2 days after IVF). The oocytes in Group 2 showed 21 and 11% of developmental rates to expanding and hatching blastocysts, respectively, which was not significantly different (p>0.05) from those (20 and 10%, respectively) of oocytes in Group 1. In conclusion, it has been clarified that a microdrop culture system without granulosa cells for in vitro maturation can support bovine embryonic development to blastocyst in vitro as readily as a granulosa cell coculture system.

• Journal of Embryo Transfer
• /
• v.22 no.4
• /
• pp.245-249
• /
• 2007

The aim of present experiment was to examine hatching rate as in vitro indicator of viability of porcine embryos before early stage embryo transfer such as zygotes or 2-cell stage embryos. Cumulus-oocyte complexes (COCs) collected from ovaries were matured in North Carolina State University 23 (NCSU-23) containing 10% porcine follicular fluid (pFF), 10 ng/ml epidermal growth factor (EGF), $10{\mu}g/ml$ follicle stimulating hormone (FSH), $35{\mu}g/ml$ luteinizing hormone (LH), and 1mg/ml cysteine. After 24 hours, the COCs were transferred to the same medium without hormones. After 65h of maturation, oocytes were exposed to phosphate buffered saline (PBS) with 7% ethanol (v/v) for 7 minutes, and then the oocytes were washed and cultured in tissue culture medium (TCM) 199 containing 5 ug/ml cytochalasin B for 5h at $38.5^{\circ}C$ in an atmosphere of 5% $CO_2$ and 95% air with high humidity. After cytochalasin B treatment, the presumptive parthenotes were cultured in porcine zygote medium (PZM)-5 and cleavage of the parthenotes was assessed at 72h of activation, Normally cleaved parthenotes were cultured for an additional 8 days to evaluate their ability to develop to blastocyst and hatching stages. The fetal bovine serum (FBS) were added at Day 4 or 5 with concentrations of 2.5, 5 or 10%. The blastocyst rates were ranged within $39.1{\sim}70%$ in each treatment. However hatching rate was dramatically decreased in non-addition group. In this experiment, embryo viability in female reproductive tract may be estimated before embryo transfer with in vitro culture adding FBS by hatching ability.

• Journal of Nutrition and Health
• /
• v.55 no.2
• /
• pp.240-249
• /
• 2022

Purpose: Glabridin (GD) is a bio-available isoflavane isolated from the root extract of licorice (Glycyrrhiza glabra L.). It exhibits a variety of pharmacological activities such as anti-inflammatory and anti-oxidant activities. However, extracellular vesicles (EVs) secretion and the anti-cancer mechanism of action remains largely unknown. The present study investigates the anticancer effects of GD by determining the inhibition of EVs secretion in the human breast cancer cell line, MDA-MB-231. Methods: Cell viability, reactive oxygen species (ROS) production, migration, invasion rate, and vascular endothelial growth factor (VEGF) concentration were assessed in MDA-MB-231 cells treated with increasing concentrations of GD (0.1, 1, 5, 10, 20 µM). Subsequently, EV secretion and exosomal DEL-1 protein expression were evaluated to determine the anticancer effects of GD. Results: The results showed that GD significantly inhibited the cell proliferation of MDA-MB-231 cells in a dose- or time-dependent manner. Also, ROS production and apoptosis marker protein cleaved caspase-3 were significantly increased in GD-treated MDA-MB-231, compared to control. Furthermore, GD exposure resulted in significantly decreased not only migration and invasion rates but also the VEGF concentration, thereby contributing to a reduction in angiogenesis. Interestingly, the concentration and number of EVs as well as EV marker proteins, such as CD63 and TSG101, were decreased in GD-treated MDA-MB-231 cells. Markedly, extracellular matrix protein DEL-1 as angiogenesis factor was decreased in EVs from GD-treated MDA-MB-231 cells. Conclusion: This study identifies that the anti-cancer molecular mechanism of GD is exerted via inhibition of angiogenesis and EVs secretion, indicating the potential of GD as a chemotherapeutic agent for breast cancer.

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.63-63
• /
• 2003

This study was carried out to investigate the effects of liquid boar sperm stored at 4$^{\circ}C$ on sperm motility, normal acrosome, and in-vitro fertilization and culture of pig oocytes matured in-vitro. The sperm-rich fraction (30~60 ml) of ejaculate was collected into an insulated vacuum bottle. Semen was slowly cooled to room temperature (20~23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min at 800$\times$g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of lactose, egg yolk and N-acetyl-D-glucosamine (LEN) diluent to provide 1.0$\times$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$ and preserved for 5 days to examine sperm motility and normal acrosome. The medium used for oocyte maturation was modified tissue culture medium (TCM) 199. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 6 h in 500 ${mu}ell$ mTBM fertilization media with 0.2, 1, 5 and 10$\times$10$^{6}$ /ml sperm concentration, respectively. At 6 h after IVF, oocytes were transferred into 500 ${mu}ell$ Hepes-buffered NCSU-23 culture medium for further culture of 6, 48 and 144 h. There were significant differences in sperm motility and normal acrosome among preservation days and incubation times, respectively. The rates of sperm penetration and polyspermy were higher in 5 and 10$\times$10$^{6}$ sperm/ml than in 0.2 and 1$\times$10$^{6}$ sperm/ml. Male pronuclear formation was lower in 0.2$\times$10$^{6}$ sperm/ml than in 1, 5 and 10$\times$10$^{6}$ sperm/ml. Mean numbers of sperm in penetrated oocyte were highest in 10$\times$10$^{6}$ sperm/ml compared with other sperm concentrations. The rate of blastocysts from the cleaved oocytes (2~4 cell stage) was highest in 1$\times$10$^{6}$ sperm/ml compared with other sperm concentrations. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend 1$\times$10$^{6}$ ml sperm concentration for in-vitro fertilization of pig oocytes.

• Journal of Nutrition and Health
• /
• v.57 no.1
• /
• pp.43-52
• /
• 2024

Purpose: Cancer is the leading cause of death in Koreans, with breast cancer being the most common among women. Breast cancer readily metastasizes, and the existing treatment processes impose a significant burden on patients. This study examined whether pomegranate-derived exosome-like nanovesicles (PNVs) have anti-cancer effects by inhibiting cell infiltration and metastasis while increasing apoptosis on breast cancer MDA-MB-231 cells. Methods: Initially, exosome-like nanovesicles were isolated from pomegranate using ultracentrifugation. Subsequently, the size range of these nanovesicles was confirmed using nanoparticle tracking analysis. The ability of breast cancer MDA-MB-231 cells to internalize these natural nanovesicles was assessed with flourescence microscope. The anti-cancer effects of the PNVs were confirmed by applying various concentrations of PNVs (10, 50, 100 ㎍/mL) to MDA-MB-231 cells and systematically assessing their impact on cell viability and migration. Results: The round shape of the lipid bilayer in the PNVs was confirmed, providing crucial insights into their structural properties. We demonstrate that PNVs-associated DiD dye can be efficiently internalized by the MDA-MB-231 cells. The data showed that the PNVs inhibited cell viability, invasion rates, and migration in MDA-MB-231 cells. In addition, PNVs were absorbed into the MDA-MB-231 cells, leading to an increased expression of apoptosis proteins, such as cleaved caspase-3 and phosphorus-JNK, in a concentration-dependent manner. Furthermore, a reduction in cell infiltration and decreased expression of the transition markers MMP-2 and MMP-9 proteins were observed. Conclusion: For the first time, this study suggests that PNVs may be useful in the prevention or treatment of breast cancer by inhibiting the infiltration and metastasis of MDA-MB-231 cells and inducing apoptosis.

• Korean Journal of Animal Reproduction
• /
• v.27 no.3
• /
• pp.249-258
• /
• 2003

The aim of this study was to investigate the effects of different hexoses in medium with IGF-I and/or EGF on in vitro development of porcine embryos. In first experiment, when the embryos were cultured in medium with concentrations of 5 ng/ml IGF-I and 10 ng/ml EGF, the morula and blastocyst rates were higher than in another culture conditions (P<0.05). In second experiment, the effect of IGF-I in medium with different hexoses during the embryo culture was examined. The higher cleavage rates were obtained in medium with glucose and IGF-I (P<0.05). However, the higher proportion of embryos developed to morula and blastocyst stages in medium with IGF-I were obtained than in medium without IGF-I in medium with glucose, mannose and galactose. In third experiment, the effect of EGF on in vitro development of porcine embryos in medium with different hexoses were examined. In the culture medium was supplemented with glucose, the higher proportions (P<0.05) of embryos developed to molura and blastocyst stages were obtained in medium with that than without EGF. However, the proportions of embryos developed to blastocyst stage were not significantly different between with and without of EGF in medium with different hexoses. In firth experiment, the effects of presence of IGF-I and EGF in medium with different hexoses on in vitro development of porcine embryos were examined. When culture medium was supplemented with IGF-I and EGF, the higher proportions of oocytes cleaved and developed to 8-cell stage were obtained in medium with glucose than mannose, galactose and fructose (P<0.05). These results show that glucose and growth factors, supported the development in vitro of porcine embryos, especially with greater embryo cleavage and development in medium with glucose added to media with IGF-I and/or EGF.

• Clinical and Experimental Reproductive Medicine
• /
• v.26 no.2
• /
• pp.231-238
• /
• 1999

The objective of this study was to examine the effect of developmental stage and embryo age of in vitro produced bovine blastocysts after vitrification and thawing. In vitro cultured day 8 blastocysts after IVF were equilibrated 20% ethylene glycol (EG) for 3 min. and were vitrified using EFS40, which is consisted of 40% EG, 18% ficoll, 0.3M sucrose and 10% FBS added in mDPBS for 30 sec. before being plunged into $LN_2$. Also, survival in vitro was assessed by re-expansion and hatching or hatched at 24 hand 48 h postwarming, respectively. The results obtained in these experiments were summarized as follows; 1) When the embryos were cultured for 8 day after IVF, 41.0% of the cleaved embryos developed to the blastocysts (early; 7.6%, expanded; 22.9%, hatching; 4.6% and hatched; 5.9%). 2) When the embryos were exposed or vitrified to the freezing solution, the re-expansion of vitrified embryos (73.3%) was significantly lower than that of control and exposed embryos (100, 97.0%) (p<0.05). But the formation rate of hatching or hatched blastocysts of vitrified embryos (66.7, 46.7%) at 48h after thawing was similar to that of exposed embryos (66.7, 39.4%) but not control (100, 100%) (p<0.01). However, in the total cell numbers of those developed hatched blastocysts, there were not significantly different among the treatment groups. 3) When the embryo survival rates by different developmental stage were examined, the re-expansion was not different among the groups $(64.5{\sim}75.6%)$. After warming 48 h, the hatching and hatched formation of early blastocysts (25.8, 9.7%) was significantly lower than those of expanded (69.7, 39.4%) and hatching blastocysts (53.3, 43.3%) (p<0.05). 4) In addition, when the expanded blastocysts at day 7, 8 and 9 were vitrified, the re-expansion of day 8 and 9 embryos was significantly lower than that of day 7 (day 7; 93.9%, day 8; 75.8% and day 9; 87.5%) (p<0.05). However, the rates of development to hatched blastocysts were no difference among the groups (day 7; 36.4%, day 8; 36.4% and day 9; 31.3%). These results suggested that in vitro produced expanded or hatching blastocysts can be efficiently cryopreserved by the two-step vitrification method using EFS40.

• Korean Journal of Animal Reproduction
• /
• v.26 no.1
• /
• pp.17-23
• /
• 2002

The present study was carried out to investigate the effects of the maturation media such as a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium on penetrability of pig oocytes by liquid boar sperm. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. When immature pig oocytes were cultured in mTCM-199, mWaymouth MB 752/1 and NCSU-23 maturation media for 44 h in 5% $CO_2$, in air at 38.5$^{\circ}C$, the germinal vesicle breakdown (CVBD) rates of the oocytes were 95.6, 94.1 and 94.9%, respectively, and the maturation rates (metaphase II) of oocytes were 92.5, 90.1 and 91.1%, respectively. No differences were observed among the maturation media. The spermrich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ for 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes, cumulus cells were removed and oocytes (30~40) coincubated far 6 h in 0.5 $m\ell$ mTCM-199 and mTBM fertilization media with 2$\times$1061$m\ell$ sperm concentration. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ mTCM-199 and NCSU-23 culture media for further culture 6 or 42 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF, and developmental ability of oocytes at 48 h after IVF were evaluated. The oocytes in combination with NCSU-23 medium for maturation and mTBM medium for IVF increased male pronuclear formation (48.0%) compared to those in combination with mTCM-199 media for maturation and IVF, and mWaymouth MB 752il medium for maturation and mTCM-199 medium far IVF. The rates of cleaved embryos (2~4 cell stage) at 48 h after IVF were 24.1% in combination with mTCM-199 media for maturation, IVF and culture, 43.6% in combination with mWaymouth MB 75211 medium fur maturation and mTCM-199 media for IVF and culture, and 71.2% in combination with NCSU-23 medium for maturation, mTBM medium for IVF and NCSU-23 medium for culture. In conclusion, we found out the oocytes matured in vitro were fertilized by liquid boar sperm stored in BTS extender at 17$^{\circ}C$ for 5 days. We recommend the simple defined NCSU-23 medium for nuclear maturation, mTBM medium and liquid boar sperm for IVF, and NCSU-23 medium for embryo culture.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.45 no.5
• /
• pp.671-679
• /
• 2016

Extracts from spinach, cabbage, and onion are known to possess various instructive characteristics, including antioxidant and anti-inflammation activities. Spinach, cabbage, and onion are consumed worldwide and represent important sources of dietary phytochemicals with proven antioxidant properties, such as flavonoids and phenolic acids. Food-derived flavonoids and phenolic compounds are expected to be promising drugs for cancer. In the present study, we investigated the effects of methanol extracts of spinach, cabbage, and onion on cell proliferation and apoptosis in human gastric and breast cancer cells. Proliferation rates of AGS, MDA-MB-231, and SK-BR-3 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The methanol extracts of spinach, cabbage, and onion inhibited proliferation of cancer cells in a dose-dependent manner. 4',6-Diamidino-2-phenylindole (DAPI) staining revealed that chromatin condensation significantly increased compared with the control. In the results of MTT assay and DAPI staining, onion extract was the most effective in inhibiting cancer cell proliferation and apoptosis. To assess changes in protein expression level by onion extract, we identified Bax (pro-apoptotic), Bcl-2 (anti-apoptotic), and poly(ADP-ribose) polymerase (PARP) protein by western blot analysis. The expression of Bax and cleaved-PARP increased, whereas expression of Bcl-2 was decreased compared with the control. These results suggest that spinach, cabbage, and onion extracts suppressed growth of human gastric cancer AGS, human breast cancer MDA-MB-231, and SK-BR-3 cells through induction of apoptosis. Among the extracts, onion extract had stronger anti-cancer and apoptosis induction effects than spinach and cabbage extracts. Further, onion extract more effectively induced apoptosis of human gastric cancer cells than human breast cancer cells. Therefore, further studies are needed to determine the anti-cancer effects of onion extracts in vivo. Onion extract can be developed as a chemopreventive or therapeutic agent for gastric cancer.

• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.65 no.3
• /
• pp.161-171
• /
• 2020

Direct seeding is one of the rice seedling establishment methods that is increasingly being practiced by farmers to save labor and reduce costs. However, this method often causes poor germination under flooding conditions after sowing. In this study, we developed japonica elite lines with quantitative trait loci (QTL) associated with anaerobic germination (AG) tolerance to overcome poor germination and seedling establishment in wet direct seeding. The QTL introgression lines were developed from a cross between weedy photoblastic rice as the AG donor and the Nampyeong variety via phenotypic and genotypic selection. Compared to Nampyeong, the survival rates of the selected lines were improved by approximately 50% and 240% under field and greenhouse conditions, respectively. To improve selection efficiency by marker assisted selection, the QTL markers associated with AG tolerance were converted to cleaved amplified polymorphic sequence markers designed based on next-generation sequence analysis. These lines retained similar agronomic traits and yield potential to the parent, Nampyeong. Among these lines, we selected the most promising line, which exhibited high survival rate and good agricultural traits under flooding conditions and named the line as Jeonju643. This line will contribute to breeding programs aiming to develop rice cultivars adapted to wet direct seeding. This study demonstrates the successful application of marker-assisted selection to targeted introgression of anaerobic genes into a premium quality japonica rice variety.