• 한국수정란이식학회지
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• 제28권3호
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• pp.265-271
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• 2013

Cryopreservation and in vitro fertilization (IVF) protocols are important in genetic studies and applications to transgenic animals. Various studies about boar sperm cryopreservation have been studied for a long time. Those were about the use of extenders, the choice of sugars, the cooling and warming rates. The factors that influence the boar sperm are the dramatic changes in temperatures, osmotic and toxic stresses, and reactive oxygen species (ROS) generation. Among these factors, ROS generation is the main damage to DNA which is a principal genetic material and the most important for the practical applications. So we wondered whether ROS generation could be reduced. In previous study, monothioglycerol (MTG) was essential for the culture of embryo stem cells. Therefore we added MTG in the freezing extender based on lactose-egg yolk (LEY) with trehalose. For the assessment of the frozen-thawed sperm, we focused onmotility, membrane integrity and DNA damage. First, we used a computer-aided sperm analysis system for overall conditions of sperm such as motility and viability. Then we performed the sperm chromatin structure assay for DNA integrity and hypo-osmotic swelling test for membrane integrity. And our result showed the existence of MTG in the freezing extender caused less damage to DNA and higher motility in frozen-thawed boar sperm. Also we checked a relative antioxidant activity of MTG in modified Modena B extender. We concluded that this reagent can activate sperm mitochondria at MTG $0.2{\mu}M$, contribute to sperm motility and DNA integrity but there was no significant difference on membrane integrity. Also antioxidant activity of MTG in modified Modena B extender was proved.

• Asian-Australasian Journal of Animal Sciences
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• 제19권3호
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• pp.335-340
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• 2006

A large number of toxicological substances and pharmacological and physical agents can cause reproductive intervention at the cellular and molecular level. The present study was designed to assess the effect of mercury ($HgCl_2$) at 50 to $550{\mu}M$ concentration ranges, in vitro, on the sperm membrane and DNA integrity, viability, and acrosomal status of normal bull spermatozoa. The samples were processed for sperm analyses using semen-diluting fluid (PBS, pH 7.2). We recorded a sharp increase in the lipid peroxidation/LPO rate; the highest was at $550{\mu}M$ mercury concentration, indicating a deleterious effect of mercury on the sperm membrane intactness. There was also a strong negative correlation between LPO rate and % viable spermatozoa (R = 0.987, p<0.001). Data obtained from a comet assay technique revealed that mercury is capable of inducing DNA breaks in the sperm nuclei. Interestingly, 92% of DNA breaks were double-stranded. The correlation between LPO rate and % DNA breaks was 0.984. Performing the gelatin test indicates that mercury is able to alter the integrity of acrosomal membranes showing an abnormal acrosome reaction. In this regard, a strong link was found between LPO rate and % halos (R = 0.990, p<0.001). Collectively, mercury proved to be a potent oxidant in the category of environmental factors affecting bull spermatozoa. Hence, considering the wide spread use of mercury and its compounds, these metals should be regarded with more concern.

• Journal of Microbiology and Biotechnology
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• 제32권1호
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• pp.37-45
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• 2022

The fungal cell wall and membrane are the principal targets of antifungals. Herein, we report that myricetin exerts antifungal activity against Candida albicans by damaging the cell wall integrity and notably enhancing the membrane permeability. In the presence of sorbitol, an osmotic protectant, the minimum inhibitory concentration (MIC) of myricetin against C. albicans increased from 20 to 40 and 80 ㎍/ml in 24 and 72 h, respectively, demonstrating that myricetin disturbs the cell wall integrity of C. albicans. Fluorescence microscopic images showed the presence of propidium iodide-stained C. albicans cells, indicating the myricetin-induced initial damage of the cell membrane. The effects of myricetin on the membrane permeability of C. albicans cells were assessed using crystal violet-uptake and intracellular material-leakage assays. The percentage uptakes of crystal violet for myricetin-treated C. albicans cells at 1×, 2×, and 4× the MIC of myricetin were 36.5, 60.6, and 79.4%, respectively, while those for DMSO-treated C. albicans cells were 28.2, 28.9, and 29.7%, respectively. Additionally, myricetin-treated C. albicans cells showed notable DNA and protein leakage, compared with the DMSO-treated controls. Furthermore, treatment of C. albicans cells with 1× the MIC of myricetin showed a 17.2 and 28.0% reduction in the binding of the lipophilic probes diphenylhexatriene and Nile red, respectively, indicating that myricetin alters the lipid components or order in the C. albicans cell membrane, leading to increased membrane permeability. Therefore, these data will provide insights into the pharmacological worth of myricetin as a prospective antifungal for treating C. albicans infections.

• 생명과학회지
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• 제33권7호
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• pp.586-592
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• 2023

돼지 정액을 저장하는 동안 산화스트레스의 발생은 정자의 질과 생존에 영향을 미치는 중요한 인자이다. 정액의 저장은 온도 변화, 동결보호제 등의 다양한 스트레스 인자에 노출되어 있다. 이러한 정자 내에서의 산화스트레스는 활성산소종의 생성에 의해 발생되며, 이는 지질, 단백질, DNA와 같은 세포를 구성하는 물질에 산화적으로 손상을 일으킨다. 활성산소종과 항산화물질의 균형있는 체계는 정자의 생존과 그 기능을 유지하는 데 중요한 역할을 한다. 정액을 장기간 보존하게 되면 활성산소종의 수준이 증가하여 정자의 운동성, 막 온전성, DNA 온전성에 영향을 미치게 된다. 또한, 활성산소종에 의해 유도된 지질과산화 반응은 정자막의 유동성과 안정성에 영향을 미쳐 정자의 운동성을 감소시킨다. 그리고, DNA의 산화적 손상은 DNA 단편화를 일으켜 정자의 DNA 온전성을 손상시킬 수 있다. 결론적으로, 정액을 보관하는 동안 발생되는 산화스트레스는 정자의 질과 기능을 유지하는 데 중요하다. 따라서, 산화스트레스의 기본적인 메커니즘과 정자의 기능에 미치는 영향을 이해하는 것은 산화스트레스로부터의 손상을 최소화하고, 효율적이고 기능적인 정자의 저장 방법을 개선하기 위한 효과적인 전략과 연구 개발을 위해 중요할 것으로 판단된다.

• 한국미생물·생명공학회지
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• 제48권4호
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• pp.429-438
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• 2020

The emergence of multi-drug resistant, pathogenic methicillin-resistant Staphylococcus aureus (MRSA) is a threat to global health and has created a need for novel functional therapeutic agents. In this study, we evaluated the underlying mechanisms of the anti-MRSA effect of an azaphilone pigment, sclerotiorin (SCL) from Penicillium sclerotiorum. The antimicrobial activity of SCL was evaluated using agar disc diffusion, broth microdilution, time-kill assays and biophysical studies. SCL exhibits selective activity against Gram positive bacteria including MRSA (range, MIC = 128-1028 ㎍/ml) and exhibited rapid bactericidal action against MRSA with a > 4 log reduction in colony forming units within three hours of administration. Biophysical studies, using fluorescent probes and laser or electron microscopy, demonstrated a SCL dose-dependent alternation in membrane potential (62.6 ± 5.0.4% inhibition) and integrity (> 95 ± 2.3%), and the release of UV260 absorbing materials within 60 min (up to 3.2 fold increase, p < 0.01) of exposure. Further, SCL localized to the cytoplasm and hydrolyzed plasmid DNA. While in vitro checkerboard studies revealed that SCL potentiated the antimicrobial activity of topical antimicrobials such as polymixin, neomycin, and bacitracin (Fractional Inhibitory Concentration Index range, 0.26-0.37). Taken together these results suggest that SCL targets the membrane and DNA of MRSA to facilitate its anti-MRSA antimicrobial effect.

• 한국수정란이식학회지
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• 제28권1호
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• pp.63-71
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• 2013

In this study, we used flow a cytometric assay to evaluate plasma membrane integrity and mitochondrial activity in post-thawed sperm that was supplemented with ginsenoside-$Rg_1$. Varying concentrations of ginsenoside-$Rg_1$ (0, 25, 50 and $100{\mu}M/ml$) were used in the extender during cryopreservation to protect the DNA of thawed sperm, thereby increasing the viability and motility rate as evaluated using a computer-assisted sperm analysis (CASA) method. The results derived from CASA were used to compare the fresh, control, and ginsenoside-$Rg_1$ groups. Sperm motility and the number of progressively motile sperm were significantly (p<0.05) higher in the $50{\mu}M/ml$ ginsenoside-Rg1 group ($61.0{\pm}4.65%$) than in the control ($46.6{\pm}7.02%$), $25{\mu}M/ml$ ($46.2{\pm}4.76%$), and $100{\mu}M/ml$ ginsenoside-$Rg_1$ ($52.0{\pm}1.90%$) groups. However, the velocity distribution of post-thawed sperm did not differ significantly. Membrane integrity and MMP staining as revealed using flow cytometry were significantly (p<0.05) higher ($91.6{\pm}0.82%$) in the $50{\mu}M/ml$ ginsenoside-$Rg_1$ group than in the other groups. Here, we report that ginsenoside-$Rg_1$ affects the motility and viability of boar spermatozoa. Moreover, ginsenoside-$Rg_1$ can be used as a protective additive for the suppression of intracellular mitochondrial oxidative stress caused by cryopreservation.

• Clinical and Experimental Reproductive Medicine
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• 제49권2호
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• pp.87-92
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• 2022

Objective: The aim of this study was to determine the effects of melatonin and selenium in freezing extenders on frozen-thawed rat sperm. Methods: Semen samples were collected from 20 adult male Wistar albino rats. Following dilution, the samples were divided into six groups: four cryopreserved groups with 1 mM and 0.5 mM melatonin and selenium supplements, and two fresh and cryopreserved control groups. The rapid freezing technique was used to freeze the samples. Flow cytometry was used to assess plasma membrane integrity, mitochondrial membrane potential, and DNA damage, while computer-assisted sperm analysis was used to assess motility. Results: Total motility was higher in the 1 mM melatonin supplementation group than in the cryopreserved control group (mean±standard error of the mean, 69.89±3.05 vs. 59.21±1.31; p≤0.05). The group with 1 mM selenium had the highest plasma membrane integrity (42.35%±1.01%). The cryopreserved group with 0.5 mM selenium had the highest mitochondrial membrane potential, whereas the cryopreserved control group had the lowest (45.92%±4.53% and 39.45%±3.52%, respectively). Conclusion: Cryopreservation of rat semen supplemented with 1 mM melatonin increased sperm motility after freeze-thawing, while supplementation with 0.5 mM selenium increased mitochondrial activity.

• 한국수정란이식학회지
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• 제31권4호
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• pp.355-365
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• 2016

The cryopreservation of sperm has become the subject of research for successful artificial insemination technologies. Antifreeze proteins (AFPs), one of the factors necessary for effective cryopreservation, are derived from certain Antarctic organisms. These proteins decrease the freezing point of water within these organisms to below the temperature of the surrounding seawater to protect the organism from cold shock. Accordingly, a recent study found that AFPs can increase the motility and viability of spermatozoa during cryopreservation. To evaluate this relationship, we performed cryopreservation of boar sperm with AFPs produced in the Arctic yeast Leucosporidium sp. AFP expression system at four concentrations (0, 0.01, 0.1, and $1{\mu}g/ml$) and evaluated motility using computer assisted sperm analysis. DNA damage to boar spermatozoa was measured by the comet assay, and sperm membrane integrity and acrosome integrity were evaluated by flow cytometry. The results showed that motility was positively affected by the addition of AFP at each concentration except $1{\mu}g/ml$ (p<0.001). Although cryopreservation with AFP decreased the viability of the boar sperm using, the tail DNA analyses showed that there was no significant difference between the control and the addition of 0.1 or $0.01{\mu}g/ml$ AFP. In addition, the percentage of live sperm with intact acrosomes showed the least significant difference between the control and $0.1{\mu}g/ml$ AFP (p<0.05), but increased with $1{\mu}g/ml$ AFP (p<0.001). Our results indicate that the addition of AFP during boar sperm cryopreservation can improve viability and acrosome integrity after thawing.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1999년도 Proceedings of the 17th Symposium on Plant Biology Environmental Stress and Photosynthesis
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• pp.68-82
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• 1999

CHilling tolereance of plants are closely correlated with the degree of fatty acid unsaturation of membrane lipids. We studied the effects of low-temperature photoinhibition on the photochemical efficiency of photosystem II in terms of fatty acid unsaturation of thylakoid membranes lipids isolated from chilling -sensitive plants and chilling -resistant ones. To directly test the chilling tolerance of photosynthetic machinery in relation to membrane lipids, we further compared wild type tobacco plants with that of transgenic tobacco plants, in which the sensitivity to chilling had been enhanced by genetic modification of fatty acid unsaturation of chloroplast membrane lipids. The transgenic tobacco plants were found to contain reduced levels of unsaturated membrane fatty acids after being transformed with cDNA for glycerol-3-phophate acyltransferase from squash. The functional integrity of photosystem II during and recovery of photosynthesis from low-temperature photoinhibition will be discussed in connection with the degree of fatty acid unsaturation of chlorophast membranes lipids.

• 한국임상수의학회지
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• 제30권6호
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• pp.442-448
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• 2013

개 정액 동결을 위한 glucose가 첨가된 glycerol-free TRIS 희석액을 개발하기 위해 glycerol-free TRIS내 알맞은 glucose의 양과 0.3 M glucose가 첨가된 glycerol-free TRIS내 정자의 적정 노출시간을 조사하였다. 여섯 마리의 수캐의 사출액을 0.04 M glucose가 첨가된 glycerol-free TRIS내에서 $4^{\circ}C$까지 100분 동안 냉각한 후 서로 다른 glucose농도 (0 M, 0,04 M, 0.1 M, 0.2 M, 0.3 M)의 glycerol-free TRIS에서 30분 동안 냉각하여 동결하였다. $37^{\circ}C$에서 25 초 동안 융해한 후 정자의 막 고유성과 첨단체 고유성을 검사하였다. 부가적으로 0.3 M glucose가 첨가된 glycerol-free TRIS내 정자의 적정 노출시간에 따른 정자의 동결 후 운동성, 생존성, DNA 고유성을 확인하였다. 막 고유성과 첨단체 고유성은 각각 6-carboxyfluoresceindiacetate(6-CFDA)/propidium iodide(PI) fluorescent staining와 Pisum sativum agglutinin conjugated-fluorescein isothiocyanate 방법에 의해 검사하였다. DNA 고유성은 terminal deoxynucleotidyl transferase dUTP nick end labeling로 염색하여 flow cytometry로 검사하였다. 0.2 M 또는 0.3 M glucose가 첨가된 glycerol-free TRIS에서 동결된 정자가 낮은 농도의 glucose가 첨가된 희석액에서 동결된 정자보다 막 고유성이 높게 나타났으며(p<0.05), 첨단체 고유성은 0.3 M 군에서 높게 나타났다(p<0.05). 운동성은 50 분 군에서 높게 나타났으나(p<0.05), DNA fragmentation index는 노출시간에 따라 차이가 없었다. 본 연구 결과 개정자가 0.3 M glucose가 첨가된 glycerol-free TRIS에서 $4^{\circ}C$, 50 분간 냉각 후 동결과 융해 후 더 높은 생존성을 나타냈다.