• Molecules and Cells
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• 제41권2호
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• pp.127-133
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• 2018

Chromatin remodeling factors are involved in many cellular processes such as transcription, replication, and DNA damage response by regulating chromatin structure. As one of chromatin remodeling factors, remodeling and spacing factor 1 (RSF1) is recruited at double strand break (DSB) sites and regulates ataxia telangiectasia mutated (ATM) -dependent checkpoint pathway upon DNA damage for the efficient repair. RSF1 is overexpressed in a variety of cancers, but regulation of RSF1 levels remains largely unknown. Here, we showed that protein levels of RSF1 chromatin remodeler are temporally upregulated in response to different DNA damage agents without changing the RSF1 mRNA level. In the absence of SNF2h, a binding partner of RSF1, the RSF1 protein level was significantly diminished. Intriguingly, the level of RSF1-3SA mutant lacking ATM-mediated phosphorylation sites significantly increased, and upregulation of RSF1 levels under DNA damage was not observed in cells overexpressing ATM kinase. Furthermore, failure in the regulation of RSF1 level caused a significant reduction in DNA repair, whereas reconstitution of RSF1, but not of RSF1-3SA mutants, restored DSB repair. Our findings reveal that temporal regulation of RSF1 levels at its post-translational modification by SNF2h and ATM is essential for efficient DNA repair.

• 한국수정란이식학회지
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• 제27권2호
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• pp.107-111
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• 2012

Miniature pig sperm cryopreservation is continually researched in biotechnology for breed conservation and reproduction. It is important to control the temperature at each stage of cryopreservation and cryoprotectant. It is also necessary to find the optimal cryoprotectant concentration and chemical elements of the extender. Recently, many studies have used various cryoprotectant materials, such as dimethyl sulphoxide (DMSO), ethylene glycol (EG), antifreeze protein (AFP), amides, and glycerol. Glycerol is a commonly used cryoprotectant. However, glycerol has critical cytotoxic properties, including osmotic pressure and it can cause irreversible damage to live cells. Therefore, We focused on membrane fluidity modifications can reduce cell damage from freezing and thawing procedures and evaluated on the positive effects of trehalose to the viability, chromatin integrity, and motility of boar sperm. Miniature pig sperm was separated from semen by washing with modified- Modena B (mMB) extender. After centrifugation, the pellet was diluted with the prepared first extender. This experiment was designed to compare the effects that sperm cryopreservation using two different extenders has on sperm chromatin. The control group used the glycerol only and it was compared with the glycerol and glycerol plus trehalose extender. Sperm viability and motility were evaluated using WST1 assays and computer-assisted semen assays (CASA). Chromatin structure was examined using acridine orange staining. For the motility descriptors, trehalose caused a significant (p<0.01) increase in total motility ($57.80{\pm}4.60%$ in glycerol vs. $75.50{\pm}6.14%$ in glycerol + trehalose) and progressive ($51.20{\pm}5.45%$ in glycerol vs. $70.74{\pm}8.06%$ in glycerol + trehalose). A significant (p<0.05) increase in VAP ($42.70{\pm}5.73{\mu}m/s$ vs. $59.65{\pm}9.47{\mu}m/s$), VSL ($23.06{\pm}3.27{\mu}m/s$ vs. $34.60{\pm}6.58{\mu}m/s$), VCL ($75.36{\pm}11.36{\mu}m/s$ vs. $99.55{\pm}12.91{\mu}m/s$), STR ($54.4{\pm}2.19%$ vs. $58.0{\pm}1.63%$), and LIN ($32.2{\pm}2.05%$ vs. $36.0{\pm}2.45%$) were also detected, respectively. The sperm DNA fragmentation index was 48.8% to glycerol only and 30.6% to glycerol plus trehalose. Trehalose added group showed higher percentages of sperm motility, stability of chromatin structure than glycerol only. In this study, we suggest that trehalose is effective in reducing freezing damage to miniature pig sperm and can reduce chromatin damage during cryopreservation.

• Animal cells and systems
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• 제13권4호
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• pp.405-409
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• 2009

When DNA double-strand breaks (DSBs) were induced in mammalian cells, many DNA damage response proteins are accumulated at damage sites to form nuclear foci called IR-induced foci. Although the formation of foci has been shown to promote repair efficiency, the structural organization of chromatin in foci remains obscure. BAF53 is an actin-related protein which is required for maintenance of chromosome territory. In this study, we show that the formation of IR-induced foci by $\gamma$-H2AX and 53BP1 were reduced when BAF53 is depleted, while DSB- activated ATM pathway and the phosphorylation of H2AX remains intact after DNA damage in BAF53 knockdown cells. We also found that DSB repair efficiency was largely compromised in BAF53 knockdown cells. These results indicate that BAF53 is critical for formation of foci by $\gamma$-H2AX decorated chromatin at damage sites and the structural organization of chromatin in foci is an important factor to achieve the maximum efficiency of DNA repair.

• Clinical and Experimental Reproductive Medicine
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• 제40권1호
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• pp.23-28
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• 2013

Objective: The aim of the present study was to examine the relationship among male age, strict morphology, and sperm chromatin structure and condensation. Methods: Sperm samples from a total of 100 men underwent semen analysis, and sperm chromatin structure and condensation were assessed with toluidine blue (TB) and aniline blue (AB) tests. Results: Prevalence of strict morphology of less than 4%, and abnormal sperm chromatin structure and condensation did not show any statistically significant differences according to male age (p=0.605, p=0.235, and p=0.080). No significant correlation was demonstrated among age of male partners, strict morphology, and abnormal sperm chromatin structure using TB and AB tests. However, abnormal sperm chromatin condensation was positively associated with sperm chromatin structure (r=0.594, p=0.000) and showed negative correlation with strict morphology (r=-0.219, p=0.029). Conclusion: The tests for sperm chromatin condensation showed a significant association with strict morphology. Further study is needed to elucidate the relationship between clinical outcome and sperm chromatin tests.

• Molecules and Cells
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• 제45권11호
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• pp.792-805
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• 2022

Repairing damaged DNA and removing all physical connections between sister chromosomes is important to ensure proper chromosomal segregation by contributing to chromosomal stability. Here, we show that the depletion of non-SMC condensin I complex subunit H (NCAPH) exacerbates chromosome segregation errors and cytokinesis failure owing to sister-chromatid intertwinement, which is distinct from the ultra-fine DNA bridges induced by DNA inter-strand crosslinks (DNA-ICLs). Importantly, we identified an interaction between NCAPH and GEN1 in the chromatin involving binding at the N-terminus of NCAPH. DNA-ICL activation, using ICL-inducing agents, increased the expression and interaction between NCAPH and GEN1 in the soluble nuclear and chromatin, indicating that the NCAPH-GEN1 interaction participates in repairing DNA damage. Moreover, NCAPH stabilizes GEN1 within chromatin at the G2/M-phase and is associated with DNA-ICL-induced damage repair. Therefore, NCAPH resolves DNA-ICL-induced ultra-fine DNA bridges by stabilizing GEN1 and ensures proper chromosome separation and chromosome structural stability.

• Journal of Ginseng Research
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• 제40권3호
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• pp.251-259
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• 2016

Background: Estrogen signaling pathways are modulated by exogenous factors. Panax ginseng exerts multiple activities in biological systems and is classified as an adaptogen. Zearalenol is a potent mycoestrogen that may be present in herbs and crops arising from contamination or endophytic association. The goal of this study was to investigate the impact of P. ginseng, zearalenol and estradiol in tests on spermatozoal function. Methods: The affinity of these compounds for estrogen receptor (ER)-alpha and beta ($ER{\alpha}$ and $ER{\beta}$)-was assessed in receptor binding assays. Functional tests on boar spermatozoa motility, movement and kinematic parameters were conducted using a computer-assisted sperm analyzer. Tests for capacitation, acrosome reaction (AR), and chromatin decondensation in spermatozoa were performed using microscopic analysis. Results: Zearalenol-but not estradiol ($E_2$)- or ginseng-treated spermatozoa-decreased the percentage of overall, progressive, and rapid motile cells. Zearalenol also decreased spontaneous AR and increased chromatin decondensation. Ginseng decreased chromatin decondensation in response to calcium ionophore and decreased AR in response to progesterone ($P_4$) and ionophore. Conclusion: Zearalenol has adverse effects on sperm motility and function by targeting multiple signaling cascades, including $P_4$, $E_2$, and calcium pathways. Ginseng protects against chromatin damage and thus may be beneficial to reproductive fitness.

• Clinical and Experimental Reproductive Medicine
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• 제45권1호
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• pp.17-24
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• 2018

Objective: To investigate sperm chromatin/DNA integrity, global DNA methylation, and DNMT mRNA transcription in men with oligoasthenoteratozoospermia (OAT) compared with normozoospermic men. Methods: Semen samples from 32 OAT patients who comprised the case group and 32 normozoospermic men who comprised the control group were isolated and purified using a standard gradient isolation procedure according to World Health Organization criteria. DNMT1, DNMT3A, and DNMT3B transcripts were then compared between groups using real-time quantitative reverse-transcription polymerase chain reaction. Global DNA methylation in sperm was determined by an enzyme-linked immunosorbent assay. Protamine deficiency and the proportion of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3), aniline blue (AB), and toluidine blue (TB) staining, as well as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The p-values < 0.05 were considered to indicate statistical significance. Results: Significantly higher proportions of AB+, TB+, CMA3+, and TUNEL+ spermatozoa, as well as DNMT3A and DNMT3B transcription, were found in the OAT group. Positive correlations were detected between sperm parameters, DNA/chromatin damage, and DNMT3A and DNMT3B transcripts. Global DNA methylation was significantly higher in the OAT patients and had a significant correlation with abnormal results of all sperm chromatin integrity tests, but was not associated with DNMT1, DNMT3A, or DNMT3B expression. Conclusion: Oligoasthenoteratozoospermic men showed abnormal sperm parameters, abnormal chromatin/DNA integrity, and a higher global DNA methylation rate, as well as overexpression of DNMT mRNA.

• Clinical and Experimental Reproductive Medicine
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• 제42권3호
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• pp.101-105
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• 2015

Objective: Several publications have established a relationship between sperm DNA damage and male factor infertility, based on data from America, Europe, and Asia. This study aimed to compare the extent of sperm DNA damage in sperm samples from Nigerian men with unexplained infertility and in sperm samples from a fertile group composed of sperm donors who had successfully impregnated a female partner naturally or through assisted conception. Methods: A total of 404 men underwent male fertility evaluation at Androcare Laboratories and Cryobank participated in this study. Semen analysis and a sperm chromatin structure assay (SCSA) were performed on all subjects. Results: The men in the unexplained infertility group were slightly older than the men in the fertile sperm group ($36{\pm}10$ years vs. $32{\pm}6$ years, p=0.051). No significant difference was observed between the two groups in semen analysis parameters ($p{\geq}0.05$). Men in the unexplained infertility group with normal semen parameters had a significantly higher DNA fragmentation index (DFI) than men in the fertile sperm group ($27.5%{\pm}7.0%$ vs. $14.1%{\pm}5.3%$, p<0.05). In the unexplained infertility group, 63% of the men had a DFI greater than 20%, compared to 4% in the fertile sperm group. In the unexplained infertility group, 15.2% of the subjects had a DFI greater than 30%, compared to 1% in the fertile sperm group. Conclusion: Our study showed that the SCSA may be a more reliable predictor of fertility potential than traditional semen analysis in cases of unexplained infertility.

• Mycobiology
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• 제41권1호
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• pp.1-12
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• 2013

Msi1-like (MSIL) proteins, which are eukaryote-specific and contain a series of WD40 repeats, have pleiotropic roles in chromatin assembly, DNA damage repair, and regulation of nutrient/stress-sensing signaling pathways. In the fungal kingdom, the functions of MSIL proteins have been studied most intensively in the budding yeast model Saccharomyces cerevisiae, an ascomycete. Yet their functions are largely unknown in other fungi. Recently, an MSIL protein, Msl1, was discovered and functionally characterized in the pathogenic yeast Cryptococcus neoformans, a basidiomycete. Interestingly, MSIL proteins appear to have redundant and unique roles in both fungi, suggesting that MSIL proteins may have evolutionarily divergent roles in different parts of the fungal kingdom. In this review, we will describe the current findings regarding the role of MSIL proteins in fungi and discuss future directions for research on this topic.

• Clinical and Experimental Reproductive Medicine
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• 제47권4호
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• pp.277-283
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• 2020

Objective: The sperm DNA fragmentation index (DFI) guides the clinician's choice of an appropriate assisted reproductive technology (ART) procedure. The DFI can be determined using commercially available methodologies, including sperm chromatin dispersion (SCD) kits and sperm chromatin structure assay (SCSA). Currently, when DFI is evaluated using SCD kits, the result is analyzed in reference to the SCSA-derived threshold for the choice of an ART procedure. In this study, we compared DFI values obtained using SCSA with those obtained using SCD and determined whether the difference affects the choice of ART procedure. Methods: We compared SCSA to two SCD kits, CANfrag (n=36) and Halosperm (n=31), to assess the DFI values obtained, the correlations between tests, the technical repeatability, and the impact of DFI on the choice of ART. Results: We obtained higher median DFI values using SCD kits than when using SCSA, and this difference was significant for the CANfrag kit (p<0.001). The SCD kits had significantly higher coefficients of variation than SCSA (p<0.001). In vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) would be chosen for a significantly higher proportion of patients if a decision were made based on DFI derived from SCD rather than DFI determined using SCSA (p=0.003). Conclusion: Our results indicate that SCD kit-specific thresholds should be established in order to avoid the unnecessary use of IVF/ICSI based on sperm DNA damage for the management of infertility. Appropriate measures should be taken to mitigate the increased variability inherent to the methods used in these tests.