• Korean journal of applied entomology
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• v.55 no.2
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• pp.177-188
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• 2016

The alpha-proteobacterium Wolbachia is one of the most important intracellular symbionts of arthropods. This Gram-negative bacterium is involved in many biological processes and is currently considered as a potential tool for biological control. Wolbachia is a cytoplasmic bacterium, maternally transferred through generations, and to facilitate its success, it has evolved several strategies that manipulate its host reproductive system to increase the number of infected individuals in the host population. The variety of Wolbachia was first recognized using genes wsp, 16S rRNA, ftsZ, gltA and groEL as molecular markers while strain genotypes of Wolbachia are determined of Multilocus sequence typing (MLST) and sequence of amino acid in region, hyper variable regions (HVRs) in protein WSP. Possible uses of the bacteria and their predominant phenotypes in control programs for agricultural pests and human disease vectors have been considered. Phenotypes are known to induce cytoplasmic incompatibility (CI), parthenogenesis induction (PI), feminization (F) and male killing (MK). Finally, applications of the bacterium in control programs of agricultural and medical insect pests have been discussed.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.2
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• pp.63-72
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• 2017

Objective: Hyperstimulation methods are broadly used for in vitro fertilization (IVF) in patients with infertility; however, the side effects associated with these therapies, such as ovarian hyperstimulation syndrome (OHSS), have not been well studied. N-glycoproteomes are subproteomes used for the remote sensing of ovarian stimulation in follicular growth. Glycoproteomic variation in human follicular fluid (hFF) has not been evaluated. In this study, we aimed to identify and quantify the glycoproteomes and N-glycoproteins (N-GPs) in natural and stimulated hFF using label-free nano-liquid chromatography/electrospray ionization-quad time-of-flight mass spectrometry. Methods: For profiling of the total proteome and glycoproteome, pooled protein samples from natural and stimulated hFF samples were selectively isolated using hydrazide chemistry to obtain the total proteomes and glycoproteomes. N-GPs were validated by the consensus sequence N-X-S/T (92.2% specificity for the N-glycomotif at p<0.05). All data were compared between natural versus hyperstimulated hFF samples. Results: We detected 41 and 44 N-GPs in the natural and stimulated hFF samples, respectively. Importantly, we identified 11 N-GPs with greater than two-fold upregulation in stimulated hFF samples compared to natural hFF samples. We also validated the novel N-GPs thyroxine-binding globulin, vitamin D-binding protein, and complement proteins C3 and C9. Conclusion: We identified and classified N-GPs in hFF to improve our understanding of follicular physiology in patients requiring assisted reproduction. Our results provided important insights into the prevention of hyperstimulation side effects, such as OHSS.

• Journal of Pharmacopuncture
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• v.19 no.4
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• pp.319-328
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• 2016

Objectives: Advancements in nanotechnology have led to nanoparticle (NP) use in various fields of medicine. Although the potential of NPs is promising, the lack of documented evidence on the toxicological effects of NPs is concerning. A few studies have documented that homeopathy uses NPs. Unfortunately, very few sound scientific studies have explored the toxic effects of homeopathic drugs. Citing this lack of high-quality scientific evidence, regulatory agencies have been reluctant to endorse homeopathic treatment as an alternative or adjunct treatment. This study aimed to enhance our insight into the impact of commercially-available homeopathic drugs, to study the presence of NPs in those drugs and any deleterious effects they might have, and to determine the distribution pattern of NPs in zebrafish embryos (Danio rerio). Methods: Homeopathic dilutions were studied using high-resolution transmission electron microscopy with selected area electron diffraction (SAED). For the toxicity assessment on Zebrafish, embryos were exposed to a test solution from 4 - 6 hours post-fertilization, and embryos/larvae were assessed up to 5 days post-fertilization (dpf ) for viability and morphology. Toxicity was recorded in terms of mortality, hatching delay, phenotypic defects and metal accumulation. Around 5 dpf was found to be the optimum developmental stage for evaluation. Results: The present study aimed to conclusively prove the presence of NPs in all high dilutions of homeopathic drugs. Embryonic zebrafish were exposed to three homeopathic drugs with two potencies (30CH, 200CH) during early embryogenesis. The resulting morphological and cellular responses were observed. Exposure to these potencies produced no visibly significant malformations, pericardial edema, and mortality and no necrotic and apoptotic cellular death. Conclusion: Our findings clearly demonstrate that no toxic effects were observed for these three homeopathic drugs at the potencies and exposure times used in this study. The embryonic zebrafish model is recommended as a well-established method for rapidly assessing the toxicity of homeopathic drugs.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.4
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• pp.323-335
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• 2002

Object: The present study was accomplished to obtain a gene expression profile of the luminal epithelium during embryo apposition in comparison of implantation (1M) and interimplantation (INTER) sites. Material and Method: The mouse uterine luminal epithelium from IM and INTER sites were sampled on day 4.5 (Day of vaginal plug = day 0.5) by Laser Captured Microdissection (LCM). RNA was extracted from LCM captured epithelium, amplified, labeled and hybridized to microarrays. Results from microarray hybridization were analyzed by Significance Analysis of Microarrays (SAM) method. Differential expression of some genes was confirmed by LCM followed by RT-PCR. Results: Comparison of IM and INTER sites by SAM identified 73 genes most highly ranked at IM, while 13 genes at the INTER sites, within the estimated false discovery rate (FDR) of 0.163. Among 73 genes at IM, 20 were EST/unknown function, and the remain 53 were categorized to the structural, cell cycle, gene/protein expression, immune reaction, invasion, metabolism, oxidative stress, and signal transduction. Of the 24 structural genes, 14 were related especially to extracellular matrix and tissue remodeling. Meanwhile, among 13 genes up-regulated at INTER, 8 genes were EST/unknown function, and the rest 5 were related to metabolism, signal transduction, and gene/protein expression. Among these 58 (53+5) genes with known functions, 13 genes (22.4%) were related with $Ca^{2+}$ for their function. Conclusions: Results of the present study suggest that 1) active tissue remodeling is occurring at the IM sites during embryo apposition, 2) the INTER sites are relatively quiescent than IM sites, and 3) the $Ca^{2+}$ may be a crucial for apposition. Search for human homologue of those genes expressed in the mouse luminal epithelium during apposition will help to understand the implantation process and/or implantation failure in humans.

• Korean Journal of Veterinary Research
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• v.44 no.4
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• pp.523-532
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• 2004

The present study was carried out to investigate the potential adverse effects of amitraz on pregnant dams after maternal exposure during the gestational days (GD) 1 through 19 in Sprague-Dawley rats. The test chemical was administered orally to pregnant rats at dose levels of 0, 3, 10, or 30 mg/kg/ day. During the test period, clinical signs, mortality, body weights, food consumption, serum biochemistry, gross findings, organ weights and reproductive findings on GD 20 were examined. In the 30 mg/kg group, an increase in the incidence of abnormal clinical signs and death, a suppression in the body weight gain, and a decrease in the food consumption were observed. A decrease in the liver weight and increases in the kidneys, adrenal glands and heart weights were also found. Serum biochemical investigations revealed increases in the aspartate aminotransferase (AST), total bilirubin, and chloride. In addition, an increase in the fetal death and decreases in the litter size and fetal body weight were seen at caesarean section. Inthe 10 mg/kg group, an increase in the incidence of abnormal clinical signs, decreases in the food consumption and liver weight, increases in the total bilirubin and chloride, and a decrease in the fetal body weight were observed. There were no adverse effects on clinical signs, mortality, body weights, food consumption, serum biochemistry, gross findings, organ weights and reproductive findings in the 3 mg/kg group. Based on the results, it was concluded that the 19-day repeated oral dose of amitraz to pregnant rats caused increases in the clinical signs, kidneys, adrenal glands and heart weights, AST, total bilirubin and chloride and decreases in the body weight gain, food consumption and liver weight at the dose levels of above 10 mg/kg/day. Under the present experimental conditions, the no-observed-adverse-effect level (NOAEL) of amitraz was considered to be 3 mg/kg/day.

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.135-139
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• 1998

Reciprocal interspecific hybrids between N. glauca(2n=24) and N. langsdorffii(2n=18) were obtained by intercrossing. One hundred percent of F$_1$ seeds was produced from intercrossing of N. glauca $\times$ N. langsdorffii, whereas the frequency of F$_1$ hybrid seed formation from N. langsdorffii $\times$ N. glauca was very low. However, all the hybrid seeds were germinated well and then grown to normal plantlets. All the plants of F$_1$ hybrids have chromosome number of interspecific hybrids (2n=21). From observation of morphological characteristic, the structure of petrol, leaf, flower, and the morphology of pollen have characteristics of F1 hybrid. Spontaneous tumors (genetic tumor) were formed from each F$_1$ hybrid; the genetic tumor arose at the reproductive phase when the maternal type of F$_1$ hybrid came from N. glauca, while the genetic tumor arose only after reproductive phase when the maternal type of F$_1$ hybrid came from N. langsdorffii. The genetic tumor actively proliferated on hormone-free medium and produced numerous teratoma shoots. In addition, normal leaf or stem explants of F$_1$ hybrid produced calli on hormone-free medium after 15 days of culture, the calli produced new numerous teratoma shoots after 30 days. The frequency of teratoma shoot formation from rnterspecific hybrid was higher in the N. glauca $\times$ N. langsdorffii than in the N. langsdorffii $\times$ N. glauca. Root development from the teratoma shoots was hardly obtained. Teratoma shoots without roots in vitro can form genetic tumor at the vegetative growth phase after tissue culture.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.3
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• pp.207-216
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• 2005

Objective: To understand the crucial requirement for the normal early folliculogenesis, we evaluated molecular as well as physiological differences during in vitro ovarian culture. Among the important regulators for follicle development, anti-Müllerian hormone (AMH) and FSH Receptor (FSHR) have been known to be expressed in the cuboidal granulosa cells. Meanwhile, it is known that c-kit is germ cell-specific and GDF-9 is also oocyte-specific regulator. To evaluate the functional requirement for the competence of normal follicular development, we investigated the differential mRNA expression of several factors secreted from granulosa cells and oocytes between in vivo and in vitro developed ovaries. Materials and Methods: Ovaries from ICR neonates (the day of birth) were cultured for 4 days (for primordial to primary transition) or 8 days (for secondary follicle formation) in ${\alpha}$-MEM glutamax supplemented with 3 mg/ml BSA without serum or growth factors. The mRNA levels of the several factors were investigated by quantitative real-time PCR analysis. Freshly isolated 0-, 4-, and 8-day-old ovaries were used as control. Results: The mRNA of AMH and FSHR as granulosa cell factors was highly increased according to the ovarian development in both of 4- and 8-day-old control. However, the mRNA expression was not induced in both of 4- and 8-day in vitro cultured ovaries. The mRNA expression of GDF-9 known to regulate follicle growth as an oocyte factor was different between in vivo and in vitro developed ovaries. In addition, the transcript of GDF-9 was expressed in the primordial follicles of mouse ovaries. The mRNA expression of c-kit was not significantly different during the early folliculogenesis in vitro. Conclusion: This is the first report regarding endogenous AMH and FSHR expression during the early folliculogenesis in vitro. In conclusion, it will be very valuable to evaluate cuboidal granulosa cell factors as functional marker(s) for normal early folliculogenesis in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.3
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• pp.132-140
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• 2017

Objective: Correlations between semen parameters and sperm DNA fragmentation index (DFI) were investigated to identify characteristics of sperm without DNA damage that could be used in selecting sperm for intracytoplasmic sperm injection (ICSI). Pregnancy outcomes were compared to determine whether in vitro fertilization (IVF) or ICSI is a better choice for patients who have sperm with a high-DFI. Methods: Semen analysis was carried out in 388 patients who visited our IVF center for the first time to investigate correlations between sperm DFI and semen parameters. In addition, 1,102 IVF cycles in 867 patients were carried out in the present study; 921 cycles in the low-DFI group (DFI < 30%) and 181 cycles in the high-DFI group ($DFI{\geq}30%$). Both the low- and high-DFI groups were subdivided into IVF and ICSI cycle groups. Results: Sperm DFI showed significant inverse correlations with sperm motility (r = -0.435, p< 0.001) and morphology (r = -0.153, p< 0.05). Sperm DFI also showed significant correlations with rapid motility (r = -0.436, p< 0.001), and the kinetic parameters of average-path velocity (r = -0.403) and linearity (r = -0.412). Although there was no significant difference in the pregnancy rates between IVF (48.6%) and ICSI (44.8%) in the low-DFI group, the pregnancy rate of ICSI cycles (44.8%, p< 0.05) was significantly higher than IVF cycles (25.0%) in the high-DFI group. No significant difference was observed in the abortion rates between the low-DFI (52 of 921, 5.6%) and high-DFI groups (7 of 181, 3.8%). Conclusion: ICSI is a better choice than IVF for improving the pregnancy outcomes of patients who have sperm with a high DFI.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.3
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• pp.122-128
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• 2018

Objective: To determine whether fragment removal on in vitro fertilization (IVF) day 2 improved the subsequent development and pregnancy outcomes of fragmented embryos compared to similar-grade embryos without fragment removal. Methods: This study was a retrospective analysis involving 191 IVF cycles in which all embryos had over 10% fragmentation (grade 3 or 4) on day 2 of the IVF-embryo transfer cycle from March 2015 to December 2017. IVF cycles were divided into the fragment removal group (n = 87) and the no fragment removal group (n = 104) as a control cohort. Before fragment removal, embryos with fragmentation on day 2 were incubated in $Ca^{2+}$- and $Mg^{2+}$-free biopsy medium under paraffin oil for 30 minutes. Microsurgical fragment removal was performed with later-assisted hatching and a handmade suction micropipette that had an outer diameter of $30{\mu}m$. Results: There were no significant differences in the characteristics of the patients between the control and the fragment removal groups. After fragment removal and subsequent in vitro culture for 24 hours, the number of blastomeres ($7.1{\pm}1.7$ vs. $6.9{\pm}1.6$) was comparable between the transferred embryos in the two groups, but the morphological grade of the embryos in the fragment removal group ($1.9{\pm}0.7$) was significantly higher than that of the control group ($3.1{\pm}0.5$, p< 0.01). The clinical pregnancy (43.7%) and implantation rates (25.8%) in the fragment removal group were significantly higher than those in the control group (28.8% and 14.0%, respectively; p< 0.05). Conclusion: Early fragment removal on day 2 significantly improved the subsequent development and pregnancy outcomes of fragmented embryos.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.33-45
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bel-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MIT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG ($2{\mu}M$) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.