Chang-Woon Kim

Dual effects of fluoxetine on mouse early embryonic development

Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50μM) for different durations. When late 2-cells were incubated with 5μM fluoxetine for 6h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetine (5μM) over 24h showed a reduction in blastocyst formation. The addition of fluoxetine (5μM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K(+) channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ~30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating.

K(+) efflux through two-pore domain K(+) channels is required for mouse embryonic development.

Numerous studies have suggested that K(+) channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K(+) channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K(+) channel blockers to identify the functional role of K(+) channels in mouse embryonic development. Voltage-dependent K(+) channel blockers, such as tetraethylammonium and BaCl(2), had no effect on embryonic development to the blastocyst stage, whereas K(2P) channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K(2P) channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ~38% compared with scrambled siRNA injection (P<0.05). The blockade of K(2P) channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K(2P) channels could improve mouse embryonic development through the modulation of gating by activators.

The effects of uterine artery embolization on ovarian reserve

OBJECTIVE To evaluate the effects of UAE for symptomatic uterine fibroids on ovarian reserve based on AMH. STUDY DESIGN This was a retrospective study conducted between March 2011 and October 2014. All women underwent UAE. At baseline and at the 3-month and 12-month follow-up visits, serum anti-Müllerian hormone (AMH), follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) levels were assessed, and ovarian volume and antral follicle count (AFC) were evaluated in each patient. RESULTS There were no statistically significant differences in serum E2, LH, or FSH levels or in ovarian volume 3 or 12 months after UAE (P=0.8194, P=0.3976, P=0.4766, and P=0.6822, respectively). However, AMH and AFC were significantly different 3 and 12 months after the procedure (P=0.00, P=0.029 and P=0.00, P=0.00, respectively). AMH levels remained low after 12 months of follow-up compared to the expected AMH levels. A statistically significant recovery of serum AMH at 12 months compared to at 3 months in those <40 years of age (P=0.00), but not in those ≥40 years (P=0.837). CONCLUSIONS Ovarian reserve appears to be affected by UAE in premenopausal women. However, younger ovaries (according to biological ovarian age) exhibit a greater capacity for recovery after ovarian damage. Therefore, larger studies are needed for more conclusive results.

H2O2-induced up-regulation of CatSper3 in mouse brain

Sperm-specific nonselective cation (CatSper) channels belong to the CatSper family of genes and are expressed only in sperm and testis (Qi et al., 2007). This family of genes contains CatSper1-4 (Ren et al., 2001). In general, gene expression profiles in the brains of humans and mice share the highest similarity with those in testis (Guo et al., 2005). Therefore, to determine if CatSper genes are expressed in the mouse brain, we performed reverse transcriptasepolymerase chain reaction (RT-PCR) and Western blotting. Primers were designed across introns to reveal genomic DNA contamination. RT-PCR was done using first-strand cDNA prepared from testis and brain. RT-PCR detected all four CatSper mRNAs in both the testis and the brain (Fig. 1A), with CatSper3 being the most highly expressed. To rule out the possibility of genomic DNA contamination during the RNA isolation, reactions without reverse transcriptase (RT ) were used as negative controls. No PCR product was detected in the RT reaction (Fig. 1B). The expression of CatSper3 mRNA identified by RT-PCR was further studied at the protein level. Consistent with RTPCR data, Western blot analysis showed that CatSper3 was expressed in the brain (Fig. 1C). As shown in Figure 1D, CatSper3 was expressed in all regions tested, specifically the cortex, hippocampus, hypothalamus, and cerebellum from 8week mouse brains. These results showed that CatSper3 was expressed in the mouse brain and the testis. In HT22 cell, a mouse hippocampal neuronal cell line, H2O2induced changes in CatSper3 expression were studied. H2O2 dramatically increased CatSper3 expression in HT22 cells in a doseand time-dependent manner (Fig. 1E). The H2O2-induced increase in CatSper3 expression was offset by the addition of the antioxidant N-acetylcysteine (NAC) (Fig. 1F), suggesting that ROS could modulate CatSper3 expression. Taken together, CatSper3 expression is not restricted to the sperm or testis. These results suggest that CatSper3 could be a potential target for the modulation of ROS.

Effects of relative thickness of the duplex-treated layer on surface properties of AlSl H13 steel

Abstract A duplex surface treatment technique based on calorizing and plasma nitriding was developed to improve the wear and oxidation resistance of H13 steel at high temperatures. The effects of the relative thickness of the calorized layer to the depth of plasma nitriding on the wear and oxidation properties at temperatures up to 900 °C were investigated in this work. High-temperature wear tests were performed at 500 °C with dry conditions in open air using a ball-on-disk type tribotest machine. Isothermal oxidation tests were performed at 900 °C for up to 100 h under controlled atmosphere. The results indicated that the specimens with a calorized layer as an intermediate phase between the surface duplex layer and the base metal showed higher wear and oxidation resistance than the specimens with a nitrided layer alone. During exposure to elevated temperatures, the aluminum in the calorized layer diffused to the surface and formed an aluminum oxide layer. This oxide layer protected the specimens from further oxidation and prevented the nitrogen from diffusing out of the surface, thus resulting in the retention of surface hardness even after exposure to high temperatures.

Uterine artery embolization using progressively larger calibrated gelatin sponge particles

Abstract Purpose To assess the effectiveness and safety of uterine artery embolization (UAE) using progressively larger calibrated gelatin sponge particles for symptomatic uterine fibroids. Material and methods Thirty patients with symptomatic uterine fibroids underwent UAE. Calibrated gelatin sponge particles were used in all patients, beginning with 355-500 μm particles, progressively increasing to 500-710 μm and finally to 710-1000 μm particles. Changes in tumor, uterine volume, and tumor infarction rate were assessed using pelvic magnetic resonance imaging (MRI). The level of complication, improvement of clinical symptoms, and Uterine Fibroid Symptom and Quality of Life (UFS-QOL) score were assessed. Results MR imaging revealed the mean largest tumor volume reduction was 56.23 ± 16.25% at three months and 72.61 ± 14.47% at 12 months after the procedure. 100% infarction of the dominant fibroids was 91.27 ± 5.02% at three months and 96 ± 5.20% at 12 months after the procedure. Menorrhagia improved markedly in all 23 patients. Bulk-related symptoms improved in 12 (92.30%) of 13 patients. The baseline UFS-QOL score was 43.13 and improved to 11.88 (p < 0.001). No major complications were observed. Conclusion UAE using progressively larger calibrated gelatin sponge particles is an effective and safe treatment for symptomatic uterine fibroids.

Identification of Differentially Expressed Genes in Bovine Follicular Cystic Ovaries

Follicular cystic ovary (FCO) is one of the most frequently diagnosed ovarian diseases and is a major cause of reproductive failure in mammalian species. However, the mechanism by which FCO is induced remains unclear. Genetic alterations which affect the functioning of many kinds of cells and/or tissues could be present in cystic ovaries. In this study, we performed a comparison analysis of gene expression in order to identify new molecules useful in discrimination of bovine FCO with follicular cystic follicles (FCFs). Normal follicles and FCFs were classified based on their sizes (5 to 10 mm and ≥25 mm). These follicles had granulosa cell layer and theca interna and the hormone 17β-estradiol (E(2))/ progesterone (P(4)) ratio in follicles was greater than one. Perifollicular regions including follicles were used for the preparation of RNA or protein. Differentially expressed genes (DEG) that showed greater than a 2-fold change in expression were screened by the annealing control primer (ACP)-based PCR method using GeneFishing™ DEG kits in bovine normal follicles and FCFs. We identified two DEGs in the FCFs: ribosomal protein L15 (RPL15) and microtubule-associated protein 1B (MAP1B) based on BLAST searches of the NCBI GenBank. Consistent with the ACP analysis, semi-quantitative PCR data and Western blot analyses revealed an up-regulation of RPL15 and a down-regulation of MAP1B in FCFs. These results suggest that RPL15 and MAP1B may be involved in the regulation of pathological processes in bovine FCOs and may help to establish a bovine gene data-base for the discrimination of FCOs from normal ovaries.

A rare case of squamous cell carcinoma in situ arising in mature cystic teratoma

Mature cystic teratoma (MCT) is the most common ovarian tumor. Secondary malignant tumors rarely arise in MCTs, and squamous cell carcinoma (SCC) is the most common form of such tumors. MCT-derived SCC in situ (CIS) is mostly found together with invasive SCC; it is seldom detected alone. A 44-year-old woman with breast cancer was found to have a left ovarian cyst (size >8 cm) before treatment. She underwent bilateral salpingo-oophorectomy, and frozen biopsy showed MCT with focal proliferation of squamous epithelium and mild atypism. However, definitive pathologic diagnosis confirmed CIS arising in MCT. In addition, germline BRCA 1/2 test and human papillomavirus test of tumor tissue yielded negative results. This report is the first case of its kind in Korea. Our report can aid in clinical decision making and serve as a basis for follow-up studies on this rare type of CIS arising in MCT.

Effects of three-area laser-assisted zona thinning in 8-cell human embryos on pregnancy outcomes in vitro fertilization

Objective This study conducted a preliminary examination of the effects of three-area laser-assisted zona thinning (LAZT) during the cleavage stage of embryo development on the hatching process in human in vitro fertilization-embryo transfer (IVF-ET) with subjects of advanced female age or frozen-thawed (FT) embryos. Methods Eight-cell stage embryos were treated with LAZT in three areas of the zona pellucida at 120° intervals. The control group was embryos without LAZT. Of the 72 consecutive fresh cycles and the 28 FT embryo transfer cycles, the patients in 55 fresh cycles and 17 FT cycles declined LAZT, and those cycles were defined as the control group. Results In the fresh cycles, the pregnancy rates were similar in the LAZT and control groups. However, in the FT cycles, the pregnancy rate was significantly higher in the LAZT group than in the control group (45.5% in the LAZT group vs. 23.5% in the control group, p<0.05). Conclusion These results show that multi-area LAZT resulted in significantly improved pregnancy outcomes in human 8-cell embryos compared to controls.

Differential expression of visfatin, leptin, stromal cell derived factor-1α, endothelial nitric oxide synthase, and vascular endothelial growth factor in human leiomyomas

Abstract Aim: This study was aimed to understand expressions of the visfatin, leptin, stromal cell derived factor (SDF)-1α, endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor (VEGF) in human uterine leiomyomas (UL) and normal myometrium. Method: This study investigated expression of visfatin, leptin, SDF-1α, eNOS and VEGF in 23 uterine leiomyoma patients and 10 normal myometrium by RT-PCR and western blot. Messenger RNA transcripts of SDF-1α, eNOS, VEGF and hypoxia inducible factor-1α (HIF-1α) were analyzed according to the size of UL by real-time PCR. Results: There were no significant differences in expressions of visfatin and leptin between UL compared with normal myometrium. However, expressions of eNOS, SDF-1α and VEGF were significantly higher in both intramural and subserosal UL compared with normal myometrium. The expression of SDF1-α was significantly increased in small UL (<5 cm) compared to the large UL (≥5 cm), whereas the expressions of eNOS, VEGF and HIF-1α were higher in large UL than small UL. Conclusions: This study shows that expression of SDF-1α, eNOS and VEGF were significantly higher in UL than myometrium with a different expression pattern according to the size of UL. However, expressions of visfatin and leptin had no significant differences between the two groups.