• Clinical and Experimental Reproductive Medicine
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• v.45 no.1
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• pp.1-9
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• 2018

Objective: To determine the localization, expression, and function of Toll-like receptors (TLRs) in fallopian tube epithelial cells. Methods: The localization of TLRs in fallopian tube epithelial cells was investigated by immunostaining. Surprisingly, the intensity of staining was not equal in the secretory and ciliated cells. After primary cell culture of fallopian tube epithelial cells, ring cloning was used to isolate colonies of ciliated epithelial cells, distinct from non-ciliated epithelial cells. The expression of TLRs 1-10 was examined by quantitative real-time polymerase chain reaction, and protein localization was confirmed by immunostaining. The function of the TLRs was determined by interleukin (IL)-6 and IL-8 production in response to TLR2, TLR3, TLR5, TLR7, and TLR9 ligands. Results: Fallopian tube epithelial cells expressed TLRs 1-10 in a cell-type-specific manner. Exposing fallopian tube epithelial cells to TLR2, TLR3, TLR5, TLR7, and TLR9 agonists induced the secretion of proinflammatory cytokines such as IL-6 and IL-8. Conclusion: Our findings suggest that TLR expression in the fallopian tubes is cell-type-specific. According to our results, ciliated cells may play more effective role than non-ciliated cells in the innate immune defense of the fallopian tubes, and in interactions with gametes and embryos.

• Environmental Analysis Health and Toxicology
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• v.24 no.1
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• pp.25-32
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• 2009

Toxicity of ocean dumping wastes(dye waste, urban sewage, food waste) were examined by observing fertilization and embryo development rates of the Sea Urchin, Strongylocentrotus nudus. Spawning was induced by injecting 1 mL of 0.5 M KCl into coelomic cavity. Males released white or cream-colored sperms and females released yellow or orange-colored eggs. Experiments were began within 30 min after the collection of both gametes. The fertilization and embryo development rates tests were performed for 10 min and 48 h after fertilization, respectively. The fertilization and embryo development rates in the control condition(not including ocean dumping wastes sludge elutriate) were greater than 90%, but markedly decreased with increasing concentrations of ocean dumping waste sludge elutriate. The fertilization and normal embryogenesis rates were significantly inhibited in all waste sludge elutriate from dye waste($EC_{50}$=5.76; $EC_{50}$=4.53), urban sewage($EC_{50}$=9.82; $EC_{50}$=9.67) and food waste($EC_{50}$=3.90; $EC_{50}$=3.27), respectively. The NOEC(>3.13%) and LOEC(3.13%) of fertiliztion and normal embryogenesis rates very similar in all waste sludge elutriate. These results suggest biological assay using the fertilization and embryo development rates of S. nudus are very useful test method for the ecological toxicity assessment of ocean dumping wastes.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.65-72
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• 2020

Fish ovarian germline stem cells (OGSCs) that have the abilities to self-renew and differentiate into functional gametes can be used in various researches and applications. A main issue to be solved for effective utilization of fish OGSCs is the development of their stable in vitro culture condition, but only few researches about fish OGSC culture have been reported so far. In this study, in order to find the clues to develop the culture condition for OGSCs from Japanese medaka (Oryzias latipes), we tried to establish somatic cell lines as a candidate for the feeder cells and evaluated its supporting effects on the culture of ovarian cell populations from O. latipes. As the results, the somatic cell lines could be established only from the embryonic tissues among three tissues derived from embryos, fins and ovaries. Three embryonic cell lines were tested as a feeder cell for the culture of ovarian cell population and all three cell lines induced cell aggregation formation of the cultured ovarian cells whereas the feeder-free condition did not. Furthermore, a significant cellular proliferation was observed in the ovarian cells cultured on two of three cell lines. As a trial to increase the capacity of the cell lines as a feeder cell that supports the proliferation of the cultured ovarian cells, we subsequently established a stable line that expresses the foreign O. latipes fibroblast growth factor 2 (FGF2) from an embryonic cell line and evaluated its effectiveness as a feeder cell. The ovarian cells cultured on FGF2 expressing feeder cells still formed cell aggregates but did not show a significant increase in cellular proliferation compared to those cultured on non-transformed feeder cells. The results from this study will provide the fundamental information for in vitro culture of medaka OGSCs.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.1
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• pp.22-29
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• 2019

Objective: As paternal age increases, the quality of sperm decreases due to increased DNA fragmentation and aneuploidy. Higher levels of structural chromosomal aberrations in the gametes ultimately decrease both the morphologic quality of embryos and the pregnancy rate. In this study, we investigated whether paternal age affected the euploidy rate. Methods: This study was performed using the medical records of patients who underwent in vitro fertilization (IVF) procedures with preimplantation genetic screening (PGS) from January 2016 to August 2017 at a single center. Based on their morphological grade, embryos were categorized as good- or poor-quality blastocysts. The effects of paternal age were elucidated by adjusting for maternal age. Results: Among the 571 total blastocysts, 219 euploid blastocysts were analyzed by PGS (38.4%). When the study population was divided into four groups according to both maternal and paternal age, significant differences were only noted between groups that differed by maternal age (group 1 vs. 3, p= 0.031; group 2 vs. 4, p= 0.027). Further analysis revealed no significant differences in the euploidy rate among the groups according to the morphological grade of the embryos. Conclusion: Paternal age did not have a significant impact on euploidy rates when PGS was performed. An additional study with a larger sample size is needed to clarify the effects of advanced paternal age on IVF outcomes.

• Development and Reproduction
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• v.24 no.4
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• pp.249-261
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• 2020

Spermatogonial stem cells (SSCs) have stemness characteristics, including germ cell-specific imprints that allow them to form gametes. Spermatogenesis involves changes in gene expression such as a transition from expression of somatic to germ cell-specific genes, global repression of gene expression, meiotic sex chromosome inactivation, highly condensed packing of the nucleus with protamines, and morphogenesis. These step-by-step processes finally generate spermatozoa that are fertilization competent. Dynamic epigenetic modifications also confer totipotency to germ cells after fertilization. Primordial germ cells (PGCs) in embryos do not enter meiosis, remain in the proliferative stage, and are referred to as gonocytes, before entering quiescence. Gonocytes develop into SSCs at about 6 days after birth in rodents. Although chromatin structural modification by Polycomb is essential for gene silencing in mammals, and epigenetic changes are critical in spermatogenesis, a comprehensive understanding of transcriptional regulation is lacking. Recently, we evaluated the expression profiles of Yin Yang 1 (YY1) and CP2c in the gonads of E14.5 and 12-week-old mice. YY1 localizes at the nucleus and/or cytoplasm at specific stages of spermatogenesis, possibly by interaction with CP2c and YY1-interacting transcription factor. In the present article, we discuss the possible roles of YY1 and CP2c in spermatogenesis and stemness based on our results and a review of the relevant literature.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.27 no.3
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• pp.243-246
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• 1982

A proposal of gene transfer scheme from diploid to hexaploid in oats was described. The main idea of this scheme are (1) use Avena magna which has two genomes partially in common with two genomes of the hexaploid Avena sativa or a common genome and the rest genomes partially commonn, and which lead to more regular pairing between them rather than AABB genome type to get 6x-amphiploid as a bridge between ploidy level. Cross between Avena strigosa and Avena magna is compatible and further give 42% seed set, (2) extract tetraploid derivatives which have in corporated desired genes from Avena strigasa to Avena magna, (3) Synthetic petaploid provide 2n=21 chromosome number in female gametes, which lead to complete pairing or nearly so in progenies with Avena sativa, (4) eventually homozygous lines will be produced by selfing the heterozygous (regarding to$A^{As}$ genome) at final step.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.110-110
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• 2003

The high incidence of polyspermic fertilization is one of the major causes lowering the overall efficiency of porcine IVF. The common procedure for IVF involves the co-culture of both gametes in the medium drop, which increases sperm concentration and incidence of polyspermy. Therefore, the present study was carried out to increase the efficiency of porcine IVF by reducing polyspermy using a modified swim-up method. This method modifies conventional swim-up washing by placing oocytes directly at the time of washing. Sperm pellet was prepared in the tube and mature oocytes were placed on cell strainer with $70 \mu m$ pore size (Falcon 2350) at the top of the tube. After insemination, the oocytes were stained for examination. Also, the developmental potential of fertilized embryos was measured to evaluate for the feasibility of this method. While having similar penetration rates in both methods ($86.67 \pm 2.36% to 83.33 \pm 1.36%$), there was a significant reduction of polyspermy in modified swim-up method ($17.50 \pm 1.60%$) compare to the control ($44.1 \pm 3.70%$ (p<0.05). Subsequent culture showed higher rate of blastocyst formation in modified swim-up method (20.44$\pm$0.99%) than the control ($15.73 \pm 3.26%$) (P<0.05), even though there was no significant difference. These results suggest that, by controlling the number of spermatozoa reaching the oocytes, porcine oocytes might be protected from polyspermy in vitro. Also, the developmental potential of the fertilized embryos using this method could be improved by increasing the pool of spermatozoa with better quality. Further optimization of the procedure required to implicate this method in routine porcine IVF.

• Korean Journal of Animal Reproduction
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• v.10 no.1
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• pp.109-120
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• 1986

This study was conducted to define the effect of addition of lysolecithin (LC) and 20% v/v rabbit serum to sperm preincubation medium on the induction of acrosome reaction (AR) an fertilizing ability in vitro of LG-added sperm. Ejaculated rabbit sperm from New Zealand White buck was washed once by centrifugation, then preincubated for 2 or 4 hrs in a chemically defined medium (DM), DM plus 20% rabbit serum or BSA-free DM plus 20% rabbit serum at 37$^{\circ}C$ water bath or CO2 incubator. At the end of preincubation LC was added to the preincubated sperm, which was stained at 0.5 to 4 hr later and examined for AR and sperm motility. For in vitro fertilization, gametes were coincubated in DM up to 24 hrs and thereafter fertilized embryos were incubated in BSM -II up to 48 hrs. Addition of LC to 4-hr preincubated sperm was more effective for the AR and sperm motility than that to 2-hr preincubated sperm and optimal concentration of LC for AR was about 80${\mu}$g/ml. A significant increase in AR occured from 20 to 30 min. after addition of 80 to 100${\mu}$g/ml in 4-hr preincubated sperm. BSA-free DM plus 20% rabbit serum showed a higher AR and sperm motility than those of DM plus 20% rabbit serum in LC-added sperm after 4-hr preincubation. The incidence of AR after 4-hr preincubation and at 30 min after 60${\mu}$g/ml LC addition varied greatly among individual bucks. Sixty ${\mu}$g/ml LC-added sperm showed a slight high cleavage rate over control levels, but 100${\mu}$g/ml LC-added sperm showed lower cleavage rate rather than 60${\mu}$g/ml LC. It is concluded that optimal concentration of LC for high AR induction and sperm motility in 4-hr preincubated sperm was about 80${\mu}$g/ml, but 60${\mu}$g/ml level was more useful for in vitro fertilization.

• Journal of Life Science
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• v.27 no.1
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• pp.100-121
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• 2017

The sexual reproduction of the unicellular green alga Chlamydomonas is reviewed for a comprehensive understanding of the complex processes. The sexual life cycle of C. reinhardtii is distinguished into five main stages: gametogenesis, gamete activation, cell fusion, zygote maturation, and meiosis and germination. Gametogenesis is induced by nitrogen starvation in the environment. C. reinhardtii has two mating types: mating type plus ($mt^+$) and mating type minus ($mt^-$), controlled by a single complex mating type locus ($MT^+$ or $MT^-$) on linkage group VI. In the early gametogenesis agglutinins are synthesized. The $mt^+$ and $mt^-$ agglutinins are encoded by the autosomal genes SAG1 (Sexual AGglutination1) and SAD1 (Sexual ADhesion1), respectively. The agglutinins are responsible for the flagellar adhesion of the two mating type of gametes. The flagellar adhesion initiates a cAMP mediated signal transduction pathways and activates the flagellar tips. In response to the cAMP signal, mating structures between two flagella are activated. The $mt^+$ and $mt^-$ gamete-specific fusion proteins, Fus1 and Hap2/Gcs1, are present on the plasma membrane of the two mating structures. Contact of the two mating structures leads to develop a fertilization tubule forming a cytoplasmic bridge between the two gametes. Upon fusion of nuclei and chloroplasts of $mt^+$ and $mt^-$ cells, the zygotes become zygospores. It is notable that the young zygote shows uniparental inheritance of chloroplast DNA from the $mt^+$ parent and mitochondrial DNA from the $mt^-$ parent. Under the favorable conditions, the zygospores divide meiotically and germinate and then new haploid progenies, vegetative cells, are released.

• Korean Journal of Environmental Biology
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• v.38 no.2
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• pp.207-215
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• 2020

Toxic assessment of antifouling agents (diuron and irgarol) was conducted using the fertilization and the normal embryogenesis rates of the sea urchin, Mesocentrotus nudus. Bioassessment began with male and female reproductive cell induction. White or cream-colored male gametes(sperm) and yellow or orange-colored female gametes (eggs) were acquired and fully washed, separately. Then, the fertilization and normal embryogenesis rates were measured after 10 min and 48 h of exposure to the toxicants, respectively. The fertilization and embryo development rates were greater than 90% in the control, validating the suitability of both endpoints. The normal embryogenesis rates were significantly decreased with increasing concentrations of diuron and irgarol, but no changes in the fertilization rates were observed in concentrations ranging from 0 to 40 mg L^-1. The EC₅₀ values of diuron and irgarol for the normal embryogenesis rates were 20.07 mg L^-1 and 22.45 mg L^-1, respectively. The no observed effect concentrations (NOEC) were <1.25 mg L^-1 and the lowest observed effect concentrations (LOEC) were 1.25 mg L^-1 and 2.5 mg L^-1, respectively. From these results, concentrations of diuron and irgarol over 1.25 mg L^-1 and 2.5 mg L^-1, respectively, can be considered to have toxic effects on invertebrates, including M. nudus. The ecotoxicological bioassay in this study using the noted fertilization and normal embryogenesis rates of M. nudus can be used as baseline data for the continued establishment of environmental quality standards for the effects of antifouling agents(especially diuron and irgarol) in a marine environment.