The conserved THO/TREX complex is participated in pre-mRNA processing and mRNA nuclear export. In Metazoa, it has been reported that THO/TREX is loaded on nascent RNA transcribed by RNA polymerase II in a splicing-dependent manner; however, how THO/TREX functions is poorly understood. To understand the role of THO/TREX in eukaryotic gene expression, we investigated the role of THO/TREX in Drosophila germline, and found that lack of THOC5, a component of THO/TREX, showed defects in the biogenesis of piRNA, a distinct class of small non-coding RNAs that control expression of transposable elements (TE) in the Drosophila germline. Genome wide RNA-seq showed that THOC5 and other TREX components are essential for the biogenesis of piRNA. THO/TREX components are enriched on piRNA precursors transcribed from dual-strand piRNA clusters and co-localize in distinct nuclear foci that overlap with sites of piRNA transcription. The localization of TREX in nuclear foci and its loading on piRNA precursor transcripts depends on Cutoff, a protein associated with chromatin of piRNA clusters. We also show that TREX is required for accumulation of nascent piRNA precursors, suggesting that TREX is required for their efficient transcription. In addition to the biogenesis of piRNA, both THO and piRNA mutants showed over-proliferation of germline stem cell-like cells outside of stem cell niche. Taken together our study reveals a novel splicing-independent mechanism for THO/TREX loading on nascent RNA and its importance in piRNA biogenesis as well as a role of piRNA in the differentiation of germline stem cell.

Spinal cord is a posterior part of central nervous system, developmentally produced by the folding of the neural plate via an embryonic process called neurulation. Defects in neurulation is one of the most common birth defects in human, raising the importance to develop in vitro model recapitulating human neurulation. The advent of organoid technology, which can produce 3D structure resembling parts of organs from human embryonic stem cells/induced pluripotent stem cells (ESCs/iPSCs), has offered new tools to model human diseases. Recently, we developed organoid model exhibiting morphogenetic features of spinal cord development, such as neural plate formation, neural folding, and neural tube closure and profound production of spinal cord-type motor neurons. Human spinal cord organoids will be a useful tool for assessing genetic and environmental factors affecting spinal cord development, and screening ‘personalized drugs’ for spinal cord diseases such as Amyotrophic Lateral Sclerosis (ALS).

Contemporary systems for in vitro culture of ovarian follicles do not recapitulate the mechanical heterogeneity in mammalian ovary. Here we report microfluidic generation of biomimetic ovarian microtissue for miniaturized three-dimensional (3D) culture of early secondary preantral follicles by using alginate (harder) and collagen (softer) to fabricate the ovarian cortical and medullary tissues, respectively. This biomimetic configuration greatly facilitates follicle development to antral stage. Moreover, it enables in vitro ovulation of cumulus-oocyte complex (COC) from the antral follicles in the absence of luteinizing hormone (LH) and epidermal growth factor (EGF) that are well accepted to be responsible for ovulation in contemporary literature. These data reveal the crucial role of mechanical heterogeneity in the mammalian ovary in regulating follicle development and ovulation. The biomimetic ovarian microtissue and the microfluidic technology developed in this study are valuable for improving in vitro culture of follicles to preserve fertility and for understanding the mechanism of follicle development and ovulation to facilitate the search of cures to infertility due to ovarian disorders.

Peroxiredoxin1 (Prdx1) is an antioxidant enzyme belonging to the peroxiredoxin family of proteins. Prdx1 catalyzes the reduction of H2O2 and alkyl hydroperoxide and plays an important role in different biological processes. Prdx1 also participates in various age-related diseases and cancers. In this study, we investigated the role of Prdx1 in pronephros development during embryogenesis. Prdx1 knockdown markedly inhibited proximal tubule formation in the pronephros and significantly increased the cellular levels of reactive oxygen species (ROS), which impaired primary cilia formation. Additionally, treatment with ROS (H2O2) severely disrupted proximal tubule formation, whereas Prdx1 overexpression reversed the ROS-mediated inhibition in proximal tubule formation. Epistatic analysis revealed that Prdx1 has a crucial role in retinoic acid and Wnt signaling pathways during pronephrogenesis. In conclusion, Prdx1 facilitates proximal tubule formation during pronephrogenesis by regulating ROS levels.

The formation of definitive endoderm (DE) is a fundamental step for the development of the gastrointestinal tract, respiratory tract and endocrine organs. We present a CRISPR-based pooled screening approach to identify genes which contribute to DE induction from hiPSCs in vitro. CRISPR-based pooled genetic screens in mammalian cell culture enable researchers to identify genes required for a cellular phenotype of interest in an unbiased way. To enable a CRISPR-based forward genetic screen for identifying regulatory genes required for DE differentiation from hiPSCs, we performed pooled screens using a human genome-scale CRISPR knockout library. In addition, we performed a transcriptional activation screen using a lentiviral CRISPRa library to identify the downstream targets of the TGF/nodal/activin signaling pathway, which is a key signaling pathway for DE specification. We identified several signaling pathways including TGFβ, Erk, JNK, and CREB pathways are involved with DE differentiation. We suggest that CRISPR-based pooled genetic screens are a useful tool to identify key signaling pathways and genes required for in vitro differentiation processes and are served as a platform to improve differentiation protocols.

The initial segment (IS) in rodents is functionally and structurally distinct from other epididymal segments and plays an important role in sperm maturation. We previously showed that, in the mouse epididymis, basal cells (BCs) extend a narrow luminal-reaching projection only in the IS, while in all other regions, they mainly nestle at the base of the epithelium. We also found that BC projections are regulated by testicular luminal factors, and the present study was aimed at characterizing the signaling pathway involved in their formation and elongation. Previous studies reported that testicular luminal factors maintain the extracellular signal-regulated kinase (ERK) in a highly phosphorylated state in the IS. We report here that the BC projections, which we call axiopodia, periodically extend and retract over time. We found that axiopodia extensions and retractions follow an oscillatory pattern. This movement is controlled by MAPK/ERK signaling pathway. Our results suggest that ERK phosphorylation plays a key role in the formation and elongation of BC projections. Such unexpected cell motility may reflect a novel mechanism by which specialized epithelial cells sample the luminal environment.

Ovarian function and implantation of embryo are critical factors in pregnancy. So, their optimal conditions and tightly regulated networks are necessary in pregnancy as well as fetal development. However, there are limit approach to cure ovarian dysfunction or improve implantation rate despite of the development of associated reproductive technologies. Recently, translational studies have been explored the therapeutic effect of stem cells in reproductive medicine. Placenta derived mesenchymal stem cells (PD-MSCs) have been reported as alternative cell source capable of overcoming the limitation of bone marrow derived MSCs (BM-MSCs) and adipose-derived MSCs (AD-MSCs), which have stemness dependent by donor age as well as invasive procedure. In addition, their activities for self-renewal and immunomodulation were higher than those of others. In this section, we will review the stem cell therapy in reproductive medicine and introduce feasibilities of PD-MSCs on a rat model with ovarian failure as well as on trophoblast invasion activity. Finally, we introduce new insights into further understanding of stem cell-based therapeutic mechanisms for reproductive system as well as new avenues to develop more efficient therapies in degenerative medicine.

In this talk, I will describe recent advances in the field of metazoan comparative genomics and regeneration, the insights into the marine animal genomic and annelid regeneration. The scientific questions arising from the ability of certain species, but not others, to massively regenerate their bodies are among the most fascinating and challenging confronting modern cell and developmental biologists today. The tremendous implications of this research area for human medicine and tissue engineering are obvious. Yet many other animals exhibit robust regenerative capabilities, including "lower" vertebrates such as amphibians, and invertebrates such as echinoderms, flatworms and annelids. In the extreme case, some species can reproduce vegetative indefinitely. Such animals must contain the operational equivalent of immortal, totipotent somatic stem cells. From invertebrates to the higher vertebrates, their metabolic pathway, developmental regulatory genes, and intercellular signaling pathways are evolutionary conserved. With these, study on regeneration is an ingenious, powerful model system for studying the post-embryonic development, innate immunity mechanisms, and primordial germ cells (PGCs).

This study analyzed the mitochondrial DNA (mtDNA) sequences of the Red-spotted grouper, Epinephelus akaara (Perciformes, Serranidae), and used for construction of molecular phylogeny and for association between maternal haplotypes and phenotypic differences of F1 progeny. This study revealed phylogenetic position of the endangered red-spotted grouper, Epinephelus akaara (Perciformes, Serranidae) based on the nucleotide sequences of complete mt genome. Complete nucleotide sequences were determined from the mt genomes of two individuals of the red-spotted grouper caught in South Korea. The mitochondrial genome had 16,795 base pairs (bp) and 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a noncoding control region. The two mt genomes were highly homologous (99.71% similarity). The two mt genomes of E. akaara determined in this study were found in Clade I in the phylogenetic tree with those of E. awoara, E. fasciatomaculosus, E. sexfasciatus, E. diacanthus, E. sticus, and E. morio, suggesting that this may be helpful to understand phylogenetic position of Epinephelus species including red-spotted grouper. The genetic structure and phylogenetic relationship were investigated in the red-spotted grouper populations using the sequence polymorphisms of the cytochrome c oxidase subunit I(COI) gene and variable number of tandem repeats (VNTRs) of the control region (CR). A total of forty-one COIhaplotypes were found from 174 COIsequences from East Asia. The Jeju Island population (n=5) had four haplotypes, and the South Sea population (n=105) had twenty-five haplotypes. The Hong Kong population had nineteen haplotypes from fifty-nine COIsequences determined in this study. Among the COIhaplotypes, EAC_03 is commonly found in all populations (Jeju Island and South Sea of Korea, China, Hong Kong and Taiwan). In addition, there were four haplotypes (EAC_12, EAC_14, EAC_28 and EAC_35) also common among the populations tested in this study and collected from NCBI database. However, twenty haplotypes were specific in the Korean populations, and fifteen haplotypes were specific in the China and Hong Kong populations. The neighbor-joining (NJ) trees constructed from the phylogenetic analyses based on the polymorphisms of the COIhaplotypes showed the monophyletic branching pattern within the genus Epinephelus, indicating that the red spotted grouper populations had evolved from common maternal ancestors. Consequently, East Asian red-spotted grouper populations are maternally related at least in part, as well as sharing the same evolutionary history, and still affected by the East Asian ocean current (Kuroshio). From the haplotype analysis for mtDNA CR, we obtained VNTR polymor-phisms in all populations tested. We found five haplotypes for the CR VNTR patterns. The 133-bp repeat units were counted two to five. Using CR VNTR haplotypes, the statistical association was examined between mtDNA haplotypes and growth traits of aquafarming young fishes of the red-spotted grouper. A total of 386 F1 progeny, which were randomly selected from a progeny population produced by artificial insemination in the farm, were genotyped and statistically compared their body length (BL), body weights (BW) and length-weight indexes (LWI) at 11-months after hatching. There haplotypes H03, H04 and H05 were detected for CR in the parents and progeny populations. The significant difference was found in the BL values among three haplotypes (p<0.05). The F1 animals with haplotype H03 had freater level of BL (19.22±2.000 cm) than those of H04 (18.64±1.964 cm) and H05 (18.86±1.512 cm). There were no significant differences in BW and LWI among haplotypes (p<0.05). These results concluded that the maternal lineages affected the growth rates during early developmental stage in the red-spotted grouper. These findings suggested that the mitochondrial background of the fertilized eggs may play an important role in the early development, and the markerassisted selection system for broodstork animals may be helpful in improving performance traits for aquaculture industry as well as for conservation biology of the endangered red-spotted grouper. However, the results from the association analysis between haplotypes and phenotypes of F1 progeny (n=1,093) at 60-days after hatching showed that there were no significant difference (p>0.05). Consequently, the results of this study may be useful information for understanding the evolutionary relation with other species and may be good genetic markers for breeding management in the red-spotted grouper aquaculture system.

척추동물의 생식선자극호르몬방출호르몬(gonadotropin-releasing hormone, GnRH)은 뇌하수체의 생식선자극호르몬 FSH 및 LH의 합성1과 분비를 촉진하여, 생식소의 발달과 배란․방정을 유도하는 신경펩티드호르몬이다. 1971년 Guillemin과 Schally가 처음으로 포유동물의 시상하부로 부터 10개의 아미노산으로 구성된 GnRH(LHRH)를 분리․동정하고 LH의 분비 촉진 기능을 확인하였다. 현재까지 원구류 등 원시적인 척추동물의 뇌뿐만 아니라, 무척추동물의 신경절에도 GnRH유사 펩티드가 존재한다고 알려져 있다. 최근, 해양성 연체동물과 절지동물에서 11~12개의 아미노산으로 구성된 GnRH유사 펩티드 및 그 전구체를 암호화하는 cDNA가 동정되어 있으나, 상세한 연구는 아직 부족한 실정이다. 본 연구는 북방전복 Haliotis discus hannai의 GnRH에 관한 기초 연구를 수행하기 위하여 GnRH cDNA를 클로닝하고, 유전자 발현을 조사하였다. 다른 연체동물의 GnRH(GnRH-V형) 전구체와 유사한 아미노산 서열을 코드하는 한 개의 cDNA를 신경절 조직으로부터 동정하였으며, 신경절 조직에서만 특이적으로 GnRH mRNA가 발현되는 결과를 얻었다. 또한, GnRH mRNA 발현은 암수, 성숙도 및 신경절 조직에 따른 차이점이 있었다. 향후, 척삭동물에서 알려진 4종류의 GnRH와 구별되는 무척 추동물의 GnRH 구조와 기능적인 차이점에 관한 연구가 필요할 것으로 사료된다.

The conserved The oocytes acquire competence to undergo complex processes, oocyte growth and oocyte maturation, and the capacity for fertilization and preimplantational embryo development, by accumulating RNAs and proteins in the ooplasm. Therefore, the identification of the genes expressed in the oocyte and its functional analysis will provide valuable resources to study molecular regulatory mechanism of oocyte maturation, fertilization and early embryogenesis. To better understand these mechanisms, a decade ago, we identified a list of differentially expressed genes between GV and MII oocytes using annealing control primer (ACP)-PCR technology. Among these genes, we selected two genes, Gas6 (growth arrest-specific 6), and Sebox (skin-embryobrain- oocyte homeobox) that expressed significantly higher levels in GV than MII and analyzed its functions by using RNA interference (RNAi). Unexpectedly, and fortunately, it turned out that both of genes are new candidate of maternal effect genes (MEGs) that is important for fertilization and/or early embryogenesis but not crucial for oocyte meiotic maturation. In particular, Gas6 is essential in maintaining the proper mitochondrial function, and biosynthesis of heparan sulfate and glutathione, which are required for normal sperm chromatin decondensation, pronuclear formation, and mainly for the sufficient cytoplasmic maturation of oocytes. We suggest that the correction in the Gas6 signaling network in oocytes may improve the embryonic developmental capacity caused by deterioration of the mitochondrial functions and/or contents during oocyte maturation. Meanwhile, Sebox is crucial for zygotic genome activation (ZGA) required for subsequent embryonic development beyond the 2-cell stage by coordinating the expression of other maternal factors, such as c-mos, Gdf9, Ube2a and Wee1. In conclusion, the observed failure of fertilization after Gas6 RNAi and the embryonic development at the 2-cell stage after Sebox RNAi was similar to the loss-of-function of the previously well-known MEGs. Based on these findings, we added Gas6 and Sebox as new mammalian MEGs. Findings of our research would broaden our knowledge regarding MEGs and a field of maternal programming in oocytes.

Spindle dynamics has a critical role in many physiological mechanisms such as genomic integrity and aging. It is also well known that these mechanisms affect oocyte quality. Because the oocyte quality affects fertility and embryo development, many efforts have been conducted to understand the molecular mechanisms that regulate spindle dynamics during oocyte maturation. Here, we show that translationally controlled tumor protein (TCTP) regulates spindle dynamics during oocyte maturation and prevents deterioration of oocyte quality during postovulatory aging. TCTP was expressed and localized at the cytoplasm with strong enrichment at the spindle microtubules during meiosis. Through the knockdown of TCTP, we found that TCTP regulated stability of the polar microtubules. Meanwhile, spindle dynamics were dramatically decreased with reduced TCTP level during the postovulatory oocyte aging both in vitro and in vivo. Knockdown of TCTP accelerated the reduction of spindle dynamics and aging-related quality deterioration of oocytes. Conversely, overexpression of TCTP rescued microtubule dynamics during postovulatory aging of oocytes and prevented aging-induced deterioration of quality. In addition, overexpression of TCTP improved fertilization competency and subsequent embryonic development. Therefore, our results demonstrate that TCTP is a microtubule-associating protein required to regulate spindle dynamics in mouse oocytes and protects oocytes from aging-related quality deterioration.

Adipogenesis is a primary energy valancing response in physiological status and critical in embryo development. One of the essential factors for initiation and maintaining of adipogenesis is the composition of extracellular matrix. Previously, we confirmed the effects of diphlorethohydroxycarmalol (DPHC), an extract of Ishige okamurae, on the antiobesity effects and ECM stability in adipose tissue. In vitro model for adipogenesis study, 3T3-L1, a precursor cell type of adipocyte, and the adipose-tissue derived stem cell (ADSC) can be used. Usually the induction period for adipocyte is shorter in 3T3-L1 than in ADSCs. However, so far, the difference of the expression patterns of ECM components in 3T3-L1 and ADSCs, and the effects of DPHC are not much known. We induced differentiation of 3T3-L1 and ADSCs into adipocyte with or without DPHC (0, 0.4, 2, 10, 50 μg/mL) and confirmed the adipogenesis with adipogenic markers (PPAR-γ, LDL). After then, the levels of collagen type 1 alpha 1 (Col1a1), collagen type 3 alpha 1 (Col3a1), collagen type 4 (Col4), collagen type 6 (Col6), Elastin (Eln) and microfibrillar associated protein 5 (Mfap5) were analyzed with real-time RT-PCR. During early adipogenesis of ADSC, the expression levels of Col1, Col3, Col6, and Mfap5 mRNA were decreased but Col4 and Eln mRNA were increased. In the matured adipocyte, the expression levels of Col1, Col3, Col4, Mfap5 mRNA were decreased but not Eln. In the case of early differentiation of 3T3-L1, the expression levels of Col1, Col3, Eln mRNA were decreased but the expression levels of Col6 and Mfap5 were increased. In matured adipocyte of 3T3-L1, the expression levels of Col1, Col3, Eln, Mfap5 mRNA were increase but the expression level of Col6 mRNA was decreased. The expression levels of Col4, Eln mRNA were suppressed by 50 mg/mL DPHC treatment during early adipogenic period of ADSC. On the other hand in 3T3-L1, the expression levels of Col3 and Col6 mRNA were not changed by the DPHC treatment during early induction period. In the matured adipocytes derived from ADSC, Col1 mRNA levels was not decreased by the treatment of 50 mg/mL DPHC. Col4 mRNA levels was not increased by DPHC treatment. In the case of matured adipocytes derived from 3T3-L1, DPHC suppressed the increase of Col1, Col3, Col6 mRNA expression and the expression of Col4 and Eln mRNA was decreased. In summary, these data show that expression levels of each ECM component types are dramatically changed with some common patterns in two cell types, and the treatment of DPHC can modify the expression patterns of some ECM components in each cell types. It is suggested that one of the reason of antiadipogenic effect of DPHC may be the ECM modification.

골 형성 단백질(Bone morphogenetic protein, BMP)은 TGF-β superfamily의 구성원 중에 하나이며, 이들은 원래 뼈 형성을 유도하는 능력에 의해 발견되었지만, 뼈 외에도 다른 세포 및 기관의 성장과 분화를 조절하는 것으로 밝혀졌다. 다기능적인 골 형성 단백질은 포유동물의 reproduction에도 매우 중요한 역할을 하는데, 예를 들어 BMP8A와 BMP8B는 생쥐의 정자 형성과 부고환에 일정한 기능을 하는 것으로 보고되었고 난소에서의 BMP15는 난포성장을 자극하고, 과립막세포(granulosa cell)의 증식시키는데 관여하는 단백질로 알려져 있다. 하지만 난관(oviduct)에서의 BMP 역할은 알려진 바가 적다. 그래서 본 연구에서는 골 형성 단백질이 착상 전 oviductal environment에 어떠한 영향을 미치는지 밝히고자 하였다. 8주령 생쥐의 Estrous cyclic oviduct을 가지고 RT-PCR과 Immunohistochemistry(IHC)를 통해 BMP2, 4의 mRNA와 단백질 발현을 확인하였다. Estrogen에 의한 BMP2, 4의 영향을 확인하고자 난소절제술을 시행한 생쥐와 Estrogen receptor alpha(ERα) Knockout 생쥐를 통해 mRNA와 단백질 발현을 확인하였다. Oviduct의 ciliated cell을 가지고 BMP2, 4의 기능을 밝히고자 siRNA실험을 진행 하였다. Estrous cyclic oviduct cDNA를 통해 RT-PCR한 결과, 이 중 Estrus 시기에 가장 높은 발현을 보인 BMP2, 4의 mRNA level은 Isthmus보다 Infundibulum과 Ampulla에 증가하였고, Immunohistochemistry (IHC)를 통해 BMP2와 BMP4 단백질은 난관 상피세포 중에서 ciliated cell에 발현되었다. 이를 ciliated cell marker인 β-tubulin과 함께 Immunofluorescence(IF)를 진행한 결과, 주로 β-tubulin-positive ciliated cell에서 발현됨을 확인하였다. 난소 절제술을 시행한 생쥐의 난관에서는 E2와 DPN (ERβ agonist)에 의해 BMP2, 4 mRNA 발현이 증가하고, ERαKO 난관의 경우 WT(Die)에 비해 BMP2, 4 mRNA, 단백질의 발현 모두 증가하였다. BMP2와 BMP4의 기능을 밝히고자 Ciliated cell line인 OA-6b를 가지고 siRNA 실험을 진행한 결과, Bmp2, Bmp4 siRNA 처리군에서 Ciliated cell marker인 FOXJ1의 발현이 줄어들고 Proliferation marker인 Ki67, Pcna의 발현이 낮아졌다. 이로써, Oviduct 내 BMP2와 BMP4 발현은 Estrogen과 양의 상관성이 있으며, Ciliated cell에서 Estrogen - ER β signaling을 통해 조절된다. Estrogen에 의해 유도되는 BMP2와 BMP4는 ciliated cell에서 Autocrine, Paracrine factor로 작용할 가능성이 있다. 더 나아가 Oviduct의 Infundibulum 및 Ampulla에 강하게 발현되는 BMP2, BMP4는 난자 및 배아에 autocrine, paracrine factor로 작용하여 oviductal cell proliferation을 조절하고, 수정을 위한 oviductal environment 조성에 중요한 역할을 하는 것으로 사료된다.

The greater horseshoe bat (Rhinolophus ferrumequinum) is distributed throughout Europe, Africa, Australia, and South Asia. It habits mainly in the cave in small groups and forming communities in late spring. It has interesting reproductive behavior because it keeps sperm for a few months in female reproductive tracts and then those sperms attend in fertilization. This breeding pattern is a sperm storage type and belongs to Rhinolophidae or Hipposideridae. The greater horseshoe also habits in Korea. However, the reasons of reproductive behaviors has not much uncovered. In this study the characters of ovary and the levels of steroid hormones were investigated from September to November. The histological, ELISA, and immunohistochemical methods were employed. The pre-ovulatory follicle was detected only at October sample. On the other hand, the blood level of testosterone was not detectable but the levels of 17β-estradiol and progesterone were exist within the detectable range. E2 and P4 levels were peak in October. Besides, the key enzymes for estradiol synthesis, CYP17 and CYP19 were localized in the theca layer and granulosa cells, respectively. October is known as mating time in this species. However, progesterone receptors could not detect at this period. Put together, it is suggested that, the increase of estrogen and the absence of progesterone receptors on preovulatory follicle is the cause of the mating without ovulation. The understanding of the expression regulation in this system will be base of the understanding the anovulation in mammals.

For successful embryo implantation, the stromal cells of the endometrium are morphologically and functionally differentiated into decidual cells. In the endometrium, estrogen induces proliferation of epithelial cells, but progesterone regulates the differentiation of epithelial cells, leading to decidualization of stromal cells. Kruppel like factor (KLF) is a zinc finger DNA binding protein that regulates transcription and has a wide range of functions in the cell cycle, cell apoptosis and differentiation control. In the uterus, KLF9, 13 plays an important role in implantation and decidual cell differentiation. KLF4 and KLF15 regulate the proliferation and differentiation of endometrial epithelial cells, but their role in stromal cells is unknown. In this study, we investigated the role of KLF4 and KLF15 in endometrial stromal cells. In mouse uterus, KLF4 was expressed in proliferative phase of glandular and luminal epithelial cells. However in endometrial stromal cells, KLF4 was highly expressed in secretory phase and secondary decidual zone after implantation. The expression of KLF15 was little in cytoplasm of luminal and glandular epithelial cells and proliferated in nucleus of secretory phase stromal cell.herefore, KLF4 and 15 are thought to be important for decidualization. To investigate the effect of estrogen and progesterone on the expression of KLF4 and KLF15, uterus of ovariectomy (OVX) mice which were injcected 17β- estradiol (E2, 0.3 mg) and progesterone (P4, 1 mg) and both ERα-knock out and wild type (diestrus, estrus) mice were used. KLF4 in OVX+E2 group was significantly higher than OVX+E2 / P4 group was lower than OVX+E2 group. There was no significant difference between ERαKO and WT diestrus group and significantly lower than WT estrus group. Expression of KLF15 was higher in the OVX+ P4 group than in the OVX group and lower in the OVX+E2 group. The OVX+E2 / P4 group was higher than the OVX+E2 group. There was no difference between ERαKO and WT diestrus. The differences in expression of KLF4 and KLF15 by P4 in OVX mouse uterine tissues may be due to the tissue specific expression pattern of epithelial (KLF4) and stromal cells (KLF15). The expression of KLF4 and KLF15 was increased by treatment of cyclic adenosine monophostphate (cAMP, 0.5 mM) and medroxyprogesterone acetate (MPA, 1μM) in human endometrial stromal cells. KLF15 siRNA increased the expression of decidualization markers (BMP2, IGFBP-1 and prolactin) with increasing progesterone receptor A/B (PR A/B), while KLF4 siRNA treatment decreased expression of decidualization markers. There was no significant difference in cell proliferation and apoptosis markers in KLF4 / 15 siRNA treatment. Therefore, progesterone induces KLF4 to promote decidualization, while normally induced KLF15 inhibits progesterone receptor expression. Expression of KLF4 in endometrial epithelium is induced by estrogen but induced by progesterone to promote decidualization, and KLF15 is mainly induced by progesterone in stromal cells and inhibit excessive PR A / B activity.

As alternatives of phthalate plasticizers harmful as endocrine disruptors, citrate esters have been considered for plasticizer in the production of cosmetics, PVC plastics, and pharmaceuticals. Though considered to be low toxic in mammals in vivo and in vitro toxicological information for citrate esters in aquatic lives remained poorly understood. In an effort to find alternative plasticizers we examined the developmental toxicity of tributyl O-acetylcitrate (ATBC), triethyl 2-acetylcitrate (ATEC) and trihexyl O-acetylcitrate (ATHC) together with dibutyl phthalate (DBP) as the positive control in Xenopus laevis embryos based on Frog Embryo Teratogenesis Assay Xenopus (FETAX). In X. laevis embryos LC50 and EC50 values of ATBC at 96 hours were calculated to be 12.7 ppm (13.3 mg/L) and 11.6 ppm (12.2 mg/L). The LC50 and EC50 values of ATEC at 96 hours were calculated to be 360.6 ppm (409.6 mg/L) and 364.3 ppm (413.8 mg/L), respectively. The LC50 values of ATHC at 96 hours were calculated to be 97.5 ppm (98.0 mg/L). The LC50 and EC50 values of dibutyl phthalate (DBP) at 96 hours were calculated to be 12.7 ppm (13.2 mg/L) and 7.1 ppm (7.4 mg/L), respectively. Developmental abnormality such as head malformation, gut malformation, bent trunk, ventral blister, abnormal tail and myotome were significantly increased by DBP at 8.9 ppm, and which was observed by citrate esters at much higher concentration (ATEC, 320 ppm; ATHC, > 75 ppm; ATBC, 15 ppm). In DBP treated embryos, overgrowth of nostrils was frequently observed and growth was inhibited at 6 ppm. ATEC and ATBC inhibited growth at 80 and 15 ppm, respectively. In ATHC treated embryos, the head and tail length were significantly increased at 14.8 ppm. Lipid peroxidation in tadpoles was significantly increased by DBP (10 ppm) but not by ATEC, ATBC, and ATHC. In tadpoles pro-apoptotic bad, bax and bak mRNA levels and DNA fragmentation were significantly increased by DBP (10 ppm) but not by citrate esters. Together, citrate esters could be considered as substitution for phthalate esters as plastic plasticizers.

환경호르몬(내분비계 교란물질)은 생체 외부에서 들어와 인간의 내분비기관에서 호르몬의 생리 작용을 교란시키는 화합물을 뜻한다. 환경호르몬은 생체 내 호르몬의 합성, 방출, 수송 등 다양한 과정에 관여해 각종 형태의 교란을 일으킴으로써 생태계 및 인간에게 영향을 주며, 생식 이상과 성장 억제 등을 초래하기도 한다. Alkylphenol ethoxylates의 일종인 4-tert-octylphenol(OP)은 호르몬과 유사한 작용을 하거나, 호르몬 작용을 방해할 수 있는 내분비계 장애물질로 알려져 있다. 비교적 약한 유사 에스트로겐 작용을 가지고 있지만, OP의 화학적 특성상 비이온성 지용성 물질로 인체에 축적된 다면 내분비 조절 기구에 영향을 미칠 수 있는 가능성을 갖고 있다. OP는 주로 가정용 및 공업용 세제 등의 계면활성제, 페인트, 살충제, 플라스틱, 합성 수지류의 산화방지제나 안정제 용도로 주로 사용되고 있다. 이러한 내분비계 장애물질인 OP는 수질오염 및 식습관에서 유발되는 환경적 요인에 의해 체내로 유입된다. 본 연구에서는 무당개구리(Bombina orientalis)와 아프리카 발톱개구리(Xenopus laevis) 의 배아를 이용하여 4-tert-octylphenol(OP)이 초기 발생에 미치는 영향을 온도와 노출 시간 그리고 종간의 차이로 분석하였다. OP 50μM이상 처리군에서 온도, 종에 차이 없이 생존율이 0%로 확인되었다. 23℃에서 embryo가 tadpole이 되기까지 Xenopus의 경우 96h, Bombina의 경우 144h이 소요되는데, 각각 OP 에 노출되는 시간의 차이가 있으므로 Xenopus는 23℃ 96h, 19℃ 144h , Bombina의 경우 26℃ 96h, 23℃ 144h으로 온도의 차이를 주어 노출시간을 같게 하여 초기 발생을 관찰 하였다. Bombina 배아에 OP 10μM 을 23℃, 144h 동안 노출시킬 경우 두부 연골의 기형을 갖는 올챙이로 성장한다. 하지만 동일한 온도조건에서 Xenopus 배아에 23℃ 96h동안 노출시킬 경우 두부 연골의 기형이 관찰되지 않고, 척추, 꼬리의 기형만이 관찰된다. 이러한 결과는 같은 온도 조건임에도 불구하고, OP이 종간에 미치는 영향이나 노출시간에 따라 영향이 다를 수 있음을 보여준다. 본 결과로서 수계에서 OP에 노출된 양서류의 종간 차이, 더 나아가 OP이 환경의 변화에 따라 양서류에 다른 영향을 줄 것으로 예상된다.