DGCR8 is a RNA-binding protein working with DROSHA to produce pre-microRNA in the nucleus, while DICER does not only mature microRNA but also endogenous siRNAs in the cytoplasm. Here, we have produced Dgcr8 conditional knock-out mice using progesterone receptor (PR)-Cre (Dgcr8flox/flox; PRcre/+ mice, Dgcr8d/d) and demonstrated that canonical microRNAs dependent of DROSHA-DGCR8 complex are required for uterine development as well as female fertility in mice. Adult Dgcr8d/d females did not undergo regular reproductive cycle and produce any pups when housed with fertile males, whereas administration of exogenous gonadotropins induced normal ovulation with corpus luteal formation in these mice. Ovulated oocytes from Dgcr8d/d mice had comparable fertilization potentials and were normally developed to the blastocyst after fertilization as compared to those in control Dgcr8f/f mice. Interestingly, PR-Cre-dependent Dgcr8 deletion showed aberrant infiltration of acute inflammatory immune cells to female reproductive organs only when Dgcr8d/d mice were mated with male mice. With respect to uterine development, gross morphology, histology, and weight of Dgcr8d/d uterus were similar to those of control at 3-week-old age. However, multiple uterine abnormalities were noticeable at 4-week-old age when PR expression is significantly increased, and these deformities became severe onwards. Gland formation and myometrial layers were significantly reduced, and stromal cell compartment did not expand and became atrophic during uterine development in these mice. These results were consistent with aberrantly reduced cell proliferation in stromal cell compartments of Dgcr8d/d mice. Collectively, our results suggest that DGCR8 dependent-canonical microRNAs are essential for development and physiology of the uterus with respect to morphogenesis, proper immune modulation, reproductive cycle, and steroid hormone responsiveness in mice.

The transcription factor, early growth response protein 1 (EGR1), act as immediate early response genes to control various cellular and reproductive events. Egr1-deficient female mice show infertility by anovulation resulting from luteinizing hormone-β (LH-β) subunit deficiency. While ovulation, fertilization and embryo development normally occur in Egr1-deficient mice treated with a superovulation regime to rescue LH deficiency, embryo implantation was completely failed. The morphology and ultrastructure of uterine tissues were observed by light and transmission electron microscopy during the peri-implantation period in Egr1-deficient mice. To examine alterations in cellular organelles, the uterine horns were fixed with 2.5% glutaraldehyde and postfixed with 1% osmium tetroxide in PBS. After dehydration and infiltration, the samples were embedded in Epon 812. Semi-thin sections 0.5 μm thick were cut with an ultramicrotome and stained with toluidine blue for light microscopy. Thin sections were cut with a diamond knife of the ultramicrotome and placed on copper grids. The sections were double stained and examined under a transmission electron microscope. The height of luminal epithelial cells was decreased and the polarity was poorly differentiated in the Egr1-deficient comparing to the wild mice. The abundant mucinous materials were observed in the surface of luminal epithelial cells of the Egr1-deficient. It was confirmed the microarray and real time qPCR data. The luminal epithelial cells of wild mice had many dense lipophilic granules and healthy mitochondria, but not in the Egr1-deficient. It may related to production and secretion of steroid hormones and prostaglandins in the luminal epithelial cells for successful implantation. These results show that Egr1 is a critical transcription factor to fine-tune subcellular morphological and functional changes for the receptive phase of peri-implantation period of uterine tissue in mice.

The Egr family of zinc finger transcription factors is rapidly induced by various mitogens and regulates cell growth, differentiation, and apoptosis. While it is clear that loss of Egr1 leads to anovulatory infertility due to LHβ deficiency in female mice, molecular function of Egr1 in male reproduction has not been clearly investigated. Here, we demonstrate that Egr1 acts as an intrinsic transcription factor in Leydig cells to regulate their proliferation and steroidogenesis in the testis as well as an extrinsic factor for male reproduction via LHβ transcription in the pituitary. Egr1 is predominantly expressed in spermatogonia and Leydig cells in immature testes and later detected in some of these cell types in mature testes. The fertility potential of Egr1(-/-) male mice is relatively deteriorated even at 2 month-old age and aggravated with aging. The incidence of abnormalities of seminiferous tubules such as Sertoli cell only was dramatically increased with aging. The number and mean size of Leydig cells were significantly reduced in Egr1(-/-) testes. The impairment of Leydig cells is consistent with significant reduction in levels of testosterone and expression of factors critical for steroidogenesis such as StAR in Egr1(-/-) testes. Exogenous administration of hCG rapidly and transiently induced Egr1 expression in Leydig cells culture in vitro. hCG could reinstate reduced mean size of Leydig cells but not reduced number of Leydig cells and aberrantly low StAR expression, suggesting that Egr1 has critical functions for Leydig cell proliferation and their steroidgenesis. In addition, daily sperm production and in vitro fertilization (IVF) competence were significantly reduced, and apoptosis was facilitated in these mice. Furthermore, hCG administration to compensate for relatively low LH levels in Egr1(-/-) males could not restore the compromised reproductive phenotypes such as IVF competence and apoptosis in these mice. Interestingly, expression of Egr2, a member of Egr family, is significantly elevated in Egr1(-/-) Leydig cells suggesting that genetic compensation of Egr2 may alleviate phenotypic aberration of Egr1(-/-) male testes. Collectively, these results suggest that Egr1 act as an intrinsic transcription factor required for proliferation and steroidogenesis of Leydig cells to govern spermatogenesis in the testis.

Early growth response 1 (Egr1) belongs to the Egr family of zinc finger transcription factors (Egr1 to Egr4) that regulates cell growth, differentiation, and apoptosis. Egr1(-/-) female mice are infertile due to anovulation resulting from luteinizing hormone β subunit (LHβ) deficiency. While it is clear that Egr1 is critical for LHβ transcription in the pituitary gland, function of Egr1 in uterus still remain unexplored. Uteri on various experimental conditions or days of pregnancy were collected for mRNA microarrays, realtime-RT-PCR, Western blotting, and histological analyses for immunofluorescence and BrdU staining. Egr1 and other Egr family members, Egr2 and Egr3 are highly expressed in the uterus on day 4 of pregnancy (Day 4). While ovulation, fertilization and embryo development normally occur in Egr1(-/-) mice treated with a superovulation regime to rescue LH deficiency, embryo implantation was completely failed. In addition to implantation failure, oviductal transport of embryos is also impaired in these mice. 17/24 Egr1(-/-) mice (71%) retained blastocysts in the oviduct as well as in the uterus of Egr1(-/-) mice on Day 4 whereas all Egr1(+/+) mice have them in the uterus. While serum levels of E2 and P4 in Egr1(-/-) mice on Day 4 were comparable to those of wildtypes, expression of E2 responsive genes which are expressed in luminal epithelium, such as Mucin 1 and lactoferrin, is aberrantly increased in Egr1(-/-) mice with embryos in the oviduct on Day 4. In contrast, P4 responsive genes such as Hoxa10 and amphiregulin are normally expressed in these mice. Collectively, these data suggest that Egr1 deficiency in the oviduct and uterus leads to estrogen hypersensitivity. BrdU incorporation experiments provided evidence that epithelial cells undergo hyperproliferation in Egr1(-/-) mice. This is consistent with microarray data that several key factors for cell cycle progression such as cyclin Ds and E2F1 are overexpressed in these mice. Furthermore, in the uteri of Egr1(-/-) mice treated with E2+P4, stromal cell proliferation is severely impaired and epithelial cells persistently proliferating. With respect to decidualization, Egr1 as well as Egr2 and Egr3 are induced immediately after decidualization stimuli were given. Although the responses were relatively less than those of wildtype mice, decidualization does occur in Egr1(-/-) mice. Relatively compromised decidualization responses seems to result from functional compensation of Egr2 and Egr3 in Egr1(-/-) deficient uteri. Collectively, our results show that Egr1 is a critical transcription factor to fine-tune estrogen responses via regulation of a spectrum of genes for embryo implantation in the uterus.

Estrogen is a primary steroid hormone to govern cell fates in the endometrium. It induces expression of a spectrum of genes such as early growth response 1 (Egr1) critical for dynamic change of uterine environments for embryo implantation. Egr1 belongs to the Egr family of zinc finger transcription factors consisting of 4 members (Egr1 to Egr4) that are co-expressed in many different tissues, suggesting that they may have some redundant functions. Bisphenol A (BPA) is a well-known endocrine disruptor with potent estrogenic activity on reproductive system. Here we have demonstrated molecular pathway(s) by which estrogen (17β estradiol, E2) and BPA regulates Egr1 in uterus. Eight-week-old female mice were ovariectomized (OVX) and rested for a week. Uteri of OVX mice treated with E2, BPA and/or progesterone (P4) were collected 2 h after hormone treatment unless otherwise indicated. ICI 182,780 [estrogen receptor (ER) antagonist] and RU486 [progesterone receptor (PR) antagonist] were pretreated 30 min before hormone treatment. Collected uteri were mainly utilized for RT-PCR, realtime-RT-PCR and Western blotting. Egr1 mRNA was rapidly induced with the highest level at 2h after E2 treatment and gradually decreased to basal levels at 12 h. Pretreatment of ICI 182,780 effectively inhibited E2-induced phosphorylation of ERK1/2 and AKT as well as Egr1 transcription. U0126 (a pharmacological ERK1/2 inhibitor), but not Watmannin (a AKT inhibitor), significantly blocked E2-induced Egr1 expression as well as ERK1/2 phosphorylation in the uterus. P4 effectively dampened E2-dependent Egr1 transcription, and its antagonistic effects were partially interfered with RU486 pretreatment. Interestingly, Egr2 and Egr3 showed similar hormone-dependent expression profiles to that of Egr1 in the uterus. BPA (100 mg/kg) was able to induce immediate expression of Egr1 as effective as E2 at 2 h after treatment. ICI 182,780 and P4 considerably reduced BPA-induced expression of Egr1. In addition, RU486 counteracted inhibitory action of P4 on BPA-induced expression of Egr1. While overall patterns of BPA- induced expression of Egr2 and Egr3 were similar to that of Egr1, BPA was not as effective as E2 for induction of Egr2 and Egr3. BPA could induce phosphorylation of ERK1/2 as well as expression of Egr family members, too. Collectively, these results strongly suggest that BPA as well as E2 can activate concurrent expression profiles of Egr family members via ER-ERK1/2 pathways in the uterus.

DGCR8 is a RNA-binding protein working with DROSHA involved in critical processes for microRNA production in the nucleus. To understand function of miRNAs in the uterus, we have produced uterus-specific Dgcr8 conditional knock-out mice using two well-known Cre mouse models, anti-Mullerian hormone receptor 2 (Amhr2)-Cre and progesterone receptor (PR)-Cre. Dgcr8flox/flox;PRcre/+ mice were mainly analyzed and considered as uDgcr8 KO in this study unless otherwise indicated as Dgcr8flox/flox;Amhr2cre/+ mice. Morphological and histological analyses, embryo cultures, genomic DNA PCR, realtime RT-PCR and Western blotting were performed. uDgcr8 KO females bred with fertile males did not produce any offspring, suggesting that these mice are infertile. Vaginal smear analyses showed that these mice do not undergo estrous cycle, whereas Dgcr8flox/flox;Amhr2cre/+ mice exhibited regular estrous cyclicity. In vitro culture of 2-cell stage embryos and histological analyses for CL in uDgcr8 KO demonstrated that they can respond to gonadotrophins to ovulate healthy oocytes with comparable fertilization potentials as compared to those in Dgcr8flox/flox mice (Control). Gross morphology, histology, and weight of uteri of uDgcr8 KO mice were similar to those of control at 3-week-old stage. However, uterus become extremely thinner and shorter from 4-week-old stage onward. Histological examination showed significant reduction in gland numbers and stromal area from 4-week-old stage. Interestingly, this phenotype is reflected by significant increase of PR expression in the uteri of 4-week-old mice. In addition, stromal cell proliferation of uDgcr8 KO is severely impaired. BrdU incorporation experiments showed that while epithelial cells undergo proliferation by E2 treatment, stromal cells do not incorporate BrdU under the uterine conditions provided with E2+P4. Collectively, these results conclude that microRNAs are essential for uterine stromal cell proliferation in mice.

Fetal bovine serum (FBS) is the most frequently used serum for the cultivation of mammalian cells. However, since animal-derived materials might not be appropriate due to safety issues, allogeneic human serum (HS) has been used to replace FBS, particularly for the culture of human cells. While there has been a debate about the advantages of HS, its precise effect on human adult stem cells have not been clarified. The present study aimed to investigate the effect of HS on the human eyelid adipose stem cells (HEACs) in vitro. When HEACs were cultivated in a medium containing 10% HS, many cells moved into several spots and aggregated there. The phenomenon was observed as early as 9 days following 10% HS treatment, and 12 days following 5% HS plus 5% FBS treatment. However, the aggregation was never observed when the same cells were cultivated with 10% FBS or bovine serum albumin. To examine whether cell density might affect the aggregation, cells were seeded with different densities on 12-well dish. Until the beginning of aggregation, cells seeded at low densities exhibited the longest culture period of 16 days whereas cells seeded at high densities showed the shortest period of 9 days to form aggregation. The number of cells was as the least for the low density group, and as the greatest for the high density group. When human cord blood serum or normal bovine serum was examined for the same effect on HEACs, interestingly, cord blood serum induced the aggregation of cells whereas bovine serum treatment has never induced. When cells were cultivated with 10% HS for 9 days, they were obtained and analyzed by RT-PCR. Compared to FBS-cultivated HEACs, HS-cultivated HEACs did not express VIM, and less expressed GATA4, PALLD. On the other hand, HS-cultivated HEACs expressed MAP2 more than FBS-cultivated HEACs. In conclusion, human adult stem cells could move and form aggregates by the treatment with human body fluids.

Early growth response 1 (Egr1) is an immediate early response gene which is induced by various external stimuli and acts as transcription factor to direct second-wave gene expression leading to cell growth, differentiation and/or apoptosis. It is well known that Egr1 regulates transcription of a cluster of genes in cancers and luteinizing hormone (LH) beta subunit in the pituitary. In addition to function of Egr1 in cancers and pituitary, we recently showed that Egr1 acts as a local master regulator to mediate estrogenic actions in the uterus. However, regulatory mechanism by which Egr1 directs transcription of its downstream target genes in the uterus remains to be yet explored. Thus, we have tried to identify direct target genes of Egr1 in the uterus by analyzing mRNA microarray data sets followed by in silico promoter analyses with chromatin immunoprecipitation (CHIP). mRNA expression profiles of Egr1(－/－) uteri and Egr1(－/－) ovaries were compared to those of wildtype mice to provide a potential list of direct target genes of Egr1 in the uterus. Whereas Egr1 is rapidly and transiently induced in the ovary and the uterus by external stimuli, LH and estrogen, respectively with a similar manner, a list of differentially expressed genes between Egr1(+/+) and Egr1(－/－) mice were barely overlapped between these two datasets. This result suggests that the transcriptional network of Egr1 in the uterus is quite different from that in the ovary. The list of differentially expressed genes in Egr1(－/－) uterus was enriched by RT-PCR. In silico analyses with MatInspector provided evidence that Egr1 binding sites are relatively enriched in －500 bp promoter regions of genes in the list. CHIP assays for Egr1 antibody with uterine tissues 2 h after estrogen treatment reinforced the possibility that genes identified in this study such as Gadd45g and Lbh could be directly regulated by Egr1 in uterine context. Collectively, we show that bioinformatic analyses of expression profiles with in silico analyses could be a useful tool to enrich potential candidates of direct target genes of transcription factors.

Early growth response 1 (Egr1) belongs to the Egr family of zinc finger transcription factors that regulates cell growth, differentiation, and apoptosis. Egr1(－/－) female mice are infertile due to anovulation resulting from luteinizing hormone β subunit (LHβ) deficiency. While it is clear that Egr1 is a critical factor to regulate transcription of LHβ in the pituitary gland, function of Egr1 and mechanisms by which estrogen (E2) and/or progesterone (P4) regulates Egr1 in uterus still remain unexplored. Using multiple approaches, here we have characterized regulatory mechanism of Egr1 induction in the uterus and uterine phenotypes of Egr1(－/－) mice. Eight-week-old female mice were ovariectomized (OVX) and rested for a week. Uteri of OVX mice treated with various concentrations of E2 and/or other hormones were collected at 2h after hormone treatment unless otherwise indicated. Collected uteri were utilized for mRNA microarrays, realtime-RT-PCR, Western blotting, and histological analyses for immunofluorescence and BrdU staining. Egr1 mRNA was rapidly induced with the highest level at 2h after E2 treatment and gradually decreased to basal levels at 12 h. E2-induced phosphorylation of ERK1/2 and AKT, and Egr1 transcription were effectively inhibited by pretreatment of ICI 182,780. Pharmacological inhibition of ERK1/2, but not AKT significantly blocked E2-induced Egr1 expression in the uterus. P4 effectively dampened E2-dependent Egr1 transcription and its antagonistic effects were partially interfered with RU486 pretreatment. Interestingly, BrdU incorporation experiments provided evidence that epithelial cells undergo hyperproliferation in Egr1(－/－) mice. This is consistent with microarray data that several key factors for cell cycle progression such as cyclin Ds and E2F1 are overexpressed in these mice. Furthermore, in the uteri of OVX Egr1(－/－) mice treated with E2+P4, stromal cell proliferation is severely impaired and epithelial cells persistently proliferating. While ovulation, fertilization and embryo development normally occur in Egr1(－/－) mice treated with a superovulation regime to rescue LH deficiency, embryo implantation is severely impaired. Blastocysts were not able to implant even on day 6 of pregnancy in Egr1(－/－) mice. In addition to embryo implantation, uterine response to artificial decidualization in hormone-primed Egr1(－/－) OVX mice was relatively less than that of wildtype mice. Collectively, our results show that Egr1, which is rapidly induced by E2-ER-ERK1/2 pathways, is a critical factor to control E2-dependent cell proliferation via regulation of a spectrum of genes for embryo implantation in the uterus.

MicroRNAs (miRNAs) are single-stranded, small non-coding RNAs which are critical for gene regulatory networks by directing the translational repression or degradation of complementary target mRNAs. They can be divided into two subclasses: canonical and non-canonical miRNAs. Canonical miRNAs are produced from long primary transcripts by sequential complex events in which RNA III enzymes such as Drosha and Dicer and accessory factors such as DGCR8 and Argonautes work cooperatively. DGCR8 is a RNA-binding protein that assists Drosha to process canonical miRNAs in the nucleus. To understand function of canonical miRNAs in uterine physiology, we have characterized uterine phenotypes of uterine-specific DGCR8 knock-out mice (uDGCR8 KO, DGCR8flox/flox; PRcre/+), and hormonal regulation of expression profiles of major factors working for miRNA biogenesis in the uterus. Gross morphological and histological analyses, immunohistochemistry, PCR and realtime RT-PCR were performed. While DGCR8 and Drosha do not seem to be regulated by ovarian steroid hormones, expression of Dicer, Exportin 5 and Argonaute 2 was transiently increased by E2 but not by P4. Combination of E2+P4 did not have any additional effects on their expression profiles. Genomic DNA PCR analyses showed that while DGCR8 gene is not completely deleted in the uterus, DGCR8 is specifically deleted in the uterus where PR is strongly expressed. uDGCR8 KO females bred with fertile males did not produce any offspring, suggesting that these mice are infertile. Vaginal smear analyses provided evidence that these mice do not undergo estrous cycle. Whereas gross morphology and histological analyses of uteri of 3-week-old uDGCR8 KO mice is similar to that of DGCR8flox/flox mice (control), uteri of 5- and 8-week-old mice are extremely thinner and shorter than those of control mice. These results were supported by significant decrease in uterine weight/body weight of uDGCR8 KO mice at 5-week-old age onward. Interestingly, this phenotype is reflected by significant increase of PR expression in the uteri of 4-week-old mice. Expression of DGCR8 and Dicer is significantly increased after birth. BrdU incorporation experiments showed that cell proliferation governed by ovarian steroid hormones does not normally occur in these mutant mice. Furthermore, artificial decidualization does not occur in these mice. Collectively, these results conclude that canonical miRNAs plays critical roles in normal uterine development and steroid hormone-dependent uterine function.

Human eyelid adipose-derived stem cells (hEAs) and amniotic mesenchymal stem cells (hAMs) are very valuable sources for the cell therapeutics. Both types of cells have a great proliferating ability in vitro and a multipotency to differentiate into adipocytes, osteoblasts and chondrocytes. In the present study, we evaluated their stem cell characteristics after long-time cryopreservation for 6, 12 and 24 months. When frozen-thawed cells were cultivated in vitro, their cumulative cell number and doubling time were similar to freshly prepared cells. Also they expressed stem cell-related genes of SCF, NANOG, OCT4, and TERT, ectoderm-related genes of NCAM and FGF5, mesoderm/endoderm-related genes of CK18 and VIM, and immune-related genes of HLA-ABC and 2M. Following differentiation culture in appropriate culture media for 2-3 weeks, both types of cells exhibited well differentiation into adipocyte, osteoblast, and chondrocyte, as revealed by adipogenic, osteogenic or chondrogenic-specific staining and related genes, respectively. In conclusion, even after long-term storage hEAs and hAMs could maintain their stem cell characteristics, suggesting that they might be suitable for clinical application based on stem cell therapy.

A new deep red colored standard rose (Rosa hybrida Hort.) cultivar, `Best Red' was developed at the Jeollanamdo Agricultural Research and Extention Services (JARES). Seedling and line selection were conducted in 2003 and its characteristics were examined three times from 2004 to 2006. 'Best Red' which was obtained by cross between 'Vital' and 'Cardinal'. It shows a red group color (RHS No. 46C), medium fragrance, and standard flower with good harmony between ray floret and flower center. The upper stem is moderate curved. The average yield during 2004-2006 was 111 stems/m2. Its vase life was 10 days. This cultivar was registered as a commercial cultivar in 2007. It is suitable for greenhouses in all regions in Korea and will be released to farmers in 2009.

A new pale pink colored standard rose (Rosa hybrida Hort.) cultivar, 'Fine Pink' was developed at the Jeollanamdo Agricultural Research and Extention Services (JARES). Seedling and line selection were conducted in 2003 and its characteristics were examined three times from 2004 to 2006. 'Fine Pink' was obtained by crosse between 'Nobless' and 'Yvessaaintlaurent'. It shows a red group color (RHS No. 56C), very weak fragrance, and standard flower with good harmony between ray floret and flower center. It has a characteristic of straight stem. The average yield during 2004-2006 was 113 stems/m2. It has 10 days vase life and resistance to powdery mildew. This cultivar was registered as a commercial cultivar in 2007. It is suitable for greenhouse in all regions in Korea and will be released to farmers in 2009.

A new Cymbidium hybrid “Bright Evening” was released by National Horticultural Research Institute (NHRI) in 2003. A cross was made in 1995 between Cymbidium “Tropical Yellow”, a brownish yellow colored petal and medium plant sized variety, and “Lucky Rainbow Rapin Hot”, a light purple-pink petal with red lip and medium sized variety. A cross was made in 1995 between Cymbidium “Tropical Yellow”, a brownish yellow colored petal and medium plant sized variety, and “Lucky Rainbow Lapin Hot”, a light purple-pink petal with red lip and medium sized variety. The eighty seven progenies were obtained after planting and acclimatization in green house. In 1999, a line (9529125) was selected and multiplicated for its flower color, leaf shape, flower stalk, and vigorous growing habit. After evaluation trial for two growing seasons, the selected line was named as “Bright Evening”. The “Bright Evening” has yellow petals and is a medium sized (average leaf length is about 62 cm and width of flower is about 6 cm) Cymbidium variety with vigorous growth and erect flower stalks. Blooming is started from November to February in optimal culture condition. High temperature (more than 30℃) and low light intensity should be avoided for the appropriate growth. In addition, flower stalks develop easily even from underdeveloped pseudobulbs.

The present study describes carbohydrate metabolism, macro-element utilization and antioxidant defenses in response to an ozone dose (100 ppb, 8d) in two rice varieties. Tolerant (cv. Jinpumbyeo) and sensitive (cv. Chucheongbyeo) varieties of rice were grown in growth chamber for 30 days after sowing. Concentrations of chloroplast pigments and non-structural carbohydrates as well as activity of antioxidant enzymes were determined to evaluate the resistance against ozone stress. Ozone caused the decrease in chlorophyll a and carotenoid contents, and also resulted in faster decomposition of non-structural carbohydrate in leaf blade and leaf sheath. The contents of nitrogen and potassium in leaves were visibly decreased in cv. Chucheongbyeo with an increase in ozone exposure, but not in cv. Jinpumbyeo. Enzymatic antioxidants against ROS in both varieties responded in the order of POD, SOD and CAT, and their capacity was stronger in cv. Jinpumbyeo.