본 연구는 이미 진정효과가 뛰어나다고 알려져 있는 클래식과 함께 진정효과가 있다고 밝혀진 레게의 효과를 비교하고자 진행되었다. 연구에 사용된 실험견들은 실험을 잘 이해하고 있는 연구원들이 현재 키우는 반려견 4마리를 섭외하여 진행되었다. 섭외 된 반려견은 말티즈 2마리, 요크셔테리어 2마리이다. 진정효과를 알 수 있는 지표로는 Lori R. Kogan외의 ‘Behavioral effects of auditory stimulation on kenneled dogs’ 논문에서 사용한 지표를 일상에서도 확인하기 쉽도록 간소화하여 짖은 횟수, 서있는 시간, 서있지 않은 시간, 수면시간의 평균을 냈다. 그 결과 짖는 횟수 외의 큰 차이는 보이지 않았고, 진정효과만은 확실한 것을 확인하였다. 다만 일상생활에서 실행하는 것과 실험견의 일정한 결과를 위해 각 가정에서 실행하여 정확도는 떨어질 수 있다. 주변 소음 등이 적은 가정에서 실행한다면 조금 더 정확한 결과를 얻을 수 있을 것이다.

Persimmon seeds contain considerable amounts of minerals, amino and organic acids, natural antioxidants and phenolic compounds. The objective of this study was to investigate quality characteristics and antioxidant potential of Korean persimmon seeds. The pH (4.88-4.94), color values, contents of minerals, free amino acids, organic acids, and phenolic compounds and DPPH free radical scavenging potentials of persimmon seed extracts significantly (p < 0.05) varied with the genotypes. This study showed that the seeds could be used as a source of different mineral elements (47.14-85.07 ㎎/㎏) without any measureable amount of heavy metals such as arsenic, cadmium, lead and mercury. Similarly, considerable amounts of organic (1550.13-2413.08 ㎎/㎏) and essential amino (50.85-54.03 ㎎/㎏) acids and total phenolic compounds (1227.91-1307.78 ㎍ gallic acid equivalent/g) were also found in the seed extracts, indicating their potential food value as a natural antioxidant. Results of the present study imply that prethanol-A, a food preservative, can be used as an effective extraction to obtain the minerals, organic and free amino acids, and phenolic compounds from the persimmon seeds, which possess a big potential to be commercially used in food, cosmetic and pharmaceutical industries.

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.

Early growth response 1 (Egr1) is a zinc-finger transcription factor to direct second-wave gene expression leading to cell growth, differentiation and/or apoptosis. While it is well-known that Egr1 controls transcription of an array of targets in various cell types, downstream target gene(s) whose transcription is regulated by Egr1 in the uterus has not been identified yet. Thus, we have tried to identify a list of potential target genes of Egr1 in the uterus by performing multi-step in silico promoter analyses. Analyses of mRNA microarray data provided a cohort of genes (102 genes) which were differentially expressed (DEGs) in the uterus between Egr1(+/+) and Egr1(–/–) mice. In mice, the frequency of putative EGR1 binding sites (EBS) in the promoter of DEGs is significantly higher than that of randomly selected non-DEGs, although it is not correlated with expression levels of DEGs. Furthermore, EBS are considerably enriched within –500 bp of DEG’s promoters. Comparative analyses for EBS of DEGs with the promoters of other species provided power to distinguish DEGs with higher probability as EGR1 direct target genes. Eleven EBS in the promoters of 9 genes among analyzed DEGs are conserved between various species including human. In conclusion, this study provides evidence that analyses of mRNA expression profiles followed by two-step in silico analyses could provide a list of putative Egr1 direct target genes in the uterus where any known direct target genes are yet reported for further functional studies.

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.