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This study aimed to evaluate the effects of thyroid hormone on the decidua and metrial gland of rats and to examine the expression of angiogenic factors. 72 adult, female rats were divided into hypothyroid, T4-treated2, and control groups. At 10, 14 and 19 days of gestation (DG), the decidua and metrial gland were collected for histomorphometric and immunohistochemical evaluation of the expression of VEGF, Flk-1 and Tie-2. Hypothyroidism reduced the area of the decidua at 10 and 19 DG. Furthermore, VEGF was increased at 10 and 14 DG, and Flk-1 only at 14 DG, but both was reduced at 19 DG in the metrial gland without significantly changing the area occupied by blood vessels. Rats treated with T4 showed an increase in the decidua blood vessels at 10 and 19 DG. However, at 10 DG, excess T4 resulted in increased of Flk-1 in the decidua and metrial gland. Hypothyroidism increased the Tie-2 at 10 and 19 DG in the decidua and metrial gland. In conclusion, hypothyroidism reduces the area of the decidua and increases the expression of VEGF, Tie-2 and Flk-1. The excess of T4 promotes tissue angiogenesis by increasing the number of vessels in the decidua because of the increased expression of Flk-1.(AU)
Este estudo teve como objetivo avaliar os efeitos dos hormônios tireoidianos sobre a decídua e a glândula metrial pela análise da expressão de fatores angiogênicos em ratas. 72 ratas adultas, fêmeas foram distribuídas nos grupos hipotiroideo, tratado com T4 e controle. Aos 10, 14 e 19 dias de gestação (DG), a decídua e a glândula metrial foram coletadas para avaliação histomorfométrica e imunoistoquímica da expressão de VEGF, Flk-1 e Tie-2. O hipotireoidismo reduziu a área da decídua aos 10 e 19 DG. Além disso, o VEGF aumentou aos 10 e 14 DG e o Flk-1 apenas aos 14 DG, mas ambos foram reduzidos aos 19 DG na glândula metrial sem alterar significativamente a área ocupada pelos vasos sanguíneos. As ratas tratadas com T4 apresentaram aumento do número de vasos sanguíneos na decídua aos 10 e 19 DG. Além disso, aos 10 DG, o excesso de T4 resultou no aumento de Flk-1 na decídua e na glândula metrial. O hipotireoidismo aumentou o Tie-2 em 10 e 19 DG na decídua e na glândula metrial. Desta forma, pode-se concluir que o hipotireoidismo reduz a área da decídua e aumenta a expressão de VEGF, Tie-2 e Flk-1. O excesso de T4 promove a angiogênese tecidual ao aumentar o número de vasos na decídua devido ao aumento da expressão de Flk-1.(AU)
Subject(s)
Animals , Female , Rats , Phenylthiourea/analysis , Thyroid Hormones/analysis , Decidua , Angiogenesis Inducing Agents/analysis , Metrial GlandABSTRACT
CCAAT/enhancer binding protein β (C/EBPβ) is a critical member of C/EBP family.C/EBPβ,based on the leucine zipper located in C terminal of the protein,can regulate transcriptional activities of downstream target genes involved in diverse cellular processes,such as proliferation and differentiation.Recently published studies have identified that C/EBPβ is essential in the decidualization of endometrial stromal cells.This review summarizes the roles and mechanisms of C/EBPβ during the courses of decidualization,which is aimed at providing novel insights for dysfunctional decidualization.
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Objective Among the factors affecting embryo implantation, oxidative stress is one that receives much medical attention. The purpose of the study was to explore the effect of H2 O2 on the endometrial stromal cells ( ESCs) in the decidual mouse model of oxidative stress of ESCs. Methods Primary ESCs cultured by enzymatic digestion with a mesh filter were divided into a control, an experimental, and a model group. Decidualization was induced in the mice of the experimental and model groups by inter-vention with 10 nM E2 and 1μmol/L P4 for 72 h in vitro. Then, the animals of the model group were treated with different concentra-tions of H2 O2 for 4 hours. The primary ESCs were identified by immunohistochemistry, the expression of dPRP mRNA determined byRT-PCR, the proliferation of the decidual ESCs treated with H2 O2 analyzed by CCK-8, and the level of ROS detected by flow cytometry. Results Primary mouse ESCs were successfully isolated, cultured, and identified, which were shaped like spindles and polygons, radial-ly aligned under the microscope. Immunofluorescence analysis showed positive expression of vimentin and negative expression of cytokeratin. The purity of the primary mouse ESCs was ( 96. 3 ± 0. 49 )%. Theexpression of dPRP mRNA was significantly higher in the ESCs treated with E2 and P4 than in the control (0.0002±0.0000 vs 1.0010±0.0011, P<0.01). H2O2 at ≥150μmol/L suppressed the proliferation of the decidual ESCs by 6.9% (P<0.05), and in a concen-tration-dependent manner, reaching the maximum inhibition rate of 70.6% at the concentration of 300μmol/L. The level of intracellular ROS was markedly increased in the ESCs treated with H2O2 at 50μmol/L (27.77±4.20) and 100μmol/L (43.57±6.58), with statisti-cally significant difference from that in the control group (17.47±0.61) (P=0.001). Conclusion H2O2 at 100μmol/L can signifi-cantly elevate the intracellar ROS level without affecting the proliferation of decidual primary ESCs, and therefore can be used for estab-lishing the model of oxidative stress of ESCs in decidual mice.
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Objective To study the expression and regulation of polycystic ovary syndrome ( PCOS) susceptibility gene Hmga2 in endometrial receptivity and decidualization. Methods Experiments including early pregnancy, delayed implantation and activation, artificial decidualization and ovarian steroid hormone treatment were performed, and Q-PCR and Western blot analyses were applied to explain the role of Hmga2 in endometrial receptivity. Results The Hmga2 ex-pression increased gradually with pregnancy. Its expression at the implantation site was significantly higher than that at the inter-implantation site,in the embryo activation group than in delayed implantation group, and in the artificial decidualiza-tion than the non-decidualization. The expression of Hmga2 was positively correlated with the expression of estrogen and progesterone, and was positively regulated by estrogen and progesterone in vivo. Conclusions Our findings indicate that the expression of Hmga2 is closely related to the embryo implantation process in early pregnancy in mice, is involved in the process of endometrial decidualization, and is influenced by the activated blastocyst and steroid hormones.
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Objective To study the expression and regulation of polycystic ovary syndrome ( PCOS) susceptibility gene Hmga2 in endometrial receptivity and decidualization. Methods Experiments including early pregnancy, delayed implantation and activation, artificial decidualization and ovarian steroid hormone treatment were performed, and Q-PCR and Western blot analyses were applied to explain the role of Hmga2 in endometrial receptivity. Results The Hmga2 ex-pression increased gradually with pregnancy. Its expression at the implantation site was significantly higher than that at the inter-implantation site,in the embryo activation group than in delayed implantation group, and in the artificial decidualiza-tion than the non-decidualization. The expression of Hmga2 was positively correlated with the expression of estrogen and progesterone, and was positively regulated by estrogen and progesterone in vivo. Conclusions Our findings indicate that the expression of Hmga2 is closely related to the embryo implantation process in early pregnancy in mice, is involved in the process of endometrial decidualization, and is influenced by the activated blastocyst and steroid hormones.
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Objective:To construct the pleiotrophin (PTN )overexpression vector,and to explore the effect of PTN on the decidualization of uterine stromal cells in the mice.Methods:The specific primers containing restriction enzyme cutting sites were designed according to the PTN gene sequences published in GenBank for PCR amplification.The amplified fragment of PTN was recovered from the agarose gel and cloned into the pGEM-T vector.The pGEMT-PTN was cut by double enzyme digestion and ligated into pcDNA3.1 (+)to construct the PTN overexpression plasmid.After transfection with PTN overexpression plasmid,the expression levels of PTN mRNA in the uterine stromal cells and the expression levels of decidualization markers Prl8a2 and Prl3c1 were detected by qRT-PCR method.The uterine stromal cells transfected with pcDNA3.1 (+)empty vector were used as control group. Results:The results of identification by double enzyme digestion indicated that the bands of PTN overexpression plasmid were consistent with those of the target gene,and the clone sequencing results suggested that it had 100% homology with mouse PTN gene sequence published in GenBank.Compared with control group, the expression levels of PTN,Prl8a2 and Prl3c1 mRNA in mouse uterine stromal cells in PTN overexpression group were significantly increased (P < 0.05).Conclusion:PTN overexpression could increase the expression levels of decidualization markers in mouse uterine stromal cells,indicating that PTN might play an enhancement effect during uterine decidualization in the mice.
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OBJECTIVE: To compare the hatching rates of the vitrified and the fresh mouse blastocysts co-cultured with decidualized or non decdualized human endometrial cells and to confirm the necessity of assisted hatching in the vitrified mouse blastocyst. METHODS: Stromal and epitherial cells isolated from human endometrial tissue were co-cultured and decidualized with TGF-beta 1 and progesterone. The vitrified and the fresh mouse blastocysts were co-cultured with human decidualized or non-decidualized endometrial cells, respectively and the hatching rates were investigated. RESULTS: Epithelial and stromal cells isolated from endometrial tissue were cultured seperately for 24 hours and stained by immunohistochemical staining for cytokeratin (epithelial cells) or vimentin (stromal cells). The immunohistochemical study was positive for cytokeratin or vimentin and confirmed that epithelial and stromal cells were isolated from endometrial tissue successfully. The co-cultured human stromal and epitherial cells were decidualized by administration of TGF-beta 1 and progesterone. The hatching rates of the fresh and the vitrified mouse blastocysts co-cultured with decidualized human endometrial cells were 89% and 31%, respectively. The hatching rates of the fresh and the vitrified mouse blastocysts co-cultured with non-decidualized human endometrial cells were 82% and 27%, respectively. The hatching rates were significantly higher in fresh mouse blastocysts than in vitrified mouse blastocysts regardless of decidualization of human endometrial cells (P<0.05). CONCLUSION: The hatching rate was significantly higher in fresh mouse blastocysts than in vitrified mouse blastocysts. This results showed that the cryopreservation procedure caused the zona hardening of mouse blastocyst and the decidualization of endometrial cells did not affect to hatching rate of the vitrified mouse blastocysts. We confirmed that assisted hatching was necessary for improving the hatching rate of cryopreserved mouse blastocysts.
Subject(s)
Animals , Humans , Mice , Blastocyst , Coculture Techniques , Cryopreservation , Herpes Zoster , Keratins , Progesterone , Stromal Cells , Transforming Growth Factor beta , VimentinABSTRACT
OBJECTIVE: To investigate the role of ERK and PPAR gamma on the TGF-beta1 induced human endometrial stromal cell decidualization in vitro. METHOD: Endometrial stromal cells are cultured under the following condition: DMEM/F12 (10% FBS, 1 nM E2 and 100 nM P4). TGF-beta1 (5 ng/ml), Rosiglitazone (50 nM), and PD98059 (20 microgram) were added according to experimental purposes. Trypan-Blue and hematocytometer were utilized to count cell number. Enzyme-linked immunosorbent assay (ELISA) and western blotting were utilized to detect proteins. RESULT: TGF-beta1 inhibited proliferation of cultured human endometrial stromal cells and induced expression of PGE2 and prolactin. This effect was mediated by Smad and ERK activation. Administration of rosiglitazone, PPAR gamma agonist, prevented TGF-beta1 effect on cell proliferation. Furthermore, Rosiglitazone inhibited TGF-beta1 induced activation of ERK, consequently reduced PGE2 and prolactin production. CONCLUSION: TGF-beta1 induced decidualization of endometrial stromal cell through Smad and ERK phosphorylation. PPAR gamma acts as a negative regulator of human endometrial cell decidualization in vitro.
Subject(s)
Humans , Blotting, Western , Cell Count , Cell Proliferation , Dinoprostone , Enzyme-Linked Immunosorbent Assay , Peroxisome Proliferator-Activated Receptors , Phosphorylation , PPAR gamma , Prolactin , Stromal Cells , Transforming Growth Factor beta1ABSTRACT
OBJECTIVE: To ascertain the expression of transforming growth factor (TGF)-beta receptors in normal human endometrium during the menstrual cycle, and the action of TGF-beta and peroxisome proliferator-activated receptor (PPAR)-gamma during endometrial decidualization using cultured human endometrial stromal cells. METHODS: Human endometrial tissues were examined immunohistochemically for the expression of TGF-beta receptors and Smad. Western blotting, confocal microscopy and viable cell counting were performed on cultured human endometrial stromal cells which were treated with TGF-beta (10 ng/mL) and PPAR-gamma agonists (Rosiglitazone(R) 50 nM). Thereafter we compared the effect of TGF-beta and PPAR-gamma on the Smad phosphorylation, prolactin expression, and cellular proliferation in vitro human endometrial decidualization. RESULTS: The results revealed significantly increased expression of both TGF-beta receptor-I and -II proteins in the secretory stromal cells compared to the epithelial cells of human endometrium. The degree of expression and translocation into the nucleus of the phosphorylated Smad2/3 was also increased in the secretory endometrium compared to the proliferative endometrium. In the stromal cell culture, the decidualization process associated with TGF-beta and pSmad is inhibited by the PPAR-gamma agonist. In contrast to the PPAR-gamma agonist, TGF-beta inhibits cellular proliferation. CONCLUSION: TGF-beta/Smad signaling pathway is essential for endometrial decidualization and closely related to cellular differentiation. PPAR-gamma plays a conflicting role by directly acting on the Smad protein and blocking the TGF-beta/Smad signaling pathway.
Subject(s)
Female , Humans , Blotting, Western , Cell Count , Cell Proliferation , Endometrium , Epithelial Cells , Menstrual Cycle , Microscopy, Confocal , Peroxisomes , Phosphorylation , Prolactin , Receptors, Transforming Growth Factor beta , Stromal Cells , Transforming Growth Factor beta , Transforming Growth FactorsABSTRACT
OBJECTIVE: Decidualization of endometrial stromal cells is essential for a successful implantation of the embryo. The process of decidualization can be modulated by sex steroids, growth factors and cytokines. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the interleukin-6 family and has different biological actions in various tissue systems. Recently, LIF has been reported as an important factor for adequate decidualization of endometrium. However, the effect on production of prolactin (PRL), known as the decidualization marker, are not clearly understood. The objective of this study was to determine whether LIF is capable of modulating prolactin production during 8-bromo (Br)-cAMP induced decidualization in vitro. METHODS: Human endometrial stromal cells were cultured and decidualization was induced by 0.5 mM 8-Br-cAMP. Phase contrast microscopy was used to verify morphological changes associated with differentiation in vitro in response to 8-Br-cAMP. Both stromal cells exposed to 8-Br-cAMP and cells not exposed to 8-Br-cAMP were also incubated with LIF (10 ng/mL). PRL levels in each supernatant were measured by a commercial PRL ELISA kit. Immunostaining and reverse transcription-polymerase chain reaction (RT-PCR) for PRL were performed. RESULTS: The concentration of PRL in the supernatant increased significantly in the cells treated with 8-Br-cAMP plus LIF (10 ng/mL) at culture day 6 compared with the others. The results of immunohistochemical staining reflected that of immunoassay. The PRL genes are expressed in the decidualized stromal cells treated with 8-Br-cAMP. No PRL mRNA was detectable in the absence of the 8-Br-cAMP. The intensity of the PCR band measured by densitometry is stronger in the cells treated with 8-Br-cAMP plus LIF than that with the others. CONCLUSION: These results suggested that LIF increased the production of PRL in decidualizing human endometrial stromal cells and may play a role in preparing the human endometrium for implantation through the promotion of PRL production.
Subject(s)
Female , Humans , Cytokines , Densitometry , Embryonic Structures , Endometrium , Enzyme-Linked Immunosorbent Assay , Immunoassay , Intercellular Signaling Peptides and Proteins , Interleukin-6 , Leukemia Inhibitory Factor , Leukemia , Microscopy, Phase-Contrast , Polymerase Chain Reaction , Prolactin , RNA, Messenger , Steroids , Stromal CellsABSTRACT
OBJECTIVES: To investigate the role of TGF (Transforming growth factor-beta) involved in the paracrinic communication during decidualization between UEC (uterine epithelial cells) and USC (uterine stromal cells), we have employed a co-culture system composed of human endometrial epithelial and stromal cells in defined hormonal conditions. DESiGN: in the co-culture, endometrial epithelial cells cultured in the matrigel-coated cell culture insert are seeded on top of the endometrial stromal cells cultured within a collagen gel. The co-culture was maintained for 48 hours under the following hormonal conditions: progesterone dominant condition (100 nM P4 and 1 nM E2) or estrogen-dominant condition (100 nM E2 and 1 nM P4). 10 ng/ ml HGF and/or 10 ng/ml TGF-beta1 are added. METHODS: RT-PCR is utilized to detect mRNAs quantitatively. Enzyme-linked immunosorbent assay (ELiSA) and immunohistochemical staining are utilized to detect proteins in the tissue. RESULTS: Prolactin mRNA is expressed in the co-cultured stromal cells under the progesterone dominant condition. TGF-beta1 and its receptors are expressed in both the co-cultured epithelial and stromal cells irrespective of the steroid present, which is in contrast with no or negligible expression of TGF-beta1 or its receptor in cells separately cultured. Both estrogen and progesterone significantly elevate the concentration of hepatocyte growth factor (HGF) in the conditioned medium of the co-culture with the value of 4,325 pg/ml in E2-dominant and 2,000 pg/ml in P4-dominant condition compare to 150 pg/ ml in no hormone. in separately cultured stromal cells, administration of HGF induces the expression of TGF receptor 1 in both hormonal conditions, but induction of TGF receptor 2 is only manifest in the P4-dominant condition. Administration of TGF-beta and HGF directly induce the decidualization marker prolactin mRNA in separately cultured stromal cells. CONCLUSION: it is likely that steroid hormones induces prolactin mRNA indirectly by promoting the cell to cell communication between the stromal and the epithelial cells. TGF-beta and HGF are two possible paracrine mediators in the human endometrial decidualization.
Subject(s)
Humans , Cell Communication , Cell Culture Techniques , Coculture Techniques , Collagen , Culture Media, Conditioned , Enzyme-Linked Immunosorbent Assay , Epithelial Cells , Estrogens , Hepatocyte Growth Factor , Progesterone , Prolactin , RNA, Messenger , Stromal Cells , Transforming Growth Factor beta , Transforming Growth Factor beta1ABSTRACT
Objective:To investigate the distribution and regulation of uterine NK cells in mouse uterus during early pregnancy. Methods:Using a unique uNK cell marker,Dolichos biflorus agglutinin (DBA) lectin,to localize NK cells in uterus of normal pregnancy,delayed implantation and artificially induced decidualization. Results:DBA staining is mainly distributed in some vascular endothelial cells during embryo pre-implantation. In D5 of pregnancy after embryo implantation,NK cells were distributed in the stroma of uterine mesometrial pole,with round,immature and small lymphocyte-like cells. During D7 and D8 of pregnancy,the immunostaining results showed that uNK cells were increasingly accumulated in the mesometrial pole of implantation sites,with their morphology changing from small round cells with scattered cytoplasm to large and mature cells with heavy cytoplasmic granules. In the artificially induced decidualization model,the distribution and maturity of NK cells are similar to those of the normal pregnancy in the mouse uteri. Conclusion:The distribution and maturation of uterine NK cells are dependent of uterine decidualization in mouse early pregnancy.
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Uterine cells carry out proliferation and differentiation for preparation the embryonic implantation during pregnancy. Therefore regulation of the cell proliferation is an essential step for uterine preparation, but there is not much information about the proliferation related genes in pregnant uterus. To identify these implantation specific genes, a PCR-select cDNA subtraction method was employed and got a few genes. One of the identified genes is a novel gene encoding oral tumor suppressor doc-1. To detect the doc-1 expression on the pregnant uterus, dot blotting, RT-PCR, and in situ hybridization were employed. Dot blotting revealed that doc-1 mRNA expression increase after implantation. During normal pregnancy, doc-1 mRNA expression was detected as early as day 1 of pregnancy with RT-PCR. Its expression was increased about 15 times after embryonic implantation. doc-1 transcript was localized in luminal epithelial cells but it was very faint during preimplantation. After starting the implantation, it localized in the stromal cells; heightened expression of doc-1 correlates with intense stromal cell proliferation surrounding the implanting blastocyst on day 6 morning. However in the decidualized cells, the intensity of localized doc-1 mRNA was weak. From those results, it is revealed that doc-1 express at pregnant uterus of the mouse. In addition it is suggested that doc-1 is the gene regulating the proliferation of the luminal epithelial cells and stromal cells during early implantation and decidualization.
Subject(s)
Animals , Mice , Pregnancy , Blastocyst , Cell Proliferation , DNA, Complementary , Epithelial Cells , Genes, vif , In Situ Hybridization , Phenobarbital , RNA, Messenger , Stromal Cells , UterusABSTRACT
Uterine cells carry out proliferation and differentiation for preparation the embryonic implantation during pregnancy. Therefore regulation of the cell proliferation is an essential step for uterine preparation, but there is not much information about the proliferation related genes in pregnant uterus. To identify these implantation specific genes, a PCR-select cDNA subtraction method was employed and got a few genes. One of the identified genes is a novel gene encoding oral tumor suppressor doc-1. To detect the doc-1 expression on the pregnant uterus, dot blotting, RT-PCR, and in situ hybridization were employed. Dot blotting revealed that doc-1 mRNA expression increase after implantation. During normal pregnancy, doc-1 mRNA expression was detected as early as day 1 of pregnancy with RT-PCR. Its expression was increased about 15 times after embryonic implantation. doc-1 transcript was localized in luminal epithelial cells but it was very faint during preimplantation. After starting the implantation, it localized in the stromal cells; heightened expression of doc-1 correlates with intense stromal cell proliferation surrounding the implanting blastocyst on day 6 morning. However in the decidualized cells, the intensity of localized doc-1 mRNA was weak. From those results, it is revealed that doc-1 express at pregnant uterus of the mouse. In addition it is suggested that doc-1 is the gene regulating the proliferation of the luminal epithelial cells and stromal cells during early implantation and decidualization.
Subject(s)
Animals , Mice , Pregnancy , Blastocyst , Cell Proliferation , DNA, Complementary , Epithelial Cells , Genes, vif , In Situ Hybridization , Phenobarbital , RNA, Messenger , Stromal Cells , UterusABSTRACT
Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in the endometrium during the menstrual cycle and in early pregnancy. Specificity of integrins is known to be different in human endometrial stromal cells and decidual cells. These shifts of integrins suggested to play an important role in embryo implantation and can be modulated by progesterone, cAMP derivatives, and cytokines. The mechanisms of decidualization and its precise physiological role are still not clearly understood and in vitro systems could provide an alternative that overcomes limitations of studying such complex biological phenomena in vivo at the time of implantation. This study was undertaken to establish an in vitro model system for human decidualization using 8-bromo-cAMP and to investigate the characteristics of stromal integrin expression in vitro by 8-Br-cAMP. Endometrial stromal cells were isolated and cultured, and then were induced to decidualize by 0.5 mM 8-Br-cAMP for 15 days. Immunofluorescence staining and flow cytometric analyses of the integrin subunits (alpha1, alpha4, alpha5, alpha6, beta1 and alpha v beta3) were performed at day 9. In the presence of 8-Br-cAMP, the staining intensity of alpha v beta3 was significantly higher than control and measurements for alpha1, alpha4, alpha5, alpha6, and beta1 were similar. Immunofluorescent localization of the integrins reflected the differences obtained from the flow cytometric analyses described above. In summary, the expression of alpha;avbeta;b3 integrin increased in stromal cells in vitro decidualized by 8-Br-cAMP and this up-regulation of alpha v beta3 integrin expression during decidualization might influence on human implantation.