scholarly journals GnRH antagonist alters the migration of endometrial epithelial cells by reducing CKB

Reproduction ◽  
2020 ◽  
Vol 159 (6) ◽  
pp. 733-743 ◽  
Author(s):  
Qian Chen ◽  
Yong Fan ◽  
Xiaowei Zhou ◽  
Zheng Yan ◽  
Yanping Kuang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Implantation Rate ◽  
Endometrial Receptivity ◽  
Ishikawa Cells ◽  
Atp Generation ◽  
And Migration ◽  
Insight Into ◽  
Endometrial Epithelial Cells

Some studies have demonstrated that the implantation rate of fresh transfer cycles is lower in the gonadotropin-releasing hormone antagonist (GnRH-ant) protocol than in the GnRH agonist (GnRH-a) protocol during in vitro fertilization (IVF). This effect may be related to endometrial receptivity. However, the mechanisms are unclear. Here, endometrial tissues obtained from the mid-secretory phase of patients treated with GnRH-a or GnRH-ant protocols and from patients on their natural cycle were assessed. Endometrial expression of B-type creatine kinase (CKB), which plays important roles in the implantation phase, was significantly reduced in the GnRH-ant group. At the same time, expression of the endometrial receptivity marker HOXA10 was considerably reduced in the GnRH-ant group. GnRH-ant exposure in endometrial epithelial cells (EECs) in vitro decreased CKB expression and ATP generation and blocked polymerization of actin. Furthermore, in vitro GnRH-ant-exposed Ishikawa cells showed enhanced F-actin depolymerization, and these effects were rescued by CKB overexpression. Similar effects were observed after CKB knockdown, and these effects were rescued by CKB overexpression. Moreover, cell migration was decreased in CKB-knockdown Ishikawa cells compared with that in control cells, and this effect was also rescued by CKB overexpression. Overall, these findings showed that GnRH-ant affected CKB expression in EECs, resulting in cytoskeletal damage and migration failure. These results provide insight into the roles and molecular mechanisms of GnRH-ant treatment in the endometrium.

scholarly journals Proteomic analysis of extracellular vesicles secreted by primary human epithelial endometrial cells reveals key proteins related to embryo implantation

2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Marina Segura-Benítez ◽  
María Cristina Carbajo-García ◽  
Ana Corachán ◽  
Amparo Faus ◽  
Antonio Pellicer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Proteomic Analysis ◽  
Embryo Implantation ◽  
Endometrial Receptivity ◽  
Biological Processes ◽  
Endometrial Cells ◽  
Isolation Methods ◽  
Ishikawa Cells ◽  
Endometrial Epithelial Cells ◽  
Implantation Success

Abstract Background Successful implantation is dependent on coordination between maternal endometrium and embryo, and the role of EVs in the required cross-talk cell-to-cell has been recently established. In this regard, it has been reported that EVs secreted by the maternal endometrium can be internalized by human trophoblastic cells transferring their contents and enhancing their adhesive and invasive capacity. This is the first study to comprehensively evaluate three EV isolation methods on human endometrial epithelial cells in culture and to describe the proteomic content of EVs secreted by pHEECs from fertile women. Methods Ishikawa cells and pHEECs were in vitro cultured and hormonally treated; subsequently, conditioned medium was collected and EVs isolated. Ishikawa cells were used for the comparison of EVs isolation methods ultracentrifugation, ExoQuick-TC and Norgen Cell Culture Media Exosome Purification Kit (n = 3 replicates/isolation method). pHEECs were isolated from endometrial biopsies (n = 8/replicate; 3 replicates) collected from healthy oocyte donors with confirmed fertility, and protein content of EVs isolated by the most efficient methodology was analysed using liquid chromatography–tandem mass spectrometry. EV concentration and size were analyzed by nanoparticle tracking analysis, EV morphology visualized by transmission electron microscopy and protein marker expression was determined by Western blotting. Results Ultracentrifugation was the most efficient methodology for EV isolation from medium of endometrial epithelial cells. EVs secreted by pHEECs and isolated by ultracentrifugation were heterogeneous in size and expressed EV protein markers HSP70, TSG101, CD9, and CD81. Proteomic analysis identified 218 proteins contained in these EVs enriched in biological processes involved in embryo implantation, including cell adhesion, differentiation, communication, migration, extracellular matrix organization, vasculature development, and reproductive processes. From these proteins, 82 were selected based on their functional relevance in implantation success as possible implantation biomarkers. Conclusions EV protein cargos are implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs. Identified proteins may define new biomarkers of endometrial receptivity and implantation success.

scholarly journals Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

Reproduction ◽  
2019 ◽  
pp. 53-64 ◽  
Author(s):  
Yumiko Miyazaki ◽  
Akihito Horie ◽  
Hirohiko Tani ◽  
Masashi Ueda ◽  
Asuka Okunomiya ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Chondroitin Sulfate ◽  
Cell Line ◽  
Human Embryo ◽  
Embryo Implantation ◽  
Cancer Cell Line ◽  
Promoting Effect ◽  
Ishikawa Cells ◽  
Endometrial Epithelial Cells

The endometrium extracellular matrix (ECM) is essential for embryo implantation. Versican, a large chondroitin sulfate proteoglycan that binds hyaluronan and forms large ECM aggregates, can influence fundamental physiological phenomena, such as cell proliferation, adhesion and migration. The present study investigated the possible role of versican in human embryo implantation. Versican V1 expression and secretion in human endometrial epithelial cells (EECs) was most prominent in the mid-secretory phase. Versican expression in EECs significantly increased after treatment with estrogen and progesterone, but not by estrogen alone. We also established versican V1-overexpressing Ishikawa (endometrial cancer cell line) cells (ISKW-V1), versican V3-overexpressing (ISKW-V3) and control GFP-overexpressing (ISKW-GFP) Ishikawa cells. By the in vitro implantation model, the attachment ratio of BeWo (choriocarcinoma cell line) spheroids to the monolayer of ISKW-V1, but not of ISKW-V3, was found significantly enhanced compared with attachment to the ISKW-GFP monolayer. The conditioned medium derived from ISKW-V1 (V1-CM) also promoted the attachment of BeWo spheroids to the ISKW monolayer. However, this attachment-promoting effect was abolished when V1-CM was pretreated with chondroitinase ABC, which degrades chondroitin sulfate. Therefore, out of the ECM components, versican V1 may facilitate human embryo implantation.

P–379 Human platelet lysate improves trophoblast spheroid attachment to primary endometrial epithelial cells from patients with recurrent implantation failure

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
T T T N Nguyen ◽  
Y S S Kwok ◽  
S Russell ◽  
C Librach
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Fluorescent Microscopy ◽  
Endometrial Receptivity ◽  
Platelet Lysate ◽  
Implantation Failure ◽  
Trophoblast Cells ◽  
Recurrent Implantation Failure ◽  
Endometrial Epithelial Cells

Abstract Study question Could non-autologous platelet lysate (PL) increase attachment of HTR–8 spheroids in vitro to primary endometrial epithelial cells (EECs) from patients with recurrent implantation failure (RIF)? Summary answer Increased quantity of HTR–8 spheroids attached to primary EECs, isolated from patients with RIF, suggests in vitro treatment with non-autologous PL could improve endometrial receptivity. What is known already Inadequate endometrial receptivity and thickness are major causes for RIF. Recent studies suggest that platelet-rich plasma (PRP) may improve pregnancy outcomes for RIF and/or thin endometrium (TE) patients. Our previous results show that a commercially sourced and non-autologous human PRP/PL (HPL) promotes EC proliferation in vitro, suggesting that HPL may help to standardize future clinical treatments. In addition to EC proliferation, HPL treatment may improve embryo attachment to primary EECs isolated from patients with a history of RIF. In vitro attachment assays with trophoblast spheroids (embryo model) could help elucidate the effect of HPL on endometrial receptivity in RIF patients. Study design, size, duration Endometrial tissue was collected from nine RIF patients at the CReATe Fertility Centre, Toronto, Canada (Veritas REB#16580): five with (RIF+TE) and four without a TE (RIF only). Primary EECs were enzymatically isolated and treated with serum-free culture media (SFM) or 1% HPL in SFM for 48 hours before performing the attachment assay. Trophoblast cells (HTR–8/SVneo) were grown in suspension on a rocker to form 70–100 uM spheroids over 24 hours before use in the assay. Participants/materials, setting, methods Spheroids were fluorescently labelled with calcein-AM for 30 minutes and size-selected to capture spheroids similar in size to a human blastocyst. Spheroids were seeded on top of EEC monolayers and calcein fluorescence was immediately measured by a spectrophotometer. Following the 1-hour incubation, unattached spheroids were aspirated, and fluorescence was measured again. Spheroids were also individually quantified by fluorescent microscopy and ImageJ™ software. The percentage of spheroid attachment was calculated for calcein fluorescence and ImageJ™ quantification. Main results and the role of chance The HTR–8/SVneo cell line, derived from human first-trimester extravillous trophoblast cells (EVT), has been shown to be a suitable cell line to assess adhesion and invasion in vitro. Trophoblast spheroids generated from this cell line visually resembled a blastocyst and maintained expression of the EVT and implantation biomarkers: GATA3, ITGA5, and LIF. Primary EECs, treated for 48 hours with SFM supplemented with 1% commercially sourced and non-autologous HPL, overall exhibited increased attachment to HTR–8 spheroids. The percentage of spheroid attachment, as measured by fluorescence alone, significantly increase from 47.98% to 64.27% (P < 0.01) of seeded spheroids in RIF+TE EEC cultures, and from 48.12% to 85.77% (P < 0.001) of seeded spheroids in RIF only EEC cultures. Quantification by fluorescent microscopy and ImageJ™ software for individual calcein-stained spheroids, revealed a significant increase in spheroid attachment, from 57.52% to 86.5% (P < 0.01) in RIF+TE EEC cultures, and from 42.58% to 68.90% (P < 0.01) in RIF only EEC cultures. Limitations, reasons for caution Although there was a positive correlation between calcein fluorescence and spheroid quantity, quantification by fluorescence alone may be unreliable due to the variable numbers of cells in each spheroid. Our data suggest a more precise increase in attachment is detected when quantified by fluorescent microscopy and ImageJ™ software. Wider implications of the findings: We report a method for functional assessment of endometrial receptivity in vitro. HPL appears to promote implantation in RIF patients in a model of embryo attachment. We predict that the observed increase in attachment is due to increased endometrial receptivity gene expression, which will be our next investigative avenue. Trial registration number N/A

scholarly journals FBXO18/Circrna211/Mir-431/CSF1 Axis Regulates the Establishment of Endometrial Receptivity Though Activating MAPK and CSF1R/PI3K/AKT/Mtor Pathways in Dairy Goats

2021 ◽  
Author(s):  
Lichun Yang ◽  
Xiaorui Liu ◽  
Lei Zhang ◽  
Danni Li ◽  
Guili Li ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Regulatory Network ◽  
Target Genes ◽  
Endometrial Receptivity ◽  
Physiological Status ◽  
Dairy Goats ◽  
In Vivo Models ◽  
Endometrial Epithelial Cells

Abstract Background: Endometrial epithelial cells proliferation and secretion of various cytokines have a strong impact on the formation of receptive endometrium, which is known as a physiological status that allows an activated embryo to attach to the endometrium for a limited time. Circular RNAs and miRNAs can be involved in the dynamic physiological changes of endometrium by regulating relevant functional target genes in the uterus. Our work presented here with the ultimate purpose of revealing the latent molecular mechanism of FBXO18/circRNA211/miR-431/CSF1 axis in the establishment of endometrial receptivity of dairy goats.Results: In vitro, we found a regulatory network of FBXO18/circRNA211/miR-431/CSF1 in goat endometrial epithelial cells that circRNA211 severed as a sponge for miR-431, resulting in weakening the inhibition of miR-431 on target genes CSF1 and FBXO18. FBXO18/circRNA211/miR-431/CSF1 axis promoted the proliferation through regulating the key proteins of Ras, Raf, MEK, ERK in MAPK pathway via CCK-8, EdU, flow cytometry and Western blot assays. Furthermore, FBXO18/circRNA211/miR-431/CSF1 axis activated the phosphorylation of key proteins PI3K, AKT and mTOR in PI3K-mTOR pathway by CSF1R, thereby promoting the establishment of endometrial receptivity. In vivo models, mice injected with miR-431 agomir showed that the endometrial thickness and the number of pinopodes were significantly decreased by HE staining and scanning electron microscope. Immunohistochemistry results showed that VEGF and OPN proteins were down-regulated and MUC1 protein was up-regulated under the treatment of miR-431 agomir. Further study demonstrated that miR-431 inhibited embryo implantation by impeding the establishment of endometrial receptivity.Conclusion: Ultimately, our study revealed a regulatory mechanism of FBXO18/circRNA211/miR-431/CSF1 axis in goat endometrial epithelial cells. This circRNA/miRNA/mRNA regulatory network presented here in vitro and in vivo models may provide a novel insight into the potentially regulating endometrium biological functions and promoting the formation of endometrium receptivity.

scholarly journals Enhancement of Endometrial Receptivity by Cnidium officinale through Expressing LIF and Integrins

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Tae-Wook Chung ◽  
Mi-Ju Park ◽  
Hoyoung Lee ◽  
Keuk-Jun Kim ◽  
Cheorl-Ho Kim ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Embryo Implantation ◽  
Implantation Rate ◽  
Endometrial Receptivity ◽  
Female Infertility ◽  
In Vivo Model ◽  
Cnidium Officinale ◽  
Ishikawa Cells

Improvement of endometrial receptivity is necessary for successful embryo implantation, and its impairment is associated with female infertility. In this study, we investigated the effect of the roots of Cnidium officinale Makino (CoM) on endometrial receptivity in both in vitro and in vivo model of embryo implantation. We found that CoM enhanced the adhesion of JAr cells to Ishikawa cells by stimulating expression of leukemia inhibitory factor (LIF) and integrins. In addition, blocking of LIFR using hLA or neutralization of integrins αV, β3, and β5 using antibodies significantly reduced the enhanced adhesion between JAr cell and CoM-treated Ishikawa cells, indicating that LIF and integrin play an important role in trophoblast-endometrium adhesion for embryo implantation. Furthermore, we identified that CoM significantly improved the implantation rate of blastocysts in the mouse model of RU-induced implantation failure. By collecting these results, here, we suggest that CoM has a therapeutic potential against female infertility associated with decreased endometrial receptivity.

Nano-Curcumin Regulates p53 Phosphorylation and PAI-1 Expression during Bleomycin Induced Injury in Alveolar Basal Epithelial Cells

2020 ◽  
Vol 16 (1) ◽  
pp. 85-89
Author(s):  
Mahesh M. Gouda ◽  
Ashwini Prabhu ◽  
Varsha Reddy S.V. ◽  
Rafa Jahan ◽  
Yashodhar P. Bhandary
Keyword(s):  
Epithelial Cells ◽  
Lung Injury ◽  
Molecular Mechanisms ◽  
A549 Cells ◽  
Plasminogen Activator Inhibitor ◽  
Underlying Mechanism ◽  
Protective Role ◽  
Alveolar Epithelial Cells ◽  
Cellular Damage

Background: Bleomycin (BLM) is known to cause DNA damage in the Alveolar Epithelial Cells (AECs). It is reported that BLM is involved in the up-regulation of inflammatory molecules such as neutrophils, macrophages, chemokines and cytokines. The complex underlying mechanism for inflammation mediated progression of lung injury is still unclear. This investigation was designed to understand the molecular mechanisms associated with p53 mediated modulation of Plasminogen Activator Inhibitor-I (PAI-I) expression and its regulation by nano-curcumin formulation. Methods: A549 cells were treated with BLM to cause the cellular damage in vitro and commercially available nano-curcumin formulation was used as an intervention. Cytotoxic effect of nano-curcumin was analyzed using Methyl Thiazolyl Tetrazolium (MTT) assay. Protein expressions were analyzed using western blot to evaluate the p53 mediated changes in PAI-I expression. Results: Nano-curcumin showed cytotoxicity up to 88.5 % at a concentration of 20 μg/ml after 48 h of treatment. BLM exposure to the cells activated the phosphorylation of p53, which in turn increased PAII expression. Nano-curcumin treatment showed a protective role against phosphorylation of p53 and PAI-I expression, which in turn regulated the fibro-proliferative phase of injury induced by bleomycin. Conclusion: Nano-curcumin could be used as an effective intervention to regulate the severity of lung injury, apoptosis of AECs and fibro-proliferation during pulmonary injury.

scholarly journals Cyr61 promotes Schwann cell proliferation and migration via αvβ3 integrin

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhenghui Cheng ◽  
Yawen Zhang ◽  
Yinchao Tian ◽  
Yuhan Chen ◽  
Fei Ding ◽  
...  
Keyword(s):  
Nerve Injury ◽  
Peripheral Nerves ◽  
Molecular Mechanisms ◽  
Αvβ3 Integrin ◽  
Promoting Effect ◽  
Ccn Family ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background Schwann cells (SCs) play a crucial role in the repair of peripheral nerves. This is due to their ability to proliferate, migrate, and provide trophic support to axon regrowth. During peripheral nerve injury, SCs de-differentiate and reprogram to gain the ability to repair nerves. Cysteine-rich 61 (Cyr61/CCN1) is a member of the CCN family of matrix cell proteins and have been reported to be abundant in the secretome of repair mediating SCs. In this study we investigate the function of Cyr61 in SCs. Results We observed Cyr61 was expressed both in vivo and in vitro. The promoting effect of Cyr61 on SC proliferation and migration was through autocrine and paracrine mechanisms. SCs expressed αvβ3 integrin and the effect of Cyr61 on SC proliferation and migration could be blocked via αvβ3 integrin. Cyr61 could influence c-Jun protein expression in cultured SCs. Conclusions In this study, we found that Cyr61 promotes SC proliferation and migration via αvβ3 integrin and regulates c-Jun expression. Our study contributes to the understanding of cellular and molecular mechanisms underlying SC’s function during nerve injury, and thus, may facilitate the regeneration of peripheral nerves after injury.

scholarly journals Effect of Slit/Robo signaling on regeneration in lung emphysema

2021 ◽  
Author(s):  
Jin-Soo Park ◽  
RyeonJin Cho ◽  
Eun-Young Kang ◽  
Yeon-Mok Oh
Keyword(s):  
Epithelial Cells ◽  
Mouse Model ◽  
Lung Epithelial Cells ◽  
Mouse Lung ◽  
Chronic Obstructive ◽  
Irreversible Damage ◽  
Lung Epithelial ◽  
And Migration ◽  
Proliferation And Migration

AbstractEmphysema, a pathological component of chronic obstructive pulmonary disease, causes irreversible damage to the lung. Previous studies have shown that Slit plays essential roles in cell proliferation, angiogenesis, and organ development. In this study, we evaluated the effect of Slit2 on the proliferation and migration of mouse lung epithelial cells and its role in regeneration in an emphysema lung mouse model. Here, we have shown that Slit2/Robo signaling contributes to the regeneration of lungs damaged by emphysema. Mouse epithelial lung cells treated with Slit2 exhibited increased proliferation and migration in vitro. Our results also showed that Slit2 administration improved alveolar regeneration in the emphysema mouse model in vivo. Furthermore, Slit2/Robo signaling increased the phosphorylation of ERK and Akt, which was mediated by Ras activity. These Slit2-mediated cellular signaling processes may be involved in the proliferation and migration of mouse lung epithelial cells and are also associated with the potential mechanism of lung regeneration. Our findings suggest that Slit2 administration may be beneficial for alveolar regeneration in lungs damaged by emphysema.

scholarly journals Endometrial regeneration with endometrial epithelium: homologous orchestration with endometrial stroma as a feeder

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ryo Yokomizo ◽  
Yukiko Fujiki ◽  
Harue Kishigami ◽  
Hiroshi Kishi ◽  
Tohru Kiyono ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Stromal Cells ◽  
Therapeutic Option ◽  
Three Dimensional ◽  
Fertility Treatment ◽  
Feeder Cells ◽  
Endometrial Stromal Cells ◽  
Thin Endometrium ◽  
Endometrial Epithelial Cells

Abstract Background Thin endometrium adversely affects reproductive success rates with fertility treatment. Autologous transplantation of exogenously prepared endometrium can be a promising therapeutic option for thin endometrium; however, endometrial epithelial cells have limited expansion potential, which needs to be overcome in order to make regenerative medicine a therapeutic strategy for refractory thin endometrium. Here, we aimed to perform long-term culture of endometrial epithelial cells in vitro. Methods We prepared primary human endometrial epithelial cells and endometrial stromal cells and investigated whether endometrial stromal cells and human embryonic stem cell-derived feeder cells could support proliferation of endometrial epithelial cells. We also investigated whether three-dimensional culture can be achieved using thawed endometrial epithelial cells and endometrial stromal cells. Results Co-cultivation with the feeder cells dramatically increased the proliferation rate of the endometrial epithelial cells. We serially passaged the endometrial epithelial cells on mouse embryonic fibroblasts up to passage 6 for 4 months. Among the human-derived feeder cells, endometrial stromal cells exhibited the best feeder activity for proliferation of the endometrial epithelial cells. We continued to propagate the endometrial epithelial cells on endometrial stromal cells up to passage 5 for 81 days. Furthermore, endometrial epithelium and stroma, after the freeze-thaw procedure and sequential culture, were able to establish an endometrial three-dimensional model. Conclusions We herein established a model of in vitro cultured endometrium as a potential therapeutic option for refractory thin endometrium. The three-dimensional culture model with endometrial epithelial and stromal cell orchestration via cytokines, membrane-bound molecules, extracellular matrices, and gap junction will provide a new framework for exploring the mechanisms underlying the phenomenon of implantation. Additionally, modified embryo culture, so-called “in vitro implantation”, will be possible therapeutic approaches in fertility treatment.

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