scholarly journals Microtubule depolymerization attenuates WNT4/CaMKIIα signaling in mouse uterus and leads to implantation failure

Reproduction ◽  
2019 ◽  
Vol 158 (1) ◽  
pp. 47-59 ◽  
Author(s):  
Vinay Shukla ◽  
Jyoti Bala Kaushal ◽  
Rohit Kumar ◽  
Pooja Popli ◽  
Promod Kumar Agnihotri ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Embryo Implantation ◽  
Signaling Cascades ◽  
Implantation Failure ◽  
Microtubule Depolymerization ◽  
Mouse Uterus ◽  
Intracellular Ca ◽  
Related Proteins ◽  
Transcription Factor Nfat ◽  
Endometrial Epithelial Cells

Microtubule (MT) dynamics plays a crucial role in fertilization and early embryonic development; however its involvement in uterus during embryo implantation remains unclear. Herein, we report the effect of microtubule depolymerization during embryo implantation in BALB/c mice. Intrauterine treatment with depolymerizing agent nocodazole at pre-implantation phase (D4, 07:00 h) in mice resulted into mitigation in receptivity markers viz. LIF, HoxA10, Integrin-β3, IHH, WNT4 and led to pregnancy failure. MT depolymerization in endometrial epithelial cells (EECs) also inhibited the blastocyst attachment and the adhesion. The decreased expression of MT polymerization-related proteins TPPP and α/β-tubulin in luminal and glandular epithelial cells along with the alteration in morphology of pinopodes in the luminal epithelium was observed in nocodazole receiving uteri. Nocodazole treatment also led to increased intracellular Ca+2levels in EECs, which indicated that altered Ca+2homeostasis might be responsible for implantation failure. Microtubule depolymerization inhibited WNT4 and Fz-2 interaction, thereby suppressing the downstream WNT4/CaMKIIα signaling cascades calmodulin and calcineurin which led to attenuation of NF-κB transcriptional promoter activity in EECs. MT depolymerization or CaMKIIα knockdown inhibited the transcription factor NFAT and NF-κB expression along with reduced secretion of prostaglandins PGE2 and PGF2α in mouse EECs. Overall, MT depolymerization impaired the WNT4/CaMKIIα signaling and suppressed the secretion of PGE2 and PGF2α in EECs which may be responsible for implantation failure in mice.

scholarly journals Mitochondrial dysfunction induced by high estradiol concentrations in endometrial epithelial cells

2019 ◽  
Author(s):  
Chia-Hung Chou ◽  
Shee-Uan Chen ◽  
Chin-Der Chen ◽  
Chia-Tung Shun ◽  
Wen-Fen Wen ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mitochondrial Dysfunction ◽  
Embryo Implantation ◽  
Ros Production ◽  
Vitro Fertilization ◽  
Equine Chorionic Gonadotropin ◽  
In Vivo Effects ◽  
Endometrial Epithelial Cells

Abstract Context A supraphysiological estradiol (E2) concentration after ovarian stimulation is known to result in lower embryo implantation rates in in vitro fertilization (IVF). Endometrial epithelial cells (EECs) apoptosis occurs after the stimulation with high E2 concentrations, and mitochondria play important roles in cell apoptosis. Objective To investigate the mitochondrial function in EECs after the stimulation with high E2 concentrations. Materials and Methods Human EECs were purified and cultured with different E2 concentrations (10-10, 10-9, 10-8, 10-7 M) in vitro, in which 10-7 M is supraphysiologically high. Eight-week-old female mouse endometrium was obtained 5.5 days after the injection of 1.25 IU or 20 IU equine chorionic gonadotropin (eCG), roughly during the embryo implantation window, to examine the in vivo effects of high E2 concentrations on mouse EECs. Results In vivo and in vitro experiments demonstrated decreased mitochondrial DNA contents and ATP formation after EECs were stimulated with supraphysiologically high E2 concentrations than those stimulated with a physiologic E2 concentration. Less prominent immunofluorescence mitochondrial staining, fewer mitochondria number under electron microscopy, lower JC-1 aggregate/monomer ratio, and greater reactive oxygen species (ROS) production were found after EECs were stimulated with supraphysiologically high E2 concentrations. The high E2-induced ROS production was reduced when EECs were pretreated with N-acetyl-cysteine (NAC) in vitro, but remained unchanged after the pretreatment with coenzyme Q10. Conclusion High E2 concentrations increase extra-mitochondrial ROS production in EECs and subsequently result in mitochondrial dysfunction.

Annexin A2 acts as an adherent molecule under the regulation of steroids during embryo implantation

2020 ◽  
Vol 26 (11) ◽  
pp. 825-836
Author(s):  
Bing Wang ◽  
Yan Shao
Keyword(s):  
Epithelial Cells ◽  
Outer Surface ◽  
Calcium Binding ◽  
Surface Membrane ◽  
Embryo Implantation ◽  
Annexin A2 ◽  
Endometrial Cells ◽  
Membrane Bound ◽  
Endometrial Epithelial Cells ◽  
Embryo Attachment

Abstract We previously showed that annexin A2 (Axna2) was transiently expressed at the embryo-uterine luminal epithelium interface during the window of implantation and was involved in mouse embryo implantation. At the same time, Axna2 was reported to be upregulated in human receptive endometrium, which was critical for embryo attachment as an intracellular molecule. Here, we identified Axna2 as a membrane-bound molecule on human endometrial epithelial cells and trophoblast cells, and the outer surface membrane-bound Axna2 was involved in human embryo attachment. In addition, physiological levels of estrogen and progesterone increased the expression of overall Axna2 as well as that in the extracellular surface membrane protein fraction in human endometrial cells. Furthermore, p11 (or S100A10, a member of the S100 EF-hand family protein, molecular weight 11 kDa) was involved in the translocation of Axna2 to the outer surface membrane of endometrial epithelial cells without affecting its overall expression. Finally, the surface relocation of Axna2 was also dependent on cell–cell contact and calcium binding. A better understanding of the function and regulation of Axna2 in human endometrium may help us to identify a potential therapeutic target for subfertile and infertile patients.

scholarly journals Implantation Failure in Female Kiss1−/− Mice Is Independent of Their Hypogonadic State and Can Be Partially Rescued by Leukemia Inhibitory Factor

Endocrinology ◽  
2014 ◽  
Vol 155 (8) ◽  
pp. 3065-3078 ◽  
Author(s):  
Michele Calder ◽  
Yee-Ming Chan ◽  
Renju Raj ◽  
Macarena Pampillo ◽  
Adrienne Elbert ◽  
...  
Keyword(s):  
Leukemia Inhibitory Factor ◽  
Hypogonadotropic Hypogonadism ◽  
Embryo Implantation ◽  
Critical Role ◽  
Inhibitory Factor ◽  
Implantation Failure ◽  
Mouse Uterus ◽  
Female Mice ◽  
Wild Type Female ◽  
Neuroendocrine Axis

The hypothalamic kisspeptin signaling system is a major positive regulator of the reproductive neuroendocrine axis, and loss of Kiss1 in the mouse results in infertility, a condition generally attributed to its hypogonadotropic hypogonadism. We demonstrate that in Kiss1−/− female mice, acute replacement of gonadotropins and estradiol restores ovulation, mating, and fertilization; however, these mice are still unable to achieve pregnancy because embryos fail to implant. Progesterone treatment did not overcome this defect. Kiss1+/− embryos transferred to a wild-type female mouse can successfully implant, demonstrating the defect is due to maternal factors. Kisspeptin and its receptor are expressed in the mouse uterus, and we suggest that it is the absence of uterine kisspeptin signaling that underlies the implantation failure. This absence, however, does not prevent the closure of the uterine implantation chamber, proper alignment of the embryo, and the ability of the uterus to undergo decidualization. Instead, the loss of Kiss1 expression specifically disrupts embryo attachment to the uterus. We observed that on the day of implantation, leukemia inhibitory factor (Lif), a cytokine that is absolutely required for implantation in mice, is weakly expressed in Kiss1−/− uterine glands and that the administration of exogenous Lif to hormone-primed Kiss1−/− female mice is sufficient to partially rescue implantation. Taken together, our study reveals that uterine kisspeptin signaling regulates glandular Lif levels, thereby identifying a novel and critical role for kisspeptin in regulating embryo implantation in the mouse. This study provides compelling reasons to explore this role in other species, particularly livestock and humans.

scholarly journals Uterine SOX17: a key player in human endometrial receptivity and embryo implantation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sophie Kinnear ◽  
Lois A. Salamonsen ◽  
Mathias Francois ◽  
Vincent Harley ◽  
Jemma Evans
Keyword(s):  
Epithelial Cells ◽  
Clinical Investigation ◽  
Embryo Implantation ◽  
Adhesive Contact ◽  
Endometrial Receptivity ◽  
Large Numbers ◽  
Yin And Yang ◽  
Hormonal Milieu ◽  
Endometrial Epithelial Cells

Abstract The yin and yang of female fertility is a complicated issue; large numbers of women/couples desire fertility and seek assisted reproduction intervention to achieve conception, while others seek to prevent pregnancy. Understanding specific molecules which control endometrial-embryo interactions is essential for both facilitating and preventing pregnancy. SOX17 has recently emerged as an important transcription factor involved in endometrial receptivity and embryo implantation. However, studies to date have examined mouse models of pregnancy which do not necessarily translate to the human. Demonstration of a role for ‘implantation factors’ in a human system is critical to provide a rationale for in depth clinical investigation and targeting of such factors. We demonstrate that SOX17is present within the receptive human endometrium and is up-regulated within human endometrial epithelial cells by combined estrogen & progesterone, the hormonal milieu during the receptive window. SOX17 localizes to the point of adhesive contact between human endometrial epithelial cells and a human ‘embryo mimic’ model (trophectodermal spheroid). Targeting SOX17 in endometrial epithelial cells using CRISPR/Cas9 knockdown or a SOX-F family inhibitor, MCC177, significantly inhibited adhesion of an trophectodermal spheroids to the epithelial cells thereby preventing ‘implantation’. These data confirm the important role of endometrial SOX17 in human endometrial receptivity and embryo implantation.

scholarly journals Activation of SGK1 in Endometrial Epithelial Cells in Response to PI3K/AKT Inhibition Impairs Embryo Implantation

2016 ◽  
Vol 39 (5) ◽  
pp. 2077-2087 ◽  
Author(s):  
Madhuri S. Salker ◽  
Jennifer H. Steel ◽  
Zohreh Hosseinzadeh ◽  
Jaya Nautiyal ◽  
Zoe Webster ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Embryo Implantation ◽  
Transport Processes ◽  
Akt Inhibitor ◽  
Down Regulation ◽  
Uterine Lumen ◽  
Akt Activation ◽  
A Cell ◽  
Phosphoinositide 3 Kinase ◽  
Endometrial Epithelial Cells

Background: Serum & Glucocorticoid Regulated Kinase 1 (SGK1) plays a fundamental role in ion and solute transport processes in epithelia. In the endometrium, down-regulation of SGK1 during the window of receptivity facilitates embryo implantation whereas expression of a constitutively active mutant in the murine uterus blocks implantation. Methods/Results: Here, we report that treatment of endometrial epithelial cells with specific inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT activity pathway results in reciprocal activation of SGK1. Flushing of the uterine lumen of mice with a cell permeable, substrate competitive phosphatidylinositol analogue that inhibits AKT activation (AKT inhibitor III) resulted in Sgk1 phosphorylation, down-regulation of the E3 ubiquitin-protein ligase Nedd4-2, and increased expression of epithelial Na+ channels (ENaC). Furthermore, exposure of the uterine lumen to AKT inhibitor III prior to embryo transfer induced a spectrum of early pregnancy defects, ranging from implantation failure to aberrant spacing of implantation sites. Conclusion: Taken together, our data indicate that the balanced activities of two related serine/threonine kinases, AKT and SGK1, critically govern the implantation process.

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.

P–289 Progesterone receptor is not downregulated in endometrial epithelial compartment during embryo receptivity phase in assisted reproductive cycles

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
W Palomino ◽  
M P Rivas ◽  
F Argandoña ◽  
L Devoto ◽  
A Fuenets ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Progesterone Receptor ◽  
Laser Capture Microdissection ◽  
Luteal Phase ◽  
Embryo Implantation ◽  
Mrna Levels ◽  
Infertile Women ◽  
Epithelial Compartment ◽  
Laser Capture ◽  
Endometrial Epithelial Cells

Abstract Study question Is progesterone receptor (PGR) downregulation disrupted within endometrial epithelial compartment, during embryo receptivity phase in assisted reproductive technology (ART) cycles? Summary answer PGR is not downregulated in endometrial epithelial cells from ART cycles during embryo receptivity phase. What is known already Progesterone (P4) promotes the downregulation of its own progesterone receptor (PGR). During the mid-luteal phase, PGR is downregulated in endometrial epithelial cells (EEC), a critical process for embryo implantation. Embryos are unable to attach to the maternal surface when PGR expression is sustained in EEC. Non-physiologic ovarian steroid produced or employed in ART cycles may alter endometrial development compromising its receptivity. Scarce information is available whether PGR is downregulated in EEC from ARTs including ovarian stimulation for in vitro fertilization (IVF) cycles or hormonal endometrial preparation for frozen thawed embryo transfer (HEP-FET). Study design, size, duration Cross sectional study including endometrial samples from fertile women during natural cycle (FNC, n = 23), from infertile women submitted to IVF (n = 19) and from infertile women who underwent mock HEP-FET (n = 35). Samples were obtained between 2018–2019. Sample size was calculated considering a power of 90%, alpha error=0.05, an expected PGR expression of 2 and 0.5 in ART and FNC groups, respectively, having a standard deviation=0.9. At least 9 patients would be necessary in each group. Participants/materials, setting, methods Endometrial samples were obtained during mid-luteal phase scheduled 7 days after ovulation in FNC, 5 days after oocyte retrieval in IVF without embryo transfer or 5 days after P4 supplementation in HEP-FET. Immunohistochemistry was employed to quantify PGR using histologic score (Hscore). PGR mRNA levels were determined by qRT-PCR from EEC dissected by laser capture microdissection. Anova test was used for comparing means of Hscore and mRNA among groups. Statistical significance was established as P < 0.05. Main results and the role of chance No statistical differences were found in demographic characteristics including age, body mass index or endometrial thickness. The PGR expression was reduced in FNC compared to IVF and HP-FET endometria (0.6 ± 0.1, 1.9 ± 0.9 and 2.2 ± 0.9 respectively; P < 0.0001). The PGR mRNA levels from ECC dissected by laser capture microdissection were higher in IVF and HP-ET cycles compared to FNC (10.6 ± 3.1, 13.6 ± 2.3 and 0.8 ± 0.1 respectively; P < 0.0001) corroborating the elevated PGR Hscore in EEC from ART cycles. Limitations, reasons for caution This is a descriptive study reporting failure of PGR downregulation in endometria from ART cycles with vaginal P4 supplementation during the luteal-phase. Whether interference or resistance to P4 signal is the mechanism involved in the failure of PGR down regulation in ART cycles needs to be determined Wider implications of the findings: PGR downregulation within EEC was shown in FNC. The retained PGR expression detected in most ART cycles may interfere with embryo implantation and might explain the restricted pregnancy success. Future studies might reveal whether PGR evaluation in EEC can predict embryo implantation. Trial registration number Not Aplicable

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