164. INTEGRIN CLEAVAGE IS MEDIATED BY PROPROTEIN CONVERTASE 6 IN HUMAN ENDOMETRIAL EPITHELIAL CELLS FOR ENDOMETRIAL RECEPTIVITY AND IMPLANTATION

2010 ◽  
Vol 22 (9) ◽  
pp. 82
Author(s):  
S. G. Paule ◽  
G. Nie
Keyword(s):  
Menstrual Cycle ◽  
Epithelial Cells ◽  
Critical Role ◽  
Integrin Αvβ3 ◽  
Endometrial Receptivity ◽  
Human Endometrium ◽  
Antibody Array ◽  
Integrin Expression ◽  
Sirna Sequence ◽  
Endometrial Epithelial Cells

Integrins are transmembrane glycoproteins composed of non-covalently associated α and β chains. Integrins participates in cell–cell interaction and binding to components of the extracellular matrix. Integrin expression changes during the establishment of receptivity for implantation. Integrins α1β1, α4β1 and αvβ3 are expressed in the endometrium during the window of implantation [1, 2] and deficiency in integrin αvβ3 is associated with idiopathic infertility and delayed endometrial maturation [1, 3, 4]. Proprotein convertases (PCs) cleave proproteins at the basic amino acids consensus motif (K/R)-(X)n-(K/R)↓ (where n = 0, 2, 4 or 6 and X is any aa) for activation. Integrins require post-translational cleavage for activation and are known to be cleaved by PCs. PC6 plays a critical role in the establishment of endometrial receptivity. We hypothesized that PC6 cleaves integrins for its activation in the endometrial epithelium for implantation. The aim of this study was to investigate the functional importance of PC6 in integrin cleavage, using a stable PC6-knockdown in HEC1A cells (HEC1A-HP) by siRNA technology.Cells transfected with a scrambled siRNA sequence (HEC1A-control) were used as the control. To determine the amount of functional integrins on the cell surface, HEC1A-PC6 and HEC1A-control were subjected to an integrin monoclonal antibody array. The amount of functional integrins α1, α3, α5, αv, αvβ3, β1, β3, β4 and α5β1 on the cell surfacewas much less in HEC1A-HP than HEC1A-control cells. Western blot analysis confirmed that cleavage of pro-integrin α5 into disulfide-linked heavy chain (110kDa) and light chain (35kDa) was greater in HEC1A-control compared to HEC1A-HP cells, suggesting that knockdown of PC6 affects integrin cleavage. Our studies imply that integrin cleavage is mediated by PC6 in endometrial epithelial cells for the establishment of receptivity for embryo implantation. (1) Lessey BA, et al., Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest, 1992. 90(1): 188–95.(2) Tabibzadeh S, Patterns of expression of integrin molecules in human endometrium throughout the menstrual cycle. Hum Reprod, 1992. 7(6): 876–82.(3) Lessey BA, et al., Further characterization of endometrial integrins during the menstrual cycle and in pregnancy. Fertil Steril, 1994. 62(3): 497–506.(4) Lessey BA, et al., Aberrant integrin expression in the endometrium of women with endometriosis. J Clin Endocrinol Metab, 1994. 79(2): 643–9.

Human endometrial epithelial cells grown on collagen in serum-free medium. Estrogen responsiveness and morphology

1991 ◽  
Vol 125 (1) ◽  
pp. 101-108 ◽  
Author(s):  
Bertil G. Casslén ◽  
Michael J. K. Harper
Keyword(s):  
Epithelial Cells ◽  
Primary Cultures ◽  
Collagen Matrix ◽  
Human Endometrium ◽  
Serum Free Medium ◽  
Free Medium ◽  
Endometrial Stromal Cells ◽  
Prolonged Incubation ◽  
Serum Free ◽  
Endometrial Epithelial Cells

Abstract. The aim of the study was to explore the possibility of using human endometrial epithelial cells in serum-free culture as a sensitive assay for hormonal effects on the human endometrium. Glands were isolated following enzymatic digestion of the endometrial tissue and plated on a collagen matrix. The epithelial cells were grown in either medium containing serum or in supplemented serum-free medium. No morphologic difference was found between cells grown in these two media for up to 5 days, using either light or scanning electron microscopy. Secretion of prostaglandin F2α (PGF2α) in response to estradiol was not lower in serum-free medium than in medium containing serum for the first 2 days of culture, whereas secretion declined after prolonged incubation in the serum-free medium. This response to estradiol was clearly dose-dependent, and it was further enhanced by addition of arachidonic acid, the precursor for prostaglandin synthesis, to the medium. Co-culture of endometrial stromal cells did not influence the secretion of PGF2α by epithelial cells. We conclude that the secretion of PGF2α from primary cultures of human endometrial epithelial cells grown on collagen in serum-free medium can be used for a limited period as an assay of estrogenic effects on the human endometrium.

scholarly journals The integrin coactivator Kindlin-2 plays a critical role in angiogenesis in mice and zebrafish

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4978-4987 ◽  
Author(s):  
Elzbieta Pluskota ◽  
James J. Dowling ◽  
Natalie Gordon ◽  
Jeffrey A. Golden ◽  
Dorota Szpak ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Critical Role ◽  
Integrin Αvβ3 ◽  
Tube Formation ◽  
Basement Membranes ◽  
Cytoskeletal Protein ◽  
Integrin Expression ◽  
Partial Reduction ◽  
Wild Type ◽  
Developmental Defects

Abstract Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Knockdown of kindlin-2 levels in endothelial cells resulted in defective adhesive and migratory responses, suggesting that angiogenesis might be aberrant even with partial reduction of kindlin-2. This hypothesis has now been tested in the kindlin-2+/− mice. RM1 prostate tumors grown in kindlin-2+/− mice had fewer blood vessels, which were thinner and shorter and supported less tumor growth compared with wild-type littermates. The vessels that did form in the kindlin-2+/− mice lacked smooth muscle cells and pericytes and had thinner basement membranes, indicative of immature vessels. VEGF-induced angiogenesis in matrigel implants was also abnormal in the kindlin-2+/− mice. Vessels in the kindlin-2+/− mice were leaky, and BM transplantation from kindlin-2+/− to WT mice did not correct this defect. Endothelial cells derived from kindlin-2+/− mice had integrin expression levels similar to WT mice but reduced αVβ3-dependent signaling, migration, adhesion, spreading, and tube formation. Developmental angiogenesis was markedly impaired by kindlin-2 morpholinos in zebrafish. Taken together, kindlin-2 plays an important role in pathologic and developmental angiogenesis, which arises from defective activation of integrin αVβ3.

Integrins in the endometrium

1995 ◽  
Vol 4 (1) ◽  
pp. 43-58 ◽  
Author(s):  
Bruce A Lessey ◽  
Arthur J Castelbaum
Keyword(s):  
Menstrual Cycle ◽  
Integrin Expression ◽  
Uterine Receptivity ◽  
Dynamic Changes ◽  
Histological Changes ◽  
Fibronectin Receptor ◽  
Science Activity ◽  
Different Types ◽  
Implantation Process ◽  
Endometrial Epithelial Cells

The endometrium expresses many of the same integrins displayed by other tissues. Endometrial epithelial cells maintain the ‘classic’ epithelial integrins, including α2, α3, α6, and β4, while the stroma expresses the fibronectin receptor, α5β1. During the menstrual cycle, the endometrium undergoes dynamic changes in morphology in preparation for implantation. With these histological changes are concomitant alterations in integrin expression that appear to ‘frame’ the window of implantation, by the co-expression of glandular αvβ3 and α4β1 during days 20 to 24 of the menstrual cycle. The changes in integrin expression shift from epithelial to stroma predominance late in the menstrual cycle, extending into early pregnancy. Decidual integrins that appear upregulated in pregnancy include α1β1, α3β1, α6β1 and αvβ3. Markers of uterine receptivity hold promise for a better understanding of the implantation process and may help to explain many different types of infertility. These markers will be essential for monitoring and improving infertility therapies. The importance of integrins in the human endometrium now seems well established and promises to be an area of great clinical and basic science activity in the future.

182 CALBINDIN-D28k IS PREDOMINANTLY EXPRESSED AND REGULATED BY ESTROGEN IN HUMAN ENDOMETRIUM DURING MENSTRUAL CYCLE

2011 ◽  
Vol 23 (1) ◽  
pp. 192
Author(s):  
K.-C. Choi ◽  
H. Yang ◽  
E.-B. Jeung
Keyword(s):  
Menstrual Cycle ◽  
Calcium Binding ◽  
Potential Role ◽  
Embryo Implantation ◽  
Endometrial Receptivity ◽  
Human Endometrium ◽  
Ishikawa Cell ◽  
Proliferative Phase ◽  
Calbindin D28k ◽  
The Brain

The human endometrium resists embryo implantation except during the window of receptivity. A change in endometrial gene expression is required for the development of receptivity. Uterine calbindin-D28k (CaBP-28k) has been shown to be involved in the regulation of endometrial receptivity by intracellular Ca2+. Nowadays, this protein has been mainly linked to the brain, kidneys, and pancreas, but potential role(s) of CaBP-28k remain to be clarified in the uterus of humans during the menstrual cycle. Thus, we demonstrated in this study that the expression of CaBP-28k in the human endometrium in more divided in the menstrual phases. During the menstrual cycle of humans, uterine expression levels of CaBP-28k mRNA and protein increased at the proliferative phase and fluctuated in these tissues, compared with other phases. We assessed the effects of the sex-steroid hormones E2 and P4 on the expression of CaBP-28k in the Ishikawa cell line. A significant increase in the expression of CaBP-9k mRNA was observed at the concentration of 17β-oestradiol (E2; 10–9 to 10–7 M). In addition, spatial expression of CaBP-28k was detected by immunohistochemistry. CaBP-28k is abundantly localised in the cytoplasm of the luminal and glandular epithelial cells during the menstrual cycle. Taken together, these results indicate that CaBP-28k, a uterine calcium-binding protein, is abundantly expressed in the human uterus, suggesting that uterine expression of CaBP-28k may be involved in reproductive functions during the menstrual cycle of humans.

203 PLASMA MEMBRANE Ca2+-PUMPING ATPase 1 IS ABUNDANTLY EXPRESSED AND DISTINCTLY REGULATED BY ESTROGEN IN HUMAN ENDOMETRIUM DURING THE MENSTRUAL CYCLE

2011 ◽  
Vol 23 (1) ◽  
pp. 201
Author(s):  
H. Yang ◽  
E.-B. Jeung
Keyword(s):  
Plasma Membrane ◽  
Menstrual Cycle ◽  
Critical Role ◽  
Human Endometrium ◽  
Cycle Phase ◽  
Menstrual Phase ◽  
Menstrual Cycle Phase ◽  
Secretory Phase ◽  
Total N ◽  
High Level

Plasma membrane Ca2+-pumping ATPases (PMCA) play a critical role in maintaining cellular Ca2+ homeostasis. The PMCA mRNA are encoded on 4 genes, designated PMCA1 to PMCA4. In a previous study, we found that both PMCA1 and PMCA4 are expressed at similar levels in astrocytes and in neurons. Although PMCA1b is expressed in the uterus of rats during the oestrous cycle, the expression of PMCA1 and its potential roles has not been elucidated during the menstrual cycle in the human endometrium. Thus, in the current study, the expression pattern of PMCA1 was examined to predict its roles in the human endometrium during the menstrual cycle. Human uterine tissues (total n = 40) were separated into 3 groups according to menstrual cycle phase: menstrual phase, proliferative phase (early, mid, late), and secretory phase (early, mid, late). Using real-time PCR and Western blot analysis, uterine expression of PMCA1 mRNA and protein increased to 1.5-fold in the early-, mid- and late-proliferative phases in the endometrium of the human uterus, compared with other menstrual phases. In addition, uterine PMCA1 was abundantly localised in the cytoplasm of the luminal and glandular epithelial cells in the menstrual phases, indicating that this protein may participate in the uterine Ca balance of the human endometrium during the menstrual cycle. Taken together, these results suggest that a high level of uterine PMCA1 expression may be involved in reproductive functions during the menstrual cycle of 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 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.

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