Formation Of Adherence Junctions Research Articles

P–259 Blastocyst quality is associated with both the start and the duration of compaction after ICSI

Abstract Study question Is the start and the total duration of compaction related to embryo quality? Summary answer The timing of the start, the end and the total duration of compaction are associated with blastocyst quality grade in the IVF laboratory. What is known already Preimplantation embryo development follows a programmed timeline during which a series of critical events take place. One event typically occurring on day 3/4 post fertilisation is the formation of adherence junctions between blastomeres in a process called compaction. It is considered the first morphological event in the differentiation process of the mammalian embryo. Evaluation of developmental events are used to optimize the selection of the most competent embryos for transfer and/or cryopreservation in the IVF laboratory. It has already been shown that the time of full compaction is indicative for high-quality blastocysts with a higher implantation rate. Study design, size, duration A single-centre retrospective observational study including 74 ICSI cycles performed in 2020. Injected oocytes were cultured in blastocyst medium (Origio) in the EmbryoScope + (Vitrolife) for 5/6 days. Embryos that reached the blastocyst stage were evaluated for the start of compaction, the time to reach full compaction and the total duration of compaction. These parameters were compared between good- and poor-quality blastocysts; the primary outcome parameter of the study was embryo quality. Participants/materials, setting, methods Only ICSI cycles with ejaculated fresh/frozen-thawed sperm and monitored in time-lapse incubator were included. All MNC, IVM and PGT cycles were excluded. Time zero was the start of ICSI. Good-quality embryos were full and expanded blastocysts with good-quality inner cell mass and trophectoderm (AA, AB, BA and BB according to Gardner and Schoolcraft (1999)). GraphPad Prism was used for statistical analysis. After testing for normality and homogeneity, unpaired t-test or Mann-Whitney test determined significant differences. Main results and the role of chance In this study, of the 528 included 2PN oocytes, 229 (43.4%) reached the blastocyst stage and 299 (56.6%) were arrested. Among the former, 131 (57.2%) blastocysts were classified in the good-quality group and 98 (42.8%) blastocysts in the poor-quality group. In general, human embryos compacted slowly while dividing further and the blastomeres moved during the compaction process. The start of compaction was heterogeneous (between 50.9 and 102.7 hours post ICSI; mean=80.0 hours), as well as the cell number at the initiation (between 4 and 18 blastomeres; mean=12 blastomeres). The time analysis showed that the embryos in the good-quality group started to compact significantly earlier than those in the poor-quality group (mean=78.6 vs 82.2 hours; R²=0.06; p < 0.01). We confirmed that blastocysts in the good-quality group reached full compaction earlier than those in the poor-quality group (mean=86.8 vs 93.8 hours; R²=0.17; p < 0.01). Furthermore, the total duration of compaction was significantly lower in the good-quality than in the poor-quality group (median=7.4 vs 10.7 hours; p < 0.01). Limitations, reasons for caution As this is a retrospective study, the influence of uncontrolled variables cannot be excluded. The absence of the pregnancy outcome and live birth rate is a shortcoming and will be subject of a larger patient-to-patient study. Wider implications of the findings: These results indicate that an earlier start and a shorter duration of compaction are associated with better blastocyst quality. These morphological events can be valuable additional parameters in selecting the embryo of better quality when using a time-lapse incubator. Trial registration number Not applicable

Rapgef2, a guanine nucleotide exchange factor for Rap1 small GTPases, plays a crucial role in adherence junction (AJ) formation in radial glial cells through ERK-mediated upregulation of the AJ-constituent protein expression

Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap1, characterized by possession of the Ras/Rap-associating domains and implicated in the etiology of schizophrenia. We previously found that dorsal telencephalon-specific Rapgef2 conditional knockout mice exhibits severe defects in formation of apical surface adherence junctions (AJs) and localization of radial glial cells (RGCs). In this study, we analyze the underlying molecular mechanism by using primary cultures of RGCs established from the developing cerebral cortex. The results show that Rapgef2-deficient RGCs exhibit a decreased ability of neurosphere formation, morphological changes represented by regression of radial glial (RG) fibers and reduced expression of AJ-constituent proteins such as N-cadherin, zonula occludens-1, E-cadherin and β-catenin. Moreover, siRNA-mediated knockdown of Rapgef2 or Rap1A inhibits the AJ protein expression and RG fiber formation while overexpression of Rapgef2, Rapgef6, Rap1AG12V or Rap1BG12V in Rapgef2-deficient RGCs restores them. Furthermore, Rapgef2-deficient RGCs exhibit a reduction in phosphorylation of extracellular signal-regulated kinase (ERK) leading to downregulation of the expression of c-jun, which is implicated in the AJ protein expression. These results indicate a crucial role of the Rapgef2-Rap1A-ERK-c-jun pathway in regulation of the AJ formation in RGCs.

Endothelial cell division in angiogenic sprouts of differing cellular architecture.

ABSTRACTThe vasculature of the zebrafish trunk is composed of tubes with different cellular architectures. Unicellular tubes form their lumen through membrane invagination and transcellular cell hollowing, whereas multicellular vessels become lumenized through a chord hollowing process. Endothelial cell proliferation is essential for the subsequent growth and maturation of the blood vessels. However, how cell division, lumen formation and cell rearrangement are coordinated during angiogenic sprouting has so far not been investigated at detailed cellular level. Reasoning that different tubular architectures may impose discrete mechanistic constraints on endothelial cell division, we analyzed and compared the sequential steps of cell division, namely mitotic rounding, cytokinesis, actin re-distribution and adherence junction formation, in different blood vessels. In particular, we characterized the interplay between cell rearrangement, mitosis and lumen dynamics within unicellular and multicellular tubes. The lumen of unicellular tubes becomes constricted and is ultimately displaced from the plane of cell division, where a de novo junction forms through the recruitment of junctional proteins at the site of abscission. By contrast, the new junctions separating the daughter cells within multicellular tubes form through the alteration of pre-existing junctions, and the lumen is retained throughout mitosis. We also describe variations in the progression of cytokinesis: while membrane furrowing between daughter cells is symmetric in unicellular tubes, we found that it is asymmetric in those multicellular tubes that contained a taut intercellular junction close to the plane of division. Our findings illustrate that during the course of normal development, the cell division machinery can accommodate multiple tube architectures, thereby avoiding disruptions to the vascular network.

Loss of AF-6/afadin induces cell invasion, suppresses the formation of glandular structures and might be a predictive marker of resistance to chemotherapy in endometrial cancer

BackgroundAF-6/afadin plays an important role in the formation of adherence junctions. In breast and colon cancer, loss of AF-6/afadin induces cell migration and cell invasion. We aimed to elucidate the role of AF-6/afadin in human endometrial cancer.MethodsMorphology and AF-6/afadin expression in endometrial cancer cell lines was investigated by 3-dimensional culture. We used Matrigel invasion assay to demonstrate AF-6/afadin knockdown induced invasive capability. Cell proliferation assay was performed to estimate chemoresistance to doxorubicin, paclitaxel and cisplatin induced by AF-6/afadin knockdown. The associations between AF-6/afadin expression and clinicopathological status were determined by immunohistochemical analysis in endometrial cancer tissues. Informed consent was obtained from all patients before the study.ResultsThe majority of cell clumps in 3-dimensional cultures of Ishikawa cells that strongly expressed AF-6/afadin showed round gland-like structures. In contrast, the cell clumps in 3-dimensional cultures of HEC1A and AN3CA cells—both weakly expressing AF-6/afadin—showed irregular gland-like structures and disorganized colonies with no gland-like structures, respectively. AF-6/afadin knockdown resulted in reduced number of gland-like structures in 3-dimensional cultures and enhancement of cell invasion and phosphorylation of ERK1/2 and Src in the highly AF-6/afadin-expressing endometrial cancer cell line. Inhibitors of MAPK/ERK kinase (MEK) (U0126) and Src (SU6656) suppressed the AF-6/afadin knockdown-induced invasive capability. AF-6/afadin knockdown induced chemoresistance to doxorubicin, paclitaxel and cisplatin in Ishikawa cells, not in HEC1A. Immunohistochemical analysis showed that AF-6/afadin expression was significantly associated with myometrial invasion and high histological grade.ConclusionsAF-6/afadin regulates cell morphology and invasiveness. Invasive capability is partly regulated through the ERK and Src pathway. The inhibitors to these pathways might be molecular-targeted drugs which suppress myometrial invasion in endometrial cancer. AF-6/afadin could be a useful selection marker for fertility-sparing therapy for patients with atypical hyperplasia or grade 1 endometrioid adenocarcinoma with no myometrial invasion. AF-6/afadin knockdown induced chemoresistance especially to cisplatin. Therefore, loss of AF-6/afadin might be a predictive marker of chemoresistance to cisplatin.

Nanoscale E-Cadherin Ligand Patterns Show Threshold Size for Cellular Adhesion and Adherence Junction Formation

The role of ligand spatial distribution on the formation of cadherin mediated cell-cell contacts is studied utilizing nanopatterns of E-cadherin ligands. Protein patches ranging in size from 100 to 800 nm prepared by colloidal lithography critically influence adhesion, spreading, and formation of adherence junctions in epithelial cells. Cells at 100 nm patterns show poor adhesion, while larger pattern sizes show good adhesion, significant spreading, and defined cortical actin. We estimate a threshold of 0.03 μm(2) for epithelial cellular attachment via E-Cadherin.

Distribution of prolactin receptors suggests an intraductal role for prolactin in the mouse and human mammary gland, a finding supported by analysis of signaling in polarized monolayer cultures

Despite the important role of prolactin (PRL) in mammary gland development and function, little is known about the distribution of the different forms of the prolactin receptor (PRLR) under various physiological circumstances. Here, the distribution of the long (LF) and the short (S3 in mouse) receptor common to both mice and rats was determined by immunofluorescence on frozen sections of virgin, pregnant and lactating mouse mammary gland. Myoepithelial cells were consistently and intensely stained for both receptors. For luminal cells at all stages (ducts and alveoli), a large proportion of PRLR staining was unexpectedly present on the apical face. In the non-lactating state, no basal staining of luminal cells was detectable. During lactation, a proportion of both receptors moved to the basolateral surface. In vitro, HC11 cells showed constitutive expression of LF but expression of S3 only upon the formation of adherent junctions. Tight junction formation was accelerated by incubation in pseudo-phosphorylated PRL, as measured by transepithelial resistance and the expression and placement of the tight junction protein, zonula occludens-1. Once an intact monolayer had formed, all LF and S3 receptors were apical (akin to the non-lactating state) and only apical application of PRL activated the Jak2-STAT5 and ERK pathways. By contrast, basolateral application of PRL resulted in a reduction in basal ERK phosphorylation, suggesting an involvement of a dual specificity protein phosphatase. Normal human breast samples also showed apical PRLRs. These results demonstrate important contextual aspects of PRL-PRLR interactions with implications for the analysis of the role of PRL in breast cancer.

N-Cadherin Expressing MM Cells Form Calcium-Dependent, Adherent Junctions That Likely Contribute to Focal Lesions and Nodular Growth of Multiple Myeloma Cells

AbstractAbstract 979E-cadherin-mediated adhesion regulates homeostasis in tissues of epithelial origin and homotypic N-cadherin interactions are central to the interaction of hematopoietic stem cells and the endosteal niche. Loss of E-cadherin in carcinomas is characteristic of the epithelial-to-mesenchymal transition and metastasis. The loss of E-cadherin, causes the release of b-catenin from the adherent complex, increased nuclear translocation, and increased transcriptional activity of b-catenin/TCF. We and others have demonstrated that alterations in the Wnt/b-catenin pathway exists in multiple myeloma, a malignancy of terminally differentiated antibody secreting plasma cells. The first evidence of this deregulation came from studies showing that MM cells secrete the potent Wnt/b-catenin signaling inhibitor DKK1. While it is now clear that DKK1 mediated suppression of Wnt/b-catenin in the bone marrow contributes to the decoupling of bone formation, the role of Wnt/b-catenin in normal plasma cell development and myelomagenesis is less clear and often controversial. Myeloma cells grow exclusively in the bone marrow. This growth is characterized as being interstitial or nodular, with most disease exhibiting a mixed pattern. Nodular growth, recognized as focal lesions (FL) on MRI, characterizes the conversion of MGUS to symptomatic MM. Consistently, DKK1 levels are highest in CD138 cells isolated from FL where nodular tumor growth and bone destruction may be linked by Wnt/b-catenin suppression in MM cells and local microenvironment. Given the central role of classical cadherins in promoting cell-cell adhesion and regulating b-catenin, we hypothesized that abnormal expression of cadherins might play a direct role in myelomagenesis.Cell lysates were prepared from 24 MM cell lines and from CD138+ cells from the BM of eight patients with MM. Immunoblotting was performed with antibodies specific to human N-cadherin protein. N-cadherin protein was observed in more than 82% of MM cell lines, with high protein levels in 55% of the cell lines. Similar levels of N-cadherin protein were seen in primary myeloma cells from the eight MM patients: two patients showed the highest levels of N-cadherin protein, two showed intermediate levels, and three weak levels; N-cadherin protein was absent in one patient. Similar results were obtained with three color-flow cytometry analysis of primary MM BM. To determine whether N-cadherin mediated MM cell interactions, a cell aggregation assay using GFP-expressing, N-cadherin+ MM cells observed under fluorescence microscopy was employed. When N-cadherin+ JJN3 cells were cultured in normal growth medium, they aggregated to form clusters. Similar results were observed in other MM cell lines, including OPM-2 and KMS-28-BM. Addition of a neutralizing N-cadherin antibody to these cultures significantly attenuated aggregation of JJN3 cells compared to control cells in normal medium or cells treated with control IgG. Homotypic N-cadherin interaction forms adherent junctions in a calcium dependent manner. To see if aggregation of MM cells results in the formation of adherent junctions, immunochemical staining was preformed to visualize the N-cadherin protein in OPM-2 cells. N-cadherin protein was clearly observed between myeloma cells in aggregation clusters that were significantly diminished in calcium-free medium. Taken together, these data suggest that N-cadherin induces homotypic adhesion of myeloma cells in a calcium-dependent manner and suggests that calcium release during bone resorption may enhance adherent junctions in MM that may in turn enhance plasma membrane localization of b-catenin. Studies are currently underway to determine whether DKK1 and N-cadherin adherent junctions cooperate to suppress b-catenin nuclear activity in MM cells and all converge to induce the nodular growth pattern and bone destruction often seen in N-cadherin-positive MM. Disclosures:No relevant conflicts of interest to declare.

The expressions of claudin-1 and E-cadherin in junctional epithelium

The epithelium provides an important barrier against microbial invasion. Tight junction structural proteins called claudins are known to contribute to the epithelial cell barrier. Junctional epithelium is located at a strategically important interface between gingival sulcus and is interconnected by desmosomes and gap junctions, but not by tight junctions. Although claudins are tight junction-associated proteins, they are also expressed in the epithelium despite its lack of tight junctions in invertebrates. Therefore, claudins may play an important role in junctional epithelium without tight junctions. E-cadherin is a key molecule in the formation of adherence junctions and desmosomes. In the present study, we aimed to investigate the expressions of claudin-1,claudin-3, claudin-7 and E-cadherin in the junctional epithelium of Fischer 344 rats. Gingival tissues from Fischer 344 rats were analyzed by immunohistochemical staining for claudin-1, claudin-3, claudin-7, and E-cadherin. Intense staining for claudin-1 and E-cadherin were observed in the junctional epithelium. In contrast to claudin-1, claudin-3 was mainly expressed in oral gingival epithelium and claudin-7 could not be detected on immunohistochemical analysis of the rat gingiva. These data suggest that claudin-1 and E-cadherin exist in the junctional epithelium and may play an important role in epithelial barrier function.

The CD155/poliovirus receptor enhances the proliferation of ras‐mutated cells

Stimulation of the CD155/poliovirus receptor, which localizes in the cell-matrix and at cell-cell junctions, inhibits cell adhesion and enhances cell migration. Necl-5, a mouse homolog of CD155, is implicated in the formation of adherence junctions. Recently, Necl-5 has also been found to enhance cell proliferation via the stimulation of serum and platelet-derived growth factor through the Ras-Raf-MEK-ERK signaling pathway. In our present study, we find that CD155 significantly enhances the serum-induced cell proliferation of NIH3T3 cells which have been transformed by an oncogenic Ras (V12Ras-NIH3T3), but not the parental cells. CD155 expression in V12Ras-NIH3T3 cells is also found to upregulate cyclin D2, downregulate p27(Kip1) and shorten the G0/G1 phase of the cell cycle. An inhibitor of focal adhesion kinase does not reduce this CD155-mediated enhancement of V12Ras-NIH3T3 cell proliferation. The expression of CD155DeltaCP, which lacks the cytoplasmic region including the immunoreceptor tyrosine-based inhibitory motif (ITIM), has a reduced ability to enhance the serum responsiveness of V12Ras-NIH3T3 cells, suggesting that the ITIM might be required for this effect of CD155. In addition, the overexpression of exogenous CD155 enhances the serum responsiveness of HT1080 cells, which harbor a mutant N-ras gene. On the other hand, siRNA-induced knockdown of endogenous CD155 and/or CD155DeltaCP expression significantly repress the serum responsiveness of DLD-1 cells, which express endogenous CD155 and harbor a mutant K-ras gene, suggesting that this mutant may function in a dominant negative manner. Taken together, our present data suggest that CD155, at least in part, enhances the proliferation of ras-mutated cells.

Activation of multiple signal transduction pathways by glucocorticoids: protection of ovarian follicular cells against apoptosis

We have recently demonstrated that glucocorticoids protect against serum-deprivation, cAMP-, TNFα-, and p53-induced apoptosis in ovarian follicular cells involved in up-regulation of Bcl-2. We demonstrated that dexamethasone, which enhances steroidogenesis by up-regulation of the p450scc enzyme system, stimulates the MAPK cascade by phosphorylation of ERK1, ERK2 as well as by Akt phosphorylation within 1–5 min with no effect on p38 MAPK phosphorylation. Moreover, glucocorticoids enhance expression of connexin 43, formation of gap junctions, expression of cadherins, and formation of adherence junctions within 24 h of hormone stimulation of ovarian granulosa cells. It is suggested that the protective effects of glucocorticoids against apoptosis are mediated by both genomic and non-genomic mechanisms. Moreover, for the first time we show that protein phosphorylation, cell–cell contact, and intracellular communication are important mediators in glucocorticoid protection against apoptosis in ovarian follicular cells.