Monensin-dependent and -independent mechanisms of cell-matrix adhesion

AbstractAttachment and spreading of human FL cells on a subcellular matrix (SCM) preparation made by treating confluent cell monolayers with deoxycholate are insensitive to the presence of monensin. However, if the cell suspension is surface-iodinated prior to adhesion using the LPO/H2O2 system, cell spreading on SCM is inhibited by 1 μM monensin. The suggested interpretation is that cell surface components required for cell spreading on SCM are inactivated by iodination and need replacement from intracellular reserves by a monensin-sensitive pathway. This pathway is not required in the absence of iodination when sufficient surface components (or a monensin-independent pathway of surface expression) are available. Support for this interpretation is obtained by means of double-iodination experiments in which surface-labelled cells adhere and spread, are detached and labelled a second time and then allowed to adhere again to SCM. Cell spreading in the second case is inhibited by ~ 80%, suggesting that both previously expressed and newly recruited receptors are inactivated

Hormone-regulated expression and distribution of versican in mouse uterine tissues

<p>Abstract</p> <p>Background</p> <p>Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination.</p> <p>Methods</p> <p>Uteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR.</p> <p>Results</p> <p>In diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus.</p> <p>Conclusion</p> <p>These results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma.</p

Transmembrane and truncated (SEC) isoforms of MUC1 in the human endometrium and Fallopian tube

The cell surface mucin MUC1 is expressed by endometrial epithelial cells with increased abundance in the secretory phase of the menstrual cycle, when it is found both at the apical cell surface and in secretions. This suggests the presence of a maternal cell surface glycoprotein barrier to embryo implantation, arising from the anti-adhesive property of MUC1. In previous work, we demonstrated alternatively spliced MUC1 variant forms in tumour cells. The variant MUC1/SEC lacks the transmembrane and cytoplasmic sequences found in the full-length variant. We now show that MUC1/SEC mRNA is present in endometrial carcinoma cell lines, endometrial tissue and primary cultured endometrial epithelial cells. The protein can be detected using isoform-specific antibodies in uterine flushings, suggesting release from endometrium in vivo. However, on the basis of immunolocalisation studies, MUC1/SEC also remains associated with the apical epithelial surface both in tissue and in cultured cells. Transmembrane MUC1 and MUC1/SEC are both strikingly localised to the apical surface of tubal epithelium. Thus MUC1 may contribute to the anti-adhesive character of the tubal surface, inhibiting ectopic implantation. The mechanism by which this barrier is overcome in endometrium at implantation is the subject of ongoing investigation

Altered protein O-GlcNAcylation in placentas from mothers with diabetes causes aberrant endocytosis in placental trophoblast cells

From Springer Nature via Jisc Publications RouterHistory: received 2021-05-28, accepted 2021-09-27, registration 2021-10-06, pub-electronic 2021-10-19, online 2021-10-19, collection 2021-12Publication status: PublishedAbstract: Women with pre-existing diabetes have an increased risk of poor pregnancy outcomes, including disordered fetal growth, caused by changes to placental function. Here we investigate the possibility that the hexosamine biosynthetic pathway, which utilises cellular nutrients to regulate protein function via post-translationally modification with O-linked N-acetylglucosamine (GlcNAc), mediates the placental response to the maternal metabolic milieu. Mass spectrometry analysis revealed that the placental O-GlcNAcome is altered in women with type 1 (n = 6) or type 2 (n = 6) diabetes T2D (≥ twofold change in abundance in 162 and 165 GlcNAcylated proteins respectively compared to BMI-matched controls n = 11). Ingenuity pathway analysis indicated changes to clathrin-mediated endocytosis (CME) and CME-associated proteins, clathrin, Transferrin (TF), TF receptor and multiple Rabs, were identified as O-GlcNAcylation targets. Stimulating protein O-GlcNAcylation using glucosamine (2.5 mM) increased the rate of TF endocytosis by human placental cells (p = 0.02) and explants (p = 0.04). Differential GlcNAcylation of CME proteins suggests altered transfer of cargo by placentas of women with pre-gestational diabetes, which may contribute to alterations in fetal growth. The human placental O-GlcNAcome provides a resource to aid further investigation of molecular mechanisms governing placental nutrient sensing

Apposition to endometrial epithelial cells activates mouse blastocysts for implantation.

How do interactions between blastocyst-stage embryos and endometrial epithelial cells regulate the early stages of implantation in an in vitro model?Mouse blastocyst apposition with human endometrial epithelial cells initiates trophectoderm differentiation to trophoblast, which goes on to breach the endometrial epithelium.In vitro models using mouse blastocysts and human endometrial cell lines have proven invaluable in the molecular characterisation of embryo attachment to endometrial epithelium at the onset of implantation. Genes involved in embryonic breaching of the endometrial epithelium have not been investigated in such in vitro models.This study used an established in vitro model of implantation to examine cellular and molecular interactions during blastocyst attachment to endometrial epithelial cells.Mouse blastocysts developed from embryonic day (E) 1.5 in vitro were hatched and co-cultured with confluent human endometrial adenocarcinoma-derived Ishikawa cells in serum-free medium. A scale of attachment stability based on blastocyst oscillation upon agitation was devised. Blastocysts were monitored for 48 h to establish the kinetics of implantation, and optical sectioning using fluorescence microscopy revealed attachment and invasion interfaces. Quantitative PCR was used to determine blastocyst gene expression. Data from a total of 680 mouse blastocysts are reported, with 3-6 experimental replicates. T-test and ANOVA analyses established statistical significance at P < 0.05, P < 0.01 and P < 0.001.Hatched E4.5 mouse blastocysts exhibited weak attachment to confluent Ishikawa cells over the first 24 h of co-culture, with intermediate and stable attachment occurring from 28 h (E5.5 + 4 h) in a hormone-independent manner. Attached embryos fixed after 48 h (E6.5) frequently exhibited outgrowths, characterised morphologically and with antibody markers as trophoblast giant cells (TGCs), which had breached the Ishikawa cell layer. Beginning co-culture at E5.5 also resulted in intermediate and stable attachment from E5.5 + 4 h; however, these embryos did not go on to breach the Ishikawa cell layer, even when co-culture was extended to E7.5 (P < 0.01). Blastocysts cultured from E4.5 in permeable transwell inserts above Ishikawa cells before transfer to direct co-culture at E5.5 went on to attach but failed to breach the Ishikawa cell layer by E6.5 (P < 0.01). Gene expression analysis at E5.5 demonstrated that direct co-culture with Ishikawa cells from E4.5 resulted in downregulation of trophectoderm transcription factors Cdx2 (P < 0.05) and Gata3 (P < 0.05) and upregulation of the TGC transcription factor Hand1 (P < 0.05). Co-culture with non-endometrial human fibroblasts did not alter the expression of these genes.None.The in vitro model used here combines human carcinoma-derived endometrial cells with mouse embryos, in which the cellular interactions observed may not fully recapitulate those in vivo. The data gleaned from such models can be regarded as hypothesis-generating, and research is now needed to develop more sophisticated models of human implantation combining multiple primary endometrial cell types with surrogate and real human embryos.This study implicates blastocyst apposition to endometrial epithelial cells as a critical step in trophoblast differentiation required for implantation. Understanding this maternal regulation of the embryonic developmental programme may lead to novel treatments for infertility.This work was supported by funds from the charities Wellbeing of Women (RG1442) and Diabetes UK (15/0005207), and studentship support for SCB from the Anatomical Society. No conflict of interest is declared

Studies of the dynamics of nuclear clustering in human syncytiotrophoblast

Syncytial nuclear aggregates (SNAs), clusters of nuclei in the syncytiotrophoblast of the human placenta, are increased as gestation advances and in pregnancy pathologies. The origins of increased SNAs are unclear; however, a better appreciation of the mechanism may give insight into placental ageing and factors underpinning dysfunction. We developed three models to investigate whether SNA formation results from a dynamic process of nuclear movement and to generate alternative hypotheses. SNA count and size were measured in placental explants cultured over 16 days and particles released into culture medium were quantified. Primary trophoblasts were cultured for 6 days. Explants and trophoblasts were cultured with and without cytoskeletal inhibitors. An in silico model was developed to examine the effects of modulating nuclear behaviour on clustering. In explants, neither median SNA number (108 SNA/mm(2) villous area) nor size (283 μm(2)) changed over time. Subcellular particles from conditioned culture medium showed a wide range of sizes that overlapped with those of SNAs. Nuclei in primary trophoblasts did not change position relative to other nuclei; apparent movement was associated with positional changes of the syncytial cell membrane. In both models, SNAs and nuclear clusters were stable despite pharmacological disruption of cytoskeletal activity. In silico, increased nuclear movement, adhesiveness and sites of cytotrophoblast fusion were related to nuclear clustering. The prominence of SNAs in pregnancy disorders may not result from an active process involving cytoskeleton-mediated rearrangement of syncytial nuclei. Further insights into the mechanism(s) of SNA formation will aid understanding of their increased presence in pregnancy pathologies

Targeted Delivery of Epidermal Growth Factor to the Human Placenta to Treat Fetal Growth Restriction

Placental dysfunction is the underlying cause of pregnancy complications such as fetal growth restriction (FGR) and pre-eclampsia. No therapies are available to treat a poorly functioning placenta, primarily due to the risks of adverse side effects in both the mother and the fetus resulting from systemic drug delivery. The use of targeted liposomes to selectively deliver payloads to the placenta has the potential to overcome these issues. In this study, we assessed the safety and efficacy of epidermal growth factor (EGF)-loaded, peptide-decorated liposomes to improve different aspects of placental function, using tissue from healthy control pregnancies at term, and pregnancies complicated by FGR. Phage screening identified a peptide sequence, CGPSARAPC (GPS), which selectively homed to mouse placentas in vivo, and bound to the outer syncytiotrophoblast layer of human placental explants ex vivo. GPS-decorated liposomes were prepared containing PBS or EGF (50–100 ng/mL), and placental explants were cultured with liposomes for up to 48 h. Undecorated and GPS-decorated liposomes containing PBS did not affect the basal rate of amino acid transport, human chorionic gonadotropin (hCG) release or cell turnover in placental explants from healthy controls. GPS-decorated liposomes containing EGF significantly increased amino acid transporter activity in healthy control explants, but not in placental explants from women with FGR. hCG secretion and cell turnover were unaffected by EGF delivery; however, differential activation of downstream protein kinases was observed when EGF was delivered via GPS-decorated vs. undecorated liposomes. These data indicate that targeted liposomes represent a safe and useful tool for the development of new therapies for placental dysfunction, recapitulating the effects of free EGF