Apical Cell Surface Research Articles

Probing Allosteric Modulation of Membrane Receptor in the Native State by Data Mining-Integrated Tracking Microscopy

Probing Allosteric Modulation of Membrane Receptor in the Native State by Data Mining-Integrated Tracking Microscopy

Microscopic focus on ependymal cells of the spinal cord of the one-humped camel (Camelus dromedarius): Histological, immunohistochemical, and transmission microscopic study.

The current study was designed to give a complete microscopic description of the ependymal cells of the one-humped camel (Camelus dromedarius) using histological, immunohistochemical, and transmission microscopic descriptions of the ependymal cells of the fresh 35 spinal cord samples immediately after their slaughtering. In our findings, the central canal of the spinal cord was lined by multilayered stratified cuboidal or columnar ependymal cells. The ependymal cells had an irregular striated border at their free surface. The ependymal cells do not exhibit a basement membrane. The simple oval nucleus was occupied a large part of the cell with spherical mitochondria. The apical surface of the ependymal cells possesses long cilia; each cilium was bounded by an evagination of the luminal plasma membrane. Some ependymal cells had minute finger-like projections on their luminal plasma membrane. In the perinuclear zone of ependymal cells, many cristiform mitochondria, free ribosomes, and Golgi complexes usually occur. Vacuoles with homogenous and clear fluid were observed. The lateral surface of the adjacent ependymal cells exhibits several tight junctions represented by zonulae occludens and adherens. There were many desmosomes between the neighboring ependymal cells. A perinuclear whorl of filaments fills the lateral part of these ependymal cells. The ependymal cells revealed a clear immunohistochemical reaction with proliferating cell nuclear antigen and nestin stain. There were no obvious differences between the different segments of the spinal cord. Our data concluded that the ependymal cells display clear differences in anatomy as well as ultrastructure that may reflect their distinct functional activity.

COVID-19 Anosmia: High Prevalence, Plural Neuropathogenic Mechanisms, and Scarce Neurotropism of SARS-CoV-2?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen of coronavirus disease 2019 (COVID-19). It is known as a respiratory virus, but SARS-CoV-2 appears equally, or even more, infectious for the olfactory epithelium (OE) than for the respiratory epithelium in the nasal cavity. In light of the small area of the OE relative to the respiratory epithelium, the high prevalence of olfactory dysfunctions (ODs) in COVID-19 has been bewildering and has attracted much attention. This review aims to first examine the cytological and molecular biological characteristics of the OE, especially the microvillous apical surfaces of sustentacular cells and the abundant SARS-CoV-2 receptor molecules thereof, that may underlie the high susceptibility of this neuroepithelium to SARS-CoV-2 infection and damages. The possibility of SARS-CoV-2 neurotropism, or the lack of it, is then analyzed with regard to the expression of the receptor (angiotensin-converting enzyme 2) or priming protease (transmembrane serine protease 2), and cellular targets of infection. Neuropathology of COVID-19 in the OE, olfactory bulb, and other related neural structures are also reviewed. Toward the end, we present our perspectives regarding possible mechanisms of SARS-CoV-2 neuropathogenesis and ODs, in the absence of substantial viral infection of neurons. Plausible causes for persistent ODs in some COVID-19 convalescents are also examined.

Quantifying force transmission through fibroblasts: changes of traction forces under external shearing

Mammalian cells have evolved complex mechanical connections to their microenvironment, including focal adhesion clusters that physically connect the cytoskeleton and the extracellular matrix. This mechanical link is also part of the cellular machinery to transduce, sense and respond to external forces. Although methods to measure cell attachment and cellular traction forces are well established, these are not capable of quantifying force transmission through the cell body to adhesion sites. We here present a novel approach to quantify intracellular force transmission by combining microneedle shearing at the apical cell surface with traction force microscopy at the basal cell surface. The change of traction forces exerted by fibroblasts to underlying polyacrylamide substrates as a response to a known shear force exerted with a calibrated microneedle reveals that cells redistribute forces dynamically under external shearing and during sequential rupture of their adhesion sites. Our quantitative results demonstrate a transition from dipolar to monopolar traction patterns, an inhomogeneous distribution of the external shear force to the adhesion sites as well as dynamical changes in force loading prior to and after the rupture of single adhesion sites. Our strategy of combining traction force microscopy with external force application opens new perspectives for future studies of force transmission and mechanotransduction in cells.

The Rho GTPase Cell Division Cycle 42 Regulates Stereocilia Development in Cochlear Hair Cells

Stereocilia are actin-based cell protrusions on the apical surface of inner ear hair cells, playing a pivotal role in hearing and balancing sensation. The development and maintenance of stereocilia is tightly regulated and deficits in this process usually lead to hearing or balancing disorders. The Rho GTPase cell division cycle 42 (CDC42) is a key regulator of the actin cytoskeleton. It has been reported to localize in the hair cell stereocilia and play important roles in stereocilia maintenance. In the present work, we utilized hair cell-specific Cdc42 knockout mice and CDC42 inhibitor ML141 to explore the role of CDC42 in stereocilia development. Our data show that stereocilia height and width as well as stereocilia resorption are affected in Cdc42-deficient cochlear hair cells when examined at postnatal day 8 (P8). Moreover, ML141 treatment leads to planar cell polarity (PCP) deficits in neonatal hair cells. We also show that overexpression of a constitutively active mutant CDC42 in cochlear hair cells leads to enhanced stereocilia developmental deficits. In conclusion, the present data suggest that CDC42 plays a pivotal role in regulating hair cell stereocilia development.

Induction of apically mistrafficked epiregulin disrupts epithelial polarity via aberrant EGFR signaling.

In polarized MDCK cells, disruption of the tyrosine-based YXXΦ basolateral trafficking motif (Y156A) in the epidermal growth factor receptor (EGFR) ligand epiregulin (EREG), results in its apical mistrafficking and transformation in vivo. However, the mechanisms underlying these dramatic effects are unknown. Using a doxycycline-inducible system in 3D Matrigel cultures, we now show that induction of Y156A EREG in fully formed MDCK cysts results in direct and complete delivery of mutant EREG to the apical cell surface. Within 3 days of induction, ectopic lumens were detected in mutant, but not wild-type, EREG-expressing cysts. Of note, these structures resembled histological features found in subcutaneous xenografts of mutant EREG-expressing MDCK cells. These ectopic lumens formed de novo rather than budding from the central lumen and depended on metalloprotease-mediated cleavage of EREG and subsequent EGFR activity. Moreover, the most frequent EREG mutation in human cancer (R147stop) resulted in its apical mistrafficking in engineered MDCK cells. Thus, induction of EREG apical mistrafficking is sufficient to disrupt selective aspects of polarity of a preformed polarized epithelium. This article has an associated First Person interview with the first author of the paper.

Primary Cilia Mediate TSH-Regulated Thyroglobulin Endocytic Pathways.

Primary cilia are sensory organelles with a variety of receptors and channels on their membranes. Recently, primary cilia were proposed to be crucial sites for exocytosis and endocytosis of vesicles associated with endocytic control of various ciliary signaling pathways. Thyroglobulin (Tg) synthesis and Tg exocytosis/endocytosis are critical for the functions of thyroid follicular cells, where primary cilia are relatively well preserved. LRP2/megalin has been detected on the apical surface of absorptive epithelial cells, including thyrocytes. LRP2/megalin on thyrocytes serves as a Tg receptor and can mediate Tg endocytosis. In this study, we investigated the role of primary cilia in LRP2/megalin expression in thyroid gland stimulated with endogenous TSH using MMI-treated and Tg-Cre;Ift88flox/flox mice. LRP2/megalin expression in thyroid follicles was higher in MMI-treated mice than in untreated control mice. MMI-treated mice exhibited a significant increase in ciliogenesis in thyroid follicular cells relative to untreated controls. Furthermore, MMI-induced ciliogenesis accompanied increases in LRP2/megalin expression in thyroid follicular cells, in which LRP2/megalin was localized to the primary cilium. By contrast, in Tg-Cre;Ift88flox/flox mice, thyroid with defective primary cilia expressed markedly lower levels of LRP2/megalin. Serum Tg levels were elevated in MMI-treated mice and reduced in Tg-Cre;Ift88flox/flox mice. Taken together, these results indicate that defective ciliogenesis in murine thyroid follicular cells is associated with impaired LRP2/megalin expression and reduced serum Tg levels. Our results strongly suggest that primary cilia harbors LRP2/megalin, and are involved in TSH-mediated endocytosis of Tg in murine thyroid follicles.

Above the Matrix: Functional Roles for Apically Localized Integrins.

Integrins are transmembrane proteins that are most typically thought of as integrating adhesion to the extracellular matrix with intracellular signaling and cell regulation. Traditionally, integrins are found at basolateral and lateral cell surfaces where they facilitate binding to the ECM and intercellular adhesion through cytosolic binding partners that regulate organization of actin microfilaments. However, evidence is accumulating that integrins also are apically localized, either endogenously or due to an exogenous stimulus. Apically localized integrins have been shown to regulate several processes by interacting with proteins such as connexins, tight junction proteins, and polarity complex proteins. Integrins can also act as receptors to mediate endocytosis. Here we review these newly appreciated roles for integrins localized to the apical cell surface.

Relocation of phosphofructokinases within epithelial cells is a novel event preceding breast cancer recurrence that accurately predicts patient outcomes

Although recurrent cancers can become life threatening, little is known about the intracellular events required for cancer recurrences. Due to this lack of mechanistic information, there is no test to predict cancer recurrences or nonrecurrences during early stages of disease. In this retrospective study, we use ductal carcinoma in situ of the breast as a framework to better understand the mechanism of cancer recurrences using patient outcomes as the physiological observable. Conventional pathology slides were labeled with anti-phosphofructokinase type L (PFKL) and anti-phosphofructokinase/fructose-2,6-bisphosphatase type 4 (PFKFB4) reagents. PFKL and PFKFB4 were found in ductal epithelial cell nucleoli from DCIS samples of women who did not experience a cancer recurrence. In contrast, PFKL and PFKFB4 may be found near the plasma membrane in samples from patients who will develop recurrent cancer. With the use of machine learning to predict patient outcomes, holdout studies of individual patient micrographs for the three biomarkers PFKL, PFKFB4, and phosphorylated glucose transporter 1 (GLUT1) demonstrated 38.6% true negatives, 49.5% true positives, 11.9% false positives, and 0% false negatives ( n = 101). A subpopulation of recurrent samples demonstrated PFKL, PFKFB4, and phosphorylated GLUT1 accumulation at the apical surface of epithelial cells, suggesting that carbohydrates can be harvested from the ducts’ luminal spaces as an energy source. We suggest that PFK isotype patterns are metabolic switches representing key mechanistic steps of recurrences. Furthermore, PFK enzyme patterns within epithelial cells contribute to an accurate diagnostic test to classify DCIS patients as high or low recurrence risk.

Human deafness-associated variants alter the dynamics of key molecules in hair cell stereocilia F-actin cores.

Stereocilia protrude up to 100µm from the apical surface of vertebrate inner ear hair cells and are packed with cross-linked filamentous actin (F-actin). They function as mechanical switches to convert sound vibration into electrochemical neuronal signals transmitted to the brain. Several genes encode molecular components of stereocilia including actin monomers, actin regulatory and bundling proteins, motor proteins and the proteins of the mechanotransduction complex. A stereocilium F-actin core is a dynamic system, which is continuously being remodeled while maintaining an outwardly stable architecture under the regulation of F-actin barbed-end cappers, severing proteins and crosslinkers. The F-actin cores of stereocilia also provide a pathway for motor proteins to transport cargos including components of tip-link densities, scaffolding proteins and actin regulatory proteins. Deficiencies and mutations of stereocilia components that disturb this "dynamic equilibrium" in stereocilia can induce morphological changes and disrupt mechanotransduction causing sensorineural hearing loss, best studied in mouse and zebrafish models. Currently, at least 23 genes, associated with human syndromic and nonsyndromic hearing loss, encode proteins involved in the development and maintenance of stereocilia F-actin cores. However, it is challenging to predict how variants associated with sensorineural hearing loss segregating in families affect protein function. Here, we review the functions of several molecular components of stereocilia F-actin cores and provide new data from our experimental approach to directly evaluate the pathogenicity and functional impact of reported and novel variants of DIAPH1 in autosomal-dominant DFNA1 hearing loss using single-molecule fluorescence microscopy.

Planar polarization of cilia in the zebrafish floor-plate involves Par3-mediated posterior localization of highly motile basal bodies.

Epithelial cilia, whether motile or primary, often display an off-center planar localization within the apical cell surface. This form of planar cell polarity (PCP) involves the asymmetric positioning of the ciliary basal body (BB). Using the monociliated epithelium of the embryonic zebrafish floor-plate, we investigated the dynamics and mechanisms of BB polarization by live imaging. BBs were highly motile, making back-and-forth movements along the antero-posterior (AP) axis and contacting both the anterior and posterior membranes. Contacts exclusively occurred at junctional Par3 patches and were often preceded by membrane digitations extending towards the BB, suggesting focused cortical pulling forces. Accordingly, BBs and Par3 patches were linked by dynamic microtubules. Later, BBs became less motile and eventually settled at posterior apical junctions enriched in Par3. BB posterior positioning followed Par3 posterior enrichment and was impaired upon Par3 depletion or disorganization of Par3 patches. In the PCP mutant vangl2, BBs were still motile but displayed poorly oriented membrane contacts that correlated with Par3 patch fragmentation and lateral spreading. Thus, we propose an unexpected function for posterior Par3 enrichment in controlling BB positioning downstream of the PCP pathway.

Nitric Oxide System and Bronchial Epithelium: More Than a Barrier.

Airway epithelium forms a physical barrier that protects the lung from the entrance of inhaled allergens, irritants, or microorganisms. This epithelial structure is maintained by tight junctions, adherens junctions and desmosomes that prevent the diffusion of soluble mediators or proteins between apical and basolateral cell surfaces. This apical junctional complex also participates in several signaling pathways involved in gene expression, cell proliferation and cell differentiation. In addition, the airway epithelium can produce chemokines and cytokines that trigger the activation of the immune response. Disruption of this complex by some inflammatory, profibrotic, and carcinogens agents can provoke epithelial barrier dysfunction that not only contributes to an increase of viral and bacterial infection, but also alters the normal function of epithelial cells provoking several lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) or lung cancer, among others. While nitric oxide (NO) molecular pathway has been linked with endothelial function, less is known about the role of the NO system on the bronchial epithelium and airway epithelial cells function in physiological and different pathologic scenarios. Several data indicate that the fraction of exhaled nitric oxide (FENO) is altered in lung diseases such as asthma, COPD, lung fibrosis, and cancer among others, and that reactive oxygen species mediate uncoupling NO to promote the increase of peroxynitrite levels, thus inducing bronchial epithelial barrier dysfunction. Furthermore, iNOS and the intracellular pathway sGC-cGMP-PKG are dysregulated in bronchial epithelial cells from patients with lung inflammation, fibrosis, and malignancies which represents an attractive drug molecular target. In this review we describe in detail current knowledge of the effect of NOS-NO-GC-cGMP-PKG pathway activation and disruption in bronchial epithelial cells barrier integrity and its contribution in different lung diseases, focusing on bronchial epithelial cell permeability, inflammation, transformation, migration, apoptosis/necrosis, and proliferation, as well as the specific NO molecular pathways involved.

Identification of Cilia in Different Mouse Tissues.

Cilia are microtubule-based hair-like organelles that extend from the cell surface. However, the existence and distribution of cilia in each organ and tissue at the postnatal stage in vivo remain largely unknown. In this study, we defined cilia distribution and arrangement and measured the ciliary lengths and the percentage of ciliated cells in different organs and tissues in vivo by using cilium dual reporter-expressing transgenic mice. Cilia were identified by the presence of ARL13B with an mCherry+ signal, and the cilium basal body was identified by the presence of Centrin2 with a GFP+ signal. Here, we provide in vivo evidence that chondrocytes and cells throughout bones have cilia. Most importantly, we reveal that: 1. primary cilia are present in hepatocytes; 2. no cilia but many centrioles are distributed on the apical cell surface in the gallbladder, intestine, and thyroid epithelia; 3. cilia on the cerebral cortex are well oriented, pointing to the center of the brain; 4. ARL13B+ inclusion is evident in the thyroid and islets of Langerhans; and 5. approximately 2% of cilia show irregular movement in nucleus pulposus extracellular fluid. This study reveals the existence and distribution of cilia and centrioles in different tissues and organs, and provides new insights for further comprehensive study of ciliary function in these organs and tissues.

Dissecting Out the Molecular Mechanism of Insecticidal Activity of Ostreolysin A6/Pleurotolysin B Complexes on Western Corn Rootworm.

Ostreolysin A6 (OlyA6) is a protein produced by the oyster mushroom (Pleurotus ostreatus). It binds to membrane sphingomyelin/cholesterol domains, and together with its protein partner, pleurotolysin B (PlyB), it forms 13-meric transmembrane pore complexes. Further, OlyA6 binds 1000 times more strongly to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with PlyB, OlyA6 has potent and selective insecticidal activity against the western corn rootworm. We analysed the histological alterations of the midgut wall columnar epithelium of western corn rootworm larvae fed with OlyA6/PlyB, which showed vacuolisation of the cell cytoplasm, swelling of the apical cell surface into the gut lumen, and delamination of the basal lamina underlying the epithelium. Additionally, cryo-electron microscopy was used to explore the membrane interactions of the OlyA6/PlyB complex using lipid vesicles composed of artificial lipids containing CPE, and western corn rootworm brush border membrane vesicles. Multimeric transmembrane pores were formed in both vesicle preparations, similar to those described for sphingomyelin/cholesterol membranes. These results strongly suggest that the molecular mechanism of insecticidal action of OlyA6/PlyB arises from specific interactions of OlyA6 with CPE, and the consequent formation of transmembrane pores in the insect midgut.

Trace Amine-Associated Receptor 1 Trafficking to Cilia of Thyroid Epithelial Cells.

Trace amine-associated receptor 1 (rodent Taar1/human TAAR1) is a G protein-coupled receptor that is mainly recognized for its functions in neuromodulation. Previous in vitro studies suggested that Taar1 may signal from intracellular compartments. However, we have shown Taar1 to localize apically and on ciliary extensions in rodent thyrocytes, suggesting that at least in the thyroid, Taar1 may signal from the cilia at the apical plasma membrane domain of thyrocytes in situ, where it is exposed to the content of the follicle lumen containing putative Taar1 ligands. This study was designed to explore mouse Taar1 (mTaar1) trafficking, heterologously expressed in human and rat thyroid cell lines in order to establish an in vitro system in which Taar1 signaling from the cell surface can be studied in future. The results showed that chimeric mTaar1-EGFP traffics to the apical cell surface and localizes particularly to spherical structures of polarized thyroid cells, procilia, and primary cilia upon serum-starvation. Moreover, mTaar1-EGFP appears to form high molecular mass forms, possibly homodimers and tetramers, in stably expressing human thyroid cell lines. However, only monomeric mTaar1-EGFP was cell surface biotinylated in polarized human thyrocytes. In polarized rat thyrocytes, mTaar1-EGFP is retained in the endoplasmic reticulum, while cilia were reached by mTaar1-EGFP transiently co-expressed in combination with an HA-tagged construct of the related mTaar5. We conclude that Taar1 trafficking to cilia depends on their integrity. The results further suggest that an in vitro cell model was established that recapitulates Taar1 trafficking in thyrocytes in situ, in principle, and will enable studying Taar1 signaling in future, thus extending our general understanding of its potential significance for thyroid autoregulation.

Fine-tuning multiciliated cell differentiation at the post-transcriptional level: contribution of miR-34/449 family members.

Cell differentiation is a process that must be precisely regulated for the maintenance of tissue homeostasis. Differentiation towards a multiciliated cell fate is characterized by well-defined stages, where a transcriptional cascade is activated leading to the formation of multiple centrioles and cilia. Centrioles migrate and dock to the apical cell surface and, acting as basal bodies, give rise to multiple motile cilia. The concerted movement of cilia ensures directional fluid flow across epithelia and defects either in their number or structure can lead to disease phenotypes. Micro-RNAs (miRNAs; miRs) are small, non-coding RNA molecules that play an important role in post-transcriptional regulation of gene expression. miR-34b/c and miR-449a/b/c specifically function throughout the differentiation of multiciliated cells, fine-tuning the expression of many different centriole- and cilia-related genes. They strictly regulate the expression levels of genes that are required both for commitment towards the multiciliated cell fate (e.g. Notch) and for the establishment and maintenance of this fate by regulating the expression of transcription factors and structural components of the pathway. Herein we review miR-34 and miR-449 spatiotemporal regulation along with their roles during the different stages of multiciliogenesis.

New Therapies to Correct the Cystic Fibrosis Basic Defect.

Rare diseases affect 400 million individuals worldwide and cause significant morbidity and mortality. Finding solutions for rare diseases can be very challenging for physicians and researchers. Cystic fibrosis (CF), a genetic, autosomal recessive, multisystemic, life-limiting disease does not escape this sad reality. Despite phenomenal progress in our understanding of this disease, treatment remains difficult. Until recently, therapies for CF individuals were focused on symptom management. The discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and its product, a protein present at the apical surface of epithelial cells regulating ion transport, allowed the scientific community to learn about the basic defect in CF and to study potential therapies targeting the dysfunctional protein. In the past few years, promising therapies with the goal to restore CFTR function became available and changed the lives of several CF patients. These medications, called CFTR modulators, aim to correct, potentialize, stabilize or amplify CFTR function. Furthermore, research is ongoing to develop other targeted therapies that could be more efficient and benefit a larger proportion of the CF community. The purpose of this review is to summarize our current knowledge of CF genetics and therapies restoring CFTR function, particularly CFTR modulators and gene therapy.

The filter-house of the larvacean Oikopleura dioica. A complex extracellular architecture: From fiber production to rudimentary state to inflated house.

While cellulose is the most abundant macromolecule in the biosphere, most animals are unable to produce cellulose with the exception of tunicates. Some tunicates have evolved the ability to secrete a complex house containing cellulosic fibers, yet little is known about the early stages of the house building process. Here, we investigate the rudimentary house of Oikopleura dioica for the first time using complementary light and electron microscopic techniques. In addition, we digitally modeled the arrangement of chambers, nets, and filters of the functional, expanded house in three dimensions based on life-video-imaging. Combining 3D-reconstructions based on serial histological semithin-sections, confocal laser scanning microscopy, transmission electron microscopy, scanning electron microscopy (SEM), and focused ion beam (FIB)-SEM, we were able to elucidate the arrangement of structural components, including cellulosic fibers, of the rudimentary house with a focus on the food concentration filter. We developed a model for the arrangement of folded structures in the house rudiment and show it is a precisely preformed structure with identifiable components intricately correlated with specific cells. Moreover, we demonstrate that structural details of the apical surfaces of Nasse cells provide the exact locations and shapes to produce the fibers of the house and interact among each other, with Giant Fol cells, and with the fibers to arrange them in the precise positions necessary for expansion of the house rudiment into the functional state. The presented data and hypotheses advance our knowledge about the interrelation of structure and function on different biological levels and prompt investigations into this astonishing biological object.

Morphological features of endometrial pinopodia formation during the luteal phase in women with previous unsuccessful implantation

Pinopodia of the receptive endometrium is one of the prerequisites for successful embryo implantation.Study objective: to analyze the morphological changes of the endometrium in infertile women, in particular to investigate the apical surface of epithelial cells and pinopodia formation. Materials and methods. 12 patients with a history of previous implantation failure were examined. Patients underwent hysteroscopy with biopsy under intravenous anesthesia on 20–24th day of the menstrual cycle. Morphological structure of pinopodia on the surface of the endometrial epithelium was evaluated by scanning electron microscopy.Results. Apical surface of epitheliocytes was damaged in 16.7% studied biopsies. Morphological picture and maturity of the endometrial glands did not correspond to the actual day of the menstrual cycle in 41.7% biopsies. Degree of glands maturity in these patients was low, so there was a delay in the menstrual cycle. Ultrastructural examination showed clear changes in the structure of secretory and ciliary cells in 33.3%. On the apical surface of the secretory cells there was no or small number of microvilli; cilia of eyelashes were shortened or sometimes fused together. Pinopodia as a one of the most important components of implantation was detected in only 50% of patients. Only in 33.3% of participants visualized fully developed, mature pinopodia. Conclusions. Analysis of own data showed that in the endometrium of women with previous unsuccessful implantation there are certain morphological changes, which are physiologically manifested by a delay in the luteinizing phase of the menstrual cycle and, possibly, as a result mismatch of endometrial structure and embryo maturity. Time of pinopodia onset in participants varied from 20 to 24 days of the menstrual cycle. It is necessary to improve the design and conduct further research to study the association between the expression of receptivity markers and ultrastructural structure.

Epithelial apical glycosylation changes associated with thin endometrium in women with infertility - a pilot observational study

BackgroundLow endometrial receptivity is one of the major factors affecting successful implantation in assisted reproductive technologies (ART). Infertile patients with thin endometrium have a significantly lower cumulative clinical pregnancy rate than patients with normal endometrium. Molecular pathophysiology of low receptivity of thin endometrium remains understudied. We have investigated composition of glycocalyx of the apical surface of luminal and glandular epithelial cells in thin endometrium of infertile women.MethodsThirty-two patients with tubal-peritoneal infertility undergoing in vitro fertilization (IVF) were included in the study. Endometrial samples were obtained in a natural menstrual cycle. Patients were divided into two groups: patients with normal endometrium (≥8 mm) and with thin endometrium (< 8 mm). Histochemical and immunohistochemical analysis of paraffin-embedded endometrial samples was performed using six biotinylated lectins (UEA-I, MAL-II, SNA, VVL, ECL, Con A) and anti-LeY and MECA-79 monoclonal antibodies (MAbs).ResultsComplex glycans analysis taking into account the adjusted specificity of glycan-binding MAbs revealed 1.3 times less expression of MECA-79 glycans on the apical surface of the luminal epithelial cells of thin endometrium compared to normal endometrium; this deficiency may adversely affect implantation, since MECA-79 glycans are a ligand of L-selectin and mediate intercellular interactions. The glycans containing a type-2 unit Galβ1-4GlcNAcβ (LacNAc) but lacking sulfo-residues at 6-OH of GlcNAcβ, and binding to MECA-79 MAbs were found; they can be considered as potential markers of endometrium receptivity.Expression of the lectins-stained glycans on the apical surfaces of the luminal and glandular epithelial cells did not differ significantly. Correlation between the expression of difucosylated oligosaccharide LeY on the apical surfaces of the luminal and glandular epithelial cells was found in patients with thin endometrium and recurrent implantation failure. A similar relationship was shown for mannose-rich glycans.ConclusionsSpecific features of key glycans expression in epithelial compartments of thin endometrium may be essential for morphogenesis of the endometrial functional layer and explain its low receptivity.