The Intestinal Tract Brush Border in Young Children Uniformly Expresses Guanylate Cyclase C.

The present study examined staining of guanylate cyclase C (GCC/GUCY2C) in the small and large intestines of children younger than age 7 years. Normal intestinal tissue from children aged 0 to 7 years was stained using GCC, uroguanylin, and villin antibodies and scored for staining intensity. A subset underwent quantitative real-time polymerase chain reaction. Data were analyzed using t test of independent means, descriptive statistics, and logistic regression. Four hundred sixty-four specimens underwent immunohistochemistry; 291 specimens underwent real-time polymerase chain reaction. GCC, villin, and uroguanylin were detected across age groups and anatomic sites. No significant differences were identifiable across age groups. GUCY2C and uroguanylin mRNA was detected in all samples, with no variability of statistical significance of either target-to-villin normalization between any age cohorts. A gradient of expression of GCC across age groups does not seem to exist.

A protocol for imaging microvilli biogenesis on the surface of cultured porcine kidney epithelial cell monolayers.

A key facet of epithelial differentiation is the assembly of actin-based protrusions known as microvilli, which amplify apical membrane surface area for various cell functions. To probe mechanisms of microvillus assembly, we developed a protocol using spinning disk confocal microscopy to directly visualize microvillus biogenesis on the surface of cultured porcine kidney epithelial cell monolayers engineered to express fluorescent proteins. This protocol offers access to the molecular details of individual protrusion growth events at high spatiotemporal resolution. For complete details on the use and execution of this protocol, please refer to Gaeta et al. (2021).

Analysis of microRNA expression in brush cytology specimens improves the diagnosis of pancreatobiliary cancer.

BACKGROUND/OBJECTIVES: Malignant pancreatobiliary strictures are in many cases clinically indistinguishable and present a major problem to endoscopy specialists. Intraductal sampling procedures such as brush cytology are commonly used for diagnosis with a sensitivity that is low for a diagnostic test used in daily clinical practice. MicroRNA (miR) alterations detected in many cancers are disease-specific, which can be utilized in clinical applications. The aim of the present study was to analyze whether determination of miR expression levels in intraductal brush cytology specimens is a feasible approach to improve the diagnosis of pancreatobiliary cancer. METHODS: Brush cytology specimens have been collected during endoscopic retrograde cholangio-pancreatography (ERCP) and analyzed by routine cytology and ancillary miR assays. Total RNA was extracted using the miRNeasy Mini Kit and the expression of miRs frequently dysregulated in pancreatobiliary cancer (miR-16, miR-21, miR-196a, miR-221) were analyzed by quantitative real-time PCR using RNU6B as internal control. RESULTS: Routine cytology resulted in no false positive diagnoses, however, the combined sensitivity remained at 53.8%. Expression (ΔCt values) of miR-16 (p = 0.0039), miR-196a (p = 0.0003) and miR-221 (p = 0.0049) showed a clear statistical significance between malignant and benign pancreatobiliary specimens (n = 35). Malignancy could be detected combining routine cytology and the miR-196a single marker expression levels with a sensitivity of 84.6% (92.9% in biliary strictures) with no false positives. CONCLUSIONS: The results offer the first direct demonstration that microRNAs are readily detectable in brush cytology specimens obtained during ERCP, and have the potential to help the cytological diagnosis of pancreatobiliary malignancy.

Impact of cell-penetrating peptides (CPPs) melittin and Hiv-1 Tat on the enterocyte brush border using a mucosal explant system.

"Cell penetrating peptides" (CPPs) are natural or synthetic peptides with the ability to interact with cell membranes in order to enter cells and/or deliver cargo. They attract considerable interest as permeation enhancers for oral delivery of therapeutic drugs with poor bioavailability, such as proteins or DNA. A main barrier is the intestinal epithelium where passage needs to proceed through a paracellular -and/or a transcellular pathway. Using an organ cultured mucosal explant model system and a selection of fluorescent polar -and lipophilic tracers, the aim of the present study was to investigate the interaction of two CPPs, melittin and Hiv-1 Tat, with the enterocyte brush border. Melittin belongs to the amphipathic class of CPPs, and within 0.5-1 h it bound to, and penetrated, the enterocyte brush border, causing leakage into the cytosol and increased paracellular passage into the lamina propria. Surprisingly, melittin also abolished endocytosis of tracers from the brush border into early endosomes in the terminal web region (TWEEs), excluding any permeation enhancing effect via such an uptake mechanism. Electron microscopy revealed that melittin caused an elongation of the brush border microvilli and a reduction in their diameter. HIV-1 Tat is a cationic CPP that is internalized by cells due to a sequence, mainly of arginines, from residue 49 to 57, and a peptide containing this sequence permeabilized enterocytes to a polar tracer by a leakage into the cytosol. In conclusion, the CPPs studied acted by causing leakage of tracers into the enterocyte cytosol, not by inducing endocytosis.

Nanoscale kinetic segregation of TCR and CD45 in engaged microvilli facilitates early T cell activation.

T cells have a central function in mounting immune responses. However, mechanisms of their early activation by cognate antigens remain incompletely understood. Here we use live-cell multi-colour single-molecule localization microscopy to study the dynamic separation between TCRs and CD45 glycoprotein phosphatases in early cell contacts under TCR-activating and non-activating conditions. Using atomic force microscopy, we identify these cell contacts with engaged microvilli and characterize their morphology, rigidity and dynamics. Physical modelling and simulations of the imaged cell interfaces quantitatively capture the TCR-CD45 separation. Surprisingly, TCR phosphorylation negatively correlates with TCR-CD45 separation. These data support a refined kinetic-segregation model. First, kinetic-segregation occurs within seconds from TCR activation in engaged microvilli. Second, TCRs should be segregated, yet not removed too far, from CD45 for their optimal and localized activation within clusters. Our combined imaging and computational approach prove an important tool in the study of dynamic protein organization in cell interfaces.

Effects of fixation on electrophysiology and structure of human jejunal villi.

The villi of human jejunum vary in size and shape during different functional conditions. In the base the lamina propria is isotonic with blood, in the tip hyperosmotic. Here we study electrophysiological and morphological effects of incubation in hypotonic, isotonic, or hypertonic solutions, and to test various isotonic fixatives for microscopy. Samples of jejunal mucosae, obtained during surgery in obese patients, were studied in Ussing chambers where electrical parameters were registered during incubation in Krebs solution at various osmolarities, and during fixation in formaldehyde, glutaraldehyde, or osmium tetroxide (OsO4 ). The same fixatives were used for other jejunal specimens that were fixed directly for light microscopy. Morphometry was carried out to determine size and height of villi, proportion of lamina propria, and surface enlargement due to villi. Ussing chamber incubation in fluids with low osmolarity resulted in increased electrical resistance and epithelial swelling. Opposite results were obtained at high osmolality. Fixation was faster in formaldehyde than in glutaraldehyde or OsO4 . In biopsies processed directly for light microscopy the proportions of lamina propria of the mucosa, and of lamina propria of villi, were significantly larger in biopsies fixed in formaldehyde than after fixation in glutaraldehyde or OsO4 . The villus tips sometimes ended with a bleb with prominent spaces between the epithelial cells. In summary, jejunal villi swell in vitro when exposed to hypotonic solutions, and shrink in hypertonic solutions. Much of the morphological changes occurring during fixation can be related to the physiological hyperosmolar milieu in villus tips.

Cry6Aa1, a Bacillus thuringiensis nematocidal and insecticidal toxin, forms pores in planar lipid bilayers at extremely low concentrations and without the need of proteolytic processing.

Cry6Aa1 is a Bacillus thuringiensis (Bt) toxin active against nematodes and corn rootworm insects. Its 3D molecular structure, which has been recently elucidated, is unique among those known for other Bt toxins. Typical three-domain Bt toxins permeabilize receptor-free planar lipid bilayers (PLBs) by forming pores at doses in the 1-50 µg/ml range. Solubilization and proteolytic activation are necessary steps for PLB permeabilization. In contrast to other Bt toxins, Cry6Aa1 formed pores in receptor-free bilayers at doses as low as 200 pg/ml in a wide range of pH (5.5-9.5) and without the need of protease treatment. When Cry6Aa1 was preincubated with Western corn rootworm (WCRW) midgut juice or trypsin, 100 fg/ml of the toxin was sufficient to form pores in PLBs. The overall biophysical properties of the pores were similar for all three forms of the toxin (native, midgut juice- and trypsin-treated), with conductances ranging from 28 to 689 pS, except for their ionic selectivity, which was slightly cationic for the native and midgut juice-treated Cry6Aa1, whereas dual selectivity (to cations or anions) was observed for the pores formed by the trypsin-treated toxin. Enrichment of PLBs with WCRW midgut brush-border membrane material resulted in a 2000-fold reduction of the amount of native Cry6Aa1 required to form pores and affected the biophysical properties of both the native and trypsin-treated forms of the toxin. These results indicate that, although Cry6Aa1 forms pores, the molecular determinants of its mode of action are significantly different from those reported for other Bt toxins.

Visualizing dynamic microvillar search and stabilization during ligand detection by T cells.

During immune surveillance, T cells survey the surface of antigen-presenting cells. In searching for peptide-loaded major histocompatibility complexes (pMHCs), they must solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet and quantum dot-enabled synaptic contact mapping microscopy to show that anomalous diffusion and fractal organization of microvilli survey the majority of opposing surfaces within 1 minute. Individual dwell times were long enough to discriminate pMHC half-lives and T cell receptor (TCR) accumulation selectively stabilized microvilli. Stabilization was independent of tyrosine kinase signaling and the actin cytoskeleton, suggesting selection for avid TCR microclusters. This work defines the efficient cellular search process against which ligand detection takes place.

ABCC2 is associated with Bacillus thuringiensis Cry1Ac toxin oligomerization and membrane insertion in diamondback moth.

Cry1A insecticidal toxins bind sequentially to different larval gut proteins facilitating oligomerization, membrane insertion and pore formation. Cry1Ac interaction with cadherin triggers oligomerization. However, a mutation in an ABC transporter gene (ABCC2) is linked to Cry1Ac resistance in Plutella xylostella. Cry1AcMod, engineered to lack helix α-1, was able to form oligomers without cadherinbinding and effectively countered Cry1Ac resistance linked to ABCC2. Here we analyzed Cry1Ac and Cry1AcMod binding and oligomerization by western blots using brush border membrane vesicles (BBMV) from a strain of P. xylostella susceptible to Cry1Ac (Geneva 88) and a strain with resistance to Cry1Ac (NO-QAGE) linked to an ABCC2 mutation. Resistance correlated with lack of specific binding and reduced oligomerization of Cry1Ac in BBMV from NO-QAGE. In contrast, Cry1AcMod bound specifically and still formed oligomers in BBMV from both strains. We compared association of pre-formed Cry1Ac oligomer, obtained by incubating Cry1Ac toxin with a Manduca sexta cadherin fragment, with BBMV from both strains. Our results show that pre-formed oligomers associate more efficiently with BBMV from Geneva 88 than with BBMV from NO-QAGE, indicating that the ABCC2 mutation also affects the association of Cry1Ac oligomer with the membrane. These data indicate, for the first time, that ABCC2 facilitates Cry1Ac oligomerization and oligomer membrane insertion in P. xylostella.

Expression of sodium-dependent dicarboxylate transporter 1 (NaDC1/SLC13A2) in normal and neoplastic human kidney.

Regulated dicarboxylate transport is critical for acid-base homeostasis, prevention of calcium nephrolithiasis, regulation of collecting duct sodium chloride transport, and the regulation of blood pressure. Although luminal dicarboxylate reabsorption via NaDC1 (SLC13A2) is believed to be the primary mechanism regulating renal dicarboxylate transport, the specific localization of NaDC1 in the human kidney is currently unknown. This study's purpose was to determine NaDC1's expression in normal and neoplastic human kidneys. Immunoblot analysis demonstrated NaDC1 expression with an apparent molecular weight of ~61 kDa. Immunohistochemistry showed apical NaDC1 immunolabel in the proximal tubule of normal human kidney tissue; well-preserved proximal tubule brush border was clearly labeled. Apical NaDC1 expression was evident throughout the entire proximal tubule, including the initial proximal convoluted tubule, as identified by origination from the glomerular tuft, and extending through the terminal of the proximal tubule, the proximal straight tubule in the outer medulla. We confirmed proximal tubule localization by colocalization with the proximal tubule specific protein, NBCe1. NaDC1 immunolabel was not detected other than in the proximal tubule. In addition, NaDC1 immunolabel was not detected in tumors of presumed proximal tubule origin, clear cell and papillary renal cell carcinoma, or in tumors of nonproximal tubule origin, oncocytoma and chromophobe carcinoma. In summary, 1) in the human kidney, apical NaDC1 immunolabel is present throughout the entire proximal tubule, and is not detectable in other renal cells; and 2) NaDC1 immunolabel is not present in renal tumors. These studies provide important information regarding NaDC1's role in human dicarboxylate metabolism.

NHERF1 in Microvilli of Vomeronasal Sensory Neurons.

In most mammals, the vomeronasal system detects a variety of (semio)chemicals that mediate olfactory-driven social and sexual behaviors. Vomeronasal chemosensation depends on G protein-coupled receptors (V1R, V2R, and FPR-rs) that operate at remarkably low stimulus concentrations, thus, indicating a highly sensitive and efficient signaling pathway. We identified the PDZ domain-containing protein, Na+/H+ exchanger regulatory factor-1 (NHERF1), as putative molecular organizer of signal transduction in vomeronasal neurons. NHERF1 is a protein that contains 2 PDZ domains and a carboxy-terminal ezrin-binding domain. It localizes to microvilli of vomeronasal sensory neurons and interacts with V1Rs. Furthermore, NHERF1 and Gαi2 are closely colocalized. These findings open up new aspects of the functional organization and regulation of vomeronasal signal transduction by PDZ scaffolding proteins.

Glycol chitosan: A stabilizer of lipid rafts in the intestinal brush border.

Chitosan is a polycationic polysaccharide consisting of ß-(1-4)-linked glucosamine units and due to its mucoadhesive properties, chemical derivatives of chitosan are potential candidates as enhancers for transmucosal drug delivery. Recently, glycol chitosan (GC), a soluble derivative of chitosan, was shown to bind specifically to lipid raft domains in model bilayers. The small intestinal brush border membrane has a unique lipid raft composition with high amounts of glycolipids cross-linked by lectins, and the aim of the present work therefore was to study the interaction of FITC-conjugated GC (FITC-GC) with the small intestinal epithelium. Using organ culture of pig jejunal mucosal explants as a model system, we observed widespread binding of luminal FITC-GC to the brush border. Only little uptake via constitutive endocytosis into apical early endosomes occurred, unless endocytosis was induced by the simultaneous presence of cholera toxin B subunit (CTB). Biochemically, GC bound to microvillus membrane vesicles and caused a change in the density profile of detergent resistant membranes (DRMs). Collectively, the results showed that FITC-GC binds passively to lipid raft domains in the brush border, i.e. without inducing endocytosis like CTB. Instead, and unlike CTB, FITC-GC seems to exert a stabilizing, detergent-protective effect on the lipid raft organization of the brush border.

Restoring effect of selenium on the molecular content, structure and fluidity of diabetic rat kidney brush border cell membrane.

Diabetic kidney disease (DKD) is a dominant factor standing for kidney impairments during diabetes. In this study, attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was used to disclose the diabetes-induced structural changes in the kidney and evaluate the effects of selenium on diabetes. The increase in the area of the olefinic band indicated increased amount of lipid peroxidation end products in diabetic kidney brush border cell membrane. Moreover, saturated lipid content of this cell membrane considerably diminished. DKD was found to disrupt lipid order and cause a decrease in membrane dynamics. However, the administration of selenium at low and medium doses was shown to improve these conditions by changing the lipid contents toward control values, restoring the ordered structure of the lipids and membrane dynamics. Curve-fitting and artificial neural network (ANN) analyses of secondary structures of proteins demonstrated a relative increase in α-helix and reduction in the ß-sheet during diabetes in comparison to the control group, which were ameliorated following selenium treatment at low and medium doses. These findings were further confirmed by applying hierarchical cluster analysis (HCA) and principal component analysis (PCA). A clear separation of the experimental groups was obtained with high heterogeneity in the lipid and protein regions. These chemometric analyses showed that the low and medium doses of selenium-treated diabetic groups are successfully segregated from the diabetic group and clustered closer to the control. The study suggests that medium and, more predominantly, low-dose selenium treatment can be efficient in eliminating diabetes-induced structural alterations.

Fluid shear triggers microvilli formation via mechanosensitive activation of TRPV6.

Microvilli are cellular membrane protrusions present on differentiated epithelial cells, which can sense and interact with the surrounding fluid environment. Biochemical and genetic approaches have identified a set of factors involved in microvilli formation; however, the underlying extrinsic regulatory mechanism of microvilli formation remains largely unknown. Here we demonstrate that fluid shear stress (FSS), an external mechanical cue, serves as a trigger for microvilli formation in human placental trophoblastic cells. We further reveal that the transient receptor potential, vanilloid family type-6 (TRPV6) calcium ion channel plays a critical role in flow-induced Ca(2+) influx and microvilli formation. TRPV6 regulates phosphorylation of Ezrin via a Ca(2+)-dependent phosphorylation of Akt; this molecular event is necessary for microvillar localization of Ezrin in response to FSS. Our findings provide molecular insight into the microvilli-mediated mechanoresponsive cellular functions, such as epithelial absorption, signal perception and mechanotransduction.

Localization of cystic fibrosis transmembrane conductance regulator signaling complexes in human salivary gland striated duct cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent protein kinase (PKA)-regulated Cl(-) channel, crucial for epithelial cell regulation of salt and water transport. Previous studies showed that ezrin, an actin binding and A-kinase anchoring protein (AKAP), facilitates association of PKA with CFTR. We used immunohistochemistry and immunogold transmission electron microscopy to localize CFTR, ezrin, and PKA type II regulatory (RII) and catalytic (C) subunits in striated duct cells of human parotid and submandibular glands. Immunohistochemistry localized the four proteins mainly to the apical membrane and the apical cytoplasm of striated duct cells. In acinar cells, ezrin localized to the luminal membrane, and PKA RII subunits were present in secretory granules, as previously described. Immunogold labeling showed that CFTR and PKA RII and C subunits were localized to the luminal membrane and associated with apical granules and vesicles of striated duct cells. Ezrin was present along the luminal membrane, on microvilli and along the junctional complexes between cells. Double labeling showed specific protein associations with apical granules and vesicles and along the luminal membrane. Ezrin, CFTR, and PKA RII and C subunits are co-localized in striated duct cells, suggesting the presence of signaling complexes that serve to regulate CFTR activity.

Molecular mechanism of regulation of villus cell Na-K-ATPase in the chronically inflamed mammalian small intestine.

Na-K-ATPase located on the basolateral membrane (BLM) of intestinal epithelial cells provides a favorable intracellular Na+ gradient to promote all Na dependent co-transport processes across the brush border membrane (BBM). Down-regulation of Na-K-ATPase activity has been postulated to alter the absorption via Na-solute co-transporters in human inflammatory bowel disease (IBD). Further, the altered activity of a variety of Na-solute co-transporters in intact villus cells has been reported in animal models of chronic enteritis. But the molecular mechanism of down-regulation of Na-K-ATPase is not known. In the present study, using a rabbit model of chronic intestinal inflammation, which resembles human IBD, Na-K-ATPase in villus cells was shown to decrease. The relative mRNA abundance of α-1 and ß-1 subunits was not altered in villus cells during chronic intestinal inflammation. Similarly, the protein levels of these subunits were also not altered in villus cells during chronic enteritis. However, the BLM concentration of α-1 and ß-1 subunits was diminished in the chronically inflamed intestinal villus cells. An ankyrin-spectrin skeleton is necessary for the proper trafficking of Na-K-ATPase to the BLM of the cell. In the present study, ankyrin expression was markedly diminished in villus cells from the chronically inflamed intestine resulting in depolarization of ankyrin-G protein. The decrease of Na-K-ATPase activity was comparable to that seen in ankyrin knockdown IEC-18 cells. Therefore, altered localization of Na-K-ATPase as a result of transcriptional down-regulation of ankyrin-G mediates the down-regulation of Na-K-ATPase activity during chronic intestinal inflammation.

Histopathological effects and immunolocalization of periplocoside NW from Periploca sepium Bunge on the midgut epithelium of Mythimna separata Walker larvae.

Periplocoside NW (PSNW) with pregnane glycoside skeleton is a novel insecticidal compound isolated from the root bark of Periploca sepium Bunge. This compound has a potent stomach poisoning activity against several insect pests. In this study, we observed the intoxication symptoms, investigated the histopathological effects and carried out immuno-electron microscopic localization of PSNW on the midgut epithelium of oriental armyworm Mythimna separata Walker larvae for better understanding its action mechanism against insects. Ultrastructural observations showed that cell damages caused by PSNW in the midgut of M. separata larvae are related to the degeneration of brush border microvilli. The dissolution of cytoskeletal structures in the interior and on the surface of microvilli was responsible for the decrease in size and eventual disappearance of microvilli when bubbles of cytoplasmic substances protrude into the midgut lumen of M. separata, thus resulting in cell death. The immuno-electron microscopic localization research showed that gold particle appeared on the microvilli layer of the midgut of M. separate larvae firstly. The density of gold particle gradually added with the time, and finally microvilli layer was destructed severely. Meantime, the gold particles were also presented to the intracellular organelle membrane and the organelles also were destructed. Therefore, we proposed that this membrane system on insect midgut epithelium cells is the initial acting site of PSNW against insects.

Evaluation of intestinal biopsies for pediatric enteropathy: a proposed immunohistochemical panel approach.

Congenital enteropathies are rare disorders with significant clinical consequences; however, definitive diagnosis based on morphologic assessment of duodenal biopsies with routine stains alone is often impossible. To determine the role of immunohistochemistry (IHC) in the evaluation for microvillous inclusion disease, congenital tufting enteropathy (intestinal epithelial dysplasia), and enteroendocrine cell dysgenesis, a series of duodenal biopsies from 26 pediatric patients with chronic/intractable diarrhea was retrospectively reviewed. IHC stains for CD10, EpCAM, chromogranin, and villin were performed on all biopsies, and the results were correlated with hematoxylin and eosin and ultrastructural findings using electron microscopy, when available. Biopsies from 2 patients diagnosed with microvillous inclusion disease at the time of original biopsy demonstrated diffuse CD10-positive cytoplasmic inclusions within enterocytes and normal expression of EpCAM and chromogranin. Biopsies from 3 patients, including 2 siblings with confirmed EPCAM mutations, demonstrated complete loss of EpCAM expression and normal expression of CD10 and chromogranin; electron microscopic evaluation revealed characteristic ultrastructural findings of tufting enteropathy. Biopsies from 1 patient with a confirmed NEUROG3 mutation demonstrated an absence of intestinal enteroendocrine cells by chromogranin staining, consistent with enteroendocrine cell dysgenesis. Four patients' biopsies displayed nonspecific staining patterns for CD10 and/or EpCAM with normal expression of chromogranin, and 16 patients' biopsies exhibited normal expression for all 3 markers. Villin stains demonstrated heterogenous brush border labeling with nonspecific cytoplasmic reactivity, a pattern variably present throughout the biopsy series. In conclusion, the routine use of an IHC panel of CD10, EpCAM, and chromogranin is warranted in patients meeting specific age and/or clinical criteria, as the morphologic findings of congenital enteropathies may be subtle, focal, or inapparent on routine stains.

Fatty acid composition in matrix vesicles and in microvilli from femurs of chicken embryos revealed selective recruitment of fatty acids.

Hypertrophic chondrocytes participate in matrix mineralization by releasing matrix vesicles (MVs). These MVs, by accumulating Ca(2+) and phosphate initiate the formation of hydroxyapatite. To determine the types of lipids essential for mineralization, we analyzed fatty acids (FAs) in MVs, microvilli and in membrane fractions of chondrocytes isolated from femurs of chicken embryos. The FA composition in the MVs was almost identical to that in microvilli, indicating that the MVs originated from microvilli. These fractions contained more monounsaturated FAs especially oleic acid than in membrane homogenates of chondrocytes. They were enriched in 5,8,11-eicosatrienoic acid (20:3n-9), in eicosadienoic acid (20:2n-6), and in arachidonic acid (20:4n-6). In contrast, membrane homogenates from chondrocytes were enriched in 20:1n-9, 18:3n-3, 22:5n-3 and 22:5n-6. Due to their relatively high content in MVs and to their selective recruitment within microvilli from where MV originate, we concluded that 20:2n-6 and 20:3n-9 (pooled values), 18:1n-9 and 20:4n-6 are essential for the biogenesis of MVs and for bone mineralization.

Increased expression of ERp57 in rat oocytes during meiotic maturation is associated with sperm-egg fusion.

Oocyte meiotic maturation is a developmental transition that starts during germinal-vesicle breakdown and ends at the arrest in metaphase of meiosis II. This transition is associated with changes to both the proteins that are synthesized and the abundance/distribution of post-translational modifications that are crucial for subsequent fertilization and embryogenesis. Here, we isolated and cultured rat oocytes in vitro during both metaphase of meiosis I (MI) and meiosis II (MII) stages, respectively, and then compared their proteomic profiles by high-resolution, two-dimensional gel electrophoresis (2DE) followed by mass spectrometry. We found that the expression of five proteins was up-regulated while six proteins were down-regulated when comparing MI to MII oocytes. The expression of ERp57, an endoplasmic reticulum chaperone, underwent a dramatic increase between MI and MII oocytes, and became concentrated in a dome-shaped area of the cell surface within the microvillar region. A similar profile was observed during spermatogenesis, and sperm ERp57 eventually localized to the head and flagellum surfaces, finally ending in the equatorial region of acrosome-reacted sperm. Given the localization pattern, we tested and found that a polyclonal antiserum created against recombinant rat ERp57 significantly inhibited spermatozoa from penetrating zona pellucida-free oocytes without affecting either sperm motility or the acrosome reaction. These results indicate that ERp57 expression on oocytes, and possibly sperm, plays an important physiological role during sperm-egg fusion.