Oocyte Transport Research Articles

Crustacean cardioactive peptides: Expression, localization, structure, and a possible involvement in regulation of egg-laying in the cuttlefish Sepia officinalis

The cuttlefish (Sepia officinalis) is a cephalopod mollusk distributed on the western European coast, in the West African Ocean and in the Mediterranean Sea. On the Normandy coast (France), cuttlefish is a target species of professional fishermen, so its reproduction strategy is of particular interest in the context of stock management. Egg-laying, which is coastal, is controlled by several types of regulators among which neuropeptides. The cuttlefish neuropeptidome was recently identified by Zatylny-Gaudin et al. (2016). Among the 38 neuropeptide families identified, some were significantly overexpressed in egg-laying females as compared to mature males.This study is focused on crustacean cardioactive peptides (CCAPs), a highly expressed neuropeptide family strongly suspected of being involved in the control of egg-laying. We investigated the functional and structural characterization and tissue mapping of CCAPs, as well as the expression patterns of their receptors. CCAPs appeared to be involved in oocyte transport through the oviduct and in mechanical secretion of capsular products. Immunocytochemistry revealed that the neuropeptides were localized throughout the central nervous system (CNS) and in the nerve endings of the glands involved in egg-capsule synthesis and secretion, i.e. the oviduct gland and the main nidamental glands. The CCAP receptor was expressed in these glands and in the subesophageal mass of the CNS. Multiple sequence alignments revealed a high level of conservation of CCAP protein precursors in Sepia officinalis and Loligo pealei, two cephalopod decapods. Primary sequences of CCAPs from the two species were fully conserved, and cryptic peptides detected in the nerve endings were also partially conserved, suggesting biological activity that remains unknown for the time being.

Delaying meiotic resumption during transportation of bovine cumulus-oocyte complexes: effects on development, apoptosis and caspases activity of in vitro-produced embryos.

This study examined the effects of meiosis inhibition during bovine oocyte transportation on developmental competence and quality of produced embryos. The transportation medium was supplemented with: 100 μM butyrolactone I (BL), 500 μM IBMX + 100 μM forskolin (mSPOM), 100 μM milrinone (MR) or follicular fluid (bFF), and was carried out in a portable incubator for 6 h. Next, oocytes were in vitro matured (IVM) for 18 h, without the meiotic inhibitors, with the exception of mSPOM group, in which was added 20 μM cilostamide. The three control groups were IVM with 10% fetal calf serum (FCS) (Control Lab FCS) or 0.6% bovine serum albumin (BSA) (Control Lab BSA) in a CO2 in air incubator or in the portable incubator with 0.6% BSA (Control Transp BSA). Higher cleavage rates (P 0.05) to the other groups (23.6 ± 3.3% to 28.8 ± 2.7%). The total number of blastomeres was higher (P 0.05) from bFF (67.7 ± 4.2). No differences (P > 0.05) were found in apoptosis by the activity of caspases (139.0 ± 3.2 to 152.4 ± 6.5, expressed in fluorescence intensity) as well as the percentage of TUNEL-positive cells (12.3 ± 2.0% to 15.7 ± 1.7%). In conclusion, the transportation of oocytes over 6 h with BL, mSPOM or bFF enabled the acquisition of developmental competence at similar rates to the Control Lab FCS group.

Are there intracellular Ca2+ oscillations correlated with flagellar beating in human sperm? A three vs. two-dimensional analysis

Are there intracellular Ca2+ ([Ca2+]i) oscillations correlated with flagellar beating in human sperm? The results reveal statistically significant [Ca2+]i oscillations that are correlated with the human sperm flagellar beating frequency, when measured in three-dimensions (3D). Fast [Ca2+]i oscillations that are correlated to the beating flagellar frequency of cells swimming in a restricted volume have been detected in hamster sperm. To date, such findings have not been confirmed in any other mammalian sperm species. An important question that has remained regarding these observations is whether the fast [Ca2+]i oscillations are real or might they be due to remaining defocusing effects of the Z component arising from the 3D beating of the flagella. Healthy donors whose semen samples fulfill the WHO criteria between the age of 18-28 were selected. Cells from at least six different donors were utilized for analysis. Approximately the same number of experimental and control cells were analyzed. Motile cells were obtained by the swim-up technique and were loaded with Fluo-4 (Ca2+ sensitive dye) or with Calcein (Ca2+ insensitive dye). Ni2+ was used as a non-specific plasma membrane Ca2+ channel blocker. Fluorescence data and flagella position were acquired in 3D. Each cell was recorded for up to 5.6 s within a depth of 16 microns with a high speed camera (coupled to an image intensifier) acquiring at a rate of 3000 frames per second, while an oscillating objective vibrated at 90 Hz via a piezoelectric device. From these samples, eight experimental and nine control sperm cells were analyzed in both 2D and 3D. We have implemented a new system that allows [Ca2+]i measurements of the human sperm flagellum beating in 3D. These measurements reveal statistically significant [Ca2+]i oscillations that correlate with the flagellar beating frequency. These oscillations may arise from intracellular sources and/or Ca2+ transporters, as they were insensitive to external Ni2+, a non-specific plasma membrane Ca2+ channel blocker. N/A. Analysis in 3D needs a very fast image acquisition rate to correctly sample a volume containing swimming sperm. This condition requires a very short exposure time per image making it necessary to use an image intensifier which also increases noise. The lengthy analysis time required to obtain reliable results limited the number of cells that could be analyzed. The possibility of recording flagellar [Ca2+]i oscillations described here may open a new avenue to better understand ciliary and flagellar beating that are fundamental for mucociliary clearance, oocyte transport, fertilization, cerebrospinal fluid pressure regulation and developmental left-right symmetry breaking in the embryonic node. This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT) (grants 253952 to G.C.; 156667 to F.M.M. and Fronteras 71 39908-Q to A.D. and Post-doctoral scholarships 366844 to P.H.-H. and 291028 to F.M.) and the Dirección General de Asuntos del Personal Académico of the Universidad Nacional Autónoma de México (DGAPA-UNAM) (grants CJIC/CTIC/4898/2016 to F.M. and IN205516 to A.D.). There are no conflicts of interest to declare.

Uricosuric targets of tranilast.

Uric acid, generated from the metabolism of purines, has both proven and emerging roles in human disease. Serum uric acid in humans is determined by production and by the net balance of reabsorption and secretion in kidney and intestine. In the human kidney, epithelial reabsorption dominates over secretion, such that in normal subjects there is at least 90% net reabsorption of filtered urate resulting in a fractional excretion of <10%. Tranilast, an anti‐inflammatory drug with pleiotropic effects, has a marked hypouricemic, uricosuric effect in humans. We report here that tranilast is a potent inhibitor of [14C]‐urate transport mediated by the major reabsorptive urate transporters (URAT1, GLUT9, OAT4, and OAT10) in Xenopus oocytes; this provides an unequivocal molecular mechanism for the drug's uricosuric effect. Tranilast was found to inhibit urate transport mediated by URAT1 and GLUT9 in a fully reversible and noncompetitive (mixed) manner. In addition, tranilast inhibits the secretory urate transporters NPT1, OAT1, and OAT3 without affecting the secretory efflux pump ABCG2. Notably, while benzbromarone and probenecid inhibited urate as well as nicotinate transport, tranilast inhibited the urate transport function of URAT1, GLUT9, OAT4, OAT10, and NPT1, without significantly affecting nicotinate transport mediated by SMCT1 (IC 50 ~1.1 mmol/L), SMCT2 (IC 50 ~1.0 mmol/L), and URAT1 (IC 50 ~178 μmol/L). In summary, tranilast causes uricosuria by inhibiting all the major reabsorptive urate transporters, selectively affecting urate over nicotinate transport. These data have implications for the treatment of hyperuricemia and gout, the pharmacology of tranilast, and the structure‐function analysis of urate transport.

Multiple cis-acting signals, some weak by necessity, collectively direct robust transport of oskar mRNA to the oocyte.

Localization of mRNAs can involve multiple steps, each with its own cis-acting localization signals and transport factors. How is the transition between different steps orchestrated? We show that the initial step in localization of Drosophila oskar mRNA - transport from nurse cells to the oocyte - relies on multiple cis-acting signals. Some of these are binding sites for the translational control factor Bruno, suggesting that Bruno plays an additional role in mRNA transport. Although transport of oskar mRNA is essential and robust, the localization activity of individual transport signals is weak. Notably, increasing the strength of individual transport signals, or adding a strong transport signal, disrupts the later stages of oskar mRNA localization. We propose that the oskar transport signals are weak by necessity; their weakness facilitates transfer of the oskar mRNA from the oocyte transport machinery to the machinery for posterior localization.

180 LONG-TERM TRANSPORTATION OF BOVINE OOCYTES WITH MEIOTIC BLOCKERS: EFFECTS ON NUCLEAR MATURATION

The aim of this study was to assess the blockade and the reversal of meiosis block in bovine oocytes treated with a cyclin-dependent kinase inhibitor (butyrolactone-I; BL) combined or not with a selective inhibitor of epidermal growth factor receptor protein (tyrphostin AG 1478; AG) in a prematuration (PM) culture during oocyte transport. Cumulus-oocyte complexes (n = 4107) were transported in PM medium (TCM-199 with bicarbonate and 0.3% BSA) supplemented with one of the following inhibitors: 50 µM BL; 100 µM BL; 1 µM AG; 50 µM BL + 1 µM AG; or 100 µM BL + 1µM AG. Cumulus-oocyte complexes were transported in well-sealed polystyrene tubes (30 oocytes/tube) containing 200 μL of PM medium covered with mineral oil and gassed with 5% O2, 5% CO2, and 90% N2. The tubes were packed in a portable incubator (Thawing Unit MT 35/42, Minitub, Tiefenbach, Germany) at 38.5°C for 22 h. Afterward, treated oocytes were removed from meiotic inhibitors, transferred to in vitro maturation (IVM) medium (TCM-199 with bicarbonate, 0.5 mg mL−1 of FSH, 100 IU mL−1 of hCG, and 10% FCS), and cultured in a bench-top incubator (Thermo Fisher Scientific, Waltham, MA, USA) under 38.5°C and 5% CO2 in air for 20, 22, 24, or 26 h. The control groups were IVM for 20, 22, 24, or 26 h in IVM medium in the bench-top incubator at 38.5°C and 5% CO2 in air (Control; C) or in the portable incubator under the same conditions used for the treated groups (Transport Control; TC). For meiosis evaluation, oocytes were stained with 1% Hoescht immediately after follicle removal (0 h), at 6 and 22 h of PM, and after 20, 22, 24, and 26 h of IVM, and were classified as immature (germinal vesicle; GV) or mature (metaphase II; MII); intermediate phases of meiosis (GV breakdown, metaphase I, anaphase I, or telophase I) were not demonstrated in this study. Data were analysed by ANOVA followed by Tukey’s test (P &lt; 0.05) and are presented as mean ± standard error of the mean. The GV rates after 6 h of transport did not differ (P &gt; 0.05) between 0-h oocytes (88.6 ± 2.3%) and the treated groups (70.3 ± 1.9% to 79.3 ± 2.2%); although GV rates of C (49.5 ± 2.4%) and TC (49.5 ± 2.4%) groups differed (P &lt; 0.05) from 0-h oocytes, they did not differ from treated oocytes with the exception of the 1 µM AG group (79.3 ± 2.2%), which differed from TC (P &lt; 0.05). After 22 h of transport, the GV rates of treated oocytes (50.3 ± 5.5 to 70.3 ± 6.6%) did not differ (P &gt; 0.05) from 0-h oocytes (88.6 ± 2.3%) and were higher (P &lt; 0.05) than C (4.6 ± 2.8%) and TC (8.3 ± 4.5%) that had the highest MII rates (68.4 ± 5.3 and 75.5 ± 2.0%, respectively, for C and TC) compared with the other groups (0 to 13.2 ± 10.2%). After meiotic inhibitors removal and IVM, meiosis block was fully reversed and there were no differences (P &gt; 0.05) in the rates of MII between treated oocytes and C and TC groups after 20 (56.6%, averaged), 22 (57.7%, averaged), 24 (66.2%, averaged), or 26 h of IVM (57.0%, averaged). In conclusion, the meiotic inhibitors were effective in maintaining the majority of treated oocytes in GV stage after 22 h of transport and the inhibitory effect was fully reverted after its removal. Research was supported by FAPESP and CAPES.

Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes.

An egg must be fertilized by a single sperm only. To prevent polyspermy, the zona pellucida, a structure that surrounds mammalian eggs, becomes impermeable upon fertilization, preventing the entry of further sperm. The structural changes in the zona upon fertilization are driven by the exocytosis of cortical granules. These translocate from the oocyte's centre to the plasma membrane during meiosis. However, very little is known about the mechanism of cortical granule translocation. Here we investigate cortical granule transport and dynamics in live mammalian oocytes by using Rab27a as a marker. We show that two separate mechanisms drive their transport: myosin Va-dependent movement along actin filaments, and an unexpected vesicle hitchhiking mechanism by which cortical granules bind to Rab11a vesicles powered by myosin Vb. Inhibiting cortical granule translocation severely impaired the block to sperm entry, suggesting that translocation defects could contribute to miscarriages that are caused by polyspermy.

Cryoprotectant and water transport in immature and mature zebrafish oocytes

Cryoprotectant and water transport in immature and mature zebrafish oocytes

An Arabidopsis ABC Transporter Mediates Phosphate Deficiency-Induced Remodeling of Root Architecture by Modulating Iron Homeostasis in Roots

The remodeling of root architecture is a major developmental response of plants to phosphate (Pi) deficiency and is thought to enhance a plant's ability to forage for the available Pi in topsoil. The underlying mechanism controlling this response, however, is poorly understood. In this study, we identified an Arabidopsis mutant, hps10 (hypersensitive to Pi starvation 10), which is morphologically normal under Pi sufficient condition but shows increased inhibition of primary root growth and enhanced production of lateral roots under Pi deficiency. hps10 is a previously identified allele (als3-3) of the ALUMINUM SENSITIVE3 (ALS3) gene, which is involved in plant tolerance to aluminum toxicity. Our results show that ALS3 and its interacting protein AtSTAR1 form an ABC transporter complex in the tonoplast. This protein complex mediates a highly electrogenic transport in Xenopus oocytes. Under Pi deficiency, als3 accumulates higher levels of Fe3+ in its roots than the wild type does. In Arabidopsis, LPR1 (LOW PHOSPHATE ROOT1) and LPR2 encode ferroxidases, which when mutated, reduce Fe3+ accumulation in roots and cause root growth to be insensitive to Pi deficiency. Here, we provide compelling evidence showing that ALS3 cooperates with LPR1/2 to regulate Pi deficiency-induced remodeling of root architecture by modulating Fe homeostasis in roots.

Oocyte aging-induced Neuronatin (NNAT) hypermethylation affects oocyte quality by impairing glucose transport in porcine

DNA methylation plays important roles in regulating many physiological behaviors; however, few studies were focused on the changes of DNA methylation during oocyte aging. Early studies showed that some imprinted genes’ DNA methylation had been changed in aged mouse oocytes. In this study, we used porcine oocytes to test the hypothesis that oocyte aging would alter DNA methylation pattern of genes and disturb their expression in age oocytes, which affected the developmental potential of oocytes. We compared several different types of genes and found that the expression and DNA methylation of Neuronatin (NNAT) were disturbed in aged oocytes significantly. Additional experiments demonstrated that glucose transport was impaired in aged oocytes and injection of NNAT antibody into fresh oocytes led to the same effects on glucose transport. These results suggest that the expression of NNAT was declined by elevating DNA methylation, which affected oocyte quality by decreasing the ability of glucose transport in aged oocytes.

Functional assessment of SLC4A11, an integral membrane protein mutated in corneal dystrophies.

SLC4A11, a member of the SLC4 family of bicarbonate transporters, is a widely expressed integral membrane protein, abundant in kidney and cornea. Mutations of SLC4A11 cause some cases of the blinding corneal dystrophies, congenital hereditary endothelial dystrophy, and Fuchs endothelial corneal dystrophy. These diseases are marked by fluid accumulation in the corneal stroma, secondary to defective fluid reabsorption by the corneal endothelium. The role of SLC4A11 in these corneal dystrophies is not firmly established, as SLC4A11 function remains unclear. To clarify the normal function(s) of SLC4A11, we characterized the protein following expression in the simple, low-background expression system Xenopus laevis oocytes. Since plant and fungal SLC4A11 orthologs transport borate, we measured cell swelling associated with accumulation of solute borate. The plant water/borate transporter NIP5;1 manifested borate transport, whereas human SLC4A11 did not. SLC4A11 supported osmotically driven water accumulation that was electroneutral and Na+ independent. Studies in oocytes and HEK293 cells could not detect Na+-coupled HCO3- transport or Cl-/HCO3- exchange by SLC4A11. SLC4A11 mediated electroneutral NH3 transport in oocytes. Voltage-dependent OH- or H+ movement was not measurable in SLC4A11-expressing oocytes, but SLC4A11-expressing HEK293 cells manifested low-level cytosolic acidification at baseline. In mammalian cells, but not oocytes, OH-/H+ conductance may arise when SLC4A11 activates another protein or itself is activated by another protein. These data argue against a role of human SLC4A11 in bicarbonate or borate transport. This work provides additional support for water and ammonia transport by SLC4A11. When expressed in oocytes, SLC4A11 transported NH3, not NH3/H.

Abstract 1007: The amino acid transporter SNAT4: Potential role as a tumor suppressor in melanoma

Abstract Cancer cells utilize amino acids to satisfy their need for nutrients and fuel their accelerated growth. The amino acid glutamine has been identified as one of the key building blocks in cancer cells, utilized for macromolecular synthesis and energy production. Due to the increased requirement for glutamine and other amino acids, cancer cells commonly increase expression of amino acid transporters, such as SLC1A5 (ASCT2). Amino acid transporters are membrane transport proteins that are used by cells to move important amino acids in and out of the cell. Interestingly, most amino acid transporters are upregulated in cancer to ensure continued access to nutrients. We have recently shown that ASCT2 plays a critical role in regulating glutamine uptake in melanoma, prostate and breast cancer. Many other glutamine transporters are upregulated in different cancers, including SLC38 family members SNAT1, SNAT2 and SNAT3. The role of SLC38A4 (SNAT4) in amino acid uptake and cancer growth, however, has not been examined. Like the other SNATs, SNAT4 is a sodium-dependent amino acid transporter that transports neutral amino acids, such as alanine, across the plasma membrane, although it has low affinity for glutamine. Interestingly, unlike the other SNAT family members, our analysis showed that SNAT4 expression is downregulated in 879/917 cancer cell lines in Oncomine. Analysis of SNAT4 mutations in patient samples using cBioPortal showed infrequent mutations in the majority of cancers. However, the TCGA melanoma data (cBioPortal) showed that 32 of 278 melanoma cases (11.5%) have a point mutation in SNAT4 suggesting an important role in melanoma. These mutations included 9 SNAT4 hotspot mutations, present in 2-4 patients each. Using a SNAT4 homology model to predict loss-of-function mutants, we selected five of these mutations to assess in a SNAT4 transport assay in Xenopus oocytes. Two of the five hotspot mutations (G31E, S76F, G78E, G150E and S371F) were able to completely inactivate SNAT4 alanine transport in oocytes. Further analysis of 52 melanoma patient samples in Oncomine showed SNAT4 downregulation in all patients, suggesting that, in contrast to most amino acid transporters, SNAT4 plays a tumor suppressor role in melanoma. We are currently examining these mutations in melanoma cell lines to determine their effects on cell growth. We are also examining how SNAT4 is involved in either the import or export of amino acids and the downstream metabolic consequences that may directly inhibit melanoma cell growth. Through this research we will gain a better understanding of the role of SNAT4- mediated amino acid metabolism in preventing melanoma cell growth. Citation Format: Nicholas J. Otte, Angelika Broer, Patrick O’Young, Michelle van Geldermalsen, Qian Wang, Charles G. Bailey, Stefan Broer, Jeff Holst. The amino acid transporter SNAT4: Potential role as a tumor suppressor in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1007.

Remodeling of bovine oviductal epithelium by mitosis of secretory cells.

Two types of oviductal epithelial cells, secretory and ciliated, play crucial roles in the first days after fertilization in mammals. Secretory cells produce various molecules promoting embryo development, while ciliated cells facilitate transport of oocytes and zygotes by ciliary beating. The proportions of the two cell types change during the estrous cycle. The proportion of ciliated cells on the oviductal luminal surface is abundant at the follicular phase, whereas the proportion of secretory cells gradually increases with the formation of the corpus luteum. In the present study, we hypothesize that the proportions of ciliated and secretory epithelial cells are regulated by mitosis. The proportion of the cells being positive for FOXJ1 (a ciliated cell marker) or Ki67 (a mitosis marker) in epithelial cells during the estrous cycle were immunohistochemically examined. Ki67 and FOXJ1 or PAX8 (a secretory cell marker), were double-stained to clarify which types of epithelial cells undergo mitosis. In the ampulla, the percentage of FOXJ1-positive cells was highest at the day of ovulation (Day 0) and decreased by about 50% by Days 8-12, while in the isthmus it did not change during the estrous cycle. The proportion of Ki67-positive cells was highest at around the time of ovulation in both the ampulla and isthmus. All the Ki67-positive cells were PAX8-positive and FOXJ1-negative in both the ampulla and isthmus. These findings suggest that epithelial remodeling, which is regulated by differentiation and/or proliferation of secretory cells of the oviduct, provides the optimal environment for gamete transport, fertilization and embryonic development.

Micromechanical Analysis of the Hyaluronan-Rich Matrix Surrounding the Oocyte Reveals a Uniquely Soft and Elastic Composition

The cumulus cell-oocyte complex (COC) matrix is an extended coat that forms around the oocyte a few hours before ovulation and plays vital roles in oocyte biology. Here, we analyzed the micromechanical response of mouse COC matrix by colloidal-probe atomic force microscopy. We found that the COC matrix is elastic insofar as it does not flow and its original shape is restored after force release. At the same time, the COC matrix is extremely soft. Specifically, the most compliant parts of in vivo and in vitro expanded COC matrices yielded Young’s modulus values of 0.5 ± 0.1 Pa and 1.6 ± 0.3 Pa, respectively, suggesting both high porosity and a large mesh size (≥100 nm). In addition, the elastic modulus increased progressively with indentation. Furthermore, using optical microscopy to correlate these mechanical properties with ultrastructure, we discovered that the COC is surrounded by a thick matrix shell that is essentially devoid of cumulus cells and is enhanced upon COC expansion in vivo. We propose that the pronounced nonlinear elastic behavior of the COC matrix is a consequence of structural heterogeneity and serves important functions in biological processes such as oocyte transport in the oviduct and sperm penetration.

Non Equilibrium Steady State Dynamics of Contractile Actin Networks

The actin cytoskeleton plays a major role during the initial stages of embryonic development. In particular, the actin cytoskeleton can switch, in a cell-cycle dependent manner, into a contractile state and exhibit large scale flows which are essential for the organization and the establishment of polarity in early embryos. For example, myosin-driven contractile flows are essential for the initial cortical polarization in many species, while bulk actin network contraction can drive directional transport in large oocytes. We developed a reconstituted model system to study the onset of contraction in actomyosin networks, and emulate these processes in artificial cells. We encapsulate Xenopus cell extracts in cell-sized water-in-oil emulsions, and introduce different types of actin nucleators at the interface or in bulk, to reconstitute cortical or bulk actin networks, respectively. Importantly, the presence of dynamic turnover in our system allows these networks to attain a dynamic steady state characterized by contractile actin flows which can persist for hours. We add single wall carbon nanotubes as inert fluorescent probes to map the network dynamics at high spatial and temporal resolution, and uncover the self-organized dynamics that lead to the formation of these steady state contractile networks.

OsHKT1;4-mediated Na(+) transport in stems contributes to Na(+) exclusion from leaf blades of rice at the reproductive growth stage upon salt stress.

BackgroundNa+ exclusion from leaf blades is one of the key mechanisms for glycophytes to cope with salinity stress. Certain class I transporters of the high-affinity K+ transporter (HKT) family have been demonstrated to mediate leaf blade-Na+ exclusion upon salinity stress via Na+-selective transport. Multiple HKT1 transporters are known to function in rice (Oryza sativa). However, the ion transport function of OsHKT1;4 and its contribution to the Na+ exclusion mechanism in rice remain to be elucidated.ResultsHere, we report results of the functional characterization of the OsHKT1;4 transporter in rice. OsHKT1;4 mediated robust Na+ transport in Saccharomyces cerevisiae and Xenopus laevis oocytes. Electrophysiological experiments demonstrated that OsHKT1;4 shows strong Na+ selectivity among cations tested, including Li+, Na+, K+, Rb+, Cs+, and NH4+, in oocytes. A chimeric protein, EGFP-OsHKT1;4, was found to be functional in oocytes and targeted to the plasma membrane of rice protoplasts. The level of OsHKT1;4 transcripts was prominent in leaf sheaths throughout the growth stages. Unexpectedly however, we demonstrate here accumulation of OsHKT1;4 transcripts in the stem including internode II and peduncle in the reproductive growth stage. Moreover, phenotypic analysis of OsHKT1;4 RNAi plants in the vegetative growth stage revealed no profound influence on the growth and ion accumulation in comparison with WT plants upon salinity stress. However, imposition of salinity stress on the RNAi plants in the reproductive growth stage caused significant Na+ overaccumulation in aerial organs, in particular, leaf blades and sheaths. In addition, 22Na+ tracer experiments using peduncles of RNAi and WT plants suggested xylem Na+ unloading by OsHKT1;4.ConclusionsTaken together, our results indicate a newly recognized function of OsHKT1;4 in Na+ exclusion in stems together with leaf sheaths, thus excluding Na+ from leaf blades of a japonica rice cultivar in the reproductive growth stage, but the contribution is low when the plants are in the vegetative growth stage.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0709-4) contains supplementary material, which is available to authorized users.

Expression of JAK3 Sensitive Na+ Coupled Glucose Carrier SGLT1 in Activated Cytotoxic T Lymphocytes

Background: Similar to tumor cells, activated T-lymphocytes generate ATP mainly by glycolytic degradation of glucose. Lymphocyte glucose uptake involves non-concentrative glucose carriers of the GLUT family. In contrast to GLUT isoforms, Na⁺-coupled glucose-carrier SGLT1 accumulates glucose against glucose gradients and is effective at low extracellular glucose concentrations. The present study explored expression and regulation of SGLT1 in activated murine splenic cytotoxic T cells (CTLs) and human Jurkat T cells. Methods: FACS analysis, immunofluorescence, confocal microscopy, chemiluminescence and Western blotting were employed to estimate SGLT1 expression, function and regulation in lymphocytes, as well as dual electrode voltage clamp in SGLT1 ± JAK3 expressing Xenopus oocytes to quantify the effect of janus kinase3 (JAK3) on SGLT1 function. Results: SGLT1 is expressed in murine CTLs and also in human Jurkat T cells. 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose uptake was significantly decreased by SGLT1-blocker phloridzin (0.2 mM) and by pharmacological inhibition of JAK3 with WHI-P131 (156 µM), WHI-P154 (11.2 µM) and JAK3 inhibitor VI (0.5 µM). Electrogenic glucose transport (I_glucose) in Xenopus oocytes expressing human SGLT1 was increased by additional expression of human wild type JAK3, active ^A568VJAK3 but not inactive ^K851AJAK3. Coexpression of JAK3 enhanced the maximal transport rate without significantly modifying affinity of the carrier. I_glucose in SGLT1+JAK3 expressing oocytes was significantly decreased by WHI-P154 (11.2 µM). JAK3 increased the SGLT1 protein abundance in the cell membrane. Inhibition of carrier insertion by brefeldin A (5 µM) in SGLT1+JAK3 expressing oocytes resulted in a decline of I_glucose, which was similar in presence and absence of JAK3. Conclusions: SGLT1 is expressed in murine cytotoxic T cells and human Jurkat T cells and significantly contributes to glucose uptake in those cells post activation. JAK3 up-regulates SGLT1 activity by increasing the carrier protein abundance in the cell membrane, an effect enforcing cellular glucose uptake into activated lymphocytes and thus contributing to the immune response.

106 COMPARISON OF IN VITRO EMBRYO PRODUCTION IN PANAMA USING HOLSTEIN OOCYTES WITH BOS INDICUS SEXED SEMEN FROM DONORS IN DIFFERENT LOCATIONS: FIELD DATA

The objective of this work was to compare in vitro embryo production of Bos taurus × Bos indicus cross embryos using oocytes from Holstein donors under different production and environment systems. This study also examined the possibility for in vitro production using oocytes imported and transported fresh between the USA and Panama. All animals were mature Holstein cows going through a normal lactation. The first group of donors was from the University of Illinois dairy herd and went through 3 ovum pickup sessions. The second group of donors were Holstein cows already adapted to Panama and went through 10 ovum pickup sessions. The Panamanian herd of Holstein donors were born and raised in Panama in an area of mountains, on average 1300 m above sea level. This environment does not have the typical hot and humid tropical weather seen in other regions of Panama. Both groups of donors were aspirated without stimulation during the years 2013 and 2014. Oocytes recovered from donors in Illinois were imported fresh under a special sanitary research protocol between Panama and the University of Illinois. The transport of fresh oocytes from the USA to Panama was done using a portable incubator set at 39°C (Minutube of America). Oocytes were matured during transport in 5-mL tubes (~30–35 oocytes per tube) containing 400 µL of maturation media (TCM-199) that had been equilibrated with 5% CO2. Oocytes recovered from donors in Panama were matured using the same media. For both groups, oocytes were inseminated 24 h after ovum pickup using sexed semen from the same bull. All embryo production procedures followed the protocols of the In vitro Brasil™ commercial system. At 72 h postinsemination, cleavage was evaluated. On Day 7 after insemination, embryo development to the blastocyst stage (early to expanded) was recorded. Data were analysed using Chi-squared. As shown in Table 1, there was no effect of oocyte collection location on embryo development. These results indicate that it is possible to produce a viable in vitro-produced embryo using fresh oocytes collected and transported from different countries. This work opens the possibility to access superior genetics and improve herds in countries seeking to increase their production systems and potentially improve their quality of life. Table 1.Effect of oocyte collection location on embryo development This project was supported by Programa de Competitividad ProCom Senacyt, Panama.

Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine

The majority of mammalian oocytes destined for in vitro maturation (IVM) have not undergone all molecular and structural changes necessary for competence acquisition to support the fertilization and early embryogenesis. In this context, different methods able to provide a transient arrest of meiosis resumption have been tested in order to improve the in vitro developmental potential of oocytes. Based on that, our study aimed to evaluate the effect of temporary meiosis inhibition using roscovitine on gene expression in sheep oocytes and cumulus cells. For this, cumulus-oocyte complexes (COCs) were cultured for 6h in modified TCM199 medium with (Rosco) and without (Control) 75μM roscovitine. Subsequently, they were in vitro matured for a further 18h in inhibitor-free TCM199 medium supplemented with gonadotropins. At 0, 6 and 24h of culture, nuclear status of oocytes and expression of selected genes were evaluated by Hoescht staining and qRT-PCR, respectively. The analysis of oocyte chromatin organization revealed that roscovitine efficiently inhibited the meiosis of sheep oocytes for 6h and its action was completely reversed after 18h of in vitro maturation in inhibitor-free medium. Besides, no detrimental effect on cumulus expansion was observed. The expression profile of most investigated genes in cumulus cells (PTX3, GREM1, GLUT1, PTGS2, ALK5, ALK6) and oocytes (ZAR1, NLRP5, SOD1, BMP15, GDF9) was similar between Control and Rosco treatments and the ratio BCL2/BAX was maintained in both cell types even in the presence of roscovitine. These results indicate that reversible meiotic arrest promoted by roscovitine, at the concentration and exposure time studied, neither impaired nor improved the expression of investigated genes in sheep oocytes and cumulus cells. Moreover, the efficiency of temporary meiotic arrest and the absence of deleterious effect on COCs suggest that roscovitine provides a useful method for transportation or manipulation of sheep oocytes at onset of maturation. However, further investigations are necessary to evaluate the benefits of roscovitine treatment on in vitro development of sheep embryos and its effects on cellular ultrastructure.

Aquaporins Mediate Silicon Transport in Humans.

In animals, silicon is an abundant and differentially distributed trace element that is believed to play important biological functions. One would thus expect silicon concentrations in body fluids to be regulated by silicon transporters at the surface of many cell types. Curiously, however, and even though they exist in plants and algae, no such transporters have been identified to date in vertebrates. Here, we show for the first time that the human aquaglyceroporins, i.e., AQP3, AQP7, AQP9 and AQP10 can act as silicon transporters in both Xenopus laevis oocytes and HEK-293 cells. In particular, heterologously expressed AQP7, AQP9 and AQP10 are all able to induce robust, saturable, phloretin-sensitive silicon transport activity in the range that was observed for low silicon rice 1 (lsi1), a silicon transporter in plant. Furthermore, we show that the aquaglyceroporins appear as relevant silicon permeation pathways in both mice and humans based on 1) the kinetics of substrate transport, 2) their presence in tissues where silicon is presumed to play key roles and 3) their transcriptional responses to changes in dietary silicon. Taken together, our data provide new evidence that silicon is a potentially important biological element in animals and that its body distribution is regulated. They should open up original areas of investigations aimed at deciphering the true physiological role of silicon in vertebrates.