Oocyte Plasma Membrane Research Articles

Impaired plasma membrane targeting or protein stability by certain ATP1A2 mutations identified in sporadic or familial hemiplegic migraine

Mutations in three different genes have been implicated in familial hemiplegic migraine (FHM), two of them code for neuronal voltage-gated cation channels, CACNA1A and SCN1A, while the third encodes ATP1A2, the α 2-isoform of the Na+/K+-ATPase's catalytic subunit, thus classifying FHM as an ion channel / ion transporter disorder. The Na+/K+-ATPase maintains the physiological gradients for Na+ and K+ ions and is therefore critical for the activity of ion channels and transporters involved in neurotransmitter uptake or Ca2+ signaling. Diverse functional abnormalities have been identified for disease-linked ATP1A2 mutations, which reach far beyond simple loss-of-function. We have shown recently that ATP1A2 mutations frequently lead to changes in the enzyme's voltage-dependent properties, kinetics, or apparent cation affinities. Here, we present functional data on a so far uncharacterized set of ATP1A2 mutations (G301R, R908Q, and P979L) upon expression in Xenopus oocytes and HEK293FT cells, and provide evidence for a novel pathophysiological mechanism. Whereas the G301R mutant was inactive, no functional changes were observed for mutants R908Q and P979L in the oocyte expression system. However, the R908Q mutant was less effectively expressed in the plasma membrane of oocytes, making it the first missense mutation to result in defective plasma membrane targeting. Notably, the P979L mutant exhibited the same cellular expression profile as the wild-type protein, both in Xenopus oocytes and in transfected HEK293FT cells grown at 28{degree sign}C, but much less P979L protein was found upon cell growth at 37{degree sign}C, showing for the first time that temperature-sensitive effects on protein stability can underlie ATP1A2 loss-of-function.

Reduced Immunolabelling of a Porcine Oocyte Membrane Protein Reflects Reduced Fertilizability of Porcine Oocytes Following Elevated Ambient Temperature

Decreased fertility in pigs is a common occurrence during summer months. An objective of the current experiments was to evaluate if elevated ambient temperature altered the oocyte plasma membrane including potential receptors for sperm. This would potentially contribute to reduced fertilizability. Treated gilts were exposed in vivo to 32 degrees C for 12 h per day and 20 degrees C for the remaining 12 h per day for 7 days; control gilts were exposed to 22 degrees C for 12 h and 20 degrees C for the remaining 12 h each day. Cumulus-oocyte complexes were also aspirated from ovaries obtained from gilts maintained at thermoneutral ambient temperature and matured in vitro at 38.5 degrees C or 40 degrees C. Relative abundance of a porcine oocyte membrane protein was examined by intensity of immunolabelling of the in vivo and in vitro matured oocytes evaluated with confocal microscopy; fertilizability of the in vitro matured oocytes was evaluated in in vitro fertilization assays. Oocytes obtained from gilts exposed to elevated ambient temperature for 7 days had reduced immunolabelling compared with oocytes from control gilts (p < 0.05). Similarly, oocytes matured in vitro for 44 h at elevated ambient temperature had reduced immunolabelling and reduced fertilizability compared with oocytes matured at 38.5 degrees C (p < 0.01 and p < 0.05). These results suggest porcine oocyte quality is reduced by elevated ambient temperature and immunolabelling of oocytes with antibodies to specific membrane proteins may be effective to evaluate some aspects of oocyte quality.

Functional Characterization Of Clc-5 Mutations Associated With Dent's Disease

The Cl−/H+ antiporter ClC-5 has been linked to Dent's disease, an X-linked renal disease associated with low molecular weight proteinuria, hypercalciuria and nephrolithiasis. ClC-5 is expressed on early endosomes of proximal tubule cells, where it plays a critical role in endosomal function. The impact of Dent's disease-causing mutations on ClC-5 function has not been yet fully investigated. Here, we have analysed an unpublished mutation K115R and three published mutations, Y272C, N340K and K546E in terms of electrical activity and trafficking at the plasma membrane in Xenopus leavis oocytes.

SGK1 Phosphorylation of IκB Kinase α and p300 Up-regulates NF-κB Activity and Increases N-Methyl-D-aspartate Receptor NR2A and NR2B Expression

Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a downstream target of phosphatidylinositol 3-kinase signaling, and it regulates various cellular and physiological functions, but the SGK1 substrate proteins and genes regulated by SGK1 are less known. Here we have identified IkappaB kinase alpha (IKKalpha) as a novel substrate of SGK1 by using biochemical and bioinformatic approaches. SGK1 directly phosphorylates IKKalpha at Thr-23 and indirectly activates IKKalpha at Ser-180. Furthermore, SGK1 enhanced nuclear factor kappaB (NF-kappaB) activity and up-regulated N-methyl-d-aspartate receptor NR2A and NR2B expression through activation of IKKalpha at Thr-23 and Ser-180, and these two residues play an equally important role in mediating these effects of SGK1. Although SGK1 does not phosphorylate IKKbeta, IKKbeta activity is still required for IKK complex activation and for SGK1 phosphorylation and activation of NF-kappaB. In addition, SGK1 increased the acetylation of NF-kappaB through phosphorylation of p300 at Ser-1834, and this also leads to NF-kappaB activation and NR2A and NR2B expression. Moreover, an endogenous stimulus of SGK1, insulin, increased IKKalpha and NF-kappaB phosphorylation as well as NF-kappaB acetylation and NF-kappaB activity, but SGK1 small interfering RNA transfection blocked these effects of insulin. In examination of the functional significance of the SGK1-IKKalpha-NF-kappaB signaling pathway, we found that transfection of the IKKalpha double mutant (IKKalphaT23A/S180A) to rat hippocampus antagonized SGK-1-mediated spatial memory facilitation. Our results together demonstrated novel substrate proteins of SGK1 and novel SGK1 signaling pathways. Activation of these signaling pathways enhances NR2A and NR2B expression that is implicated in neuronal plasticity.

Regulation of the Na +-coupled glutamate transporter EAAT3 by PIKfyve

The Na +,glutamate cotransporter EAAT3 is expressed in a wide variety of tissues. It accomplishes transepithelial transport and the cellular uptake of acidic amino acids. Regulation of EAAT3 activity involves a signaling cascade including the phosphatidylinositol-3 (PI3)-kinase, the phosphoinositide dependent kinase PDK1, and the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether PIKfyve participates in the regulation of EAAT3 activity. To this end, EAAT3 was expressed in Xenopus oocytes with or without SGK1 and/or PIKfyve and glutamate-induced current ( I glu) determined by dual electrode voltage clamp. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed I glu. Coexpression of either, SGK1 or PIKfyve, significantly enhanced I glu in EAAT3 expressing oocytes. The increased I glu was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane. I glu and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressing EAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant K127NSGK1 did not significantly alter I glu in EAAT3 expressing oocytes and completely reversed the stimulating effect of PIKfyve coexpression on I glu. The stimulating effect of PIKfyve on I glu was abolished by replacement of the serine by alanine in the SGK consensus sequence ( S318APIKfyve). Moreover, additional coexpression of S318APIKfyve significantly blunted I glu in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.

Role of multiple phosphorylation sites in the COOH-terminal tail of aquaporin-2 for water transport: evidence against channel gating

Arginine vasopressin (AVP)-regulated phosphorylation of the water channel aquaporin-2 (AQP2) at serine 256 (S256) is essential for its accumulation in the apical plasma membrane of collecting duct principal cells. In this study, we examined the role of additional AVP-regulated phosphorylation sites in the COOH-terminal tail of AQP2 on protein function. When expressed in Xenopus laevis oocytes, prevention of AQP2 phosphorylation at S256A (S256A-AQP2) reduced osmotic water permeability threefold compared with wild-type (WT) AQP2-injected oocytes. In contrast, prevention of AQP2 single phosphorylation at S261 (S261A), S264 (S264A), and S269 (S269A), or all three sites in combination had no significant effect on water permeability. Similarly, oocytes expressing S264D-AQP2 and S269D-AQP2, mimicking AQP2 phosphorylated at these residues, had similar water permeabilities to WT-AQP2-expressing oocytes. The use of high-resolution confocal laser-scanning microscopy, as well as biochemical analysis demonstrated that all AQP2 mutants, with the exception of S256A-AQP2, had equal abundance in the oocyte plasma membrane. Correlation of osmotic water permeability relative to plasma membrane abundance demonstrated that lack of phosphorylation at S256, S261, S264, or S269 had no effect on AQP2 unit water transport. Similarly, no effect on AQP2 unit water transport was observed for the 264D and 269D forms, indicating that phosphorylation of the COOH-terminal tail of AQP2 is not involved in gating of the channel. The use of phosphospecific antibodies demonstrated that AQP2 S256 phosphorylation is not dependent on any of the other phosphorylation sites, whereas S264 and S269 phosphorylation depend on prior phosphorylation of S256. In contrast, AQP2 S261 phosphorylation is independent of the phosphorylation status of S256.

Conversion of the 2 Cl−/1 H+ antiporter ClC-5 in a NO3−/H+ antiporter by a single point mutation

Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent's disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl(-)/1 H+. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO3(-)/H+ antiporter atClCa, led to coupled NO3(-)/H+ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO3(-)/H+ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of approximately 7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.

The Drosophila RNA-binding protein Lark is required for localization of Dmoesin to the oocyte cortex during oogenesis

The RNA-binding protein Lark has an essential maternal role during Drosophila oogenesis. Elimination of maternal expression results in defects in cytoplasmic dumping and actin cytoskeletal organization in nurse cells. The function of this protein is dependent on the activity of one or more N-terminal RNA-binding domains. Here, we report the identification of Dmoesin (Dmoe) as a candidate RNA target of Lark during oogenesis. In addition to actin defects in the nurse cells of lark mutant ovaries, we observed mislocalization of posteriorly localized mRNAs including oskar and germ cell less in the developing oocyte. Anteriorly and dorsally localized mRNAs were not affected. In addition, we observed displacement of the actin cytoskeleton from the oocyte plasma membrane. These phenotypes are reminiscent of mutations in Dmoe and suggested that this RNA maybe a potential target of Lark. We observed a significant decrease in Dmoe protein associated with the membrane of the developing oocyte with no changes in expression or localization within the nurse cells. Evidence for an association between Lark protein and moe RNA during oogenesis comes from results of a microarray-based Ribonomics approach to identify Lark RNA targets. Thus, our results provide evidence that Dmoe RNA is a target of Lark during oogenesis and that it likely regulates either the splicing or translation of this RNA.

A possible role of progesterone receptor in mouse oocyte in vitro fertilization regulated by norethisterone and its reduced metabolite

Background The contraceptive effect of the progestogen norethisterone (NET) and its main metabolites 5α-NET and 3β,5α-NET has been demonstrated in several species, and most studies have focused on the effects of these compounds in the uterus. We previously reported that 5α-NET inhibits the progesterone (P 4)-induced acrosome reaction in pig and mouse spermatozoa and induces severe morphological damage in two-cell fertilized mouse oocytes. Study Design The main goal of this study was to analyze the possible role of P 4 receptor (PR) in the effects of NET and 5α-NET on the oocyte fertilization process. Different steroid treatments were used with or without cumulus-enclosed oocytes. Results It was demonstrated that NET increases the percentage of fertilized oocytes in the same manner as P 4 does, while 5α-NET reduces the percentage of fertilized oocytes. This effect was not reversed by P 4 in the same concentrations. Conclusion A possible molecular mechanism for the effects of 5α-NET may be through a PR localized in the oocyte plasma membrane.

Back from the Dead

Autism comprises a complex spectrum of neurodevelopmental disorders, likely caused by a number of different factors, most of which are unknown. The receptors for γ-aminobutyric acid (GABA) and glutamate are implicated in the development of autism, but the study of these and other neurotransmitter receptors in brain tissue is technically challenging. Limon et al . used two techniques to investigate proteins derived from postmortem brain tissue from age-matched normal and autistic subjects. First, the authors isolated poly(A + ) RNA from small quantities of brain tissue and microinjected these samples into Xenopus oocytes. Samples were taken from the cerebellum and the temporal cortex because these areas show abnormal neuronal organization in autistic patients. Even though the brains had been in storage for 2 to 5 years, they still contained intact mRNAs that gave rise to a number of different neurotransmitter receptors and voltage-operated ion channels in the injected oocytes, as assessed by electrophysiology experiments. In a second strategy, called microtransplantation, the authors made membrane preparations from the brain tissues, microinjected them into oocytes, and allowed the brain-derived membranes to fuse with the oocyte plasma membrane in a process similar to exocytosis. Electrophysiology experiments showed that the native receptors from the postmortem tissue were functional. Although some trends were observed, no significant molecular differences were found between autistic subjects and the controls. However, as Spitzer discusses, the real strengths of this study are the demonstration that native membrane proteins can still be assayed after long postmortem intervals and the adaptability of this experimental system to the study of other disease states. A. Limon, J. M. Reyes-Ruiz, R. Miledi, Microtransplantation of neurotransmitter receptors from postmortem autistic brains to Xenopus oocytes. Proc . Natl . Acad . Sci . U . S . A . 105 , 10973-10977 (2008). [Abstract] [Full Text] N. C. Spitzer, A Rosetta stone for analysis of human membrane protein function. Proc . Natl . Acad . Sci . U S A . 105 , 10641-10642 (2008). [Full Text]

WNK4 regulates the secretory pathway via which TRPV5 is targeted to the plasma membrane

TRPV5 and TRPV6 are two closely related epithelial calcium channels that mediate apical calcium entry in the transcellular calcium transport pathway. TRPV5, but not TRPV6, is enhanced by protein kinase WNK4 when expressed in Xenopus laevis oocytes. We report that the majority of human TRPV5 exogenously expressed in the Xenopus oocyte plasma membrane was complexly N-glycosylated whereas that for human TRPV6 was core-glycosylated. Unglycosylated N358Q mutants of TRPV5 and TRPV6 were able to be expressed in the plasma membrane albeit with decreased abilities in mediating calcium uptake. Syntaxin 6, a SNARE protein in the trans-Golgi network, blocked the complex glycosylation of TRPV5 and TRPV6, rendered the channels in core-glycosylated form. Blocking complex glycosylation of TRPV5 either by syntaxin 6 or by N358Q mutation abolished the enhancing effect of WNK4 on TRPV5. Thus the difference in membrane expression of TRPV5 and TRPV6 explains the selective effect of WNK4 on TRPV5, which is likely on the secretory pathway involving complex glycosylation of channel proteins.

Cyclic QDE peptide increases fertilization rates and provides healthy pups in mouse

To evaluate the effects of cyclic QDE peptide (cQDE), derived from sperm fertilin beta (ADAM2), in mouse in vitro fertilization (IVF) and its harmlessness on pups after embryo transfer. Prospective in vivo and in vitro study in mice. Murine model in an academic research environment in France. Normal B6CBF1 mice. Indirect immunofluorescence was used to evaluate cQDE binding to oolemma. The IVF assays were run in presence of the species-specific tripeptide (cQDE at 100 microM), and embryos were transferred in pseudopregnant mice. Controls were obtained by natural mating and IVF in regular medium followed by embryo transfer. Evaluation of fertilization, litter size, pup fertility and health. Biotinylated cQDE peptide binds to oocyte plasma membrane and increases gamete fusion in cumulus-intact IVF assay. Litter size as well as pup development after embryo transfer showed no statistically significant differences compared with controls. Pups born from embryos that were incubated with the peptide were as healthy and normally fertile as control mice over at least three generations. Cyclic QDE is harmless and improves mouse IVF pregnancy rates. A randomized prospective clinical trial to evaluate the beneficial effect of cyclic FEE on human IVF is required before its clinical use.

Tick vitellogenin receptor reveals critical role in oocyte development and transovarial transmission ofBabesiaparasite

A cDNA encoding the vitellogenin receptor of the ixodid tick, Haemaphysalis longicornis Neumann (HlVgR) was cloned and characterized. The full-length cDNA is 5631 bp, including an intact ORF encoding an expected protein with 1782 amino acids. The deduced amino acid sequence of the HlVgR cDNA revealed two ligand-binding domains with four class A cysteine-rich repeats in the first domain and eight in the second domain similar to those of insect VgRs. The immunoblot analysis detected approximately 197 kDa protein in both tick ovary and egg. The developmental expression profile demonstrated that HlVgR mRNA exists throughout the ovarian development, and the transcriptional level is especially high in the previtellogenic period. Immuno electron microscopy analysis demonstrated that the localization of HlVgR is detected on the external surface of oocyte plasma membrane. RNAi showed that eggs of HlVgR dsRNA-injected adult ticks had not developed into fully mature oocytes and laid abnormal eggs. The Babesia parasite DNA was not detected in the eggs of HlVgR dsRNA-injected tick that fed on Babesia gibsoni infected dog, whereas it was detected in the eggs of PBS-injected ticks and noninjected ticks. Expression of HlVgR was increased by the vitellogenic hormone 20-hydroxyecdysone. These results indicate that HlVgR, which is produced by the developing oocytes, is essential for Vg uptake, egg development in the H. longicornis tick, and transovarial transmission of Babesia parasites.

GABAA receptor properties in catastrophic infantile epilepsy

Catastrophic epilepsy due to cortical dysplasia is often intractable to anticonvulsant treatment. Many of the medications used unsuccessfully in treating this disorder are thought to exert at least a portion of their action through enhancement of inhibitory GABA(A) neurotransmission. In the present study, GABA(A) receptor properties in resected brain tissue from four infants with infantile spasms and intractable epilepsy due to cortical dysplasia were measured to determine if this clinical resistance to pharmacologic treatment correlates with alterations in receptor function. Results from epileptic cortex were compared with those from autopsy control samples. To perform these studies, we utilized the technique of injection of brain cellular membrane preparations into the Xenopus oocyte, which results in the incorporation of human GABA(A) receptors in their native configuration into the oocyte plasma membrane. Two-electrode voltage-clamp electrophysiology analysis was then performed to assess GABA(A) receptor pharmacologic properties. The intrinsic properties of affinity, reversal potential, current decay, and current rundown were unchanged in the epileptic infants. Current enhancement by benzodiazepines was also unaltered, as was the response to barbiturates. However, a significant decrease was found in the degree of GABA(A) current enhancement by neurosteroids in the epileptic infants, along with an increase in current inhibition by zinc. These findings may contribute to the mechanisms of intractability in catastrophic infantile epilepsy due to cortical dysplasia, and suggest alternative therapeutic approaches.

Proteomic analysis of the chicken egg vitelline membrane

The avian vitelline membrane (VM) is a multilayered proteinaceous structure separating egg white from yolk. The innermost layer of the VM, deposited onto the oocyte plasma membrane in the ovary, corresponds to the mammalian zona pellucida (ZP). The outer layer is produced in the infundibulum, the first section of the oviduct. Using high-throughput, high-end LC-MS(n) 137 proteins were identified, only 13 of which were known previously to be components of the VM. Depending on the washing protocol, two largely overlapping, but not identical, sets of identified proteins were produced from water-washed and salt-washed VMs. Most of the components of the VM were known previously from other egg compartments, such as, for instance, the egg white proteins lysozyme C, ovalbumin, ovotransferrin, and ovomucin. Specific components of the VM not identified previously in other egg compartments included eight ZP proteins, oviductin protease, and two ATPases. The vitelline outer membrane protein (VMO) VMO II was identified as beta-defensin-11. The list of VM proteins presented in this report is by far the most comprehensive dataset available at present and complements proteomic analyses of chicken egg compartments published previously.

Synergy and specificity of two Na+–aromatic amino acid symporters in the model alimentary canal of mosquito larvae

The nutrient amino acid transporter (NAT) subfamily is the largest subdivision of the sodium neurotransmitter symporter family (SNF; also known as SLC6; HUGO). There are seven members of the NAT population in the African malaria mosquito Anopheles gambiae, two of which, AgNAT6 and AgNAT8, preferably transport indole- and phenyl-branched substrates, respectively. The relative expression and distribution of these aromatic NATs were examined with transporter-specific antibodies in Xenopus oocytes and mosquito larval alimentary canal, representing heterologous and tissue expression systems, respectively. NAT-specific aromatic-substrate-induced currents strongly corresponded with specific accumulation of both transporters in the plasma membrane of oocytes. Immunolabeling revealed elevated expressions of both transporters in specific regions of the larval alimentary canal, including salivary glands, cardia, gastric caeca, posterior midgut and Malpighian tubules. Differences in relative expression densities and spatial distribution of the transporters were prominent in virtually all of these regions, suggesting unique profiles of the aromatic amino acid absorption. For the first time reversal of the location of a transporter between apical and basal membranes was identified in posterior and anterior epithelial domains corresponding with secretory and absorptive epithelial functions, respectively. Both aromatic NATs formed putative homodimers in the larval gut whereas functional monomers were over-expressed heterologously in Xenopus oocytes. The results unequivocally suggest functional synergy between substrate-specific AgNAT6 and AgNAT8 in intracellular absorption of aromatic amino acids. More broadly, they suggest that the specific selectivity, regional expression and polarized membrane docking of NATs represent key adaptive traits shaping functional patterns of essential amino acid absorption in the metazoan alimentary canal and other tissues.

Atomic force microscopy imaging of Xenopus laevis oocyte plasma membrane purified by ultracentrifugation

Atomic force microscopy (AFM) was used to investigate the native plasma membrane of Xenopus laevis (X. laevis) oocyte purified by means of ultracentrifugation on sucrose gradient and subsequently adsorbed on mica leaves through a physisorption process. Reproducible AFM topography images were collected, analyzed, and compared. AFM images showed the presence of large single or double bilayer membrane sheets covered with protein complexes. The lateral dimension and height of protein complexes imaged in air showed a normal distribution centred on 15.4 +/- 0.4 nm (mean +/- SE; n = 59) and 3.9 +/- 0.2 nm (mean +/- SE; n = 57), respectively. A density of about 270 protein complexes per square micron was calculated. Less frequently, ordered nanometer domains with densely packed protein complexes arranged in hexagonal patterns were also visualized in AFM images, confirming previously published data. Their lateral dimension and height showed a normal distribution centred on 23.0 +/- 0.4 nm (mean +/- SE; n = 42) and 1.5 +/- 0.6 nm (mean +/- SE; n = 90), respectively. A density of about 870 protein complexes per square micrometer was calculated. Advantages and drawbacks of this new sample preparation for AFM imaging are discussed.

Estrogen Signaling Characteristics of Atlantic Croaker G Protein-Coupled Receptor 30 (GPR30) and Evidence It Is Involved in Maintenance of Oocyte Meiotic Arrest

Human G protein-coupled receptor 30 (GPR30) mediates estradiol-17beta (E2) activation of adenylyl cyclase in breast cancer cells and displays E2 binding typical of membrane estrogen receptors (mERs). We identified a mER in Atlantic croaker ovaries with characteristics similar to those of human GPR30. To confirm the proposed role of GPR30 as a mER in this distantly related vertebrate group, we cloned GPR30 from croaker ovaries and examined its distribution, steroid binding, and signaling characteristics. Western blot analysis showed the GPR30 protein (approximately 40 kDa) is expressed on the plasma membranes of croaker oocytes and HEK293 cells stably transfected with GPR30 cDNA. Plasma membranes prepared from croaker GPR30-transfected cells displayed high-affinity, limited-capacity, and displaceable binding specific for estrogens, characteristic of mERs. Consistent with previous findings with human GPR30, estrogen treatment of plasma membranes from both croaker ovaries and GPR30-transfected cells caused activation of a stimulatory G protein (Gs) resulting in increased cAMP production. Treatment with E2 as well as G-1, a specific GPR30 ligand, significantly reduced both spontaneous and progestin-induced maturation of both croaker and zebrafish oocytes in vitro, suggesting a possible involvement of GPR30 in maintaining oocyte meiotic arrest in these species. Injection of antisense oligonucleotides to GPR30 into zebrafish oocytes blocked the inhibitory effects of estrogen on oocyte maturation, confirming a role for GPR30 in the control of meiotic arrest. These findings further support our previous suggestion that GPR30 is a vertebrate mER. In addition, the results suggest GRP30 may play a critical role in regulating reentry into the meiotic cell cycle in fish oocytes.

Alpha-bungarotoxin binding to target cell in a developing visual system by carboxylatednanodiamond

Biological molecules conjugating with nanoparticles are valuable for applicationsincluding bio-imaging, bio-detection, and bio-sensing. Nanometer-sized diamondparticles have excellent electronic and chemical properties for bio-conjugation.In this study, we manipulated the carboxyl group produced on the surface ofnanodiamond (carboxylated nanodiamond, cND) for conjugating with alpha-bungarotoxin(α-BTX), a neurotoxin derived from Bungarus multicinctus withspecific blockade of alpha7-nicotinic acetylcholine receptor(α7-nAChR). The electrostaticbinding of cND–α-BTX was mediated by the negative charge of the cND and the positive charge of theα-BTXin physiological pH conditions. Sodium dodecyl sulfate-polyacrylamide gel analysis and matrix-assistedlaser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) spectra displayedthat α-BTX proteins were conjugated with cND particles via non-covalent bindings. The green fluorescenceof the cND particles combining with the red fluorescence of tetramethylrhodamine-labeledα-BTX presented a yellow color at the same location, which indicated thatα-BTX proteins were conjugated with cND particles. Xenopus laevis’s oocytes expressed the humanα7-nAChR proteins by microinjection withα7-nAChR mRNA. ThecND–α-BTX complexeswere bound to α7-nAChR locating on the cell membrane of oocytes and human lung A549cancer cells analyzed by laser scanning confocal microscopy. The choline-evokedα7-nAChR-mediated inward currents of the oocytes were blocked bycND–α-BTX complexes in a concentration-dependent manner using two-electrodevoltage-clamp recording. Furthermore, the fluorescence intensity ofcND–α-BTXbinding on A549 cells could be quantified by flow cytometry. These results indicate that cND-conjugatedα-BTX still preserves its biological activity in blocking the function ofα7-nAChR, and provide a visual system showing the binding ofα-BTXto α7-nAChR.

Ultrastructure of the oocyte envelopes of some Eurasian acipenserids

Summary The cellular and acellular envelopes of oocytes and follicles at the ultrastructural level are described for some acipenserids. The objective was to identify whether morphological features exist that can assist in discrimination of caviar products in trade originating from various species. The follicular envelopes include one thecal and one follicular cell layer separated by a basal lamina. The oocyte is surrounded by four acellular layers: (i) the internal zona radiata directly in contact with the oocyte plasma membrane; (ii) the external zona radiata; (iii) a layer of alveolar material; and (iv) a thin adhesive layer probably secreted by the follicular cells. These structures are similar in the eggs of genus Acipenser and Huso. Comparison of descriptions and definitions from different authors reveal a confusing terminology and sometimes different terms are used for similar structures. A terminology for the envelopes of Acipenseriform eggs is proposed. The authors conclude that morphological characters of sturgeon eggs are less valuable for discrimination of caviar.