Membrane Potential In Oocytes Research Articles

Lipopolysaccharide impairs the in vitro growth, steroidogenesis, and maturation of oocyte-cumulus-granulosa cell complexes derived from bovine early antral follicles

In postpartum dairy cows, lipopolysaccharide (LPS) derived from gram-negative bacteria causes uterine or mammary inflammation, resulting in low fertility. The present study aimed to investigate the effect of LPS on the in vitro growth (IVG), steroidogenesis, and maturation of oocyte-cumulus-granulosa cell complexes (OCGCs) derived from bovine early antral follicles. OCGCs were isolated from bovine early antral follicles (0.5–1 mm in diameter) and cultured in vitro for 12 days using media containing 0 (control), 0.01, or 1 μg/mL of LPS. The viability, cavity formation, and oocyte diameter of the OCGCs, as well as the concentrations of estradiol (E2) and progesterone (P4) in the IVG culture media, were determined. After IVG culture, oocytes collected from viable OCGCs were matured in vitro (IVM) in a medium without LPS. The nuclear maturation rate and the mitochondrial membrane potential of oocytes were determined. Bovine oocytes and cumulus-granulosa complexes derived from early antral follicles expressed genes encoding LPS receptor complex, such as toll-like receptor 4 (TLR4). Immunohistochemistry analysis further localized TLR4 expression predominantly in follicular granulosa and theca cells of early antral follicles. The viability of OCGCs and cavity formation in OCGCs were lower in the 0.01 and 1 μg/mL LPS groups than in the control group. No significant difference in oocyte diameter was observed between the treatment groups throughout the culture period. Moreover, E2 production was suppressed in the 0.01 and 1 μg/mL LPS groups from Days 4–8, whereas P4 production increased in the 1 μg/mL LPS group from Days 0–8. The nuclear maturation rate after IVM was lower in the 0.01 and 1 μg/mL LPS groups than in the control group. The mitochondrial membrane potential of post-IVM oocytes was lower in the 0.01 and 1 μg/mL LPS groups than in the control group. Taken together, these results indicate that LPS inhibited the growth and steroidogenesis of OCGCs and the meiosis and mitochondrial function of oocytes derived from early antral follicles. This study suggests that the detrimental effects of LPS on developing oocytes may contribute to long-term decreased fertility in postpartum dairy cows.

Dendrobine enhances bovine oocyte maturation and subsequent embryonic development and quality

Strategies for improving the quality of oocytes have important theoretical and practical significance for increasing the efficiency of livestock breeding. In this respect, the accumulation of reactive oxygen species (ROS) is a major factor affecting the development of oocytes and embryos. This study investigated the effects of Dendrobium nobile extract (DNE) on the in vitro maturation of bovine oocytes and embryonic development after IVF. DNE is an extract from Dendrobium rhizomes that contains alkaloids with anti-inflammatory, anti-cancer and anti-ageing functions. Various concentrations of DNE (0, 5, 10, 20 and 50 μmol/L) were added during oocyte maturation in vitro, and we found that 10 μmol/L of DNE remarkably increased the oocyte maturation rate, the subsequent blastocyst formation rate and embryo quality. Further, we found that DNE treatment decreased the frequency of spindle/chromosome defects and ROS and increased the oocyte glutathione and mitochondrial membrane potential in oocytes. Moreover, DNE upregulated the expression of oxidative stress-related genes (Sirt1, Sirt2, Sirt3 and Sod1) in oocytes and apoptosis-related genes (Caspase-3, Caspase-4, Bax, Bcl-xl and Survivin) in blastocysts. These results suggest that DNE supplementation can promote oocyte maturation and subsequent embryonic development by regulating redox reactions and inhibiting embryonic apoptosis.

Growth Hormone Promotes Oocyte Maturation In Vitro by Protecting Mitochondrial Function and Reducing Apoptosis.

Some studies have been conducted to explore the influence of growth hormone (GH) on oocytes in in vitro maturation (IVM); however, previous studies reporting showed different results, and the specific mechanisms were not clear. In the present study, GH supplementation improved oocyte maturation rate. The rate of germinal vesicle breakdown (GVBD) in the GH group was 83.9%, which was significantly higher than that (72.1%) in the control group (p = 0.001). The maturation rate of the GH group (79.2%) was significantly higher than that (65.4%) of the control group (p = 0.000). The fertilization (68.6 vs. 59.3%) and blastocyst (30 vs. 25.3%) rates showed an increasing trend in the GH group compared to those in controls. The dynamic parameters of nuclear maturation of oocytes were recorded by time-lapse monitoring system; oocytes in the GH group completed nuclear maturation earlier than did those in the control group. GH reduced cAMP levels to promote oocyte maturation. Single-cell RNA sequencing analysis revealed that the majority of differentially expressed genes (DEGs) involved in mitochondrial oxidative phosphorylation was upregulated in the GH group. Furthermore, the mitochondrial membrane potential of oocytes significantly increased, and the levels of intracellular reactive oxygen species (ROS) and Ca2+ largely decreased in the GH group. Finally, single-oocyte transcriptome analysis indicated that GH decreased the expression of apoptosis-related genes in oocytes. GH treatment reduced the expression of γH2AX and caspase-3. Therefore, GH improves the developmental potential of immature oocytes by reducing cAMP levels more rapidly within 0.5h, protecting mitochondrial function, and reducing DNA damage and apoptosis.

MitoQ Protects Ovarian Organoids against Oxidative Stress during Oogenesis and Folliculogenesis In Vitro.

Ovarian organoids, based on mouse female germline stem cells (FGSCs), have great value in basic research and are a vast prospect in pre-clinical drug screening due to their properties, but the competency of these in vitro-generated oocytes was generally low, especially, in vitro maturation (IVM) rate. Recently, it has been demonstrated that the 3D microenvironment triggers mitochondrial dysfunction during follicle growth in vitro. Therefore, therapies that protect mitochondria and enhance their function in oocytes warrant investigation. Here, we reported that exposure to 100 nM MitoQ promoted follicle growth and maturation in vitro, accompanied by scavenging ROS, reduced oxidative injury, and restored mitochondrial membrane potential in oocytes. Mechanistically, using mice granulosa cells (GCs) as a cellular model, it was shown that MitoQ protects GCs against H2O2-induced apoptosis by inhibiting the oxidative stress pathway. Together, these results reveal that MitoQ reduces oxidative stress in ovarian follicles via its antioxidative action, thereby protecting oocytes and granulosa cells and providing an efficient way to improve the quality of in vitro-generated oocytes.

Iloprost affects in vitro maturation and developmental competence of bovine oocytes

Prostacyclin (PGI2) is synthesised in oviductal fluid and enhance the embryo development during the preimplantation period. The objective of the present study was to determine the effect of an analogue of prostacyclin (iloprost) on the in vitro maturation (IVM) and the developmental competence of bovine oocytes. Cumulus oocyte complexes (COCs) were cultured in maturation medium with iloprost (0.5 μM) for 24 h. We found that iloprost assisted maturation rates and cumulus cell expansion of bovine oocytes, and it increased the mRNA expression of genes related to cumulus expansion: ADAM17, AREG, and TNFAIP6 and cathepsin genes (CTSK and CTSS). Moreover, iloprost reduced the occurrence of apoptosis in COCs and promoted an antiapoptotic balance in the transcription of genes involved in apoptosis (BAX and BCL2). COCs treatment with iloprost during IVM also reduced intracellular reactive oxygen species (ROS) levels, while glutathione (GSH) levels and the mRNA expression of antioxidant genes CAT and GPx4 were markedly increased. We also showed that an analogue of PGI2 influenced the mitochondrial status via distribution rates of mitochondria and mitochondrial membrane potential in oocytes. Although, iloprost-enhanced maturation had no direct effect on number of embryos cleaved, it increased blastocyst rates of bovine embryos as well as proportion of expanded blastocysts. These results indicate that the supplementation of maturation medium with iloprost is beneficial for the maturation efficiency and developmental competence of bovine oocytes.

Progesterone Increases Mitochondria Membrane Potential in Non-human Primate Oocytes and Embryos

Mitochondrial activity is critical and correlates with embryo development. The identification of a novel human mitochondrial progesterone receptor (PR-M) that increases cellular respiration brings into question a role for progesterone in oocyte and preimplantation embryo development. Oocytes and embryos were generated from three Rhesus non-human primates (Macaca mulatta) undergoing in vitro fertilization. Immunohistochemical (IHC) staining for the progesterone receptor and mitochondria, RT-PCR with product sequencing for a mitochondrial progesterone receptor, and mitochondrial membrane determination with JC-1 staining were performed. IHC staining with selective antibodies to the progesterone receptor showed non-nuclear staining. Staining was absent in mouse control embryos. RT-PCR with product sequencing demonstrated PR-M transcript in Rhesus oocytes and embryos, which was absent in mouse embryos. Treatment of Rhesus oocytes and embryos with progesterone showed increased mitochondrial membrane potential, which was absent in mouse embryos. Our results support that progesterone increases mitochondrial membrane potential in oocytes and developing embryos. This is likely an in vivo mechanism to support preimplantation embryo development, and brings up the possibility of in vitro manipulation of culture media for optimization of growth.

Detrimental effects of oxidative stress in bovine oocytes during intracytoplasmic sperm injection (ICSI)

Intracytoplasmic sperm injection (ICSI) is an essential technology in animal and human reproduction. However, the developmental competence and pregnancy rate of embryos derived from ICSI are still lower than that from the conventional in vitro fertilization technique. In this report, we focused on reactive oxygen species (ROS) as a potential detrimental factor for ICSI. Experiment 1 was conducted to evaluate the effect of oxidative stress by two different oxygen concentrations (20%: control vs. 5%) in ICSI on the developmental competence (blastocyst rate: day 7, DNA fragmentation rate: day 4) and, ROS concentration and mitochondrial membrane potential of oocytes in ICSI. In the 5% O2 group, the blastocyst rate (29.5%) was higher and DNA fragmentation rate (4.8 ± 1.0%) was lower than those in the control group significantly (12.7% and 18.2 ± 2.4%, respectively, P < 0.05). Also, ROS concentration in the 5% O2 group (12.8 ± 0.7) was significantly lower than that in the control group (47.8 ± 6.9, P < 0.05). In experiment 2, we examined the supplementation of media with reduced glutathione (GSH) during ICSI procedure in an attempt to reduce the oxidative stress. The addition of GSH to the culture medium improved the blastocyst rate (17.6% vs. 30.4%, P < 0.05), and decreased the ROS levels in the oocytes (70.0 ± 7.4 vs. 23.9 ± 4.0, P < 0.05). In conclusion, our present study revealed that oocytes are under oxidative stress in ICSI procedure. Reduction of the oxygen concentration to 5% in the culture environment, or the addition of GSH in to the medium during ICSI procedure can promote the normal embryo development following the ICSI.

SIRT2 Inhibition Results in Meiotic Arrest, Mitochondrial Dysfunction, and Disturbance of Redox Homeostasis during Bovine Oocyte Maturation.

SIRT2, a member of the sirtuin family, has been recently shown to exert important effects on mitosis and/or metabolism. However, its roles in oocyte maturation have not been fully clarified. In this study, SIRT2, located in the cytoplasm and nucleus, was found in abundance in the meiotic stage, and its expression gradually decreased until the blastocyst stage. Treatment with SIRT2 inhibitors resulted in the prevention of oocyte maturation and the formation of poor-quality oocytes. By performing confocal scanning and quantitative analysis, the results showed that SIRT2 inhibition induced prominent defects in spindle/chromosome morphology, and led to the hyperacetylation of α-tubulin and H4K16. In particular, SIRT2 inhibition impeded cytoplasmic maturation by disturbing the normal distribution of cortical granules, endoplasmic reticulum, and mitochondria during oocyte meiosis. Meanwhile, exposure to SirReal2 led to elevated intracellular reactive oxygen species (ROS) accumulation, low ATP production, and reduced mitochondrial membrane potential in oocytes. Further analysis revealed that SIRT2 inhibition modulated mitochondrial biogenesis and dynamics via the downregulation of TFAM and Mfn2, and the upregulation of DRP1. Mechanistically, SIRT2 inhibition blocked the nuclear translocation of FoxO3a by increasing FoxO3a acetylation, thereby downregulating the expression of FoxO3a-dependent antioxidant genes SOD2 and Cat. These results provide insights into the potential mechanisms by which SIRT2-dependent deacetylation activity exerts its effects on oocyte quality.

5-Diphosphoinositol pentakisphosphate (5-IP7) regulates phosphate release from acidocalcisomes and yeast vacuoles

Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate-sodium symporter (TbPho91 and Pho91p in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (Pi) transport are unclear. We found here that Tbpho91-/- trypanosomes have a lower growth rate under phosphate starvation and contain larger acidocalcisomes that have increased Pi content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and Pi conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na+/Pi-loaded giant vacuoles prepared from WT or from TbPHO91-expressing pho91Δ strains but not from the pho91Δ yeast strains or from the pho91Δ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar Pi and Na+ efflux and that myo-inositol polyphosphates stimulate the Na+/Pi symporter activities through their SPX domains.

Two Distinct Allosteric Binding Sites at α4β2 Nicotinic Acetylcholine Receptors Revealed by NS206 and NS9283 Give Unique Insights to Binding Activity-associated Linkage at Cys-loop Receptors

Positive allosteric modulators (PAMs) of α4β2 nicotinic acetylcholine receptors have the potential to improve cognitive function and alleviate pain. However, only a few selective PAMs of α4β2 receptors have been described limiting both pharmacological understanding and drug-discovery efforts. Here, we describe a novel selective PAM of α4β2 receptors, NS206, and compare with a previously reported PAM, NS9283. Using two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, NS206 was observed to positively modulate acetylcholine (ACh)-evoked currents at both known α4β2 stoichiometries (2α:3β and 3α:2β). In the presence of NS206, peak current amplitudes surpassed those of maximal efficacious ACh stimulations (Emax(ACh)) with no or limited effects at potencies and current waveforms (as inspected visually). This pharmacological action contrasted with that of NS9283, which only modulated the 3α:2β receptor and acted by left shifting the ACh concentration-response relationship. Interestingly, the two modulators can act simultaneously in an additive manner at 3α:2β receptors, which results in current levels exceeding Emax(ACh) and a left-shifted ACh concentration-response relationship. Through use of chimeric and point-mutated receptors, the binding site of NS206 was linked to the α4-subunit transmembrane domain, whereas binding of NS9283 was shown to be associated with the αα-interface in 3α:2β receptors. Collectively, these data demonstrate the existence of two distinct modulatory sites in α4β2 receptors with unique pharmacological attributes that can act additively. Several allosteric sites have been identified within the family of Cys-loop receptors and with the present data, a detailed picture of allosteric modulatory mechanisms of these important receptors is emerging.

Amphetamine Activates an Amine-gated Chloride Channel to Generate Behavioral Effects in Caenorhabditis elegans

Amphetamine is a highly addictive psychostimulant, which is thought to generate its effects by promoting release of dopamine through reverse activation of dopamine transporters. However, some amphetamine-mediated behaviors persist in dopamine transporter knock-out animals, suggesting the existence of alternative amphetamine targets. Here we demonstrate the identification of a novel amphetamine target by showing that in Caenorhabditis elegans, a large fraction of the behavioral effects of amphetamine is mediated through activation of the amine-gated chloride channel, LGC-55. These findings bring to light alternative pathways engaged by amphetamine, and urge rethinking of the molecular mechanisms underlying the effects of this highly-addictive psychostimulant.

ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o- WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells.

Fractional contribution of major ions to the membrane potential of Drosophila melanogaster oocytes

In ovarian follicles of Drosophila melanogaster, ion substitution experiments revealed that K(+) is the greatest contributor (68%) in setting oocyte steady-state potential (E(m)), while Mg(2+) and a metabolic component account for the rest. Because of the intense use made of Drosophila ovarian follicles in many lines of research, it is important to know how changes in the surrounding medium, particularly in major diffusible ions, may affect the physiology of the cells. The contributions made to the Drosophila oocyte membrane potential (E(m)) by [Na(+)](o), [K(+)](o), [Mg(2+)](o), [Ca(2+)](o), [Cl(-)](o), and pH (protons) were determined by substitutions made to the composition of the incubation medium. Only K(+) and Mg(2+) were found to participate in setting the level of E(m). In follicles subjected to changes in external pH from the normal 7.3 to either pH 6 or pH 8, E(m) changed rapidly by about 6 mV, but within 8 min had returned to the original E(m). Approximately half of all follicles exposed to reduced [Cl(-)](o) showed no change in E(m), and these all had input resistances of 330 kOmega or greater. The remaining follicles had smaller input resistances, and these first depolarized by about 5 mV. Over several minutes, their input resistances increased and they repolarized to a value more electronegative than their value prior to reduction in [Cl(-)](o). Together, K(+) and Mg(2+) accounted for up to 87% of measured steady-state potential. Treatment with sodium azide, ammonium vanadate, or chilling revealed a metabolically driven component that could account for the remaining 13%.

Characterization of the Transport Mechanism and Permeant Binding Profile of the Uridine Permease Fui1p of Saccharomyces cerevisiae

The uptake of Urd into the yeast Saccharomyces cerevisiae is mediated by Fui1p, a Urd-specific nucleoside transporter encoded by the FUI1 gene and a member of the yeast Fur permease family, which also includes the uracil, allantoin, and thiamine permeases. When Fui1p was produced in a double-permease knock-out strain (fur4Deltafui1Delta) of yeast, Urd uptake was stimulated at acidic pH and sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone. Electrophysiological analysis of recombinant Fui1p produced in Xenopus oocytes demonstrated that Fui1p-mediated Urd uptake was dependent on proton cotransport with a 1:1 stoichiometry. Mutagenesis analysis of three charged amino acids (Glu(259), Lys(288), and Asp(474) in putative transmembrane segments 3, 4, and 7, respectively) revealed that only Lys(288) was required for maintaining high Urd transport efficiency. Analysis of binding energies between Fui1p and different Urd analogs indicated that Fuip1 interacted with C(3')-OH, C(2')-OH, C(5)-H, and N(3)-H of Urd. Fui1p-mediated transport of Urd was inhibited by analogs with modifications at C-5', but was not inhibited significantly by analogs with modifications at C-3', C-5, and N-3 or inversions of configuration at C-2' and C-3'. This characterization of Fui1p contributes to the emerging knowledge of the structure and function of the Fur family of permeases, including the Fui1p orthologs of pathogenic fungi.

Cloning of a unique electrogenic bicarbonate transporter from the squid giant fiber lobe

The squid (Loligo pealei) giant axon is a well-studied model system for intracellular pH (pHi) regulation, but the molecular identities of the proteins responsible for HCO3 transport in this system are still unclear. To date, only one HCO3 transporter—an electroneutral Na-driven Cl/HCO3 exchanger (sqNDCBE), related to the SLC4 family—has been cloned and characterized from squid giant fiber lobe (GFL) cDNA. Our goal was to identify and characterize another. Using PCR with degenerate primers, followed by 5′ and 3′ RACE, we identified a full-length clone (SF3) that consists of 1,162 amino acids with 50% identity to sqNDCBE, 46–48% identity to human electroneutral NBCs, and 40–44% identity to human electrogenic NBCs. Phylogenetic analysis of the SF3 amino acid sequence reveals that SF3 clusters with sqNDCBE and other electroneutral NBCs, suggesting that SF3 is electroneutral. However, when Xenopus oocytes were injected with SF3 cRNA and exposed to a buffer containing 5% CO2/33 mM HCO3, the oocyte membrane potential showed a large, rapid hyperpolarization, followed by a slow relaxation, indicating that SF3 is electrogenic. Two-electrode voltage clamping and pHi measurements demonstrate that the current and pHi recovery via SF3 depend on extracellular Na+ but not Cl−, and are inhibited by DIDS. In conclusion, we have cloned from squid GFL cDNA an electrogenic Na-dependent HCO3 cotransporter (sqNBCe), which to our knowledge is the first electrogenic NBC characterized from an invertebrate.

CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR-ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain.

Effect of 3-O-Octanoyl-(+)-catechin on the Responses of GABAA Receptors and Na+/Glucose Cotransporters Expressed in Xenopus Oocytes and on the Oocyte Membrane Potential

Recently, 3-O-octanoyl-(+)-catechin (OC) was synthesized from (+)-catechin (C) by incorporation of an octanoyl chain into C in the light of (-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg), which are the major polyphenols found in green tea and have strong physiological activities. OC was found to inhibit the response of ionotropic gamma-aminobutyric acid (GABA) receptors (GABA(A) receptors) and Na+/glucose cotransporters expressed in Xenopus oocytes in a noncompetitive manner more strongly than does C. OC also induced a nonspecific membrane current and decreased the membrane potential of the oocyte, and thus death of the oocyte occurred even at lower concentrations than that induced by C or EGCg. Although EGCg produced H2O2 in aqueous solution, OC did not. This newly synthesized catechin derivative OC possibly binds to the lipid membrane more strongly than does C, Ecg, or EGCg and as a result perturbs the membrane structure.

Preclinical evaluation of anticonvulsant activity of N-(p-aminobenzoyl)-1,2,3,4-tetrahydroquinoline

Anticonvulsant activity of N-(p-aminobenzoyl)-1,2,3,4- tetrahydro-quinoline (NTQ), a newly synthesized ameltolide analog, was investigated with regards to efficacy and toxicity in mice. Additionally, its effect on NMDA receptors was also performed on NR1a/NR2B expressed in Xenopus oocytes. NTQ, given intraperitoneally, was able to protect the animals in both maximal electroshock seizure and pentylenetetrazole (PTZ) tests, while ameltolide was effective only in the MES. NTQ was less potent than ameltolide, exhibiting the median effective dose (ED50) of 13.33 mg/kg body weight (B.W.), whereas the corresponding value of ameltolide was 0.96 mg/kg B.W. Ameltolide seemed to possess better safety profile than NTQ as indicated by the relative safety margin (LD50/ED50) of 65.28 for ameltolide and 44.97 for NTQ. Furthermore, based on the finding that the median neurotoxic dose (TD50) established by rotarod test was 37.28 and 7.18 mg/kg B.W. for ameltolide and NTQ respectively, ameltolide appeared to have more favorable protective index (TD50/ED50) than NTQ. Further experiment using Xenopus oocyte with NMDA receptor composing of NR1a and NR2B subunits was performed to probe for the effect of NTQ and ameltolide on NMDA-induced current. While NTQ and ameltolide did not induce any changes in current or a shift in membrane potential of oocyte, both of them significantly inhibited NMDA-induced current demonstrating the IC50 of 0.10 and 0.12 ?M respectively. Therefore, it is likely that inhibition of excitation of NMDA receptor may, at least in part, accounted for its anticonvulsant activity. In conclusion, the present study has identified NTQ as a broad-spectrum anticonvulsant agent, albeit its lower safety margin and protective index. Inhibition of NMDA receptor may partly contribute to anticonvulsant activity of these two compounds. Further structural modification of NTQ to improve its safety while preserving a broad-spectrum anticonvulsant activity should be carried out.

Teleost Fh14-3-3a protein protects Xenopus oocytes from hyperosmolality.

We have previously cloned and characterized a novel 14-3-3 gene from the euryhaline telost Fundulus heteroclitus, Fh14-3-3a (Kültz et al., 2001). The corresponding gene product is osmoregulated and most highly expressed in gill epithelium of this fish. In the present study we have expressed Fh14-3-3a cRNA in Xenopus laevis oocytes and investigated the survival and electrophysiological parameters of Xenopus oocytes in isosmotic and various hyperosmotic media. Xenopus oocytes expressing Fh14-3-3a show no mortality after a 16 hour exposure to hyperosmolality in the form of elevating medium K(+), Na(+), polyethylene glycol, or sorbitol concentrations up to 444 mosmol/kg. In contrast, 16 hours of the same hyperosmolality caused 100% mortality in control Xenopus oocytes injected with water. As a result of hyperosmolality the Xenopus oocyte membrane potential decreased between 10 and 70% in oocytes expressing Fh14-3-3a whereas it was completely abolished in control oocytes. We report that one potential cause for the osmoprotective effect of Fh14-3-3a on Xenopus oocytes could be its inhibition of an endogenous chloride current. Hyperosmotic urea was not as harmful to Xenopus oocytes as hypertonicity and maybe acting through a different mechanism. Coexpression of Fh14-3-3a with a human calcium channel in Xenopus oocytes did not affect the electrophysiological properties of this exogenous channel. Thus, the osmoprotective effect of Fh14-3-3a may prove a valuable tool for the characterization of exogenous ion channels in Xenopus oocytes exposed to hyperosmotic conditions.

Hamster oocyte membrane potential and ion permeability vary with preantral cumulus cell attachment and developmental stage.

BackgroundIn vitro maturation of mammalian oocytes is an area of great interest due to its potential application in the treatment of infertility. The morphological and physiological changes that occur during oocyte development are poorly understood, and further studies are needed investigating the physiological changes associated with oocyte maturation. In this study we evaluated the membrane potential and the sodium/potassium permeability ratio of oocytes acutely isolated, and cumulus-oocyte complexes in metaphase II and preantral follicle stages.ResultsIntracellular electrical recordings revealed that cumulus-enclosed oocytes have a membrane potential significantly more negative at the preantral follicle stage than at metaphase II stage (-38.4 versus -19.7 mV, p < 0.0005). The membrane potential of the cumulus-free oocytes was not different between the preantral and metaphase II stages. The membrane potential of the cumulus cells forming preantral stage follicles was shown to be significantly different from that of the oocyte within the follicle (-28.6 versus -38.4 mV, p < 0.05). The sodium/potassium permeability measured in cumulus-enclosed oocytes at the preantral stage equaled a mean value of 0.33. The ratio was significantly lower when measured in oocytes denuded of cumulus cells or cumulus-enclosed metaphase II oocytes, 0.76, 0.79, 0.77 respectively (p < 0.001).ConclusionsThese data show a change in the membrane potential and Na+/K+ permeability ratio during ooycte development from the preantral stage oocyte to the metaphase II stage. We have also demonstrated a change in the preantral oocyte membrane potential when surrounding cumulus cells are removed; either due to membrane changes or loss of cumulus cells.