Receptors In Oocytes Research Articles

Melatonin decreases excessive polyspermy for single oocyte in pigs through the MT2 receptor

Melatonin supplementation during in vitro maturation (IVM) improves porcine oocyte maturation and embryonic development by exerting antioxidative effects. Nevertheless, the mechanism by which melatonin prevents polyspermy after in vitro fertilization (IVF) remains unclear. Here, we examined the effects of melatonin on cytoplasmic maturation and the incidence of polyspermic penetration in porcine oocytes. No statistically significant difference was observed in the rate of first polar body formation between the groups (Control, Melatonin, Melatonin + Luzindole, and Melatonin + 4-P-PDOT). Interestingly, melatonin supplementation significantly improved the cytoplasmic maturation of porcine oocytes by enhancing the normal distribution of organelles (Golgi apparatus, endoplasmic reticulum and mitochondria) and upregulating organelle-related gene expressions (P < 0.05). However, these promotional effects were counteracted by melatonin antagonists, suggesting that melatonin enhances cytoplasmic maturation through its receptors in porcine oocytes. Melatonin supplementation also significantly improved the rate of diploid and blastocyst formation after IVF by promoting the normal distribution of cortical granules (P < 0.05). In conclusion, melatonin supplementation during in vitro maturation of porcine oocyte improves fertilization efficiency and embryonic developmental competence by enhancing cytoplasmic maturation.

P-667 The RANK-RANKL-OPG System - A new player in Oocyte Maturation

Abstract Study question Does the RANK-RANKL-OPG system play a role in oocyte maturation and ovulation? Summary answer RANKL, a new ovulatory gene expressed by cumulus and mural granulosa cells activates RANK receptors in oocytes, promoting oocyte maturation through oocyte-granulosa cell communication. What is known already The receptor activator of nuclear factor-kB (RANK), its ligand RANKL, and the soluble decoy osteoprotegerin (OPG) system are known due to their role in bone biology, the immune system, and other tissues. It was recently shown that RANKL was a highly upregulated gene in our ovulation-associated gene library of cumulus cells (CCs) from mature and non-mature oocytes. However, its ligand, RANK was undetected in both samples. The goal of this study was to elucidate the expression and function of the RANK-RANKL-OPG system in the ovary during the preovulatory period, and ovulation. Study design, size, duration In vitro and in vivo experiments using mice and human oocytes and granulosa cells. Participants/materials, setting, methods RANKL regulation experiments involve granulosa cells isolated from follicular fluid as well as mice oocytes. Analysis included cell culture, qPCR, and western blot. To examine RANK-RANKL-OPG function upon ovulation and oocyte maturation, super-ovulated mice and human oocytes were used. Main results and the role of chance We demonstrated for the first time that RANK mRNA is expressed in murine oocytes. Moreover, RANKL expression was demonstrated in human MGCs and CCs and is regulated by hCG/LH and PGE2. The addition of RANKL peptide, to the murine and human oocyte during IVM procedure, induces the maturation of GV oocytes. OPG (natural RANKL inhibitor) injection to mice during super-ovulation reduced oocyte maturation rate. Limitations, reasons for caution Animal model results may not fully translate to humans. Additional studies are needed to confirm the effects of RANKL/RANK-OPG system on fertility and long-term health. Wider implications of the findings Our observations suggest that RANKL expressed by granulosa cells acts on the RANK receptor found in the oocyte and plays an important role in oocyte-granulosa cell communication activating oocyte maturation. This research contributes to new insights in understanding pre-ovulatory processes and may improve the procedure of in vitro maturation (IVM). Trial registration number not applicable

Improvement of oocyte competence and in vitro oocyte maturation with EGF and IGF-I in Guinea pig model

In vitro maturation (IVM) system is an alternative method to superovulation protocols to obtain mature oocytes. Epidermal Growth Factor (EGF) and Insulin-like Growth Factor I (IGF-I) have been widely used in IVM medium in different species. Although the guinea pig is a valuable animal model for reproductive studies, IVM is rarely used. We aimed to establish a suitable in vitro production system using EGF and/or IGF-I during IVM to improve oocyte competence. Firstly, immunolocalization of EGF and IGF-I receptors in the ovary was assessed. An IVM dose-response experiment was performed with cumulus-oocyte complexes (COCs) supplemented with: 1) EGF [0, 10, 50, 100 ng/mL or 10% fetal calf serum (FCS)]; 2) IGF-I [0, 50, 100, 200 ng/mL or 10% FCS]; or 3) the concentrations of EGF and IGF-I which showed the best IVM index in the previous experiments, with or without Fetal Calf Serum (FCS). Cortical granule and mitochondria distribution patterns were determined in in vivo and in vitro-matured oocytes for the first time in this species. Apoptotic rate after IVM and oocyte competence by in vitro embryo development were evaluated. Immunohistochemistry results showed positive immunostaining of EGF and IGF receptors in corpus luteum, oocytes, granulosa and theca cells in follicles in all stages of development. Supplementation of IVM medium with 50 ng/mL EGF or 100 ng/mL IGF-I or their combination with FCS successfully led to oocyte nuclear and cytoplasmic maturation and reduced the apoptotic rate. Both growth factors improved oocyte competence during IVM in this species since early embryos were in vitro developed, showing better results when FCS was used in the IVM medium.

L-theanine attenuates nicotine reward and withdrawal signs in mice

BackgroundL-theanine, 2-amino-4-(ethylcarbamoyl) butyric acid, an amino acid detected in green tea leaves, is used as a dietary supplement to attenuate stress and enhance mood and cognition. Furthermore, L-theanine induces anxiolytic effects in humans. Recently, L-theanine was reported to reduce morphine physical dependence in primates, suggesting the potential usefulness of L-theanine for drug dependence intervention. ObjectiveThe aim of this study is to determine whether L-theanine attenuates nicotine-withdrawal (somatic and affective signs) and nicotine reward in mice. We also investigated the effects of L-theanine on nicotinic receptors binding and function. MethodsICR male mice rendered dependent to nicotine through implanted subcutaneous osmotic minipumps for 14 days undertook precipitated nicotine withdrawal by mecamylamine on day 15. Anxiety-like behaviors using LDB, somatic signs observation and hot plate latency were assessed consecutively after treatment with L-theanine. Furthermore, we examined the effect of L-theanine on acute nicotine responses and nicotine conditioned reward in mice and on expressed nicotinic receptors in oocytes. Key findingsL-theanine reduced in a dose-dependent manner anxiety-like behavior, hyperalgesia and somatic signs during nicotine withdrawal. Also, L-theanine decreased the nicotine CPP, but it did not affect the acute responses of nicotine. Finally, L-theanine did not alter the binding or the function of expressed α4β2 and α7 nAChRs. ConclusionOur results support the potential of L-theanine as a promising candidate for treating nicotine dependence.

Role of calcium-sensing receptor in regulating activation susceptibility of postovulatory aging mouse oocytes.

The mechanisms underlying postovulatory oocyte aging (POA) remain largely unknown. The expression of the calcium-sensing receptor (CaSR) in mouse oocytes and its role in POA need to be explored. Our objective was to observe CaSR expression and its role in the susceptibility to activating stimuli (STAS) in POA mouse oocytes. The results showed that, although none of the newly ovulated oocytes were activated, 40% and 94% of the oocytes recovered 19 and 25 h after human chorionic gonadotropin (hCG) injection were activated, respectively, after ethanol treatment. The level of the CaSR functional dimer protein in oocytes increased significantly from 13 to 25 h post hCG. Thus, the CaSR functional dimer level was positively correlated with the STAS of POA oocytes. Aging in vitro with a CaSR antagonist suppressed the elevation of STAS, and cytoplasmic calcium in oocytes recovered 19 h post hCG, whereas aging with a CaSR agonist increased STAS, and cytoplasmic calcium of oocytes recovered 13 h post hCG. Furthermore, the CaSR was more important than the Na-Ca2+ exchanger in regulating oocyte STAS, and T- and L-type calcium channels were inactive in aging oocytes. We conclude that the CaSR is involved in regulating STAS in POA mouse oocytes, and that it is more important than the other calcium channels tested in this connection.

Reconstitution of an N-AChR from Brugia malayi, an evolved change in acetylcholine receptor accessory protein requirements in filarial parasites.

Neurotransmission is an important target for anthelmintic drugs, where receptor characteristics and response can be examined through reconstitution ex vivo in Xenopus laevis oocytes. The homomeric ACR-16 nicotine sensitive acetylcholine receptors (N-AChRs) of several helminth species have been characterized in this way. Our efforts to reconstitute the N-AChR from the clade III filarial parasite, Brugia malayi using similar conditions, initially produced no detectable response. A robust response to acetylcholine is obtained from the closely related clade III parasite Ascaris suum, suggesting that specific changes have occurred between Ascaris and Brugia. N-AChRs from three species intermediate between A. suum and B. malayi were characterized to provide information on the cause. Maximal response to acetylcholine did not change abruptly, consistent with a discrete event, but rather decreased progressively from A. suum through Dracunculus medinensis, Gonglylonema pulchrum and Thelazia callipaeda. Receptor responses to the characteristic nicotine, and other agonists were generally similar. The decrease in maximal current did correlate with a delayed time to reach larger response. Together, this suggested that the failure to reconstitute the B. malayi N-AChR was one extreme of a progressive decrease and that an issue with synthesis of the receptor in oocytes was responsible. Addition of accessory proteins EMC-6, NRA-2 and NRA-4, in addition to RIC-3, produced a small, but measurable B. malayi N-AChR response. Pharmacological properties of a chimeric B. malayi N-AChR were equivalent to the other species, confirming the receptor response remains unchanged while its production is increasingly dependent on accessory proteins. One possibility is that loss of many subunits for acetylcholine receptors from the filarial nematode genome is linked to new subunit combinations that lead to such a dependence. This novel phylogenetic approach allowed the first characterization of a B. malayi AChR ex vivo and in doing so, provides a framework for the successful characterization of other receptors that have yet to be reconstituted.

Expression of Mutant Glycine Receptors in Xenopus Oocytes Using Canonical and Non-Canonical Amino Acids Reveals Distinct Roles of Conserved Proline Residues.

Pentameric ligand-gated ion channels (pLGIC) play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain. The binding of the agonist to the ECD induces a structural change that is transduced to the TMD to open the channel. Molecular details of this process are emerging, but a comprehensive understanding is still lacking. Proline (Pro) is one amino acid that has attracted much interest; its unusual features generate bends in loops and kinks and bulges in helices, which can be essential for function in some pLGICs. Here, we explore the roles of four conserved Pros in the glycine receptor (GlyR), creating substitutions with canonical and noncanonical amino acids, characterizing them using two electrode voltage clamp electrophysiology in Xenopus oocytes, and interpreting changes in receptor parameters using structural data from the open and closed states of the receptor. The data reveal that for efficient function, the Pro in the α1β1 loop is needed to create a turn and to be the correct size and shape to interact with nearby residues; the peptide bond of the Pro in the Cys-loop requires the cis conformation; and the Pros in loop A and M1 allow efficient function because of their reduced hydrogen bonding capacity. These data are broadly consistent with data from other pLGICs, and therefore likely represent the important features of these Pros in all members of the family.

Macromolecules Influence Cellular Competence and Expression Level of IGFs Genes in Bovine Oocytes In Vitro.

Simple SummaryThe macromolecule content of culture media can affect the maturation competence of oocytes, which influences the subsequent in vitro development of embryos. This study was designed to determine the effects of macromolecules on cellular competence and the transcript level of insulin-like growth factors (IGF1, IGF2) and their receptors in bovine oocytes. The current study showed that bovine serum albumin (BSA) and fetal calf serum (FCS) improved nuclear maturation and protein biosynthesis (especially FCS). Polyvinyl alcohol did not support the antioxidant defense mechanism due to decreased glutathione peroxidase enzyme activity. The expression of the IGF1 gene could not be detected in all experimental groups, but BSA and FCS increased the transcript level of the IGF2 gene. Moreover, oocyte maturation with BSA increased the transcript level of the IGF1R gene, whereas the transcript level of the IGF2R gene was similar among macromolecule supplementation groups. The BSA and FCS could improve in vitro bovine oocyte development due to supporting cellular characteristics.In vitro maturation (IVM) of mammalian oocytes, which influences subsequent in vitro development of embryos, is affected by the macromolecule content in culture media for the success of oocyte maturation competence, in which the cytoplasmic and nuclear reprogramming events occur. The insulin-like growth factor family (IGFs) promotes the maturation of bovine oocytes and the expansion of cumulus cells and also inhibits apoptosis. This study was, therefore, designed to examine the effects of macromolecules (bovine serum albumin, BSA; fetal calf serum, FCS; and polyvinyl alcohol, PVA) on in vitro nuclear maturation, total cellular protein, glutathione peroxidase (GPx) enzyme activity, and the gene expression level of IGF1, IGF2, and their receptor in bovine oocytes. Oocytes obtained from bovine ovaries were cultured in bicarbonate-buffered medium 199 supplemented with 4 mg/mL BSA, 10% FCS, 1 mg/mL PVA, and without macromolecule supplement (control) during 22 h in the air with a humidified atmosphere and 5% CO2 at 38.5 °C temperature. Supplementation of BSA and FCS increased (χ2 = 9.84; p < 0.05) the percentages of oocytes that reached metaphase II compared to the control and PVA. The amount of protein per ml of cell extracts of oocytes matured in FCS supplemented culture media was higher (p < 0.05) than the oocytes in the PVA and control. The levels of GPx enzyme activity in cell extracts isolated from oocytes in each experimental group did not change over time, but the GPx enzyme activity in oocytes matured in PVA-supplemented culture media was lower (p < 0.05) than in oocytes in the other experimental groups. Transcript for the IGF1 gene was not detected in all experimental groups, but the supplementation of BSA and FCS significantly elevated the transcript level of the IGF2 gene. In addition, the maturation of oocytes with BSA-supplemented media increased the transcript level of the IGF1R gene, whereas the transcript level of the IGF2R gene was similar among macromolecule supplementation groups. The current study concluded that BSA and FCS could improve in vitro bovine oocyte development due to supporting nuclear maturation and increasing the total cellular protein content, GPx enzyme, and transcript activity.

Different efficiency of auxiliary/chaperone proteins to promote the functional reconstitution of honeybee glutamate and acetylcholine receptors in Xenopus laevis oocytes.

Heterologous expression systems (e.g., Xenopus laevis oocytes) are useful to study the biophysical properties and pharmacology of ionotropic receptors such as ionotropic glutamate (iGLuRs) and nicotinic acetylcholine (nAChRs) receptors. However, insect receptors often require the co‐expression of chaperone proteins to be functional. Only few iGluRs and nAChRs have been successfully expressed in such systems. Here, we compared the efficiency of chaperone proteins to promote the functional expression of one Apis mellifera iGluR and several nAChR subunit combinations (α1α8β1, α7, α2α8β1 and α2α7α8β1) in Xenopus oocytes. To this end, we cloned a new iGluR (GluR‐1) and potential chaperone proteins (e.g., SOL‐1, Neto, NACHO) and tested more than 40 combinations of human, nematode and honeybee proteins. We obtained robust expression of GluR‐1 and α1α8β1 when co‐expressed with honeybee chaperone proteins and found that nAChR expression critically depended on the α1 subunit N‐terminal sequence. We recorded small ACh‐gated currents in few oocytes when the α7 subunit was co‐expressed with Caenorhabditis elegans RIC‐3, but none of the chaperone proteins allowed efficient expression of α2α8β1 or α2α7α8β1. Our results show that only some protein combinations can reconstitute functional receptors in Xenopus oocytes and that protein combination efficient in one species is not always efficient in another species.

Kisspeptin stimulates oocyte maturation, and food deprivation modulates the abundance of kisspeptin system in zebrafish gonads

Kisspeptin is a hormone involved in the neuroendocrine control of reproduction in fish. We hypothesized that kisspeptin stimulates oocyte maturation and modulates other reproductive hormones in zebrafish; and the gonadal kisspeptin system in zebrafish is affected during energy unavailability. The main goals of this research were to test in vitro effects of kisspeptin on oocyte maturation and mRNA abundance in zebrafish ovarian follicles and determine how short-term feed restriction affects kisspeptin and its receptors in zebrafish testis and ovary. This study demonstrates the presence of kisspeptin and its receptors in zebrafish (Danio rerio) gonads and its direct action on ovarian follicles in vitro. Kisspeptin (10 ng/mL) induced oocyte maturation, as indicated by germinal vesicle break down at 18- and 24- hours post-treatment. Kisspeptin significantly increased the abundance of mRNAs encoding reproductive hormones and its receptors in zebrafish oocytes. This suggests that kisspeptin-10 affects ovarian functions by modulating other hormones. Reproduction is a process that requires energy. Therefore, whether energy availability affects the kisspeptin system in zebrafish gonads was determined. Food deprivation modulated kisspeptin expression differently in zebrafish testis and ovary. Kiss2 and kiss1ra were upregulated while kiss1rb was downregulated in the testis post-food deprivation. Meanwhile, no changes in kiss in the ovary were found after food deprivation. However, kiss1rb was downregulated in unfed fish at 3- and 7-days post-food deprivation. Overall, our results suggest sex- and tissue-specific changes in the gonadal abundance of the kisspeptin system in zebrafish. The fine tuning of reproduction during energy fluctuations in fish is likely mediated via changes in hormones, including kisspeptin as shown in this research.

Characterisation of thymol effects on RDL receptors from the bee parasite Varroa destructor

A major contributor to bee colony decline is infestation with its most devastating pest, the mite Varroa destructor. To control these mites, thymol is often used, although how it achieves this is not understood. One well-documented action of thymol is to modulate GABA-activated ion channels, which includes insect RDL receptors, a known insecticidal target. Here we have cloned two Varroa RDL subunits, one of which is similar to the canonical RDL subunit, while the other has some differences in M4, and, to a lesser extent, M2 and its binding site loops. Expression of this unusual RDL receptor in Xenopus oocytes reveals GABA-activated receptors, with an EC50 of 56 μM. In contrast to canonical RDL receptors, thymol does not enhance GABA-elicited responses in this receptor, and concentration response curves reveal a decrease in GABA Imax in its presence; this decrease is not seen when similar data are obtained from Apis RDL receptors. We conclude that an M2 T6′M substitution is primarily responsible for the different thymol effects, and suggest that understanding how and where thymol acts could assist in the design of novel bee-friendly miticides.

Efficient visible/NIR light-driven uncaging of hydroxylated thiazole orange-based caged compounds in aqueous media.

In photoactivation strategies with bioactive molecules, one-photon visible or two-photon near-infrared light-sensitive caged compounds are desirable tools for biological applications because they offer reduced phototoxicity and deep tissue penetration. However, visible-light-sensitive photoremovable protecting groups (PPGs) reported so far have displayed high hydrophobicity and low uncaging cross sections (εΦ < 50) in aqueous media, which can obstruct the control of bioactivity with high spatial and temporal precision. In this study, we developed hydroxylated thiazole orange (HTO) derivatives as visible-light-sensitive PPGs with high uncaging cross sections (εΦ ≈ 370) in aqueous solution. In addition, 2PE photolysis reactions of HTO-caged glutamate were achieved using a NIR laser (940 nm). Moreover, HTO-caged glutamate can activate N-methyl-d-aspartic acid receptors in Xenopus oocytes and mammalian cells with green-light illumination, thus allowing optical control of biological functions.

The avermectin/milbemycin receptors of parasitic nematodes

Glutamate-gated chloride channels are the most important target of ivermectin and related compounds in parasitic nematodes. A small family of genes encode subunits of these channels, allowing the assembly of multiple channel subtypes; the subunit composition of most of the native receptors is unknown. The members of the gene family vary between species, making extrapolation from C. elegans to parasites difficult. Expression of recombinant receptors in Xenopus oocytes can identify subunits that have the ability to co-assemble into novel channels, but localisation data, ideally at the single-cell level, is required to confirm that these subunits are expressed in the same cells and tissues. Fortunately, recent advances in this area are starting to make this information available; this information is adding to our understanding of how the drugs act and of the possible subunit combinations that create their targets in vivo.

A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism.

Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission and are crucial drug targets. Their gating mechanism is triggered by ligand binding in the extracellular domain that culminates in the opening of a hydrophobic gate in the transmembrane domain. This domain is made of four α-helices (M1 to M4). Recently the outer lipid-facing helix (M4) has been shown to be key to receptor function, however its role in channel opening is still poorly understood. It could act through its neighboring helices (M1/M3), or via the M4 tip interacting with the pivotal Cys-loop in the extracellular domain. Mutation of a single M4 tyrosine (Y441) to alanine renders one pLGIC—the 5-HT3A receptor—unable to function despite robust ligand binding. Using Y441A as a proxy for M4 function, we here predict likely paths of Y441 action using molecular dynamics, and test these predictions with functional assays of mutant receptors in HEK cells and Xenopus oocytes using fluorescent membrane potential sensitive dye and two-electrode voltage clamp respectively. We show that Y441 does not act via the M4 tip or Cys-loop, but instead connects radially through M1 to a residue near the ion channel hydrophobic gate on the pore-lining helix M2. This demonstrates the active role of the M4 helix in channel opening.

Nicotinic acetylcholine receptors: Ex-vivo expression of functional, non-hybrid, heteropentameric receptors from a marine arthropod, Lepeophtheirus salmonis.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mostly located in the post-synaptic membrane of cholinergic synapses. The natural neurotransmitter is acetylcholine, but they are also the direct targets for neonicotinoids, chemicals widely used against ectoparasites, arthropod vectors and agricultural pests. There are significant concerns regarding adverse effects of neonicotinoids on beneficial insects. In arthropods, functional nAChRs made of α subunits have been expressed from Drosophila genes, and hybrid receptors (sometimes also referred to as chimeric receptors) using species-specific α subunits and vertebrate β subunits have been expressed ex-vivo. Arthropod-specific nAChRs made of both α and β subunits from the target species have not been expressed ex-vivo. The aim of the current study was to express such receptors in Xenopus oocytes using only genes from Lepeophtheirus salmonis, to characterize them and study their modulation. Genes encoding α and β subunits of the nAChRs and three ancillary proteins, RIC-3, UNC-50 and UNC-74 were identified in the L. salmonis genome, subjected to RACE-PCR, cloned into an expression vector and the cRNA produced was then injected into Xenopus laevis oocytes. Co-expression of the ancillary proteins was essential for the successful expression of the L. salmonis nAChRs with both α and β subunits. Two functional nAChRs were identified: Lsa-nAChR1 consisting of α1, α2, β1 and β2 subunits, reconstituted to one distinct receptor, while Lsa-nAChR2, consisting of α3, β1 and β2 subunits reconstitutes receptors with two distinct characteristics. Out of seven neonicotinoids tested, six worked as partial agonist of Lsa-nAChR1 while only three did so for Lsa-nAChR2. Four non-neonicotinoid compounds tested had no effect on either of the nAChRs. The study demonstrated that fully functional, non-hybrid nAChRs containing both α and β subunits from an arthropod can be reconstituted ex-vivo by co-expression of essential ancillary proteins. Such models would be valuable for in-depth studies of effects by neonicotinoids and other compounds on target pests, as well as for studies of adverse effects on non-target arthropods.

Fam70A binds Wnt5a to regulate meiosis and quality of mouse oocytes.

ObjectivesLittle is known about the roles of integral membrane proteins beyond channels, carriers or receptors in meiotic oocytes. The transmembrane protein Fam70A was previously identified as a likely “female fertility factor” in Fox3a‐knockout mouse ovaries where almost all follicles underwent synchronous activation and the mice became infertile very early. However, whether Fam70A functions in oocyte meiosis remains unknown. Therefore, the present study aimed to address this question.Materials and MethodsCo‐immunoprecipitation, immunogold labelling‐electron microscopy, co‐localization and yeast two‐hybrid assays were used to verify the interaction. Antibody or small interfering RNA transfection was used to deplete the proteins. Immunofluorescence, immunohistochemistry and live tracker staining were used to examine the localization or characterize phenotypes. Western blot was used to examine the protein level.ResultsFam70A was enriched in oocyte membranes important for normal meiosis. Fam70A depletion remarkably disrupted spindle assembly, chromosome congression and first polar body extrusion, which subsequently increased aneuploidy and abnormal fertilization. Moreover, Fam70A directly bound Wnt5a, the most abundant Wnt member within oocytes. Depletion of either Fam70A or Wnt5a remarkably increased adenomatous polyposis coli (APC), which stabilizes active β‐catenin and microtubules. Consequently, depletion of either Fam70A or Wnt5a remarkably increased p‐β‐catenin (inactive form) and acetylated tubulin, while APC knockdown remarkably decreased these two. Furthermore, Fam70A depletion remarkably reduced Akt phosphorylation.ConclusionsFam70A regulates meiosis and quality of mouse oocytes through both canonical and non‐canonical Wnt5a signalling pathways.

Characterization of AN6001, a positive allosteric modulator of α6β2-containing nicotinic acetylcholine receptors

α6β2-Containing nicotinic acetylcholine receptors (α6β2* nAChRs) are predominantly expressed in midbrain dopaminergic neurons, including substantia nigra pars compacta (SNc) neurons and their projections to striatal regions, where they regulate dopamine release and nigrostriatal activity. It is well established that nAChR agonists exert protection against dopaminergic neurotoxicity in cellular assays and parkinsonian animal models. Historically, drug development in the nAChR field has been mostly focused on development of selective agonists and positive allosteric modulators (PAMs) for the predominant neuronal nAChRs, α7 and α4β2. Here, we report the discovery and characterization of AN6001, a novel selective α6β2* nAChR PAM. AN6001 mediated increases in both nicotine potency and efficacy at the human α6/α3β2β3V9’S nAChR in HEK293 cells, and it positively modulated ACh-evoked currents through both α6/α3β2β3V9’S and a concatenated β3-α6-β2-α6-β2 receptor in Xenopus oocytes, displaying EC50 values of 0.58 µM and 0.40 µM, respectively. In contrast, the compound did not display significant modulatory activity at α4β2, α3β4, α7 and muscle nAChRs. AN6001 also increased agonist-induced dopamine release from striatal synaptosomes and augmented agonist-induced global cellular responses and inward currents in dopaminergic neurons in SNc slices (measured by Ca2+ imaging and patch clamp recordings, respectively). Finally, AN6001 potentiated the neuroprotective effect of nicotine at MPP+-treated primary dopaminergic neurons. Overall, our studies demonstrate the existence of allosteric sites on α6β2* nAChRs and that positive modulation of native α6β2* receptors strengthens DA signaling. Hence, AN6001 represents an important tool for studies of α6β2* nAChRs and furthermore underlines the therapeutic potential in these receptors in Parkinson’s disease.

Sphingosine‐1‐phosphate (S1P) analog phytosphingosine‐1‐phosphate (P1P) improves the in vitro maturation efficiency of porcine oocytes via regulation of oxidative stress and apoptosis

Phytosphingosine-1-phosphate (P1P) is a signaling sphingolipid that regulates various physiological activities. However, little is known about the effect of P1P in the context of reproduction. Thus, we aimed to investigate the influence of P1P on oocyte maturation during porcine in vitro maturation (IVM). Here, we report the expression of S1PR1-3 among P1P receptors (S1PR1-4) in cumulus cells and oocytes. When P1P was administered at concentrations of 10, 50, 100, and 1,000 nM during IVM, the metaphase II rate was significantly increased in the 1,000 nM (1 μM) P1P treatment group. Maturation rate improvement by P1P supplementation was observed only in the presence of epidermal growth factor (EGF). Oocytes under the influence of P1P showed decreased intracellular reactive oxygen species levels but no significant differences in glutathione levels. In our molecular studies, P1P treatment upregulated gene expression involved in cumulus expansion (Has2 and EGF), antioxidant enzymes (SOD3 and Cat), and developmental competence (Oct4) while activating extracellular signal-regulated kinase1/2 and Akt signaling. P1P treatment also influenced oocyte survival by shifting the ratio of Bcl-2 to Bax while inactivating JNK signaling. We further demonstrated that oocytes matured with P1P displayed significantly higher developmental competence (cleavage and blastocyst [BL] formation rate) and greater BL quality (total cell number and the ratio of apoptotic cells) when activated via parthenogenetic activation (PA) and in vitro fertilization. Despite the low levels of endogenous P1P found in animals, exogenous P1P influenced animal reproduction, as shown by increased porcine oocyte maturation as well as preimplantation embryo development. This study and its findings are potentially relevant for both human and animal-assisted reproduction.

Tonic Calcium-Activated Chloride Current Sustained by ATP Release and Highly Desensitizing Human P2X1 Receptors

Extracellular adenosine triphosphate (ATP) participates in maintaining the vascular tone in the CNS, particularly in the retina, via the tonic activity of ligand gated activated P2X1 receptors. P2X1 receptors are characterized by their high affinity for ATP and their strong desensitization to concentrations of ATP that are 200-fold lower than their EC50. The mechanism behind P2X1 tonic activity remains unclear. In this study, we expressed human P2X1 (hP2X1) homomeric receptors in Xenopus oocytes to explore the relationship between ATP release from oocytes at rest, hP2X1, and Ca2+-activated Cl− channels. Our results indicate that Xenopus oocytes release ATP at rest via vesicular exocytosis, and this process is a constitutive phenomenon independent of extracellular Ca2+. Our results also indicate that hP2X1 receptors are able to sustain a tonic activity of Ca2+-activated Cl− channels. In the presence of extracellular Ca2+ the activity of hP2X1 receptors is greatly amplified by its coupling with Ca2+-activated Cl− channels. Future studies addressing the relationship between hP2X1 receptors and Ca2+-activated Cl− channels in vascular smooth muscle cells should provide information about additional mechanisms that regulate the vascular tone and their potential as pharmaceutical targets. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Role of Type 1 Inositol 1,4,5-triphosphate Receptors in Mammalian Oocytes.

The ability of oocytes to undergo normal fertilization and embryo development is acquired during oocyte maturation which is transition from the germinal vesicle stage (GV), germinal vesicle breakdown (GVBD) to metaphase of meiosis II (MII). Part of this process includes redistribution of inositol 1,4, 5-triphosphate receptor (IP3R), a predominant Ca2+ channel on the endoplasmic reticulum membrane. Type 1 IP3R (IP3R1) is expressed in mouse oocytes dominantly. At GV stage, IP3R1 are arranged as a network throughout the cytoplasm with minute accumulation around the nucleus. At MII stage, IP3R1 diffuses to the entire cytoplasm in a more reticular manner, and obvious clusters of IP3R1 are observed at the cortex of the egg. This structural reorganization provides acquisition of [Ca2+]i oscillatory activity during fertilization. In this review, general properties of IP3R1 in somatic cells and mammalian oocyte are introduced.