Receptors In Oocytes Research Articles

Temporal and Spatial Distribution of the Cannabinoid Receptors (CB1, CB2) and Fatty Acid Amide Hydroxylase in the Rat Ovary

Although the effects of Delta(9)-tetrahydrocannabinol (THC) on ovarian physiology have been known for many decades, its mechanism of action in the rat ovary remains poorly understood. The effects of THC and endocannabinoids on many cell types appear to be mediated through the G-protein-coupled CB(1) and CB(2) receptors. Evidence also suggests that the concentration of the endocannabinoid anandamide is regulated by cellular fatty acid amide hydrolase (FAAH). Therefore, we examined the rat ovary for the presence of CB(1) and CB(2) receptors and FAAH. The CB(1) receptor was present in the ovarian surface epithelium (OSE), the granulosa cells of antral follicles, and the luteal cells of functional corpus luteum (CL). The granulosa cells of small preantral follicles, however, did not express the CB(1) receptor. Western analysis also demonstrated the presence of a CB(1) receptor. In both preantral and antral follicles, the CB(2) receptor was detected only in the oocytes. In the functional CL, the CB(2) receptor was detected in the luteal cells. FAAH was codistributed with CB(2) receptor in both oocytes and luteal cells. FAAH was also present in the OSE, subepithelial cords of the tunica albuginea (TA) below the OSE, and in cells adjacent to developing preantral follicles. Western analysis also demonstrated the presence of FAAH in oocytes of both preantral and antral follicles. Our observations provide potential explanation for the effects of THC on steroidogenesis in the rat ovary observed by earlier investigators and a role for FAAH in the regulation of ovarian anandamide.

Parthenogenic Blastocysts Derived from Cumulus-Free In Vitro Matured Human Oocytes

BackgroundApproximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis.Methodology/Principal FindingImmature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1.Conclusions/SignificanceHuman cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer.

Multifunctional Merkel cells: Their roles in electromagnetic reception, finger-print formation, Reiki, epigenetic inheritance and hair form

Merkel cells are located in glabrous and hairy skin and in some mucosa. They are characterized by dense-core secretory granules and cytoskeletal filaments. They are attached to neighboring keratinocytes by desmosomes and contain melanosomes similar to keratinocytes. They are excitable cells in close contact with sensory nerve endings but their function is still unclear. In this review, following roles are attributed for the first time to the Merkel cells: (1) melanosomes in Merkel cells may be involved in mammalian magnetoreception. In this model melanosome as a biological magnetite is connected by cytoskeletal filaments to mechanically gated ion channels embedded in the Merkel cell membrane. The movement of melanosome with the changing electromagnetic field may open ion channels directly producing a receptor potential that can be transmitted to brain via sensory neurons. (2) Merkel cells may be involved in finger-print formation: Merkel cells in glabrous skin are located at the base of the epidermal ridges the type of which defines the finger-print pattern. Finger-print formation starts at the 10th week of pregnancy after the arrival of Merkel cells. Keratinocyte proliferation and the buckling process observed in the basal layer of epidermis resulting in the epidermal ridges may be controlled and formed by Merkel cells. (3) Brain-Merkel cell connection is bi-directional and Merkel cells not only absorb but also radiate the electromagnetic frequencies. Hence, efferent aspects of the palmar and plantar Merkel nerve endings may form the basis of the biofield modalities such as Reiki, therapeutic touch and telekinesis. (4) Adaptive geographic variations such as skin color, craniofacial morphology and hair form result from interactions between environmental factors and epigenetic inheritance system. While environmental factors produce modifications in the body, they simultaneously induce epigenetic modifications in the oocytes and in this way adaptive changes could be passed onto the next generations. Merkel cells are multisensorial cells that can receive almost all environmental stimuli including electromagnetic and ultraviolet radiations, temperature, humidity and food type and they seem to transfer the environmental information to oocytes by affecting nuclear receptors in oocytes. (5) Hair form is categorized as straight, wavy and spiral. Merkel cells found at the bulge region of hair follicles may determine the hair form with their different paracrine secretions related to hair cycle producing variations between populations. In conclusion, Merkel cells are multifunctional cells which may close the gap between orthodox medicine and complementary medicine such as acupuncture and Reiki.

Influence of leptin on in vitro maturation and steroidogenic secretion of cumulus–oocyte complexes through JAK2/STAT3 and MEK 1/2 pathways in the rabbit model

Extreme body mass indexes may impair reproductive outcome in assisted reproductive technologies. Leptin reflects the amount of body fat and could act as a modulator of oocyte quality through activation of specific transcription factors. The aim of this work was to establish whether: 1) leptin influences meiotic and cytoplasmic oocyte maturation; 2) STAT3 and MAPK mediate the effects of leptin and 3) leptin modulates steroid secretion by cumulus-oocyte complexes (COC) during in vitro maturation (IVM). We confirmed immunolocalisation of leptin receptor in oocytes, cumulus/granulosa cells during the peri-ovulatory period. The confocal study showed that COC supplemented with 1, 10 and 100 ng/ml leptin had a significantly higher metaphase II (MII) percentage than those IVM without leptin (P<0.05) and a similar MII index compared to the group supplemented with 10% FCS. Leptin did not increase the percentage of cytoplasmically matured oocytes in terms of cortical granule migration rate, whereas a significantly higher index was found in the FCS group (P<0.001). Oestradiol concentrations in spent media were higher in the FCS group compared to other treatments (P<0.001). Leptin-stimulated nuclear oocyte maturation was significantly impaired when leptin-induced JAK2/STAT3 and MEK 1/2 activation was suppressed by the inhibitors (P<0.001). Steroid secretion of COC was not affected by leptin activation of JAK2/STAT3 or MEK 1/2 pathways. In conclusion, JAK2/STAT3 and MEK 1/2 pathways mediate the enhancement of nuclear oocyte maturation by leptin; however, neither cytoplasmic oocyte maturation nor steroidogenic response of COC were improved in the present rabbit model.

An Acetylcholine Receptor Keeps Muscles in Balance

Muscle contraction is controlled by receptors in the muscle cell membranes that respond to the neurotransmitter acetylcholine when it is released from motor neurons. Acetylcholine receptors are also found on neurons, where they perform a variety of important functions, including modulating cognition and addiction. In a new study in PLoS Biology, Yishi Jin and colleagues have identified and characterized a neuronal acetylcholine receptor in the Caenorhabditis elegans that allows the tiny worm to wriggle about. The receptor regulates the balance between excitation and inhibition in muscles, and thus contributes to the coordinated contraction and relaxation of muscles on opposite sides of the body that results in locomotion. An acetylcholine receptor consists of five subunits, and there are many subunit types (29 in C. elegans) from which a receptor can be assembled. The subunit composition of a receptor, and, in particular, of its transmembrane pore, which is lined with one transmembrane domain from each subunit, determines how it responds to acetylcholine and what effects this response has on the cell. Because of the number of possible subunit combinations, it is very difficult to identify the cell-specific composition of an acetylcholine receptor. In this study, the authors began by identifying a mutant strain of C. elegans in which the muscles were overstimulated, causing the worms to ‘‘shrink’’, as all their muscles contracted when they were touched. Molecular characterization of the mutation revealed that it consisted of an activating mutation in an acetylcholine receptor subunit called ACR-2. Specifically, the mutation is in the pore-forming transmembrane domain, in a position that is thought to influence the ion selectivity of the channel. When the authors used reporter genes in which fluorescent proteins were controlled by the acr-2 promoter, they discovered that the ACR-2 subunit is expressed in cholinergic motor neurons in the worm’s ventral cord. Expression of wildtype acr-2 or a ‘‘mini-gene’’ containing part of the gene in the mutant worms reversed the ‘‘shrinking’’ defect, confirming that the defect resulted from the acr-2 mutation. Worms in which acr-2 contained a loss-of-function or null mutation do not show hypercontraction of muscles, but rather moved slowly, and electrophysiological analysis showed that the release of acetylcholine from the motor neurons was reduced in these animals. In worms with the activating mutation of acr-2, the release of acetylcholine from motor neurons was increased. In addition, neurotransmission from inhibitory GABAreleasing motor neurons was reduced. However, ACR-2 is not found in GABAergic neurons, so this reduction in GABA neurotransmission is likely to be an indirect result of the mutation’s effects on cholinergic neurons. To find out which other subunits combine with ACR-2 to make a functional receptor on cholinergic motor neurons, the authors looked for mutations in other genes that suppressed the effects of the activating acr-2 mutation. Several such mutations were found, and most of these mutations mapped to three other acetylcholine receptor subunit genes—acr-12, unc-38, and unc-63. Other suppressor mutations mapped to genes that are required for transport of the acetylcholine receptor to the cell surface. To confirm the subunit composition of the acetylcholine receptor, the authors reconstituted the receptor in Xenopus oocytes and discovered that in addition to the ACR-2, ACR-12, UNC-38, and UNC-63 subunits, the functional receptor also required ACR-3. The acr-3 gene is very close to acr-2, so that the two subunits are likely to be coexpressed. Together, these results show that the ACR-2 neuronal acetylcholine receptor manages the interplay between excitation and inhibition in the muscles in C. elegans. They also demonstrate that a gain-offunction mutation in the pore-forming domain of a receptor subunit can influence the pharmacological function of the receptor channel so that transmitter release from the receptor-bearing neuron is increased. Finally, the authors show how the analysis of suppressor mutations can be used to address the challenge of defining the subunit composition of a heteromeric receptor. Further study of how ACR-2 modulates the excitation and inhibition of muscles might give insight into how this balance is maintained in other neuronal contexts,

332 GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) ENHANCES CUMULUS CELLS EXPANSION OF IN VITRO-MATURED BOVINE CUMULUS - OOCYTE COMPLEXES IN A CHEMICALLY DEFINED MEDIUM

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine that stimulates proliferation, differentiation and function in different cells types. We have previously demonstrated (Bücher DD et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 146 abst.) that both subunits of GM-CSF receptor are expressed in granulosa cells from antral follicles in bovine ovaries. Also, we determined that the cytokine enhances glucose uptake through facilitative hexose transporters in granulosa cells in primary culture. The goals of the present study were to characterize the expression of GM-CSF receptor in cumulus cells and oocytes from bovine antral follicles and to determine its effects on in vitro-matured bovine COCs in a chemically defined medium. To determine the presence of a and |5 subunits of GM-CSF receptor, COCs were aspirated from follicles &lt;8 mm in diameter, fixed, and submitted to immunocytochemistry. To study the effect of GM-CSF on in vitro maturation of oocytes, COCs (n =481) were cultured using serum-free medium (SOF) containing 0, 1, 10, and 100 ng mL-1 of human recombinant GM-CSF (R&amp;D Systems, Inc., Minneapolis, MN, USA) for 22 h at 39°C, 5% CO2 in humidified air. Nuclear stage, cumulus expansion, cumulus cell number, and viability were analyzed after in vitro maturation. Cumulus expansion was assessed using the cumulus expansion index (CEI) (Fagbohun C and Down S 1990 Biol. Reprod. 42, 413-423). Nuclear stage was evaluated using aceto-orcein stain. To determine cumulus cell viability and number, COCs (n = 10-12 per group) were transferred into an Eppendorf tube and cumulus cells were removed by vortexing for 3 min, stained with trypan blue and counted with a hemocytometer. The study was conducted in 6 replicates. Data from cumulus expansion and cell number were analyzed by Kruskal-Wallis analysis. Data for nuclear stage and cell viability were analyzed by chi-square analysis and one way ANOVA, respectively. Both receptor subunits were present in cumulus cells and oocytes from COCs. COCs cultured in 10 and 100 ng mL-1 GM-CSF had CEI scores (0.8 and 1.22, respectively) greater (P &lt; 0.01) than controls (0.2), but the proportion of COCs displaying second metaphase did not differ (P = 0.5) among treatment groups. GM-CSF at a concentration of 100 ng mL-1 increased (P &lt; 0.01) cumulus cell viability by more than 20% compared to the control group. Similarly, GM-CSF at concentrations of 10 and 100 ng mL-1 increased (P &lt; 0.05) cumulus cell number by more than 20% and 45%, respectively, from the control group. The use of a specific inhibitor of PI3 kinase (Ly294002; 10 and 100 μM) blocked the stimulatory effect of GM-CSF on cumulus expansion, cell viability, and cell number. In conclusion, the results of the study suggest a plausible modulator role of GM-CSF in the metabolism and function of cumulus cells and oocytes during in vitro maturation. Funding from Faculty of Veterinary Sciences, Universidad Austral de Chile, MECESUP AUS-0005, AUS-0601, and DID D-2006-24 and from Universidad Católica de Temuco, research grant 2007 DGI-CDA-04.

Neurotrophin NT3 promotes ovarian primordial to primary follicle transition

Neurotrophins are growth factors that are known to have a role in promoting cell survival and differentiation. The focus of the current study is to examine the role of neurotrophins in regulating ovarian primordial follicle development. Ovaries from 4-day old rats were placed into organ culture and cultured for 10 days in the absence or presence of neurotrophin-3 (NT3), brain-derived neurotrophic factor (BDNF), or nerve growth factor (NGF). Treatment of ovaries with NT3 resulted in a significant (P<0.01) increase in primordial follicle development (i.e. primordial to primary follicle transition). Treatment with BDNF at high doses of 100-250 ng/ml also significantly (P<0.01) increased primordial follicle development, but NGF had no effect. Immunohistochemical studies determined that NT3 was present in granulosa cells, interstitial tissue, and in the oocytes of primordial and primary follicles. The NT3 receptor NTRK3 was present in oocytes at all stages of development. Analysis of ovaries that contain predominantly primordial follicles demonstrated the transcripts for NT3, NTRK3, NGF, and the BDNF/neurotrophin-4 (NT4) receptor NTRK2 are expressed, while BDNF, NT4, and the NGF receptor NTRK1 are not detectable. Inhibition of the NTRK3 receptor with the tyrphostin AG 879 resulted in oocyte death and a significant (P<0.01) reduction in follicle pool size. Inhibition of the NTRK receptors with K252a slowed primordial to primary follicle transition. A microarray analysis demonstrated that a small number of genes were differentially expressed after NT3 treatment. Observations indicate that the neurotrophin NT3, acting through the NTRK3 receptor in oocytes, promotes the primordial to primary follicle transition.

Neurotrophin Nt3 Promotes Ovarian Primordial to Primary Follicle Transition.

Neurotrophins are growth factors that are known to have a role in promoting cell survival and differentiation. The focus of the current study is to examine the role of neurotrophins in regulating ovarian primordial follicle development. Ovaries from 4-day old rats were placed into organ culture and cultured for 10 days in the absence or presence of neurotrophin-3 (NT3), brain-derived neurotrophic factor (BDNF), or nerve growth factor (NGF). Treatment of ovaries with NT3 resulted in a significant (P<0.01) increase in primordial follicle development (i.e. primordial to primary follicle transition). Treatment with BDNF at high doses of 100-250 ng/ml also significantly (P<0.01) increased primordial follicle development, but NGF had no effect. Immunohistochemical studies determined that NT3 was present in granulosa cells, interstitial tissue, and in the oocytes of primordial and primary follicles. The NT3 receptor NTRK3 was present in oocytes at all stages of development. Analysis of ovaries that contain predominantly primordial follicles demonstrated the transcripts for NT3, NTRK3, NGF, and the BDNF/neurotrophin-4 (NT4) receptor NTRK2 are expressed, while BDNF, NT4, and the NGF receptor NTRK1 are not detectable. Inhibition of the NTRK3 receptor with the tyrphostin AG 879 resulted in oocyte death and a significant (P<0.01) reduction in follicle pool size. Inhibition of the NTRK receptors with K252a slowed primordial to primary follicle transition. A microarray analysis demonstrated that a small number of genes were differentially expressed after NT3 treatment. Observations indicate that the neurotrophin NT3, acting through the NTRK3 receptor in oocytes, promotes the primordial to primary follicle transition.

Effects of triterpenoids from Poria cocos Wolf on the serotonin type 3A receptor-mediated ion current in Xenopus oocytes

Poria cocos Wolf ( P. cocos Wolf) is used to treat chronic gastritis, edema, nephrosis, gastric atony, acute gastroenteric catarrh, dizziness, emesis and vomiting. Triterpenoids are a class of natural compounds produced by P. cocos Wolf that contain acyclic 30-carbon precursors. In this study, we investigated the effect of triterpenoids (PA, Pachymic acid; DA, dehydroeburicoic acid; HA, 3β-hydroxylanosta-7,9(11),24-trien-21-oic acid) on human 5-hydroxytryptamine 3A (5-HT 3A) receptor channel activity, which is one of the ligand-gated ion channel families. The two-electrode voltage-clamp technique was used to examine the 5-HT3A mediated current. The inhibitory effect of triterpenoids on 5HT-induced inward current ( I 5-HT) occurred in a concentration dependent and reversible manner. Furthermore, the half-inhibitory concentrations (IC 50) of PA, DA and HA were 3.2 ± 0.2, 5.5 ± 0.6 and 1.4 ± 0.2 µM, respectively. This corresponded to an order of potency for the inhibition of I 5-HT in oocytes expressing human 5-HT 3A receptor of HA > PA > DA. Finally, inhibition of I 5HT by triterpenoids occurred in a non-competitive manner, while inhibition by HA and PA showed more voltage-dependency. Taken together, these results indicate that triterpenoids may regulate the expressed 5-HT 3A receptors in Xenopus oocytes. Furthermore, this regulation of the ligand-gated ion channel activity by triterpenoids may be one of the pharmacological actions of P. cocos Wolf.

Xenopus laevis Oocytes Endogenously Express All Subunits of the Ionotropic Glutamate Receptor Family

Xenopus laevis oocytes are commonly used as a heterologous expression system for the electrophysiological characterization of ionotropic glutamate receptors (iGluRs). Recently, however, several glutamate receptor subunits of the N-methyl-d-aspartate receptor subfamily have been found to be expressed endogenously in Xenopus oocytes, thus limiting the use of this expression system for such receptors. We therefore screened oocytes for the Xenopus homologs of all iGluR subunits known to be expressed in mammals to investigate which additional subunits may be present endogenously in oocytes and what, if any, influence such proteins might have on the functional analysis of heterologously expressed receptors. We found Xenopus homologs of every mammalian iGluR subunit to be expressed at the mRNA level. We then cloned, from oocytes, full-length copies of the four Xenopus alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunits XenGluR1 through XenGluR4 and, additionally, XenGluR6 as a representative subunit for the kainate receptor subfamily and electrophysiologically characterized them. Upon analysis, we found only minor functional differences between homologous subunits from Xenopus and rat. Next, we investigated whether endogenous iGluR subunits can be detected electrophysiologically in oocytes. We found no indication for any functional glutamate receptors in native oocytes; however, after heterologous expression of the auxiliary subunit stargazin, endogenous alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors were detected. These data demonstrate that Xenopus oocytes express glutamate receptor subunits endogenously, albeit at very low levels. Such endogenous receptor proteins can, under certain circumstances, become electrophysiologically detectable and then might influence electrophysiological recordings performed on recombinant receptors in oocytes.

Triton x-100 inhibits agonist -induced currents and suppresses benzodiazepine modulation of GABA A receptors in Xenopus oocytes

Changes in lipid bilayer elastic properties have been proposed to underlie the modulation of voltage-gated Na + and L-type Ca 2+ channels and GABA A receptors by amphiphiles. The amphiphile Triton X-100 increases the elasticity of lipid bilayers at micromolar concentrations, assessed from its effects on gramicidin channel A appearance rate and lifetime in artificial lipid bilayers. In the present study, the pharmacological action of Triton-X 100 on GABA A receptors expressed in Xenopus laevis oocytes was examined. Triton-X 100 inhibited GABA A α 1β 3γ 2S receptor currents in a noncompetitive, time- and voltage-dependent manner and increased the apparent rate and extent of desensitization at 10 μM, which is 30 fold below the critical micelle concentration. In addition, Triton X-100 induced picrotoxin-sensitive GABA A receptor currents and suppressed allosteric modulation by flunitrazepam at α 1β 3γ 2S receptors. All effects were independent of the presence of a γ 2S subunit in the GABA A receptor complex. The present study suggests that Triton X-100 may stabilize open and desensitized states of the GABA A receptor through changes in lipid bilayer elasticity.

Molecular Weight and Volume at Position 46 in Transmembrane Domain 1 (TM1) are Important Determinants for Ethanol Sensitivity in P2X4 Receptors

Purinergic P2X receptors (P2XRs) are a family of fast acting, cation-permeable ligand-gated ion channels, which are gated by synaptically released adenosine 5′-triphosphate (ATP). Building evidence supports the notion that P2XRs play a role in mediating and/or modulating behavioral effects of ethanol. However, this work is in the early stages and the sites and mechanisms of ethanol action in P2XRs are poorly understood. Recently, we identified an amino acid located in TM1 that was conserved across P2X3 and P2X4Rs that appeared to represent an important site for the action of ethanol (i.e., Trp41 and Trp46, respectively). The current study tests the hypothesis that physical-chemical properties of the residue at position 46 play a significant role in determining ethanol sensitivity in P2X4Rs. We expressed wildtype and mutated receptors in Xenopus oocytes and determined changes in ethanol sensitivity (200mM) using an 8-channel two-electrode voltage clamp system (OpusXpress 6000; -70mV). Exchanging Trp46 residue with other aromatic residues did not significantly alter ethanol sensitivity whereas replacing Trp46 with aliphatic residues significantly reduced the action of ethanol. Correlation analysis determined that molecular weight and volume of the residue at position 46 were significantly correlated with ethanol sensitivity whereas polarity or hydrophobicity was not. The findings suggest that the size and aromaticity of the residue at position 46 in TM1 play an important role in determining ethanol sensitivity of P2X4Rs. Identifying physical-chemical properties of residues that are important for ethanol sensitivity will increase our knowledge regarding structural requirements that are necessary for ethanol to cause changes in modulation and/or transduction of agonist action in P2X4Rs. Support: NIAAA/NIH F31 AA017029, KO1 AA017243-01A1, AA013922, AA03972 and USC School of Pharmacy.

Developmental regulation of the expression of the transferrin receptor and Ki67 in oocytes of the baboon fetal ovary by estrogen

We previously showed that estrogen regulates baboon fetal ovarian follicle development and oocyte integrity. Because iron incorporated into cells by the transferrin receptor is essential for cell/nuclear function, we determined whether fetal oocyte expression of transferrin receptor and the nuclear protein Ki67 were developmentally regulated by estrogen and associated with DNA integrity/fragmentation. Transferrin-receptor expression was minimal at midgestation and abundant in late gestation and localized to the cytoplasm and surface of oocytes of primordial follicles. Expression of transferrin receptor, however, was negligible in oocytes in fetuses in which serum estradiol-17beta levels were suppressed (>95%) by daily maternal treatment between mid- and late gestation with the aromatase inhibitor letrozole and partially restored by treatment with letrozole and estradiol benzoate. Ki67 was localized to pregranulosa and germ cells at midgestation and throughout the oocyte nucleus in late gestation in estrogen-replete fetuses. In contrast, in estrogen-suppressed fetuses, Ki67 was localized to a limited number of foci around the oocyte nucleus. Apoptosis detected in pregranulosa and germ cells at midgestation was not observed in late gestation in estrogen-replete/-suppressed fetuses. We conclude that estrogen regulates fetal oocyte transferrin-receptor expression and that inhibition of receptor development is associated with alterations in Ki67 expression by the oocyte but not apoptosis. Collectively, these results and our previous studies further define the essential role of estrogen in regulating development of follicles comprised of healthy oocytes by the baboon fetal ovary.

Gene expression and protein distribution of leptin and its receptor in bovine oocytes and preattachment embryos produced in vitro

Many investigations point out the important role of leptin during the preimplantation development. Transcripts for the leptin gene (LEP) and its receptor (LEPR) have been identified in several tissues related to reproduction (e.g. ovaries, testis and oviduct) in both human and mouse. This work shows for the first time the expression and distribution patterns of LEP and LEPR in bovine oocytes and in vitro-produced embryos. Gene expression was analysed by reverse transcription PCR and real-time PCR, and the proteins were localised by immunostaining. This study included immature and mature oocytes, zygotes, two-, four-, eight- to 16-cell embryos, morulae and blastocysts and the LEP transcript was identified throughout all stages of bovine preimplantation development. However, mRNA for the LEPR gene was detected at all stages, excluding four-cell embryos. Expression of both LEP and LEPR genes was reduced at the eight- to 16-cell stage. This in addition to the absence of LEPR mRNA in four-blastomere embryos may suggest that maternally derived transcripts degenerate towards the eight- to 16-cell stage coinciding with embryonic genome activation at eight- to 16-cell stage and subsequent appearance of embryonic mRNA. Immunofluorescent staining demonstrated that LEP and LEPR proteins form a spherical rim beneath the oolemma. After maturation, however, the proteins became evenly distributed within the cytoplasm. In two- to eight-cell embryos, fluorescence was observed in the apical surface of the blastomeres, and from 10- to 16-cell stage in the apical region of outer blastomeres. This pattern persisted to the blastocyst stage, leading to LEP and LEPR distribution within trophoblast cells, but not in the inner cell mass. These results support previous findings on polar distribution of proteins within mammalian oocytes and embryos, as well as suggests leptin’s potential role during early mammalian development and implantation.

Structural insights into phenylethanolamines high-affinity binding site in NR2B from binding and molecular modeling studies

BackgroundPhenylethanolamines selectively bind to NR2B subunit-containing N-methyl-D-aspartate-subtype of ionotropic glutamate receptors and negatively modulate receptor activity. To investigate the structural and functional properties of the ifenprodil binding domain on the NR2B protein, we have purified a soluble recombinant rat NR2B protein fragment comprising the first ~400 amino acid amino-terminal domain (ATD2B) expressed in E. coli. Spectral measurements on refolded ATD2B protein demonstrated specific binding to ifenprodil. We have used site-directed mutagenesis, circular dichroism spectroscopy and molecular modeling to obtain structural information on the interactions between critical amino acid residues and ifenprodil of our soluble refolded ATD2B proteins. Ligand-induced changes in protein structure were inferred from changes in the circular dichroism spectrum, and the concentration dependence of these changes was used to determine binding constants for ifenprodil and its analogues.ResultsLigand binding of ifenprodil, RO25,6981 and haloperidol on soluble recombinant ATD2B determined from circular dichroism spectroscopy yielded low-to-high micromolar equilibrium constants which concurred with functional IC50 measurement determined in heterologously expressed NR1/NR2B receptors in Xenopus oocytes. Amino acid residue substitutions of Asp101, Ile150 and Phe176 with alanine residue within the ATD2B protein altered the recombinant protein dissociation constants for ifenprodil, mirroring the pattern of their functional phenotypes. Molecular modeling of ATD2B as a clam-shell-like structure places these critical residues near a putative ligand binding site.ConclusionWe report for the first time biochemical measurements show that the functional measurements actually reflect binding to the ATD of NR2B subunit. Insights gained from this study help advance the theory that ifenprodil is a ligand for the ATD of NR2B subunit.

CFTR is activated through stimulation of purinergic P2Y2 receptors

It has been reported that the cystic fibrosis transmembrane conductance regulator (CFTR) can be activated through cAMP- and protein kinase A-independent pathways involving GTP-binding proteins and an unknown kinase. In this study, we further examined how G protein-coupled pathways regulate CFTR. We demonstrate that stimulation of purinergic P2Y(2) receptors in CFTR-expressing oocytes and in airway epithelial cells activates CFTR Cl(-) currents. Activation of CFTR Cl(-) currents via P2Y(2) was inhibited by CFTR(inh)-172 and was independent of intracellular Ca(2+), protein kinase C, or calmodulin-dependent kinase (CAMK). However, activation of CFTR was suppressed by inhibition of phospholipase C and by the nonselective protein kinase inhibitor staurosporine. Activation of CFTR through P2Y(2) receptors was enhanced when G(i) proteins were inhibited by pertussis toxin. Inhibition of protein kinase A and of protein kinases downstream of P2Y(2) receptors such as mitogen-activated protein kinases, tyrosine kinase, or c-src kinase did not interfere with activation of CFTR. The present results demonstrate an antagonistic regulation of CFTR by P2Y(2) receptors: CFTR is inhibited by stimulation of G(i) proteins and is activated by stimulation of G(q/11)/PLC and an unknown downstream protein kinase.

Nicotine-induced Ca2+-myristoyl switch of neuronal Ca2+ sensor VILIP-1 in hippocampal neurons: a possible crosstalk mechanism for nicotinic receptors.

Visinin-like protein (VILIP-1) belongs to the neuronal Ca2+ sensor family of EF-hand Ca2+-binding proteins that regulate a variety of Ca2+-dependent signal transduction processes in neurons. It is an interaction partner of alpha4beta2 nicotinic acetylcholine receptor (nAChR) and increases surface expression level and agonist sensitivity of the receptor in oocytes. Nicotine stimulation of nicotinic receptors has been reported to lead to an increase in intracellular Ca2+ concentration by Ca2+-permeable nAChRs, which in turn might lead to activation of VILIP-1, by a mechanism described as the Ca2+-myristoyl switch. It has been postulated that this will lead to co-localization of the proteins at cell membranes, where VILIP-1 can influence functional activity of alpha4-containing nAChRs. In order to test this hypothesis we have investigated whether a nicotine-induced and reversible Ca2+-myristoyl switch of VILIP-1 exists in primary hippocampal neurons and whether pharmacological agents, such as antagonist specific for distinct nAChRs, can interfere with the Ca2+-dependent membrane localization of VILIP-1. Here we report, that only alpha7- but not alpha4-containing nAChRs are able to elicit a Ca2+-dependent and reversible membrane-translocation of VILIP-1 in interneurons as revealed by employing the specific receptor antagonists dihydro-beta-erythroidine and methylallylaconitine. The nAChRs are associated with processes of synaptic plasticity in hippocampal neurons and they have been implicated in the pathology of CNS disorders, including Alzheimer's disease and schizophrenia. VILIP-1 might provide a novel functional crosstalk between alpha4- and alpha7-containing nAChRs.

Multiple Modes for Conferring Surface Expression of Homomeric β1 GABAA Receptors

The gamma-aminobutyric acid type A (GABA(A)) receptor assembles from individual subunits to form ligand-gated ion channels. Human (h) beta3 subunits assemble to form homomeric surface receptors in somatic cells, but hbeta1 subunits do not. We have identified three distinct sets of amino acid residues in the N-terminal extracellular domain of the hbeta1 subunit, which when mutated to the homologous residue in hbeta3 allow expression as a functional homomeric receptor. The three sets likely result in three modes of assembly. Mode 1 expression results from a single amino acid change at residue hbeta1 Asp-37. Mode 2 expression results from mutations of residues between positions 44 and 73 together with residues between positions 169 and 173. Finally, mode 3 results from the mutations A45V and K196R. Examination of homology-based structural models indicates that many of the residues are unlikely to be involved in physical inter-subunit interactions, suggesting that a major alteration is stabilization of an assembly competent form of the subunit. These mutations do not, however, have a major effect on the surface expression of heteromeric receptors which include the alpha1 subunit.

RNA editing regulates insect gamma-aminobutyric acid receptor function and insecticide sensitivity

A-to-I pre-mRNA editing by adenosine deaminase enzymes has been reported to enhance protein diversity in the nervous system. In Drosophila, the resistance to dieldrin (RDL) gamma-aminobutyric acid (GABA) receptor subunit displays an editing site (R122) that is close to the putative GABA-binding site. We assessed the functional effects of editing at this site by expressing homomeric RDL receptors in Xenopus oocytes. After replacement of arginine 122 with a glycine, both agonist and fipronil potencies were shifted to the right in either fipronil-sensitive receptors or mutated resistant receptors (A301G/T350M). These data provide the first insight on the influence of RNA editing on GABA receptor function.

Meiotic Maturation: Receptor Trafficking Is the Key

Meiotic maturation and ovulation rates in Caenorhabditis elegans are regulated by a sperm-released gradient of major sperm protein (MSP). Recent work has provided insights into the modulation of the MSP signal by the trafficking of its receptor in oocytes.