Coordinate control of translation and localization of Vg1 mRNA in Xenopus oocytes

Vg1, a member of the transforming growth factor-β family involved in mesoderm induction, is translated subsequent to the localization of its mRNA to the vegetal pole of Xenopus oocytes. Whereas the localization of Vg1 mRNA is known to be directed by the 3′ untranslated region (UTR), the basis of its translational regulation is unknown. We show here that the 3′ UTR of Vg1 causes translational repression of two different reporter mRNAs in Xenopus oocytes. A 350-nucleotide region of the 3′ UTR, which is distinct from the localization element, is necessary and sufficient for mediating translational repression and specifically binds to a 38-kDa polypeptide. The translational repression activity is found throughout the oocyte and at all stages of oogenesis. These results suggest that factors colocalized with Vg1 mRNA at the vegetal pole relieve translational repression to allow expression of Vg1 protein.

Oocytes are a source of catecholamines in the primate ovary: Evidence for a cell–cell regulatory loop

Catecholamines, thought to derive from the extrinsic innervation of the ovary, participate in the regulation of ovarian development and mature gonadal function. Recently, intraovarian neurons containing tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, were described in the ovary of nonhuman primates. We now show that the primate ovary expresses both the genes encoding TH and dopamine β-hydroxylase (DBH), the key enzymes in norepinephrine (NE) biosynthesis. Ovarian neurons were identified as a site of TH and DBH gene expression, and surprisingly, oocytes were identified as an exclusive site of DBH synthesis. Oocytes contain neither TH mRNA nor protein, indicating that they are unable to synthesize dopamine (DA). They did, however, express a DA transporter gene identical to that found in human brain. The physiological relevance of this transporter system and DBH in oocytes was indicated by the ability of isolated oocytes to metabolize exogenous DA into NE. Isolated follicles containing oocytes—but not those from which the oocytes had been removed—responded to DA with an elevation in cAMP levels; this elevation was prevented by propranolol, a β-adrenoreceptor antagonist. The results suggest that oocytes and somatic cells are linked by a neuroendocrine loop consisting of NE synthesized in oocytes from actively transported DA and cAMP produced by somatic follicular cells in response to NE-induced β-adrenoreceptor activation.

Transport of uncharged organic solutes in Xenopus oocytes expressing red cell anion exchangers (AE1s)

When expressed in Xenopus oocytes, the trout red cell anion exchanger tAE1, but not the mouse exchanger mAE1, elicited a transport of electroneutral solutes (sorbitol, urea) in addition to the expected anion exchange activity. Chimeras constructed from mAE1 and tAE1 allowed us to identify the tAE1 domains involved in the induction of these transports. Expression of tAE1 (but not mAE1) is known to generate an anion conductance associated with a taurine transport. The present data provide evidence that (i) the capacity of tAE1 and tAE1 chimeras to generate urea and sorbitol permeability also was associated with an anion conductance; (ii) the same inhibitors affected both the permeability of solutes and anion conductance; and (iii) no measurable water transport was associated with the tAE1-dependent conductance. These results support the view that fish red blood cells, to achieve cell volume regulation in response to hypotonic swelling, activate a tAE1-associated anion channel that can mediate the passive transport of taurine and electroneutral solutes.

Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes

Na+/Cl−-dependent neurotransmitter transporters form a superfamily of transmembrane proteins that share 12 membrane-spanning regions. To gain information about the quaternary structure of these transporter proteins, we heterologously expressed the glial glycine transporter GlyT1 and its neuronal homolog GlyT2 in Xenopus oocytes. By using metabolic labeling with [35S]methionine or surface labeling with a plasma membrane impermeable reagent followed by affinity purification, we separately analyzed the total cellular pools of newly synthesized GlyTs and its functional plasma membrane-bound fractions. Upon blue native gel electrophoresis, the surface-localized transporter proteins were found to exist exclusively in complex-glycosylated monomeric form, whereas a significant fraction of the intracellular GlyT1 and GlyT2 was core-glycosylated and oligomeric. In contrast, even after treatment with the crosslinker glutaraldehyde, surface GlyTs failed to migrate as oligomeric proteins. These results indicate that plasma membrane-bound GlyT1 and GlyT2 are monomeric proteins. Thus, Na+/Cl−-dependent neurotransmitter transporters do not require oligomerization for substrate translocation.

The Polo-like Kinase Plx1 Is Required for Activation of the Phosphatase Cdc25C and Cyclin B-Cdc2 in Xenopus Oocytes

In the Xenopus oocyte system mitogen treatment triggers the G2/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, we have utilized a cell-free oocyte extract that carries out the signaling events of oocyte maturation after addition of the heat-stable inhibitor of PKA, PKI. PKI stimulated the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos did not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delayed, but did not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide had a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibited activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21Cip1 had no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity.

The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway.

Expression of a renal type I sodium/phosphate transporter (NaPi-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions.

Two distinct molecular types (I and II) of renal proximal tubular brush border Na+/Pi cotransporters have been identified by expression cloning on the basis of their capacity to induce Na+-dependent Pi influx in tracer experiments. Whereas the type II transporters (e.g., NaPi-2 and NaPi-3) resemble well known characteristics of brush border Na+/Pi cotransport, little is known about the properties of the type I transporter (NaPi-1). In contrast to type II, type I transporters produced electrogenic transport only at high extracellular Pi concentrations (> or =3 mM). On the other hand, expression of NaPi-1 induced a Cl- conductance in Xenopus laevis oocytes, which was inhibited by Cl- channel blockers [5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) > niflumic acid >> 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid]. Further, the Cl- conductance was inhibited by the organic anions phenol red, benzylpenicillin (penicillin G), and probenecid. These organic anions induced outwardly directed currents in the absence of Cl-. In tracer studies, we observed uptake of benzylpenicillin with a Km of 0.22 mM; benzylpenicillin uptake was inhibited by NPPB and niflumic acid. These findings suggest that the type I Na+/Pi cotransporter functions also as a novel type of anion channel permeable not only for Cl- but also for organic anions. Such an apical anion channel could serve an important role in the transport of Cl- and the excretion of anionic xenobiotics.

Identification of a protein that confers calcitonin gene-related peptide responsiveness to oocytes by using a cystic fibrosis transmembrane conductance regulator assay.

An expression-cloning strategy was used to isolate a cDNA that encodes a protein that confers calcitonin gene-related peptide (CGRP) responsiveness to Xenopus laevis oocytes. A guinea pig organ of Corti (the mammalian hearing organ) cDNA library was screened by using an assay based on the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR is a chloride channel that is activated upon phosphorylation; this channel activity was used as a sensor for CGRP-induced activation of intracellular kinases. A cDNA library from guinea pig organ of Corti was screened by using this oocyte-CFTR assay. A cDNA was identified that contained an open reading frame coding for a small hydrophilic protein that is presumed to be either a CGRP receptor or a component of a CGRP receptor complex. This CGRP receptor component protein confers CGRP-specific activation to the CFTR assay, as no activation was detected upon application of calcitonin, amylin, neuropeptide Y, vasoactive intestinal peptide, or beta-endorphin. In situ hybridization demonstrated that the CGRP receptor component protein is expressed in outer hair cells of the organ of Corti and is colocalized with CGRP-containing efferent nerve terminals.

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

A corticosteroid-induced gene expressing an "IsK-like" K+ channel activity in Xenopus oocytes.

Screening a rat colon cDNA library for aldosterone-induced genes resulted in the molecular cloning of a cDNA whose corresponding mRNA is strongly induced in the colon by dexamethasone, aldosterone, and a low NaCl diet. A similar mRNA was detected in kidney papilla but not in brain, heart, or skeletal muscle. Xenopus laevis oocytes injected with cRNA synthesized from this clone, designated CHIF (channel-inducing factor), express a K(+)-specific channel activity. The biophysical, pharmacological, and regulatory characteristics of this channel are very similar to those reported before for IsK (minK). These include: slow (tau > 20 s) activation by membrane depolarization with a threshold potential above -50 mV, blockade by clofilium, inhibition by phorbol ester, and activation by 8-bromoadenosine 3',5'-cyclic monophosphate and high cytoplasmic Ca2+. The primary structure of this clone, however, shows no homology to IsK. Instead, CHIF exhibits > 50% similarity to two other short bitopic membrane proteins, phospholemman and the gamma subunit of Na+K(+)-ATPase. The data are consistent with the possibility that CHIF is a member of a family of transmembrane regulators capable of activating endogenous oocyte transport proteins.

Incorporation of acetylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes.

A method was developed to transplant assembled nicotinic acetylcholine receptors (AcChoRs) and Cl- channels from the electric organ of Torpedo to the membrane of Xenopus oocytes. Membrane vesicles from Torpedo electroplaques were injected into the oocytes and, within a few hours, the oocyte membrane acquired AcChoRs and Cl- channels. The mechanism of expression of these receptors and channels is very different from that which follows the injection of mRNA, since the appearance of receptors after membrane injection does not require de novo protein synthesis or N-glycosylation. This, and other controls, indicate that the foreign receptor-bearing membranes fuse with the oocyte membrane and cause the appearance of functional receptors and channels. All this makes the Xenopus oocyte an even more powerful tool for studies of the structure and function of membrane proteins.

Modulation of nicotinic acetylcholine receptors by strychnine

Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (α1β1γδ, α1β1γ, and α1β1δ) and neuronal (α2β2 and α2β4) nicotinic acetylcholine receptors (AcChoRs) expressed in Xenopus oocytes. Strychnine alone (up to 500 μM) did not elicit membrane currents in oocytes expressing AcChoRs, but, when applied before, concomitantly, or during superfusion of acetylcholine (AcCho), it rapidly and reversibly inhibited the current elicited by AcCho (AcCho-current). Although in the three cases the AcCho-current was reduced to the same level, its recovery was slower when the oocytes were preincubated with strychnine. The amount of AcCho-current inhibition depended on the receptor subtype, and the order of blocking potency by strychnine was α1β1γδ > α2β4 > α2β2. With the three forms of drug application, the Hill coefficient was close to one, suggesting a single site for the receptor interaction with strychnine, and this interaction appears to be noncompetitive. The inhibitory effects on muscle AcChoRs were voltage-independent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltage-dependent, with an electrical distance of ≈0.35. We conclude that strychnine regulates reversibly and noncompetitively the embryonic type of muscle AcChoR and some forms of neuronal AcChoRs. In the former case, strychnine presumably inhibits allosterically the receptor by binding at an external domain whereas, in the latter case, it blocks the open receptor-channel complex.

The β subunit of CKII negatively regulates Xenopus oocyte maturation

CKII (formerly known as casein kinase II) is a ubiquitously expressed enzyme that plays an important role in regulating cell growth and differentiation. The β subunit of CKII (CKIIβ) is not catalytic but forms heterotetramers with the catalytic subunit α to generate an α2β2 holoenzyme. In Xenopus oocytes, CKIIβ also associates with another serine/threonine kinase, Mos. As a key regulator of meiosis, Mos is necessary and sufficient to initiate oocyte maturation. We have previously shown that the binding of CKIIβ to Mos represses Mos-mediated mitogen-activated protein kinase (MAPK) activation and that the ectopic expression of CKIIβ inhibits progesterone-induced Xenopus oocyte maturation. We have now used an antisense oligonucleotide technique to reduce the endogenous CKIIβ protein level in Xenopus oocytes, and we find that oocytes with a reduced content of CKIIβ are more sensitive to low doses of progesterone and show accelerated MAPK activation and germinal vesicle breakdown. Furthermore, ectopic expression of a Mos-binding fragment of CKIIβ suppressed the effect of antisense oligonucleotide. These results suggest that the endogenous CKIIβ normally sets a threshold level for Mos protein, which must be exceeded for Mos to activate the MAPK signaling pathway and induce oocyte maturation.