Xenopus oocytes can synthesise but do not secrete the Z variant of human alpha 1-antitrypsin

Human liver mRNA was prepared from a patient homozygous for alpha 1-antitrypsin deficiency (PiZZ) and from a normal subject (PiMM). Both liver RNAs were microinjected into Xenopus oocytes and alpha 1-antitrypsin identified by immunoprecipitation. The normal M variant of alpha 1-antitrypsin is synthesised and secreted by Xenopus oocytes, the abnormal Z protein is not secreted and an intracellular form accumulates in the oocytes. In the presence of tunicamycin an unglycosylated form of M alpha 1-antitrypsin appears in the incubation medium but no corresponding unglycosylated version of the Z protein is secreted.     

Translation of human interleukin 2 mRNA in Xenopus laevis oocytes

Poly(A)-positive mRNA extracted from tonsillar mononuclear cells stimulated with phytohemagglutinin-M and 12-o-tetradecanoyl phorbol 13-acetate was successfully translated into biologically active interleukin 2 (IL-2) in Xenopus laevis oocytes, and secreted into the incubation medium. In control experiments, the extract of oocytes injected with either poly(A)-negative RNA or buffer did not show any IL-2 activity. By sucrose density gradient centrifugation analysis, IL-2 mRNA was found as a single peak corresponding to a sedimentation coefficient of 10-11S.     

The production of active human thyroid-stimulating hormone from alpha and beta mRNAs in Xenopus laevis oocytes

Active human thyroid-stimulating hormone (hTSH) was produced by Xenopus laevis oocytes following injection of an mRNA mixture of hTSH beta and alpha subunits synthesized by T3 RNA polymerase. Some of the hTSH molecules were secreted into the medium, while others remained in the cells. The active molecules consisted of alpha and beta subunits and were in highly glycosylated form. The Xenopus laevis oocyte-produced hTSH stimulated the rat thyroid cell line FRTL-5 to produce and secrete the cyclic AMP as does authentic hTSH.     

Secreted alkaline phosphatase: an internal standard for expression of injected mRNAs in the Xenopus oocyte

The Xenopus oocyte is widely used to study the various aspects of eukaryotic cell structure and function. It is also being used increasingly in expression cloning of cDNAs encoding proteins for which there are no structural data. One of the drawbacks of the Xenopus oocyte system is that individual oocytes taken at the same time from the same frog vary considerably in the amount of protein synthesized from the same amount of injected mRNA. In this report we describe the preparation and use of the mRNA for a secreted mutant form of human placental alkaline phosphatase as an internal, coinjected standard to monitor translation in oocytes. Secreted alkaline phosphatase can be readily determined in the medium of cultured oocytes by using a standard colorimetric assay. The amounts of alkaline phosphatase secreted into the medium were shown to parallel the level of expression of two membrane proteins. This permits rapid identification and selection of those oocytes that efficiently express injected mRNAs. The procedure yields more precise data and results in an enormous saving of time and expense, especially in investigations that involve complex measurements on individual oocytes.     

Expression of rat renal sodium/phosphate cotransporter in Xenopus laevis oocytes.

In an attempt to identify the renal Na+/Pi cotransporter, Xenopus laevis oocytes were used to express mRNA isolated from the renal cortex of rat kidney. Na(+)-dependent uptake of Pi in oocytes, injected with mRNA, resulted in an increase of 2-4-fold as compared to oocytes injected with water. Both the new expressed and endogenous Na(+)-dependent Pi uptake activity were inhibited with 2 mM phosphonoformic acid (PFA). Expression of Pi uptake into oocytes was dose-dependent with the amount of mRNA injected. When mRNA was fractionated on a sucrose gradient, a mRNA fraction of 2.5 kilobases expressed the Na+/Pi cotransport activity in oocytes. This fraction resulted in a 6-fold stimulation of Na(+)-dependent Pi transport when compared to oocytes injected with water. The Km and Vmax for Na(+)-dependent Pi uptake were 0.18 mM and 118 pmol/oocyte per 30 min, respectively.     

Secretion of zona pellucida glycoprotein mZP2 by growing oocytes from mZP3+/+ and mZP3−/− mice

The mouse egg extracellular coat, or zona pellucida (ZP), is composed of three glycoproteins, called mZP1–3, which are synthesized and secreted concomitantly by growing oocytes. Disruption of the mZP3 gene by targeted mutagenesis yields mice that are homozygous nulls (mZP3^−/−). Growing oocytes from mZP3^−/− mice do not synthesize mZP3 mRNA or protein and, as a result, do not assemble a ZP. Here, we examined secretion of mZP2 by growing oocytes and eggs from mZP3^−/− mice, as well as incorporation of mZP2 into the ZP of oocytes from mZP3^+/+ mice. Laser scanning confocal microscopy (LSCM) of antibody‐labeled samples showed that, indeed, mZP2 was synthesized and secreted by oocytes isolated from mZP3^−/− mice and cultured in vitro. Nascent mZP2 was found in the culture medium, associated with the surface of the plasma membrane of growing oocytes, and in the oocyte cytoplasm. By contrast, mZP2 was barely detectable at any of these sites when ovulated eggs from mZP3^−/− mice were examined. Examination of oocytes from wild‐type (mZP3^+/+) mice showed that, while a portion of nascent mZP2 was assembled into the ZP (approximately 40%), here too a significant fraction was secreted into the culture medium (approximately 60%). Similar results also were obtained when intact pre‐antral follicles were isolated from mZP3^+/+ mice and cultured in vitro. Several of these observations are consistent with previous results obtained with oocytes from heterozygous null mice (mZP3^+/−). Furthermore, the results suggest that ZP assembly from nascent glycoproteins may be a stochastic process that requires the presence of both mZP2 and mZP3 and occurs completely outside the growing oocyte. Dev. Genet. 25:95–102, 1999. © 1999 Wiley‐Liss, Inc.     

Molecular cloning and partial characterization of medaka fish stromelysin-3 and its restricted expression in the oocytes of small growing follicles of the ovary.

A cDNA clone (2755-bp) for stromelysin-3 was isolated by screening the cDNA library and by 3'- and 5'-rapid amplification of cDNA ends using ovary RNA of the medaka fish Oryzias latipes. The clone encodes a protein of 492 amino acids. Stromelysin-3 mRNA was detected only in the ovary. In situ hybridization analysis revealed that stromelysin-3 mRNA was localized in the oocyte cytoplasm of small growing follicles. RT-PCR analysis of total RNAs isolated from various-sized follicles and ovulated oocytes was conducted in order to determine the mRNA levels during oocyte growth. The stromelysin-3 mRNA level was the highest in the small follicles, and the mRNA levels decreased as the follicles grew. No significant stromelysin-3 mRNA was detected in the ovulated oocytes or immature ovaries. The fish stromelysin-3 cDNA was expressed in COS-1 cells in order to characterize the intracellular localization of the protein. A 56 kDa protein was synthesized and secreted into the culture medium. The secreted stromelysin-3 exhibited gelatin-degrading activity.     

Primary muscle cells cultivated in medium conditioned by spinal cord cells show changes in messenger RNA as detected by translation in ovo accompanied by synthesis of extracellular matrix components

Primary muscle cell cultures were prepared from rat embryos and maintained in normal (unconditioned) and spinal cord (conditioned) media. In order to relate translational regulation to the morphological changes associated with each culture condition, mRNA was isolated from both culture variants and subjected to in ovo translation. Xenopus oocytes were injected with mRNA and their incubation media checked for the presence of newly formed proteins coded by the mRNAs and secreted into the medium. Electrophoretograms indicated mRNA-dependent synthesis of several proteins in the molecular mass range of 30-260 kD. To further characterize these proteins, antisera directed against several extracellular matrix proteins were used for immunoprecipitation: antigens recognized by anti-heparan-sulfate-proteoglycan and anti-fibronectin were enhanced in conditioned cells, whereas laminin was found to be reduced.     

Synthesis of surface immunoglobulin E receptor in Xenopus oocytes by translation of mRNA from rat basophilic leukemia cells

Immunoglobulin E-binding activity was expressed in Xenopus oocytes injected with mRNA from rat basophilic leukemia cells which possess abundant immunoglobulin E (IgE) receptor. Such activity was demonstrated with intact oocytes by their binding of 125I-labeled mouse monoclonal IgE. Binding activity was specific as shown by the total inhibition of 125I-IgE binding by unlabeled IgE but not by unlabeled IgG1. The relevance of the IgE-binding activity to the IgE receptor was also supported by the absence of this activity in oocytes injected with mRNA from cells lacking surface IgE receptors. mRNA coding for the IgE-binding activity was enriched in fractions sedimenting at 13.5 S in sucrose density gradients. From oocytes injected with rat basophilic leukemia mRNA, two major polypeptides were isolated by affinity purification on IgE immunoadsorbent. One (Mr = 31,000) is equivalent in size to the previously identified "receptor-associated protein;" the other (Mr = 40,000) is speculated to be a partially glycosylated or unglycosylated form of the alpha subunit of the IgE receptor. The binding of IgE-coated fluorescent microspheres by oocytes injected with rat basophilic leukemia mRNA demonstrated the surface expression of the IgE-binding proteins.     

Expression of Functional Bradykinin Receptors in Xenopus Oocytes

mRNA prepared from various tissues and cultured cells was injected into Xenopus laevis oocytes. Three to five days after injection, the response of the oocytes to the peptide bradykinin was monitored. The oocytes were voltage clamped and the membrane currents generated on application of agonist were recorded. mRNA from NG108-15, rat uterus, and human fibroblast cell line WI38 gave similar responses to bradykinin (1 microM), with an initial inward current (10-20 nA) followed by a prolonged period of membrane current oscillations. The same pattern of response was given by total RNA from rat dorsal root ganglia. No response to bradykinin (10 microM) was recorded from oocytes injected with rat brain mRNA, although these oocytes gave peak inward currents of about 75 nA in response to serotonin (10 microM). mRNA from both NG108-15 cells and rat uterus was fractionated on sucrose gradients. This resulted in an approximately five-fold increase in the size of the response compared to that given by unfractionated mRNA. The largest responses were given by mRNA fractions with a size of approximately 4.5 kb. Data were obtained consistent with the expression of both B1 and B2 receptors by WI38 human fibroblasts and with the expression of only the B2 type of receptor by NG108-15 cells.     

Expression of rat liver glutamine transporters in Xenopus laevis oocytes.

As a first step in attempting to isolate the Na(+)-dependent System N transporter from rat liver we have investigated the use of prophase-arrested oocytes from Xenopus laevis for the functional expression of rat liver glutamine transporters. Individual oocytes, defolliculated by collagenase treatment, were injected with 50 nl of a 1 mg.ml-1 solution of poly(A)+ RNA (mRNA) isolated from rat liver. 50 microM L-[3H]glutamine uptake was measured 1-5 days post-injection: after 48 h, poly(A)+ RNA-injected oocytes showed a 60 +/- 12% increase in Na(+)-dependent glutamine uptake compared to controls. This increased uptake showed characteristic features of hepatic System N: that is, it tolerated Li(+)-for-Na+ substitution and was inhibited by the System N substrate L-histidine (5 mM) in Li medium, unlike endogenous Na(+)-dependent glutamine transport. In subsequent experiments rat liver poly(A)+ RNA, size-fractionated by density gradient fractionation, was injected into oocytes. Injection of poly(A)+ RNA of 1.9-2.8 kilobases (kb) in size resulted in a significant stimulation of Na(+)-dependent glutamine transport to 0.362 +/- 0.080 pmol.min-1/oocyte from 0.178 +/- 0.060 pmol.min-1/oocyte in vehicle-injected oocytes (p less than 0.01). A lighter fraction, with poly(A)+ RNA of less than 1.9 kilobases size resulted in a similar increase in Na(+)-dependent glutamine uptake which was largely Li(+)-tolerant: Li(+)-stimulated glutamine uptake in oocytes injected with this fraction increased to 0.230 +/- 0.070 pmol.min-1/oocyte from 0.098 +/- 0.029 pmol.min-1/oocyte in controls (p less than 0.05). This enhanced rate of Li(+)-stimulated glutamine uptake was inhibited 28 and 70%, respectively, by 1 and 5 mM L-histidine. Na(+)-independent uptake of glutamine rose by 72 +/- 12% in oocytes injected with poly(A)+ RNA of 2.8-3.6 kb (p less than 0.001). These results demonstrate that glutamine transporters, with characteristics associated with hepatic Systems N, L, and A (or ASC), can be expressed in X. laevis oocytes injected with specific size fractions of rat liver mRNA.     

Assembly, glycosylation, and secretion of the oligomeric rat prostatic binding protein in Xenopus oocytes

Prostatic binding protein (PBP), a hormonally controlled oligomeric glycoprotein secreted by the rat ventral prostate, is composed of three different polypeptide chains, C1, C2, and C3. Microinjection of prostate mRNA into Xenopus laevis oocytes results in the synthesis, processing, and correct assembly of these three components, and also in the export of PBP into the medium. The glycosylation of component C3--the only glycopeptide of PBP--by the oocyte enzymes does not lead to the same result as in the native prostate tissue. The intracellular oocyte component contains an incompletely processed oligomannosyl core unit. Upon secretion this sugar core is further processed, probably at random because the carbohydrate chains attached to the exported C3 molecules are heterogeneous; they are also different from the oligosaccharide unit of authentic C3. However, tunicamycin experiments show that glycosylation is neither a prerequisite for secretion nor for the assembly of PBP, at least in oocytes.     

A qualitative difference in the interleukin 2 (IL-2) requirement of helper and cytotoxic T lymphocytes

A qualitative difference in the requirement of mouse helper and cytotoxic T lymphocytes for interleukin 2 (IL-2) was revealed by offering such cells IL-2 synthesized in Xenopus laevis oocytes that had been microinjected with messenger RNA (mRNA) encoding human IL-2. While both helper and cytotoxic mouse T-cell lines proliferate in response to the IL-2 present in medium conditioned by stimulated human lymphocytes, only helper-T-cell lines respond to human IL-2 secreted from oocytes. This result demonstrates a difference between helper and cytotoxic T lymphocytes in their response to IL-2. The growth response of murine cells shows that IL-2 secreted from human cells has properties not found in the IL-2 secreted from oocytes, even though a monoclonal antibody directed against the human IL-2 receptor blocks the activity of both types of IL-2. Quite possibly, this difference results from a post-translational modification.     

Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.     

Expression of foreign proteins in a <Emphasis Type="Italic">Vibrio cholerae</Emphasis> vaccine strain using the stationary phase hemagglutinin/protease promoter

The use of the hemagglutinin(HA)/protease promoter and secretion signals to drive expression and secretion of a foreign antigen in a live genetically attenuated cholera vaccine candidate is demonstrated. A Vibrio cholerae vaccine strain, containing a HA/protease-tetanus toxin C fragment (TCF) fusion, produced soluble-and cell-associated TCF. The fraction of TCF secreted to the culture medium was degraded unless expressed in a HA/protease-defective vaccine strain. Comparison of the hapA promoter with the strong Tac promoter using quantitative real time PCR revealed that at least five times more TCF mRNA was produced when expressed from the hapA promoter.     

Hyaluronic acid synthesis and secretion by rat liver fat storing cells (perisinusoidal lipocytes) in culture

The ability of rat liver fat storing cells to synthesize and to secrete hyaluronic acid was examined in monolayer cultures. The cells produce [3H] glucosamine-labeled hyaluronic acid, of which about 80% are secreted into the medium. The synthesis rate per cell (mg DNA) of labeled total glycosaminoglycans and hyaluronic acid in the medium increases significantly with culture time, but hyaluronic acid expressed as fraction of total glycosaminoglycans declines from about 0.70 in early cultures (up to the 4th day) down to 0.20 in advanced cultures. Cycloheximide increases and beta-D-xylopyranoside decreases significantly the fraction of hyaluronic acid in the medium, colchicine up to 5 microM was without effect. The synthesis of hyaluronic acid is a newly recognized function of this special type of sinusoidal liver cells. The results suggest that fat storing cells are likely to be a major source of hyaluronic acid in normal and probably also in injured liver.     

Thyroid hormone regulates type I deiodinase messenger RNA in rat liver

Conversion of the prohormone T4 to the active hormone T3 is catalyzed by 5'-deiodinases, enzymes that have not been purified. Previous studies have shown that modulating thyroid status results in changes in type I deiodinase activity in the rat liver. We have quantitated type I deiodinase mRNA in liver by an expression assay using Xenopus laevis oocytes. We report here that changes in enzyme activity correlate closely with changes in levels of the mRNA for this enzyme, indicating that thyroid hormone regulates type I deiodinase at a pretranslational step. Using the oocyte system to express size-fractionated mRNA, we have also determined that the mRNA coding for this protein is between 1.9-2.4 kilobases in length. It has been proposed that protein disulfide isomerase (PDI) is closely related to the rat type I 5'-deiodinase. Our results indicate that this is not the case, since injection of in vitro transcribed PDI mRNA into oocytes did not result in expression of deiodinase activity, and the deiodinase mRNA could be physically separated from the 2.8-kilobase mRNA species hybridizing to rat PDI cRNA by size fractionation.     

Synthesis and secretion of ovalbumin by mouse-growing oocytes following microinjection of chick ovalbumin mRNA

Mouse-growing oocytes were injected with chick ovalbumin mRNA. The oocytes were cultured for 18 h in the presence of [³H]leucine and the labeled ovalbumin was measured by immunoprecipitation. Two types of ovalbumin were precipitated by antibody to ovalbumin; one co-migrated with authentic, glycosylated ovalbumin in an 18% polyacrylamide gel and was estimated to be 45 000 D, whereas the other migrated faster with an apparent MW of 41 500 D. Both types of ovalbumin were also detected in the culture medium. This study demonstrates that mouse-growing oocytes can translate exogenous mRNA coding for a secreted protein and secrete two forms of the product.     

mRNA-induced expression of the cardiac Na+-Ca2+ exchanger in Xenopus oocytes

Xenopus oocytes were injected with total mRNA isolated from hearts of 1-day-old chicks. After 5 days of incubation the follicular cell layers were removed and the oocytes were loaded with Na+ by incubation in hypertonic EGTA solution at 37 degrees C. The Na+-loaded oocytes accumulated 45Ca2+ from a Na+-free medium at a 3-18-fold higher rate than noninjected oocytes or oocytes injected with control solution containing no mRNA. Oocytes not subjected to the Na+-loading procedure showed no mRNA-dependent 45Ca2+ uptake. Size fractionation of the mRNA using sucrose density gradient centrifugation under denaturing conditions led to the identification of a 25 S fraction competent for induction of the Na+-Ca2+ exchange system.