Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry

In order to understand the importance of the cytosolic and nuclear-specific O-linked N-acetylglucosaminylation (O-GlcNAc) on cell cycle regulation, we recently reported that inhibition of O-GlcNAc transferase (OGT) delayed or blocked Xenopus laevis oocyte germinal vesicle breakdown (GVBD). Here, we show that increased levels of the long OGT isoform (ncOGT) accelerate X. laevis oocyte GVBD. A N-terminally truncated isoform (sOGT) with a similar in vitro catalytic activity towards a synthetic CKII-derived peptide had no effect, illustrating the important role played by the N-terminal tetratrico-peptide repeats. ncOGT microinjection in the oocytes increases both the speed and extent of O-GlcNAc addition, leads to a quicker activation of the MPF and MAPK pathways and finally results in a faster GVBD. Microinjection of anti-OGT antibodies leads to a delay of the GVBD kinetics. Our results hence demonstrate that OGT is a key molecule for the timely progression of the cell cycle.     

Microinjection of a 19-kDa guanine nucleotide-binding protein inhibits maturation of Xenopus oocytes

ADP-ribosylation factors (ARFs) are 19-21-kDa proteins purified from bovine brain that bind guanosine 5'-triphosphate (GTP). They exhibit GTP-dependent activity as activators of cholera toxin-catalyzed ADP-ribosylation of the alpha-subunit of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system (Gs alpha). ARF, which interacts directly with the catalytic subunit of cholera toxin, has no known physiologic role. Intracellular microinjection of ARF was employed to investigate the effect of ARF on progesterone- and insulin-stimulated maturation of Xenopus oocytes. Maturation was inhibited by injection of ARF 3-8 h before exposure of oocytes to progesterone or insulin. ARF inhibition was dependent on progesterone concentration but not on insulin concentration. Inhibition was enhanced by concomitant injection of GTP and to a greater extent by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) which, in the absence of ARF, inhibited somewhat at early time points. The demonstration of this effect of ARF on both progesterone- and insulin-stimulated oocyte maturation may provide a clue to the physiologic role of this guanine nucleotide-binding protein.     

Microinjection of Xenopus oocytes: An automated device for volume control in the nanoliter range

Injection of Xenopus oocytes is a technique widely used in studies of gene expression for both qualitative and quantitative analysis of DNA and mRNA. Published methods for controlling and assessing volumes transferred into oocytes are tedious, time consuming, and require considerable expertise. We describe the construction of an apparatus which automatically controls nanoliter injection volumes. For injection volumes from 50 to 10 nl, the mean volume injected into 10 oocytes was within 10% of the expected value and often better, and even for 1-nl injections was within 20% of the expected value. The precision of volume dispensing into individual oocytes decreased with decreasing volume; the standard deviation was about 10–15% of the mean for volumes in the 30- to 50-nl range and about 30–40% of the mean for volumes in the 1- to 2-nl range.     

Stability and movement of mRNAs and their encoded proteins in Xenopus oocytes

The stability and movement of several polyadenylated (poly A+) and nonpolyadenylated (poly A-) mRNAs in Xenopus oocytes have been examined. At least 50% of the poly A+ mRNA molecules (9S rabbit globin mRNA, chicken ovalbumin, and lysozyme) were stable in oocytes over a 48-h period, irrespective of the amount injected. About 50% of injected poly A- reovirus mRNAs was degraded within the first 24 h of injection, irrespective of the amount injected, although no further degradation was observed over an additional 24 h. The movement of all poly A+ mRNAs injected at either the animal or vegetal pole of the oocyte was very slow. Little movement of RNA from the animal half to the vegetal half was observed even 48 h after injection. In contrast, similar amounts of mRNA were present in both halves 48 h after vegetal pole injection. Similar results were obtained after injection of poly A- reovirus mRNAs. The movement of the proteins encoded by the poly A+ mRNAs was studied in the 6-h period after injection when little mRNA movement had occurred. 85% of the globin synthesized accumulated in the animal half irrespective of injection site. The movement of the sequestered secretory proteins ovalbumin and lysozyme in the same oocytes as globin was much slower; very little lysozyme appeared in the half of the oocyte opposite the site of injection.     

Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes.

When DNA molecules are injected into Xenopus oocyte nuclei, they can recombine with each other. With bacteriophage lambda DNAs, it was shown that this recombination is stimulated greatly by introduction of double-strand breaks into the substrates and is dependent on homologous overlaps in the recombination interval. With plasmid DNAs it was shown that little or no recombination occurs between circular molecules but both intra- and intermolecular events take place very efficiently with linear molecules. As with the lambda substrates, homology was required to support recombination; no simple joining of ends was observed. Blockage of DNA ends with nonhomologous sequences interfered with recombination, indicating that ends are used directly to initiate homologous interactions. These observations are combined to evaluate possible models of recombination in the oocytes. Because each oocyte is capable of recombining nanogram quantities of linear DNA, this system offers exceptional opportunities for detailed molecular analysis of the recombination process in a higher organism.     

Microinjection of living adherent cells by using a semi-automatic microinjection system

Testing in vitro is an alternative to animal experimentation. The capillary pressure microinjection technique is a supporting technology for efficient in vitro testing. The main benefit of the technique is the possibility of injecting large molecules into a single living cell. The ultimate goal of the research discussed in this paper is to increase the cell survival rate in capillary pressure microinjection. A method to reliably evaluate cell survival rate is therefore needed. A three-phase evaluation process is presented in this paper. The first phase determines the success rate of the injection capillary to penetrate the cell membrane. The second phase studies the success rate of delivering the injection substance inside the cell, while the third phase studies cell survival after the microinjection. In addition to the three-phase evaluation process, this paper describes the initial results of penetration and injection tests performed by using a semi-automatic capillary pressure microinjection system developed by the research group. Three adherent cell lines, namely, retinal pigment epithelial cells, MCF-7 human breast cancer cells and SH-SY5Y neuroblastoma cells, were used in the experiments. The results of the penetration tests show that the average success rate of penetrating the cell membrane using the micromanipulator was 87%. The goal of the injection tests was to demonstrate the successful microinjection of living cells and to study the injection success rate. Fluorescein dextran was injected into MCF-7 cells, and preliminary results showed an injection success rate of 49%. In the survival tests, the neuronal cells were microinjected with KCl. During long-term observation after the microinjection, the microinjected cells first decreased their adhesion to the plate, but later adhered to the bottom of the plate and even grew some dendrites. In the next phase of the study, more tests will be performed in order to obtain a statistically reliable value for the survival rate.     

Release of acetylcholine by Xenopus oocytes injected with mRNAs from cholinergic neurons.

Xenopus laevis oocytes were injected with poly(A)+ mRNAs extracted from the electric lobes of Torpedo marmorata. The electric lobes contain the perikarya of approximately 120,000 cholinergic neurons that innervate the electric organs and are homologous to motor neurons. The injected oocytes accumulated acetylcholine and were able to synthesize [14C]acetylcholine from 1-[14C]acetate. With KCl depolarization and upon treatment with a Ca2+ ionophore, they released their endogenous as well as the radiolabelled neurotransmitter in a Ca(2+)-dependent manner. No synthesis or release were obtained from control oocytes. With respect to their dependency upon Ca2+ concentration, the oocytes injected with Torpedo electric lobe mRNAs released acetylcholine in a manner which closely resembled that found in the native synapses. In contrast to the controls, primed oocytes were also able to release [14C]acetylcholine that was injected a few hours prior to the release trial. Immunoblot analysis demonstrated that the 15 kd proteolipid antigen of the purified mediatophore, a 200 kd presynaptic protein able to translocate acetylcholine, was expressed in the ACh-releasing oocytes but not in the controls. The present observation may provide a useful approach for investigating the proteins involved in the release of acetylcholine and of other neurotransmitter substances.     

Changes in intracellular calcium and in membrane currents evoked by injection of inositol trisphosphate into Xenopus oocytes

Intracellular calcium was monitored by the use of aequorin in voltage-clamped oocytes of Xenopus laevis. Injection of inositol trisphosphate (IP3) into oocytes elicited slowly rising and decaying aequorin/calcium signals and produced oscillatory chloride membrane currents. These responses did not depend upon extracellular calcium, since they could be elicited in calcium-free solution and after addition of cobalt or lanthanum to block calcium channels in the surface membrane. We conclude that IP3 causes the release of calcium from intracellular stores in the oocyte. Injections of calcium gave aequorin and membrane current responses that were more transient than those seen with IP3.     

Microinjection of pp60v-src into Xenopus oocytes increases phosphorylation of ribosomal protein S6 and accelerates the rate of progesterone-induced meiotic maturation.

Microinjection of purified pp60v-src into Xenopus oocytes caused the phosphorylation of ribosomal protein S6 on serine residues and also increased total protein phosphorylation, with almost a two-fold increase in the percentage of phosphotyrosine present. In addition, pp60v-src accelerated the time course of progesterone-induced oocyte maturation, suggesting that the biochemical pathway influenced by pp60v-src is related to that induced by progesterone.     

Activin incorporation into vitellogenic oocytes of Xenopus laevis.

Activin uptake into Xenopus oocytes was studied by several complementary methods. Immunocytochemistry of adult ovary localized activin and follistatin in the cytoplasm of vitellogenic oocytes and surrounding follicle cells. Surface plasmon resonance analysis of protein interaction kinetics indicated that while follistatin or a complex of activin-follistatin bound to yolk vitellogenin, activin alone did not. Radioactive tracer analysis measured specific incorporation of activin by viable oocytes in vitro. Together, the results suggest that vitellogenic oocytes can import activins from follicle cells and that follistatin may act as a chaperone for binding activin to vitellogenin in yolk platelets.     

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.     

Direct injection of cell-free Kir1.1 protein into Xenopus oocytes replicates single-channel currents derived from Kir1.1 mRNA

The development of integral membrane protein cell-free synthesis permits in-vitro labeling of accessible cysteines for real-time FRET and LRET measurements. The functional integrity of these synthetic ion channel proteins has been verified at the whole oocyte level by direct injection into, and recording from, Xenopus oocytes. However, the microscopic single-channel properties of cell-free translated protein have not been systematically examined. In the present study, we compare patch-clamp currents originating from cell-free protein with currents derived from mRNA injection, using the same (single-Cys) inward rectifier DNA template (C189-Kir1.1b). Results indicate that cell-free Kir protein, incorporated into liposomes and injected into oocytes, is trafficked to the plasma membrane where it inserts in an outside-out orientation and exhibits single-channel characteristics identical to that derived from a corresponding mRNA.