Microinjection of Xenopus Laevis Oocytes Article doi: 10.3791/1106. 2 We show that X. tropicalis oocytes possess an intracellular Ca 2+ store that is … ZERO BIAS - scores, article reviews, protocol conditions and more The frog's ventral surface is solid yellowish white to spotted. After co-injection of Xenopus egg extracts and single mouse cumulus cells into enucleated mouse oocytes, statistically higher pronucleus formation and development rates were observed in the egg Extract− co-injected group compared with those in the no egg extract-injected (NT) group (38–66% vs 18–34%, P <0.001). The search-image is essentially a uniform pigmentation of the animal pole of stage V and VI oocytes (9). D. Bertrand, ... M. Ballivet, in Methods in Neurosciences, 1991. Preparation. Indeed, it is now thought that X. laevis (for example) is, in fact, tetraploid (Kobel and Du Pasquier, 1986; Cannatella and de Sá, 1993), making X. ruwenzoriensis a dodecaploid (Kobel and Du Pasquier, 1986; Tymowska, 1991). The expression cloning approach is unconstrained by the nonavailability of nucleotide or amino acid sequence information of the desired genes or gene products and can be, theoretically, applied to clone any gene encoding a protein of detectable function. Every ovary contains hundreds of oocytes so only a fraction are removed at any one time. This example emphasizes that much more flexibility may exist in sex determination systems in animals than assumed in the models of Muller (1925) and Orr (1990). National Xenopus Resource (NXR) - USA +1 508 289 7627: Marko Horb: European Xenopus Resource Centre (EXRC) - UK +44 2392 842327: Matt J. Guille: Biological Resource Center Xenpus (CRB) - FR +33(0)2 23 23 52 51 +33(0)2 23 23 67 82: Contact: National BioResource Project (NBRP) - JP +82 424 4782 +82 424 0739: Masayuki Sumida: X. laevis Research Resource for Immunobiology - NY USA +1 … Depending on quality of local supply, water may be used directly, mixed with deionized water, or may be aged in large containers for several days. Expression of recPfHly III in X. laevis oocytes resulted in sensitivity to hypotonic lysis in a time-dependent manner similar to that seen with hAQP1-expressing oocytes. In compliance with the UK Animals (Scientific Procedures) Act 1986, female Xenopus were anesthetized with benzocaine (ethyl 4-aminobenzoate) and minimum amounts of oocytes were used. Female X. laevis laevis are larger than males and average 110 mm in length (Kobel et al., 1996). The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins. 1997 ), except that the defolliculation procedure was performed in … In Xenopus laevis, vitellogenin (the yolk precursor) is synthesized in the liver and transported via the circulatory system to the ovary. The eyes of the clawed frog are located more dorsally on the head, are lidless, have a convex cornea, and are adapted for vision in air rather than water. exocytosis in Xenopus laevis oocytes and eggs in response to cytosolic Ca ~÷. If the wash solution around the defolliculated oocytes is very cloudy even after several rinses and becomes even more cloudy after 30 min in culture, then the oocytes are probably unusable. Using the two-electrode voltage-clamp technique, the current induced by inorganic phosphate (IPi) was measured and taken as an index of NPT2a-mediated Pi transport . Twenty species are currently described within it. Michael E. Zuber, in Current Topics in Developmental Biology, 2010. Emed Res 2: 100008. Xenopus laevis oocytes. The activity is doubled by the presence of 200–400 mM NaCl, KCl, LiCl, or NaF; NaNO3 also stimulates activity at 100 mM but this effect is lost at higher concentrations [5]. (A) Development of ICSI embryos to each stage is summarized. Since then, most of the polyploids have become functional diploids in terms of gene expression patterns. Xenopus oocytes are widely used for heterologous expression of cRNAs encoding cytosolic enzymes, immunoglobulins, membrane receptors, ion channels, ion pumps, and membrane transporters. Their large nuclei and mitochondrial masses are clearly visible in the intact oocyte. Xenopus laevis (African clawed frog) Status. The channel adopts an open conformation at low or zero membrane potential and a closed conformation at potentials above 30-40 mV. 3. During oogenesis, Xenopus oocytes accumulate large amounts of storage proteins that—after fertilization—provide the developing embryo with building blocks and energy metabolites. The open state has a … A last and strong argument in favour of membrane as a target for triggering meiosis reinitiation has been brought about by a series of injection experiments. Furthermore, several pharmacological membrane active agents were able to mimick progesterone action. The Xenopus oocyte has been called a “living test tube” (de Robertis et al. B., Lane, C. D., Woodland, H. R., et al. Epub 2020 May 25. . The lack of multivalents during meiosis (at least in the tetraploids), an absence of closely related diploids, observations of mixed interbreeding, and the relatively common generation of polyploid oocytes in female hybrids all point to allopolyploidy in Xenopus species (Tymowska, 1991; Kobel, 1996). John P. Leonard, in Methods in Neurosciences, 1989. Oocytes were individually separated, as previously described ( Chen et al. Figure 2: Xenopus laevis oocytes before and after maturation. For this reason, and despite decades of work on X. laevis, research in developmental genetics is shifting to the smaller, faster-developing, and genetically much more convenient diploid Xenopus tropicalis (Vogel, 1999). Since ATP synthase activity is redox regulated, we explored a regulatory role for reversible thiol oxidation. Dev Biol 36:669- cleavage furrow formation in Xenopus laevis oocytes and eggs. The coevolutionary influence of polyploidy goes a step further in this regard, because it appears that allopolyploidy may confer some resistance to certain species of these parasites (Jackson and Tinsley, 2003). This subspecies has a fifth toe that is much longer than the tibia. Females have large cloacal papillae, and males develop dark inner surfaces on their forearms (nuptial pads). It is thus concluded that the first step of meiosis induction is linked to the interaction of any inducer with membrane constituents, which can be phospholipids (binding Ca2+) and/or enzymes (such as CaATPase or adenylate cyclase). The enzyme retains 90% of activity upon freezing when in 20% glycerol [5]. The oocytes are then denuded of overlying follicle cells by treatment with 2 mg/ml collagenase (Sigma type I-A), in Ca2+-free solution of the following composition (mM): NaCl, 82.5; KCl, 2.0; MgCl2, 1.0; HEPES, 5.0 (pH 7.4). pus (zĕn′ə-pəs) n. Any of various aquatic, tongueless, clawed frogs of the genus Xenopus of southern Africa, certain species of which, especially the African clawed frog, are widely used for biological research. Whether its action is also related to membrane interaction is not elucidated yet. Two species of Xenopus are regularly used by biologists, Xenopus laevis and Xenopus tropicalis. Activity in Xenopus laevis Oocytes. Characteristics Cytoplasm. Mediation ofanion transport in oocytes ofXenopus laevis by biosynthetically inserted band3 protein from mouse spleen erythroid cells M.Morgan. In addition to Fe(III)–DMA, HvYSL2 can transport Zn(II)–DMA, Ni(II)–DMA, Cu(II)–DMA, Mn(II)–DMA and Co(II)–DMA at fairly similar levels, which makes it functionally more similar to ZmYS1 than HvYS1, the latter of which has a narrow substrate specificity (Harada, Sugase, Namba, Iwashita, & Murata, 2007; Murata et al., 2006). Xenopus laevis oocytes are a powerful tool for the characterization of signal transduction pathways leading to the induction of DNA synthesis. Engrailed 2 (Xen2) is normally expressed in the caudal midbrain and midbrain/hindbrain border. 2.4D). It was observed that OsYSL2 can transport Fe(II)–NA and Mn(II)–NA, but not Fe(III)–DMA or Mn(II)–DMA, among various other metals tested (Koike et al., 2004). Xenopus laevis oocytes were digested in ORII solution, which contained 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 5 mM HEPES, pH 7.4, and 1.5 mg/mL collagenase, for 90 min at room temperature. 2003;227:245-58. 5 ). In vitro transcribed poly(A)-cRNA is microinjected into the cytoplasm of the oocyte and on the next day the function of the encoded protein can be measured. The lungs, heart, and liver of Xenopus are large, and the urinary bladder is spherical. Abstract Xenopus oocytes are a versatile expression system particularly suited for membrane transporters and channels. Even when the frogs are properly tended, there are times when healthy oocytes are not being produced. Test potentials that ranged between -80 mV to +60 mV applied in 10mV increments were applied to injected oocytes. Comparative Medicine 50:206-211. Oocytes. Table 1. Recherche d'information médicale. This procedure does not affect the electrophysiological responses of the oocytes (measured at least 36 hr later) or in any way alter its ability to translate ion channel mRNA (reviewed in Ref. Figure 3: Development of ICSI embryos, produced from in vitro matured oocytes. Function i. As the very first commercial service company Ecocyte Bioscience has supported customers in Europe with high quality defolliculated Xenopus laevis oocytes since 2006. Poly(A)+ RNA was functionally expressed in Xenopus oocytes 4 to 7 d after injection. 2.4E). Interestingly, systematists now consider X. tropicalis and X. epitropicalis to warrant placement into their own genus (Silurana) on the basis of morphological characteristics (e.g., Cannatella and Trueb, 1988; de Sá and Hillis, 1990; Cannatella and de Sá, 1993; Rödel, 2000; Frost, 2004). carsten.wagner@uni-tuebingen.de Because OsYSL18 is expressed in phloem parenchyma, companion cells, flowers and developing seeds, it may be involved in long distance transport of Fe(III)–DMA via phloem, and thus Fe(III)–DMA may also contribute to seed Fe loading in rice. In either case, the (sub)genus Xenopus itself would therefore consist entirely of polyploids, potentially making it the only group of amphibians not directly linked to related diploids. The collagenase treatment should be terminated when about 50% of the oocytes are stripped of overlying follicle cells (1.5–3 hr). (1998) Characterization of the monocarboxylate transporter 1 expressed in. HvYS1 and OsYSL15 have also been shown to transport Fe(III)–MA (Murata et al., 2006) and Fe(III)–DMA (Inoue et al., 2009), respectively, using electrophysiology. It is known that this ion does not penetrate the cells and interferes with Ca2+-movements from membrane sites and/or adenylate cyclase activity (15). This is a preview of subscription content, Stegen, C., Matskevich, I., Wagner, C. A., et al. Alijevic O, McHugh D, Rufener L, Mazurov A, Hoeng J, Peitsch M (2020) An electrophysiological characterization of naturally occurring tobacco alkaloids and their action on human alpha4beta2 and alpha7 nicotinic acetylcholine receptors. Their large size facilitates both injection of heterologous cRNA and subsequent electrophysiological recordings of ion channel currents. Both species are fully aquatic, and are easy to maintain in captivity. Life span of X. laevis in the wild is reported to be greater than 10 years. Immature eggs (oocytes) of the South African clawed frog Xenopus laevis are considered an ideal expression system for such studies. (Biochemical changes during progesterone-induced maturation in Xenopus laevis oocytes. Test potentials that ranged between -80 mV to +60 mV applied in 10mV increments were applied to injected oocytes. Using this method, we show here for the first time, that standard Amos and Croc-type asbestos fibres significantly modify the electrophysiological properties of the XLO plasma membrane starting very shortly after exposure. Two Xenopus laevis females were injected with 450 U of hCG (human chorionic gonadothropin) hormone and ovulated oocytes were obtained 12 hours after stimulation by gentle manual squeezing. For injection (11, 12), we use untreated females during most of the year. 678 J Cell Biol 108:852-892 Kline D (1988) Calcium-dependent events at fertilization of the Bement WM, Capco DG (1990 a) Transformation of the amphibian frog egg: Injection of a calcium buffer blocks ion channel open- oocyte into the egg: structural and biochemical events. This service is more advanced with JavaScript available, Membrane Transporters Preparation. Cells to be avoided include those with diffuse lightening at the tip of the animal pole. Even tadpoles develop and utilize lungs as well as gills for breathing (Deuchar, 1975). The system is also suitable for functional analysis by a variety of electrophysiological techniques due to the large size of oocytes, which permits introduction of electrodes into the cell with considerable ease. Among those inhibitors were gammexane (19), an inositol analogue, which indeed can interfere with membrane phospholipid metabolism and/or Ca2+-gating, some inhibitors of phospholipid methylation (such as SIBA = 5′-deoxy-5′-S-(2-methyl-propyl)-5′-thioadenosine (SIBA), a methylase inhibitor (20)) which modify the responses to several hormones and/or mediators as well as compounds able to change the redox state of the membrane (for example dithiothreitol) (21). Agents interacting with meiosis in Xenopus laevis oocytes, Some steroids: progesterone, cortisol, testosterone, pregnenolone, Some organo-mercurials: parachloromercuribenzoate, mersalyl, Some ions: lanthanum, zinc, calcium*† magnesium*, Some antiarrythmic drugs: D-600(isoptin), D-200(verapamil), Some O-receptor adrenergic antagonists: isomers of propranolol, alprenolol, dichloroisoproterenol, Some local anesthetics: dibucaine, tetracaine, Some CNS acting drugs: barbital, imipramine, chlorpromazine, fluphenazine, fenfluramine, cycloheximide (inhibitor of protein synthesis), theophylline, IBMX (inhibitors of phosphodiesterases), dithiothreitol (protecting agent for SH groups), gammexane (inhibitor of phospholipid metabolism). Oocytes have little background activity and therefore offer a very high signal-to-noise ratio for transporter and channel characterization. L.L Lindsay, in Handbook of Proteolytic Enzymes (Third Edition), 2013. (We first tested rosiglitazone (RGZ), a TZD, on human ENaC activity in Xenopus laevis oocytes. epitropicalis and those of other species like X. laevis are so different as to make direct comparisons difficult (Tymowska, 1991). The oocyte is the cellular precursor for the development of specialized cells. This volume presents the first comprehensive treatment of the wide range of uses for Xenopus laevis oocytes and embryos in cell and molecular biology. Xenopus laevis (frog) embryos are well-suited to model congenital kidney diseases and to explore the mechanisms that cause these developmental defects. Among those, only compounds unable to enter the cells were active meiosis inducers. Oviductin is inhibited by DFP, aprotinin, soybean trypsin inhibitor, leupeptin, antipain EDTA, and EGTA, and also weakly by TLCK, TPCK, and 1,10-phenanthroline; pepstatin, E-64, and iodoacetamide do not inhibit [5]. Microinjection of size-fractionated mRNA from the tissue sources, followed by subsequent functional assays, narrows down the potential target mRNAs to a relatively smaller subpopulation. Stage I consists of small (50 to 100 μ) colorless oocytes whose cytoplasm is transparent. Levis, R. A. and Rae, J. L. (1992) Constructing a patch clamp setup. BACKGROUND. Xenopus laevis oocytes are arrested in prophase of the first meiotic division. The first step of this physiological process seems to involve a target site at the oocyte membrane, as shown by a variety of experimental data (4). The relatively broad expression domain and range of phenotypes observed following its inactivation indicate that XSix3 is required not only for eye field specification, but also for much of rostral brain formation. Recording electrode resistances were between 0.5-1.0 MΩ. Xenopus laevis oocytes. This approach effectively reduces the size of the cDNA library to be screened and increases the probability of successful isolation of the target cDNA. Cite as, Table 1 Xenopus laevis Oocytes. They are described to displace calcium from membrane sites, to inhibit CaATPase or to regulate the activity of adenylate cyclase (4,17). The widespread use of the oocyte expression system is based upon the fact that oocytes can not only translate exogenous RNA from various sources efficiently and faithfully, but also carry out posttranslational modifications, such as precursor processing, phosphorylation, glycosylation, intracellular trafficking and protein targeting into the plasma membrane, complex subunit assembly, and even exporting secretory proteins. XSix3 misexpression has dose-dependent effects. Phytochemistry 170:112187. J Am Assoc Lab Anim Sci … Such primed females can be used four or more months after ovulation. In this review, we describe the basics of using X. laevis as a model organism for studying cell division and outline experimental approaches for imaging cytoskeletal components in vivo in X. laevis embryos and eggs. Xenopus laevis oocytes have been used widely in the field of electrophysiology to study the structure and the function of numerous ion channels and to screen for selective blockers. A full characterization of a specific protein will o … Sarah Cohen 1, Shelly Au 1, Nelly Pant é 1. The first agents tested were the organo-mercurials (13). In line with the latter finding is the fact that Zn2+ (as ZnSO4) was also an effective agent mimicking progesterone action (unpublished) and indeed it was shown to inhibit adenylate cyclase in other systems(16). These findings were something of a surprise, given that the relatively low opacity of Xenopus eggs and embryos was assumed to make them poor subjects for live-cell imaging. This particular subpopulation of mRNA can then be used to construct an expression cDNA library for subsequent screening using the same oocyte expression technique. The Xenopus oocyte, through injection of exogenous RNA, a method now more than 40 years old (Gurdon et al., 1971), is one of the major expression system for membrane proteins such as ion channels (Cens and Charnet, 2007) as well as other potential partners or regulators of the SOCE process. In addition, they lack transcriptional activity until the mid-late blastula stage. Sabine Schorderet-Slatkine, ... Etienne-Emile Baulieu, in Endocrinology, Neuroendocrinology, Neuropeptides: Volume 13, 1981. They are induced to resume meiosis when exposed to a steroid hormone, progesterone. Xenopus laevis laevis has a yellowish to darker, spotted to marbled dorsal coloring. Wagner, C. A., Ott, M., Klingel, K., et al. A 1–2 cm incision is made in the abdomen and several ovarian lobes removed (6). School of Biochemistry and Molecular Biology, https://doi.org/10.1385/1-59259-387-9:245. Sterile, 70% ethanol-treated surgical instruments are used for all procedures. Xenopus Laevis Oocytes, supplied by Integrated DNA Technologies, used in various techniques. MPF can directly induce the breakdown of germinal vesicle (GVBD) when injected into oocytes which have not been exposed to progesterone (2,3,7,8). (2001) Inhibition of the glutamate transporter EAAC1 expressed in. Exocytosis of chromatfin granule membranes was detected by the appearance of dopamine-/3-hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Xenopus oocytes are widely used for heterologous expression of cRNAs encoding cytosolic enzymes, immunoglobulins, membrane receptors, ion channels, ion pumps, and membrane transporters. Part of Springer Nature. In fact, X. tropicalis is the only known diploid in the genus (with 2n = 20), but even here there is at least one associated cryptic tetraploid species, X. epitropicalis (4x = 40), which differs from X. tropicalis primarily with regard to mating call characteristics and some additional karyotypic features (Loumont, 1983; Tymowska, 1991). Single cell experimentation in Xenopus laevis oocytes is a routine for experimental biologists. Xenopus laevis has long been a popular model for studies of development and, based on the use of cell-free extracts derived from its eggs, as a model for reconstitution of cell cycle regulation and other basic cellular processes. buffer. Such cells are undergoing spontaneous maturation. We have used rhodamine-conjugated phalloidin and confocal microscopy to examine the organisation of filamentous actin (F-actin) during oogenesis in Xenopus laevis.F-actin was restricted to a thin shell in the cortex of oogonia and post-mitotic oocytes less than 35 μm in diameter. In studies of purified oviductin substrate specificity, the artificial substrate Boc-Phe-Ser-Arg↓NHMec was found to be preferred [5]. DI-fusion Ultrastructure of Xenopus laevis oocytes after injection of. Yolk DNA behaves like nuclear DNA, and not like mitochondrial DNA, upon renaturation after denaturation. It is estimated that the protein content is about 500 ug in each oocyte and approximately 10-50 mg/mL in the extract. 8). The females are killed by decapitation after 10–15 min of anesthesia in 0.2% MS222 (Sandoz, Basel, Switzerland). BACKGROUND. On the other hand, OsYSL18 can transport Fe(III)–DMA but not Fe(II)–NA, Zn(II)–DMA or Zn(II)–NA (Aoyama et al., 2009). An increase in eye field and eye size is observed at low doses, while structures both anterior and posterior to the eye field have altered fates at high doses. bonjour j ai 14 tetard de xenopus albinos on ma donner de l aquatic code veggys comme nourriture est ce bon? Surgical Laparotomy to obtain oocytes: In Xenopus, oogenesis is divided into six stages (I-VI) and oocytes are obtained by surgical laparotomy from anesthetized female X. laevis. Sarah S. Conte, Elsbeth L. Walker, in Current Topics in Membranes, 2012. We have found it best to send oocytes out as dissected ovary lobes on wet ice but can provide defolliculated oocytes if necessary, albeit at extra cost. Xenopus laevis RIC‐3 enhances the functional expression of the C. elegans homomeric nicotinic receptor, ACR‐16, in Xenopus oocytes. This contributes to reducing the number of female Xenopus used for oocyte injection experiments. In Xenopus laevis, vitellogenin (the yolk precursor) is synthesized in the liver and transported via the circulatory system to the ovary. Learn more, Over 10 million scientific documents at your fingertips. On the other hand, some inhibitors of progesterone-induced meiosis were also acting at membrane level, thus on the first step of the cascade of events triggered by the steroid (Table 1). The frog's skin is kept moist during surgery to prevent acidification. Oogenesis in the anuran Xenopus laevis can be divided into six stages based on the anatomy of the developing oocyte. Xenopus laevis Oocytes. Thus, excessive XSix3 appears to impart retinal character to part of the midbrain (Bernier et al., 2000). Thus, OsYSL2 maintains a fairly narrow substrate specificity and is likely to function in phloem transport of Fe and Mn. Page 3 of 12 Volume 2, Article ID: 100008 extracellular KCM-211 solution at a flow rate of 75 mL/hour. Here we show that: oligomycin sensitive ATP synthase activity is significantly greater (~170 vs. 20 nmol/min −1 /mg −1) in testes compared to oocytes in Xenopus laevis (X. laevis). To investigate whether CFTR expression interacts with NPT2a function, we co-expressed both proteins in Xenopus laevis oocytes. Please Specify Uncurling arrive around 38 NF