Genomic potential of erythroid and leukocytic cells of Rana pipiens analyzed by nuclear transfer into diplotene and maturing oocytes

In order to determine whether differentiated somatic cells maintain genetic totipotency, nuclear transplantations from several differentiated somatic cell types into eggs and oocytes were performed previously in Rana pipiens and Xenopus laevis. The formation of postneurula embryos and tadpoles under the direction of the test nuclei demonstrated their genetic multipotency. In addition, Rana erythrocyte nuclei transplanted to oocytes directed more extensive tadpole development than those injected into eggs. We have extended our studies of the genomic potential of differentiated somatic nuclei from the peripheral blood of Rana pipiens. First, we show that the developmental potential of erythrocyte nuclei injected into oocytes at first meiotic metaphase was greater than those injected into diplotene oocytes. Second, we demonstrate that erythroblast and leukocyte nuclei transplanted to oocytes at first meiotic metaphase promoted more advanced tadpole development than those previously injected into Xenopus eggs. Third, erythrocyte nuclei were more successful in promoting advanced tadpole development compared with erythroblast and leukocyte nuclei. The results show that differentiated somatic nuclei transferred to the cytoplasm of oocytes at first meiotic metaphase display enhanced genomic and developmental potential over those transplanted to diplotene oocytes and eggs, at least for the three nuclear cell types tested from the peripheral blood.     

Xenopus white papers and resources: folding functional genomics and genetics into the frog

The frog Xenopus has been vital for biomedical science for over 80 years, contributing to diverse fields from cell signaling, cell and developmental biology, to ion channel physiology and toxicology. Its experimentally manipulable oocytes and embryos provide abundant material for molecular and biochemical approaches for a wide range of gene discovery and protein function studies. In recent years, the Xenopus community has invested in key resources for functional genomics, including genome-wide full-length cDNA collections and genome assemblies as well as genetic tools. These assets combine with Xenopus' extensive range of functional assays to create exciting new research avenues with medical as well as basic applications. This review describes how these resources were developed and what new tools are on the horizon.     

Posttranscriptional regulation of c-myc RNA during early development of Xenopus laevis

The remarkable stability of c-myc during oogenesis contrasts with its degradation during the early developmental period in Xenopus laevis. Three evolutionary conserved motifs found in the 3'-untranslated region of Xenopus c-myc RNAs have been analyzed for a possible role in c-myc RNA degradation. No specific degradation was observed when these sequences were cloned downstream of a reporter gene and the corresponding RNAs were injected into fertilized eggs. The relation between polyadenylation and degradation of c-myc mRNA has been examined during early development. c-myc is adenylated during early oogenesis, and a dramatic de-adenylation occurs in full grown oocytes. Consequently, the de-adenylation of c-myc mRNA that occurs in eggs might be a requirement for its degradation after fertilization, but is not sufficient to trigger its degradation.     

Vimentin expression in oocytes, eggs and early embryos of Xenopus laevis

Immunocytochemical studies using a monoclonal anti-porcine vimentin antibody reveal a well-organized pattern of staining in Xenopus laevis oocytes, eggs and early embryos. The positions of Xenopus vimentin and desmin in two-dimensional (2D) polyacrylamide gels were first established by immunoblotting of muscle Triton extracts with anti-intermediate filament antibodies (anti-IFA), which cross-react with all intermediate filament proteins (IFPs). The anti-porcine vimentin reacts with vimentin and desmin in muscle 2D immunoblots, but only reacts with one polypeptide in oocyte blots in the position predicted for vimentin (Mr 55 x 10(3), pI 5.6). Using an anti-sense probe derived from a Xenopus vimentin genomic clone in RNase protection assays, we show that expression of vimentin begins in previtellogenic oocytes. The level of expression remains constant throughout oogenesis and in unfertilized eggs. These data suggest that vimentin is expressed in oocytes and eggs. Most interestingly, the immunocytochemical results also show that vimentin is present in the germ plasma of oocytes, eggs and early embryos. It is therefore possible that vimentin has an important role in the formation or behaviour of early germ line cells.     

Evidence for direct membrane retrieval following cortical granule exocytosis in Xenopus oocytes and eggs

Rapid exocytosis is typically followed by rapid resorption of exocytosed membrane; however, whether membrane retrieval occurs via indirect endocytosis of numerous small vesicles or direct resealing of the original, larger exocytotic vesicles is controversial. Here we show that cortical granule (CG) exocytosis in Xenopus oocytes and eggs is followed by rapid formation of endosomes as large as the CGs. Large endosomes are translucent, and their formation has the same developmental and pharmacological profile as CG exocytosis. Time course analyses show that large endosomes are not derived from small endosomes. Large endosome formation is triggered by stimuli that do not trigger increases in intracellular-free calcium and is insensitive to perturbation of microtubules by treatment with nocodazole. Perturbation of the f-actin cytoskeleton with latrunculin, however, sharply reduces large endosome formation. We conclude that CG membrane is directly retrieved in Xenopus oocytes and eggs and suggest that this retrieval is not directly dependent on an increase in intracellular-free calcium, but is dependent on the actin cytoskeleton.     

Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase

It has been demonstrated that the Xenopus homolog of the fission yeast cdc2 protein is a component of M phase promoting factor (MPF). We show that the Xenopus cdc2 protein is phosphorylated on tyrosine in vivo, and that this tyrosine phosphorylation varies markedly with the stage of the cell cycle. Tyrosine phosphorylation is high during interphase (in Xenopus oocytes and activated eggs) but absent during M phase (in unfertilized eggs). In vitro activation of pre-MPF from Xenopus oocytes results in tyrosine dephosphorylation of the cdc2 protein and switching-on of its kinase activity. The product of the fission yeast suc1 gene (p13), which inhibits the entry into mitosis in Xenopus extracts, completely blocks tyrosine dephosphorylation and kinase activation. However, p13 has no effect on the activated form of the cdc2 kinase. These findings suggest that p13 controls the activation of the cdc2 kinase, and that tyrosine dephosphorylation is an important step in this process.     

Cysteine string proteins are associated with cortical granules of Xenopus laevis oocytes

Cysteine string proteins (csps) are associated with secretory organelles in a wide range of eukaryotic cells. Functional studies of these proteins indicate that they subserve one or more vital steps in the pathway of regulated exocytosis. Here, we document the presence of csps in fully grown (stage VI) oocytes of the frog, Xenopus laevis. Both Northern and immunoblot data support the conclusion that csps are expressed in these cells. In addition, immunoreactive csp is seen even at the earliest stage of oocyte development, namely, in stage I oocytes. Finally, immunoblot and immunocytochemical results indicate that csps are associated with cortical granules of stage II-VI oocytes. These observations suggest that csps participate in the cortical reaction that underlies the sustained block to polyspermy in Xenopus eggs. Moreover, because of the relative ease of manipulating cells as large as Xenopus oocytes, this system harbors considerable promise as a model for studying the role of csps and other proteins in exocytotic events.     

The maternal gene product D7 is not required for early Xenopus development.

The Xenopus D7 gene codes for a novel protein whose expression is restricted to early development. D7 protein is synthesized for the first time during oocyte maturation (1988, Genes Dev. 2, 1296-1306). Injection of D7 RNA into the full-grown oocyte and its subsequent translation into D7 protein neither induced oocyte maturation nor affected the kinetics of hormone-induced maturation. Overexpression of D7 protein by 20-fold in the early Xenopus embryo by injection of D7 RNA into fertilized eggs did not affect subsequent development. Oocytes specifically lacking D7 mRNA were generated by oligodeoxynucleotide-mediated RNA destruction within the oocyte. Unfertilized eggs generated from such oocytes lacked detectable D7 protein, but nevertheless could be activated and fertilized. Embryos generated from such eggs, estimated to contain less than 5% of wildtype levels of D7 protein, developed normally up to the tailbud stage. Thus the D7 protein, the product of a maternal mRNA that is under strict translational repression in oocytes, appears not to be required for oocyte maturation, activation, fertilization or early embryonic development in Xenopus.     

Ribonucleotide reductase activity during amphibian development.

Uniformly labeled [3H] uridine is incorporated into DNA by dissociated Pleurodeles blastulae; the label is found in cytosine and to a much lesser extent in thymine. Ribonucleotide reductase activity cannot be detected in full grown oocytes of Xenopus and Pleurodeles, but is present in unfertilized egg. The enzyme is synthesized (or activated) when maturation is induced in Xenopus oocytes by in vitro hormonal treatment. The enzymatic activity increases after fertilization and reaches a peak at the 2--4 cell stage; it decreases at the blastula, gastrula and neurula stages to the low level initially present in unfertilized eggs. The enzyme is no longer detectable in swimming tadpoles. Addition of hydroxyurea (1 mg/ml) to fertilized eggs leads to complete loss of ribonucleotide reductase activity: cycloheximide (20 mug/ml) inhibits the rise in activity characteristic of early cleavage, while actinomycin D (20 mug/ml) has no effect. The significance of these results in discussed.     

Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4

Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near‐native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4‐M23) was expressed in the X. laevis oocytes following their injection with AQP4‐M23 cRNA. AQP4‐M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4‐M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over‐expressed AQP4‐M23, the membranes from AQP4‐M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher‐order arrays of AQP4‐M23. In addition, but only infrequently, AQP4‐M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.     

Xtr, a plural tudor domain-containing protein, is involved in the translational regulation of maternal mRNA during oocyte maturation in Xenopus laevis

Xtr in the fertilized eggs of Xenopus has been demonstrated to be a member of a messenger ribonucleoprotein (mRNP) complex that plays a crucial role in karyokinesis during cleavage. Since the Xtr is also present both in oocytes and spermatocytes and its amount increases immediately after spematogenic cells enter into the meiotic phase, this protein was also predicted to act during meiotic progression. Taking advantage of Xenopus oocytes' large size to microinject anti-Xtr antibody into them for inhibition of Xtr function, we examined the role of Xtr in meiotic progression of oocytes. Microinjection of anti-Xtr antibody into immature oocytes followed by reinitiation of oocyte maturation did not affect germinal vesicle break down and the oscillation of Cdc2/cyclin B activity during meiotic progression but caused abnormal spindle formation and chromosomal alignment at meiotic metaphase I and II. Immunoprecipitation of Xtr showed the association of Xtr with FRGY2 and mRNAs such as RCC1 and XL-INCENP mRNAs, which are involved in the progression of karyokinesis. When anti-Xtr antibody was injected into oocytes, translation of XL-INCENP mRNA, which is known to be repressed in immature oocytes and induced after reinitiation of oocyte maturation, was inhibited even if the oocytes were treated with progesterone. A similar translational regulation was observed in oocytes injected with a reporter mRNA, which was composed of an enhanced green fluorescent protein open reading frame followed by the 3' untranslational region (3'UTR) of XL-INCENP mRNA. These results indicate that Xtr regulates the translation of XL-INCENP mRNA through its 3'UTR during meiotic progression of oocyte.     

A cadherin-like protein in eggs and cleaving embryos of Xenopus laevis is expressed in oocytes in response to progesterone

A new cadherin-like protein (CLP) was identified in oocytes, eggs, and cleavage stage embryos of Xenopus laevis. As a probe for detecting new cadherin proteins, an antiserum was raised to a 17 amino acid peptide derived from a highly conserved region in the cytoplasmic domain of all cadherins which have been sequenced to date. This antipeptide antibody recognized Xenopus E-cadherin and a polypeptide in Xenopus brain extracts similar to N-cadherin, which were independently identified by specific mAbs. In extracts of eggs and midblastula stage embryos the antipeptide antibody recognized specifically a 120-kD glycoprotein that migrated faster on SDS gels than the 140-kD E- and N-cadherin polypeptides. This 120-kD polypeptide was not recognized by the mAbs specific to E- and N-cadherin. In fact, E- and N-cadherin were not detectable in eggs or midblastula stage embryos. The possible relationship of CLP to P-cadherin, which has been identified in mouse tissues, has not yet been determined. CLP was synthesized by large, late stage oocytes. When oocytes were induced to mature in vitro with progesterone it accumulated to the same level found in normally laid eggs. It did not accumulate further to any significant extent during the early cleavage stages. CLP was detected on the surface of stage 8 blastomeres by cell surface biotinylation, but only after the tight junctions of the blastula epithelium were opened by removal of Ca2+. We conclude that CLP is a maternally encoded protein that is the major, if not only, cadherin-related protein present in the earliest stages of Xenopus development, and we propose that it may play a role in the Ca2(+)-dependent adhesion and junction formation between cleavage stage blastomeres.     

Factors affecting oogenesis in the South African clawed frog (Xenopus laevis)

Xenopus laevis, commonly known as the South African Clawed frog, is a hardy adaptable species that is relatively easy to maintain as a laboratory animal. Gametogenesis in wild Xenopus laevis is continuous and under ideal conditions, reproduction can occur year round. This unique aspect of amphibian reproduction offers an advantage over mammalian model systems: the eggs and oocytes collected from laboratory maintained Xenopus laevis provide an abundant and readily obtainable supply of material for cellular and biological research. However, many investigators report that laboratory Xenopus laevis go through periods of unexplained inefficient or complete failure of oocyte production or the production of poor quality oocytes. This results in experimental delays, inability to reproduce data, and ultimately the use of more animals. There is a lack of evidenced based information regarding the housing conditions that are necessary to optimize the health and fecundity of this species in captivity, but studies of wild Xenopus laevis have shown that temperature, age of the female, and nutrition are of key importance. The objective of this report is to review oogenesis with a special emphasis on these factors as they pertain to laboratory Xenopus laevis maintained for the purpose of providing a steady supply of eggs and oocytes. Harvesting methods and other experimental techniques that affect the quality of eggs and oocytes are also discussed.     

Disappearance of a novel protein component of the 26S proteasome during Xenopus oocyte maturation

We have prepared polyclonal antibodies against Xenopus 20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development of Xenopus laevis. A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.     

Aster formation in eggs of Xenopus laevis. Induction by isolated basal bodies

We have assayed various materials for their ability to induce aster formation by microinjection into unfertilized eggs of Xenopus laevis. We have found that purified basal bodies from Chlamydomonas reinhardtii and Tetrahymena pyriformis induce the formation of asters and irregular cleavage furrows within 1 h after injection. Other microtubule structures such as flagella, flagellar axonemes, cilia, and brain microtubules are completely ineffective at inducing asters or cleavage furrows in unfertilized eggs. When known amounts of sonicated Tetrahymena and Chlamydomonas preparations are injected into unfertilized eggs, 50% of the injected eggs show a furrowing response at approximately 3 cell equvalents for Chlamydomonas and 0.1 cell equivalent for Tetrahymena. These results are close to those expected if basal bodies were the effective astral-inducing agent in these cells. Other materials effective at inducing asters in unfertilized eggs, such as crude brain nuclei, sperm, and a particulate fraction from brain known to induce parthenogenesis in eggs of Rana pipiens, probably contain centrioles as the effective agent. Our experiments provide the first functional assay to indicate that centrioles play an active role in aster initiation. None of the injected materials effective in unfertilized eggs produced any observable response in fully grown oocytes. Oocytes and eggs were found to have equal tubulin pools as judged by colchicine-binding activity. Therefore, the inability of oocytes to form asters cannot be due to a lack of an organizing center or to a lack of tubulin. Experiments in which D2O was found to stimulate aster-like fibrous areas in eggs but not oocytes suggest that the inability of oocytes to form asters may be due to an inability of tubulin in oocytes to assemble.     

Calmodulin triggers the resumption of meiosis in amphibian oocytes

The calcium-binding protein, calmodulin, has been purified from Xenopus laevis oocytes. This 18,500-dalton protein, pl 4.3, has two high-affinity calcium-binding sites per mole protein having a dissociation constant of 2.8 x 10(-6) M. Full-grown Xenopus oocytes, arrested in late G2 of the meiotic cell cycle, resumed meiosis when microinjected with 60-80 ng (3-4 pmol) of calmodulin in the form of a calcium-calmodulin complex. The timing of the meiotic events in these recipient oocytes was the same as that normally induced by progesterone. Xenopus ovarian calmodulin stimulated bovine brain phosphodiesterase (PDE) 3- to 10-fold in a calcium-dependent manner, but it had no apparent effect on ovarian PDE activity. A calcium-calmodulin-dependent protein kinase has been isolated from Xenopus oocytes using a calmodulin-Sepharose 4B affinity column. The possible role for this kinase in regulating the G2-M transition in oocytes has been discussed.