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.     

Particulate DNA polymerase from the cytoplasm of Xenopus laevis oocytes

A DNA polymerase has been partially purified and characterized from Xenopus laevis stage 6 oocytes. The enzyme is present only in the cytoplasm and has been shown to be able to copy Poly(A) x oligo(dT), to be sensitive to N-ethylmaleimide, and to sediment faster than 4 S in high salt glycerol gradient. The enzyme can be extracted from particulate material which has a density in sucrose gradient ranging from 1.200 to 1.225 g/cc. This particulate material is identified by its ability to use Poly(A) x oligo(dT) as template in an exogenous DNA polymerase reaction and by its endogenous DNA synthesizing capacity.     

The number of mitochondria in Xenopus laevis ovulated oocytes

An attempt was made to estimate the total number of mitochondria in Xenopus laevis ovulated oocytes. For this purpose the necessary basic parameters were calculated employing planimetry and simple mathematical formulas. It was found that the number of mitochondria in the ovulated oocyte of Xenopus is of the order of 10(7). The significance of this finding is discussed.     

Characterization of DNA-protein complexes from the mitochondria of Xenopus laevis oocytes

Mitochondrial DNA (mtDNA)-protein complexes (nucleoids) from Xenopus laevis oocytes were purified either on rate-zonal sucrose or isopyknic metrizamide gradients. From electron microscopic studies and staphylococcal nuclease digestion experiments mtDNA appears to be packaged into regular beaded structures. Protein electrophoretic analysis and M banding results show that mtDNA is associated with the membrane structures and also with few specific proteins including one acid-soluble polypeptide of 28 kD.     

Heterogeneous distribution and replication activity of mitochondria in Xenopus laevis oocytes

In early diplotene oocytes of Xenopus laevis mitochondria are not dispersed all over the cytoplasm but gathered in a well described mitochondrial mass [18]. When tracing these organelles during active vitellogenesis we observe that some of them are involved in the elaboration of a cortical layer at the vegetative hemisphere of the cell while others stay around the nucleus. The latter contribute to the transient formation of a mitochondrial crown throughout active mitochondriogenesis. Autoradiographic studies of thymidine incorporation into mtDNA suggest a differential participation of each organelle to the final population of a full-grown oocyte according to its position in the cytoplasm.     

Microinjection of cytoplasm or mitochondria derived from somatic cells affects parthenogenetic development of murine oocytes

Cloned mammals are readily obtained by nuclear transfer using cultured somatic cells; however, the rate of generating live offspring from the reconstructed embryos remains low. In nuclear transfer procedures, varying quantities of donor cell mitochondria are transferred with nuclei into recipient oocytes, and mitochondrial heteroplasmy has been observed. A mouse model was used to examine whether transferred mitochondria affect the development of the reconstructed oocytes. Cytoplasm or purified mitochondria from somatic cells derived from the external ear, skeletal muscle, and testis of Mus spretus mice or cumulus cells of Mus musculus domesticus mice were transferred into M. m. domesticus (B6SJLF1 and B6D2F1) oocytes to observe parthenogenetic development through the morula stage. All B6D2F1 oocytes injected with somatic cytoplasm or mitochondria showed delayed development when compared to oocytes injected with buffer. The developmental rates were not different among injected cell sources, with the exception of testis-derived donor cells injected into B6SJLF1 oocytes (P < 0.01). The developmental rate of B6D2F1 oocytes injected with buffer alone (98.8% survival) was different from those injected with somatic cytoplasm (60.8% survival) or somatic mitochondria (56.5% survival) (P < 0.01). Conversely, injection of ooplasm into B6D2F1 oocytes did not affect parthenogenetic development (100% survival). Our results indicate that injection of somatic cytoplasm or mitochondria affected parthenogenetic development of murine oocytes. These results have further implications for in vitro fertilization protocols employing ooplasmic transfer where primary oocyte failure is not confirmed.     

Expression of the brown fat mitochondria uncoupling protein in Xenopus oocytes and important into mitochondrial membrane

Non shivering thermogenesis of brown adipose tissue is due to the uncoupling protein (UCP), located in the inner mitochondrial membrane, which functions as a proton translocator and can thus uncouple mitochondrial respiration. We describe here the expression of UCP in Xenopus laevis oocytes after injection of UCP mRNA, which was transcribed in vitro. UCP seems to be correctly transported into mitochondria and integrated into the membrane, but we were not able to establish definitely the functionality of this UCP. We conclude that this expression system could be suitable for the study of the mitochondrial import mechanism but not for the examination of physiological properties of UCP.     

Actin in Xenopus oocytes

It has been found that a high-speed supernatant fraction from Xenopus oocytes extracted in the cold will form a clear, solid gel upon warming. Gel formation occurs within 60 min at 18 degrees-40 degrees C, and is, at least initially, temperature reversible. Gelation is strictly dependent upon the addition of sucrose to the extraction medium. When isolated in the presence of ATP, the gel consists principally of a 43,000-dalton protein which co-migrates with Xenopus skeletal muscle actin on SDS-polyacrylamide gels, and a prominent high molecular weight component of approx. 250,000 daltons. At least two minor components of intermediate molecular weight are also found associated with the gel in variable quantities. Actin has been identified as the major consituent of the gel by ultrastructural and immunological techniques, and comprises roughly 47% of protein in the complex. With time, the gel spontaneously contracts to form a small dense aggregate. Contraction requires ATP. In the absence of exogenous ATP, a polypeptide which co-migrates with the heavy chain of Xenopus skeletal muscle myosin becomes a prominent component of the gel. This polypeptide is virtually absent from gels which have contracted in ATP-containing extracts. It has also been found that Ca++ is required for gelation in oocyte extracts. At both low and high concentrations of Ca++ (defined as a ratio of Ca++/EGTA in the extraction medium), gelation is inhibited.     

Structural and functional relationships between Ca2+ puffs and mitochondria in Xenopus oocytes

Ca²⁺ uptakeand release from endoplasmic reticulum (ER) and mitochondrialCa²⁺ stores play important physiological and pathologicalroles, and these processes are shaped by interactions that depend onthe structural intimacy between these organelles. Here we investigate the morphological and functional relationships between mitochondria, ER, and the sites of intracellular Ca²⁺ release inXenopus laevis oocytes by combining confocal imaging oflocal Ca²⁺ release events ("Ca²⁺ puffs")with mitochondrial localization visualized using vital dyes andsubcellularly targeted fluorescent proteins. Mitochondria and ER arelocalized in cortical bands ~6-8 µm wide, with the mitochondria arranged as densely packed "islands" interconnected bydiscrete strands. The ER is concentrated more superficially thanmitochondria, and the mean separation between Ca²⁺ puffsites and mitochondria is ~2.3 µm. However, a subpopulation ofCa²⁺ puff sites is intimately associated with mitochondria(~28% within <600 nm), a greater number than expected ifCa²⁺ puff sites were randomly distributed. Ca²⁺release sites close to mitochondria exhibit lower Ca²⁺ puffactivity than Ca²⁺ puff sites in regions with lowermitochondrial density. Furthermore, Ca²⁺ puff sites inclose association with mitochondria rarely serve as the sites forCa²⁺ wave initiation. We conclude that mitochondria playimportant roles in regulating local ER excitability, Ca²⁺wave initiation, and, thereby, spatial patterning of globalCa²⁺ signals.

     

The cytoplasm of Xenopus oocytes contains a factor that protects double-stranded RNA from adenosine-to-inosine modification.

Here we describe studies of double-stranded RNA (dsRNA) adenosine deaminase in Xenopus laevis, in particular during meiotic maturation, the period during which a stage VI oocyte matures to an egg. We show that dsRNA adenosine deaminase is in the nuclei of stage VI oocytes. Most importantly, we demonstrate that the cytoplasm of stage VI oocytes contains a factor that protects microinjected dsRNA from deamination when dsRNA adenosine deaminase is released from the nucleus during meiotic maturation. Our data suggest that the protection factor is a cytoplasmic dsRNA-binding protein or proteins that bind to dsRNA in a sequence-independent manner to occlude dsRNA from binding to dsRNA adenosine deaminase. The cytoplasmic double-stranded RNA-binding protein(s) does not bind to other nucleic acids and can be titrated at high concentrations of dsRNA. These studies raise the question of whether all dsRNA-binding proteins share endogenous substrates and also suggest potential means of regulating dsRNA adenosine deaminase in vivo.     

A synthetic histone pre-mRNA-U7 small nuclear RNA chimera undergoing cis cleavage in the cytoplasm of Xenopus oocytes.

The 3' processing of histone pre-mRNAs is a nuclear event in which the U7 small nuclear ribonucleoprotein (snRNP) participates as an essential trans-acting factor. We have constructed a chimeric histone-U7 RNA that when injected into the cytoplasm of Xenopus laevis oocytes assembles into a snRNP-like particle and becomes cleaved at the correct site(s). RNP assembly is a prerequisite for cleavage, but, since neither the RNA nor the RNP appreciably enter the nucleus, cleavage occurs mostly, if not exclusively, in the cytoplasm. Consistent with this, cleavage also occurs in enucleated oocytes or in oocytes which have been depleted of U7 snRNPs. Thus all necessary components for cleavage must be present in the oocyte cytoplasm. The novel cleavage occurs in cis, involving only a single molecule of chimeric RNA with its associated proteins. This reaction is equally dependent upon base pairing interactions between histone spacer sequences and the 5'-end of the U7 moiety as the natural in trans reaction. These results imply that U7 is the only snRNP required for histone RNA processing. Moreover, the chimeric RNA is expected to be useful for further studies of the cleavage and assembly mechanisms of U7 snRNP.     

Interchromatin granule clusters in vitellogenic oocytes of the flesh fly, Sarcophaga sp

Insect oocyte nuclei contain different extrachromosomal nuclear bodies including Cajal bodies and interchromatin granule clusters (IGCs). In the present study, we describe IGC equivalents in the vitellogenic oocytes of the flesh fly, Sarcophaga sp. These structures were found to consist of 20-40-nm granules and also include the fibrillar areas of high and low electron density. Immunogold labeling electron microscopy revealed IGC marker protein SC35, Sm proteins, and trimethylguanosine cap of small nuclear (sn) RNAs in these bodies. Antibody against the non-phosphorylated RNA polymerase II selectively labeled the fibrillar areas of low electron density located inside the IGCs.     

Gene expression in Xenopus oocytes

1. Gene expression in Xenopus oocytes is now an integral part of many molecular cloning strategies. 2. For some genes, such as those encoding the ion channels, this system has emerged as the only available means to authenticate and examine the biological activities of the cloned DNA. 3. This review discusses some of the current applications of Xenopus oocytes in modern molecular biology.     

Segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in Xenopus oocytes: Identification of an ovalbumin signal sequence

The intramolecular signals for chicken ovalbumin secretion were examined by producing mutant proteins in Xenopus oocytes. An ovalbumin complementary DNA clone was manipulated in vitro, and constructs containing altered protein-coding sequences and either the simian virus 40 (SV40) early promoter or Herpes simplex thymidine kinase promoter, were microinjected into Xenopus laevis oocytes. The removal of the eight extreme N-terminal amino acids of ovalbumin had no effect on the segregation of ovalbumin with oocyte membranes nor on its secretion. A protein lacking amino acids 2 to 21 was sequestered in the endoplasmic reticulum but remained strongly associated with the oocyte membranes rather than being secreted. Removal of amino acids 231 to 279, a region previously reported to have membrane-insertion function, resulted in a protein that also entered the endoplasmic reticulum but was not secreted. Hybrid proteins containing at their N terminus amino acids 9 to 41 or 22 to 41 of ovalbumin fused to the complete chimpanzee α-globin polypeptide were also sequestered by oocyte membranes. We conclude that the ovalbumin “signal” seque?ce is internally located within amino acids 22 to 41, and we speculate that amino acids 9 to 21 could be important for the completion of ovalbumin translocation through membranes.     

In vivo uptake of mitochondrial malate dehydrogenase from watermelon by mitochondria of Xenopus laevis oocytes

The heterologous in vivo translation system of Xenopus laevis oocytes was used to translate messenger RNA isolated from water-melon cotyledons. Immunocytochemistry was used to localize the translation products in situ within the oocyte. In addition, the translation products were immunoprecipitated from homogenized oocytes, separated on SDS-polyacrylamide electrophoresis and visualized by fluorography. A variety of watermelon proteins encoded in the injected mRNA were translated within the oocytes. Among them was the mitochondrial isoenzyme of malate dehydrogenase (mtMDH). The mtMDH was correctly imported into the mitochondria of the oocytes, as detected by immunocytochemistry.