Stockpiling of DNA polymerases during oogenesis and embryogenesis in the frog, Xenopus laevis

The amounts of the various forms of DNA polymerase (alpha 1, alpha 2, beta, and gamma) have been determined in oocytes, eggs, and embryos of the frog, Xenopus laevis. During oogenesis the relative proportions and absolute levels of all forms changed dramatically. In stage I (early) oocytes, DNA polymerase-gamma, the "mitochondrial" polymerase, was the predominant form. During oocyte growth, DNA polymerase-alpha 1 and -alpha 2 increased by more than 100-fold, DNA polymerase-beta by 15-fold, and DNA polymerase-gamma by only 8-fold. During oocyte maturation and ovulation, the levels of all forms of DNA polymerase roughly doubled. The mature stage VI oocyte contained 5 orders of magnitude more DNA polymerase activity than is found in an individual somatic cell. DNA polymerase-alpha 1 and -alpha 2, the "replicative" polymerases, were the predominant forms in mature oocytes and ovulated unfertilized eggs. During fertilization, the relative proportions and absolute levels of the four forms remained constant. During subsequent stages of embryogenesis, the total amounts of DNA polymerase-alpha 1 and -alpha 2 declined slightly from cleavage through gastrulation, the stages of most rapid chromosomal DNA replication. The rapid increase in cell number during early embryogenesis establishes the same levels of DNA polymerase/cell as are present in adult somatic cells. After neurulation, the absolute levels of DNA polymerase-alpha 1 and -alpha 2 increased in proportion to increases in cell number. The absolute levels of DNA polymerase-beta remained constant, and the levels of DNA polymerase-gamma increased 2-fold throughout embryogenesis.     

Developmentally regulated RNA binding proteins during oogenesis in Xenopus laevis

During oogenesis, Xenopus oocytes synthesize and accumulate all types of RNA. In particular, they store poly(A+) RNA to such an extent that only about 5% is actually translated in the oocyte. Using a protein blotting and in vitro binding assay, we have identified proteins which are associated with poly(A+) RNA and perhaps other RNAs as well. Two groups of binding proteins were identified. The first group accumulates during oogenesis, generally is less than 50,000 molecular weight, and sediments in the 80 S and polysome regions of a gradient. These proteins most likely include ribosomal proteins. A second group of proteins is oocyte-specific, sediments less than 80 S as well 80 S and slightly heavier, generally has molecular weights greater than 50,000, and diminishes in amount as oogenesis progresses. In addition, these proteins are retained by oligo(dT)-cellulose when ribonucleoproteins are analyzed by chromatography and, when challenged with several different types of RNA in vitro, bind poly(A+) RNA preferentially. The possibility that some of these proteins might regulate the stability or translatability of mRNAs during oogenesis is discussed.     

A restriction map of Xenopus laevis mitochondrial DNA

The mitochondrial DNA from Xenopus laevis is a 17.4 x 10(3)-base-pair circular DNA molecule. The mapping of this DNA, using 19 different restriction endonucleases is reported here. The sites are as follows: 1 for BamHI, PstI, SacI, SalI, BalI; 2 for BglII, SacII, EcoRI, ClaI, 3 for XhoI, 4 for AvaI, XbaI, PvuII, 5 for HindIII, 6 for HhaI, BclI, HpaI, 10 for AvaII and 11 for HincII. The same sites (except for one of the two ClaI sites) are observed in the molecule cloned in pBR322 DNA. The fragments corresponding to 62 cleavage sites have all been ordered and precisely located. They provide suitable conditions for further investigations connected with the study of replication and nucleotide sequence determination of this molecule.     

The development of competence for meiotic maturation during oogenesis in Xenopus laevis

Meiotic maturation of large, 1.2-1.4 mm in diameter, stage VI oocytes of Xenopus laevis can be induced to mature in vitro by exposure to progesterone or by microinjection of maturation-promoting factor (MPF). Small, 0.95 mm in diameter, stage IV oocytes do not respond to progesterone but do undergo germinal vesicle breakdown (GVBD) in response to microinjection of MPF. The possibility that small oocytes are nonresponsive to progesterone due to a specific defect in an event known to occur with large oocytes is investigated. Both large and small oocytes possess a plasma membrane steroid receptor (Mr = 110,000) as measured by photoaffinity labeling with [3H]R5020, but the density of receptors in small oocytes is only 20% of that in large oocytes. Adenylate cyclase activity stimulated by guanyl-5'-yl-imidodiphosphate is equally inhibited by steroid (50%) in plasma membranes from both large and small oocytes with an apparent IC50 of 2 X 10(-7) M progesterone. Microinjection of the heat-stable inhibitor protein of cAMP-dependent protein kinase induces GVBD in large but not in small oocytes. These results indicate that the nonresponsiveness of small, stage IV oocytes to progesterone is due to a deficiency in an event(s) subsequent to cAMP fluctuations but prior to MPF action.     

Expression of DNA ligases I and II during oogenesis and early development of Xenopus laevis.

We have analyzed the expression of DNA ligase I protein during oogenesis and early development of Xenopus laevis. The protein is already present in stage I oocytes and then accumulates throughout oogenesis to reach a steady state level by stage VI. It remains at this level at least until tadpole stage. In stage VI oocytes DNA ligase I protein is almost exclusively localized in the germinal vesicle. We have partially purified a DNA ligase II activity from stage VI oocytes, unfertilized eggs, and stage 8 embryos. An 80-kDa polypeptide can be specifically adenylated in all three purified extracts. It is not recognized by antibodies directed against DNA ligase I and is active on oligo(dT)-poly(rA) substrate. It could therefore represent DNA ligase II protein. The presence of both DNA ligases I and II in oocytes and embryos is inconsistent with the DNA ligase model that had been previously proposed for amphibia.     

Poly(A)+ RNA metabolism during oogenesis in Xenopus laevis.

In this study, we have measured the synthesis and turnover of oligo(dT)cellulose-bound RNA [poly(A)⁺ RNA] in Xenopus laevis oocytes at the maximal lampbrush chromosome stage (stage 3) and at the completion of oocyte growth (stage 6). Oocytes at both stages are shown to be active in the synthesis of poly(A)⁺ RNA. In stage 6 oocytes, the mean rate of synthesis of stable poly(A)⁺ RNA is 15% the instantaneous rate of synthesis, while the mean half-life of the unstable component is 1.6 hr. In contrast, the instantaneous rate of synthesis in stage 3 oocytes is about one-third that seen in stage 6, and most of it is devoted to the production of unstable species with an average half-life of 5 hr. Studies on the nuclear versus the cytoplasmic distribution of the newly synthesized poly(A)⁺ RNA demonstrated that by the end of a 12-hr labeling period for stage 3 oocytes and a 24-hr labeling period for stage 6 oocytes, approximately half of the material was cytoplasmic. This cytoplasmic material had the same electrophoretic mobility as bulk poly(A)⁺ RNA. Similarly, as with bulk poly(A)⁺ RNA, little, if any, of the newly synthesized material was found to be polysomal. Also, poly(A) labeling studies indicated that the newly synthesized poly(A)⁺ RNA was associated with the synthesis of poly(A) of the same length as that appearing on bulk poly(A)⁺ RNA. Studies on the content of bulk oligo(dT)cellulose-bound RNA indicated that about 86 ng is present in both stage 3 and stage 6 oocytes. The continual synthesis of poly(A)⁺ RNA throughout oogenesis in the absence of its accumulation led to the conclusion that it must be turning over. These data are discussed in relation to the hypothesis that bulk levels of poly(A)⁺ RNA are maintained by continually changing rates of synthesis and degradation.     

The synthesis and storage of histones during the oogenesis of Xenopus laevis

Further data, including two-dimensional gel electrophoresis and peptide mapping of newly synthesized proteins, confirms the view that oocytes make several types of histone. The newly synthesized histone is present in both nucleus and cytoplasm, but at a higher concentration in the oocyte nucleus and in great excess over the DNA binding sites. The unfertilized egg seems to contain a pool of histones detectable on two-dimensional electrophoretograms. The peptide maps of these proteins are consistent with their identification as histones. The egg contains enough histone to support nuclear replication through most of cleavage.     

Circular mitochondrial DNA from Xenopus laevis and Rana pipiens

Mitochondrial DNA from oocytes of Xenopus laevis and Rana pipiens and from the liver of Gallus domesticus was studied by electron microscopy using the Kleinschmidt technique. A high percentage of circular molecules, either highly twisted or open, was observed in all preparations. The mean contour length of circles from X. laevis was 5.40 , from R. pipiens 5.56 and from G. domesticus 5.26 . Highly twisted circles were found in greater abundance in a fresh preparation than in preparations left standing for 3 months. These molecules are considered to be the native form of mitochondrial DNA.     

Localized protein synthesis during oogenesis of Xenopus laevis: analysis by in situ translation

An in situ translation assay was developed to examine localized protein synthesis activity on the subcellular level of fixed and sectioned material by autoradiography. Our data indicate that in situ translation produced polypeptides also synthesized in vivo and is dependent on protein synthesis from messenger RNA preserved in the fixed material. Applied to oocytes of Xenopus laevis at different stages, the in situ translation assay revealed localized protein synthesis activity in the cortical region of vitellogenic and postvitellogenic oocytes. The spatial pattern of protein synthesis activity observed in stage 6 oocytes disappeared when oocytes were induced to mature.     

Contractile proteins and nonerythroid spectrin in oogenesis of Xenopus laevis

The distribution of contractile proteins, actin and myosin, and an actin-binding protein, spectrin, was studied in oogenesis of Xenopus laevis. These proteins are present in oocytes already at the previtellogenic stages, which are characterized by their diffuse distribution. The localization of proteins changed with the beginning of vitellogenesis. At all vitellogenic stages, including the fully grown oocyte, animal–vegetal differences were noted in localization of actin and myosin: in the animal hemisphere they appear as fibrillar-like structures, while in the vegetal one they are localized around the yolk platelets. By the end of the oocyte's growth, a cortical gradient appeared: predominant localization of actin and myosin in the cortical area. As the oocyte maturation proceeded, the distribution of actin and myosin again became diffuse and nonuniform, so that a cortical gradient appears. At the beginning of vitellogenesis spectrin is distributed as a network all over the ooplasm, while in the fully grown oocyte it is localized mostly in teh subcortical area of the animal hemisphere and, as individual inclusions, in other regions of the oocyte. No spectrin is found by the end of maturation. Actin, myosin, and spectrin are also present in the oocyte's nuclei. Changes in the distribution of contractile proteins and spectrin during oocyte maturation are discussed with respect to the development of cortical contractility, as well as to the changes in spatial distribution of yolk platelets and regional sensitivity of the maturing oocyte to cytochalasin B. © 1994 Wiley-Liss, Inc.