Morphological observations on cultured lampbrush-stage Rana pipiens oocytes.

Early lampbrush-stage oocytes are characterized by small lampbrush chromosome loops, a small amount of ribonucleoprotein (RNP) matrix on the loops, small nucleoli, few RNP particles in the nucleoplasm, and a smooth germinal vesicle contour. In vitro culture of these oocytes in serum-free culture medium for 24 hr at 18°C promotes a number of morphological changes in the oocytes: The lampbrush loops increase in diameter and acquire extensive RNP matrix, the nucleoli increase in size and complexity, the nucleoplasm accumulates numerous polymorphic RNP particles, and the germinal vesicle envelope acquires a sacculated contour. These characteristics are typical of the in vivo maximum lampbrush stage, and their appearance is due to an apparent in vitro acceleration of the lampbrush phase. Two possible interpretations of these observations are discussed.     

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.     

Nuclear structures in the telotrophic ovarioles of nocturnal ground beetles (Tenebrionidae, Polyphaga). II. The oocyte nucleus of Blaps lethifera and Gnaptor spinimanus. Light optical data]

Changes in the nuclear structures and their participation in RNA synthesis in the growing oocytes were followed in two species of beetles Blaps lethifera and Gnaptor spinimanus. In the oocytes of both the species, the chromosomes join into the karyosphere following the short-term lampbrush stage. A large capsule appears around the karayosphere which consists of the fibrous substance, granules and karyosphere nucleoli. The latter form in the karyosphere and contain RNP but they are not true nucleoli since they do not include 3H-uridine. RNA synthesis on the chromosomes, active at the lampbrush stage, falls markedly following their joining into the karyosphere. The oocyte nuclei of these beetles are, thus, characterized by the absence of RNA synthesizing nucleolar system and, as compared with the trophocytes, by the low level of RNA synthesis on the chromosomes.     

Quantitative and qualitative analysis of RNA synthesis in stage 6 and stage 4 oocytes of Xenopus laevis

RNA synthesis has been studied in oocytes taken from Xenopus laevis females which have not recently ovulated. Such females contain a population of large (stage 6) oocytes which exhibit white equatorial bands and which are considered to represent the terminal stage of oocyte development. Rates of RNA synthesis in these “banded” oocytes were measured by analyzing the kinetics of incorporation of ³H-guanosine into acid-precipitable, alkaline-labile material, and changes in precursor pool (GTP) specific activity during incubations. In additional experiments, rates of RNA synthesis were measured after ³H-GTP was injected directly into stage 6 oocytes. For comparison, rates of RNA synthesis were measured in lampbrush chromosome stage oocytes (stage 4; 0.5–0.6 mm diameter). The results show that, under the in vitro conditions employed, stage 6 oocytes are not metabolically dormant, but synthesize total RNA at a rate at least as great as the stage 4 oocytes.Qualitative studies on newly synthesized RNA in the two oocyte classes have been performed using sucrose density gradient centrifugation and acrylamide gel electrophoresis. Both stage 4 and stage 6 oocytes exhibited similar patterns, and the bulk of the RNA synthesized and accumulated during 12-hr pulses appears to be ribosomal. These observations are discussed in terms of existing concepts concerning synthetic activity in stage 6 oocytes.     

An autoradiographic and cytophotometric study of oogenesis in a pulmonate snail,Planorbarius corneus

Summary The spatial and temporal patterns of macromolecular syntheses in oocytes and somatic auxiliary cells of the snail Planorbarius corneus have been investigated by autoradiography and cytophotometry. Oogenesis has been divided into three stages, comprising early meiosis up to diplotene (stage I), previtellogenetic growth phase (stage II), and vitellogenesis (stage III). No DNA synthesis was found in any oocyte stage. In stage-I oocytes, only nucleoli were found labelled with ³H-uridine. Oocyte nuclei of stage II and III actively synthesize RNA in nucleoli and chromosomes. The most intense incorporation of uridine in chromatin probably occurs during the previtellogenesis — vitellogenesis transition period during which cytological findings suggest well developed lampbrush chromosomes. RNA synthesis in amphinucleoli of stage-III oocytes is restricted to basophilic nucleolar parts, whereas acidophilic parts (protein bodies) neither synthesize nor store RNA. During vitellogenesis oocytes incorporate amino acids into yolk platelet proteins. Radioactive proteins are found in yolk platelet precursors 5 h after injection of the tracer and in yolk platelets 3 h thereafter. The labelling pattern suggests that oocytes synthesize certain hitherto unidentified yolk components. No evidence for the participation of follicle cells in synthesis and transport of vitellogenic proteins has been obtained from autoradiography. Cytological findings suggest an important role for these cells in oogenesis. They are highly active in RNA and protein synthesis. Cellular differentiation is accompanied by polyploidization of the nuclei which attain a highest DNA content of 256 c. Polyploidization probably occurs in incremental steps as indicated by complete endomitotic chromosomal cycles. Autoradiographs show that, during vitellogenesis, oocytes do not incorporate significant amounts of glucose, and only certain follicle cells were labelled with glucose, probably indicating the synthesis of glycogen.     

Funktionsstrukturen im Oocytenkern von Locusta migratoria

Examination of living oocyte nuclei of Locusta migratoria has revealed the presence of thread-like struktures. They are paired and are thought to be the uncoiled chromosomes since they are broken into fragments by treatment with DNase. The greater part of the threads carries lateral loops like the lampbrush chromosomes of Amphibia (Fig. 14). A smaller part has no loops hut bears a series of conspicious granules with bright appearance under positive phase contrast optics (pearl-string segments) (Fig. 2). — The visibility of the chromosomes has been investigated in solutions with several ions. In hypertonic media the chromosomes contract, the granules fuse, and the pearl-string segments become lumpy (Fig. 21). In nitrogenous atmosphere and if kept at low temperature the pearl-string structures are likewise transformed into a few lumps (Fig. 19). After return to normal conditions they reconstitute their characteristic beaded appearance. — In autoradiographs obtained by injection of H³-uridine into the body cavity and by incubation of isolated nuclei in vitro, a rather uniformly distributed labelling occurs over the oocyte nuclei up to 30 min incubation time (Fig. 23). With prolonged incubation the activity of the pearl-string segments becomes more intense than the labelling of the lampbrush chromosomes (Fig. 24). After treatment with actinomycin RNA synthesis is stopped, the pearl-string axes and the lampbrush chromosomes contract, and the granules disappear more and more (Figs. 25-28). — The pearlstring segments look very much like the nucleoli in the oocytes of Amphibia, where the nucleolar substance is likewise distributed as a series of beads of rather uniform size on an axis. Therefore, the pearl-string structures may have nucleolar function in Locusta too. If so, the only difference to the Amphibian nucleoli would be the continued attachment to the lampbrush chromosomes.     

The location of 5S RNA genes in lampbrush polytene chromosomes from Drosophila

Drosophila polytene chromosomes were transformed into lampbrush-like structures by exposure to solutions of alkali-urea. In this process, the chromosomes shorten and widen, and the bands (chromomeres) extend laterally into loops leaving a central core between the paired homologues. The expanded polytene chromosomes are very similar in appearance to the true lampbrush chromosomes of amphibian oocytes and to ordinary chromosomes in pachytene. The denaturing effects of alkali-urea were partially counteracted by return of the treated chromosomes to Ringer solution. These observations are interpreted in terms of recent findings on protein backbones in chromosomes, and indicate that chromosomes generally may have very similar basic organization, despite differences due to species, polyteny and degree of condensation. To gain more information on the specific location of a structural gene, ¹²⁵I-labelled low molecular weight (containing 5S RNA) was hybridized in situ to normal and lampbrush-like polytene chromosomes. Autoradiography showed silver grain distribution for 5S RNA consistent with hybridization primarily to the loop regions of the lampbrush chromosomes rather than the core. This provides further indirect evidence that structural genes like 5S RNA may be located on the bands (chromomeres) and not the interbands of normal polytene chromosomes.     

RNA transcription and translation in sea urchin oocytes and eggs

The steady-state concentrations and absolute rates of synthesis of ribosomal RNA (rRNA) molecules were measured in oocytes, eggs, embryos, and larvae of the Hawaiian sea urchin Tripneustes gratilla. The steady-state concentration per genome of the RNA precursor sequences measured by hybridization to a cloned rDNA fragment was approximately 100- to 300-fold greater in the RNA obtained from oocytes and eggs than in the RNA extracted from embryos and larvae. Since the rate of processing of the rRNA precursor at different stages is not greatly different, the rates of rRNA synthesis must be considerably greater in oocytes than in embryo cells. The absolute rate of RNA synthesis in oocytes and embryos was determined from the incorporation of [³H]guanosine into cellular GTP pools and into both precursor and mature rRNA species. The data indicate an approximately 40-fold higher rate of rRNA synthesis in oocytes than that measured in embryos or previously in larvae (J. Griffith and T. Humphreys, 1979, Biochemistry18, 2178–2185). Together these results indicate that the ribosomal genes are transcribed much more rapidly during sea urchin oogenesis than during embryogenesis or larval stages.     

Identification of the lampbrush chromosome loops which transcribe 5S ribosomal RNA in Notophthalmus (Triturus) viridescens

The loops which transcribe 5S ribosomal RNA in lampbrush chromosomes of the newt, Notophthalmus (Triturus) viridescens, were identified by hybridizing purified 5S DNA to nascent 5S RNA in situ. The genes which code for 5S RNA were found near the centromeres of chromosomes 1, 2, 6, and 7 by hybridizing iodinated 5S RNA to denatured lampbrush and mitotic chromosomes in situ. These genes and their intervening spacer DNA were isolated from Xenopus laevis using sequential silver-cesium sulfate equilibrium centrifugations. This purified 5S DNA was iodinated and hybridized to non-denatured lampbrush chromosomes in situ, where it bound to nascent 5S RNA on loops at the base of the centromeres of chromosomes 1, 2, 6, and 7. The number of 5S genes present in the haploid chromosome complement of N. viridescens was determined. — The 5S loops were chosen for study, since (1) the synthesis of 5S RNA has been demonstrated during the lampbrush stage, (2) both 5S RNA and 5S DNA could be isolated in pure form, and (3) the localization of the repetitive 5S genes could be verified by conventional in situ hybridization procedures. These methods may be applicable to the identification of other loops, leading to a better understanding of lampbrush chromosome function.