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.     

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.     

Comportement des chromosomes surnuméraires et relation avec le taux de mortalité dans une population africaine de Locusta migratoria migratorioides

Transmission of supernumerary chromosomes is studied in adults and embryos of an African population of Locusta migratoria migratorioides; 34% of the animals show one or two B chromosomes (24% one, 10% two). This percentage is the same in both sexes. During mitosis, B chromosomes are very stable. At meiosis, in some cases they show pairing, in other cases they enter as univalents. The eventuality of synapsis or association is discussed. These two kinds of behaviour could not be explained by the presence of two different B chromosomes as is shown from the study of parthenogenetic progeny. Repartition of B chromosomes in the progeny is different depending on whether the male or the female parent supplies them; so the behaviour of these supernumerary should be conditioned, not by their own structure, but by a connection with the cell. — One B chromosome apparently is neutral. When there are two B chromosomes, in case of synapsis, the number of animals with two B in the progeny is slightly lower than previous; in case of asynapsis, there is a higher lethality of individuals and oocytes with two B chromosomes.     

SUPERNUMERARY CHROMOSOMES IN SAXIFRAGA VIRGINIENSIS (SAXIFRAGACEAE)

Acetocarmine squashes of root tips have demonstrated that 2n = 20 and 38 in Saxifraga virginiensis. These contrast with the earlier reported count of 2n = 28 for this species. In several populations supernumerary chromosomes were detected. Both intrapopulational and interpopulational variation in supernumerary chromosome number were detected, with the largest number of supernumerary chromosomes observed being six. Because these supernumerary chromosomes are equal in size to many of the smaller A chromosomes during mitotic metaphase, the presence of supernumerary chromosomes in this species could not be ascertained by analysis of mitotic metaphase preparations alone. During mitotic prophase, however, the supernumerary chromosomes of S. virginiensis are highly heterochromatic, appearing more densely coiled and darkly stained than the A chromosomes. This characteristic facilitated the recognition of supernumerary chromosomes in this species. The similarity in size of A and supernumerary chromosomes during mitotic metaphase and the observation of six supernumerary chromosomes in one population suggest that the count of 2n = 28 reported earlier for S. virginiensis may actually be a misinterpretation of 2n = 20 plus 8 supernumerary chromosomes. Furthermore, these findings and the observation of this same supernumerary chromosome phenomenon in other species of Saxifraga raise the possibility that some of the many disparate chromosome counts attributed to aneuploidy in the large genus Saxifraga may also be the result of misinterpretations of supernumerary chromosomes as A chromosomes.     

THE GENUS COLLINSIA. XVI. SUPERNUMERARY CHROMOSOMES

Dhillon , T. S., and E. D. Garber . (U. Chicago, Chicago, Ill.) The genus Collinsia. XVI. Supernumerary chromosomes. Amer. Jour. Bot. 49 (2) : 168–170. 1962.—The pairing and transmission of supernumerary chromosomes in Collinsia solitaria and in hybrids between C. sparsiflora subsp. arvensis and C. bruceae were studied. Two supernumerary chromosomes usually formed a bivalent with 1 chiasma at metaphase I; 4 supernumerary chromosomes occasionally yielded a trivalent and univalent at this stage. Plants with 2–4 supernumerary chromosomes were fertile and plants with 5–8, sterile. Plants with a given number of supernumerary chromosomes when used as seed or pollen parent gave gametes with a higher number of such chromosomes.     

DEVELOPMENT OF THE NUCLEAR STRUCTURE AND METABOLISM DURING OOGENESIS OF PERINEREIS CULTRIFERA (ANNELIDA,POLYCHAETA)

An ultrastructural study of the nucleus was carried out, during oogenesis of Perinereis cultrifera, accompanied by an autoradiographic and biochemical study of the syntheses of RNA. The nucleus encloses formations deriving from the dispersal of meiotic chromosomes and a voluminous nucleolus. The latter undergoes morphological development of which each stage is characteristic of a stage of oogenesis. The autoradiographic study shows that the synthesis of RNA of extra-nucleolar origin is highly intense in young oocytes (during the stages of previtellogenesis and vitellogenesis) and that it decreases in older oocytes. The synthesis of RNA of nucleolar origin is very weak during previtellogenesis, increases during vitellogenesis, which is the stage at which it reaches its peak, and then decreases during the stages of the development of cortical alveoli and of maturity. These autoradiographic results are confirmed by a biochemical study which shows that once an oocyte diameter of 80 μm is reached (mid-vitellogenesis), the specific radioactivity of 18 and 28 S rRNA and of 4 and 5 S RNA decreases progressively up to the end of oogenesis.     

Struktur und Funktion der Oocytenchromosomen und Nukleolen sowie der Extra-DNS während der Oogenese panoistischer und meroistischer Insekten

In panoistic ovaries (without nurse cells) there are three predominating structures: lampbrush chromosomes, multiple nucleoli, and the hitherto undescribed endobody (Binnenkörper). Nucleoli are always multiple during the growth period of the oocyte of panoistic ovaries. This is true even in the case of Blattella which seems to possess only one big nucleolus, if examined in the light microscope (cf. Figs. 2 and 14b).—In the meroistic type of ovary (with nurse cells) the development of nucleoli and lampbrush chromosomes in the oocyte is very reduced. Only in the early growth stages of the oocyte the chromosomes despiralisize in a speciesspecific degree before they condense to a karyosphere (Pigs. 8, 9). On the other hand the endobody is bigger in the meroistic than in the panoistic ovary (Figs. 5, 8,14). — Lampbrush chromosomes and multiple nucleoli are sites of a very intensive RNA-synthesis (Fig. 1). The nucleoli are built up by granules measuring 125 Å in diameter (Figs. 15, 16). In the endobody, no RNA-metabolism could be demonstrated (Figs, 1a, b, 8c). The endobody is very homogeneous in electron microscope pictures and clearly distinct from the granular nucleoli (Fig. 17). The labelling pattern after incubation with ³H-amino acids suggests a permanent exchange of protein molecules between the karyoplasm and the endobody. — In the meroistic type of ovary the oocyte obtains RNA from the nurse cells, and RNA-synthesis in the oocyte nucleus is decreased in the same measure as its chromosomes are condensed. — The water-beetles Dytiscus and Acilius possess extra-DNA and deviate from the rule of restricted RNA-synthesis in the oocyte nucleus of the meroistic ovary albeit their chromosomes form a karyosphere too (Fig. 11) and RNA streams also from the nurse chamber into the ooplasm (Fig. 10). The extra-DNA resolves itselve into a network of fine fibrils no longer stainable by the Feulgen reaction. True multiple nucleoli develop on the fibrils suggesting the extra-DNA contains a huge mass of nucleolus organizers. The case of Dytiscus is very similar to the development of the multiple nucleoli in Gryllus.     

Studies of oogenesis in the rotifer,Asplanchna

Summary Oocyte development in Asplanchna brightwelli was studied by observation through the transparent body wall of living females and by electron microscopy. During oogenesis, which requires four to six hours, the oocyte increases in volume approximately 1000-fold. Most of its cytoplasm appears to be derived from the vitellarium by direct flow through the cytoplasmic bridge. This flow is rapid enough to be easily observed in the living female at low magnifications. Ribosomes, mitochondria, cortical granules, and lipid droplets were observed in the bridge area in electron micrographs.RNA synthesis during oogenesis was studied by means of autoradiography. Very little synthesis could be demonstrated in oocyte nuclei at any period of oogenesis, whereas the vitellarium nuclei show active incorporation of ³H-uridine throughout the reproductive life of the adult female. Most of this RNA is subsequently transferred to developing oocytes.This research was supported by USPHS Grant GM 121183 to Dr. C. W. Birky, Jr.     

Lampenbürstenchromosomen in den Oocytenkernen von Sepia officinalis

The nuclei of the growing oocytes of Sepia officinalis contain lamp-brush chromosomes with clearly demonstrable chromomeres bearing pairs of lateral loops (Fig. 4). Similar to the findings of Callan and Lloyd (1960) in amphibian oocytes the chromosomes of Sepia exhibit some specially organized loops which can be used as landmarkers for recognizing certain chromosomes (Fig. 5). Maximal loop despiralization occurs only in nuclei of follicles during their initial growth phase. The following developmental period is characterized by a successive contraction of loops and chromosome axes (Fig. 7), despite the fact that the diameter of the oocyte nucleus enlarges still three times. In the oldest nuclei hundreds of nucleolus-like granules arise near the chromosomes which later spread over the entire nucleus (Fig. 9).

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Herrn Professor Dr. K. Bier () in dankbarer Verehrung gewidmet.     

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.     

Die experimentelle Beeinflussung des Puffmusters von Riesenchromosomen

By treating larvae and prepupae of Ch. thummi with 2 mg/ml oxytetracycline (OTC) about 30 puffs not present in normal development are induced in the salivary gland chromosomes. Already existing puffs become enlarged (cf. Fig. 4). A considerable number of induced puffs appeared in heterozygous condition (cf. Fig. 1a-c). The species Ch. strenzkei does not react in any way to the same treatment. Other inhibitors of protein synthesis such as cycloheximide and chloramphenicole do not influence the puffing pattern in both species. — Animals which had been treated with OTC for 2 hrs show the first signs of puffing. Fully developed OTC-induced puffs are detectable 20 hrs after treatment. At this time the Balbiani rings and the nucleolus are mostly regressed. — Both the induced puffing pattern and the number of heterozygous puffs depend on the genetic constitution of the animals. Animals derived from different locations can be shown to possess different specific spectra of induced puffs. The induced puffing pattern of animals bred from single egg masses is reduced, and heterozygous puffs are rare or absent. — OTC-induced puffs show a strong uptake of tritiated uridine (cf. Fig. 2). Heterozygous puffs are labelled only in the puffed half of the band (cf. Fig. 3).     

The supernumerary segment system of Stethophyma

Three species of the genus Stethophyma have been cytologically examined and all three show variation both for supernumerary heterochromatic segments and for the distribution of standard heterochromatin among the autosomes. The European species, S. grossum, for example, shows considerable interpopulation variation for standard heterochromatin while two of the populations, from Spain and Austria, show supernumerary segment polymorphism. The segments are located interstitially on the S₁₁ chromosome but occupy different positions in the different populations. — In all species, the presence of the extra heterochromatic segments increases the mean chiasma frequency. Moreover, the influence of the segments upon mean chiasma frequency is different in different populations and in different species. In the Spanish population, the increase is both intra- and interchromosomal whereas in Austria the influence of the segment is completely interchromosomal. — In the American species, S. gracile and S. lineatum, where supernumerary heterochromatic segments are carried on both S₁₀ and S₁₁ chromosomes, the effect on chiasma frequency shows a dosage relationship, an increase in the number of segments per individual being correlated with an increase in mean chiasma frequency. It is suggested that the interstitial segments found in all species have originated by direct duplication of chromosome material. By contrast the terminal segments in S. lineatum and S. gracile may be derived by translocation from a B-chromosome since such a chromosome has been found in one individual of the former species. — The variation in segment structure and the distribution of standard heterochromatin, among the European species of S. grossum suggests that these systems have evolved independently in different populations.On educational leave from the Forest Research Laboratory, Fredericton, N. B. Canada.     

Electrophoretic analysis of newly synthesized and stored maternal RNA during oogenesis ofCalliphora erythrocephala (Dipt.)

Summary Ovaries ofC. erythrocephala synthesize large amounts of poly(A)⁺ and poly(A)^– RNA during early and middle stages of oogenesis as shown by labelling with³H-uridine in vivo. After incubation for 1 h, a striking difference in the electrophoretic pattern of newly synthesized labelled poly(A)⁺ RNA and the poly(A)⁺ RNA present in sufficient amounts for optical density measurements (steady state poly(A)⁺ RNA) was observed. During early and mid-oogenesis, in the poly(A)^– RNA fraction, 4S predominantly mature rRNA, 5S RNA and tRNA were labelled. These fractions were no longer synthesized during late oogenesis, whereas poly(A)⁺ RNA was labelled continously During oogenesis stage specific differences in the size distribution of newly synthesized and steady state poly(A)⁺ RNA were not obvious. However, different sizes of labelled poly(A)⁺ RNA species were detected in 0–2h old preblastoderm embryos, after injection of³H-uridine into females either 3–4 days (stage 3–4 of oogenesis) or 24 h before oviposition (stage 5–6 of oogenesis). This difference in RNA synthesis was related to the presence of active nurse cell nuclei. The poly(A)⁺ RNA fraction represents about 2–3% of the total RNA in both ovaries and freshly laid eggs as judged by measurements of optical density and radioactivity bound to oligo(dT). The length of poly(A)-segments in ovarian poly(A)⁺ RNA varied from about 30 to 200 nucleotides.     

The structure of the chromosomes in human primordial oocytes

Primordial oocytes (oocytes in primordial follicles) from human ovaries aged 51/2 months post conception to 11 3/4 years post partum were examined in: (a) squash preparations of fresh and fixed tissue; (b) histological preparations; and (c) thin sections by electron microscopy, in order to study the structure of the chromosomes. — The light microscope shows that the chromosome consists of a thread bearing numerous fine lateral appendages. Cytochemical tests indicate that the thread contains DNA, and is surrounded by material containing RNA and protein. — The electron microscope shows that there are three main structural components in the chromosome: (i) an axis or core containing at least two longitudinal strands about 200 Å thick; (ii) a surrounding sheath composed of coiled fibrils which form symmetrically arranged columns and loops, and (iii) clusters of large granules which are associated with the outer parts of the sheath. Small nucleoli and other granular bodies are also present. — These observations indicate the presence of lampbrush chromosomes in the human oocyte. The significance of this type of chromosome in mammals is discussed in relation to the differential radiosensitivity of the oocytes, and to the form of chromosomes at the dictyate stage in rodents.     

Oogenesis in Campodea sp. (Diplura)

Summary The egg chamber of Campodea consists of a group of nurse cells and an oocyte, and is surrounded by a simple, markedly flattened follicular epithelium. Three types of yolk occur in the oocytes: type I appears within elements of the rough endoplasmic reticulum; type II is produced by specific complexes of endoplasmic reticulum and dictyosomes; type III is incorporated by micropinocytosis. Histochemical tests show that mature yolk spheres contain proteins and polysaccharides. The main function of the nurse cells is to synthesize RNA, but they also produce small amounts of type I yolk. Phylogenetic conclusions are drawn from this and other studies of oogenesis in apterygote insects.The author is grateful to Dr. F. Kaczmarski of the Medical Academy, Kraków, for the use of EM facilities in his laboratory     

Cell-free translation of messenger RNA from oocytes of Xenopus laevis

The poly(A)⁺ RNA which accumulates during oogenesis in the amphibian Xenopus laevis is shown to be functional mRNA; the RNA was active in the mRNA-dependent “shift assay” for initiation sites in the rabbit reticulocyte lysate, and was an efficient template for protein synthesis in the wheat-germ cell-free system. Analysis of the in vitro protein products showed no differences between the coding properties of poly(A)⁺ RNA extracted from oocytes at all stages of development from previtellogenesis to maturity. In previtellogenic oocytes, the in vitro products of polysomal and of mRNP-associated poly(A)⁺ RNA were also identical. Neither was there any evidence for changes in the coding properties of the poly(A)⁺ mRNA of the oocyte. However, the patterns of oocyte in vivo protein synthesis changed markedly during early vitellogenesis. We conclude that the mRNP-associated poly(A)⁺ RNA present in mature oocytes constitutes the stored maternal mRNA, and that during oogenesis the coding composition of the poly(A)⁺ mRNA synthesised does not change markedly, while some form of translational control operates to direct the changing pattern of protein synthesis.     

Supernumerary chromosomes in the second outbreeding species of the wheat group

Meiosis was examined in PMC of 138 plants of the outbreeder Triticum speltoides (=Aegilops speltoides) and 175 plants of the closely related T. longissimum (= Ae. longissima), which is a selfer. Nine of the T. speltoides plants, originating fra two of the five Israeli populations under study, contained B chromosomes in addition to the normal complements (2n = 14). No supernumerary chromosomes were found in T. longissimum. — Plants were found carrying 1, 2 or 3 B chromosomes, which seem to be stable among PMC. Their size is 2/3 of the average length of the regular chromosomes, but otherwise they are similar in appearance. Pairing between Bs is common as bivalents and trivalents, but whether this association is ehiasmatic is not clear. There is a slight reduction in chiasma frequency of the regular complement in B-carriers compared to other plants from the same population. — The only other species in the wheat group reported to have B chromosomes in natural populations is also an outbreeder, namely T. tripsacoides (=Ae. mutica). The connection between outbreeding and B chromosomes might be significant.     

The <Emphasis Type="Italic">Drosophila</Emphasis> RNA-binding protein Lark is required for localization of Dmoesin to the oocyte cortex during oogenesis

The RNA-binding protein Lark has an essential maternal role during Drosophila oogenesis. Elimination of maternal expression results in defects in cytoplasmic dumping and actin cytoskeletal organization in nurse cells. The function of this protein is dependent on the activity of one or more N-terminal RNA-binding domains. Here, we report the identification of Dmoesin (Dmoe) as a candidate RNA target of Lark during oogenesis. In addition to actin defects in the nurse cells of lark mutant ovaries, we observed mislocalization of posteriorly localized mRNAs including oskar and germ cell less in the developing oocyte. Anteriorly and dorsally localized mRNAs were not affected. In addition, we observed displacement of the actin cytoskeleton from the oocyte plasma membrane. These phenotypes are reminiscent of mutations in Dmoe and suggested that this RNA maybe a potential target of Lark. We observed a significant decrease in Dmoe protein associated with the membrane of the developing oocyte with no changes in expression or localization within the nurse cells. Evidence for an association between Lark protein and moe RNA during oogenesis comes from results of a microarray-based Ribonomics approach to identify Lark RNA targets. Thus, our results provide evidence that Dmoe RNA is a target of Lark during oogenesis and that it likely regulates either the splicing or translation of this RNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.