RNA synthesis in pig follicular oocytes. Autoradiographic and cytochemical study

RNA synthesis in pig oocytes was studied using autoradiography and silver staining of the nucleolus organizing region. Both methods confirmed that oocytes from the smallest follicles (0.5-0.7 mm in diam.) very intensely synthesize nuclear and nucleolar RNA. The nucleolar area of oocytes originating from follicles of 1.6-2.2 mm in diam. was labelled mainly on its periphery. After short pulse labelling (15 min) of oocytes from follicles of 5-6 mm in diam. only the nucleoplasm was labelled. The nucleolus had no significant labelling. The possibility that labelling of the compact nucleolus after a longer pulse represents migration of the newly synthesized nuclear RNA into the compact nucleolus, is discussed. The quantity of silver-positive material in dictyate oocytes significantly decreased as pig follicles enlarged in diam. from 2 mm to 5-6 mm.     

RNA polymerase activity in germinating onion seeds

Frozen sections of endosperm cut from dry unimbibed onion seed were immersed in an aqueous solution of tritium labelled triphosphate; nucleolar RNA polymerase (ribonucleoside triphosphate: RNA nucleotidyltransferase E.C. 2.7.7.6) activity was detected by autoradiography after soaking for 10–15 min in the solution of the radioactive nucleotide. Throughout germination, activity appears to be mainly confined to the nucleolus with chromatin incorporation being very low or non-existent. In the embryo, in contrast to the endosperm, chromatin activity is initiated after 1 hr presoaking, while the nucleolus displays a lag of several hours. No incorporation could be detected in vivo before 18 hr.     

SUBCELLULAR LOCALIZATION OF NEWLY-FORMED [3H]ACETYLCHOLINE IN RAT CEREBRAL CORTEX IN VITRO

—Slices from rat brain cortex were incubated for either 5 or 60 min in a medium containing [³H]choline and 4·7 or 25 mm -KCl. Bioassayable ACh and labelled ACh were determined in the incubation medium, in the total tissue homogenate and in subcellular fractions. Raising the KCl concentration from 4·7 to 25 mm stimulated the release and synthesis of total and of labelled ACh. In medium containing 25 mm -KCl the amounts of ACh decreased in the tissue and in the nerve ending cytoplasm, but remained constant in the synaptic vesicles. After incubation in 25 mm -KCl medium the ACh in the vesicles was labelled to the same extent as the cytoplasmic ACh but after incubation in 4·7 mm -KCl medium vesicular ACh was labelled less than cytoplasmic ACh. During 5 min incubation in medium containing 25 mm -KCl the ratio of labelled to total ACh was much higher in the medium than in the homogenate, the vesicles or the cytoplasm. During the last 15 min of the 60 min incubation the ratio of labelled to total ACh in the medium was still higher than that in the tissue fractions, but less so than during the 5 min incubation. It is concluded that the vesicular and cytoplasmic fractions are not identical with the store in the tissue from which newly-synthesized ACh is preferentially released.     

d-β-HYDROXYBUTYRATE: A MAJOR PRECURSOR OF AMINO ACIDS IN DEVELOPING RAT BRAIN

Abstract— D-β-hydroxybutyrate (β-OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3-¹⁴C]β-OHB or [2-¹⁴C]glucose was injected subcu-taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3-¹⁴C]β-OHB rapidly labelled brain amino acids at all ages whereas [2-¹⁴C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3-¹⁴C]β-OHB from birth-15 days of age whereas brain alanine was more heavily labelled by [2-¹⁴C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β-OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [¹⁴C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2-^,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3-¹⁴C]β-OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [¹⁴C]label from [2-¹⁴C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [¹⁴C]label into the brain amino acids of the 15-day-old [3-¹⁴C]β-OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?-OHB at 6 days was 19 8 min and at 15 days was 12-2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3-¹⁴C]β-OHB injected rats compared to the [2-¹⁴C]glucose injected rats despite a heavier labelling of brain glutamate in the [3-¹⁴C]β-OHB injected group. We interpreted these data to mean that β-OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.     

THE PREDOMINANT ROLE OF THE SPLEEN IN LYMPHOCYTE RECIRCULATION

Autologous blood lymphocytes from three normal pigs were labelled with ³H-uridine and retransfused before and after splenectomy. Frequent samples for up to 150 min after retransfusion were evaluated autoradiographically to determine the rate of disappearance of labelled lymphocytes from the blood. In one pig retransfusion was performed before and after sham-splenectomy. In all preoperative experiments the pattern of disappearance of labelled lymphocytes was very similar. After a first rapid decline (halving time on average 8 min) a short rise of the labelling index was observed from 10 to 15 min after retransfusion. Then a second more gradual decrease of labelled lymphocytes followed. The mean halving time during this period was less than 32 min. From 60 min onwards the labelling index remained nearly constant. Retransfusions performed 3 days after splenectomy revealed only one nearly constant decline of the labelling index (halving time on average 129 min). After sham-splenectomy the pattern of disappearance was similar to the preoperative experiment. One hour after the end of retransfusion the labelling index had decreased by three-quarters of the initial value in normal pigs and by only one-third in the splenectomized ones. These results indicate that in the pig the total rate of recirculation is at least 4 times faster with the spleen in situ than without the spleen.     

RADIOAUTOGRAPHIC STUDIES IN SYNCHRONIZED CULTURES OF OEDOGONIUM CARDIACUM

Synthesis of deoxyribonucleic acid (DNA) in synchronized cultures of Oedogonium cardiacum has been studied by radioautography. The germinated zoospores are pulse-labelled for 15 min with thymidine-2-C¹⁴. Use of penicillin in the medium reduces the background in the radioautographs by suppressing the growth of bacteria on Oedogonium cells. Incorporation of labelled precursor is greatly enhanced by growing the cells in a conditioned inorganic medium. The length of the DNA synthesis period (S), as estimated from the curve of percentage of labelled cells versus age during the first cell cycle is about 5-7 hr. The rate of labelling in the nucleus is non-uniform showing a dip during the mid S period. Concomitant with the doubling of DNA in the nucleus a fourfold increase in the nuclear volume is observed.     

Development of the large nucleolus in the oocytes of the copepod Acanthocyclops vernalis: an electron microscope study

A central feature of oogenesis in the copepod crustacean, Acanthocyclops vernalis, is the development of a very large nucleolus in the oocytes. This nucleolus appears to be the only source of rRNA for the oocyte, as no helper cells are present. Previous work has suggested that ribosomal DNA sequences other than those found at the morphological nucleolar organizers are participating in the elaboration of this nucleolus. It has been hypothesized that chromatin diminution, which occurs during early embryonic development, may involve the loss of these rDNA sequences, which are needed only for the production of ribosomes during oogenesis. The present study examines the development of the large oocyte nucleolus at the electron microscopic level. Nucleologenesis in A. vernalis was found to proceed through 5 stages. During the first 3 stages nucleolar morphology resembled that described in other organisms. In the last 2, however, nucleolar morphology changed radically and the nucleolus was seen to increase greatly in size while breaking up into multiple subunits. The subunits initially resemble active nucleoli, although in the last stage, synthesis appears to stop, as the nucleolus was found to consist only of dense areas containing ribosome-like particles. These observations are consistent with the hypothesis that diminuted DNA contains ribosomal RNA genes.     

玉米双受精过程的细胞学观察

1.玉米双受精属于有丝分裂前配子融合的类型。2.授粉后21至24小时,大部分雌、雄性核发生融合。雌、雄性核融合时,雄性核仁的出现存在两种情况,其一是精核染色质逐渐分散的同时出现雄性核仁,其二是精核染色质逐渐分散后,约经2至4小时才出现雄性核仁。3.合子内的雌、雄性核仁若发生融合,一般在合子分裂前进行。合子以一个大核仁的形式进入分裂期;雌、雄性核仁不融合的合子同样可以进入分裂期。授粉后30至33小时,合子进行第一次分裂。合子静止期大约为9小时左右。4.初生胚乳核内的雌、雄性核仁不发生融合。授粉后24至26小时,初生胚乳核进行第一次分裂。初生胚乳核的静止期为2—4小时。5.人工授粉条件下,玉米果穗受精过程的进行有一定的顺序;即自果穗上部逐渐向下部完成受精作用。     

TRANSLOCATION IN POLYTRICHUM COMMUNE (BRYOPHYTA) I. CONDUCTION AND ALLOCATION OF PHOTOASSIMILATES

Leafy stems and connecting underground rhizomes of Polytrichum commune Hedw. contain leptome tissues similar in structure to phloem. Isolated stems in clonal groupings were pulse labelled with ¹⁴CO₂. Labelled sugar, mostly sucrose, glucose, and fructose, appeared in the pulse labelled stems 30 min after treatment. A small amount (3.3%) of labelled sugar was transported to neighboring stems. Silver grain deposition in microautoradiographs of interconnecting rhizomes occurred predominantly over leptome tissues. Increased amounts of translocated radioactivity appeared in starch and cell wall polysaccharide pools one week and six weeks after treatment. These results 1] indicate that transport of photoassimilate occurs through the leptome of perennating rhizomes, 2] demonstrate that translocated carbon is subsequently utilized or stored, and 3] raise important questions about the significance of long distance transport in the life strategy of this complex clonal moss.     

TRANSPORT OF 14C INCORPORATED PROTEIN IN THE CORTICOSPINAL TRACT OF THE RAT BRAIN

Abstract— The origin of fibres of a corticospinal pathway in rat brain was located by cortical ablation and Marchi staining and also by electrical stimulation of the motor cortex.
Enzyme changes investigated histochemically over 0–14 days post-injection of 5 μ10.15 m NaCl into the neocortex indicated that very little apparent disturbance of nerve cell metabolism beyond a narrow band adjacent to the path of the microneedle within the cortex had occurred. [¹⁴ C]Leucine as a precursor of protein synthesis was used to study incorporation of the amino acid into protein. At the site of injection the maximum level of labelled protein was recorded at 30 min post-injection, the level decreasing to less than 2 per cent of this at 6 hr.
The subsequent axonal flow of labelled protein along the corticospinal pathway was investigated during the period 15 min to 21 days post-injection. Within 24 hr increasing amounts of labelled proteins were measured caudally, but not more than 6 per cent had migrated beyond 5 mm from the site of injection. At 3 days this percentage had increased to 14.6 per cent, the labelled proteins being distributed in progressively decreasing amounts to a further 13 mm caudal. Very little change from this position was seen during the following 18 days.     

DEPENDENCE OF FAST AXOPLASMIC TRANSPORT IN NERVE ON OXIDATIVE METABOLISM

—A crest of labelled activity moving down the sciatic nerve at 401 ± 35 mm/day after injection of the L7 dorsal root ganglion of the cat with L-[³H]leucine characterizes fast axoplasmic transport of materials and has been studied with regard to its dependence on oxidative metabolism. Transport of labelled materials in vitro occurred if the nerve was supplied with O₂ or 95 % O₂+ 5 % CO₂. Transport was not dependent upon continuity of the fibres with the ganglionic soma. Asphyxiation (N₂) rapidly blocked fast transport in vitro. Likewise NaCN or dinitrophenol in an O₂ atmosphere both effectively block fast transport within 15 min. Tetrodotoxin and procaine, agents which block excitation of the membrane, had no effect on fast transport. The inference is that oxidative metabolism supplies the energy required by the molecular mechanism underlying fast axoplasmic transport.     

In situ hybridization of “Nick-translated” 3H-ribosomal DNA to chromosomes from salamanders

A technique is described for preparation of ³H-labelled DNA by nick-translation employing deoxyribonuclease I and DNA polymerase I. The labelled DNA can be obtained in high yield with specific activities of 10⁶ cpm/g or more. Ribosomal DNA, isolated from ovaries of young Xenopus laevis, and whole DNA from Plethodon cinereus were labelled in this way. The rDNA was used for in situ hybridization to meiotic chromosomes from P. cinereus, P. vehiculum and P. dunni. Autoradiographs of in situ hybrids were exposed for 5 to 10 days, by which time nucleolus organizer regions on the chromosomes of all 3 species were clearly and specifically labelled. In all eases, labelling was confined to a short region near the middle of the short arm of both halves of a medium length bivalent. It is concluded that nick-translation is a useful and altogether efficient method of labelling nucleic acids for subsequent use in experiments involving in situ hybridizations.     

GLYCEROL KINASE AND DIHYDROXYACETONE KINASE IN RAT BRAIN

—The enzymatic phosphorylation of glycerol and dihydroxyacetone by ATP to sn-glycerol-3-phosphate and dihydroxyacetone phosphate respectively in various subcellular fractions of rat brain was studied. A sensitive radiochemical assay was used where the labelled phosphorylated products were separated from the radioactive substrates by high voltage paper electrophoresis and the radioactivity in these compounds determined. Using this assay the glycerol kinase (EC 2.7.1.30) activity was found to be associated with the mitochondrial fraction of the brain. Under optimum conditions 2.45 nmol of glycerol was phosphorylated/min per mg of protein. The K_m for glycerol was 70 μm at pH 7. This mitochondrial enzyme, like other glycerol kinases from different sources, also phosphorylated dihydroxyacetone. Under optimum conditions 1.7 nmol of dihydroxyacetone phosphate was formed/min per mg of mitochondrial protein. The K_m for dihydroxyacetone was 0.6 mm . Glycerol kinase activity was also present in the cytoplasm of brain. However, the specific activity of this enzyme in cytosol is about 15% of the mitochondrial glycerol kinase. Compared to glycerol, dihydroxyacetone was phosphorylated by ATP in cytoplasm at a much higher rate. The pH optimum for this soluble dihydroxyacetone kinase was much lower (pH 6.5) than that of the soluble or mitochondrial glycerol kinase (pH 10.0). Using ammonium sulfate, brain cytoplasm was fractionated to yield a fraction in which the dihydroxyacetone kinase was enriched 2–3 fold with no glycerol kinase activity. Under optimum conditions 1.0 nmol of dihydroxyacetone was phosphorylated/min per mg protein. The K_m for dihydroxyacetone was 60 μm . This cytosol fraction was also found to phosphorylate d -glyceraldehyde and l -glyceraldehyde at a rate of 30–40% to that of the dihydroxyacetone phosphorylation. The properties and the possible metabolic role of these enzymes in brain are discussed.     

Der Nucleolus und seine Beziehungen zu den Ribosomen des Cytoplasmas. Eine Untersuchung an den Malpighischen Gefässen von Drosophila melanogaster

Summary The investigation described herein is concerned with the structure and the behavior of the nucleolus of the cells of the Malpighian tubules of Drosophila melanogaster (see Fig. 16).Primarly the nucleolus consists of ribonucleic-acid granules about 150 Å in diameter which, with electron microscope, are not distinguishable from the ribosomes of the cytoplasm; they lie on or within the structural network of the nucleolus (Fig. 9). During the stages of the life history of the insect the nucleolus shows correspondingly characteristic peculiarities in structure which reflect the varying functional stages of the cell: The structure is loose in the cells of the larva (Fig. 2) and imago (Fig. 12), whereas it is compact in the pupa (Fig. 8, 9). In pupa sufficiently mature for emergence, the nucleolus contains a large central vacuole (Fig. 8, 9). The nucleolus of the larva has a similar appearance after a decrease in assimilation induced by starvation (Fig. 10, 11).There are smaller vacuoles in the nucleolus which mostly are filled with parts of the nucleolus-chromosome. In the pupal stage some of the peripheral constituents of the nucleolus separate and move to the nuclear membrane through which they pass via pores into the cytoplasm (Fig. 3, 4).Larger pieces reach the cytoplasm by local dissolution of the nuclear membrane (Fig. 5). During this process the extruded nucleolar material is dispersed into its structural components. Because the cytoplasm becomes richer in ribosomes during the period of nucleolar extrusion (Fig. 1b), it can be assumed, that the nucleolus must be a source of ribosomes, and that through such an extrusion which has been demonstrated to be a natural process (Fig. 7) the cytoplasm can be provided with ribosomes rapidly.The importance of this phenomenon in the synthesis of imaginal body is discussed.In the nucleus there are many vacuoles with a diameter of about 425 Å (Fig. 15a, b) which pass through the pores of membrane in the cytoplasm (Fig. 15c).

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Die Untersuchung wurde mit Unterstützung der Deutschen Forschungsgemeinschaft durchgeführt.     

Fluxes and accumulation of ethirimol in haustoria of Erysiphe pisi and protoplasts of Pisum sativum

Isolated purified fractions containing haustorial complexes and mesophyll protoplasts were used to investigate the relative affinities in vitro of the host-parasite interface and host for the mildew specific fungicide, ethirimol. Compounds labelled with [¹⁴C] were used throughout this study. Isolated haustorial complexes accumulated fungicide against a concentration gradient and, in 15 min, to much higher concentrations than they accumulated glucose, sucrose, uracil, glycine, ethanolamine hydrochloride, inulin carboxylic acid, thiocyanate ions or uridine diphosphate glucose. Accumulation of ethirimol ceased after 30 min when the internal concentration was over twenty times greater than in the ambient medium. Similar quantities were absorbed in 15 min at pH 4.2 and 6–2. The quantities absorbed during 1 h incubation were directly related to the ambient concentration (4–400 μm). Ethirimol introduced into haustorial complexes was easily removed by washing; one third was removed in the first 5 min and only one sixth of the original remained after 3 h. Analysis of the kinetics of ethirimol efflux showed that it was located in two compartments within the complexes; thus it most probably entered the fungal cytoplasam. Ethirimol entered pea mesophyll protoplasts showing biphasic kinetics with considerable uptake in the first 30 min and a more gradual influx during the following 18 h. It was not accumulated against a concentration gradient until 6 h and after 18 h the internal concentration exceeded that of the ambient by only 1.74 fold. Extraction and chromatography showed that only small proportions of the ethirimol were degraded by haustorial complexes and protoplasts during the experimental periods. These in vitro experiments indicate competitive accumulation of ethirimol by the host-parasite interface in vivo.     

Gonadal morphogenesis and sex differentiation in cultured Ussuri catfish Tachysurus ussuriensis

The objective of this study was to investigate the optimal developmental time to perform sex reversal in Ussuri catfish Tachysurus ussuriensis, to develop monosex breeding in aquaculture. Systematic observations of gonadal sex differentiation of P. ussiriensis were conducted. The genital ridge formed at 9 days post fertilization (dpf) and germ cells begin to proliferate at 17 dpf. The ovarian cavity began forming on 21 dpf and completed by 25 dpf while presumptive testis remained quiescent. The primary oocytes were at the chromatin nucleolus stage by 30 dpf, the peri‐nucleolus stage by 44 dpf and the cortical alveoli stage by 64 dpf. The germinal vesicle migrated towards the animal pole (polarization) at 120 dpf. In presumptive testis, germ cells entered into mitosis and blood vessels appeared in the proximal gonad on 30 dpf. The efferent duct anlage appeared on 36 dpf and formation of seminal lobules with spermatogonia and lobules interstitium occurred at 120 dpf. Therefore, gonadal sex differentiation occurred earlier in females than in males, with the histological differentiation preceding cytologic differentiation in T. ussuriensis. This indicates that undifferentiated gonads directly differentiate into ovary or testis between 17 and 21 dpf and artificial induction of sexual reversal by oral steroid administration must be conducted before 17 dpf.     

Nuclear Division in the Ameboflagellate Adelphamoeba galeacystis

Nuclear division in synchronized cultures of the ameboflagellate Adelphamoeba galeacystis has been described. Division in this organism is typically promitotic. It occurs within an intact nuclear membrane and is characterized by the persistence of the nucleolus and its transformation into 2 polar masses. The nucleolus is stained with pyronin-Y by the methyl green pyronin-Y technic, and with Heidenhain's hematoxylin, but is unstained by the Feulgen reaction. The reaction with these stains is removed after digestion of the nucleolus by ribonuclease. During mitosis the nucleolus undergoes an orderly series of vacuolizations before forming the polar masses. The chromatin is Feulgen positive, stains with methyl green by the methyl green pyronin-Y technic and undergoes a series of characteristic changes during the division process. Synchronization of amebae grown on coverglasses was accomplished by transfer of cells from 30 to 38.5 C for a period of 100 min. A temporal sequence of nucleolar and chromatin participation in the nuclear division of this organism is suggested.     

THE NUCLEOLUS OF PARACENTROTUS LIVIDUS DURING THE DEVELOPMENT IN THE PRESENCE OF ACTINOMYCIN D*

An ultrastructural study of the nucleolus of embryos of Paracentrotus lividus was carried out after treatment with Actinomycin D. It was shown that the fibrillar component of the nucleolus persists in the embryos treated with Actinomycin D in the mesenchyme blastula stage and fixed 24 and 48 hr after fertilization. The results are discussed in relation to the synthesis of RNA.