Immunofluorescent distribution of postribosomal particles during oogenesis and early development of an insect (Dysdercus intermedius, Heteroptera, Pyrrhoc.).

During previtellogenesis, the oocytes of the telotrophic meroistic ovary ofDysdercus are provided with ribosomes and ribonucleoprotein (RNP) particles by the nurse cells. At the end of vitellogenesis, the oocyte itself becomes active as shown by autoradiography. The proteins synthesized by the oocyte are stored in cytoplasmic postribosomal particles which are preformed by the tropharium. The proteins of these particles were separated by SDS polyacrylamide gels and their endogenous oocyte proteins revealed by fluorography. The synthesis, transport, and storage of the postribosomal particles are demonstrated by indirect immunofluorescence. The young oocytes of previtellogenic follicles show a diffuse distribution of these particles. In late vitellogenesis, fluorescence becomes more and more concentrated in spots throughout a distinct region in the middle part of the oocyte. Thus, in freshly laid eggs, the periplasm is free of fluorescence. During migration of the cleavage nuclei the postribosomal particles were shifted into the cortex. Fluorescence is then most intense in the periplasmic region. During blastoderm formation, however, fluorescence decreases.

[Endogenous synthesis of short-lived messenger-RNA-species in oocytes ofDysdercus intermedius Dist. after termination of vitellogenesis]. / Endogene Synthese kurzlebiger Messenger-RNS in der Oocyte vonDysdercus intermedius Dist. nach Abschluß der Vitellogenese.

Non-ribosomal RNA is synthesized in oocytes of the telotrophicmeroistic ovary ofDysdercus intermedius Dist. during late oogenesis, 4-14 h before they become mature eggs. RNA is labelled "in vivo" by radioactive RNA precursors from a nucleotide pool which is established in the ooplasm prior to chorion-formation.RNA synthesized by late oocytes is characterized by a high turnover rate and appears first as a high molecular weight precursor which is converted during a few hours to smaller non-ribosomal RNA species of 30-5 S. In newly-laid eggs RNA molecules synthesized by the oocyte are no longer present. Their degradation products can be found within the nucleotide fraction. Thus, in contrast to RNA synthesized by trophocytes and stored in eggs in a stable form, endogeneously-formed RNA is not conserved for use in embryogenesis.Endogeneous RNA of oocytes is found to have a high content of poly(A)-segments. Fifty-seven percent of these are hybridizable with poly(U) immobilised on glassfiber filters. A few hours before the maturation of eggs, shortlived RNA synthesized by the oocyte is found in association with polysomes. By incubation of such polysomes in an "in vitro" protein-synthesizing system, polypeptides are formed which show a characteristic banding pattern after separation on SDS-polyacrylamide gels. The apparent molecular weights for the 4 main proteins are 65.000, 48.000, 44.000, and 40.000. There is no evidence for identity of any one of these proteins with a chorion protein.Some of the heterogeneous population of RNA molecules synthesized by late oocytes are characterized by a short lifetime, a relatively high content of poly(A) and the ability to activate protein synthesis "in vivo" or "in vitro", thus suggesting a function like that of mRNA.

[RNA-Synthesis in telotrophic meroistic ovarioles of dysdercus intermedius DIST. (heteroptera, pyrrhoc.)]. / Ribonucleinsäuresynthese in der telotroph-meroistischen Ovariole vonDysdercus intermedius Dist. (Heteroptera, Pyrrhoc.).

RNA in insect ovaries was investigated by polyacrylamide gel electrophoresis to study site of synthesis, transportation and incorporation in mature eggs. Telotrophic-meroistic ovarioles ofDysdercus intermedius were selected for this work since they could be dissected in distinct portions: the apical one with polyploid nurse cells and the vitellarium with oocytes covered by a follicular epithelium. RNA was labeled by injecting radioactive precursors or incubating isolated ovarioles in vitro.In nurse cells labeled rRNA, tRNA and non-ribosomal RNA were found and evidence was presented for the processing of a 39s rRNA precursor molecule in 2 steps (36s and 32s) into 28s rRNA while the 18s rRNA was directly derived from the 36s molecule. A low concentration (0.5 Μg/ml) of α-Amanitin inhibited synthesis of non-ribosomal RNA thereby revealing a more distinct processing pattern for rRNA, while a high concentration (5 Μg/ml) blocked processing at the 36s molecule and led to its accumulation. Appropriate concentrations of Actinomycin D inhibited rRNA synthesis while distinct peaks of non-ribosomal RNA became apparent.RNA synthesized in the follicular cells was similar to the RNA made in the nurse cells only in the high molecular weight RNA. Some material, tentatively named "nucleotide fraction", with a mobility higher than tRNA was detected and could be localized in yolk granules. It was separable from RNA by chromatography on Sephadex G 100 and had an absorption maximum at 255 run. The "nucleotide fraction" could be rapidly labeled. It seemed to be utilized in RNA synthesis during embryogenesis. Mature eggs contained a relatively high amount of the "nucleotide fraction". If a single injection with precursor was done 4 days before analysing the RNA of mature eggs most of the label was found in the "nucleotide fraction". Eggs collected 5-8 days after injection contained only high molecular labeled RNA. This observation correlated well with the timing of egg maturation. It takes 4 days for a group of oocytes to reach the distal part of the vitellarium where yolk production and maturation go on for another 4 days. It is apparent, therefore, that the follicular epithelium contributes the "nucleotide fraction" to the oocyte, while most of the other RNA, including a stable polydisperse class of RNA, is made in the nurse cells and is transported into the oocyte.

Enzymatic patterns in the embryonic development of the cricket,Acheta domesticus L.

The activity of the enzymes G6PDH, 6PGDH, GAPDH, LDH, CS, MDH, NADP-linked ICDH, GOT, GPT, and GLDH was tested in different stages of developing eggs and in just hatched larvae of crickets. A malic synthase could not be found. The enzymatic pattern changes considerably during this period. Considering this pattern, and especially the quotient (GAPDH)/CS, it seems justified to state that, up to the formation of the germ band, the energy is derived mainly from oxidation of glucose in the citric acid cycle. Later on, lipids substitute partly for glucose. Anaerobic energy production seems to be limited to the stage of the dorsal closure. By inhibiting the formation of the germ band, the activity of GAPDH, and CS is not changed significantly. The rise in activity of G6PDH is correlated with the increased rate of r-RNA synthesis. The increase of activity of MDH and NADP-linked ICDH corresponds to the differentiation of the germ band. These enzymes, together with GOT, GPT, and GLDH should be involved in the synthesis of amino acids from carbohydrates during the later part of development. In the hatched larvae the activity of all enzymes has risen considerably. This stage is characterized by the fact that G6PDH, 6PGDH, GAPDH, LDH, CS, and NADP-linked ICDH display the same activity.