Polyribosomes in different stages of the life cycle of the water mold Allomyces arbuscula

Synchronous gametogenesis in the water mold Allomyces arbuscula is blocked by actinomycin D added at the onset of the process. Formation of the male gametangium can be selectively inhibited by administering actinomycin one hr after the induction of gametogenesis. The polyribosome pattern obtained after density gradient centrifugation remains virtually unchanged throughout gametogenesis until a stage immediately preceding maturation of the gametes. When ribosomes from gametes and swarming zygotes are analyzed on gradients, some RNase-sensitive material is found to band in the heavier portion of the gradient. Its presence suggests that some messenger RNA associated with ribosomes is conserved in the swarming cells. During gametogenesis RNA is de novo synthesized and becomes associated with the polyribosomes.     

Untersuchungen zur Entwicklung des PhycomycetenAllomyces arbuscula

InAllomyces arbuscula formation of gametes occurs within 80 min in isolated gametangia. Gametogenesis shows to be sensitive to cycloheximide at 50 and 100 g/ml, while actinomycin D at 25 and 50 g/ml fails to inhibit gametogenesis. Synthesis ofrRNA can be profoundly inhibited by 3×10^-3 M 5-fluorouracil prior to gametogenesis, without any effect on differentiation of gametes. It is shown by polyacrylamide-gel electrophoresis that during gametogenesis radioactive phosphate is incorporated intotRNA, but not intorRNA. The results indicate that formation of gametes is dependent uponmRNA already present in the gametangia before induction of gametogenesis. It is concluded further that protein synthesis on cytoplasmic ribosomes is finished 20–30 min after induction and thatrRNA synthesis seems not to be a prerequisite for the differentiation of gametes.

     

Preferential digestion of chloroplast DNA in male gametangia during the late stage of gametogenesis in the anisogamous algaBryopsis maxima

Summary The fate of chloroplast nuclei (cp-nuclei) and mitochondrial nuclei (mt-nuclei) was followed during gametogenesis in male and female coenocytic thalli in the anisogamous algaBryopsis maxima by epifluorescence microscopy, after staining with 4,6-diamidino-2-phenylindole (DAPI), by quantification of chloroplast DNA (cp-DNA) by fluorimetry using a video-intensified, photon-counting system (VIMPICS), and by CsCl density gradient centrifugation. The male and female coenocytic thalli, 48 h before the release of gametes, contain a large number of chloroplasts, each of which is larger in size than the cell nucleus and the mitochondria and contains about 150 cp-nuclei. The size of each chloroplast in the female and male gametangia decreases markedly during gametogenesis as a result of continuous divisions till about 10 h before the release of gametes and, eventually, the numbers of cp-nuclei per chloroplast in the male and female gametangia fall to about 20 and 5, respectively. Two hours later, as the preferential digestion of cp-DNA in the male gametangium occurs, the number of cp-nuclei in the chloroplast of each male gamete falls to zero while the number of cp-nuclei in female gamete does not change, even after release of female gametes. Several mt-nuclei are observed in all of the female gametes. By contrast, the mt-nuclei in the bulk of the male gametes disappear but those in a few gametes remain. The profiles after CsCl density gradient centrifugation of DNAs extracted from male and female plants and gametes support the cytological data. The results suggest that the preferential digestion of cp-DNA in male plants occurs about 8 h before the release of gametes and that there is differential digestion of cp-DNA and mitochondrial DNA (mt-DNA).     

GAMETOGENESIS AND GAMETE RELEASE OF ULVA MUTABILIS AND ULVA LACTUCA (CHLOROPHYTA): REGULATORY EFFECTS AND CHEMICAL CHARACTERIZATION OF THE “SWARMING INHIBITOR”1

Gametophytes of Ulva mutabilis Føyn and Ulva lactuca L. were artificially induced to form gametangia by removal of sporulation inhibitors. After this treatment, U. mutabilis gametes were ready for swarming on the third morning after induction, while U. lactuca gametangia needed 1–2 d longer for maturation. Release of gametes of U. lactuca was dependent solely upon exposure to the first light in the morning. Gametangia of U. mutabilis, however, also required sufficient dilution of the swarming inhibitor (SWI). SWI was excreted transiently by both Ulva species early during gametogenesis. While the SWI concentration in U. mutabilis medium remained above the inhibitory concentration until the gametangia were mature, the concentration of U. lactuca‐SWI dropped rapidly below this level. In the presence of sufficient SWI, mature gametes of U. mutabilis remained motionless within the gametangia despite light and open exit pores. However, using SEM, an additional seal was detected within these pores, which probably prevented premature swarming until dilution of SWI and exposure to light. Observations by time lapse microscopy and experiments with the myosin kinase inhibitor BDM suggest that the gametes may be either extruded by the gametangium or leave the exit pore by active gliding motion, driven by a myosin‐like motor protein. The SWIs were purified from both Ulva species, and mass spectral analysis showed their molecular masses (292 Da) were identical.     

Structure and reproduction of Hormidiella bharatiansis sp. nov.

The structure and reproduction of Hormidiella bharatiansis sp. nov. is described. Asexual reproduction is by zoospores: sexual reproduction is by morphologically similar gametes, some of which, after a brief period of swarming, settle down, round off, enlarge and act as females gametes, the others remaining active and functioning as male gametes. Fertilisation takes place about six to eight hours after liberation: one to two percent of the zygotes are abortive.     

Untersuchungen zur Entwicklung des Phycomyceten Allomyces arbuscula

Polysomes were isolated both from growing gametophytes of Allomyces arbuscula and from gametangia prepared from mycelia at different periods during gametogenesis. Analysis of polysomes by sucrose gradients showed that ribosomes present in the gametangia monosome pool were shifted into polysomes. This shift was found to be correlated with gametangia differentiation. The ribosome distribution remained virtually unchanged during the early stage of gamete formation. In mature gametes and swarming zygotes a low level of polysomes was detected. Labeling of rRNA by ³²PO₄ demonstrated a de novo synthesis of monosomes throughout the period of gametangia differentiation. No incorporation of ³²PO₄ was found to be present in ribosomes prepared from gametangia after onset of gamete formation. On the basis of these labeling experiments it is concluded that radioactivity in polysomes extracted from mature gametes and swarming zygotes can be attributed in part to conserved mRNA.Synchronous formation of gametangia was induced by transferring the vegetative mycelia from growth medium into a low salt buffer. Under these conditions the incorporation of either ³²PO₄ or ³H-uridine into RNA, particularly into rRNA, was found to be markedly decreased. This obviously indicates a shutdown of RNA synthesis. rRNA from induced mycelia examined by polyacrylamide gel electrophoresis was found to be severely degraded. In contrast to this, rRNA isolated from ribosomes of developing gametangia and from gametes exhibited no degradation products. It is suggested that endonucleases cause rRNA hydrolysis in the hyphal cytoplasm during gametangia differentiation. Ribosomes compartmentalized in gametangia seem to be inaccessible to nucleases during the later process of gametogenesis.

---

Abkürzungen MAK Methyl-Albumin-Kieselgur - PAA Polyacrylamid - stains all 4,5,4,5-Dibenzo-3,3-diäthyl-9-methylthiacarbocyaninbromid (Serva, Heidelberg)     

LIGHT AND ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF HEPATIC CELL NUCLEOLI IN MICE TREATED WITH ACTINOMYCIN D

Nucleolar partition induced by actinomycin D was used to demonstrate some aspects of nucleolar RNA synthesis and release in mouse hepatic cells, with light and electron microscopic radioautography. The effect of the drug on RNA synthesis and nucleolar morphology was studied when actinomycin D treatment preceded labeling with tritiated orotic acid. Nucleolar partition, consisting of a segegration into granular and fibrillar parts was visible if a dosage of 25 µg of actinomycin D was used, but nucleolar RNA was still synthesized. After a dosage of 400 µg of actinomycin D, nucleolar RNA synthesis was completely stopped If labeling with tritiated orotic acid preceded treatment with 400 µg of actinomycin D, labeled nucleolar RNA was present 15 min after actinomycin D treatment while high resolution radioautography showed an association of silver grains with the granular component. At 30 min after actinomicyn D treatment all labeling was lost. Since labeling was associated with the granular component the progressive loss of label as a result of actinomycin D treatment indicated a release of nucleolar granules. The correlation between this release and the loss of 28S RNA from actinomycin D treated nucleoli as described in the literature is discussed.     

Ribosomal RNA on the surfaces of nonleukemic mouse ascites tumor cells

RNAs on the cell surfaces of two nonleukemic and two leukemic strains of mouse ascites tumor cells were studied by fractionating the RNAs released from the cell surface by gentle pronase treatment after sucrose density gradient centrifugation, by indirect membrane immunofluorescence that used anti-RNA antibody and by cell electrophoresis. RNA was extracted from the cell supernatants of Ehrlich ascites tumor and sarcoma 180 cells (nonleukemic) that had been treated or not treated with pronase (1 microgram/ml, 37 degrees C, 20 min) followed by sucrose density gradient centrifugation. It was clearly demonstrated that the amounts of ribosomal RNA (18S and 28S) released after pronase treatment were approximately 80% greater than that of nonpronase-treated cells. Ehrlich ascites tumor cells that had been treated with actinomycin D (100 micrograms/kg body weight of mouse, 16 h) in vivo released an amount of ribosomal RNA after pronase treatment only 20% greater than the value for untreated cells. Actinomycin D treatment greatly reduced both the cell surface negative charge and the cell surface immunofluorescence when rabbit anti-RNA antibody was used. Under the same experimental conditions with actinomycin D, only ribosomal RNA synthesis showed preferential inhibition, not the syntheses of poly A-containing messenger RNA, 4S or other small-size RNAs. In contrast, L1210 and C1498 cells (leukemic) showed no change in the amounts of ribosomal RNA released after pronase treatment. L1210 cells also showed no change in the surface negative charge after being treated with actinomycin D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a selective destabilization of an integral membrane protein, the cytochrome b6/f complex, during gametogenesis in Chlamydomonas reinhardtii.

We studied the process of photosynthetic inactivation during gametogenesis of the unicellular green alga Chlamydomonas reinhardtii. We show that it is caused by the selective destabilization of a single transmembrane protein complex, the cytochrome b6/f complex, which is initially accumulated in the thylakoid membranes of vegetative cells. This protein destabilization is controlled by the intracellular energy sources available in the gametes, i.e. the coupled electron flow in the mitochondria and the amount of starch accumulated in the chloroplast. It nevertheless requires the expression of gamete-specific proteins. The loss of cytochrome b6/f complexes during gametogenesis is prevented by the addition of cycloheximide, but is chloramphenicol insensitive. Therefore, it is likely to involve some translation product of nuclear origin, specifically expressed during gametogenesis. Among the new polypeptides specifically found in the gametes, we detected a soluble polypeptide M alpha (approximate molecular mass of 63 kDa), which shared common epitopes with cytochrome f. Its synthesis displays an antibiotic sensitivity typical of a nuclear-encoded polypeptide and is controlled by the same intracellular signals which control the destabilization of the cytochrome b6/f complexes in the thylakoid membranes.     

Gametic differentiation in Chlamydomonas reinhardtii: light dependence and gene expression patterns

Gametes of Chlamydomonas reinhardtii synthesize numerous proteins not observed in vegetative cells and vice versa. Gametogenesis induced changes in gene expression were confirmed by SDS-PAGE of in vitro translation products using total RNA from gametes and vegetative cells. Vegetative cells and gametes thus represent two cell types with distinct patterns of gene expression. The generation of mature gametes from liquid cultures of asynchronously growing vegetative cells was dependent on light. This light requirement could not be substituted for by an organic source of energy and carbon, indicating that light serves as a signal in gametogenesis. The light signal was shown to become effective only after preincubation in nitrogen-free medium. This delayed competence for light indicates that the two external signals — nitrogen-starvation and light —may function in sequence. Execution of the light dependent step in gamete formation required cytoplasmic protein synthesis and RNA synthesis.Abbreviations CAM chloramphenicol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSII photosystem II - TAP Tris acetate phosphate - TMP Tris minimal phosphate This paper is dedicated by C. F. Beck to Professor John L. Ingraham, teacher and friend, on the occasion of his 65th birthday     

Epifluorescent microscopic studies on the mechanism of preferential destruction of chloroplast nucleoids of male origin in young zygotes ofChlamydomonas reinhardtii

Summary The studies on the kinetics of nucleoid destruction reported here showed that destruction of chloroplast nucleoids (ct nucleoids) of male origin began to occur at about 30 minutes after mixing of male (mt^–) and female (mt⁺) gametes. The timing of initiation of the destruction differed among zygotes but usually occurred during 50–120 minutes after mixing. About 10 minutes was required for complete digestion of the ct nucleoids. UV irradiation on young zygotes or addition of an RNA-synthesis inhibitor, actinomycin D, to the incubation medium during the first 0–30 minutes after mixing almost completely inhibited the incorporation of³H uridine into the cell nuclei and the preferential destruction without inhibiting cell nuclear fusion. These results suggest that soon after mating,de novo RNA synthesis is concerned for the preferential destruction of ct nucleoids. To determine in which of the two cell nuclei in the zygotes the RNA is synthesized, each gamete (mt^–, mt⁺) was irradiated with UV and mated with unirradiated gametes of opposite mating type. This treatment of the male gametes had no effect on the incorporation of³H uridine into cell nuclei and the preferential destruction of ct nucleoids but UV irradiation of female gametes almost completely inhibited the incorporation of³H uridine into cell nuclei and the preferential destruction of ct nucleoids. Similar phenomena occurred in other crosses. The UV effect was photoreactivated in about 50% by white light, suggesting that the UV target is DNA. Thus, RNA synthesized in the cell nucleus of female origin soon after mating may be responsible for the preferential destruction of ct nucleoids of male origin     

On the evolution of anisogamy from isogamous monoecy and on the origin of sex

A new hypothesis is established for the evolution of anisogamy in isogamous monoecious haploid unicells. Contrary to present concepts, it is assumed that in such isogamous ancestors the genetically identical gametes are bipolarly different as (+) and (?). The gametic differentiation of a monoecious taxon may be perceived as a phenocopy of that of a related dioecious form. The two gametic phenotypes result from alternative pathways of sexual differentiation. The alternative must be controlled by a sensible switch mechanism that responds to minor differences between vegetative cells when gametogenesis is triggered. Cell-size dependent factors are assumed to be able to influence the switch mechanism causing bigger cells to be of one sex, smaller cells of the other. Fertilization then occurs selectively between big and small gametes because of their sexual difference. Size-different gametes within one species may arise from different types of gametogenesis depending on how many gametes one vegetative cell produces. These assumptions, i.e. the bipolarity of isogametes, in monoecious taxa, size-dependent sex determination, and different types of gametogenesis within one taxon, are justifiable since they are realized independently from each other in various species. It is postulated that mutational fixation of the size-dependent sex determination in such monoecious species creates cell lineages producing sex-different macro- and microgametes and establishes constitutional anisogamous dioecy. The proposed hypothesis clearly separates the evolution of anisogamy as a problem of sexual reproduction from the evolution of sex per se, sex being defined as a bipolarity effecting fertilization.     

Light is a crucial signal for zoosporogenesis and gametogenesis in some green microalgae

Patterns of reproduction were investigated in some microalgal species of Chlorophyceae (Botryosphaerella sudetica, Neochloris aquatica, Neochloris vigensis, Bracteacoccus minor). Under continuous light, the microalgae reproduced asexually producing autospores. However, appropriate manipulation of external conditions led to a change in the reproduction pattern towards production of zoospores or gametes. Production of zoospores and gametes was inhibited by light; motile cells emerged when microalgae were cultivated in darkness. The period of dark treatment necessary for zoosporogenesis or gametogenesis differed substantially among species that were tested. Sexual reproduction was observed in Neochloris vigensis and Bracteacoccus minor, whose generative life cycle had not been previously reported. The morphology of motile cells, the mode of sexual reproduction, and the efficiency of both the production of motile stages and mating events, were investigated. In order to gain detailed insights into patterns of reproduction, Botryosphaerella sudetica was selected for investigation under different light treatments. Non-actinic red light applied in the early phase of dark cultivation (up to 2 h) suppressed both zoosporogenesis and gametogenesis. However, after a 3-h dark pre-treatment, red light treatment had no effect on zoosporogenesis or gametogenesis. In contrast, non-actinic blue light did not block zoosporogenesis or gametogenesis, regardless of the time of treatment. The possible role of a red-light photoreceptor in zoosporogenesis and gametogenesis is discussed.     

Origin and early development of female germ cells in Eoleptestheria ticinensis Balsamo-Crivelli, 1859 (Crustacea, Branchiopoda, Conchostraca)

We have studied the early stages of the development of the female germ cells in the Conchostraca Eoleptestheria ticinensis Balsamo-Crivelli, 1859. The gametes originate in a scattered way throughout the tubular units of the gonad, with no development gradient recognizable. The female germ cells arise from successive karyokineses not followed by cytokinesis, within an unorganised area in which a sort of plasmodium is formed. Each primordial nucleus of this germarium develops and then forms an individual plasmic membrane. Subsequently, the usual organules differentiate in the cytoplasm. The presence of synaptinemal complexes and the beginning of the endogenous vitellogenesis by the rough endoplasmic reticulum and the Golgi apparatus qualify the previtellogenesis. The general characteristics of this early development phase of the gametes, as well as several substantial differences in the gametogenesis with respect to the other Phyllopoda studied, lead us to suggest the systematic positioning of the Leptestheriidae.     

Origin of the actinomycin D insensitive RNA species in Aedes albopictus cells.

In the presence of actinomycin D or a combination of actinomycin D and either camptothecin or alpha-amanatin. Aedes albopictus cells synthesize a variety of single stranded RNA species. These actinomycin D resistant species are ethidium bromide sensitive and they are present in the cell cytoplasm in an RNase resistant structure which has the sedimentation and buoyant density characteristics of mitochondria. Twelve actinomycin D insensitive RNA species can be detected by electrophoresis in 7M urea and 11 of these bind to oligo(dT)-cellulose. An identical set of oligo(dT)-cellulose binding RNA species is obtained when A. albopictus cells are labeled in the presence of camptothecin alone. The actinomycin D insensitive RNA species which bind to oligo(dT)-cellulose hybridize to mitochondrial DNA. These data indicate that the actinomycin D insensitive RNA species have a mitochondrial origin and are not associated with the replication of an inapparent contaminating virus.     

Expression of inhibin α-subunit gene during mouse gametogenesis

Mammalian gametogenesis is regulated through complex interactions between germ and somatic cells. To investigate the mechanism underlying the differentiation of functional gametes, some genes specifically expressed during gametogenesis have been isolated and characterized. In a search for further examples of such genes, we have isolated from a newborn mouse testis cDNA library, a clone corresponding to mouse inhibin alpha-subunit. Although it is known that the inhibin alpha-subunit molecule is abundantly produced in ovarian follicle and in testicular Sertoli cells, the spatial and temporal patterns of expression of this gene remain to be elucidated. In this study, the patterns of expression of inhibin alpha-subunit mRNA during mouse gametogenesis were examined by RNA blot, cytoplasmic dot and in situ hybridization techniques. In the testis, the concentration of inhibin alpha-subunit mRNA increased from about 16 dpc (days post coitum), peaked at birth and then gradually decreased, paralleling testicular development. Inhibin alpha-subunit mRNA was localized in Sertoli cells of wild type as well as W/Wv testes. In adult testis, mRNA was restricted to the perinuclear cytoplasm of Sertoli cells. Inhibin alpha-subunit mRNA was expressed in follicle cells of adult ovary more abundantly than in adult testis. Analysis of expression during folliculogenesis showed that the accumulation of this mRNA began in preantrum follicles and the level of expression reached a maximum in Graafian follicles.     

Dissection of the Blue-Light-Dependent Signal-Transduction Pathway Involved in Gametic Differentiation of Chlamydomonas reinhardtii

Gametogenesis of the green alga Chlamydomonas reinhardtii may be viewed as a two-step process that is controlled by the environmental cues of nitrogen deprivation and blue light. Initiation of gametogenesis is induced by nitrogen deprivation, resulting in mating-incompetent pregametes, when cells are kept in the dark. For the completion of gametic differentiation light is required. Pregametes were treated with pharmacological compounds to influence the light-dependent conversion to mature gametes. Dibutyryl-cyclic 3[prime]5[prime] adenosinemonophosphate, papaverine, and genistein were found to inhibit the progression of gametogenesis in the light. Treatment of pregametes in the dark with either staurosporine or papaverine resulted in their conversion to mature gametes. Apparently, papaverine has different effects in the dark and in the light; the effect of staurosporine suggested that a protein kinase C-like component inhibits the conversion of pregametes to gametes, a block that normally is relieved by illumination. This hypothesis was corroborated by the observation that activators of protein kinase C, N-heptyl-5-chloro-1-naphthalenesulfonamide, N- (6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, and the phorbolester phorbol-12-myristate 13-acetate inhibited gametogenesis in the light. Genistein and dibutyryl-cyclic 3[prime]5[prime] adenosinemonophosphate were able to inhibit the dark activation caused by staurosporine treatment, suggesting that their targets work downstream from the "protein kinase C-like" kinase. Surprisingly, staurosporine and papaverine worked synergystically on the activation of pregametes in the dark.     

Synthesis of actinomycin-insensitive RNA during the first post-irradiation mitotic cycle,in the synchronously mitotic plasmodia of Physarum polycephalum

A sucrose density gradient analysis of³H-uridine pulse-labelled RNA from the first postirradiation mitotic cycle ofPhysarum polycephalum shows that all the density classes of RNA synthesized during this period are resistant to the peptide-antibiotic, actinomycin D. In fact, the synthesis is found to be greater in the presence of the drug. The heterogenously sedimenting synthetic activity here may represent a single species of RNA and its precursors or more than one kind of RNA. Further characterization of this RNA is meaningful in view of the actinomycin insensitivity of the postirradiation mitotic cycle itself to this antibiotic.     

Rapid and slow mechanisms for loss of cell adhesiveness during fertilization in Chlamydomonas

Although vegetative cells, gametes, and zygotes of the biflagellated alga Chlamydomonas bear flagella, only the flagella of mt+ and mt- gametes are adhesive. The molecules responsible for adhesiveness, mt+ and mt- agglutinins, are long rod-shaped glycoproteins displayed on the flagellar membrane. These flagellar agglutinins, which gametes use both as adhesion and signaling molecules during the early events of fertilization, are lost from the flagella during adhesion. Flagellar adhesiveness can be maintained, however, by recruitment and activation of preexisting, inactive agglutinins from the plasma membrane of the cell body (Hunnicutt et al, 1990, J. Cell Biol. 111, 1605-1616) unless the gametes of opposite mating types fuse to form zygotes. Upon cell fusion, flagellar adhesiveness is lost. In the studies presented here, we have employed an in vitro bioassay to measure agglutinins in both cell bodies and flagella at various times during gametogenesis, during fertilization, and after zygote-formation. By use of the bioassay, which can detect agglutinins that are functionally inactive in vivo, we found that vegetative cells are devoid of agglutinins. These adhesion molecules appear only after gametogenesis is underway with the cell body agglutinins appearing first and then the flagellar agglutinins. Surprisingly, 30 min after zygote formation, when the zygotes' flagella are no longer adhesive, the flagellar agglutinin activity detectable with the bioassay remains high. One interpretation of these results is that zygotes continue to recruit agglutinins from the cell body to the flagella, but cell fusion abrogates activation of the agglutinins. Within 45-90 min after fusion both the cell body and flagellar agglutinins are lost and can be detected in the medium. These mechanisms, which render the zygotes nonadhesive to other zygotes and unmated gametes, contribute to the Chlamydomonas equivalent of a block to polyspermy.