Changes in respiration,photosynthesis and protein composition during induced synchronous formation of gametes and zoospores in Ulva mutabilis Føyn

In the marine green alga Ulva mutabilis Føyn, large daily fluctuations in respiratory and photosynthetic rates are found after the induction of synchronous formation of gametes and zoospores. A sharp reduction in photosynthetic capacity preceeds the zooid-forming cell divisions. During the same period, samples for polyacrylamide electrophoresis show an initial increase followed by a decrease in the amount of ribulose-1,5-bisphosphate carboxylase. Some other transient protein changes were also noted. A new protein band with MW 51,400 appears in the 48-h sample and becomes a prominent constituent of gametes and zoospores; it may represent tubulin.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulphate To whom correspondence should be addressed     

Conspecificity of the model organism Ulva mutabilis and Ulva compressa (Ulvophyceae,Chlorophyta)

As one of the most abundant and ubiquitous representatives of marine and brackish coastal macrophytobenthos communities, the genus Ulva is not only an important primary producer but also of ecological and morphogenetic interest to many scientists. Ulva mutabilis became an important model organism to study morphogenesis and mutualistic interactions of macroalgae and microorganisms. Here, we report that our collections of Ulva compressa Linnaeus (1753) from Germany are conspecific with the type strains of the model organism U. mutabilis Føyn (1958), which were originally collected at Olhão on the south coast of Portugal and have from that time on been maintained in culture as gametophytic and parthenogenetic lab strains. Different approaches were used to test conspecificity: (i) comparisons of vegetative and reproductive features of cultured material of U. mutabilis and German U. compressa demonstrated a shared morphological pattern; (ii) gametes of U. compressa and U. mutabilis successfully mated and developed into fertile sporophytic first‐generation offspring; (iii) molecular phylogenetics and species delimitation analyses based on the Generalized Mixed Yule‐Coalescent method showed that U. mutabilis isolates (sl‐G[mt+]) and (wt‐G[mt‐]) and U. compressa belong to a unique Molecular Operational Taxonomic Unit. According to these findings, there is sufficient evidence that U. mutabilis and U. compressa should be regarded as conspecific.     

Isolation of gametes and zygotes from Setaria viridis

Setaria viridis, the wild ancestor of foxtail millet (Setaria italica), is an effective model plant for larger C₄ crops because S. viridis has several desirable traits, such as short generation time, prolific seed production and a small genome size. These advantages are well suited for investigating molecular mechanisms in angiosperms, especially C₄ crop species. Here, we report a procedure for isolating gametes and zygotes from S. viridis flowers. To isolate egg cells, ovaries were harvested from unpollinated mature flowers and cut transversely, which allowed direct access to the embryo sac. Thereafter, an egg cell was released from the cut end of the basal portion of the dissected ovary. To isolate sperm cells, pollen grains released from anthers were immersed in a mannitol solution, resulting in pollen-grain bursting, which released sperm cells. Additionally, S. viridis zygotes were successfully isolated from freshly pollinated flowers. Isolated zygotes cultured in a liquid medium developed into globular-like embryos and cell masses. Thus, isolated S. viridis gametes, zygotes and embryos are attainable for detailed observations and investigations of fertilization and developmental events in angiosperms.

     

Human gametes and zygotes studied by nonradioactive in situ hybridization

A nonradioactive in situ hybridization technique was applied to human gametes and abnormally fertilized or developed zygotes. Using haptenized chromosome-specific probes, visualization was obtained using immunocytochemistry to achieve a fluorescent stain on specific hybrids. Using a chromosome 1-specific DNA probe, almost all spermatozoa gave a positive result, i.e., one hybridization signal per cell could be observed. Furthermore, it was possible to identify sperm cells with two spots, suggesting nondisjunction. Two cleavage arrested embryos from different patients showed both: two brightly fluorescent spots and two weaker spots with the same DNA probe. Using a Y-specific DNA probe the percentages of positive spermatozoa from the normal males ranged between 48.1% and 49.1%. In an embryo with four grossly haploid chromosome sets, three fluorescent spots were obtained with the Y-specific DNA probe, indicating the penetration of three spermatozoa.     

In vitro fertilization and expression of transgenes in gametes and zygotes

The in vitro fertilization system of maize is the well characterized model system for the fertilization process and early zygotic embryogenesis of higher plants. Application of molecular methods to the in vitro fertilization system led to the isolation of new genes and uncovered specific expression patterns of cell cycle regulators. Recent studies showed that expression of transgenes is possible in gametes and zygotes, thus transgenic approaches might offer an opportunity to unravel the roles of genes during fertilization and early development. The competence of gametes and zygotes to express transgenes will also enable the expression of GFP based reporter genes for the visualization of subcellular components in these cells in vivo. This review focuses on the data concerning the expression of transgenes in gametes and zygotes and describes some examples of recent developments in transgenic technology illustrating the emerging possibilities in experimental design by combining this technology with in vitro fertilization. Received: 20 December 2001 / Revision accepted: 6 June 2001     

Amino acid pools in developing Chlamydomonas reinhardtii: vegetative cells, gametes, and mature zygotes.

Free amino acid pools were examined for cultures of vegetative cells, gametes, and mature zygotes of the unicellular green alga Chlamydomonas reinhardtii (Dangeard). The total pool of amino acids found in premature gametes of strains 137c+ (10.0 pmol-micrograms protein-1) and 137c- (10.8 pmol.micrograms protein-1) decreased to levels about half that seen in vegetative 137c- cells (19.8 pmol.micrograms protein-1). Following light activation, amino acid pools in these gametes increased to 18.7 pmol.micrograms protein-1 in 137c+ cells and 20.0 pmol.micrograms protein-1 in 137c- cells. With the exception of cystine, individual amino acid pools in these cells had increased once more to levels similar to those seen in vegetative cells grown in liquid medium. Levels of cystine remained one to two orders of magnitude lower than that seen in vegetative cells. Mature 137c+ and 137c- gametes mixed in solutions of either 2 mM cystine or 2 mM cysteine (half-cystine) suffered a 52-64% reduction, respectively, in the number of vis-à-vis conjugative pairs formed. This suggests that pools of endogenous cystine may play a role in the onset of mating. In zygotes levels of all amino acid pools, except histidine, were depressed; levels of cystine, valine, and phenylalanine were nondetectable in these cells.     

Genetic control of morphogenesis in the multicellular alga Ulva mutabilis

Distinct ultrastructural differences between the cell walls of the mutant lumpy (lu) and wild-type are demonstrated. The polyglucan fibrils in the wild-type wall appear straight and densely packed compared to the more uneven and loosely woven fibrils in the mutant wall. The mutant grows as an aggregate of loosely connected cells. The three cell types seen in the wild type do not differentiate, and consequently no blade and holdfast forms. It is suggested that the defect in the cell wall production takes away a necessary condition for normal morphogenesis. The lumpy mutant shows epistasis over two other chromosomal mutants, bu and Sl, described earlier to be morphogenic.     

The effect of chloramphenicol and cycloheximide on meiotic zoosporogenesis and mitotic gametogenesis in Ulva mutabilis.

Ulva mutabilis cells induced to form zoospores or gametes were exposed to chloramphenicol or cycloheximide during the 48 hours preparatory period. Chloramphenicol inhibited protein and RNA accumulation to the same degree at all concentrations, and appeared to have a general toxic effect. Cycloheximide at low concentrations inhibited primarily protein accumulation, while at higher concentrations it had also an effect on the net synthesis of RNA. Most of the inhibited cells were found in interphase, but a small percent of vegetative mitoses were observed and may indicate a return to this type of division. The gametophyte appeared to be more sensitive to chloramphenicol than the sporophyte. The effect of cycloheximide on the two processes seems to be more or less the same. A transition period, after which the sporulation process could no longer be inhibited, was found about 10 hours before the cells entered meiotic prophase in zoosporogenesis.     

On the genetic control of cell cycles during morphogenesis in Ulva mutabilis

Development of the wild type and two temperature-sensitive mutants of the multicellular green alga Ulva mutabilis is compared. The mutants develop normal phenotypes at 22°C and abnormal phenotypes at 15°C. Normal development starts by formation of a filament consisting of a row of cells. The growth rate, the generation times, and the cell length at division change in a coordinated manner according to the positions of the cells within the filament. In the mutant cs₂ transfer to 15°C inhibits all cytoplasmic divisions during early development. In the mutant cs₆ the first three divisions proceed normally. Then cytoplasmic division is blocked in the most distal cells, while the proximal cells continue to divide according to a branched pattern. In the cs₂ mutant cell determination seems to occur at 15°C, while the differentiation of the determined cells can only occur at 22°C. In the mutant cs₆ the cells are not determined at 15°C. The cs₆⁺ gene, as well as the previously described Slender-like genes, presumably has a short period of activity and is concerned with more fundamental epigenetic processes than the cs₂⁺-gene and the previously described precocious-like genes, which seem to have more prolonged periods of activity.     

Global characterization and target identification of piRNAs and endo-siRNAs in mouse gametes and zygotes

A set of small RNAs known as rasRNAs (repeat-associated small RNAs) have been related to the down-regulation of Transposable Elements (TEs) to safeguard genome integrity. Two key members of the rasRNAs group are piRNAs and endo-siRNAs. We have performed a comparative analysis of piRNAs and endo-siRNAs present in mouse oocytes, spermatozoa and zygotes, identified by deep sequencing and bioinformatic analysis. The detection of piRNAs and endo-siRNAs in the spermatozoa and revealed also in zygotes, hints to their potential delivery to oocytes during fertilization. However, a comparative assessment of the three cell types indicates that both piRNAs and endo-siRNAs are mainly maternally inherited. Finally, we have assessed the role of the different rasRNA molecules in connection with amplification processes by way of the “ping-pong cycle”. Our results suggest that the ping-pong cycle can act on other rasRNAs, such as tRNA- and rRNA-derived fragments, thus not only being restricted to TEs during gametogenesis.     

Efficiency of induced mutagenesis at early stages (gametes,zygotes, proembryos) of ontogenesis in Nigella damascena L.

Summary An original technique has been developed of treating gametes, zygotes and early embryos of Nigella damascena L. with chemical and physical mutagens. A delay in fertilization and a decrease in the rate of cell division of the embryo and the endosperm after mutagen treatment have been found. Our method of treating gametes, zygotes and proembryos with chemical and physical mutagens is, by all criteria, superior to that of treating dry seeds. Treatment applied at early stages of ontogenesis not only induced a much higher mutation ratio compared with dry seeds, but also gave a broader mutation spectrum. The 55 types of hereditary change obtained affect the structure of vegetative and reproductive organs. Mutations which change the structure of the reproductive organs of flowers are of specific interest.The optimum dose for this object and the method of treatment which induces high mutation ratio (up to 96 % of families with changes) is 0.003p (16 hrs) for ethylenimine and 0.005p or 0.008p (16 hrs) for nitrosomethylurea. Treatment of dry seeds turned out to be much less effective.     

Some observations on the fine structure of the gametes and zygotes of Acetabularia

Summary Gametes and zygotes of Acetabularia have been studied with the electron microscope. The two flagella of the gamete enter the cell obliquely and the roots run near the surface of the organism. No connexion between the eyespot and the flagella was found nor was there regular relationship between the two eyespots of the zygote. When zygotes were fixed some hours after fusion of the gametes the eyespots appeared to be within the same chloroplast.Some zygotes had two nuclei or dumb-bell shaped nuclei suggesting stages of fusion. The initial contact between nuclei appeared to be through a narrow gap formed by fusion of the two nuclear membranes.