Zygote Maturation Research Articles

Chloroplast gene transmission in Chlamydomonas reinhardtii. A model for its control by the mating-type locus

In sexual crosses of Chlamydomonas reinhardtii, genes residing in the chloroplast (cp) are most often transmitted from the mating-type plus (mt+) parent only. Galloway and Goodenough (1985) proposed a model in which the mt locus (linkage group VI) is a complex region containing several genes involved in the control of both gametic differentiation and cp inheritance. The mt+ locus contains: the sfu locus necessary for sexual fusion between gametes; the upp locus (uniparental plus) which controls cp gene inheritance and also perhaps zygote maturation; and the sad locus which functions in sexual adhesion. The mt− locus also contains a sad locus as well as a regulatory element (mid) necessary for the minus dominance in mt+/mt− diploid gametes. This model has been extended to include new genetic functions linked to the mt+ or mt− locus. In this new system, there is a group of genes (maps for mating-type plus structure), present in both plus and minus strains, controlling some mt+ phenotypical traits as well as the synthesis of an activator of the cp DNA nuclease; two genes contained in the mt− locus — one (cge for cp genomic elimination) coding for a nuclease (in an inactive form) located in the chloroplast and another (mid) coding for a repressor of maps; one gene (upp) contained in the mt+ locus, coding for a substance preventing the synthesis or the activity of the nuclease and perhaps also acting on the maps product. The model can be applied to explain the complex dominance/ recessivity relationships observed between the mt+ and mt− “alleles” in heterozygous diploid gametes, the basis for mitotic vs meiotic zygote formation and the differences in transmission observed for these two physiologically distinct zygote classes. Application of the model for prediction of mutant phenotypes and the design of future experiments is also described.

Genetic analysis of mating locus linked mutations in Chlamydomonas reinhardii.

The mating-type (mt) locus of Chlamydomonas reinhardii has been analyzed using four mutant strains (imp-1, imp-10, imp-11 and imp-12). All have been shown, or are shown here, to carry mutations linked to either the plus (mt+) or the minus (mt-) locus, and their behavior in complementation tests has allowed us to define several distinct functions for each locus. Specifically, we propose that the mt+ locus contains the following genes or regulatory elements: a locus designated sfu, which is necessary for sexual fusion between gametes; a locus designated upp (uniparental plus), which controls aspects of chloroplast gene inheritance and perhaps also zygote maturation; and a locus designated sad, which functions in sexual adhesion. The mt- locus also contains a sad locus as well as a gene or regulatory element designated mid, which is necessary for the minus dominance in mt+/mt- diploids.

The formation and consumption of lipid droplets in the zygote and germinated cells ofClosterium ehrenbergii

The formation and consumption of lipid droplets was observed with an electron microscope in the zygote and the germinated cells of the green alga,Closterium ehrenbergii. The lipid droplets were formed in lysosomal vesicles during zygote maturation following conjugation. In the germinated cells, they were enclosed in ERs and gradually consumed in them. This consumption occurred in the cells at the early stages of expansion. The derivative substances may possibly be used for cell surface expansion.

Observations of in vitro gametangial copulation and oosporogenesis in Lagenidium giganteum

An in vitro liquid culture oospore production method yielding 5 × 10 3 oospores/ml was used to follow the sequential events of gametangial copulation and oospore formation in Lagenidium giganteum. Observations were made with Nomarski differential interference microscopy and scanning electron microscopy. After septation and division of fungal thalli into a chain-like series of links, certain individual subthalli differentiated into gametaniga, oogonia, and antheridia. Antheridia issued a fertilization tube which made contact with, and fused to a single oogonium. Copulative behavior was relatively synchronous and necessitated physical contact between thalli. Sexual reproduction was manifested by the migration and condensation of gametes. Plasmogamy was achieved following the introduction of the male gamete into the oogonium. The fused gametes gave rise to a zygote. Small amounts of periplasm remained in the oogonium. Zygote maturation into a fully developed oospore was characterized by the deposition of a multilaminated oospore wall, the coalescence of lipids into a highly refractive central reserve globule surrounded by a layer of fine-grained cytoplasm.

Cell walls, plasma membranes, and dictyosomes during zygote maturation ofClosterium ehrenbergii

The cell wall formation and its correlation with the plasma membrane and dictyosome were investigated by an electron microscope in the zygote cells ofClosterium ehrenbergii. During zygote maturation, six wall layers were formed outside the plasma membrane. Wall layer III was the thickest layer and consisted of microfibril bundles. Dictyosomes produced flat vesicles during formation of wall layer III. Hexagonal arrays of rosette particles appeared in the plasma membrane in this period, thus confirming the simultaneous occurrence of flat vesicles and hexagonal particle arrays in the formation of microfibril bundles even at different stages of the life cycle. Wall layer VI was second in thickness and consisted of single microfibrils. Neither flat vesicles nor hexagonal particle arrays were observed during formation of this layer.

Genetics of Chlamydomonas reinhardtii diploids. II. The effects of diploidy and aneuploidy on the transmission of non-Mendelian markers.

The transmission of two non-Mendelian drug resistance markers has been studied in crosses of Chlamydomonas reinhardtii involving diploids and aneuploids with different mating type genotypes. Under normal laboratory conditions for gametogenesis, mating and zygote maturation, the transmission pattern of the non-Mendelian markers sr-u-1 (resistance to streptomycin) and spr-u-1-27-3 (resistance to spectinomycin) is primarily determined by the mating type genotypes of the parental cells. Our results confirm and expand an earlier observation suggesting that an apparent codominant function of the female (mt+) allele in regulating chloroplast gene transmission in meiosis appears to be distinct and separate from its recessive function in regulating mating behavior. The chloroplast DNA complement (as indexed by the number of extranuclear DNA-containing bodies) may exert a secondary effect on the transmission of these markers. Within a mating type group (mt+/mt- or mt-/mt-) a cell line with more chloroplast DNA tended to transmit its non-Mendelian markers more frequently than a cell line with less chloroplast DNA.

The fate of chloroplast DNA during cell fusion, zygote maturation and zygote germination in Chlamydomonas reinhardi as revealed by DAPI staining

Chlamydomonas reinhardi, a haploid isogamous green alga, presents a classic case of uniparental inheritance of chloroplast genes. Since the molecular basis of this phenomenon is poorly understood, an examination of the cytology of the C. reinhardi plastid DNA was made in gametes, newly formed zygotes, maturing zygotes, and at zygote germination. The single plastid per cell of Chlamydomonas contains a small number of DNA aggregates (‘nucleoids’) which can be seen after staining with DNA-binding fluorochromes. In zygotes formed by pre-stained gametes, the fluorescing nucleoids disappear from the plastid of mating type minus (male) gamete plastids but not from the plastid of mating type plus (female) gamete plastids about 1 h after zygote formation. Subsequently, nucleoids aggregate slowly to a final average of two or three in the single plastid of the mature zygote. Quantitative microspectrofluorimetry indicates that gametes of both mating types have equal amounts of plastid DNA, and that zoospores arising from zygotes have 3.5 × as much as gametes. Assuming degradation of male plastid DNA, there must be a very major synthesis of plastid DNA between zygote formation and zoospore release when zygotes produce the typical 8–16 zoospores. That synthesis appears to occur at germination, where there is a massive increase in plastid DNA and nucleoid number beginning just prior to meiosis. The results support the theory that uniparental inheritance results from degradation of plastid DNA entering the zygote via the male gamete and suggest further studies, using mutants and altered conditions, which might explain how male plastid DNA sometimes survives.

Cytological demonstration of chloroplast DNA behavior during gametogenesis and zygote formation in Chlamydomonas reinhardtii

We used the flourescent dye DAPI to visualize nucleoids of chloroplast DNA and follow their behavior through sexual reproduction by counting nucleoids in fixed cells at various stages. Nucleoid number varied greatly among cells at each stage. The mean number of nucleoids per cell was similar in mt (+) and mt (-) vegetative cells, and declined similarly during gametogenesis. Longer periods of nitrogen starvation reduced the mean nucleoid number further. Mean nucleoid number declined again in mating pairs, and continued to drop in zygotes up to the latest stage that can be examined (24-h zygotes). The oldest zygotes had means of about 2 to 3 nucleoids in different experiments, significantly fewer than in the mt (+) gametes (usually 4 to 5). The quantitative data on nucleoid number, mating efficiency, and germination efficiency allowed us to show that the decrease in nucleoid number is not limited to gametes that do not mate, or to zygotes that do not germinate. These data are consistant with earlier biochemical studies showing loss of chloroplast DNA during gametogenesis in both mating types, and with the degradation of paternal chloroplast DNA detected biochemically and (in non-quantitative studies) by DAPI staining. There may also be some fusion of nucleoids, although if it occurs it is not complete by 24 h of zygote maturation.

Cytological detection of the basis of uniparental inheritance of plastid DNA in Chlamydomonas moewusii

By using the fluorochrome 4'-6-diamidino-2-phenylindole (DAPI) to stain DNA one can follow the pattern of events affecting plastid DNA which occur in the formation and maturation of individual zygotes of the green flagellate Chlamydomonas moewusii. This species, like C. reinhardi, expresses uniparental inheritance of plastid DNA characters among zygote progeny, and is particularly favorable for cytological observation because the locale of the contribution of each gamete can still be recognized in mature zygotes. Gametes contribute equal numbers of DNA nucleoids, and amounts of plastid DNA (as measured by DAPI-DNA micro spectrofluorometry), to the zygote at fusion. Starting at nine hours, coincident with the further fusion of cell contents, plastid DNA disappears from the plastid contributed by one gamete. Further slow coalescence of nucleoids leads to a final nucleoid number per zygote approximately 1/3 of the sum of the 2 gametes.The DNA loss from one gamete plastid may require plastid contact to be initiated. Both light and nutrient availability affect the final number and distribution of plastid DNA nucleoids in the mature zygote. These observations are related to known genetic and biochemical data on uniparental inheritance of plastid characters.

Disintegration of chloroplasts during zygote formation inSpirogyra verruculosa

The chloroplast disintegration during zygote maturation inSpirogyra verruculosa was investigated by electron microscopy. In the seven-day-old zygote about half of the chloroplasts commenced to disintegrate and to turn yellow, losing starch grains, and, then, were torn into fragments of various sizes, which had mostly vesiculated thylakoids and plastoglobules increasing in both size and number. At about two weeks after conjugation, in the cytoplasm, electron-dense structures, linear in section, appeared and vacuoles of various sizes developed. Each of the dense linear structures lying around a fragment seemed to form a cavity of crescent shape in section, and these cavities fused mutually into a large one, leading to the separation of the fragment from the bulk of cytoplasm. The vacuoles seemed to be, involved in the sequestration of the fragments by their fusion with the cavities and by the invagination, of tonoplast. The fragments entrapped by the vacuoles were rapidly broken down into the aggregation of residual membrane pieces, plastoglobules, and undigested starch grains. The maintained chloroplasts changed little in structure compared with the chloroplast of the vegetative cell, and were transmitted to the germling. It is suggested that the eliminated chloroplasts are derived exclusively from the male gamete.

SELF‐STERILE AND MATURATION‐DEFECTIVE MUTANTS OF THE HOMOTHALLIC ALGA, CHLAMYDOMONAS MONOICA (CHLOROPHYCEAE).1

ABSTRACTHomothallic sexual reproduction in Chlamydomonas monoica Strehlow culminated in the formation of mature, chloroform‐resistant zygospores (zygotes) in clonal culture. Early in the zygote maturation process, a distinctive “primary zygote wall” was released into the culture medium where it remained stable for at least several days. This wall appeared as a rigid, darkly‐outlined, and often multilayered structure, as viewed by phase contrast microscopy. From a sample, of 2500 individual clones isolated after ethyl methanesulfonate mutagenesis, five maturation‐defective strains (zym) produced abnormal zygotes which failed to release a primary zygote wall, failed to develop the normal reticulate zygospore wall, and disintegrated within five days. These strains were utilized to identify additional mutants which were sexually competent, but self‐sterile (het). Mixed cultures of the zym and het mutant strains were found to contain numerous, fully‐matured, chloroform‐resistant zygospores and discarded primary zygote walls. In combination, the two types of mutants provided a useful system for the selective recovery of heterozygous zygospores, thus facilitating genetic studies on a homothallic Chlamydomonas.

Cytoplasmic mixing in Saccharomyces cerevisiae: Studies on a grande X neutral petite mating and implications for the mechanism of neutrality

We have studied zygotic cytoplasmic mixing in two neutral petite by grande matings. Cytoplasmic mixing in zygotes and zygotic buds was followed histochemically and genetically. Both techniques showed that in most zygotes rapid cytoplasmic mixing occurred after early zygote formation and before first bud completion. The progeny mitochondrial genotypes produced from each cross were compatible with a model assuming random segregation of petite and grande inputs between the zygote and its first bud.While both crosses produced only grande zygotic colonies and grande diploid progeny when assayed by classical genetic methods, a class of events producing petite zygotes, retaining petite mitochondrial DNA, with grande first diploid buds was discovered. The results show that the expression of neutrality in our strains requires efficient cytoplasmic mixing during zygote maturation. The results suggest that the expression of mitochondrial genome neutrality requires competition or interaction between petite and grande parental mitochondrial DNA molecules. The possible nature of these phenomena is discussed.

Inhibition of zygote germination in Chlamydomonas moewusii (Chlorophyceae, Volvocales) by nitrogen deficiency and sodium citrate

Abstract An investigation was conducted on the effects of nitrogen deficiency and presence of sodium citrate during zygote maturation on subsequent zygote germination in Chlamydomonas moewusii. Maturation of zygotes on medium containing 1 · 7 mM sodium citrate adversely affected germination. The degree of inhibition was related to the type of medium preparation-sterilization regime employed, and varied inversely with the number of times the citrate was autoclaved. The citrate inhibition mechanism is not known, but does not involve inhibition by sodium ions or chelation of exogenous divalent metals. Inclusion of other Krebs cycle intermediates (1 · 7 mM) in the maturation medium did not adversely affect zygote germination. Sodium citrate inhibits growth of both mating-type strains, but not to the same degree as zygote germination. A maturation regime involving nitrogen deficiency and the presence of light, previously reported to promote zygote germination in C. reinhardtii, does not promote C. moewusii zyg...

Novel glycopolypeptide synthesis induced by gametic cell fusion in Chlamydomonas reinhardtii.

Within the first hour of zygote maturation, Chlamydomonas reinhardtii cells stop synthesizing certain polypeptides that characterize the vegetative and gametic stages of the life cycle and initiate the synthesis of novel, zygote-specific polypeptides. At least six of these polypeptides are secreted into the medium, and fine-structural studies indicate that they represent components of the cell wall that is synthesized and secreted early in zygote development. We conclude that a new program of protein synthesis, and possibly also gene transcription, is initiated shortly after gametic cells fuse, a program that appears highly suited to cell-differentiation studies.

Nuclear and mitochondrial DNA replication during zygote formation and maturation in yeast.

Nuclear and mitochondrial DNA replication were monitered during the development of synchronous yeast zygotes. Purified first zygotic buds were also analyzed. Nuclear DNA replicated discontinuously but coincidently with bud initiation, while mitochondrial DNA replicated throughout the zygotic formation and maturation period. First zygotic buds contained the diploid level of both nuclear and mitochondrial DNA.

CONJUGATION IN SPIROGYRA1

SUMMARYConjugating filaments of Spirogyra were examined with both light and electron microscopes. Initially 2 or more filaments of Spirogyra were attached by mucilagenous material. Papillae appeared first in one filament and then in adjacent positions on the other filament. Subsequent growth of papillae separated the conjugating filaments; wall microtubules disappeared in papillae as they elongated. Golgi activity then increased markedly only in the male filament; mucilage production by these Golgi coincided with contraction of the male gamete from its cell wall and may be responsible for its subsequent migration. The end walls separating papillae dissolved to form the conjugation tube, allowing gamete union. The male protoplast then migrated through the tube and further cytoplasmic condensation formed an elliptical‐shaped zygote. During the migration phase, zygote wall formation was initiated and numerous active Golgi apparently contributed material to it. Early zygote maturation was characterized by rapid wall formation and an increase in lipid droplets.

CONJUGATION IN THE DESMIDCLOSTERIUM LITTORALE1

SUMMARYConjugation inClosteriumhas been studied using sectioned material prepared for light and electron microscopy. Prior to conjugation, cells become dense and accumulate lipid droplets. Conjugation commences in paired cells with the formation through a point of mutual contact of a circumferential strip of papilla wall material which is not necessarily centrally situated in each cell. Some microtubules are initially present, near this specialized wall which grows all around each cell but asymmetrically so that the area of papillae in contact increases and each papilla balloons out toward the other, kinking the semicells. Vesicles probably contribute material to this wall. Microtubules disappear from near the papilla as it expands; they are then often found around the older semicell wall immediately adjacent to the papilla. An enlarging vacuole is always formed in each papilla. Cytoplasmic shrinkage is first evident from an accumulation of mucilage between the plasmalemma and wall at the tip of each semicell; the terminal vacuoles collapse and disappear. During further shrinkage of the protoplast, alveolate structures are common on the plasmalemma, and the papilla wall material separating the protoplasts thins out and disappears. Meanwhile, a profound general change renders the cytoplasm far less osmiophilic and stainable for both light and electron microscopy, as it becomes relatively homogeneous and granular, revealing a considerable loss in minor cytoplasmic structures; grana in the chloroplasts become bloated. These changes are not considered processing artefacts, but may result from a breakdown of vegetative cell structures no longer needed for zygote formation. After fusion, the zygote protoplast tends to round up. Zygote maturation commences with deposition of a multilayered wall and shrinkage of the protoplast. Then the very thick zygote wall proper is slowly secreted; meanwhile, very considerable cytoplasmic condensation now renders the cell increasingly osmiophilic and dense until ultrastructural detail becomes totally obscured. Fairly consistent changes in the appearance and probably in the function of the golgi bodies were noted. The results are discussed in terms of the possible function of cell organelles and their role in the mechanics of conjugation.

Studies on DNA replication during synchronized vegetative growth and gametic differentiation in Chlamydomonas reinhardtii

DNA synthesis occurs just prior to nuclear division in synchronized vegetative cultures of Chlamydomonas reinhardtii. The above timing of DNA replication is also observed during gametic differentiation which involves the division of a vegetative cell into four gametes in nitrogen-free medium. When vegetative cells divide to produce four vegetative daughter cells within a single cell cycle, a round of DNA replication appears to precede each of the two consecutive mitotic events. If synchronized vegetative cultures are grown under conditions of limiting light intensity, two rounds of DNA replication may occur while only one nuclear division follows. The vegetative cells produced by such a division contain twice the normal vegetative complement of DNA within a single nucleus. These cells divide without DNA synthesis about 24 h later yielding two cells with the normal vegetative amount of DNA. 15N transfer experiments [14, 17]were performed in order to corroborate the chemical determinations of DNA synthesis and to establish conclusively the exact number of replications of the cellular genome during vegetative growth, gametic differentiation and gametic dedifferentiation. In all cases, DNA replication was by a semiconservative mechanism as previously observed by Sueoka [17]. No appreciable synthesis and accumulation of “M-band” DNA, characteristic of zygote maturation, was observed in these other phases of the Chlamydomonas reinhardtii life cycle. Furthermore, the DNA content per cell never reached the lower limit observed in zoospores produced after meiosis which contain one half the amount of DNA present in gametes and vegetative cells. The results of the present study are discussed in terms of the Chlamydomonas life cycle with particular reference to previous studies of DNA replication during meiosis in this strain of Chlamydomonas.

Replication of chromosomal and cytoplasmic DNA during mitosis and meiosis in the eucaryote Chlamydomonas reinhardi.

Both meiotic and mitotic replication of chromosomal and cytoplasmic DNAs in a unicellular green alga, Chlamydomonas reinhardi, have been studied by isotopic transfer experiments coupled with density-gradient centrifugation analysis. It has been shown that during the vegetative cycle (1) the replication mode of chloroplast DNA, as well as γ DNA, is semiconservative; (2) in synchronized culture these two cytoplasmic DNA satellites replicate coordinately with a high degree of synchrony; (3) the replication of chromosomal DNA is independent of and separable from that of the two cytoplasmic DNAs. It has been shown that during the sexual cycle (1) the chloroplast DNA replicates semiconservatively during zygote maturation, at which time there is neither chromosomal DNA replication nor nuclear division; (2) another DNA component (M-band DNA) replicates extensively and appears in large quantity; (3) meiosis occurs during zygote germination and is accompanied by one round of semiconservative chromosomal DNA replication; (4) the M-band DNA disappears in the early germination period, and the degraded substance is not incorporated into the newly replicated chromosomal DNA. A possible origin of the M-band DNA and a few properties of the cytoplasmic DNA satellites have been elucidated. Genetic recombination and regulation of incoordinate replication in an eucaryotic system have been discussed in terms of the data obtained.

Deoxyribonucleic acid replication in meiosis of Chlamydomonas reinhardi: I. Isotopic transfer experiments with a strain producing eight zoospores

DNA replication in meiosis of Chlamydomonas reinhardi has been studied by 15N density-labeling experiments coupled with cytological observations. It has been shown that (1) during zygotic maturation, there is neither chromosomal DNA replication nor meiotic division; (2) during zygospore germination, meiosis occurs, and the first meiotic division is preceded by a single chromosomal DNA replication; (3) the replication of chromosomal DNA associated with meiosis is semiconservative; (4) only hybrid chromosomal DNA molecules are detected at the end of zygote germination, when eight zoospores emerge from each zygote, indicating that only one DNA replication occurs in the entire period between the mating of gametes and the germination of zygotes; (5) a DNA component (M-band DNA) replicates extensively and appears in large quantity during zygote maturation.