Nuclear membrane behavior during mitosis in normal and heteroploid Myxomycetes

Summary Photomicrographic evidence is presented of the difference in behavior of nuclear membranes during mitosis in amoebae, zygotes and plasmodia of Myxomycetes. One of the species was cultured on bacteria and possessed a normal cycle of plasmogamy and karyogamy between the amoebal and plasmodial phases. The second species was axenically grown in liquid media and had become highly heteroploid and lacked the ability to develop into plasmodia, existing only in the amoeboid form. The significance of the amoeboid form of mitosis in the heteroploid axenically cultured strain is discussed in relation to the difference in nuclear membrane behavior and the possible significance of such behavior.     

Mutations affecting the initiation of plasmodial development in Physarum polycephalum

In the heterothallic myxomycete Physarum polycephalum, uninucleate amoebae normally differentiate into syncytial plasmodia following heterotypic mating. In order to study the genetic control of this developmental process, mutations affecting the amoebal-plasmodial transition have been sought. Numerous mutants characterized by self-fertility have been isolated. The use of alkylating mutagens increases the mutant frequency over the spontaneous level but does not alter the mutant spectrum. Three spontaneous and 14 induced mutants have been analyzed genetically. In each, the mutation appears to be linked to the mating type locus. In three randomly selected mutants, the nuclear DNA content is the same in amoebae and plasmodia, indicating that amoebal syngamy does not precede plasmodium development in these strains. These results indicate that a highly specific type of mutational event, occurring close to or within the mating type locus, can abolish the requirement for syngamy normally associated with plasmodial differentiation. These mutations help define a genomic region regulating the switch from amoebal to plasmodial growth.     

Cytoskeletal and nuclear organization in mouse embryos derived from nuclear transfer and ICSI: a comparison of agamogony and syngamy before and during the first cell cycle

In this study, somatic cell nuclear transfer (SCNT) and intracytoplasmic sperm injection (ICSI) are used as models of agamogony and syngamy, respectively. In order to elucidate the reasons of low efficiency of somatic cell cloning, cytoskeletal and nuclear organization in cloned mouse embryos was monitored before and during the first cell cycle, and compared with the pattern of ICSI zygote. A metaphase-like spindle with alignment of condensed donor chromosomes was assembled within 3 hr after NT, followed by formation of pronuclear-like structures at 3-6 hr after activation, indicating that somatic nuclear remodeling depends on microtubular network organization. The percentage of two (pseudo-) pronuclei in cloned embryos derived from delayed activation was greater than that in immediate activation group (68.5% vs. 30.8%, P<0.01), but similar to that of ICSI group (68.5% vs. 65.5%, P>0.05). The 2-cell rate in NT embryos was significantly lower than that in zygotes produced by ICSI (64.8% vs. 82.5%, P<0.01). Further studies testified that the cloned embryos reached the metaphase of the first mitosis 10 hr after activation, whereas this occurred at 18 hr in the ICSI zygotes. Comparision of the pattern of microfilament assembly in early NT embryos with that in syngamic zygotes suggested that abnormal microfilamental pattern in cloned embryos may threaten subsequent embryonic development. In conclusion, agamogony, in contrast to syngamy, displays some unique features in respect of cytoskeletal organization, the most remarkable of which is that the first cell cycle is initiated ahead distinctly, which probably leads to incomplete organization of the first mitotic spindle, and contributes to low efficiency of cloning.     

Studies on fertilization of the teleost. II. Nuclear behavior and changes in histone H1 kinase

In order to understand the dynamic responses of gamete nuclei upon fertilization in the fish, Oryzias latipes, the relationship between changes in the activity of histone H1 kinase and nuclear behavior was examined during fertilization. Kinase activity rapidly decreased concomitant with the initiation of the propagative exocytosis of cortical alveoli following sperm attachment to the egg plasma membrane post-insemination (PI). Activity again increased 30 min PI. Similar changes in kinase activity, migration and syngamy of pronuclei, and subsequent cleavage were observed with aphidicolin or actinomycin D treatment, except that formation of abnormal metaphase chromosomes was retarded in aphidicolin-treated zygotes. Pretreatment of unfertilized eggs with cycloheximide or 6-dimethylaminopurine (6-DMAP) caused no nuclear changes. The activity of histone H1 kinase in these eggs rapidly declined following sperm penetration and exocytosis, but did not undergo subsequent increase in the presence of these inhibitors. In these eggs with low histone H1 kinase activity, the fertilization process from sperm penetration to syngamy occurred normally, but the pronuclear membrane did not break down and the chromosomes did not condense. The present data suggest that in fish eggs, DNA replication as well as the synthesis and phosphorylation of proteins, especially cyclin B, are required for normal formation of metaphase chromosomes at the first cleavage, but not for fertilization events from sperm penetration through to nuclear migration resulting in syngamy.     

Apogamic development of plasmodia in the myxomycetePhysarum polycephalum: a cinematographic analysis

Summary Strain CL ofPhysarum polycephalum forms multinucleate plasmodia within clones of uninucleate amoebae. The plasmodia have the same nuclear DNA content as the amoebae. Analysis of plasmodial development, using time-lapse cinematography, showed that binucleate cells were formed as a result of nuclear division in uninucleate cells. Binucleate cells developed into plasmodia by further nuclear divisions and cell fusions. No fusions involving uninucleate cells were observed. A temporary increase in cell and nuclear size occurred at the time of binucleate cell formation.     

Protein synthesis and the cell cycle: centrosome reproduction in sea urchin eggs is not under translational control

The reproduction, or duplication, of the centrosome is an important event in a cell's preparation for mitosis. We sought to determine if centrosome reproduction is regulated by the synthesis and accumulation of cyclin proteins and/or the synthesis of centrosome-specific proteins at each cell cycle. We continuously treat sea urchin eggs, starting before fertilization, with a combination of emetine and anisomycin, drugs that have separate targets in the protein synthetic pathway. These drugs inhibit the postfertilization incorporation of [35S]methionine into precipitable material by 97.3-100%. Autoradiography of SDS-PAGE gels of drug-treated zygotes reveals that [35S]methionine incorporates exclusively into material that does not enter the gel and material that runs at the dye front; no other labeled bands are detected. Fertilization events and syngamy are normal in drug-treated zygotes, but the cell cycle arrests before first mitosis. The sperm aster doubles once in all zygotes to yield two asters. In a variable but significant percentage of zygotes, the asters continue to double. This continued doubling is slower than normal, asynchronous between zygotes, and sometimes asynchronous within individual zygotes. High voltage electron microscopy of serial semithick sections from drug-treated zygotes reveals that 90% of the daughter centrosomes contain two centrioles of normal appearance. From these results, we conclude that centrosome reproduction in sea urchin zygotes is not controlled by the accumulation of cyclin proteins or the synthesis of centrosome-specific proteins at each cell cycle. New centrosomes are assembled from preexisting pools of ready-to-use subunits. Furthermore, our results indicate that centrosomal and nuclear events are regulated by separate pathways.     

STUDIES ON DIPLOID STRAINS OF DICTYOSTELIUM DISCOIDEUM

Ross, Ian K. (Yale U., New Haven, Conn.) Studies on diploid strains of Dictyostelium discoideum. Amer. Jour. Bot. 47 (1) : 54—59. Illus. 1960.–Three strains of Dictyostelium discoideum having the diploid number of chromosomes (14) at all stages of their life cycle were examined. No evidence of sexuality as shown by syngamy or meiosis was found in the diploid strains. Two of the diploid strains were unstable and reverted to the haploid type with 7 chromosomes. These haploid strains had a sexual phase in which syngamy and meiosis were observed. The nuclear behavior of both the diploid and haploid strains differed from that reported in previous papers.     

WHAT DO WE KNOW ABOUT CHAROPHYTE (STREPTOPHYTA) LIFE CYCLES?1

The charophyte algae are the closest living relatives of land plants. Their life cycles are usually characterized as haploid with zygotic meiosis. This conclusion, however, is based on a small number of observations and on theoretical assumptions about what kinds of life cycle are possible. Little is known about the life cycles of most charophytes, but unusual phenomena have been reported in comparatively well‐studied taxa: Spirogyra and Sirogonium are reported to produce diploid gametes with synapsis of homologous chromosomes before fusion of gametic nuclei; Closterium ehrenbergii is reported to undergo chromosome reduction both before and after syngamy; and zygotes of Coleochaete scutata are reported to replicate their DNA to high levels before a series of reduction divisions. All of these phenomena require confirmation, as does the conventional account.     

Life Cycle of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Plasmodiphora brassicae is a soil-borne obligate parasite. The pathogen has three stages in its life cycle: survival in soil, root hair infection, and cortical infection. Resting spores of P. brassicae have a great ability to survive in soil. These resting spores release primary zoospores. When a zoospore reaches the surface of a root hair, it penetrates through the cell wall. This stage is termed the root hair infection stage. Inside root hairs the pathogen forms primary plasmodia. A number of nuclear divisions occur synchronously in the plasmodia, followed by cleavage into zoosporangia. Later, 4–16 secondary zoospores are formed in each zoosporangium and released into the soil. Secondary zoospores penetrate the cortical tissues of the main roots, a process called cortical infection. Inside invaded roots cells, the pathogen develops into secondary plasmodia which are associated with cellular hypertrophy, followed by gall formation in the tissues. The plasmodia finally develop into a new generation of resting spores, followed by their release back into soil as survival structures. In vitro dual cultures of P. brassicae with hairy root culture and suspension cultures have been developed to provide a way to nondestructively observe the growth of this pathogen within host cells. The development of P. brassicae in the hairy roots was similar to that found in intact plants. The observations of the cortical infection stage suggest that swelling of P. brassicae-infected cells and abnormal cell division of P. brassicae-infected and adjacent cells will induce hypertrophy and that movement of plasmodia by cytoplasmic streaming increases the number of P. brassicae-infected cells during cell division.     

Acceleration of mitosis induced by mitotic stimulators of Physarum polycephalum

Extracts of the myxomycete Physarum polycephalum exhibit an accelerating effect on nuclear division which fluctuates during the synchronous nuclear division cycle. Extracts from late G2 phase plasmodia can advance mitosis in recipient test plasmodia by up to 30% of the length of the control cycle. The advancing capacity of extracts is heat- and ammonium sulphate-precipitable, non-dialysable and destroyed by pronase, suggesting that the active substance is a protein. The advance of mitosis is in strong correlation with the applied dose of stimulatory material.     

A Comparative Study of Mitosis in Amoebae and Plasmodia of the True Slime Mold Didymium nigripes

SYNOPSIS. Studies comparing mitosis in amoebae and plasmodia of the true slime mold Didymium nigripes reveal that at the time of differentiation pronounced changes occur in the mitotic process. Not only does the amount of time required for division of the 2 stages differ, but plasmodial mitosis is characterized by persistence of the nuclear membrane and the apparent lack of centrioles. The origin of multinucleate plasmodia from uninucleate cells which have already undergone cytoplasmic differentiation is described. Division time in a population of amoebae becomes more uniform after those cells which are destined to form plasmodia have differentiated.
The observations and data presented indicated that differences in mitotic behavior also occur between amoebae of 3 stocks with differences in plasmodial structure and behavior. Comparison of mitosis in the plasmodia of these 3 stocks revealed no significant differences.     

Comparative Study of Ultrastructure of Interlamellar and Intralamellar Types of Henneguya exilis Kudo from Channel Catfish*†

SYNOPSIS. The inter- and intralamellar types of Henneguya exilis Kudo (Myxosporida) infections from channel catfish are similar in spore structure and sporogenesis, but differ in the structure of their plasmodium wall and surface coat and in their relationship with the host cells. The 2 clinical types differ also in the sites of development and growth patterns of plasmodia within a gill filament. Interlamellar plasmodia are limited by 2 outer unit membranes which give rise to both single-and double-membraned pinocytic canals. Intralamellar plasmodia are limited by a single outer unit membrane which gives rise to single-membraned pinocytic canals. Interlamellar plasmodia are covered by a fine granular coat of highly variable thicknesses; in some regions there is direct contact between the parasite and cells of the host. There is some evidence that host cell cytoplasm as well as interstitial material are taken in by interlamellar plasmodia. In contrast, intralamellar plasmodia are covered by a fine granular coat of almost uniform thickness, which prevents direct contact between the parasite and cells of the host; probably only interstitial material is taken by these plasmodia.     

Patterns of oscillation during mitosis in plasmodia ofPhysarum polycephalum

Summary The rhythmic contraction pattern in plasmodia ofPhysarum polycephalum was studied to determine whether characteristic changes occur during the synchronized nuclear division. An electrical method that measures the contraction rhythm in situ during several cell cycles was used. Biopsies of the plasmodia were taken at 17 min intervals for precise determination of the cell cycle stages and were correlated with the simultaneously measured contraction rhythm. All measurements were performed in a temperature controlled environment (27 °C) at 100% relative humidity with the plasmodia (less than 24 h old) growing on a semi-defined agar medium. A total of 14 different plasmodia have been examined, and on one occasion the plasmodium was followed through 3 subsequent mitoses. The mitotic stages were identified with aceto-orcein coloring techniques and by fluorescence methods. Except for a few cases where a mitotic asynchrony of 2–3 min was observed, the mitotic events occurred simultaneously in the nuclei within a single plasmodium. Both the occurrence of the first mitosis after inoculation and the intermitotic times were highly variable. Our study indicates that the contraction rhythm in plasmodia ofPhysarum is unperturbed during the synchronized nuclear division. However, in 5 of the 17 examined mitoses an amplitude decay was observed. We discuss possible explanations for the obtained results with emphasis on the applied techniques, interpretation of the oscillation patterns, and possible restrictions in the cell itself.     

Cellular events during sexual development from amoeba to plasmodium in the slime mould Physarum polycephalum

Time-lapse cinematography and immunofluorescence microscopy were used to study cellular events during amoebal fusions and sexual plasmodium development in Physarum polycephalum. Amoebal fusions occurred frequently in mixtures of strains heteroallelic or homoallelic for the mating-type locus matA, but plasmodia developed only in the matA-heteroallelic cultures. These observations confirmed that matA controls development of fusion cells rather than cell fusion. Analysis of cell pedigrees showed that, in both types of culture, amoebae fused at any stage of the cell cycle except mitosis. In matA-heteroallelic fusion cells, nuclear fusion occurred in interphase about 2 h after cell fusion; interphase nuclear fusion did not occur in matA-homoallelic fusion cells. The diploid zygote, formed by nuclear fusion in matA-heteroallelic fusion cells, entered an extended period of cell growth which ended in the formation of a binucleate plasmodium by mitosis without cytokinesis. In contrast, no extension to the cell cycle was observed in matA-homoallelic fusion cells and mitosis was always accompanied by cytokinesis. In matA-homoallelic cultures, many of the binucleate fusion cells split apart without mitosis, regenerating pairs of uninucleate amoebae; in the remaining fusion cells, the nuclei entered mitosis synchronously and spindle fusion sometimes occurred, giving rise to a variety of products. Immunofluorescence microscopy showed that matA-heteroallelic fusion cells possessed two amoebal microtubule organizing centres, and that most zygotes possessed only one; amoebal microtubule organization was lost gradually over several cell cycles. In matA-homoallelic cultures, all the cells retained amoebal microtubule organization.     

Selective disappearance of maternal centrioles after fertilization in the anisogamous brown alga Cutleria cylindrica (Cutleriales, Phaeophyceae): Paternal inheritance of centrioles is universal in the brown algae

The behavior of centrioles in zygotes and female gametes developing parthenogenetically in the anisogamous brown alga Cutieria cyiindrica Okamura was studied using electron and immunofluorescence microscopy. Two pairs of centrioles, detected using anti-centrin antibody, were observed in the vicinity of the male and female nuclei, respectively, just after plasmogamy. The fluorescence intensity of one of the two centrin foci became weak 6 h after plasmogamy and finally disappeared. It was impossible to determine whether the male- or female-derived centrioles disappeared in zygotes, because there was nothing to detect morphological differences between the two centrioles. However, a prominent anti-centrin staining focus was located at the condensed male nucleus in zygotes in which karyogamy had not occurred yet. As a result, it was considered that the maternally inherited centrioles had selectively disappeared during development in C. cylindrica. The paternal inheritance of centrioles in zygotes was also confirmed by electron microscopy. Considering previous observations from oogamous and isogamous species of brown algae, we concluded that the paternal inheriance of centrioles could be universal in the brown algae.     

A Proposed Life Cycle of Minchinia nelsoni (Haplosporida, Haplosporidiidae) in the American Oyster Crassostrea virginica

SYNOPSIS. A developmental sequence is proposed for the haplosporidan Minchinia nelsoni Haskin, Stauber and Mackin, 1966, based on study of oyster infections over the past 5 years in Chesapeake Bay. Uninucleate stages develop by nuclear division into multinucleate plasmodia which proliferate in the tissues by plasmotomy. Relatively small plasmodia containing what are considered to be gametic nuclei originate by unequal plasmotomy of large plasmodia. These have been interpreted to aggregate and fuse to form large plasmodia which contain prozygotes. Pairing and fusion of nuclei occur within each plasmodium to produce zygote nuclei (synkaryons) which undergo division, possibly meiotic, to form sporonts. Sporoblasts differentiate into spores with the development of spore walls and opercula. Cystoid plasmodia develop during times of unfavorable conditions. An anomalous but common sequence involving sexuality and mitosis is described, and the occurrence of various life cycle stages within the host thruout the year is discussed.     

CRUCIFORM NUCLEAR DIVISION IN WORONINA PYTHII (PLASMODIOPHOROMYCETES)

Somatic nuclear divisions in sporangiogenous plasmodia of Woronina pythii Goldie-Smith were studied with transmission electron microscopy. During metaphase, each nucleus formed a cruciform configuration as chromatin became aligned at the equatorial plate perpendicular to the persistent nucleolus. Except for polar fenestrations, the original nuclear envelope remained intact throughout the mitotic division. Intranuclear membranous vesicles appeared to bleb off the inner membrane of the original nuclear envelope, adhered to the surfaces of the separating chromatin, and eventually formed new daughter nuclear envelope within the original nuclear envelope. During the first 24 hr of vegetative plasmodial growth, each telophase nucleus exhibited an obvious constriction of the original nuclear envelope in the interzonal region. Similar constrictions were not evident in telophase nuclei found in 24–36-hr-old plasmodia. This variation in the ultrastructural morphology of cruciform division appears to be related to the age and size of each sporangiogenous plasmodium, and is the first to be documented within this group of fungal pathogens.     

Meiosis in the aquatic fungusCatenaria allomycis

Summary Catenaria allomycis Couch (Blastocladiales) is an endobiotic fungal parasite primarily of species of the genusAllomyces. The life cycle ofC. allomycis contains both sexual and asexual phases. Synaptonemal complexes have been found in young developing resistant sporangia (RS) suggesting that meiosis occurs within the thick walled RS prior to syngamy. Ultrastructural evidence suggests that meiosis proceeds through pachytene in the developing RS and is arrested in diplotene of prophase I until the sporangia are induced to germinate at which time the meiotic process is completed. Quantitative nuclear counts in developing RS support the ultrastructural observations. Meiotic nuclei are characterized by polar fenestrae in the nuclear envelope and intranuclear plaque-like microtubule organizing centers (MTOC).Portion of a Ph.D. dissertation submitted by the senior author to the Graduate School, University of Georgia.     

In vitro Development of Murine Embryos Using Different Types of Microinjections

We studied the effects of different types of microinjections, such as the mechanical damage to cytoplasmic and nuclear membranes of the zygote and the injection of various gene-engineering constructs or buffer solutions into the cytoplasm or the pronucleus, on the preimplantation of murine embryos (CBA × C57BL)F₁. The survival rate of the embryos was estimated by their capacity to develop in vitro to the blastocyst or hatched blastocyst stages. Puncture of the cytoplasm using a microneedle and injection of buff or foreign DNA did not affect the zygotes capacity for further in vitro development. But, the puncture of the pronucleus and microinjection of gene-engineering constructs or buffer into it reliably decreased the survival rate of embryos, as compared to the control. The differences were found in the capacity of murine zygotes for in vitro development after injection with gene-engineering constructs.     

CRUCIFORM NUCLEAR DIVISION IN SOROSPHAERA VERONICAE

Sorosphaera veronicae Schroet. is an endobiotic, holocarpic, obligately parasitic fungus presently classified in the Plasmodiophoromycetes. The ultrastructure of nuclear envelope formation in somatic nuclear division in cystosoral plasmodia was studied. The inner membrane of the nuclear envelope during prophase appears to invaginate and blebb off intranuclear membranous vesicles. The intranuclear membranous vesicles become associated with the surface of the separating chromatin in anaphase and eventually are involved in the formation of daughter nuclear envelopes within the original nuclear envelope. The sequence of nuclear envelope breakdown and reformation in S. veronicae is noteworthy because it emphasizes alternate methods of nuclear envelope formation other than the generally considered “typical” formation described in Allium cepa L.