OBSERVATIONS ON THE FINE STRUCTURE OF OEDOGONIUM. V. EVIDENCE FOR THE DE NOVO FORMATION OF PYRENOIDS IN ZOOSPORES OF OE. CARDIACUM123

Ultrastructural studies of the chloroplasts of zoospores and developing zoospores of Oedogonium carcliacum have disclosed the occurrence of numerous incipient pyrenoids. A single developing zoospore may possess several score of these structures which appear to arise de novo in the chloroplast stroma and seem to lack any direct association with mature pyrenoids which are also present in the cells. The incipient pyrenoids lack the associated starch grains and the membrane-limited channels characteristic of mature pyrenoids, but they are readily recognized in the chloroplasts since they demonstrate a greater granularity and electron density than the surrounding chloroplast stroma. The granularity and electron density of the incipient pyrenoids match the ultra-structural appearance of the matrix of mature pyrenoids. The smallest of the incipient pyrenoids examined from serial sections had a maximum diameter of less than 0.3 μ. This may be compared with the size of mature pyrenoids, many with a maximum diameter of over 5.0 μ. In all the zoospores and developing zoospores examined, only one mature pyrenoid was observed in an apparent stage of division.     

FURTHER OBSERVATIONS ON THE FINE STRUCTURE OF THE MACRONUCLEUS IN TOKOPHRYA INFUSIONUM

In a previous paper (8) an organized structure was described in the macronuclei of certain old organisms of Tokophrya infusionum. It was found that the same honeycomb structure appears in great abundance in the macronuclei of overfed organisms. This permitted a better three-dimensional reconstruction of the described structure. Since the defined structure may be experimentally induced, it offers an opportunity for further more detailed studies as to its nature and meaning.     

OBSERVATIONS ON THE FINE STRUCTURE OF CALLIXYLON WOOD

Secondary wood of three species of Callixylon of Lower Mississippian age, preserved by three different modes (fusinization, silicification, and phosphatization), have been studied and characterized in detail. Problems of interpretation at the SEM level of permineralized woods, both containing (silicified wood of Callixylon erianum) and essentially lacking (phosphatized wood of C. arnoldii) original organic cell wall material, are analyzed and discussed. In particular, it is concluded that the flat to curved surfaces showing no evidence of apertures, observed in bordered pit pairs, commonly represent pit membrane surfaces. It is accepted, however, that some concave surfaces might be the mineral accretion surfaces of incomplete pit cavity casts as proposed by Leo and Barghoorn (1976). Regions between groups of pits, previously interpreted as crassulae, may be artifacts of preservation. The fusinized wood has the general appearance of charcoal, but unlike commercially produced charcoal provides evidence of its original microfibrillar structure. The origin of fusain in the fossil record is discussed, and it is concluded that it probably had several origins, including forest fire. Since charcoal can be produced naturally in the absence of O₂ (Brown and Davis, 1973), the suggestion that fusain (charcoal) in the geologic column provides a basis for “assessing oxygen levels in paleoatmospheres” (Cope and Chaloner, 1980) is not supported. Natural sites of fusain production in the absence of O₂ are regions of vulcanism and organic sediments inhabited by anaerobic microorganisms. A circular pattern of crystal orientation in the pit borders of C. arnoldii is interpreted to represent the original microfibrillar pattern. Pit apertures in C. arnoldii are shown to be circular to slightly elliptical. Interpretive evidence of heterogeneous pit membranes in C. arnoldii suggests but does not prove the presence of a torus. The distinctive central region in some pit membranes of the fusinized wood of Callixylon sp. might represent accumulations of waste metabolites. It is argued that a torus would be highly adaptive in large pits with circular apertures.     

OBSERVATIONS ON THE FINE STRUCTURE OF THE MACRONUCLEUS OF TOKOPHRYA INFUSIONUM

The macronucleus in Tokophrya infusionum is composed of numerous Feulgen-positive chromatin bodies (about 0.5 µ in diameter) which appear in thin sections as a dense spongework, homogeneous throughout. The same appearance characterizes metaphase chromosomes of higher forms. Some chromatin bodies of the macronucleus were found to possess a highly organized structure in certain old organisms. This structure appears in cross-sections as a honeycomb and in longitudinal sections as parallel lines about 120 A in diameter evenly spaced (about 230 A). As far as is known this is the first time a regular structure has been found in bodies of chromosomal character at the dimensional level presently explored by electron microscopy. The demonstration that OsO₄ can preserve order in chromatin material is another significant aspect of these findings.     

OBSERVATIONS ON THE FINE STRUCTURE OF SPHEROPLASTS OF RHODOSPIRILLUM RUBRUM

Spheroplasts of the photosynthetic bacterium Rhodospirillum rubrum were prepared from cultures grown in either the presence or absence of light. Cells were converted into spheroplasts by using lysozyme and Versene and fixed in a sucrose-veronal-acetate buffer mixture containing osmium tetroxide. Some preparations were shadow-cast and examined whole; others were embedded in Epon 812 and sectioned. The action of lysozyme and Versene appears to result in removal of the cell wall in strips. The relationship of the chromatophores to the cytoplasmic membrane is readily visualized in sections of broken spheroplasts, and in areas the chromatophores are seen to be continuous with the membrane. In all preparations examined, no definite connections between individual chromatophores were observed. In some cells large spherical granules were evident which either possessed or lacked a clearly visible limiting membrane. On serial sectioning, all granules appeared bounded by a single membrane 40 A wide. The granule membrane was well defined only if the section came from the center of the granule. Sections at other levels showed either a diffuse membrane or no membrane at all. The reasons for this are discussed.     

RADIOAUTOGRAPHIC STUDIES IN SYNCHRONIZED CULTURES OF OEDOGONIUM CARDIACUM

Synthesis of deoxyribonucleic acid (DNA) in synchronized cultures of Oedogonium cardiacum has been studied by radioautography. The germinated zoospores are pulse-labelled for 15 min with thymidine-2-C¹⁴. Use of penicillin in the medium reduces the background in the radioautographs by suppressing the growth of bacteria on Oedogonium cells. Incorporation of labelled precursor is greatly enhanced by growing the cells in a conditioned inorganic medium. The length of the DNA synthesis period (S), as estimated from the curve of percentage of labelled cells versus age during the first cell cycle is about 5-7 hr. The rate of labelling in the nucleus is non-uniform showing a dip during the mid S period. Concomitant with the doubling of DNA in the nucleus a fourfold increase in the nuclear volume is observed.     

THE FINE STRUCTURE OF DIFFERENTIATING XYLEM ELEMENTS

Differentiating xylem elements of Avena coleoptiles have been examined by light and electron microscopy. Fixation in 2 per cent phosphate-buffered osmium tetroxide and in 6 per cent glutaraldehyde, followed by 2 per cent osmium tetroxide, revealed details of the cell wall and cytoplasmic fine structure. The localized secondary wall thickening identified the xylem elements and indicated their state of differentiation. These differentiating xylem elements have dense cytoplasmic contents in which the dictyosomes and elements of rough endoplasmic reticulum are especially numerous. Vesicles are associated with the dictyosomes and are found throughout the cytoplasm. In many cases, these vesicles have electron-opaque contents. "Microtubules" are abundant in the peripheral cytoplasm and are always associated with the secondary wall thickenings. These microtubules are oriented in a direction parallel to the microfibrillar direction of the thickenings. Other tubules are frequently found between the cell wall and the plasma membrane. Our results support the view that the morphological association of the "microtubules" with developing cell wall thickenings may have a functional significance, especially with respect to the orientation of the microfibrils. Dictyosomes and endoplasmic reticulum may have a function in some way connected with the synthetic mechanism of cell wall deposition.     

OBSERVATIONS ON THE FINE STRUCTURE OF THE CRYPTOPHYCEAE. I. THE GENUS CRYPTOMONAS1,2

The fine structure of 3 members of the genus Cryptomonas, C. rostrella, C. reticulata, and C. calceiformis, has been examined. These species exhibit the features typical of the class Cryptophyceae. The presence of subpellicular trichocysts causes a regular folding of the periplast, and larger trichocysts are present within the gullet. The plastid contains thylacoids arranged in pairs and a prominent pyrenoid; both structures are enclosed in 4 membranes, the outermost of which is a rough endoplasmic reticulum. The nucleus, Golgi apparatus and the Corps de Maupas occupy characteristic positions within the cell, and are the most characteristic features. All these structures indicate that this algal class occupies a unique phylogenetic position.     

FINE STRUCTURE OF DWARF MALES IN OEDOGONIUM PLUVIALE (CHLOROPHYCEAE)

Oedogonium pluviale Nordstedt is nannandrous, described as possessing single-celled dwarf males. In some studies the dwarf males appear to have a reduced, lens-shaped pedicular portion whose nature and structure remain unclear. The present study aims to: 1) establish whether dwarf males of O. pluviale are single- or two-celled, 2) describe the different developmental stages of dwarf males, and 3) understand the nature of the pedicular portion. Mitotic division of the dwarf male was observed, but neither upwards phycoplast migration nor synthesis of a gametangial cell wall occurred. Instead, the phycoplast and cell plate migrated downwards, producing a small pedicular portion whose content then degenerated. The antheridium occupied most of the dwarf male wall. Gametogenesis thus took place inside the wall of the young dwarf male (internal gametangium). Because the pedicular cell degenerated, we define this type of dwarf male as pseudo-single-celled to differentiate it from a true single-celled dwarf male. The antheridial nucleus underwent one mitotic division to give rise to two gametes. Instead of a true wall between gametes, mucilage condensed and was observed as a discrete layer that disappeared during gamete release.     

OBSERVATIONS ON THE FINE STRUCTURE OF DICTYOCHA FIBULA EHRENBERG. II. THE PROTOPLAST1

Dictyocha fibula in exponential phase cultures displays a range of morphological variants of which the “sunburst” is most common. In this form, the perinuclear cytoplasm (perikaryon) contains an average of 72 dictyosomes, assorted vesicles, mitochondria, endoplasmic reticulum, and ribosomes. Cytoplasmic processes, globose to irregular, extend on fine cytoplasmic strands from the perikaryon into an extensive, viscous wall, structureless in electron micrographs except for scattered electron-opaque leaflets near the perikaryon. Mitochondria with tubular cristae occur within the globose process and occasionally within the connecting strands. Chloroplasts, with 3-disk lamellar bands and with pyrenoids not crossed by lamellae, are confined to the cytoplasmic processes in the sunburst from. A structure which may be the “flagellum” occasionally occurs attached to the perikaryon. However, no flagellar structures containing microtubules, nor flagellar root structures, have been found.     

SOME CHARACTERISTICS OF THE SPERM ATTRACTANT FROM OEDOGONIUM CARDIACUM1

A bioassay for the sperm attractant from Oedogonium cardiacum was developed. The attractant was confirmed to be highly water soluble, destroyed by heating to 100 C and by dilute acid or alkali. It was fractionated by gel filtration with the attractant accompanied by yellow pigments. It has it molecular weight between 500 and about 1500, based on gel filtration, ultrafiltration, and dialysis. Freezing changes this to both larger and smaller molecules. It appears to have cationic groups. A paper chromatographic separation was found that divided the pigment into at least 3 major bands, one of which was closely associated with the attractant. An absorption spectrum of this pigment showed 1 peak in the visible spectrum at 417 mμ and 3 well-defined peaks in the ultraviolet region. The lack of a peak at 280 mμ indicates the attractant is not a polypeptide.     

CINEMATIC OBSERVATIONS ON THE GROWTH AND DIVISION OF CHLOROPLASTS IN NITELLA

As it elongates from about 0.2 to 80 mm, the Nitella internodal cell shows an increase in plastid number from a few thousand to about 4 million. The increase takes place by plastid division. A continuous motion picture record followed a population of 8 plastids in an elongating cell until their progeny numbered 18, a span longer than 1 fission cycle for some of the plastids. One complete fission-fission cycle was about 22 hr. The highly directed nature of chloroplast expansion (elongation) is lost when cell wall strain (expansion) is mechanically inhibited by pressing the cell between glass plates. The plastids then expand about equally in all directions in the plane of the cell surface. When a new direction of maximum strain is introduced by the mechanical induction of a lateral in the cell, the plastids elongate in this new direction. The direction of the protoplasmic stream does not show this striking response to strain but tends to follow the lines of the chloroplast chains, not the long axis of individual plastids.     

SOME OBSERVATIONS ON THE FINE STRUCTURE OF THE LUTEIN CELLS OF X-IRRADIATED RAT OVARY

The ovaries of 10- to 13-week-old rats were exteriorized and irradiated with sterilizing doses of X-rays. Following treatment, the animals entered a phase of constant vaginal cornification. Animals were killed 8 to 12 wk after the onset of this phase, and their ovaries were prepared for electron microscopy. Tissue was fixed in glutaraldehyde, postfixed in osmium tetroxide (Millonig's, phosphate-buffered), and embedded in Epon. Lutein cells from these ovaries were compared with those from sham-irradiated controls. The cytoplasm of lutein cells from experimental animals was characterized by an increase in the amount of agranular endoplasmic reticulum and by an increase in the number of mitochondria. These mitochondria are more variable in external form and often possess increased numbers of villiform cristae. Other features noted were a decrease in the amount of cytoplasmic lipid granules and an increase in cell size and surface irregularity. The significance of the morphological findings is discussed in relation to ovarian hormone production in animals sterilized by X-irradiation.     

THE EFFECTS OF ISOPROPYL N-PHENYL CARBAMATE ON THE GREEN ALGA OEDOGONIUM CARDIACUM : I. Cell Division

Cell division in vegetative filaments of the green alga Oedogonium cardiacum is presented as an experimental system. We report on how we have used this system to study the effects of isopropyl N-phenylcarbamate (IPC) on the mitotic apparatus and on the phycoplast, a planar array of cytokinetic microtubules. Polymerization of microtubules was prevented when filaments, synchronized by a light/dark regime and chilled (2°C) while in metaphase or just before phycoplast formation, were exposed to 5.5 x 10^-4 M IPC and then returned to room temperature. Spindles reformed or phycoplasts formed when these filaments were transferred to growth medium free of IPC. However, the orientation of both microtubular systems was disturbed: the mitotic apparatus often contained three poles, frequently forming three daughter nuclei upon karyokinesis; the phycoplast was often stellate rather than planar, and it sometimes was displaced to the side of both daughter nuclei, resulting in a binucleate and an anucleate cell upon cytokinesis. Our results suggest that IPC (a) prevents the assembly of microtubules, (b) increases the number of functional polar bodies, and (c) affects the orientation of microtubules in O. cardiacum. High voltage (1,000 kV) electron microscopy of 0.5-µm thick sections allowed us to visualize the polar structures, which were not discernible in thin sections.