OBSERVATIONS ON THE FINE STRUCTURE OF OEDOGONIUM. III. MICROTUBULAR ELEMENTS IN THE CHLOROPLASTS OF OE. CARDIACUM12

The expansive, reticulate cliloroplasts of the filamentous green alga Oedogonium tardiacum were found to contain microtubular elements with an outside diameter of about 270 A. These microtubules have been examined in the chloroplast stroma of vegetative cells, zoospores, zoospore germlings, and eggs. They may occur singly or, more commonly, in groups or bundles of 2 to as many as 30. In longitudinal section these microtubules show a regular pattern of slightly oblique cross striations and the margins appear rough. They bear little structural resemblance to the more familiar and slightly smaller microtubules found in the cytoplasm external to the cliloroplasts. Their overall appearance suggests that each is constructed of 2 helically wound, ribbon-like units that are approximately 155-175 A broad and 45-55 A thick. The 2 units of this proposed double helix appear to lie in juxtaposition at a pitch of 17-27 deg. The dimensions of each unit, or helix, are such that it could conceivably be constructed of 3 filamentous subunits similar to those already described in the literature for other microtubular elements.     

OBSERVATIONS ON THE FINE STRUCTURE OF OEDOGONIUM. II. THE SPERMATOZOID OF O. CARDIACUM

Hoffman , L. R., and Irene Manton . (U. Leeds, England.) Observations on the fine structure of Oedogonium. II. The spermatozoid of O. cardiacum. Amer. Jour. Bot. 50(5): 455–463. Illus. 1963.—Salient features of the fine structure of the spermatozoid of Oedogonium cardiacum are described and illustrated as they appear in whole mounts and in sections. There is a close resemblance to the zoospore of the same species (Hoffman and Manton, 1962) though the gamete is smaller and in some respects simpler. The flagella, though similar in length to those on the zoospore, are fewer (ca. 30 instead of ca. 120 per cell). The construction of the flagellar ring is similar though there is less mechanical material associated with the flagellar bases in the gamete. Compound “roots” alternating with the flagellar bases are identical in structure and relative position in both types of motile cells; there is no direct connection with the nucleus. Other details of resemblance and difference between the spermatozoid and the zoospore are discussed.     

ON THE DE NOVO FORMATION OF THE CENTRIOLE IN THE ACTIVATED SEA URCHIN EGG

Eggs of Pseudocentrotus depressus were activated artificially by Loeb's "double treatment method". 50 min after activation, a number of asters were produced in the eggs. It was confirmed by electron microscopy that centrioles with a typical fine structure were present in artificially induced asters.
An unfertilized egg of Hemicentrotus pulcherrimus was divided into 2 halves, nucleated and non-nucleated, by centrifugation on a sucrose bed. Each half was activated by the same method as mentioned above. Several asters were produced in both halves after a certain period of incubation. The presence of bodies considered to be centrioles were demonstrated in the asters in both nucleated and non-nucleated halves.
The results add probability to the view that the centrioles are produced de novo in artificially activated eggs and fragments.     

BIOPTERIN V. DE NOVO SYNTHESIS OF DIHYDROBIOPTERIN: EVIDENCE FOR ITS QUINONOID STRUCTURE AND LACK OF DEPENDENCE OF ITS REDUCTION TO TETRAHYDROBIOPTERIN ON DIHYDROFOLATE REDUCTASE

Samples of quinonoid-l -erythrodihydrobiopterin (q-BH₂) and quinonoid-6-methyl-dihydro-pterin (q-6-MPH₂) were prepared by oxidation of l -erythro-5,6,7,8-tetrahydrobiopterin (BH₄) and 5,6,7,8-tetrahydro-6-methylpterin (6-MPH₄) and separated from ^D-erythro-7,8-dihydrobiopterin (7,8-BH₂) and 6-methyl-7,8-dihydropterin (7,8-6-MPH₂) as well as from the tetrahydropterins on phosphocellulose column by high-pressure liquid chromatography. The quinonoid dihydropterins were identified and quantitated by scan of their ultraviolet absorption and fluorescence emission spectra through their rearrangement to their 7,8-tautomer and also by gas chromatography of their rapidly synthesized trimethylsilyl derivative. Identification was also achieved by the enzymatic reduction of [³H]q-BH²to [³H]BH₄ by dihydrofolate reductase (DHFR). Direct proof for the enzymatic synthesis of the q-BH² from GTP or from 2-amino-6-(5′-triphosphoribosyl)-amino-5- or -6-formamido-6-hydroxypyrimi-dine (FPyd-P₃) was obtained by isolation of the compound which was identical in all respects to the q-BH₂ obtained by chemical synthesis from BH₄. The reduction of enzymatically synthesized q-BH² by dihydropteridine reductase (DHPR) to BH⁴ was not inhibited by methotrexate (MTX). When the enzymatically synthesized q-BH² was converted to 7,8-BH₂, it was reduced only by DHFR. This reduction, however, was inhibited by MTX. On the biosynthetic pathway from GTP to dihydrobiopterin, the enzyme responsible for the appearance of the quinonoid structure is the d -erythro-dihydroneopterin triphosphate synthetase, the product of which (quinonoid d -erythro-dihydroneopterin triphosphate) is converted to quinonoid dihydrobiopterin by l -erythro-dihydrobiopterin synthetase. Experiments in vivo established that DHFR does not participate in the reduction of dihydrobiopterin to tetra-hydrobiopterin when the former is synthesized from GTP de novo. MTX at 5 × 10^?6M exerted no inhibition on the reduction of the biosynthetic dihydrobiopterin to tetrahydrobiopterin in vivo, yet completely inhibited the reduction of intraventricularly injected tritiated dihydrofolate ([³H]FH₂) to tritiated tetrahydrofolate ([³H]FH₄).     

OBSERVATIONS ON THE FINE STRUCTURE OF LUTEIN CELLS

Corpora lutea from the period of delayed implantation and from early postimplantation stages of the armadillo, mink, and rat were fixed in buffered osmium tetroxide-sucrose or potassium permanganate. After rapid dehydration, the portions of the corpora lutea were embedded in either methacrylate or epoxy resin. Examination of the lutein cells by electron microscopy revealed the presence, in the better preserved material, of an extensive development of tubular agranular endoplasmic reticulum. Although the membranes of the endoplasmic reticulum are the most striking feature of the lutein cells of both stages of the three animals examined, very numerous large mitochondria with cristae that exhibit a variety of forms tending toward villiform, and protrusions and foldings of the lutein cell margins on the pericapillary space are also characteristic of these cells. Certain minor differences in the lutein cells of the species examined are also noted. No indications of conversion of mitochondria into lipid, of accumulation of lipid in the Golgi area, or of the protrusion of lutein cells into spaces between the endothelial cells, as suggested by other authors, were noted in these preparations. Some of the difficulties inherent in the visualization of the secretory activity of cells producing steroid hormones are briefly discussed.     

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.     

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 THE DEVELOPING SPERMATID IN THE DOMESTIC CHICKEN

The kinetic apparatus, the acrosome and associated structures, and the manchette of the spermatid of the domestic chicken have been studied with the electron microscope. The basic structural features of the two centrioles do not change during spermiogenesis, but there is a change in orientation and length. The proximal centriole is situated in a groove at the edge of the nucleus and oriented normal to the long axis of the nucleus and at right angles to the elongate distal centriole. The tail filaments appear to originate from the distal centriole. The plasma membrane is invaginated along the tail filaments. A dense structure which appears at the deep reflection of the plasma membrane is identified as the ring. The fine structure of the ring has no resemblance to that of a centriole and there is no evidence that it is derived from or related to the centrioles. The tail of the spermatid contains nine peripheral pairs and one central pair of tubular filaments. The two members of each pair of peripheral filaments differ in density and in shape: one is dense and circular, and the other is light and semilunar in cross-section. The dense filaments have processes. A manchette consisting of fine tubules appears in the cytoplasm of the older spermatid along the nucleus, neck region, and proximal segment of the tail. The acrosome is spherical in young spermatids and becomes crescentic and, finally, U-shaped as spermiogenesis proceeds. A dense granule is observed in the cytoplasm between acrosome and nucleus. This granule later becomes a dense rod which is interpreted as the perforatorium.     

OBSERVATIONS ON THE FINE STRUCTURE OF PHARYNGEAL MUSCLE IN THE PLANARIAN DUGESIA TIGRINA

Pharyngeal muscle of the planarian Dugesia tigrina was studied by electron microscopy after osmium tetroxide fixation. The muscle cell was observed to contain one myofibril or bundle of myofilaments parallel to its longitudinal axis. The myofilaments were of two types, different in size and distribution. No Z lines or myofilament organization into cross or helical striations were seen. Dense bodies were seen as projections from an invagination of the plasma membrane and as dense lines parallel to the myofilaments. The muscle cells are surrounded by a plasma membrane which is structurally associated with dense body projections, with vesicles and cisternae of sarcoplasmic reticulum, and with synaptic nerve endings. The cell has sarcoplasmic projections perpendicular to its long axis; these projections are seen to contain the nucleus or mitochondria and granules. Mitochondria and granules are also seen in a sarcoplasm rim around the fibril. The dense bodies may serve as attachment for thin myofilaments and function in transmission of stimuli from plasma membrane to the interior of the fibril.     

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.     

SOME OBSERVATIONS ON THE FINE STRUCTURE OF THE SYMPATHETIC GANGLION OF BULLFROG

Electron microscope observations of abdominal sympathetic ganglia of American bullfrogs, Rana catesbiana, have demonstrated the presence of specific areas of cytoplasm in the superficial zone of the perikaryon which are devoid of granulated endoplasmic reticulum. These areas are occupied almost exclusively by granules 200 to 400 A in diameter which can be stained intensely with lead hydroxide but faintly with uranyl acetate. Each granule shows subgranular internal structure after the lead staining. Granules of similar properties are found in synapses also, and may be glycogen. From the satellite cell there extends a number of leaf- or finger-like cytoplasmic projections around the root portion of the nerve process. Some of these projections directly cover the surface of the nerve process. Many others, however, are separated from the neuron by a fairly wide interspace. Multivesicular bodies of the neuron are occasionally observed in a configuration which suggests that they are being extruded from the root of the nerve process into the interspace. Filaments about 100 A in thickness are found in the satellite cell cytoplasm. They are arranged more or less parallel to each other and are especially well developed around synapses and nerve fibers.     

FURTHER OBSERVATIONS ON THE FINE STRUCTURE OF THE PARIETAL EYE OF LIZARDS

An electron microscopical study of the third eye of the Western Fence Lizard, Sceloporus occidentalis, fixed with 1 per cent osmium tetroxide, pH 7.4–7.6, for 16 to 20 hours at 0°C., revealed the following new facts. The fibrillar system of the retinal photoreceptor consists of nine double fibrils enclosed in a sheath. Pigment cells and lens cells possess similar systems. Two short cylindrical centrioles are associated with the fibrillar apparatus: one, from which striated rootlets extend inward, lies at the base of the fibrils, with the other at an oblique angle to the axis of the system. A Golgi complex, whorls of endoplasmic reticulum, lipid (?) droplets, and other organelles and inclusions in the photoreceptors are described. An axon leads from the base of the photoreceptor into the nervous layer of the retina which consists of many nerve fibers and large ganglion cells. Although the pattern of neural connections has not yet been determined, some synapses were found. The parietal nerve consists of about 250 non-medullated fibers. The capsule of the eye usually has a layer of iridocytes, which contain rows of guanine (?) rods. A few parietal eyes of the Granite Night Lizard, Xantusia henshawi, were also examined. Large lipid (?) droplets occur in the bases of their receptoral processes.     

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.     

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.     

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 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.