THE DEMONSTRATION OF THE SUCCINIC DEHYDROGENASE SYSTEM IN BACILLUS SUBTILIS USING TETRANITRO-BLUE TETRAZOLIUM COMBINED WITH TECHNIQUES OF ELECTRON MICROSCOPY

Activity of the succinic dehydrogenase system was studied in Bacillus subtilis utilizing combined techniques of cytochemistry and electron microscopy. Organisms were incubated in a medium containing tetranitro-blue tetrazolium (TNBT) which served as an electron acceptor. Enzymatic activity, as evidenced by deposition of TNBT-formazan, was found on membranous organelles associated with the cytoplasmic membrane and septal plasma membrane, the nuclear area, and the plasma membrane. Flagella, ~190 A in diameter, with thorn-like projections protruded through the cell wall. Tangential-oblique sections of the cell wall showed many pores ~220 A in diameter with a center-to-center spacing of ~450 A.     

INTRACELLULAR LOCALIZATION OF ENZYMES IN SPLEEN : I. REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE CYTOCHROMEc REDUCTASE, CYTOCHROMEc OXIDASE, AND SUCCINIC DEHYDROGENASE IN THE RAT AND GUINEA PIG

1. The intracellular distribution of nitrogen, DPNH cytochrome c reductase, succinic dehydrogenase, and cytochrome c oxidase has been studied in fractions derived by differential centrifugation from rat and guinea pig spleen homogenates. 2. In the spleens of each species, the nuclear fraction accounted for 40 to 50 per cent of the total nitrogen content of the homogenate, and the mitochondrial, microsome, and supernatant fractions contained about 8, 12, and 30 per cent of the total nitrogen, respectively. 3. Per mg. of nitrogen, DPNH cytochrome c reductase was concentrated in the mitochondria and microsomes of both rat and guinea pig spleens. Seventy per cent of the total DPNH cytochrome c reductase activity was recovered in these two fractions. The reductase activity associated with the nuclear fraction was lowered markedly by isolating nuclei from rat spleens with the sucrose-CaCl₂ layering technique. The lowered activity was accompanied by the recovery of about 90 per cent of the homogenate DNA in the isolated nuclei, indicating that little, if any, of the reductase is present in spleen cell nuclei. 4. Per mg. of nitrogen, succinic dehydrogenase was concentrated about 10-fold in the mitochondria of rat spleen, and 65 per cent of the total activity was recovered in this fraction. 5. Cytochrome c oxidase was concentrated, per mg. of nitrogen, in the mitochondria of both rat and guinea pig spleens. The activity associated with the nuclear fraction was greatly diminished when this fraction was isolated from rat spleens by the sucrose-CaCl₂ layering technique. Only 50 to 70 per cent of the total cytochrome c oxidase activity of the original homogenates was recovered among the four fractions from both rat and guinea pig spleens, while the specific activities of reconstructed homogenates were only 55 to 75 per cent of those of the original whole homogenates. This was in contrast to the results with DPNH cytochrome c reductase and succinic dehydrogenase where the recovery of total enzyme activity approached 100 per cent, and the specific activities of reconstructed homogenates equalled those of the original homogenates. The recovery of cytochrome c oxidase was greatly improved when only the nuclei were separated from rat spleen homogenates. 6. Data were presented comparing the concentrations (ratio of activity per mg. of nitrogen of the fraction to activity per mg. of nitrogen of the homogenate) of DPNH cytochrome c reductase in mitochondria and microsomes derived from different organs of different animals. 7. Data were presented comparing the activities per mg. of nitrogen of DPNH cytochrome c reductase in homogenates from several organs of various animals.     

DEVELOPMENT OF THE NEUROMUSCULAR JUNCTION : II. Cytological and Cytochemical Studies on the Neuromuscular Junction of Dedifferentiating Muscle in the Regenerating Limb of the Newt Triturus

Following amputation of the limb of the newt, Triturus viridescens, muscle fibers dedifferentiate giving rise to mesenchymal cells. The earliest changes detected in neuromuscular junctions of dedifferentiating muscle fibers are the appearance of a few vacuoles and decrease in density of the terminal axoplasm. Later, synaptic vesicles become tightly clustered in the axon termination, and their content appears denser than normal. Then, vesicles diminish in number until few are seen in the ending. While these changes are occurring, the area of contact of nerve with muscle becomes smaller. Junctional folds persist only where the nerve maintains contact with muscle, but these are shorter than normal and appear as slight ridges on the muscle surface. Subsequently, the nerve withdraws from the muscle cell and is completely invested by Schwann cell cytoplasm, and all traces of junctional folds are lost at the former region of contact. Cholinesterase activity was localized with the thiolacetic acid-lead nitrate method. Even before marked morphological changes occur in the junction, DFP- and physostigmine-sensitive activity in the cleft between nerve and muscle is decreased in intensity. Activity continues to decrease as the area of nerve-muscle contact diminishes and junctional folds disappear. When the nerve has withdrawn from the muscle surface, only a few small deposits of lead are left in the intervening region. These results show that as muscle becomes less specialized during dedifferentiation, the neuromuscular junction also loses the cytological and cytochemical specializations associated with synaptic function.     

DEVELOPMENT OF THE NEUROMUSCULAR JUNCTION : I. Cytological and Cytochemical Studies on the Neuromuscular Junction of Differentiating Muscle in the Regenerating Limb of the Newt Triturus

Development of the neuromuscular junction on differentiating muscle was investigated in the regenerating limb of the newt Triturus. Motor end-plate formation begins when vesicle-filled axon terminations approach differentiating muscle cells that have reached the stage of a multinucleate cell containing myofibrils. Slight ridges or elevations occur on the muscle surface, and there is an increase in density of the cytoplasm immediately beneath the plasma membrane of the elevation. The axon becomes more closely approximated to the muscle cell and comes to lie in a shallow depression or gutter on the surface of the muscle. The surface ridges increase in length and constrict at their bases to form junctional folds. In the axon terminal, focal accumulations of vesicles are found where the axon contour projects slightly opposite the secondary synaptic clefts. Cholinesterase activity in the developing junctions was demonstrated by the thiolacetic acid-lead nitrate method. Enzymatic activity is not found on intercellular nerve fibers or the muscle surface prior to close approximation of axon endings and muscle. Eserine- and DFP-sensitive activity appears concurrently with morphological differentiation. Activity occurs in membranous tubulovesicles in the sarcoplasm subjacent to the neuromuscular junction and in association with the sarcolemma. The largest reaction deposits occur at the tips of the emerging junctional folds. Smaller and less numerous localizations occur on the axon membrane and within the axoplasm. It is concluded from these studies that the nerve endings have an inductive effect on both the morphological and chemical specializations of the neuromuscular junction.     

THE SYNTHESIS OF DNA, RNA, AND NUCLEAR PROTEIN IN NORMAL AND TUMOR STRAIN CELLS : III. Mouse Ascites Tumor Cells

Interferometric and photometric measurements have been made on replicating mouse ascites tumor cell cultures. From a study of the relations between successive physical measurements on individual cells, it was found that whereas the net syntheses of nuclear RNA and nuclear protein are closely associated during interphase, they are dissociated from DNA replication to a significant extent. These results agree with others reported in replicating cell strains derived from tumors. In auxiliary experiments an attempt was made to block the initiation of DNA synthesis by X-irradiation: although large amounts of nuclear protein accumulated in some cells in the absence of DNA synthesis, the inability to hold the DNA block for an interphase time prevented a quantitative analysis of the results.     

Isolation and Characterization of Tumor Cells from the Ascites of Ovarian Cancer Patients: Molecular Phenotype of Chemoresistant Ovarian Tumors

Tumor cells in ascites are a major source of disease recurrence in ovarian cancer patients. In an attempt to identify and profile the population of ascites cells obtained from ovarian cancer patients, a novel method was developed to separate adherent (AD) and non-adherent (NAD) cells in culture. Twenty-five patients were recruited to this study; 11 chemonaive (CN) and 14 chemoresistant (CR). AD cells from both CN and CR patients exhibited mesenchymal morphology with an antigen profile of mesenchymal stem cells and fibroblasts. Conversely, NAD cells had an epithelial morphology with enhanced expression of cancer antigen 125 (CA125), epithelial cell adhesion molecule (EpCAM) and cytokeratin 7. NAD cells developed infiltrating tumors and ascites within 12–14 weeks after intraperitoneal (i.p.) injections into nude mice, whereas AD cells remained non-tumorigenic for up to 20 weeks. Subsequent comparison of selective epithelial, mesenchymal and cancer stem cell (CSC) markers between AD and NAD populations of CN and CR patients demonstrated an enhanced trend in mRNA expression of E-cadherin, EpCAM, STAT3 and Oct4 in the NAD population of CR patients. A similar trend of enhanced mRNA expression of CD44, MMP9 and Oct4 was observed in the AD population of CR patients. Hence, using a novel purification method we demonstrate for the first time a distinct separation of ascites cells into epithelial tumorigenic and mesenchymal non-tumorigenic populations. We also demonstrate that cells from the ascites of CR patients are predominantly epithelial and show a trend towards increased mRNA expression of genes associated with CSCs, compared to cells isolated from the ascites of CN patients. As the tumor cells in the ascites of ovarian cancer patients play a dominant role in disease recurrence, a thorough understanding of the biology of the ascites microenvironment from CR and CN patients is essential for effective therapeutic interventions.     

Interferon Treatment of Ehrlich Ascites Tumor Cells: Effects on Exogenous mRNA Translation and tRNA Inactivation in the Cell Extract

We reported earlier that in cell extracts that were prepared from interferon-treated Ehrlich ascites tumor cells and preincubated and passed through Sephadex G-25 (S30_INT), the translation of exogenous mRNA (viral and host) was impaired and the impairment could be overcome to a large extent by adding a crude tRNA preparation from Ehrlich ascites tumor cells but not from Escherichia coli. We find now that the rate of inactivation of some tRNA's (especially those specific for leucine, lysine, and serine) but not those of many others is faster in S30_INT than in corresponding extracts from control cells. This increased rate of tRNA inactivation may perhaps account for the need for added RNA to overcome at least partially the impairment of translation in S30_INT. The relationship of the increased rate of tRNA inactivation to the antiviral effect of interferon is unclear. So far no significant difference has been detected in the amount of tRNA needed to overcome the impairment of encephalomyocarditis virus RNA translation in S30_INT between tRNA from interferon-treated cells and tRNA from control cells. Furthermore, no difference was found in the rate of inactivation in S30_INT between leucine-specific tRNA's from interferon-treated and from control cells. tRNA's specific for leucine and lysine were not inactivated (unless very slowly) during incubation under our conditions in an extract from interferon-treated (or from control) cells unless the extract had been passed through Sephadex G-25 or dialyzed. The translation of exogenous mRNA was, however, impaired in an extract from interferon-treated cells that had not been passed through Sephadex G-25. This impairment was apparently not overcome by added tRNA.     

Studies on the Growth in Culture of Plant Cells: XI. THE INFLUENCE OF SHAKING RATE ON THE GROWTH OF SUSPENSION CULTUEES

The influence of shaking rates (expressed as revolutions permin) on orbital shaking platforms (1 in (2.54 cm) diam. rotarymotion) on the growth of cell suspension cultures of Acer pseudoplatanusL. and Atropa belladonna cultivar lutea Döll are described.By following cell growth and respiration and the levels of oxygenand carbon dioxide in the media during the progress of incubationit is concluded that the reduction of growth at sub-optimalshaking rates is not due to oxygen deficiency or toxic accumulationof carbon dioxide. The growth of the Atropa cell suspensionin ‘closed systems’ has been studied by the developmentof modified culture vessels and evidence obtained that the reducedgrowth in the systems is due to the formation by the culturesof an unidentified volatile growth inhibitor and not to eitheroxygen depletion or toxic accumulation of either carbon dioxideor ethylene. It is suggested that the reduced growth in ‘opensystems’ cultures at sub-optimal shaking speeds is eitherdue to retention of this volatile inhibitor or to restrictionof nutrient uptake by the existence of a stationary liquid-phaseboundary to the cells.     

Studies on the Growth in Culture of Plant Cells: XII. A VERSATILE SYSTEM FOR THE LARGE SCALE BATCH OR CONTINUOUS CULTURE OF PLANT CELL SUSPENSIONS

A versatile large-scale batch culture unit has been developedfor the growth of plant cell suspension cultures. This unithas been modified to permit of intermittent or continuous renewalof culture medium and, in a modified form, incorporated intoopen continuous culture systems of the chemostat and turbidostattype. A fully automatic culture sampler has been incorporatedinto the basic culture unit. The culture systems described havebeen successfully operated using a cell suspension derived fromAcer pseudoplatanus and results are presented demonstratingsynchronous growth in batch culture, prolonged logarithmic increassein cell number under conditions of high aeration and culturemedium renewal, and steady states of growth resulting from automaticregulation of the optical density of the cell suspension andfrom fixed rates of displacement of cell suspension by new medium.The potentialities of the culture systems are discussed in thelight of the experimental results presented.     

Responses of Preneoplastic Epidermal Cells to Tumor Promoters and Growth Factors: Use of Promoter-Resistant Variants for Mechanism Studies

The JB6 mouse epidermal cell model system is being used to study the mechanism of promotion of transformation. Promotion of anchorage independence in JB6 cells occurs in response to second-stage but not first-stage promoters, and is inhibited by inhibitors of second-stage not first-stage promotion. A number of variants that are resistant to the phorbol diester TPA have been derived. Some are resistant to plateau density mitogenic stimulation by TPA; others are resistant to promotion of anchorage independence by TPA. Some of the mitogen-resistant variants were promotable by TPA, thus ruling out a requirement for TPA mitogenesis in promotion of transformation in JB6 cells. TPA promotable clones were also sensitive to mezerein and EGF while the TPA nonpromotable variants were also resistant to mezerein and EGF, suggesting that sensitivity to promoters in these JB6 cells is determined at a level distal to receptor binding. Promotion sensitivity did not require available EGF receptors since two TPA promotable variants were EGF receptorless. The mitogenic response of JB6 cells to TPA may however be mediated by EGF since four of four mitogen-resistant variants showed low to zero levels of EGF binding. Tumor promoting phorbol esters produce specific changes in cellular gangliosides. Certain of these changes occur in promotable but not nonpromotable variants of JB6 cells, suggesting that ganglioside changes may be involved in the process of promotion of transformation.     

Studies in the Physiological Action of 2,2-Dichloropropionic Acid: II. THE EFFECTS OF LIGHT AND TEMPERATURE ON THE FACTORS RESPONSIBLE FOR THE INHIBITION OF GROWTH

When Lemna minor and Salvinia natans, grown in a constant environment,are subjected to sub-lethal concentrations of 2,2-dichioropropionicacid (DCPA), the relative growth-rates are progressively reduced.These cumulative reductions, which are greater for S. natans,are correlated with decreases in (1) the rate of leaf or frondformation, (2) the mean area per leaf or frond, and (3) thenet assimilation rate. Of these components, the first is themost important and the third is the least. The effects of light intensity (300, 600, 900 f.c.), temperature(20, 25, and 30°C), and concentration of DCPA on both therelative growth-rate and rate of leaf or frond multiplicationhave been examined in multi-factorial experiments. Over theconcentration range selected (100, 200, and 400 mg/l for S.natans and 100, 300, and 6oo mg/l for L. minor) there are positiveeffects of light intensity, temperature, and concentration.For each concentration the order of the depression is maximalunder a combination of the highest temperature and the greatestintensity. Using radioactive DCPA it has been established that uptake isalso a cumulative process, and that S. natans has a greatercapacity to absorb DCPA. The rate of uptake is independent ofthe light intensity but increases with temperature and concentration. DCPA brings about morphological and structural changes. In S.natans, many of the leaves become submerged and the proportionis positively dependent on light, temperature, and concentration.This failure to float is associated with a reduction in thedensity of epiderrnal hairs. It is concluded that the inhibitory effects of DCPA are maximalunder conditions which are optimal for both meristematic activityand accumulation.     

Studies in the Principles of Phytotoxicity: II. EXPERIMENTAL DESIGNS AND TECHNIQUES OF STATISTICAL ANALYSIS FOR THE ASSESSMENT OF TOXICITY

Some of the more important statistical methods used in the analysisof experiments concerned with studies of phytotoxicity aredescribed, and are illustrated by data from field and laboratoryexperiments undertaken within the Department of Agricultureof the University of Oxford. Most attention has been given toa consideration of quantal effects, such as the proportionatemortality. Adjustments to allow for natural mortality or theappearance of additional plants during the course of the experimentare outlined, together with the conditions under which the datashould be transformed before analysis. Since the relationshipbetween the proportionate response and some function of thedose or concentration of the toxicant generally follows a normalsigmoid law, the methods of probit analysis are appropriatefor presize estimations. In this connexion, the design of experimetsis discussed and the calculations involved in such an analysisare illustrated. In investigations where quantitative measurements are recorded,the dose-response relationehip may also be of the normal sigmoidform, so that the data be treated by a modification of the probittechnique. The methods of statistical treatment demanded whenthe dose-response relationship does not conform to a normalsigmoid are briefly discussed.     

Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin: IDENTIFICATION OF THE HEPARIN-BINDING MOTIF OF p17 AS A TARGET FOR THE DEVELOPMENT OF MULTITARGET ANTAGONISTS*

Once released by HIV⁺ cells, p17 binds heparan sulfate proteoglycans (HSPGs) and CXCR1 on leukocytes causing their dysfunction. By exploiting an approach integrating computational modeling, site-directed mutagenesis of p17, chemical desulfation of heparin, and surface plasmon resonance, we characterized the interaction of p17 with heparin, a HSPG structural analog, and CXCR1. p17 binds to heparin with an affinity (K_d = 190 nm) that is similar to those of other heparin-binding viral proteins. Two stretches of basic amino acids (basic motifs) are present in p17 N and C termini. Neutralization (Arg→Ala substitution) of the N-terminal, but not of the C-terminal basic motif, causes the loss of p17 heparin-binding capacity. The N-terminal heparin-binding motif of p17 partially overlaps the CXCR1-binding domain. Accordingly, its neutralization prevents also p17 binding to the chemochine receptor. Competition experiments demonstrated that free heparin and heparan sulfate (HS), but not selectively 2-O-, 6-O-, and N-O desulfated heparins, prevent p17 binding to substrate-immobilized heparin, indicating that the sulfate groups of the glycosaminoglycan mediate p17 interaction. Evaluation of the p17 antagonist activity of a panel of biotechnological heparins derived by chemical sulfation of the Escherichia coli K5 polysaccharide revealed that the highly N,O-sulfated derivative prevents the binding of p17 to both heparin and CXCR1, thus inhibiting p17-driven chemotactic migration of human monocytes with an efficiency that is higher than those of heparin and HS. Here, we characterized at a molecular level the interaction of p17 with its cellular receptors, laying the basis for the development of heparin-mimicking p17 antagonists.     

Studies on the Growth in Culture of Plant Cells: IV. THE INITIATION OF DIVISION IN SUSPENSIONS OF STATIONARY-PHASE CELLS OF ACER PSEUDOPLATANUS L.

Techniques are described whereby a culture medium can be ‘conditioned‘by separation from a dense cell suspension either by a sinteror by a dialysis membrane. The enhanced growth-promoting activityof the conditioned, as compared with a new medium, is revealedby using a low density of cells (15 x 10³ or less cells perml) to initiate the test cultures from a stationary-phase suspension.The optimum pH of the conditioned medium is c6.4. To obtaina conditioned medium of high activity it is necessary to usean appropriate volume ratio of culture medium to conditioningcell suspension and to limit the conditioning period. Conditioningof the culture medium reduces by a factor of 10 (i.e. down toc. 1000 cells per ml) the minimum effective cell density neededfor self-sustaining growth. There therefore exists a population-dependentrequirement which is not met by the conditioned medium as nowprepared. The retention of the activity of the conditioned mediumin various situations has been studied as a preliminary to workon the chemical basis of conditioning.     

Shrinkage-induced Activation of the Na+/H+ Exchanger in Ehrlich Ascites Tumor Cells: Mechanisms Involved in the Activation and a Role for the Exchanger in Cell Volume Regulation

Amiloride-sensitive, Na⁺-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na⁺ influx is also observed. This indicates that hypertonic challenge activates a Na⁺/H⁺ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pH _i , but the overall picture confirms the results from cell suspensions. Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells. Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization. Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of intracellular free calcium with BAPTA. At 25 mm HCO⁻ ₃, but not in nominally HCO⁻ ₃-free medium, Na⁺/H⁺ exchange contributes significantly to regulatory volume increase in Ehrlich cells. Under isotonic conditions, the Na⁺/H⁺ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage. Received: 2 March 1995/Revised: 29 September 1995     

Studies on the Growth in Culture of Plant Cells: VIII. THE PRODUCTION OF ETHYLENE BY SUSPENSION CULTURES OF ACER PSEUDOPLATANUS, L.

Sycamore cell suspension cultures in a synthetic medium releaseethylene; during a 24-day incubation period a single culture(initial volume 70 ml) produces c. 4 µ moles. There isa very sharp peak of ethylene production between day 10 andday 14 of culture; at the peak of production c. 2 nmoles ethyleneare released per million cells in 24 h. Evidence is presentedthat 2,4-D enhances ethylene production independently of itseffects on culture growth. Under the standard conditions of culture (250-ml Erlenmeyerflasks closed with aluminium foil and containing 70 ml cellsuspension) the concentration of ethylene in the gas phase ofthe cultures rises above 10 ppm. No evidence was obtained thatthis ethylene is inhibitory to culture growth or that a criticallevel of ethylene is necessary to initiate cell division incultures at a critically low cell density. The low rate of ethylene release by stationary phase culturesis temporarily enhanced by the addition of various solutes andfurther depressed by dilution with water.     

Structure and Function of Transfer Cells in the Sporophyte Haustorium of Funaria hygrometrica Hedw: II. KINETICS OF UPTAKE OF LABELLED SUGARS AND LOCALIZATION OF ABSORBED PRODUCTS BY FREEZE-SUBSTITUTION AND AUTORADIOGRAPHY

Excised sporophytes of the moss Funaria hygrometrica Hedw. absorbexternally applied sugar through their basal haustorium. Influxof [³H]sucrose is inhibited by metabolic uncouplers, darkness,and by the photosynthetic inhibitor DCMU. The kinetics of uptakeof glucose and sucrose suggest a biphasic mechanism of absorption.Uptake of 3-O-methyl [³H]glucose shows no saturation characteristicsand a passive mechanism is indicated. Externally applied glucoseis rapidly converted to sucrose. Good retention of productsof short-term absorption and metabolism of [³H]glucose was achievedby freeze-substitution. Autoradiography showed dense and uniformlabelling of the transfer cells of the haustorium. V_max valuesfor uptake of sucrose and glucose, expressed in terms of theweight and external surface area of haustorium, are considerablygreater than typical values from other plant systems. However,if the surface area amplification that is brought about by thedevelopment of wall ingrowths in the transfer cells is takeninto account, fluxes per unit area of plasma membrane are reducedinto the range of typical values. The hypothesis that the surfacearea amplification that characterizes transfer cells is relatedfunctionally to processes of solute transport is therefore supportedby the data.     

THE ORIGIN OF PROTEIN AND FATTY YOLK IN RANA PIPIENS : II. Electron Microscopical and Cytochemical Observations of Young and Mature Oocytes

Electron microscope studies of young oocytes have demonstrated that the plate-like, hexagonally shaped yolk bodies previously observed in living cells are wholly within the substance of oocyte mitochondria and that they remain within these mitochondria while increasing in size. These bodies possess a crystalline structure consisting of what appear to be lines, with a spacing of 70 to 85 A, and appear very dense in the electron microscope. After formalin fixation such bodies give an intense positive test for protein, and when viewed in the electron microscope are only slightly less dense than after OsO₄ fixation. Evidence is presented for the origin of these crystals within a single crista. The clusters of yolk globules previously studied in living cells are seen to consist of several types of bodies, but an irregular dense droplet predominates. This dense material is apparently secreted by small spherical bodies which, the evidence suggests, originate from the breaking up of filamentous mitochondria and which possess an outer double membrane and sometimes internal cristalike membranes. When thin sections of young oocytes are immersed in xylol the dense globules of the clusters are dissolved, but the hexagonal bodies are unaffected, indicating that the globules are of a predominantly fatty nature, while the hexagonal bodies are of a predominantly protein nature. Examination of mature or almost mature oocytes has revealed that the main body of the yolk platelets is crystalline in nature and is surrounded by a thick matrix which, in light microscope study, masks the fact that the face view of the main body of the platelets is often hexagonal. The spacing within the main body is found to be 70 to 85 A. The crystal laminae of this material can be resolved quite clearly into rows of particles. Dense globules of varying sizes are found in the cytoplasm between the platelets. When thin sections of these OsO₄-fixed oocytes are immersed in xylol, the material of the globules is extracted and the crystalline material of the platelets remains unaffected, indicating the fatty nature of the globules and the protein nature of the platelets. The platelets of the mature egg resemble the hexagon bodies, previously described in young oocytes, in their protein nature, their crystalline spacing, and their hexagonal outline. This is given as strong evidence for the origin of the mature platelets by the growth of the intramitochondrial hexagon bodies. The biochemical implications of this study are discussed.