bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytofluorimetric analysis of nuclear dna during meiosis, fertilization and macronuclear development in the ciliate Tetrahymena pyriformis, syngen 1
Authors: Doerder F. P. , Bault L. E. De
Published: April 2021

Fluorescence cytophotometry was used to study nuclear DNA content and synthesis patterns during meiosis, fertilization and macronuclear development in the ciliated protozoon, Tetra-hymena pyriformis, syngen i. It was found that cells entered conjugation with a G1 (45C) macronucleus and a G2 (4C) micronucleus. During meiosis the micronucleus was reduced to 4 haploid nuclei, each with a 1C amount of DNA; each meiotic product then replicated to 2C, but only the nucleus next to the attachment membrane in each conjugant divided to form the two 1C gametic nuclei. The gametic nuclei replicated to 2C prior to fertilization; hence there was no S-period in the 4C fertilization nucleus (synkaryon). The first postzygotic division products immediately entered an S-period to become 4C, and at the second postzygotic division, each of the two 4C nuclei in each conjugant divided to form one 2C micronucleus and one 2C macro-nuclear Anlage. The macronuclear Anlagen began DNA synthesis immediately and were about 8C at the completion of conjugation; the micronuclei did not undergo rapid DNA doubling and measured between 2C and 3C when the conjugants separated. The old macronucleus did not participate in any S-period during conjugation and began to decompose after the second postzygotic division; it contained an average of 24C at the end of conjugation. From this sequence of nuclear divisions a pattern emerges that, unless a general cytoplasmic signal for DNA synthesis is suppressed, DNA synthesis always occurs in micronuclear division products immediately following separation of sister chromatids.

Nuclear development continued in the first two cell cycles after conjugation. In exconjugants (the first cycle), macronuclear Anlagen underwent two rounds of DNA synthesis to become 32C and both micronuclei also underwent DNA synthesis. However, prior to the first cell division, one micronucleus and the old macronucleus completely disintegrated, and at the first cell division the remaining 4C micronucleus divided and one macronuclear Anlage was distributed to each resulting caryonide. At the end of the second cell cycle, the dividing macronucleus of each caryonide contained about 128C.

These results relate to the question of ploidy of macronuclear subunits. It is argued that the G1 macronucleus contains 22 or 23 diploid subunits, each subunit being a copy of the diploid micronuclear genome. It is suggested that unequal macronuclear division relates to the question of subunit ploidy by playing a role in the phenomenon of macronuclear assortment.
Observations on the sorting-out of embryonic cells in monolayer culture
Authors: Steinberg M. S. , Garrod D. R.
Published: April 2021

Two problems are raised concerning the movement of cells during tissue-specific sorting-out of chick embryo cells in mixed aggregates, (i) A possible expectation from the hypothesis of ‘contact inhibition’ is that cells which are entirely surrounded by other cells in monolayer should be held stationary. Cells within solid aggregates, being totally surrounded by others, might also not be expected to move. How is it then that cell movement takes place within solid aggregates during sorting-out ? (ii) Are the movements of cells within sorting aggregates ‘passive’, being driven by adhesive differentials, or ‘active’, being merely guided by such differentials ?

In order to study these questions, sorting-out experiments with chick embryonic limb bud mesenchyme and liver cells were carried out in monolayer culture, permitting direct observation of cell movements. Cell behaviour was observed by time-lapse cinematography. Sorting-out of these cells in monolayer began before and continued after the cells had spread to confluency. During sorting, liver cells showed ruffling activity even when they appeared to be totally surrounded by other cells.

Both cell types showed contact inhibition as judged by the criterion of monolayering, for they did not move over each other but remained attached to the substratum. Yet the cells in the confluent monolayer were not immobilized. Because of this, we suggest that the observed restraint against overlapping did not result from an inhibition of movement.

Several considerations, detailed in the text, suggest that cell movement during sorting-out involves active locomotion.

Previous work suggests that sorting-out configurations are determined by the relative intensities of intercellular adhesive strengths, the more cohesive of 2 cell populations tending to adopt the internal position. While limb bud cells form internal islands surrounded by liver cells in solid aggregates, the reverse was found to be the case in these monolayers. This suggests that, in the monolayer, limb bud cohesiveness is depressed relative to liver cell cohesiveness. This is consistent with the observation that the limb bud cells flattened themselves markedly against the substratum, significantly decreasing their area of mutual apposition.
Transformation of plastids in the leaves of Acer negundo L. var. odessanum (H. rothe)
Authors: Wrischer M. , Ljubešlć N. , Devidé Z.
Published: April 2021

The leaves of Acer negundo L. var. odessanum (H. Rothe), if permanently exposed to strong sunlight, do not green, but remain yellow and finally become bleached. In yellow leaves the plastids contain single thylakoids and no grana. In plastids of bleached leaves, however, only vesicles are present. The concentration of chlorophylls and photosynthetic activity are much lower in those leaves than in the green ones.

If the illumination is reduced (e.g. by shading) both the yellow and the bleached leaves become greenish, and even fully green after a few days at a sufficiently low light intensity. The plastids of yellow-green leaves contain small grana. In dark green leaves the thylakoid system of the chloroplasts is normally developed forming true grana, regardless of whether the leaves were originally green, or became green by shading the yellow or bleached ones. Their pigment concentration and photosynthetic activity are also normal.

If green leaves are exposed to sunlight they do not yellow or bleach. During a 3-week period the structure of the thylakoid system did not perceivably change, with the exception that large plastoglobules formed in the stroma.
The localization of retinol-binding protein in rat liver by immunofluorescence microscopy
Authors: Poole A. R. , Dingle J. T. , Mallia A. K. , Goodman DeW. S.
Published: April 2021

The localization of immunoreactive retinol-binding protein (RBP) in rat liver was studied by immunofluorescence microscopy. The study employed specific antisera to rat RBP prepared in a rabbit and in a sheep. The indirect, two-stage method of localizing tissue antigens was employed, and livers of both normal and vitamin A-deficient rats were examined. Fab′ fragments of immunoglobulins were used, to minimize non-specific labelling of the frozen sections of liver. With these techniques, the specific immune staining of RBP was observed within liver parenchymal cells. This staining appeared as both particulate and diffuse within the cytoplasm of the parenchymal cells, and was not concentrated within one region of the liver cell or lobule. Staining for RBP was not observed in nuclei or in cells other than parenchymal cells. Similar particulate and diffuse immune staining for RBP was observed in liver sections from both vitamin A-deficient and normal rats. More intense immune staining appeared to be present in the sections of vitamin A-deficient animals, in good correlation with the expected higher levels of RBP in deficient as compared to normal liver.

When liver sections were exposed to an antiserum to rat albumin, instead of one to rat RBP, immune cytoplasmic staining was observed which was entirely of a diffuse nature, and did not appear particulate or granular. The findings suggest that RBP, unlike albumin, is localized in part within cytoplasmic vesicles or granules which are large enough to be detected with immunofluorescence, and which are present in livers of both normal and vitamin A-deficient animals. The nature of these putative RBP-containing particles remains to be explored.
Colony-stimulating factor (csf) in the uterus of the pregnant mouse
Authors: Rosendaal M.
Published: April 2021

During the growth of the pregnant uterus of inbred Balb/c mice uterine colony-stimulating factor (CSF) content increased 800-fold. This corresponded to a 75-fold increase in CSF concentration. The size of the increase was the same in the uteri of inbred Balb/c mice and the uteri of F1 hybrid C57Bl/Balb/c mice mated with F1 males. It was about four times greater in these uteri than it was in the uteri of randomly mated TO mice. After unilateral Fallopian tubal ligation of TO mice the concentration of CSF was as great in the non-gravid uterine horns as it was in the gravid uterine horns.

Physicochemical characterization of CSF from extracts of pregnant uterus showed that it differed from that found in foetal extracts and that found in the circulation. Its function in the pregnant uterus is discussed.
The arrangement of germ cells in the rat seminiferous tubule: an electron-microscope study
Authors: Moens P. B. , Hugenholtz A. D.
Published: April 2021

The spermatogonia and early spermatocytes of 13 samples of rat seminiferous epithelium (about 0·05 mm2 each) were mapped from electron micrographs of serial sections. Clones of cells, connected by cytoplasmic bridges (syncytia of 2–100 cells), in various stages of spermatogenic development were identified. Maps of 7 separate areas are illustrated. It is concluded that, contrary to the models of spermatogonial proliferation based on light-microscope observations, regions of seminiferous epithelium which are identical in terms of spermatid and spermatocyte criteria have, in fact, quantitative and qualitative differences in their spermatogonial population. The data are interpreted that for a given epithelial area there is a periodic build-up of spermatogonia which then produce several successive quanta of spermatocytes and when the spermatogonia are depleted the process repeats.

That cell numbers less than double following a mitotic cycle has generally been attributed to systematic degeneration. Evidence from electron microscopy indicates, however, that at the mitotic peaks not all the syncytia undergo division but that some remain arrested. Similarly, within a dividing syncytium a few cells do not divide while they advance developmentally with the syncytium as a whole. The observed large size of spermatocyte syncytia further argues against systematic degeneration with its attendant fragmentation of syncytia.
Macronuclear differentiation during oral regeneration in Stentor coeruleus
Authors: Paulin J. J. , Brooks A. Susan
Published: April 2021

The moniliform macronucleus of Stentor coeruleus coalesces and renodulates during division, reorganization and regeneration. These nuclear events are spatially and temporally synchronized with oral primordium development occurring at stages six and seven of membranellar morphogenesis. Coalesced, elongating and early renodulating macronuclei at stages six and seven contained microtubules within double membrane-bound channels, passing through the nucleus parallel to the long axis. The number of microtubules per channel varied between 4 and 23. Microtubules were also found in the perinuclear cytoplasm at these stages, forming a loose network around the nucleus. The microtubules and channels are absent in control cells and macronuclei of regenerating cells prior to stage six. These transient microtubules and channels appearing in late stage six and stage seven may provide the axial plane on which elongation of the macronucleus proceeds.
Ultrastructural changes and a second mode of flagellar degeneration during ageing of Phytophthora palmivora sporangia
Authors: Hemmes D. E. , Hohl H. R.
Published: April 2021

Ageing of sporangia in Phytophthora palmivora is accompanied by a gradual breakdown of flagella, a transformation of the ‘fingerprint’ vacuoles, extensive vacuolization of the cytoplasm, accumulation of bundles of microtubules (mastigonemes) contained within cisternae of the rough endoplasmic reticulum, formation of a germination wall lining the inside of the sporangial wall and production of secondary sporangia. The mechanism of fiagellar degeneration differs from that observed in directly germinating sporangia. In ageing sporangia and in sporangia induced to germinate directly with germ tubes, there is a close correlation between loss of competence for zoosporogenesis and breakdown of the flagella.
Pattern of incorporation of [3H]uridine into rna of amphibian oocyte nucleoli
Authors: Macgregor H. C.
Published: April 2021

Amphibian oocytes were incubated in vitro in the presence of [3H]uridine, and autoradiographs were made of nucleoli isolated from these oocytes and of sections of oocytes.

After incubations of 2 h or less the nucleoli of oocytes larger than 0·6 mm diameter are asymmetrically labelled. With longer incubations nucleoli from oocytes of 0·6 tori mm diameter become more uniformly labelled. Those of oocytes larger than 1·2 mm diameter remain asymmetrically labelled whatever the incubation time. Autoradiographs of 1-μ sections through oocytes larger than 0·6 mm diameter show, after short incubations, asymmetrically labelled nucleoli. In these autoradiographs silver grains are concentrated over a distinct component of each nucleolus which is eccentrically placed towards the nuclear envelope. Thin sections of oocytes show nucleoli consisting of core and cortex. The core material is always concentrated into the half of the nucleolus which, lies nearer the nuclear envelope. Autoradiographs of separated nucleolar cores and cortices from oocytes larger than 0·6 mm diameter show, after short incubations, silver grains over cores but not over cortices. Similar autoradiographs prepared from oocytes of 0·6 to 1·1 mm diameter, after longer incubations, show grains over cores and cortices.

These results appear to indicate that nucleolar RNA is synthesized in the nucleolar core, in association with the nucleolar DNA, and is thence transferred to the cortex where it is built into ribonucleoprotein particles. Initial asymmetrical labelling is a consequence of the eccentric location of the nucleolar core. The nucleoli of oocytes smaller than 0·6 mm diameter always label symmetrically; such nucleoli consist entirely of core material. It is suggested that the nucleoli of oocytes larger than 1·2 mm diameter always label asymmetrically because transfer of RNA from core to cortex proceeds more slowly than in smaller oocytes.
Fine structure of the eye of a nudibranch mollusc, hermissenda crassicornis
Authors: Eakin R. M. , Westfall Jane A. , Dennis M. J.
Published: April 2021

The eye of a nudibranch, Hermissenda crassicornis, was studied by light and electron microscopy. Three kinds of cells were observed : large sensory cells, each bearing at one end an array of microvilli (rhabdomere) and at the other end an axon which leaves the eye by the optic nerve ; large pigmented supporting cells ; and small epithelial cells, mostly corneal. There are five sensory cells, and the same number of nerve fibres in the optic nerve. The receptor cells contain an abundance of small vesicles, 600–800 A in diameter. The lens is a spheroidal mass of osmiophilic, finely granular material. A basal lamina and a capsule of connective tissue enclose the eye. In some animals the eye is ‘infected’ with very small bodies, 4–5 μin diameter, thought to be symbionts.
Spindle Birefringence of Isolated Mitotic Apparatus Analysed by Pressure Treatment
Authors: Forer A. , Zimmerman A. M.
Published: April 2021

Sea-urchin zygote mitotic apparatus (MA) isolated in a glycerol/dimethylsulphoxide medium were treated with pressure. Pressure treatment had no effect on spindle birefringence when MA were in full-strength isolation medium. After placing MA in quarter-strength isolation medium, pressures of 4·0 × 103– 1·8 × 104 Ibf in.−2 (2·76 × 104–1·24 × 105 kN m−2) for 15 min caused reduction of birefringence which occurred in 2 steps: firstly 20–30% of the birefringence was lost, and then, at higher pressures, the rest of the birefringence was lost. Electron microscopy suggested that pressure-induced changes were in non-microtubule material. Pressure treatment had no effect on MA isolated with hexylene glycol when the MA were pressurized in hexylene glycol; but pressure treatment did cause loss of birefringence when MA isolated in hexylene glycol were transferred immediately into glycerol/dimethylsulphoxide medium and were subsequently treated with pressure (after dilution into quarter-strength glycerol/dimethylsulphoxide). We discuss the differences in response between isolated MA and in vivo MA, and we discuss the possibility that 2 components contribute to MA birefringence.
Spindle Birefringence of Isolated Mitotic Apparatus Analysed by Treatments With Cold, Pressure, and Diluted Isolation Medium
Authors: Forer A. , Zimmerman A. M.
Published: April 2021

Mitotic apparatus (MA) were isolated from sea-urchin zygotes using glycerol-dimethyl-sulphoxide. Cold treatment had no effect on MA birefringence when MA were in isolation medium, but caused a 10–15% reduction of MA birefringence when MA were in quarter-strength isolation medium. Pressure treatment also caused a reduction in MA birefringence, but the cold and pressure treatments were not additive, suggesting that both treatments affected the same MA component. MA were not stable in quarter-strength isolation medium, and birefringence gradually decayed, with a half-life of about 40 h. Electron microscopy after cold treatment, or after decay of 55 % of the MA birefringence showed abundant, normal-looking microtubules, suggesting that alterations in non-microtubule components cause the reductions in birefringence. Addition of EGTA eliminates the effect of cold treatment, suggesting that Ca*+ has a role in maintenance of spindle structure. We discuss possible reasons why isolated MA do not respond to cold treatment like MA in vivo.
A comparison of membrane fracture faces of fixed and unfixed glycerinated tissue
Authors: Breathnach A. S. , Gross M. , Martin B. , Stolinski C.
Published: April 2021

Fixed (glutaraldehyde, 3%) and unfixed specimens of rat buccal epithelium, striated muscle, and liver, were cryoprotected with glycerol, freeze-fractured, and replicated without sublimation. A comparison of fracture faces of general plasma membranes, nuclear membranes, mitochondrial membranes, and membranes of rough endoplasmic reticulum revealed no significant differences as between fixed and unfixed material. Apart from some membranes of liver endoplasmic reticulum, there was no evidence of aggregation or redistribution of intramembranous particles in the unfixed material. The results demonstrate that chemical prefixation of tissues for freeze-fracture is not always necessary, or even desirable, and that glycerol may not be as deeply or directly implicated in particle aggregation as previously thought. Fixation with glutaraldehyde alters the cleaving behaviour of plasma membrane at desmosomes and tight junctions, but not at gap junctions.
A morphometric and biochemical investigation of the vesiculation of kidney microvilli
Authors: Booth A. G. , Kenny A. J.
Published: April 2021

The vesiculation of kidney proximal tubule microvilli has been examined in tissue slices, isolated brush borders and isolated microvilli. Vesiculation could be induced in tissue slices by 2,4-dinitrophenol and anoxia. Cycloheximide and fluoride had no effect. In brush borders and microvilli, the vesiculation was found to be essentially temperature-dependent. Whilst an osmotic swelling could be produced by hypo-osmolar media, the temperature-dependent vesiculation could not be prevented in hyper-osmolar media. Of a wide variety of reagents tested, only glutaraldehyde, mercuric chloride and mersalyl were effective in arresting the vesiculation. Electron micrographs show that vesiculation involves a collapse of the internal structure of the microvillus. However, the collapse was not associated with depolymerization of the microvillus actin filaments. Rather it appeared to be due to the parting of cross-bridges between the membrane and the actin filaments. The nature of these cross-bridges is discussed: it is suggested that α-actinin possesses the characteristics for the cross-bridging protein and that vesiculation might be explained by the displacement of α-actinin by tropomyosin.
Assay of intercellular adhesiveness using cell-coated sephadex beads as collecting particles*
Authors: Vosbeck Klaus , Roth Stephen
Published: April 2021

A simple, rapid and precise method, based on a previous method, for measuring relative rates of intercellular adhesion is described. DEAE-Sephadex beads were treated with nitrocellulose in order to allow cells to grow on their surfaces. Balb/c 3T3 and Balb/c 3T12 cells were used to characterize the assay. They formed confluent cell layers on nitrocellulose-treated DEAE-Sephadex. These cell-coated beads were employed to collect 32P-labelled cells from single cell suspensions. Since they formed statistically uniform, large collecting surfaces, the collection of labelled cells was markedly improved as compared to the original assay. The cell-coated beads collected a large percentage of the labelled cells in a short time. The percentage of cells collected was independent of the concentration of labelled cells in the assay mixture, and the collection was linear for approximately 60 min. The variability between replicate assays was usually ± 5 %. The assay allows the rapid and precise determination of intercellular adhesion in large numbers of individual samples. These features make it useful to screen for effects of different treatments on intercellular adhesion.
Mechanisms of cell adhesion: Early-forming junctions between aggregating fibroblasts
Authors: Lloyd C. W. , Rees D. A. , Smith C. G. , Judge F. J.
Published: April 2021

When cultured fibroblasts (16C) are mildly dissociated with EGTA or trypsin/EDTA, they aggregate rapidly. The formation of aggregates has been found to involve junctions of the gap and adhaerens types which are seen by electron microscopy within minutes of allowing cells to come together. The process of adhesion between freshly dissociated, transformed 16C fibroblasts is therefore organized and establishes its usefulness as a model for studying cellular interactions in relation to supracellular organization.
Biochemical studies of mammalian oogenesis: Protein synthesis during oocyte growth and meiotic maturation in the mouse
Authors: Schultz Richard M. , Wassarman Paul M.
Published: April 2021

Using oocytes isolated from juvenile and adult mice, we have examined the qualitative patterns of protein synthesis during growth and during meiotic maturation of these oocytes. Oocytes were cultured in a defined medium in the presence of [35S]methionine and radioactively labelled proteins were separated by SDS-polyacrylamide gel electrophoresis and detected by fluorography. The results of these studies demonstrate that: (i) the patterns of protein synthesis are very similar in individual oocytes which are at the same stage of growth or of meiotic maturation, indicating a high degree of biochemical homogeneity in a given population of isolated mouse oocytes, (ii) the linear increase in protein content of growing mouse oocytes (with respect to oocyte volume) is accompanied by significant qualitative changes in the size classes of proteins synthesized, and (iii) meiotic maturation (germinal vesicle dissolution and nuclear progression to the second metaphase) is characterized by several discrete qualitative changes in the pattern of protein synthesis in the oocyte, especially during the period following germinal vesicle breakdown. Experiments carried out with oocytes cultured in the presence of drugs which have been shown to inhibit meiotic maturation at specific stages of nuclear progression suggest that: (i) protein synthesis is not required for germinal vesicle breakdown to take place; (ii) mixing of the oocyte’s nucleoplasm and cytoplasm must occur in order for those changes in the pattern of protein synthesis which characterize meiotic maturation to take place; and (iii) failure of nuclear progression to proceed beyond the circular bivalent stage does not prevent those changes in the pattern of protein synthesis which characterize meiotic maturation from taking place. The latter observations suggest that there are basic differences in the control of meiotic maturation in oocytes isolated from mammalian, as compared to non-mammalian, animal species.
The self-assembly of synthetic filaments of myosin isolated from Chaos Carolinensis and Amoeba Proteus
Authors: Condeelis John S.
Published: April 2021

Synthetic myosin thick filaments were formed from preparations of electrophoretically homogeneous myosin isolated from Chaos carolinensis and Amoeba proteus when dialysed to physiological ionic strength and pH. Myosin dialysed directly against low ionic strength buffers formed native-like thick filaments in the presence and absence of exogenous divalent cations. The average dimensions of the synthetic filaments grown under these conditions were 455 nm long and 16 nm wide with a distinct bare central zone 174 nm long.

Myosin predialysed against EGTA-EDTA solutions at high ionic strength and then dialysed to low ionic strength formed native-like filaments only in the presence of 1 mM Mg2+. 1 mM Ca2+ could not be substituted for Mg2+ under these conditions to achieve native-like filaments. Filaments grown from predialysed myosin in the absence of Mg2+ resembled EGTA-dissociated myosin filaments observed in EGTA-treated cytoplasm and were highly branched, poorly formed filaments lacking a distinct bare central zone. The average dimensions of the filaments grown from predialysed myosin in the absence of Mg2+ were 328 nm long, 13 nm wide with a bare central zone 111 nm long.

Under the conditions tested, myosin isolated from these amoebae did not demonstrate a divalent cation requirement for thick filament formation. The results obtained with myosin isolated from the 2 organisms were identical.
Accumulation of gene products in the oocytes of the dragonfly Cordulia Aenea: II. Induction of annulate lamellae within dense masses during diapause
Authors: Halkka Liisa , Halkka O.
Published: April 2021

During the 3 years of nymphal development of the dragonfly Cordulia aenea, gene products slowly accumulate in the cytoplasm of the previtellogenic oocytes. Nuclear extrusions separate into nematosome and dense-mass components. The dense masses, RNP-containing globules of tightly packed fibrillar material, disperse throughout the oocyte cytoplasm.

The last instar nymph experiences an obligatory diapause, after which oocyte development passes from mid to late previtellogenesis, and eventually to vitellogenesis proper in the adult. During diapause, annulate lamellae are induced within the dense masses. This process is continued during late previtellogenesis. The formation of annulate lamellae may be related to activation of some genetic message stored in the dense masses. The possibility of tubulin synthesis is discussed.
Cytochalasin B-induced pseudo cleavage of mouse oocytes In Vitro: II. studies of the mechanism and morphological consequences of pseudocleavage
Authors: Wassarman P. M. , Ukena T. E. , Josefowicz W. J. , Letourneau G. E. , Karnovsky M. J.
Published: April 2021

Mouse oocytes are induced by cytochalasin B to undergo ‘pseudocleavage’ in vitro into 2 compartments, only one of which possesses microvilli. It has been found that this particular response to cytochalasin B is related to oocyte size and, possibly, to the acquisition of meiotic competence by the oocyte during its growth phase. Certain of the morphological events which characterize pseudocleavage have been determined using transmission and scanning electron microscopy. These events include : (i) an initial withdrawal of microvilli from the surface of the oocyte, together with the concomitant disappearance of microfilaments normally associated with the microvilli; (ii) the subsequent formation of a pseudocleavage furrow and contractile ring; and (iii) the reappearance of microvilli and associated microfilaments in one of the two resulting oocyte compartments. These changes in surface architecture are reflected in the distribution of fluorescein-conjugated lectins bound to the oocyte surface during pseudocleavage.
Electron-microscopic autoradiography of tritiated testosterone in rat testis
Authors: Frederik P. M. , Molen H. J. Van Der , Klepper D. , Galjaard H.
Published: April 2021

The feasibility of a technique for autoradiography of diffusible substances has been further tested by analysing the localization of steroids in rat testes with the light- and electron-microscope. Testes of rats were perfused with tritiated testosterone (3 min) followed by 15-min perfusion with buffer containing a 100-fold excess of unlabelled testosterone. Tissue samples were frozen, freeze dried, fixed in osmium vapour and embedded in Epon. To exclude extraction of steroids, contact with water and other solvents was prevented during cutting of thin sections on an ultracryotome and further treatment for autoradiography. Light- and electron- microscopic observations indicate that the highest concentration of labelled testosterone was present within the basal parts of the Sertoli cell cytoplasm and in lipid inclusions of Sertoli cells within the seminiferous tubules. This is the first account of autoradiography of steroids at the electron-microscope level.
Mass isolation of polytene nuclei from Chironomus salivary glands
Authors: Mähr R. , Lezzi M. , Eppenberger H. M.
Published: April 2021

In this paper we describe a method for the rapid mass isolation of polytene nuclei from Chironomus salivary glands. The procedure for the isolation of glands involves 5 principal steps; (a) freezing Chironomus larvae in liquid propane; (b) breaking open frozen animals in a precooled mortar; (c) thawing the fragments in sucrose medium, free of divalent cations; (d) pressing the suspension of broken animals through a system of regularly spaced capillary constrictions to free organs; and (e) enrichment of glands by differential sedimentation and removal of contaminating material under a dissecting microscope. The nuclear isolation procedure is a large scale modification of a method previously described by Robert, using digitonin as a non-ionic detergent to solubilize cytoplasma and secretion without affecting the nuclear membrane. Nuclei obtained by this method show structural integrity and an unchanged chromosomal banding pattern. Their incorporation of UTP is within the same range as reported by other authors for nuclei isolated by hand dissection.
The fixation of nuclei in glutaraldehyde
Authors: Skaer R. J. , Whytock Susan
Published: April 2021

The production of an artifactual network in the nuclear sap of the salivary glands of Drosophila has been investigated. Mechanical stress to the cells, 3 % glutaraldehyde containing more than 4 mM calcium, tannins, or cacodylate buffer or whose temperature is above 10 °C all enhance this artifactual effect. Over the range pH 6· 65–7·4 there is no significant effect of pH. The inclusion of sucrose, and adenine nucleotides or other ‘chelating’ agents in the fixative reduces the effect, especially at low temperature (6–10 °C). These influences are additive and can abolish the artifactual effect. So too can 8 % glutaraldehyde, but this is irrespective of temperature.
Structural cross-bridges between microtubules and mitochondria in central axons of an insect (Periplaneta americana)
Authors: Smith David S. , Järlfors Ulla , Cayer Marilyn L. , Cameron Bruce F.
Published: April 2021

The distribution of microtubules and mitochondria in central axons of an insect (Periplaneta americana) is assessed by comparison between counts on micrographs and computed axon random ‘models’. These studies show that the observed multiple association of microtubules with individual mitochondria is statistically highly significant. Electron micrographs of thin sections show that linkage is effected by physical cross-bridges, possibly comprising components from the microtubule and mitochondrion. Linear particle arrays are described on the outer mitochondrial membrane in freeze-fracture replicas, and tentatively related to the bridges seen in thin sections. The results are discussed in terms of proposed roles of microtubules in neurons and other cells.
Novel behaviour of the mitotic clock in physarum
Authors: Wille John J. , Scheffey Carl , Kauffman Stuart A.
Published: April 2021

Surface plasmodia of the myxomycete, Physarum polycephalum were subjected to heat shocks at 38 °C for varying durations either in late G2, or at M + 4 h, and the excess delay to the next synchronous mitosis observed. Three-hour heat shocks applied in late G2 yielded mitotic excess delays which clustered at peaks of 4–5, 11–12 and 22–23 h. Hence plasmodia can skip part of a cycle, one full cycle, or even two full cycles, yet continue to count cycle times up to two cycles. This is one line of evidence that mitosis itself is not a necessary condition for timing the length of the mitotic cycle. In a large number of late G2 shocks, only a fraction of the nuclei divided at 4–5 h excess delay, and then went through their first full synchronous mitosis at 12 h excess delay. These partial mitoses also were observed for plasmodia which had their first full synchronous mitosis at 22–24 h excess delay. The fraction of nuclei which undergo partial mitosis is nearly independent of the mitotic interval occurring between the partial mitosis and the first full synchronous mitosis, indicating that nuclear:cytoplasmic ratio is unlikely to be the sole factor governing mitotic cycle length. The dependence of mitotic excess delay on: (1) the exact time at which heat shock began shortly before mitosis; (2) the condition of the culture from which a set of plasmodia were plated; (3) the average cycle length; (4) the occurrence of nucleolar breakdown; and (5) the extent of plasmodial death, is minimal. Six-hour heat shocks applied in late G2, also yielded rhythmical mitotic excess delay responses with peaks at 6–8, 14–16, and 26–27 h excess delay. Nine-hour heat shocks given in late G2 produced the greatest mean excess delays (17·5 h). Finally, plasmodia exposed to 12-h heat shocks in late G2, yielded mitotic excess delays with a mean of 10· 7 h, considerably less than either the 6–or 9–h heat shocks. In contrast, plasmodia given a 3–, 6– 9-, or 12–h heat shock at M+4 h showed only a monotonically increasing mitotic excess delay, which never exceeded one full cycle of delay.

The above results are consistent with and predicted by the mitotic oscillator model in which the periodicity of the mitotic cycle is underlain by a continuous biochemical limit cycle oscillation, which drives mitosis but which continues to oscillate even when mitosis is suppressed.
The abdominal epidermis of the blowfly Calliphora erythrocephala II. imaginal histoblasts during larval life
Authors: Pearson Murray J.
Published: April 2021

Abdominal histoblasts in Calliphora (Díptera) are situated within the larval epidermis, and secrete larval cuticle throughout the 3 larval instars. An optical and electron-microscopic study of histoblasts and epidermal sensilla suggests that the latter have been previously confused with histoblast Anlagen in the first instar.

A steady rate of division until the mid-third instar leads to Anlagen growth, which is not seen in larval Drosophila. After a relatively quiescent period, division begins again, at a higher rate, at the time of pupariation (at the time when division first begins in Drosophila’). During prepuparial larval life, the ventral, dorsal, and postero-dorsal Anlagen increase from approximately 6, 8 and 4 cells to about 350, 500 and 150 cells respectively.

At the time of the second (puparial) mitotic phase there is also extensive reorganization of cellular fine structure in which large secondary lysosomes play an important part. The endoplasmic reticulum and Golgi subsequently present a different profile. At the same time numerous chromatic droplets appear in the rapidly dividing histoblast Anlagen. This division and the appearance of chromatic droplets are shown (and the subcellular reorganization is suggested) to depend upon the hormonal factors which are responsible for pupariation.
The dissociation of nuclear proteins from superhelical DNA
Authors: Levin Jeremy Max , Jost Erich , Cook Peter Richard
Published: April 2021

Structures retaining many of the morphological features of nuclei may be released by gently lysing human cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain superhelical DNA. Using a double-labelling procedure we have compared, at different salt concentrations, the amounts and types of protein associated with human nucleoids containing superhelical or relaxed DNA. We find that the slightly lysine-rich histones (H2A and H2B) but not the arginine-rich histones (H3 and H4) dissociate more slowly from nucleoids containing superhelical DNA than from those containing relaxed DNA. A protein of apparent molecular weight of 22000 also binds more tightly to superhelical DNA. We conclude that this protein and the slightly lysine-rich histones transmute free energy of supercoiling into binding energy when they bind to superhelical DNA.
Endocytosis and streaming of highly gelated cytoplasm alongside rows of arm-bearing microtubules in the ciliate Nassula
Authors: Tucker J. B.
Published: April 2021

The microtubular cytopharyngeal basket acts as a jet engine when Nassula ingests filaments of blue-green algae. Prolonged and highly directed cytoplasmic streaming forms the main mechanical basis for propulsion of algal filaments through the basket. Cytoplasm surrounding such filaments streams at the same rate as that at which filaments travel through the basket’s interior. This cytoplasm appears to be very highly gelated. The arm-bearing surfaces of certain rows of microtubules seem to be located in the active shear zone where the forces responsible for cytoplasmic streaming are generated.

Algal ingestion represents a rapid and large scale form of endocytosis. An algal filament is contained in a long membranous invagination from the ciliate’s cell surface as it is drawn through the basket. The invagination moves through the basket at the same velocity as the algal filament and surrounding cytoplasm. The arm-bearing microtubules do not appear to bind directly to the invagination which consists of two unit membranes. One of these is probably an invagination of the cell surface membrane. The origin of the other membrane has not been ascertained.

There are indications that microtubule sliding and a contractile ring of microfilaments operate antagonistically during feeding to evert and re-invaginate a cell surface depression where the top of the basket is located.
Fine-structural aspects of fertilization in Chlamydomonas Reinhardi
Authors: Friedmann I. , Colwin A. L. , Colwin Laura H.
Published: April 2021

Gametes of C. reinhardi lack the cell wall which vegetative cells possess. Just below the cell apex gametes form a. fertilization tubule which is up to 2 μ long and 0·2 μ in diameter; its plasma membrane and that of the apex have slender tubular projections. At the base of the fertilization tubule regularly lies the choanoid body, a collar-shaped cytoplasmic organelle; the plasma membrane overlying the body appears as an electron-dense ring. Gametes possess two ‘free’ basal bodies in addition to the basal bodies of the two flagella.

In the initial stage of union the conjugating cells are connected by the fertilization tubule whose plasma membrane is continuous with that of both copulants. At one end of the tubule lies a conspicuous choanoid body, but at the other end is a small structure which possibly is a homologue of the choanoid body. Subsequently, the fertilization tubule shortens and widens until finally no tubule exists and the apical ends of the two protoplasts adjoin. The merging cells then bend like a jack-knife and lateral alignment of the protoplasts occurs. This four-flagellated zygote becomes motile at about the time when the flagellar bases of the former gametes seem to approach each other and when fibrillar elements of the flagellar roots come into contact. In the motile zygote the nuclei do not fuse but remain ensheathed in the cup-shaped plastids of the two gametes.

A mating of plus (+) and minus (−) strains cultured, respectively, for high and low starch content suggested that gametes of only the plus (+) mating type contain the choanoid body. Since it appears that the gamete containing the choanoid body also produces the fertilization tubule, it is inferred that gametes of only the plus (+) mating type produce the fertilization tubule. Should further investigation support this inference, it would be established that there is a structural basis for designating the plus (+) mating type as male and the minus (−) type as female.

Fertilization involves fusion of the gamete membranes through the mediation of a specialized structure (the fertilization tubule) and in this respect there are similarities to certain aspects of fertilization in animal phyla. The relation of the fertilization tubule to the protoplasmic bridge of other species of Chlamydomonas is discussed.
The distribution of the protein-polysaccharide complex in the nucleus pulposus matrix in young rabbits
Authors: Smith J. W. , Serafini-Fracassini A.
Published: April 2021

The distribution and relationship of the collagen and the proteinpolysaccharide complex in the extracellular matrix of the nucleus pulposus of the young rabbit were studied by electron microscopy. The polysaccharide was demonstrated by treatment of the tissue with either 0·5 % bismuth nitrate at pH 2 or a mixture of equal volumes of 1 % lead nitrate and 2 % osmium tetroxide at pH 5. The protein part of the complex was observed in tissue fixed in osmium tetroxide at pH 7·2 and stained with 1 % phosphotungstic acid. The collagen of the tissue was apparent in all preparations. The water-extracted proteinpolysaccharide was studied after coagulation of a concentrated solution in 0·5 % bismuth nitrate, whereas the isolated fibrous fraction was examined by negative staining of sprayed material.

Collagen is present in the intact tissues as native collagen fibrils of about 300 Å diameter, and as sheets, 400-Å thick, of parallel, separate, filaments of 50–60 Å diameter. Usually several such sheets lie close, and approximately parallel, to one another. After extraction of the proteinpolysaccharide from the tissue with water, all the collagenous components tend to aggregate into large native collagen fibrils.

Some of the proteinpolysaccharide complex of the intact tissue is free in the matrix. The polysaccharide moiety is visualized as 270 Å particles, serially attached to a protein core in the form of beaded filaments about 40 Å in diameter. The rest of the complex is attached to the sheets of collagen filaments in regular periodic zones which are 270 Å wide and centred 670 Å apart. None of the complex appears to be associated with the larger collagen fibrils.

The staining reactions of the polysaccharides in several connective tissues are discussed and the relationships of the proteinpolysaccharide complexes to collagen in cartilage, the vitreous body and the nucleus pulposus are compared.
Further ultrastructural observations on polysaccharide localization in plant cells
Authors: Pickett-Heaps J. D.
Published: April 2021

Standard periodic acid/Schiff (PAS) techniques have not shown the existence of aldehyde groups in sections of glutaraldehyde-fixed, Araldite-embedded root-tip tissue; peroxidation of such sections resulted in a typical PAS staining pattern. Permanganate-fixed root tips also gave a weak PAS reaction which was intensified by prior peroxidation of the sections. At the ultrastructural level, silver hexamine was used to detect aldehyde groups produced in poly saccharide by permanganate and/or periodate oxidation. Golgi vesicles and slime material in root-cap cells always reacted strongly; the cell wall proper was less reactive. A marked increase in the stainability of the vesicles was evident, the further removed they were from Golgi bodies. This also occurred in root epidermal cells. In both these types of cells, smallersized vesicles and/or the contents of reticulate Golgi cisternae showed evidence of histochemical staining. In meristematic root tip cells, vesicles closely apposed to Golgi bodies did not stain convincingly, though cell walls stained readily. During cell-plate formation, however, both smaller (possibly Golgi) and larger vesicles (phragmoplasts) stained strongly. The walls of permanganate-fixed sieve-tube cells also stained quite strongly, but callose did not unless the tissue block had been treated with periodate before being embedded. In glutaraldehyde-fixed xylem cells, older wall thickenings reacted very strongly even when the sections had been blocked with iodoacetate and bisulphite (which rendered the rest of the section unreactive). If similar sections of younger xylem cells were peroxidized after such blocking reactions, the primary cell wall and the wall thickenings stained, as did many of the Golgi vesicles. The results are related to other experimental observations, both ultrastructural and histochemical, on plant cells.
Spermatogenesis and the structure of the mature sperm in Nucella Lapillus (L)
Authors: Walker Muriel , Macgregor H. C.
Published: April 2021

The testis of Nucella consists of numerous tubules, all directed inwards and joining to form a common testicular duct. In a single tubule the spermatogonia lie round the periphery. Mature sperm line the lumen of the tubule. Cells in the same stage of spermatogenesis are grouped together and all members of a group pass through spermatogenesis in phase.

Staining with fast green before and after treatment with Van Slyke reagent indicates a change from lysine-rich to arginine-rich histone in the maturing spermatid.

Sperm of Nucella are motile throughout their length. The sperm are thread-like and about 80 μ long. The head is Feulgen-positive and about 40 μ long. The mid-piece lies behind the head and is about 8 μ long. The flagellum runs from the front end of the head to the tip of the tail; in the head it is completely surrounded by the nucleus.

The spermatogonia contain two centrioles situated near the nucleus and a conspicuous Golgi complex. There are synaptinemal complexes in spermatocyte nuclei in the synapsis stage. In the early spermatid the centriole pushes a tube through the nucleus. This tube is lined by nuclear membrane and is occupied by the anterior portion of the flagellar shaft. The nucleus elongates and the nucleoprotein condenses into strands arranged helically along the long axis of the nucleus. These strands fuse to form lamellae, which disappear in the mature sperm. Mitochondria aggregate at the base of the early spermatid nucleus and form a loose spiral around the flagellar shaft. The outer mitochondrial membranes fuse. The mid-piece of the mature sperm consists of a large tubular mitochondrion enclosing a portion of the flagellar shaft. At the early spermatid stage a pro-acrosomal granule is formed from a large Golgi complex. From this the acrosome develops; it consists of a cone and an acrosome granule. There are two sets of microtubules associated with the acrosome, one lying within the cone, the other outside the cone and separated from it by a ‘ragged membrane’. The microtubules of the outer set extend backwards along the head for two-thirds of its length. The centriole which gives rise to the flagellar shaft lies at the anterior end of the head and is separated from the acrosome by a thin layer of nucleoprotein and a double layer of nuclear envelope. There is no second centriole or derivative thereof in the mature sperm. In the tail groups of coiled fibres are associated with each pair of the peripheral flagellar fibrils.