A note on the behaviour of spindle fibres at mitosis

Summary Measurements done on mitosis in plant endosperm with the use of Dr. Inoué's polarizing microscope indicate that the whole chromosomal spindle fibre is shifted during anaphase, i.e. the distance between the spindle pole and the polar end of the spindle fibre decreases during anaphase. Consequently in endosperm mitosis the chromosomes move faster to the poles than the chromosomal spindle fibres decrease in length. As the chromosomal spindle fibres in animal materials presumably extend to the spindle poles already from the beginning of anaphase, the chromosomes will here approach the poles with the same speed, as the spindle fibres contract.This paper is dedicated to Professor Franz Schrader on the occasion of his seventieth birthday.     

Further studies on the neurohypophysis of the hedgehog (Erinaceus europaeus)

Summary Further studies of the neurohypophysis of the hedgehog have confirmed the presence of two distinct types of neurosecretory fibre in the infundibular process. A third type of fibre, possibly containing catecholamines, is found in the upper infundibular stem.Lesions of the paraventricular nuclei result in vacuolation of type I nerve fibres, which contain crystal-like and aggregate bodies. Type II fibres, containing membranous inclusions, with or without osmiophilic material, apparently do not degenerate. The evidence suggests that type I fibres arise in both the paraventricular and supraoptic nuclei.Dedicated to Professor Berta Scharrer on the occasion of her 60th birthday.     

Behaviour of chromosomal spindle fibres in living cells

Summary Mitosis in endosperm of Haemanthus katherinae was studied in vitro by the use of 16 mm time-lapse microcinematography. In several cells, chromosomal fibres were seen in phase contrast microscope. The fibres are more convergent in late metaphase and anaphase than in prometaphase. There exist two distinct fibre attachments at each daughter kinetochore, i.e. there are four in metaphase chromosome.Details of the division of a nearly cytoplasm-free mitotic apparatus are also reported as well as some data concerning the elongation of the spindle and activity of the phragmoplast.Dedicated to Professor H. Bauer on the occasion of his sixtieth birthday.     

Lamellate bodies in the nuclei of dragonfly (odonata) oocytes

Summary Concentrically lamellate spherical bodies have been observed in the oocyte nuclei of the anisopterous dragonflyCordulia aenea. In their ultrastructure, these bodies resemble the cortical granules reported from the cytoplasm of the sea urchin oocytes. The lamellate bodies ofCordulia are compared with other lamellate systems known to exist in nuclei. It is suggested that the nuclear lamellate spheres ofCordulia may be homologous with the somewhat less regular lamellate bodies described from maize microspores.The competent help of MissRiitta Lallukka, M. A., is acknowledged with many thanks. We are grateful to ProfessorsESko Suomalainen andAntti Telkkä for helpful suggestions. Grants for the study have been received from the National Research Council for Sciences, from the University of Helsinki and from the Emil Aaltonen Foundation.     

Some cytological aspects of antagonism in synthesis of nucleic acid

Summary The effects of solutions of benzimidazole and 5-aminouracil onAllium cepa root meristems immersed in them have been studied cytologically. Benzimidazole and 5-aminouracil, analogues of purines and pyrimidines respectively, reduce the number of nuclear divisions being initiated. A marked decline in frequency of cells in mitosis occurs at the beginning of treatment with benzimidazole (300 p.p.m.) but not until 6 or more hours later with 5-aminouracil (125 p.p.m.).After treatment with 5-aminouracil, small interstitial portions of chromosomes are Feulgen-negative and become extended during anaphase into thin strands. The distal portion of these chromosomes may lag during anaphase and contribute to malformations of the surface of the sister nuclei and to micronuclei-like bodies lying along their adjacent surfaces. However, stickiness resulting in chromosome bridges was not observed. After treatments with benzimidazole, chromosomes are more contracted at metaphase and anaphase than in the control. At the concentrations used, neither component was observed to affect the spindle mechanism of the divisions in progress at the beginning of treatment nor of the divisions which escape inhibition.Reversal of these effects by the introduction of metabolites which these analogues resemble structurally, was investigated. Adenine sulphate (800 p.p.m.) acts antagonistically to benzimidazole (300 p.p.m.) by delaying the marked decline in frequency of mitoses by more than 6 hrs. By 12 hrs. the frequency of cells in mitoses was very low; thus, reversal is not permanent. Half the inhibition of growth caused by 5-aminouracil (50 p.p.m.) is reversed by thymine (50 p.p.m.) for the 24 hrs. of the test period. When 5 p.p.m. folic acid was added to this mixture, root growth exceeded that of the controls. Feulgen-negative gaps, however, are still present in chromosomes at anaphase.Estimations of Feulgen content of nuclei to determine the effect of these treatments on DNA synthesis were made by means of a microphotometer. In control roots there are two classes of nuclei at interphase according to the amount of DNA per nucleus. The amount doubles during interphase before the next division of the nuclei in the root meristem. In roots treated with 5-aminouracil this doubling is inhibited, so that interphase nuclei with the lower amount of DNA accumulate. In roots treated with benzimidazole the relative frequencies of the two classes of nuclei is unaltered; however, mitotic activity is inhibited. This compound apparently affects not only processes concerned with doubling the amount of DNA per nucleus but also processes necessary to the division itself. There was, however, no unquestionable evidence of a nucleus being reconstituted after it had started a division.Contribution from the Program in Cytology, Department of Botany University of Wisconsin, Madison, supported in part by grants to Dr.C. Leonard Huskins from the American Cancer Society, Rockefeller Foundation, and Research Committee of the Graduate School with funds supplied by the Wisconsin Alumni Research Foundation.     

Chromosomal interchange inEleutherine plicata Herb

Chromosome behaviour at metaphase I and anaphase I of meiosis inEleutherine plicata Herb. (2n=14) is studied. Cells with chromosome associations comprising an association of four long chromosomes, in addition to five bivalents were observed more frequently than those with seven bivalents. it is concluded that the ring of four is due to a segmental interchange between the two long non-homologous chromosome pairs. The ring of four at anaphase I showed delayed disjunction, bridge formation and irregular separation of chromosomes in a number of cells while the behaviour of the other bivalents was normal.     

A gradient of activity in the hypophysis of Rana temporaria

Summary In normal pars distalis of Rana temporaria, a gradient of activity of the P.A.S.-positive cells is always present. In animals, with complete and permanent interruption of all nervous pathways to the median eminence and hypophysis and without disturbance of the normal blood supply of the hypophysis, a subtotal disappearance of the gradient of activity occurred and the total activity of the P.A.S.-cells of the pars distalis was lowered. The activity of the P.A.S.-positive cells regressed in a typical way, suggesting a humoral mechanism.In animals with marked regeneration of the interrupted nerve fibres, a normal gradient of activity was present. From these and previous experiments, it is concluded that the gradient of activity of the P.A.S.-positive cells of the pars distalis of Rana temporaria is largely dependent on the A.F.-negative fibres of the median eminence, not originating from the magnocellular neurosecretory preoptic nuclei. It is suggested that these nerve fibres exert their influence on the activity of the P.A.S.-positive cells via substances released in the blood capillaries of the median eminence.The gradient of activity and its variations are mainly related to the gonadotropic function of the pars distalis.I am indebted to miss M. T. Bakeeff and miss C. Everaert who were so kind to check the karyometric results.     

Meiotic behavior of an unequal bivalent in the grasshopper Calliptamus palaestinensis Bdhr.

Summary Unequal bivalents were found in two of four populations of the short horned grasshopper, Calliptamus palaestinensis bdhr. sampled in Israel.The inequality of the homologues was due to an extra segment which was heterochromatic and apparently terminal. Pairing configurations at pachytene and position of chiasmata at later stages revealed however that the extra segment was interstitial, and the long member terminated in a minute segment homologous to the terminal part of its normal partner. The percentage of reductional divisions at anaphase I corresponded well with the percentage of terminal chiasmata (i.e. distal to the extra segment) observed at diakinesis.The assumption of an interstitial position of the extra segment would explain the previously puzzling examples of unequal bivalents in Orthoptera whose reductional divisions have not been readily accounted for otherwise.     

Fluorescence studies on neural structures and endocrine cells in the pancreas of the cat

Summary With the use of the Falck-Hillarp histochemical technique for the detection of monoamines, nerve fibre fluorescence is observed throughout the tail of the pancreas of the cat and the arrangement and distribution of the nerve fibres can be studied in both the exocrine and endocrine tissue. In the exocrine pancreas, adrenergic nerve fibres innervate arterioles, larger veins and major pancreatic ducts. Adrenergic nerve fibres also appear to terminate on the non-adrenergic nerve cell bodies of the intrapancreatic ganglia. In the islets of Langerhans, adrenergic nerve fibres innervate both the endocrine cells and blood vessels. Some of the islet cells exhibit fluorescence with the Falck-Hillarp technique and these cells have been identified as alpha cells. In animals treated with reserpine, the fluorescence in nerve fibres and in alpha cells is absent.The author wishes to thank ProfessorG. C. Schofield and Dr.G. C. Smith for their encouragement and valuable criticism during the course of this study. The assistance of MissJ. Bennett and MissW. Kemp and the photographic help of Mr.J. S. Simmons, F.R.P.S., are gratefully acknowledged. The diagram was drawn by MissS. Flett.     

The three-dimensional architecture of chromosome fibres in the crane fly

Microtubules of two chromosome fibres in metaphase and two in anaphase of the first meiotic division of Pales ferruginea spermatocytes were traced in serial sections. The number and lengths of microtubules constituting the fibres were determined and the fibres reconstructed. The chromosome fibres were found to contain only about 20% (in metaphase) and 9% (in anaphase) kinetochore microtubules (kMT), taking the sum of all individual microtubule lengths as the measure. The remainder consisted of free microtubules (fMT), a certain percentage of which were oriented obliquely with respect to the kMTs (skew fMTs). Between metaphase and anaphase most kMTs and fMTs shortened, causing a decrease in mean length of about onehalf in the case of kMTs. The proportion of skew fMTs increased considerably in anaphase, leading to more disorder in the fibre. The absolute mass of microtubules in the fibre seems to be irrelevant for spindle function. This can be deduced from the observed variability of absolute mass. A statistical correlation was found between the total mass of microtubules (kMTs+fMTs) and the number of kMTs, suggesting that some functional interrelation exists between these parameters.Dedicated to Prof. Hans Bauer on the occasion of his 80th birthday     

The function of the hypophysis without preoptic neurosecretory control

Summary In a large number of adult female and male specimens of Rana temporaria, a total extirpation of the magnocellular neurosecretory preoptic nuclei was performed. Several months after operation, no regeneration of the neurosecretory nuclei had occurred. The results showed 1. that all A.F.-positive neurosecretory nerve fibres of the hypothalamo-hypophysial region originate from nerve cell bodies localized in the magnocellular preoptic nuclei; 2. that the magnocellular preoptic nuclei do not play an important part in the inhibitory control of the activity of the pars intermedia of Rana temporaria; 3. that the possible role of the magnocellular preoptic nuclei in the seasonal development of the gonads and of the secondary sexual characteristics of Rana, could be only of secondary importance; 4. that, in accordance with previous experiments, in Rana, ovulation is dependent on preoptic hypothalamic structures. Some results of the present and of other experiments can be interpreted as supporting the hypothesis of a possible dual higher control over the gonadotropic centre (Dierickx, 1965b, 1966, 1967) of the pars ventralis of the tuber cinereum.A part of these studies has been reported previously in abstract form (Dierickx, 1963a, 1963b, 1965a).     

Endopolyploidy and structures of the nuclei in the endosperm of theCucurbitaceae

Summary In the endosperm ofEchinocystis lobata the endopolyploid nuclei show structural changes of chromocentres typical of the process of endomitosis in Angiosperms.The cytological differentiation of the endosperm proper and the chalazal haustorium in three further representatives of theCucurbitaceae: Sicyos angulata, Cyclanthera pedata andCitrullus colocynthis is also connected with endomitotic polyploidisation. The nuclei of the endosperm proper attain in the different species different levels of polyploidy, 384n, as a maximum. In the nuclei of the chalazal haustorium the degree of polyploidy is lower (as a maximum 24n).The structure of polyploid nuclei which did not undergo postendomitotic mitoses shows some differences in the species studied in the course of the present work.Sicyos angulata has prochromosomic diploid nuclei, whereas those ofCitrullus colocynthis andCyclanthera pedata can be assigned to chromomeric nuclei with a dense structure. Endopolyploid nuclei of all three species studied are characterized by the occurence of compact chromocentres of different size; besides in some nuclei ofSicyos angulata andCyclanthera pedata the chromocentres within the resting nucleus reveal their chromomere structure. In addition, inCitrullus colocynthis nuclei with numerous thin threads of various degrees of spiralisation have been observed. — In all three species the endopolyploid nuclei after mitotic divisions have notably higher numbers of nucleoli and small chromocentres.Dedicated to Professor L.Geitler on the occasion of his 70th birthday.     

Mitosis in the protostelidPlanoprotostelium aurantium

Summary Mitosis in the protostelidPlanoprotostelium aurantium Olive andStoianovich is characterized by an open, centric spindle. The nuclear envelope breaks down prior to metaphase, begins to reform during late anaphase, and is complete by telophase. Centrioles are present at the poles throughout mitosis and are devoid of rootlet microtubules from metaphase to late anaphase. Chromosomes are small and numerous and are attached to single kinetochore microtubules during metaphase and early anaphase. Chromosome separation takes place by a presumed shortening of the chromosome to pole spindle followed by a lengthening of the interzonal spindle. Mitosis inP. aurantium is similar to that of certain other protostelid amoebae and to myxomycete amoebae, but it is considerably different from that of dictyostelid amoebae. The phylogenetic significance of this is discussed.This research represents part of a Ph.D. dissertation presented to the University of North Carolina.     

Some observations on the mode of division of somatic nuclei of Mucor and Allomyces

Summary A series of successive photographs of the division of the living nucleus in a germinating sporangiospore of Mucor fragilis has been obtained. In this sequence the nucleus is seen to divide directly by elongation and constriction. The nucleolus divides at the same time and in the same way. These observations agree with the finding, first made by Léger (1896) and several times confirmed since then, that the nuclei of Mucorales apparently divide without first arranging their chromosomes in a metaphase plate and without the help of a spindle apparatus.In stained preparations of Mucor chromosomes are not normally visible as separate entities but they can be clearly seen in Feulgen preparations of dividing somatic nuclei of Allomyces arbuscula. In contrast to Mucor the nucleolus of Allomyces is dissolved during division. The chromosomes seem to sort themselves out on their own and form new nucleoli. Metaphase plates and spindles have not been encountered.To Professor Dr. E. G. Pringsheim, teacher and friend, on his 80th bithday.     

Ultrastructural localization ofl -fucose residues in nuclei of root primordia of the green peaPisum sativum

Summary Sugars have been demonstrated in animal cell nuclei, but only a few studies have mentioned their presence in plant cell nuclei. In this studyl-fucose residues were localized at the ultrastructural level, usingUlex europeaus agglutinin I lectin, during the early stages of germination ofPisum sativum and in mature root tip cells. This sugar was present after 1 h of germination, and its concentration was found to vary during 3 to 6 h imbition; after 72 h of imbition its concentration had more than doubled. Furthermore, labelling was particularly abundant in the nucleolus, nucleolus-associated bodies and dense nuclear bodies. The possibility that some of thel-fucose residues are associated with proteins is discussed.     

Remarkable adrenergic nerves in the exocrine pancreas

Summary The adrenergic nerves in the pancreas of mice, rats, guinea-pigs, rabbits, and cats were investigated with the fluorescence method of Falck and Hillarp. The relations between the adrenergic fibres and the vessels were studied by the injection of india ink into the vessels.Besides the normal manifestation of adrenergic fibres at the large vessels, some vessels of capillary size were also accompanied by adrenergic fibres. These fibres had a very weak fluorescence, and showed up regularly only when the animal had been treated with Nialamide and L-DOPA or dopamine to increase the catecholamine content of the adrenergic fibres. The weakness of the fluorescence is perhaps due to low transmitter concentration or to small size of the nerve fibres, or to both. A rough estimate indicated that either the transmitter concentration of the nerve fibre is at least approximately 100 times below that seen in adrenergic nerves in other tissues, or that the radius of the varicosities of the nerve fibres is less than 0.2 . Neither alternative has previously been recognized.The secretory acini of the pancreas seem to lack a direct adrenergic supply. In the intrapancreatic ganglia, non-fluorescent nerve cells were reached by adrenergic terminals. No adrenergic nerve cells were detected in the pancreas of rats and cats. Small intensely fluorescent catecholamine-containing cells were observed in connexion with the intrapancreatic ganglia of rats.The research reported in this document has been sponsored by the Air Force Office of Scientific Research under grant AF EOAR 67-15 through the European Office of Aerospace Research (OAR), United States Air Force, by the United States Public Health Service (grant NB 06701-01) by the Swedish Medical Research Council (project B 67-12X-712-02A), and by the Faculty of Medicine, University of Lund, Sweden.     

Deoxyribonucleic acid synthesis in isolated polytene nuclei of Drosophila hydei

DNA synthesis has been studied in polytene nuclei isolated from larval salivary glands of Drosophila hydei. The incubation conditions employed promote maximum incorporation of TTP-H³ and retention of normal polytene chromosome morphology. The chromosome structure is sensitive to the Mg²⁺ concentration; a normal banding pattern is observed between 4 and 10 mM Mg²⁺. At the optimum pH of 7.8, incorporation continues for over an hour. All four deoxyribonucleoside triphosphates are required for maximum incorporation. The reaction is stimulated by 0.6 mmATP and strongly inhibited at higher ATP concentrations. Competition experiments demonstrate that either TDP or TTP is the effective labeled precursor. The labeled product is sensitive to DNase and has a density identical to that of nuclear DNA. Autoradiographs prepared from spread chromosomes demonstrate that discontinuous and continuous labeling patterns observed in vivo are also produced with isolated nuclei in the absence of cytoplasmic factors. Incubation of the isolated nuclei results in a low level of uniform incorporation that is superimposed on the normal autoradiographic pattern obtained after in vivo labeling. This background incorporation can be greatly increased by prior irradiation of the glands. The presence of exogenous DNA during nuclear incubation stimulates total incorporation. These observations demonstrate that the isolated nuclei possess a reserve synthetic capacity. About 20% of the isolated nuclei are inactive in DNA synthesis.This investigation was supported by PHS Research Grant No. 5 R01 GM 16298 from the National Institute of General Medical Sciences.     

Light,fluorescence and electron microscopic studies on “neurosecretion” in tritonia diomedia bergh (Mollusca,Nudibranchia)

Summary Membrane-limited electron-dense inclusions designated as elementary neurosecretory granules have a characteristic distribution in cerebropleural ganglia of the nudibranch snail Tritonia diomedia. They occur in the neuropile and also in individual nerve fibres, connectives and commissures. These granules have been found neither in perikarya of nerve cells nor in proximal segments of their processes.Specific fluorescence obtained in Tritonia preparations with Sterba's pseudoisocyanin method for neurosecretory products has the same pattern of location.The distribution of stainable material in preparations prepared with ordinary neurosecretory procedures (chrome haematoxylin-phloxin after Gomori-Bargmann and paraldehydefuchsin after Gomori-Gabe) is similar to that described by different authors in other gastropods, but strongly differs from the locationof elementary neurosecretory granules and of pseudoisocyanin-positive material. The adequacy of different histological methods for studying neurosecretion in gastropods is discussed.     

Inversion heterozygosity and sub-chromatid exchange in Agave stricta

A plant of Agave stricta Salm. (2n=60) has a bimodal complement of 10 L, 4 M and 46 S chromosomes. It is heterozygous for a paracentric inversion which involves the middle third of the long arm of one of the L chromosomes. It produces at anaphase I bridges and fragments and also loops and fragments, both single and double. Breakage and reunion at the sub-chromatid and at the chromatid level produce side-arm bridges and bridges and fragments respectively at anaphase I. A method is given, based on chiasma frequency, which will in certain cases of inversion heterozygosity provide a reasonable estimate of the position and the length in genetic map units of an inverted segment with respect to the whole chromatid arm.     

The effect of starvation on the ultrastructure of the red and white myotomal muscles of the crucian carp (Carassius carassius)

Summary The effect of 16 weeks total starvation on the ultrastructure of the red and white myotomal muscles of the crucian carp (Carassius Carassius) has been investigated. In the white fibres the amount of myofibrillar material fell from 89.6% to 70.7% of the total fibre volume whilst in the red fibres the fall was from 72.2% to 70.3%. The sarcoplasmic reticulum appeared to have become swollen during starvation in both fibre types. In the white fibres the terminal cisternae of some triads seem to have fused. The volume of the red fibres occupied by mitochondria was reduced from 16.2 % to 5.9 %. The concentration of mitochondria in the white fibres was too low to detect any quantitative changes. A marked reduction in the amount of euchromatin material was observed in most white fibre nuclei and many red fibre nuclei. Many of the ultrastructural changes noted in the present study can be correlated with biochemical changes known to occur in the red and white myotomal muscles of fish during starvation. This work was supported by a grant from the Natural Environmental Research Council.