Ultrastructural and autoradiographic studies of non-generative cells in the antheridium of Chara vulgaris. I. Manubria

During the development of an antheridium DNA content in manubria gradually increases to 8C-16C level. 3H-thymidine incorporation into the nuclei of the manubria lasts till the stage of quantitative predominance of the 16 celled antheridial filaments. The nucleus of the manubria is characterized by the low content of the condensed chromatin and the presence of nucleoli with the nucleolonema-like structure the number of which increases from 6-8 to 32-38 along with the increase of DNA content in a nucleus. In the cytoplasm of the manubria there are numerous secretive vesicles filled with fine-granular substance discharged outside plasmalemma, active Golgi apparatus, well-developed rough ER, numerous polysomes, mitochondria with the condensed structure and plastids with granar and inter-granar thylakoids as well as plastoglobules which increase in number and size along with the development of the antheridium. During spermiogenesis the cells are vacuolated, the number of the secretive vesicles decreases whereas the electron density of their content increases, smooth ER appears while rough ER is reduced. The manubria actively incorporate 3H-uridine, 3H-tryptophane and 3H-leucine. The increase of the incorporation activity is gradual in the period of increasing polyploidy of the manubria and rapid during the initiation of the spermatozoid differentiation. It has been suggested that the manubria should play an important role in the process of spermatogenesis and the induction of spermatozoid differentiation.     

Ultrastructural and autoradiographic studies of non-generative cells in the antheridium of Chara vulgaris. II. Capitular cells

In young antheridia, the structure of capitular cells is typical of meristematic cells. The cytoplasm is characterized by poorly developed ER system, numerous free ribosomes, active Golgi apparatus and plastids at the stage of proplastids. In the period of mitotic divisions, i.e. during formation of the initial cells of antheridial filaments, the nuclei of capitular cells have a changing structure. When capitular cells stop budding leading to the formation of successive antheridial filaments. DNA content in the nucleus is at 2C-4C level. The nucleolus with nucleolonema-like structure becomes gradually smaller in the course of the development of the anteheridium. During spermiogenesis capitular cells are vacuolated, cytoplasm contains numerous polysomes, mitochondria assume condensed structure, the incorporation of 3N-uridine and of labelled aminoacids increases. It has been suggested that capitular cells collaborate with other antheridial cells in the regulation of the course of spermiogenesis.     

Ultrastructural and autoradiographic studies of non-generative cells in the antheridium of Chara vulgaris L. III. Shield cells

Shield cells form the antheridium envelopes. At the first stage of spermatogenesis they grow intensively in the tangential direction, which is stopped during the period of spermatozoid differentiation. The increase in shield cell volumes is associated with the increase in DNA level in the nucleus up to 16-32 C. 3H thymidine incorporation occurs in about 30% of shields at younger developmental stages and lasts until the stage in which 16 celled antheridial filaments predominate. At first stage of spermatogenesis the intensity of 3H leucine incorporation increases as DNA amount in the nuclei increases, reaching the maximum value at the end of this period. During spermiogenesis it gradually decreases. Shield nuclei are characterized by low content of condensed chromatin, the presence of numerous nucleoli with nucleolonema-like structure as well as the occurrence of bands of intranuclear microtubules. It has been suggested that these microtubules are associated with cyclical changes in the shapes of nuclei. During DNA replication the nuclei have the form of flat discs which between successive endoreplication cycles become ring shaped. Peripheral zone of shield cells is compartmentalized through incomplete walls. They support the radial walls of shields increasing the contact surface of plasmalemma with a cell wall. During spermiogenesis the increase in plasmalemma surface results from the growth of shields in the radial direction. The shield cells contain plastids placed close to each other at the inner tangential wall. They are orange in colour and have fully formed system of grana and intergrana thylakoids, like the plastids of the thallus. The number and sizes of the plastoglobules increase as the anteridium develops. Dictiosomes are surrounded with numerous smooth and coated vesicles. Mitochondria exhibit poorly condensed structure. Microbodies adjoining the plastids are sporadically encountered. It has been assumed that changes in structural organization as well as growth character of shield cells constitute the factor regulating the exchange with external environment, determine light spectrum penetrating to the antheridium and the volume of antheridial space.     

Determination of GA3 in Chara vulgaris by capillary electrophoresis system

Method for simultaneous measurement of gibberellic acid was applied using capillary zone electrophoresis. Gibberellic acid was identified in extracts of apical part of thallus of Chara vulgaris L. The amount of gibberellins measured on the basis of activity determined by the micro-drop bioassay (59.8 mg·kg⁻¹; with gibberellic acid as a standard) was comparable with that estimated by capillary electrophoresis (54.9 mg·kg⁻¹).     

Detection of changes in the mouse circadian rhythm induced by stress

Mice were subjected to three types of acute stress (cold, forced swimming and tail hanging) in order to investigate the effects of stress on the motor activity circadian rhythm. This rhythm was studied using the cosinor method and spectral analysis. A statistically significant decrease in the amplitude and the power content of the circadian harmonic was found after stress application. These decreases could be due to a desynchronization of the circadian oscillators which drive the rhythm. The use of the power content of the circadian harmonic is proposed for the detection of the alterations due to stress.     

Further ultrastructural research of Chara vulgaris spermiogenesis: endoplasmic reticulum,structure of chromatin, 3H-lysine and 3H-arginine incorporation

On the basis of morphological features, 10 consecutive structural phases of spermatids were identified in Chara vulgaris spermiogenesis. They were schematically presented. In early and middle spermiogenesis, i.e. during the period preceding formation of fibrillar structure of mature spermatozoid nucleus, a slight remodelling of chromatin, accompanied by proplastid transformation into an amyloplast as well as by development of 2 flagella and a microtubular manchette, is observed. First, condensed chromatin concentrates around the nuclear envelope (phases III-V) and then it transforms into a network-like structure (phase VI). This change in chromatin structure is preceded by nucleolar extrusion to the cytoplasm where nucleoli become degraded (phase IV) and by a dynamic development of rough endoplasmic reticulum (RER) (phase V) which is continuous with the nuclear envelope and with RER of the adjacent spermatids via plasmodesmata. The inner membrane of the nuclear envelope invaginates into the nucleoplasm in which "nuclear reticulum" appears. It all happens during increased 3H-arginine and 3H-lysine incorporation into proteins which are rapidly translocated into the nucleus. In medium-late spermiogenesis (phases VI-VIII), network-like condensed chromatin disappears. Next, the structure of the nucleus changes dramatically. Short, randomly positioned fibrils (phase VII) appear and gradually become longer (phase VIII), thicker (phase IX) and more distinct, lying parallel to the surface of elongating and curling nucleus. Membranes of the nuclear envelope become closer to each other and a distinct dark layer--probably lamin--appears adhering to the inner membrane of the nuclear envelope. Towards the end of spermiogenesis (phase X), very densely packed parallel helices, ca 2 nm in diameter, are visible. The surfaces of flagella and the spermatozoid are covered with diamond-shaped larger and smaller scales, respectively. Helically coiled spermatozoids are liberated from antheridial filament cells through earlier created (phase VIII) "liberation pores" with pads of unknown nature.     

Cellular stabilization of the melatonin rhythm enzyme induced by nonhydrolyzable phosphonate incorporation

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) controls daily changes in the production and circulating levels of melatonin. Here, the significance of the phosphorylation of AANAT was studied using a semisynthetic enzyme in which a nonhydrolyzable phosphoserine/threonine mimetic, phosphonomethylenealanine (Pma), was incorporated at position 31 (AANAT-Pma31). The results of studies in which AANAT-Pma31 and related analogs were injected into cells provide the first direct evidence that Thr31 phosphorylation controls AANAT stability in the context of the intact cells by binding to 14-3-3 protein. These findings establish Thr31 phosphorylation as an essential element in the intracellular regulation of melatonin production. The application of Pma in protein semisynthesis is likely to be broadly useful in the analysis of protein serine/threonine phosphorylation.     

Multiscale modeling of tumor growth induced by circadian rhythm disruption in epithelial tissue

We propose a multiscale chemo-mechanical model of cancer tumor development in epithelial tissue. The model is based on the transformation of normal cells into a cancerous state triggered by a local failure of spatial synchronization of the circadian rhythm. The model includes mechanical interactions and a chemical signal exchange between neighboring cells, as well as a division of cells and intercalation that allows for modification of the respective parameters following transformation into the cancerous state. The numerical simulations reproduce different dephasing patterns—spiral waves and quasistationary clustering, with the latter being conducive to cancer formation. Modification of mechanical properties reproduces a distinct behavior of invasive and localized carcinoma.     

Altered circadian rhythm of the clock genes in fibrotic livers induced by carbon tetrachloride

Disruption in circadian rhythms either by mutation in mice or by shiftwork in people, is associated with an increased risk for the development of multiple organ diseases. In turn, organ disease may influence the function of clock genes and peripheral circadian systems. Here we showed that hepatic fibrosis induced by carbon tetrachloride in mice leads to alterations in the circadian rhythms of hepatic clock genes. Especially, we found an impaired daily Cry2 rhythm in the fibrotic livers, with markedly decreased levels during the day time while compared with control livers. Associatively, the expressions of two important clock-regulated genes peroxisome proliferator-activated receptor alpha and cytochrome P450 oxidoreductase lost circadian rhythm with significantly decreased levels during the light-dark (12/12 h) cycle in fibrotic livers.     

The effects of testosterone administered neonatally on the incorporation of 3H-leucine into protein by thyroid gland of rat: a neonatal priming.

Rats were castrated on day 2 after birth, given one injection of testosterone pronate (TP: 2.5 mg) or oil just after operation and then received TP or oil when adult. 3H-Leucine was injected intravenously 2.5 hours before killing. The incorporation of labelled amino acid into protein was investigated in the thyroid glands. Males receiving TP both neonatally and when adult incorporate twice as much labelled leucine into protein as compared to their counterparts. Grain density was highest in the thyroid gland of early hormone treated rats when challanged with testosterone in adulthood. Thyroid gland priming by early hormone treatment has been discussed.     

Effect of different fixation methods and partial chemical sympathetic denervation on cell size and 3H-leucine incorporation by rat sympathetic neurocytes

Dimensions of sympathetic nerve cells and of their nuclei were studied using various methods of fixation and embedding media, as well as autoradiographic indices of the protein synthesized system of neurocytes in normal rats, and in rats partially sympathectomized at an early age. On the ground of different changes in the average size of neurocytes seen under various methods of fixation, and of decreased labeling intensity of large neurocytes after the injection of the labeled protein biosynthesis precursor there is some reason to believe that large neurocytes may appear in the late reproductive age mainly due to the nerve cell cytoplasm swelling as a consequence of exhaustion of their growth resources.     

Phase shifts of the circadian locomotor rhythm induced by pigment-dispersing factor in the cricket Gryllus bimaculatus

Pigment-dispersing factors (PDFs) are octadeca-peptides widely distributed in insect optic lobes and brain. In this study, we have purified PDF and determined its amino acid sequence in the cricket Gryllus bimaculatus. Its primary structure was NSEIINSLLGLPKVLNDA-NH(2), homologous to other PDH family members so far reported. When injected into the optic lobe of experimentally blinded adult male crickets, Gryllus-PDF induced phase shifts in their activity rhythms in a phase dependent and dose dependent manner. The resulted phase response curve (PRC) showed delays during the late subjective night to early subjective day and advances during the mid subjective day to mid subjective night. The PRC was different in shape from those for light, serotonin and temperature. These results suggest that PDF plays a role in phase regulation of the circadian clock through a separate pathway from those of other known phase regulating agents.     

DNA-polymerase activity detected in situ. Relationship between incorporation of 3H-deoxytriphosphates and 3H-actinomycin D binding during the cell cycle in antheridial filaments of Chara vulgaris L. Effect of auxin and cytokinins

DNA-polymerase activity during the cell cycle (S + G2 + M + C type) in antheridial filaments cells of Chara vulgaris was studied using the autoradiographic method. Incorporation of 3H-deoxytriphosphates (3H-dTPs) during the whole of interphase indicates, that the cell cycle is not accompanied by distinct changes in enzyme activity. Incorporation of 3H-dTPs was also observed in spermatids and in early stages of spermatogenesis. Intensity of 3H-dTPs incorporation during interphase and spermatogenesis is similar to the intensity of 3H-actinomycin D (3H-AMD) binding. Auxin (IAA) and kinetin stimulate both 3H-AMD binding and 3H-dTPs incorporation; benzyladenine does not affect any of these processes. The in situ autoradiographic method of detecting DNA-polymerase activity reveals availability of DNA template for the enzyme rather than DNA polymerase activity itself.     

GA3 content in antheridia of Chara vulgaris at the proliferative stage and in spermiogenesis estimated by capillary electrophoresis

In male sex organs of Chara vulgaris L., the gibberellic acid (GA3), was identified by capillary zone electrophoresis. The antheridia at cell division stage of antheridial filaments leading to formation of spermatids contain 0.09 microg GA3 per antheridium, i.e. 5.3 times more than antheridia at differentiation stage of spermatozoids (spermiogenesis). Spermiogenesis is not regulated by gibberellins.