Reversibility of hepatocyte nuclear modifications in mice fed on genetically modified soybean

In the literature, the reports on the effects of a genetically modified (GM) diet are scanty and heterogeneous; in particular, no direct evidence has so far been reported that GM food may affect human or animal health. Hepatocytes represent a suitable model for monitoring the effects of a GM diet, the liver potentially being a primary target. In a previous study, we demonstrated that some modifications occur in hepatocyte nuclei of mice fed on GM soybean. In order to elucidate whether such modifications can be reversed, in the present study, 3 months old mice fed on GM soybean since their weaning were submitted to a diet containing wild type soybean, for one month. In parallel, to investigate the influence of GM soybean on adult individuals, mice fed on wild type soybean were changed to a GM diet, for the same time. Using immunoelectron microscopy, we demonstrated that a one-month diet reversion can influence some nuclear features in adult mice, restoring typical characteristics of controls in GM-fed animals, and inducing in control mice modifications similar to those observed in animals fed on GM soybean from weaning. This suggests that the modifications related to GM soybean are potentially reversible, but also that some modifications are inducible in adult organisms in a short time.     

Nucleoli undergo structural and molecular modifications during hibernation

The nucleolus is a very dynamic structure able rapidly to adapt its activity to the cellular metabolic state. An interesting physiological model characterized by drastic modifications of cellular metabolism is represented by hibernating animals. In the present study we investigated the hepatocyte nuclei of euthermic and hibernating edible dormice (Glis glis) with the aim of revealing, by means of ultrastructural and immunocytochemical analyses, possible modifications of nucleolar components during hibernation. Our observations demonstrate that, in deep hibernation, nucleoli undergo structural and molecular modifications: (a) they show numerous nucleoplasmic invaginations and clumps of dense fibrillar component extend from the nucleolar surface; (b) they are frequently in contact with coiled bodies and fibro-granular material, two nuclear bodies usually occurring in the nucleoplasm; (c) the dense fibrillar component contains significant amounts of small nuclear ribonucleoproteins, splicing factors usually distributed in the nucleoplasm. Taken together, these results suggest that during hibernation complex relationships are established between the nucleolus and nucleoplasm, probably related to functional activities peculiar to this physiological phase. However, since no evident nucleolar modification was found in early hibernating dormice, it seems likely that the particular structural and molecular arrangement of nucleoli establishes progressively during hibernation, becoming evident only in the deepest phase, and then disappears upon arousal.     

Ultrastructural analysis of testes from mice fed on genetically modified soybean

We have considered the possible effects of a diet containing genetically modified (GM) soybean on mouse testis. This organ, in fact, is a well known bioindicator and it has already been utilized, for instance, to monitor pollution by heavy metals. In this preliminary study, we have focussed our attention on Sertoli cells, spermatogonia and spermatocytes by means of immunoelectron microscopy. Our results point out that the immunolabelling for Sm antigen, hnRNPs, SC35 and RNA Polymerase II is decreased in 2 and 5 month-old GM-fed mice, and is restored to normal at 8 months. In GM-fed mice of all ages considered, the number of perichromatin granules is higher and the nuclear pore density lower. Moreover, we found enlargements in the smooth endoplasmic reticulum in GM-fed mice Sertoli cells. A possible role played by traces of the herbicide to which the soybean is resistant is discussed.     

Study of RNA synthesis in the livers of aging mice by means of electron microscopic radioautography.

The application of 3H-uridine radioautography results in labeling of the liver cells in which RNA is synthesized at various ages of the mouse. Quantitative changes of RNA synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. The silver grains were mainly located in the nucleoli and nuclei and a few in the mitochondria and rough surfaced endoplasmic reticulum of almost all of the cell populations at various ages. The number of silver grains in the hepatocyte gradually increased after birth, reached the maximum at 14 days of postnatal age, then decreased to 24 months with aging. The number of silver grains of the euchromatin was more than those of the heterochromatin of the hepatocyte nuclei at various ages. The number of silver grains of the granular components was more than those of the fibrillar components of the hepatocyte nucleoli at various ages. However, the ratio of silver grains among euchromatin, heterochromatin, granular components and fibrillar components remained approximately constant.     

A comparative ultrastructural study of hepatocyte nucleolar structures observed in physiological and pathological conditions

Aggregates of fibrillar material were observed within the meshes of the nucleolonema and at the periphery of hepatocyte nucleoli of 7-day-old untreated C3Hf and BUA mice. These aggregates had a lower density and smaller diameter than microspherules induced in mice of the same age and strains by urethane, a chemical that has been shown to inhibit RNA and DNA synthesis. However, aggregates shared some features with the microspherules since both structures had a similar fibrillar appearance and an identical localization, were easily digested by pronase and pepsin, and were bleached more than chromatin after being subjected to Bernhard's preferential staining. A close correlation between aggregates and microspherules is postulated.     

Electron microscopic study of endopolyploid nuclei in rat trophoblast giant cells. IV. Changes in nucleolar ultrastructure during cell differentiation

The ultrastructure of the nucleolus of highly differentiated trophoblast giant cells has been studied on the 17th day of the foetus development. Changes in its morphology have been followed in relation to the degree of nuclear chromatin condensation and to the cell differentiation level. The nucleoli have a reticular structure in the nuclei with dispersed and condensed chromatin. In both the cases the nucleoli involve the four components: fibro-granular, fibrillar (of moderate and normal density) and lacunar regions; fibrillar centres are distinguished within the regions. In the nucleoli with condensed chromatin, unlike those with dispersed chromatin, the perinuclear chromatin is clearly seen, and the penetration of nucleolus-organizer threads along lacunae and deep into the nucleolus can be easily followed. The fibrillar centres are more obvious. With the run of a progressive differentiation of the trophoblast cells, the number of granules is reduced; first, the fibro-granular component covers a significant part of the nucleolus, then granules become visible only in the cortical zone of the nucleolus; in the nuclei with strongly condensed chromatin no granules are seen in the nucleolus.     

Ultrastructural organization of nucleoli in benign naevi and malignant melanomas

We have studied the ultrastructural organization of nucleoli in benign naevi and malignant melanomas. In benign naevus cells the nucleoli displayed a compact ribonucleoprotein distribution, with one or two large fibrillar centres. In malignant melanoma cells the nucleoli were large with an irregular, nucleolonema-like ribonucleoprotein distribution and they exhibited numerous, small fibrillar centres. Statistical evaluation of the size of fibrillar centres indicated a mean value of 0.482 micron 2 +/- 0.136 SD for naevi and 0.221 micron 2 +/- 0.128 SD for malignant melanomas. These features, together with the more dispersed chromatin pattern of malignant melanoma nuclei compared with those of benign naevus cells, are proposed as diagnostic parameters which differentiate benign naevi from malignant melanomas at the ultrastructural level.     

Oogenesis in turbellarians of the genus Geocentrophora studied by light and electron microscopy. V. Oocyte nuclear structures containing factors of pre-mRNA splicing and pre-rRNA processing

An immunoelectron study of nuclear distribution of pre-mRNA splicing and pre-rRNA processing factors was carried out for oocytes of two turbellarian species: the Baikal endemic Geocentrophora wagini and a cosmopolitan G. baltica. Using monoclonal antibodies against Sm-epitope of small nuclear RNPs (snRNPs) and SR-protein SC35, it has been shown that on different stages of oocyte growth splicing factors (snRNPs and SC35) are distributed within the whole nucleus. A fibrogranular material located near heterochromatin clumps is labeled with these antibodies. A fibrillar part of this material seems to represent perichromatin fibrils. The features of intranuclear distribution of splicing factors in Geocentrophora oocyte nuclei and their ultrastructural features suggest that pre-mRNA synthesis and splicing may occur up to the end of diplotene. In Geocentrophora oocyte nuclei a few nuclear bodies (NBs) were found. Splicing factors (snRNPs and SC35) and fibrillarin were revealed in these NBs. Homology of Geocentrophora oocyte NBs to coiled bodies of oocyte and somatic cell nuclei of other animals is discussed. During diplotene, Geocentrophora oocyte nucleoli were found to lose their granular component and to change to large fibrillar structures named "postnucleoli". The postnucleoli contain both fibrillarin and non-nucleolar spliceosomal components (snRNPs and SC35). Geocentrophora oocyte postnucleoi are compared with similar structures of mammalian oocyte nuclei, taken as an example of morphological convergence of nuclear structure organization in phylogenetically distant animal species.     

Histochemical and morpho-metrical study of mouse intestine epithelium after a long term diet containing genetically modified soybean

Diet can influence the structural characteristics of both small and large intestine. In this study, we investigated the duodenum and colon of mice fed on genetically modified (GM) soybean during their whole life span (1–24 months) by focusing our attention on the histological and ultrastructural characteristics of the epithelium, the histochemical pattern of goblet cell mucins, and the growth profile of the coliform population. Our results demonstrate that controls and GM-soybean fed mice are similarly affected by ageing. Moreover, the GM soybean-containing diet does not induce structural alterations in duodenal and colonic epithelium or in coliform population, even after a long term intake. On the other hand, the histochemical approach revealed significant diet-related changes in mucin amounts in the duodenum. In particular, the percentage of villous area occupied by acidic and sulpho-mucin granules decreased from controls to GM-fed animals, whereas neutral mucins did not change.Key words: diet, genetically modified soybean, intestine, goblet cell mucins, coliform bacteria.     

Correlation of nucleolar activity and nucleolar vacuolation in plant cells

In root meristematic cells nucleolar structure varies with the cell cycle. Apart from normal meristematic nucleoli one finds nucleoli with a big central vacuole surrounded by a loose cortex with individual fibrillar centres [22] clearly visible within it. There are also intermediate structures between both nucleolar types. In Pisum sativum nuclear tissue, the structure of the vacuolated nucleoli is similar and appears in periods of high metabolic activity during megasporogenesis. In both tissues, vacuolated nucleoli incorporate tritiated uridine more actively than 'normal' nucleoli. In this work the structure of spontaneous nucleolar vacuoles is compared with that induced by drugs such as cordycepin, and FUdR. The vacuolated nucleolus with its increased surface corresponds to a transient structure which not only shows higher metabolic activity but also supplies a storing and/or transporting mechanism for nucleolar products.     

Sphingolipids of the nucleus and their role in nuclear signaling

Sphingolipids have important signaling and regulatory roles in the nuclei of all vertebrate cells examined to date. Sphingomyelin (SM) is the most abundant of this group and occurs in the nuclear envelope (NE) as well as intranuclear sites. The primary product of SM metabolism is ceramide, whose release by nuclear sphingomyelinase triggers apoptosis and other metabolic changes in the nucleus. Further catabolism results in free fatty acid and sphingosine formation, the latter being capable of conversion to sphingosine phosphate by action of a specific nuclear kinase. Finally, glycosphingolipids such as gangliosides occur in the NE where GM1, one member of the gangliotetraose family, influences Ca(2+) flux by activation of a Na(+)/Ca(2+) exchanger located in the inner membrane of the NE. The tightly associated GM1/exchanger complex was shown to exert a cytoprotective role in neurons and other cell types, as absence of this nuclear complex rendered cells vulnerable to apoptosis. A striking example of this mode of Ca(2+) regulation is the greatly enhanced seizure activity in knockout mice lacking gangliotetraose gangliosides, involving programmed cell death in the CA3 region of the hippocampus. In this model, Ca(2+) homeostasis was restored most effectively with LIGA-20, a membrane-permeant derivative of GM1 that entered the NE and activated the nuclear Na(+)/Ca(2+) exchanger.     

Control of nucleolar growth during interphase in higher plant meristem cells

Summary The evolution of nuclear and nucleolar sizes throughout interphase have been studied in synchronous caffeine-labeled binucleate cells of onion root meristems by using silver impregnation and stereological methods over semithin sections. Nucleus and nucleolus grow independently, since nucleolus enlarges at its fastest rate in G 1, while nucleus grows mostly in two periods: onset of replication and G 2. Nucleolar size in the cycle seems to be a genecontrolled function, hardly affected by protein synthesis inhibition. Hence, there is a biphasic response to cycloheximide (CHM) in the fast growing nucleoli of both early and late G 1 with an initial stimulation later counterbalanced by a depressed rate, so that nucleolar size in S was similar to control shortly afterwards the start of the CHM treatment. The initial enlargement under CHM was due to an increase of all nucleolar structural components, i.e., fibrillar, granular, vacuolar, and lacunar regions. No cycloheximide effect whatsoever was detected in S and G 2 nucleoli.Abbreviations CHM cycloheximide - F fibrillar component - G granular component - L lacunae - V vacuoles - VN nuclear volume - VNu nucleolar volume - VvNu volume density of the nucleoli