Big Nucleus Research Articles

Anatomy and ultrastructure of osmophores of Cymbidium tracyanum Rolfe (Orchidaceae)

The intense smell secreted by flowers of <i>Cymbidium tracyanum</i> Rolfe (<i>Orchidaceae</i>) derives from osmophores situated on the axipetal surface, mainly at the petals' base and the margin of labellum. The epiderm in those places created vesicular or somewhat elongated glandular cells, particularly on the labellum. In the production of smell 2-3 layers of subepidermal cells also take part. Submicroscopic examinations showed that those cells were characterized by the presence of a big nucleus. There were also numerous granules of starch and plastoglobules in plastids, a great amount of mitochondria and smooth-surfaced endoplasmic reticula. The traces of secretion products are visible on the surface of glandular cells. The above mentioned features are typical for osmophore cells.

Abstract P3-01-02: Oxalate microcalcifications induce breast cancer tumors

Abstract One shared characteristic of breast cancer is the appearance of mammographic upon mammography examination. Microcalcifications are routinely used to detect breast cancer in its early stages, which is of key importance due to the possibility that early detection gives for the application of more conservative therapies and for a better patient outcome. Mammary microcalcifications are composed of calcium oxalate or calcium phosphate. Oxalate has been considered as an inert end product of the metabolism. However, its accumulation is thought to be toxic to living tissues because it induces some pathological circumstances. Several results support the hypothesis that Oxalates are a secretion product that is produced by many kinds of cells, including apocrine cells, kidney cells and liver cells, among others. Exposure of renal epithelial cells to oxalate leads to diverse events that include, among others, induction of immediate early genes (IEG) expression, re-initiation of DNA synthesis, cell growth, and up or down-regulation of more than 1000 genes. Not enough research has been carried out directed to investigate the impact the presence of oxalates has within the breast tumor microenvironment. Neither are the interactions between oxalates breast-epithelial cells nor the signal transduction pathways involved in it, well understood. Herein, we have obtained good evidence showing that oxalates excreted from apocrine cells induce alterations in normal breast epithelial cells, and that the exposure of breast epithelial cells to excreted oxalate in a chronic way, sets the stage for transformation from normal to fully developed breast tumors. Oxalate induces DNA synthesis and IEG overexpression in MCF7 and MDAMB231 human breast cancer cells in culture. When these cells were treated with oxalate, the IEG c-Fos and Fra-1 were over-expressed as determined by Western Blot. Additionally, in human breast cancer tissues we found a strict correlation between oxalate concentration and overexpression of c-Fos and Fra-1. Paired adjacent non-tumor tissues linked with the tumor tissues have neither high levels of oxalate nor c-Fos/Fra-1 over-expression. Two groups of female mice (n = 20 each) periodically received an injection of oxalate or of saline solution (controls) into a mammary fatpad during a month. Surprisingly, the mice that were injected with oxalate generated very aggressive tumors. c-Fos and Fra-1 were also found over-expressed in these tumors. Furthermore, the final oxalate concentrations in the mice tumors were analogous to those found in the human breast tumor tissues whereas oxalate was almost undetectable in the non-pathological control tissues. Mice breast tumors were palpable within three months in 18/20 of oxalate treated as compared to the 0/20 control animals. Eight tumors were very aggressive and showed very fast growth rates; in these tumors, it was possible to appreciate high rate of atypical mitosis upon haematoxylin/eosin staining. Five tumors were very invasive and were found multi-localized in all breast areas. These five tumors also showed an atypically high mitotic rate, big pleomorphic nuclei, and macro and multi nucleoli. We expect that if we can control oxalic production or even better, control their action, we will significantly reduce the development of human breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-01-02.

Enrichment of epidermal stem cells of rats by Vario magnetic activated cell sorting system

Epidermal stem cells (ESCs) play an important role in skin homeostasis, wound repair, and tumorigensis which have great potential in scientific research and clinical application. So, the efficient isolation of these infrequent stem cells is very important for researchers to solve the problem of low purity and insufficient quantity of stem cells in vitro. The aim of this study was to investigate a method for the enrichment of ESCs by magnetic activated cell sorting system. The isolation strategy was CD71 depletion followed by α6-integrin positive selection. The percentage of α6(bri)CD71(dim) cells in isolated cells was 94.59%. Transmission electron microscopy results revealed that α6(bri) CD71(dim) cells exhibited some typical characteristics like progenitor cells, such as big nucleus, obvious nucleolus, large nuclear-cytoplasm ratio, and few organelles in cytoplasm. When cultured in vitro, the α6(bri)CD71(dim) cells had greater proliferating potential and higher colony-forming ability, and high levels of epidermal stem cell markers were expressed in our positive cells. ESCs have been successfully isolated from neonatal epidermis using Vario MACS and cultured in vitro. This isolation method is simple, fast, and inexpensive, providing an important tool for tissue engineering and cell transplantation studies.

Developmentally Regulated Linker Histone H1c Promotes Heterochromatin Condensation and Mediates Structural Integrity of Rod Photoreceptors in Mouse Retina

Mature rod photoreceptor cells contain very small nuclei with tightly condensed heterochromatin. We observed that during mouse rod maturation, the nucleosomal repeat length increases from 190 bp at postnatal day 1 to 206 bp in the adult retina. At the same time, the total level of linker histone H1 increased reaching the ratio of 1.3 molecules of total H1 per nucleosome, mostly via a dramatic increase in H1c. Genetic elimination of the histone H1c gene is functionally compensated by other histone variants. However, retinas in H1c/H1e/H1(0) triple knock-outs have photoreceptors with bigger nuclei, decreased heterochromatin area, and notable morphological changes suggesting that the process of chromatin condensation and rod cell structural integrity are partly impaired. In triple knock-outs, nuclear chromatin exposed several epigenetic histone modification marks masked in the wild type chromatin. Dramatic changes in exposure of a repressive chromatin mark, H3K9me2, indicate that during development linker histone plays a role in establishing the facultative heterochromatin territory and architecture in the nucleus. During retina development, the H1c gene and its promoter acquired epigenetic patterns typical of rod-specific genes. Our data suggest that histone H1c gene expression is developmentally up-regulated to promote facultative heterochromatin in mature rod photoreceptors.

Isolation, Culture and Characterization of Chicken Primordial Germ Cells

Nowadays, production of recombinant proteins in eukaryotes is gaining good deal of attention. Transgenic chicken as a eukaryotic system has a high potential for producing recombinant proteins. Post-translational changes, especially glycosylation, are characteristic of the eukaryotic proteins. In practice we need to choose a proper expressing host when considering over-expression of a recombinant protein. Chickens are among the well-considered candidates for such application. Production of transgenic chickens could be achieved in different ways, including application of primordial germ cells. Primordial germ cells are progenitor of sperm and ovum. These cells are round, with a big nucleus and a cytoplasm with lipid and glycogen particles. The first step for having transgenic chickens is isolation and culture of the primordial gem cells. In the present study, these cells were isolated by centrifugation method in presence of ficoll and using magnetic cell sorting, and were cultured in optimal culture medium. These cells were finally characterized with defined methods, like Periodic acid-schiff staining, alkaline phosphates activity assessment, and antibody staining.

5-Aminolevulinic acid ameliorates cadmium-induced morphological, biochemical, and ultrastructural changes in seedlings of oilseed rape

Due to its prolific growth, oilseed rape (Brassica napus L.) can be grown successfully for phytoremediation of cadmium (Cd)-contaminated soils. Nowadays, use of plant growth regulators against heavy metals stress is one of the major objectives of researchers. The present study evaluates the ameliorate effects of 5-aminolevulinic acid (ALA, 0, 0.4, 2, and 10 mg/l) on the growth of oilseed rape (B. napus L. cv. ZS 758) seedlings under Cd stress (0, 100, and 500 μM). Results have shown that Cd stress hampered the seedling growth by decreasing the radical and hypocotyls length, shoot and root biomass, chlorophyll content, and antioxidants enzymes. On the other hand, Cd stress increased the level of malondialdehyde (MDA) and production of H2O2 and accumulation of Cd in the shoots. The microscopic study of leaf mesophyll cells showed that toxicity of Cd totally destroyed the whole cell structure, and accumulation of Cd also appeared in micrographs. Application of ALA at lower dosage (2 mg/l) enhanced the seedling growth and biomass. The results showed that 2 mg/l ALA significantly improved chlorophyll content under Cd stress and decreased the level of Cd contents in shoots. Application of ALA reduced the MDA and H2O2 levels in the cotyledons. The antioxidants enzymes (ascorbate peroxidase, peroxidase, catalase, glutathione reductase, and superoxide dismutase) enhanced their activities significantly with the application of 2 mg/l ALA under Cd stress. This study also indicated that higher dosage of ALA (10 mg/l) imposed the negative effect on the growth of oilseed rape. Microscopic study showed that application of ALA alleviated the toxic effects of Cd in the mesophyll cell and improved the cell structure. Use of 2 mg/l ALA under 500 μM Cd was found to be more effective, and under this dosage, cell structure was clear, with obvious cell wall and cell membrane as well as a big nucleus, which was found with well-developed two or more nucleoli. Chloroplast was almost round in shape and contained thylakoids membranes and grana, but starch grains were not found in chloroplast comparatively to other treatments. On the basis of our results, we can conclude that ALA has a promotive effect which could improve plant survival under Cd stress.

Morphological and molecular characterization of Sarcocystis miescheriana from pigs in the central region of China

Sarcocystosis is an important food-borne parasitosis in humans and various animals. Sarcocystis miescheriana and Sarcocystis suihominis are pathogenic to pigs; S. suihominis is also distinctly pathogenic to humans. Intermediate and final hosts can harbor more than one Sarcocystis species, so the exact identification for Sarcocystis infection in various hosts is essential to control sarcocystosis in humans and important economic animals including pigs. In this study, four isolates of sarcocysts from slaughtered pigs (SmJY1-SmJY4) in the central region of China, in Henan province, were collected and examined by transmission electron microscopy and 18S rRNA sequence analysis to identify the Sarcocystis species in pigs in China. The results showed that cysts in the diaphragm muscles have a thick cyst wall with a number of palisade-like protrusions up to 4.38μm in length. Inside these protrusions, there were 13-16 fibrils per protrusion. Bradyzoites in cysts showed typical characteristics of Apicomplexa including a conoid, many micronemes, dense bodies, one big nucleus, and a number of amylopectin granules. These ultrastructural results suggest that characteristics of tissue cysts of the isolates SmJY1-SmJY4 were similar to those of S. miescheriana. The sequence similarities of SmJY1-SmJY4 with S. miescheriana were 99-99.5%, and the sequence similarities of SmJY1-SmJY4 with S. suihominis were much lower. Results of the ultrastructural observation in combination with molecular characterization based on the 18S rRNA sequence represent the first demonstration of S. miescheriana in pigs in China. In addition, results of the histological examination showed that the cysts of S. miescheriana had two types of cyst wall, a palisade-like thick wall and another smoothly thin wall, and could cause obvious atrophy, degeneration, and necrosis of muscle fibers in the diaphragm of naturally infected pigs. These findings will provide an important reference for the examination of Sarcocystis species in the slaughter quarantine of live pigs and in the control of sarcocystosis in pigs.

Expression of the gap junction protein connexin-36 in the adult big brown bat cochlear nucleus may be involved in temporally precise biosonar processing

The big brown bat uses biosonar to orient, navigate, and forage. Successful prey capture requires sonar emissions and returning echoes to be encoded and compared in the central auditory system with precision. Behavioral experiments show that E. fuscus can discriminate echo delay, relative timing of harmonics, and echo phase on the order of 3μs or less. The neurobiological specializations underlying this perceptual acuity remain elusive. New evidence shows that principal cells in the bat's cochlear nucleus (CN) express the neuronal gap junction protein connexin-36. During early postnatal development, connexin-36 expression is seen throughout the AVCN and PVCN of both big brown bats and mice. In adult bats, but not mice, cx36 expression is restricted to a specific population of cells in the ventral AVCN. The retention of cx36 in a discrete population of cells in the adult bat CN suggests that electrical transmission could be involved in processing echolocation sounds with temporal accuracy. Support: NSF grant 0843522 and ONR grant N00014-09-1-0691 to James Simmons.

Morphological and histological observations on the induction of anther calluses and embryos in loquat (Eriobotrya japonica Lindl.)

Morphological and histological studies were conducted systematically for the first time on the induction ofanther derived calluses and embryos in loquat (Eriobotrya japonica Lindl.). The results showed that the calluses were derived from microspores and both microspores and capillaments, when capillaments were not completely scraped off. Upon transfer onto the embryo differentiation medium, two types of calluses were formed within 5 to 7 days: 90% non-embryogenic calluses (NECs) and 10% embryogenic calluses (ECs). The NECs and ECs differed significantly in morphological and histological features. NECs were compact with a smooth surface and there was no evidence of the formation of adventitious buds or embryos, while ECs were uneven with a crisp texture and loose structure and were beginning to form salient multicellular structures on the surface. NECs cells had little cytoplasm that stained only lightly, few large vacuoles, no or only a small nucleus and wide intercellular spaces. NECs had a very low cell division capability and turned brown and eventually became necrotic. EC cells were smaller, globular and abundant in cytoplasm, with one or two big nuclei located in the centre of tightly aligned heavily stained cells. They had a high capability for cell division and continued to divide and produced somatic pro-embryos which could develop further through the typical globular, heart, torpedo and cotyledon stages. Although only 10% of ECs were induced they had a high differentiation rate and produced 12.7 globular, 8.1 heart-shape, 3.8 torpedo and 10.7 cotyledonary embryos per EC after 48 days of induction. Key words: Eriobotrya japonica Lindl., non-embryogenic callus (NEC), embryogenic callus (EC),morphology, histology.

Morphology, anatomy and ultrastructure of yarrow (Achillea millefolium L.) floral nectaries

The studies focused on the morphological and anatomical features as well as those related to the ultrastructure of nectary cells <i>Achillea millefolium</i> Asteraceae family. The nectary presence was confirmed only in the disk flowers at the pistil style base. The micromorphology of nectaries was investigated in SEM, and structure was observed in a light and transmission electron microscopes. A number of layers composing a gland, the size and shape of epidermal and glandular cells were determined. The secretory cell ultrastructure was analyzed. The discoidal nectary gland observed from above had a pentagonal shape, 181.5 µm height and 299.4 µm diameter. It was built of the monolayer epidermis and 6 layers of the secretory cells on average. The glandular cells appeared to be bigger (27 µm) than the epidermal cells (22 µm), a cell shape in both tissues differed as well. The nectar secretion occured through the modified stomata. The stomata cells were at distinguishable greater size and raised above the surface of epidermis. The nectaries were supplied by the vascular bundles running from the pistil style up to the nectary base, not getting into the gland. In the cells of the nectary epidermis observed in TEM the big cell nuclei, numerous plastids, mitochondria and vacuoles with fibrous secretion deposits and vesicular structures were found. In the cells of the nectary secretory tissue there were dense cytoplasm, many plastids, mitochondria, Golgi bodies and the extensive network of the endoplasmic reticulum.

PULSARS: GIGANTIC NUCLEI

What is the real nature of pulsars? This is essentially a question of the fundamental strong interaction between quarks at low-energy scale and hence of the non-perturbative quantum chromo-dynamics, the solution of which would certainly be meaningful for us to understand one of the seven millennium prize problems (i.e., "Yang-Mills Theory") named by the Clay Mathematical Institute. After a historical note, it is argued here that a pulsar is very similar to an extremely big nucleus, but is a little bit different from the gigantic nucleus speculated 80 years ago by L. Landau. The paper demonstrates the similarity between pulsars and gigantic nuclei from both points of view: the different manifestations of compact stars and the general behavior of the strong interaction.

Efficient differentiation of newly derived human embryonic stem cells from discarded blastocysts into hepatocyte‐like cells

To establish an optimal method to isolate and culture human embryonic stem (hES) cells from discarded blastocysts and to differentiate these cells into hepatocyte-like cells. Discarded human blastocysts of days 5-6 were cultured on mouse embryonic fibroblast cell feeder layers. Cells from the inner cell mass were isolated and subsequently cultured in vitro. To induce the formation of embryoid bodies (EB), dissociated ES cells were cultured in hanging drops for 5 days. The resulting EB were plated onto 100 mm plastic gelatin-coated dishes and allowed to attach for the outgrowth culture in the presence of hepatocyte growth factor for an additional 15 days. The hES cells had typical morphological characteristics: round or elliptic nest-like colonies with distinct cell borders and a smooth surface, significant swelling growth, compact cell aggregation, and relatively big nucleus compared to a small cytoplasm. The hES cells were identified by positive alkaline phosphatase staining. All had normal karyotypes and expressed octamer-binding transcription factor and cell surface markers, including stage-specific embryonic antigens SSEA-3, SSEA-4, and tumor rejection antigens TRA-1-60 and TRA-1-81. The cells could be differentiated to form teratomas in vivo. The hES cells could be induced to differentiate into hepatocyte-like cells and 70-80% of the cells expressed liver-associated proteins. Three hES cell lines have been successfully isolated and cultured from discarded human blastocysts. The hES cells can be efficiently differentiated into hepatocyte-like cells using the EB system. These cells possess the potential for treatment of liver diseases.

Micromorphology of trichomes of Thymus malyi (Lamiaceae)

Micromorphological, ultrastructural and morphometric investigations of the trichomes of Thymus malyi were carried out using a light microscope, a scanning electron microscope and a transmission electron microscope. Unbranched non-glandular trichomes, peltate and capitate glandular trichomes were described. The leaves of Thymus malyi bear non-glandular and glandular trichomes on both sides. Estimates of the volume density (i.e. their volume fraction per unit volume) of non-glandular trichomes were higher as compared to volume density of peltate and capitate glandular trichomes. Estimates of the number of these trichomes per area on sections showed that the capitate trichomes were the most abundant. Ultrastructural analyses of cell inner structure have shown numerous mitochondria, big nuclei and plastids with lipid globules and starch grains.

Human Embryonic Stem Cells-Derived Hemangioblasts Express HLA Antigens and Genes Related to Hematopoeisis and Endopoiesis

The early hematopoietic/endothelial progenitor (hemangioblast) has been recently described; however, HLA antigen expression of this progenitor was not characterized as yet. We have isolated blast-like cells (BLC) from ES/EB-based cultures and characterized their HLA-expression and ability to form hematopoietic and endothelial colonies. We used the two-step differentiation technology to obtain the bipotential blast cells from human embryonic stem cells (hES). This involved short differentiation in embryoid body (EB) system followed by differentiation in semisolid culture medium (Methocult H4436), supplemented with mixture of cytokines (BMP-4, VEGF, SCF, Flt3l, and TPO). First, we have shown that the occurrence of hemangioblast (BL-CFC) during EB differentiation (day 0–6) is transient and peaks on day 3. On this day about 125±35 out of 2400 EB cells form blast colonies. The emergence of this event was associated with the expression of T (early mesoderm gene) and was suppressed by the development of endoderm layer in subsequent days in culture. Similarly, the highest HB occurrence was associated with dramatic increase in expression of early hematopoietic/endothelial genes: CD34, CD31 and KDR. The similar pattern of expression of Patch1 and Gli1 genes suggests association with hedgehog (Hh) pathway activation during this process. The BL-CFC colonies were composed of 30–50 blast cells on day 6 of culture and had homogenous morphology in Wright-Giemsa stain with big nucleus containing disorganized chromatin and narrow rim of cytoplasm filled with large-size granules. However, as shown by FACS staining, they were quite heterogenous and expressed markers of both immature hematopoietic and endothelial progenitors (CD31, CD34, VE-cadherin, Flt-1) and mature differentiated cells (CD45, CD33, CD146). At least some of them expressed fetal and embryonic globin genes as shown by RT-PCR. Moreover, they could be characterized by high expression of HLA class I molecules by immunofluorescence, when compared to hES and EB cells. The ES cells, EB-derived cells and blast-like cells (BLC) were stained with FITC-labeled anti-HLA-A2 antibody BB7.2 and cells were observed under fluorescent microscope. While undifferentiated ES cells were negative for HLA-A2, the EB cells showed slight expression and the blast-like cells were strongly positive for HLA-A2 expression. These blast-like cells (BLC) could be successfully differentiated to hematopoietic cells in standard CFU assay. In these conditions, blast cells formed CFU-M colonies (63.4±0.8%) containing macrophages, BFU-E (19.5±3.5%) containing nucleated red blood cells and CFU-EM colonies (17.1±2.7%) composed of macrophages and nucleated erythrocytes. The cells of CFU-EM and BFU-E expressed both epsilon- and gamma-globin genes, but not adult-type beta-globin. When in endothelial cell culture conditions, the blast cells differentiated to endothelial cells which had the ability to uptake Dil-Ac-LDL and form complicated vascular networks in Matrigel. Concluding:Hemangioblasts transiently exist in early embryonic development and can form single cell-derived colonies,differentiation of hemangioblasts can be tracked by the use of chosen molecular markersblast-like colonies (BL-CFC) consist of cells having properties of endothelial and hematopoietic precursors, however, the issue if they can maintain dual properties over time needs to be further exploredthese blasts-like cells can be potentially used in regenerative medicine, however, strong expression of HLA molecules may limit their clinical applications in the future.

Prognostic Significance of Cytologic Features in Fine-Needle Aspiration Cytology Samples of Papillary Thyroid Carcinoma: Preliminary Report

The influence of clinicopathologic features on prognosis of papillary thyroid cancer has been examined. However, the relationship between cytologic features obtained by fine-needle aspiration (FNA) and prognosis has been minimally studied. We retrospectively studied 85 patients with usual-type papillary thyroid cancer diagnosed by FNA who underwent total or near-total thyroidectomy, had disease confirmed by pathological study, and were followed for more than 10 years except for one patient with metastasis at diagnosis. Cytologic features of FNA and recurrence of cancer were examined statistically. Four cytologic features were significantly related to recurrence: bigger nuclei, more irregularly shaped nuclei, nuclear atypia, and simultaneous presence of lighter staining nuclei and apparent nucleoli. A prognostic score of 1 (light nuclei and apparent nucleoli) plus 1.5 (irregularly shaped nuclei away from fibrous cord) plus 1 (high nucleus-to-cytoplasm ratio) plus 1 (nuclear atypia) was generated statistically. Sensitivity and specificity using a cut point of 2.5 in predicting outcome were 83.33% and 77.55%, respectively. Cytologic features of FNA samples have prognostic significance. If the prognostic score of cytologic features is high, a more radical surgical procedure and aggressive treatment plan should be considered.

Effect of Static Electric Fields in Root Cells of Vicia faba (Fabaceae)

Serial electron microscopic sections were prepared from half-ripened meristematic root cells of Vicia faba (Fabaceae) which had been exposed gradually to 700, 1000, 2500, 3500, and 5000 V/m static electric fields during seven days with and without Zn and Cd electrodes. At the end of five weeks, wall loosenings and very small nuclei were observed in those root cells which were exposed to static electric currents from the lower side of the medium without electrodes, while abnormalities in cell formation, e.g., two cells with one nucleus, and GER occurrence were present in an electrolytic (Cd upward and Zn downward) medium. The cells exposed to a static current from the upper side of the medium had small nuclei and abnormal cell divisions in the electrolyte, but in a non-electrolyte very large nuclei and thicker cell walls were observed, the cytoplasm was dense with GER, pinocytosis was seen filled with mitochondria, and protoplast formation with big nuclei was seen in exocytosis.

Dynamics and cytochemistry of oogenesis in Leporinus striatus Kner (Teleostei, Characiformes, Anostomidae) from the Rio Sapucaí, Minas Gerais State, Brazil

Oogenesis involves a sequense of transformations which are undergone by female germ cells. These cells change into oogonias and then into mature oocytes. Sexually mature females were collected monthly, during one year, from the Rio Sapucaí, tributary of the Rio Grande, which is part of the Furnas Reservoir system in the state of Minas Gerais. The observed material showed that oogonias were small spherical cells, had a big spherical nucleus, with a single nucleolus, and weakly stained cytoplasm with eosinophilic granules (FG stained), which indicate their protein content. The primary oocytes showed a big basophilic nucleus, with a large peripheral nucleolus, and several smaller nucleoli. They show a reduced cytoplasmic content. The previtellogenic oocytes presented voluminous cytoplasm and nucleus with several small peripheral nucleoli. The oocytes underwent vitellogenesis with the development of the zona radiata and the follicle cells. The zona radiata had two layers, the outer and the inner, which showed its protein content when stained with CM and FG techniques. TB pH 2.5 and pH 4.0 staining showed that oocytes undergoing vitellogenesis presented weakly stained cytoplasm and peripheral cytoplasmic vesicles. The follicle cells that were squamous became cuboidal. In mature oocytes, the yolk granules that filled the cytoplasm became green and blue when stained with FG and CM techniques, indicating their protein content. The perivitclline region showed rosy stained vesicles (TB pH 2.5 and pH 4.0) spread among the weakly stained peripheral vesicles, which seemed to be the cortical alveoli. The zona radiata cells, CM and FG stained, still showed two layers like the oocytes from the previous stage, but thicker.

A uterine choriocarcinoma in a virgin Donryu rat.

A uterine choriocarcinoma was found in a 49-wk-old virgin Donryu rat given intrauterine administration of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). The tumor was macroscopically present as a bloody cystic mass and microscopically composed of 2 kinds of cells: small basophilic cells similar to cytotrophoblasts in the rat placenta and large cells with big nuclei resembling giant trophoblasts. The giant cells were positive for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), although immunohistochemical examination was negative or nonspecific for chorionic gonadotrophins, including beta human chorionic gonadotrophin (beta hCG), human placental lactogen (hPL), and placental glycoprotein (SP-1), or for placental alkaline phosphatase (ALP). However, the animal exhibited a slight mammotropic status. The results thus suggest a choriocarcinoma nature for this uterine tumor and that the tumor cells secrete a weakly mammotrophic hormone.

MORPHOLOGICAL AND CHEMICAL CAMOUFLAGE OF THE MEDITERRANEAN NUDIBRANCH DISCODORIS INDECORA ON THE SPONGES IRCINIA VARIABILIS AND IRCINIA FASCICULATA

The nudibranch Discodoris indecora shows a perfect camouflage on its prey, the dictyoceratid sponge Ircinia variabilis. Shape and colour of the nudibranch are remarkably similar to that of the sponge which is widespread in the shallow waters of the Mediterranean Sea. The metabolite pattern of /. variabilis is dominated by unusual molecules, the sesterterpenoids palinurin and variabilin, containing 25 carbons and displaying at the ends a ^-substituted furan ring and a tetronic acid. However, a chemical polymorphism has been recently described for /. variabilis. Some specimens contain either palinurin or variabilin whereas others possess a mixture of both sestertepenoids. During the study of Mediterranean sponges, a linear sesterterpenoid related to palinurin and variabilin was found in Ircinia fasciculata, that from a taxonomic point of view is closely relatedto Ircinia variabilis. Discodoris indecora shows a typical defence behaviour when it is disturbed, secreting a copious white slime that contains large amounts of palinurin and variabilin. Most likely the animal is able to transfer the sponge metabolites from the digestive glands to mantle glands. Electron micrographs of the nudibranch mantle showed the presence of unicellular glands characterized by a cylindrical vacuole with a big nucleus on the basis. The glands were concentrated in the most conspicuous tubercles of the dorsal mantle. Probably, the sponge metabolites are stored in these glands. Comparative chemical analysis of mucous secretions, dorsal tubercles, mantle, foot, digestive glands and hepatopancreas of D. indecora confirmed this hypothesis. Palinurin and fasciculation were, of course, present in the digestive glands but they were also found in the mucus and in the mantle sections enriched by the above described unicellular glands. The defensive role of palinurin and variabilin, strongly suggested by their anatomical location, was

Cytoplasmic Localization of Cyclin D3 in Seminiferous Tubules during Testicular Development

Using a newly developed polyclonal antibody against murine cyclin D3, we have found that protein levels of cyclin D3 were highly detectable only in thymus and testis in rats. Since testis offer unique opportunities to examine the cell cyclein vivo,we examined the temporal and spatial expression of cyclin D3 and the DNA synthesis indicator, proliferating cell nuclear antigen (PCNA), in the rat testis during development. The protein levels of cyclin D3 protein in testis from 7 days to 3 months old were almost constant and then decreased gradually thereafter. The protein levels of cyclin D1 and PCNA were high in the testis of 7- and 14-day-old rats and decreased during testicular development. In the seminiferous tubules of 7-day-old newborns, cyclin D3 was surprisingly located in cytoplasm of stem cells that had bigger nuclei than the nuclei of surrounding cells. Interestingly, cyclin D3 immunopositive cells did not immunostain with PCNA in nuclei. In the adult testis, anti-cyclin D3 antibody strongly stained the cytoplasm of early stage primary spermatocytes, lightly stained pachytene spermatocytes, but did not stain elongated spermatids. There was no detectable cyclin D3 in Sertoli cells, interstitial cells, or fibroblasts within seminiferous tubules, or in blood vessels within the interstitial matrix. The known cyclin D3 partner, cyclin dependent kinase 4, was located mainly in nuclei of spermatogonia and in early stage primary spermatocytes. Strong PCNA immunopositive staining was located in the nuclei of spermatogonia in adult testis. These results indicate that cyclin D3 is detectable in meiotically active male germ cells (PCNA-negative cells), but is conspicuously absent from mitotically active spermatogonia (PCNA-positive cells). Moreover, in contrast toin vitroreports, cyclin D3 is not located in the nucleus, but rather in the cytoplasm of male germ cellsin vivo.Taken together, the presence of cyclin D3 in spermatocytes and its location in the cytoplasm lead us to speculate that cyclin D3 may have functions in male germ cells other than mitosis.