Age-related alterations in the size of human hepatocytes

Age-related alterations in the size of human hepatocytes (both mononuclear and binucleate forms), were studied in histological sections and in separated cells and nuclei using cytophotometrical and microspectrophotometrical methods. The following results were obtained: 1. The volume of nuclear DNA increased in proportion to nuclear size. The increase occurred in a group pattern reflecting nuclear polyploidization. 2. Cell size increased in proportion to nuclear size. Tetraploid cells (4C) were roughly two times greater than diploid cells (2C). 3. In most of the binucleate cells examined, the ploidy class of the two nuclei in a binucleate cell was observed to be equal. Heterogeneity of the ploidy class among the nuclei of a binucleate cell was present in less than 1% of total binucleate cells examined. The nuclear DNA volume of individual nuclei in binucleate cells appeared to be the same as that of mononuclear cells. 4. The cell size of binucleate cells corresponded with that of mononuclear cells whose ploidy class was the same as the sum of the ploidy classes of two nuclei of a binucleate cell. 5. The incidence of binucleate cells in the lobular periphery was about 4 to 6% in the third decade, and increased slightly with age up to 5 to 7% in the tenth decade. 6. The incidence of binucleate cells in the liver at different ages followed a similar pattern to that observed in mononuclear cells whose ploidy class was half of the sum of ploidy classes of the two nuclei of the binucleate cell.     

红豆草根瘤侵染细胞核在细胞凋亡中的超微结构变化

用透射电镜观察红豆草根瘤侵染细胞核在细胞凋亡过程中的超微结构,以探讨红豆草根瘤侵染细胞核在发育过程中的超微结构变化及其与细胞凋亡的关系.结果表明,红豆草根瘤侵染细胞核的超微结构随细胞发育程度不同而不同.在幼龄侵染细胞中,细胞核体积较大,近似圆形.在即将成熟和成熟的侵染细胞中,细胞核膜有内陷现象,其核仁常具有核仁泡和核仁联合体.在早期凋亡的侵染细胞中,细胞核体积减小,形状变得不规则,核膜出现大量内陷,在其表面形成许多大的突起和深的沟槽,有时还有内质网、线粒体、小液泡和细菌等位于核膜的内陷处,而且核仁也开始裂解.在后期凋亡的侵染细胞中,除细菌解体外,还出现核仁消失,核膜破裂,核质外流,并在细胞质中形成一些电子密度很高,无一定形状的团块状物质.     

Ca2+-dependent contractility of isolated and demembranated macronuclei in the hypotrichous ciliate Euplotes aediculatus

The hypotrichous ciliated protozoan Euplotes aediculatus possesses a characteristic C-shaped somatic nucleus (macronucleus) within the cytoplasm, which shows dynamic shape change during the cell cycle. It is shown that isolated macronuclei possess Ca(2+)-dependent contractility. Macronuclei were isolated, stuck fast on the glass surface, and subjected to different concentrations of Ca(2+) in a Ca(2+)-EGTA buffer. The nuclei became expanded at [Ca(2+)]<10(-7)M, and they contracted on subsequent addition of higher concentrations of Ca(2+). Cycles of expansion and contraction of the nucleus could be repeated many times by alternate addition of EGTA and Ca(2+), indicating that the size of isolated nuclei can be regulated by [Ca(2+)] alone. The nuclear contraction was observed in all phases of the cell cycle, but contractility was less evident around replication bands in the S phase. In addition to the hypotrichous ciliate Euplotes, similar Ca(2+)-dependent nuclear contractility was found to exist in other cell types, including protozoans of different taxa (a heliozoon Actinophrys sol and a peniculine ciliate Paramecium bursaria), and also mammalian culture cells (HeLa cells). Our findings suggest a possibility that Ca(2+)-dependent nuclear contractility may be shared among diverse eukaryotic organisms.     

The protective influence of ascorbic acid against the genotoxicity of waterborne lead exposure in Nile tilapia Oreochromis niloticus (L.)

The present study investigated the effects of lead (Pb) and ascorbic acid (AA) on Nile tilapia Oreochromis niloticus using the micronucleus (MN) and nuclear abnormality (NA) tests for periods of 7, 14 and 21 days. The MN frequencies in the erythrocytes, gill, liver and fin cells were analysed comparatively to evaluate the sensitivity and suitability of these different cell types. The NA shapes in erythrocytes were scored into blebbed nuclei (BL), lobed nuclei (LB), notched nuclei (NT) and binuclei (BN). It was observed that fish showed significant sensitivity to the different treatments. In general, the highest value of both MN and NA cells were significantly increased in the Pb-treated group followed by the combination of the Pb and AA-treated group. On the other hand, the MN and NA frequencies in erythrocytes were the most sensitive to the treatment and could provide more valuable information than those in gill, liver and fin cells. The frequencies of each NA shape in erythrocytes of all treatments were observed in the following ranked order NT > LB > BN > BL. The results demonstrated the efficacy of AA in reducing genotoxicity in fish induced by Pb. They showed the sensitivity and suitability of MN and NA frequencies in erythrocytes as pollution biomarkers.     

Preliminary observations on the distribution of serotoninergic and cerebellar-projecting neurons of the raphe nuclei of the brain stem

Cerebellar projection from raphe nuclei were investigated in rabbit by using retrograde transport of HRP and serotonergic mapping by direct fluorescence. A close topographical correlation between the HRP labeled cells and the serotonergic neurons has been observed. The current study has demonstrated the presence of paramedian and lateral cells whose cytoarchitecture is identical with midline cells of many raphe nuclei. All of the raphe nuclei except the linear nuclei, contained serotonergic perikarya. The midline and paramedian portions of the nuclei raphe obscurus, pallidus, magnus, and nucleus raphe dorsalis contained principally serotonergic neurons; the lateral portions of the medullary raphe nuclei and the nuclei raphe pontis and centralis superior contained a significant number of non-fluorescent cells. In these regions, fluorescent sections often revealed the size, shape, and orientation of the perikarya and dendrites; further verification of cytoarchitectural characteristics of these neurons depended heavily upon these clues.     

Types of thalamo-cortical relay neurons in the anteroventral nucleus of the cat

Summary Neurons displaying a thalamo-cortical projection were marked by means of the retrograde transport of horseradish peroxidase (HRP), and the labeled elements were compared with neurons impregnated by the Golgi technique. Injections of HRP into the posterior area of the limbic cortex resulted in its uptake by various anterior thalamic nuclei, especially the anteroventral nucleus. HRP-positive cells are characterized by their position, dendritic orientation, and the shape and size of their somata. On the basis of the combined HRP- and Golgi-analysis three different types of thalamo-cortical relay neurons can be distinguished.     

Two Plasmodium species of the crocodile skink Tribolonotus gracilis from Irian Jaya, Indonesia

Two undescribed Plasmodium species were present in 1 of 8 New Guinea skinks, Tribolonotus gracilis. Plasmodium tribolonoti n. sp. is characterized by rounded or oblong schizonts 6.1 x 5.3 microm (5-7 x 4-7) that produce on average 14.3 merozoites (10-21), in no particular arrangement of nuclei. All parasitized proerythrocytes and had no effect upon dimensions of host cells or their nuclei. Gametocytes, mostly erythrocytic, averaged 7.2 x 6.3 (6.5-9.0 x 5.5-7.5), with length x width (LW) 45.5 microm2 (38-63) and L/W 1.15 (1.0-1.5). Gametocytes are not sexually dimorphic in size or shape and had no effect upon dimensions of host erythrocytes or their nuclei. Schizonts of P. gracilis n. sp. averaged 4.3 x 3.5 (3-6 x 3-5), with 4.9 merozoites (3-8) usually arranged as a fan, and had no effect upon dimensions of host erythrocytes or their nuclei. Gametocytes averaged 5.9 x 5.5 (5.0-6.6 x 5.0-6.0), with LW 31.9 microm2 (25-40) and L/W 1.06 (1.0-1.2). Gametocytes are not sexually dimorphic in size or shape and had no effect upon dimensions of host erythrocytes or their nuclei.     

中华鲟外周血细胞组成及形态观察

利用光镜和透射电镜技术对北京海洋馆养殖的40尾中华鲟(Acipenser sinensis)(介于4—30龄以上, 包括野生、子一代和子二代共7个龄组)外周血细胞组成、大小、显微和超微结构进行研究。结果表明, 在外周血细胞中可区分出以下六类细胞。形态结果: 红细胞卵圆形, 胞质内可见少量线粒体; 淋巴细胞多圆形, 有明显伪足样或指状胞凸, 核质比大, 可明显分为大淋巴和小淋巴; 嗜中性粒细胞核型多样, 胞质细胞器丰富, 含有大小不等的特殊颗粒; 嗜酸性粒细胞多为规则圆形, 表面大量细小指状突起, 胞质细胞器丰富, 含有大量个体较大的嗜酸性颗粒; 单核细胞变形现象多, 胞质内大量空泡, 核型多样; 血栓细胞形状多样, 胞质内大量小的空泡, 散布或成团出现, 常见直接分裂现象。各类血细胞从大到小依次为: 单核细胞、嗜中性粒细胞、嗜酸性粒细胞、大淋巴细胞、红细胞、血栓细胞和小淋巴细胞, 各龄组间无显著差异。外周血红细胞总数(RBC)平均为(5.56±1.19)×108/mL, 18龄和11龄与其他龄组之间存在显著性差异(P<0.05); 白细胞总数(WBC)平均为(16.53±4.94)×106/mL, 18龄与4龄间存在显著差异, 且分别与其他龄组间存在显著性差异(P<0.05); 血栓细胞总数(15.53±15.82)×106/mL。白细胞分类计数(DLC)中大淋巴细胞、小淋巴细胞、嗜中性粒细胞、嗜酸性粒细胞和单核细胞所占百分比分别为: (5.26±3.95)%、(77.74±11.73)%、(9.40±7.98)%、(1.90±2.06)%、(5.50±4.00)%, >30龄和4龄间显著差异, 且分别与其他龄组间存在显著性差异( P<0.05)。结论认为中华鲟血细胞进化地位低, 免疫系统为淋巴细胞系为主, 主要包括淋巴细胞、粒细胞和单核细胞, 结果对中华鲟的健康评价与保育研究有重要的参考意义。     

Observations of the primordial germ cells in blood samples from the chick embryo

Primoridal germ cells (PGCs) from the circulating blood in 2-day chick embryos were observed by histochemical techniques in smear preparation and by phase contrast microscopy in fresh samples. In the blood smear, PGCs were readily distinguished from blood cells by their large size (15–20 μm in diameter) and by the round and large nuclei (8–10 μm in diameter) occupying eccentrically the greater parts of the cells. Histochemically, they were demonstrated to contain abundant glycogen, a lesser amount of yolk granules (by the PAS reaction), and a large amount of lipid droplets (by the Oil red O and Sudan black B stains) in their cytoplasm. Alkaline phosphatase activity was proved slightly in their cytoplasm by the simultaneous diazo coupling method. In phase contrast microscopy, living PGCs in the blood and from the primitive gonad of 3-day embryos in parts of the examinations were observed to change their shape in somewhat amoeboid fashion, suggesting their capability for locomotion as a possible mechanism of the migration of PGCs in vivo.     

An ultrastructural stereological analysis of the aortic body chief cell of adult rabbits

Summary The chief cells of the aortic body (subclavian body) of adult New Zealand white rabbits were examined by ultrastructural stereological analysis. The chief cell nuclei occupy 26.5% of the total volume. Dense-core vesicles account for 16.5% of the cytoplasmic volume, followed by mitochondria (11.6%), endoplasmic reticulum (3.3%), and Golgi apparatus (0.6%). The dense-core vesicles measure approximately 131.6nm in diameter (corrected) and exhibit a heterogeneous size distribution. Both perivascular adrenergic nerve terminals and presumptive afferent terminals presynaptic to the chief cells are observed. The mean synaptic vesicle size of the terminals adjacent to chief cells is 54 nm. The heterogeneous size distribution of the dense-core vesicles of chief cells may indicate the storage of different biogenic amines and/or different secretion or maturation states within the chief cells.Supported by a Grant-in-Aid from the American Heart Association (77630) and with funds contributed in part by the Texas Affiliate. The author wishes to thank Ms. Teri Heitman for her excellent technical assistance     

Effects of iron-, manganese-, or magnesium-deficiency on the growth and morphology of Euglena gracilis

Iron-, manganese-, or magnesium-deficiency has been induced in Euglena gracilis. Each arrests cell proliferation, decreases the intracellular content of the deficient metal, and increases that of several other metals. Light and electron microscopy of stationary phase cells reveal that Fe-deficient (-Fe) cells are similar in size and shape to control organisms. Magnesium-deficient (-Mg) cells, however, are larger, and approximately 14% are multilobed, containing 2 to 12 lobes of equal size emanating from a central region. Individual (-Mg) cells and each lobe of multilobed cells contain a single nucleus. Manganese-deficient (-Mn) organisms are morphologically more heterogeneous than (-Fe) or (-Mg) cells. Most are spherical and larger than controls. Approximately 15% are multilobed but, unlike (-Mg) cells, contain lobes of unequal size with either zero, one, or several nuclei present in each. Nuclei of (-Mn) cells differ in size and shape from those of control, (-Fe), or (-Mg) cells. All three deficient cell types accumulate large quantities of paramylon. Other cytoplasmic structures, however, appear normal. Addition of Fe, Mn, or Mg to the respective deficient stationary phase cultures reverses growth arrest and restores normal morphology. The results suggest that Fe-, Mn-, and Mg-deficiencies affect different stages of the E. gracilis cell cycle.     

Flowering plant sperm cells: Isolation from pollen of Gerbera jamesonii (asteraceae)

Sperm cells were isolated from pollen of Gerbera jamesonii by gentle grinding in a medium containing buffer, osmoticum, protectants and ions. Sperm nuclei were identified by means of the DNA fluorochrome DAPI. The sperm cells were observed by Nomarski optics and by scanning electron microscopy. Isolated sperm were osmotically sensitive and excluded Evans Blue. The structure of the isolated sperm cells was similar in shape and size to that of sperm cells extruded from fresh pollen and in fixed pollen.     

Seasonal changes in the structure and secretory activity of the androgenic gland of Travancoriana schirnerae

This study investigated the seasonal variation in the structure and secretory activity of the androgenic gland (AG) in the freshwater crab: Travancoriana schirnerae. The androgenic gland is an elongate structure, attached to one side on the wall of the ejaculatory duct. Histological studies showed the presence of three cell types, which differ in size, shape of nuclei, and presence or absence of secretory vesicles. Type I cells are small with large nuclei whereas type II cells are large with small nuclei. Type III cells are intermediate in size and exhibited streak-like nuclei and transparent cytoplasm. Seasonal changes were discerned in the morphology, histology and secretory activity of the gland. March-June appeared to be the active season with type II cells containing secretory vesicles. The mode of release of secretion found to be holocrine. The secretory activity almost completed by July-August (the mating season) with vacuolization of type II cells. The gland remained inactive from September-December with abundance of vacuoles, scattered pycnotic nuclei, indistinct cell membranes and total cellular degeneration. January-February was the revival period with type I cell proliferation. The present study revealed that the secretory activity of the gland is in tune with the male reproductive cycle.     

Occurrence of amitotic division of trophoblast cell nuclei in blastocysts of the western spotted skunk (Spilogale putorius latifrons)

A cytogenetic examination of spreaded cells of diapausing and early activated blastocysts obtained from 7 female western spotted skunks was performed. Mitosis was not observed in 1626 cells obtained from 9 diapausing blastocysts; however, 12 (1.5%) figures of diploid mitosis were seen in 851 cells from 5 early activated embryos. Diameter of the cell nuclei varied from 4 to 29 microm during diapause, and from 5 to 40 microm in activated blastocyst, and the heterogeneity in nuclear size was significantly different between diapausing and activated embryos (P<0.01). About 80% of nuclei from diapausing blastocysts measured 9 to 16 microm, whereas a similar percentage of nuclei from activated blastocysts ranged from 15 to 27 microm. Many enlarged nuclei exhibited morphological features characteristic of mammalian polytene (i.e. endopolyploid with polytenic organization of chromosomes) trophoblast cells. The number of silver stained nucleoli in all the nuclei did not exceed 2, which corresponds to the number of nucleolus organizers in the diploid karyotype in this species of skunk and suggests the polytene organization of chromosomes in enlarged nuclei. About 10% of large interphase nuclei were observed to undergo amitosis, i.e. direct division by constriction. The resulting nuclear fragments in diapausing blastocysts usually had normal morphology and active nucleoli. In activated embryos, nearly 15% of amitotically divided nuclei appeared to be dividing into fragments of unequal size, one of which had normal cell nuclear morphology and extremely large silver positive nucleoli, and the other fragment exhibited signs of cell death. We interpret these data as indicating that 1) amitotic division of trophoblast endopolyploid cell nuclei in the skunk blastocysts may generate new trophoblast cells which contribute to increased cell number during both diapause and activation stages, and 2) activation of blastocysts after diapause is related to the production of trophoblast cells with enhanced synthetic capabilities.     

Effects of Iron-, Manganese-, or Magnesium-Deficiency on the Growth and Morphology of Euglena gracilis1

Iron-, manganese-, or magnesium-deficiency has been induced in Euglena gracilis. Each arrests cell proliferation, decreases the intracellular content of the deficient metal, and increases that of several other metals. Light and electron microscopy of stationary phase cells reveal that Fe-deficient (-Fe) cells are similar in size and shape to control organisms. Magnesium-deficient (-Mg) cells, however, are larger, and approximately 14% are multilobed, containing 2 to 12 lobes of equal size emanating from a central region. Individual (-Mg) cells and each lobe of multilobed cells contain a single nucleus. Manganese-deficient (-Mn) organisms are morphologically more heterogeneous than (-Fe) or (-Mg) cells. Most are spherical and larger than controls. Approximately 15% are multilobed but, unlike (-Mg) cells, contain lobes of unequal size with either zero, one, or several nuclei present in each. Nuclei of (-Mn) cells differ in size and shape from those of control, (-Fe), or (-Mg) cells. All three deficient cell types accumulate large quantities of paramylon. Other cytoplasmic structures, however, appear normal. Addition of Fe, Mn, or Mg to the respective deficient stationary phase cultures reverses growth arrest and restores normal morphology. The results suggest that Fe-, Mn-, and Mg-deficiencies affect different stages of the E. gracilis cell cycle.     

An application of mathematical morphology to analysis of the size and shape of nuclei in tissue sections of non-Hodgkin's lymphoma

Using principles from the theory of mathematical morphology, a semiautomatic analysis of the size and shape of cell nuclei on tissue sections was carried out on a Leitz Texture Analysis System (Leitz-TAS). The four parameters proposed here are more discriminatory than conventional shape evaluation by the form factor (FF), which is based on the ratio of perimeter squared to area. The parameters quantified, respectively, nuclear elongation (ND), narrow (R1) and wide (R2) irregularities, and the distribution of R1 and R2 along the nuclear contour (ID). The properties of these parameters were tested nucleus-by-nucleus on 24 nuclear models. The methodology was then illustrated by a study of lymph node nuclei in non-Hodgkin's lymphoma (NHL). Prior to analysis, 45 lymphomas were classified into five categories of nuclear size and shape according to the International Working Formulation (IWF). Two hundred nuclei were measured on each lymph node section. Statistical interpretation was based upon an analysis of the nuclear surface area on sections and upon the mean values of R1, R2, and ND, the standard deviations of R1 and R2, and the percentage of cleaved nuclei detected by ID. The mean value of R2 discriminated best between the two sets of populations with regular and irregular nuclear contours, respectively. Parameters R1, ND, and ID permitted the distinction of certain NHL cases among populations with irregular nuclei. Nuclear invaginations decreased in depth as the nuclear area became greater. The median surface area was well correlated to the IWF, and the skewness coefficient (third statistical moment of the nuclear surface area distribution) was related to the number of nuclear size or shape subpopulations.     

Distribution of myonuclei and microtubules in live muscle fibers of young, middle-aged, and old mice.

We have recently published a new technique for visualizing nuclei in living muscle fibers of intact animals, based on microinjection of labeled DNA into single myofibers, excluding satellite cells (Bruusgaard JC, Liestol K, Ekmark M, Kollstad K, and Gundersen K. J Physiol 551: 467-478, 2003). In the present study, we use this technique to study fiber segments of soleus and extensor digitorum longus (EDL) muscles from mice aged 2, 14, and 23 mo. As the animals maturing from 2 to 14 mo, they displayed an increase in size and number of nuclei. Soleus showed little change in nuclear domain size, whereas this increased by 88% in the EDL. For 14-mo-old animals, no significant correlation between fiber size and nuclear number was observed (R2=0.18, P=0.51) despite a fourfold variation in cytoplasmic volume. This suggests that size and nuclear number is uncoupled in middle-aged mice. When animals aged from 14 to 23 mo, EDL IIb, but not soleus, fibers atrophied by 41%. Both EDL and soleus displayed a reduction in number of nuclei: 20 and 16%, respectively. A positive correlation between number of nuclei and size was observed at 2 mo, and this reappeared in old mice. The atrophy in IIb fibers at old age was accompanied by a disturbance in the orderly positioning of nuclei that is so prominent in glycolytic fibers at younger age. In old animals, changes in nuclear shape and in the peri- and internuclear microtubule network were also observed. Thus changes in myonuclear number and distribution, perhaps related to alterations in the microtubular network, may underlie some of the adverse consequences of aging on skeletal muscle size and function.     

Role of morphological methods in the analysis of chemically induced colon cancer in rats.

The role of selected histopathological methods was evaluated for the characterization of tumourous tissues resulting from chemically induced carcinogenesis in rats. Colon cancer was induced by treating rats with a direct carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), alone or with subsequent treatments with secondary bile acids. Morphological studies showed that the tumorigenic effect of MNNG resulted in changes in the shape of the crypts, disappearance of mucous (goblet) cells, and marked increases in the mitotic index, the size of nuclei and the number of aberrant nuclei. A significant number of lymphocytes infiltrated between the crypts and into their lumens. A sharp decrease in concentrations of neutral mucopolysaccharides and sulfomucins and an increase in the amount of vic-glycol groups and nonsulfated mucosubstances were demonstrated in tumourous epithelial cells. A moderate positive reaction to tissue polypeptide antibody was observed in these cells. The tumor-promoting effects of secondary bile acids were detected morphologically by an increase in the amount of connective tissue that infiltrated newly formed tumors. The increased number of micronuclei in otherwise histologically unaltered colon tissues adjacent to a tumor suggests that these regions represent a precancerous stage in the development of tumors. The important role of morphological methods in the evaluation of the effects of carcinogen and tumor promoters and in the detection of various phases in experimental carcinogenesis was discussed.