Subcellular distribution of titanium in the liver after treatment with the antitumor agent titanocene dichloride. A study using electron spectroscopic imaging

In the present study, the subcellular distribution of titanium in the liver of mice was determined 24 and 48 h after application of a therapeutic (ED100; ED = effective dose) and a toxic (LD25; LD = lethal dose) dose (60 and 80 mg/kg, respectively) of the antitumor agent titanocene dichloride by electron spectroscopic imaging at the ultrastructural level. At 24 h, titanium was mainly accumulated in the cytoplasm of endothelial and Kupffer cells, lining the hepatic sinusoids. Titanium was detected in the nucleoli and the euchromatin of liver cells, packaged as granules together with phosphorus and oxygen. One day later titanium was still present in cytoplasmic inclusions within endothelial and Kupffer cells, whereas in hepatocyte nucleoli only a few deposits of titanium were observed at 48 h. At this time titanium was mainly accumulated in the form of highly condensed granules in the euchromatin and the perinucleolar heterochromatin. It was found in the cytoplasm of liver cells, incorporated into cytoplasmic inclusion bodies which probably represent lysosomes. Sometimes these inclusions were situated near bile canaliculi and occasionally extruded their content into the lumen of bile capillaries. This observation suggests a mainly biliary elimination of titanium-containing metabolites. These results confirm electron spectroscopic imaging to be an appropriate method for determining the subcellular distribution of light and medium-weight elements within biological tissues. Insights into the cellular mode of action of titanocene complexes or titanocene metabolites can be deduced from the findings of the present study.     

Intracellular localization of titanium within xenografted sensitive human tumors after treatment with the antitumor agent titanocene dichloride

In the present study, the intracellular localization of titanium was analyzed in three xenografted human adenocarcinomas of the colon sigmoideum (S 90), the stomach (M-Stg 4), and the lung (L 261) in dependence on the time after application of a single therapeutic dose (80 mg/kg) of the organometallic antitumor agent titanocene dichloride (C5H5)2TiCl2. The investigations were performed by use of electron energy loss spectroscopy (EELS), a method which allows microanalysis in ultrathin sections, in combination with electron spectroscopic imaging (ESI), which offers the possibility to image the two-dimensional localization and distribution of light- and medium-weight elements in animal tissues. In all three tumors which were studied, titanium was at first detected within the nucleus and, some hours and days later, it was additionally found in cytoplasmic lysosomes. In the colon and lung tumors S 90 and L 261, already 12 hr after treatment, titanium was traceable as tender granules in the nuclear chromatin. During the following days, it was then accumulated in certain areas of the nuclear heterochromatin and, in the case of the L 261 tumor, also in the nucleolus. Maximum concentrations were attained in the nuclei and nucleoli at 48 hr after substance application. Thereafter, titanium was increasingly incorporated into cytoplasmic lysosomes which are known to be involved in intracellular degrading and digesting processes and which occurred in increased numbers in treated tumor cells. Regarding the stomach carcinoma M-Stg 4, titanium was recognized in the nuclear heterochromatin only 1 and 2 days after application of titanocene dichloride. At 48 hr, it was additionally detected in cytoplasmic lysosomes. In all cases where titanium was found accumulated in the nucleus and in lysosomes, phosphorus was simultaneously enriched in a similar local distribution and a concentration which even exceeded that within phosphorus-rich areas, e.g., the nuclear heterochromatin and cytoplasmic ribosomes. These results confirm a primary interaction of titanium-containing metabolites deriving from titanocene complexes with nucleic acid molecules, especially with nuclear DNA. They suggest the formation of aggregates between nucleic acids and titanium-containing metabolites which are obviously extruded out of the nuclei and incorporated into cytoplasmic lysosomes, known to be involved in intracellular digesting processes.     

Liver ultrastructure and a new cell type in the Japanese newt, Cynops pyrrhogaster.

The liver of the Japanese newt, Cynops pyrrhogaster, has been investigated using light, scanning, and transmission electron microscopy. Hepatic parenchyma was composed of clusters and cords or tubules of polyhedral cells separated by a sinusoidal net. Hepatocytes had spherical, euchromatic nuclei with one or more nucleoli and stacked mitochondria with sparse cristae and dense bodies. Rough endoplasmic reticula formed peribiliary stacks and diffusely scattered vesicles and tubules. Smooth endoplasmic reticula were more pronounced in glycogen-rich hepatocytes. Most hepatocytes contained peroxisomes, Golgi complexes and large numbers of fat droplets within the cytoplasm along with glycogen. Some cells were mainly glycogen-storing and contained few or no fat droplets. A special feature of the newt liver was biliary atresia. Bile canaliculi had short, stout microvilli which were entirely atretic in some canaliculi. Canaliculi were sealed off by junctional complexes including zonulae occludentes and maculae adherentes. The latter showed extraordinary wider desmosomal gaps in the vicinity of the atretic bile canaliculi. The sinusoid wall was non-distinctive and contained fenestrated endothelial cells connected to Kupffer cells by zonulae occludentes. A distinctive new cell type (OG cell) was observed in the newt liver. These cells were found individually or in small clusters in proximity with the sinusoidal surfaces. They had small nuclei, a paucity of cytoplasmic organelles, but numerous, unique, osmiophilic granules of two distinct types. Less numerous Type I granules contained homogeneous electron-dense material, and a predominant Type II granule contained circumferentially arranged subparticulation. Granules of both types were detected within the cytoplasm of endothelial cells and within sinusoids together with blood elements. The function of this secretory type cell remains obscure, though it may represent a stage of melanophore.     

Localization of cathepsin D in rat liver. Immunocytochemical study using post-embedding immunoenzyme and protein A-gold techniques

Light and electron microscopic localization of cathepsin D in rat liver was investigated by post-embedding immunoenzyme and protein A-gold techniques. By light microscopy, cytoplasmic granules of parenchymal cells and Kupffer cells were stained for cathepsin D. Weak staining was also noted in sinusoidal endothelial cells. In the parenchymal cells many of positive granules located around bile canaliculi. In the Kupffer cells and the endothelial cells, diffuse staining was noted in the cytoplasm in addition to granular staining. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were seen in typical secondary lysosomes and some multivesicular bodies of the parenchymal cells and Kupffer cells. The lysosomes of the endothelial cells and fat-storing cells were weakly labeled. Quantitative analysis of the labeling density in the lysosomes of these three types of cells demonstrated that the lysosomes of parenchymal cells and Kupffer cells are main containers of cathepsin D in rat liver. The results suggest that cathepsin D functions in the intracellular digestive system of parenchymal cells and Kupffer cells but not so much in that of the endothelial cells.     

Localization of cathepsin D in rat liver

Summary Light and electron microscopic localization of cathepsin D in rat liver was investigated by post-embedding immunoenzyme and protein A-gold techniques. By light microscopy, cytoplasmic granules of parenchymal cells and Kupffer cells were stained for cathepsin D. Weak staining was also noted in sinusoidal endothelial cells. In the parenchymal cells many of positive granules located around bile canaliculi. In the Kupffer cells and the endothelial cells, diffuse staining was noted in the cytoplasm in addition to granular staining. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were seen in typical secondary lysosomes and some multivesicular bodies of the parenchymal cells and Kupffer cells. The lysosomes of the endothelial cells and fat-storing cells were weakly labeled. Quantitative analysis of the labeling density in the lysosomes of these three types of cells demonstrated that the lysosomes of parenchymal cells and Kupffer cells are main containers of cathepsin D in rat liver. The results suggest that cathepsin D functions in the intracellular digestive system of parenchymal cells and Kupffer cells but not so much in that of the endothelial cells.     

Cytochemical Observations on Proteins,Alkaline and Acid Phosphatases,Adenosine Triphosphatase,and 5′-Nucleotidase in Chick Liver Cell Cultures Infected with Trichomonas vaginalis*, †

SYNOPSIS. By means of the ninhydrin-Schiff method for proteins a diffuse reaction as well as one localized in granular inclusions can be shown in the cytoplasm of fibroblasts, epithelial cells, and macrophages in trypsin-dispersed chick liver cell cultures. Nuclei and nucleoli also take the specific stain. A progressive loss of cytoplasmic and nuclear staining occurs in the fibroblasts in cultures infected with a relatively pathogenic strain of T. vaginalis. A loss occurs in epithelial cells in advanced stages of degeneration, but in less damaged cells, while the diffuse reaction disappears, the number and staining intensity of the cytoplasmic inclusions remain unchanged or possibly may increase somewhat. The intensity of the diffuse reaction and the number and size of the characteristic inclusions increase in the active, parasite-free, experimental macrophages, but phagocytes with trichomonads closely applied to their external surfaces and those containing the flagellates within their cytoplasm typically retain only a few weak-staining inclusions. Similar distribution of alkaline and acid phosphatases occurs in preparations treated according to Gomori's and Burstone's methods, except that no nuclear staining is obtained with the latter. Activity of both enzymes is localized primarily in inclusions which are dispersed thruout the cytoplasm of fibroblasts and epithelial cells and tend to accumulate along the cell membranes and around the nuclei. In the course of infection with T. vaginalis there is a progressive loss of alkaline phosphatase from both cell types; however, the acid phosphatase activity increases. In the control macrophages both enzymes are localized in mostly rather large, rounded cytoplasmic inclusions. The number of such inclusions increases in the parasite-free experimental macrophages, but only a few weak-staining granules remainin phagocytes with engulfed trichomonads and in those whose external surfaces are in direct contact with the parasites. The loss of the inclusions is less apparent in macrophages containing degenerated flagellates than in the ones with healthy trichomonads, but regardless of the condition of the parasites, the highest enzymatic activity is found around them. ATPase and 5′-nucleotidase are localized in small granules dispersed thruout the cytoplasm of fibroblasts and epithelial cells. The granules tend to accumulate along the periphery of the cells and around the nuclei. A diffuse cytoplasmic reaction is present in preparations processed for 5′-nucleotidase. Nuclei and nucleoli give positive reactions for both enzymes. In the course of infection with trichomonads, activity of the 2 enzymes declines in both culture cell types. Control macrophages have diffuse cytoplasmic reaction for ATPase and 5′-nucleotidase and these enzymes are localized also in rounded cytoplasmic inclusions. Activity of both enzymes increases in the parasite-free experimental phagocytes, but little if any diffuse staining and only a few characteristic inclusions are left in macrophages with engulfed healthy trichomonads and in those whose external surfaces are invested with the flagellates. The ninhydrin-Schiff-positive inclusions found in the macrophages appear to be the same as some of those which have acid phosphatase activity and may well be identical with the glycolipoprotein bodies noted by us previously. On the grounds of their chemical constitution and behavior it seems likely that the inclusions are lysosomes.     

THE LOCALIZATION OF ALBUMIN AND FIBRINOGEN IN HUMAN LIVER CELLS

Human liver sections were stained with anti-human serum albumin and/or anti-human fibrin monomer fluorescent conjugates. Approximately 10 per cent of the hepatic cells stained specifically for human serum albumin,1 per cent for fibrinogen, and 0.1 per cent for both. Approximately 18 per cent of the Kupffer cells stained specifically for human serum albumin and 33 per cent for fibrinogen. Staining of both cell types was mainly cytoplasmic, although albumin was found in the nuclei of some parenchymal cells, depending on the method of fixation. Cytoplasmic granules staining specifically for fibrinogen could be seen just inside the cell membrane facing the bile caniculi in many more parenchymal cells than the 1 per cent showing diffuse cytoplasmic staining. The technical difficulties involved in preparing fluorescent conjugates against these antigens and in the fixation of these antigens for immunofluorescent staining are discussed.     

Effect of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells. Immunocytochemical observations

We studied the effects of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells using the direct immunoenzyme technique. Livers were perfusion-fixed 0.5, 1, and 2 h after injection of colchicine. Vibratome sections of the fixed liver were stained using peroxidase-conjugated Fab' of anti-albumin or anti-fibrinogen. By light microscopy, reaction deposits showing albumin and fibrinogen were observed in the cytoplasmic granules of hepatocytes. Such stained granules decreased 30 min after injection, but later increased gradually and crowded in the cytoplasm. The Golgi complex stained for the proteins decreased after 30 min but increased in the juxtanuclear region after 60 min. The analysis of serial sections showed that colchicine severely disturbed the spatial relationship between the Golgi apparatus and the bile canaliculus. We obtained similar results by electron microscopy; a positive reaction for albumin and fibrinogen was observed in a small number of the cytoplasmic granules after 30 min. After 1 h of treatment, most of the Golgi complexes were fragmented and lost their stacked cisternae. However, they reappeared accompanied with vacuolated cisternae and secretory granules, which were partially stained for albumin and fibrinogen. After 2 h, the secretory granules positive for both proteins accumulated further. Some of them lined a long the plasma membrane, and others made a cluster in the cytoplasm. The profiles showing exocytosis were very rarely seen. These results showed that in the first 30 min, colchicine primarily disturbs partially the Golgi assembly but does not affect the post Golgi secretory pathway much. Later, the drug affects both the post Golgi pathway and the Golgi assembly, and it causes a marked accumulation of secretory granules.     

Presence of angiotensin II in the adult male rat anterior pituitary gland: immunocytochemical study after cryoultramicrotomy

Angiotensin II (AII)-like immunoreactivity and binding sites have recently been demonstrated at the pituitary level. This peptide also exerts a stimulatory effect on anterior pituitary hormone release. Immunocytochemistry on ultrathin sections obtained by cryoultramicrotomy was used with the aim of localizing endogenous AII-like material at the cellular and subcellular levels of the anterior pituitary gland. AII-like immunostaining was observed only in gonadotrophs, lactotrophs, and corticotrophs. In gonadotrophs, AII-like immunoreactivity was restricted only to secretion granules. In the two other immunoreactive cells, lactotrophs and corticotrophs, immunostaining was observed in the cytoplasm and in the nucleus. In the cytoplasm, AII-like material was visualized in the cytoplasmic matrix and in the secretory granules. In the nucleus, immunostaining was distributed in the euchromatin in the vicinity of the heterochromatin. AII-like immunoreactivity was also seen at the plasma membrane, but only scarcely. No reaction product was found when anti-AII serum preincubated with AII was used. These immunocytochemical results (1) provide evidence that gonadotrophs are only a site of synthesis and/or storage of AII-like material, (2) indicate that lactotrophs and corticotrophs are cells for AII and (3) provide cytological evidence for a direct participation of AII in the regulation of the lactotropic and corticotropic function.     

Ultrastructural localization of rRNA in HeLa cells, rat liver cells and Xenopus laevis oocytes by means of the monoclonal antibody--protein a--gold technique

Summary The postembedding localization of rRNA was investigated in ultrathin sections of HeLa cells, rat liver andXenopus laevis oocytes by means of the monoclonal antibody to rRNA and protein A-gold technique. The incidence of gold particles was highest in nucleoli and cytoplasmic areas containing ribosomes. The chromosomes were labelled less than the surrounding cytoplasm in mitotic HeLa cells. In nucleoli of HeLa cells and rat hepatocytes, the labelling of areas containing ribonucleoprotein components was greater than the labelling of fibrillar centres. In segregated nucleoli ofX. laevis oocytes, the labelling of the granular region substantially exceeded that of the fibrillar regions. The incidence of nucleoplasmic gold particles in interphasic HeLa cells was found to be slightly increased in the vicinity of nucleoli. The labelling of clusters of interchromatin granules in rat hepatocytes was not significantly different from that of the rest of the nucleophasmic interchromatin spaces.A part of this study was presented as the poster and abstract at the 8th European Congress on Electron Microscopy 1984 in Budapest.     

Demonstration of glucose-6-phosphate dehydrogenase in rat Kupffer cells by a newly-developed ultrastructural enzyme-cytochemistry

Although various tissue macrophages possess high glucose-6-phosphate dehydrogenase (G6PD) activity, which is reported to be closely associated with their phagocytotic/bactericidal function, the fine subcellular localization of this enzyme in liver resident macrophages (Kupffer cells) has not been determined. We have investigated the subcellular localization of G6PD in Kupffer cells in rat liver, using a newly developed enzyme-cytochemical (copper-ferrocyanide) method. Electron-dense precipitates indicating G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of Kupffer cells. Cytochemical controls ensured specific detection of the enzymatic activity. Rat Kupffer cells abundantly possessed enzyme-cytochemically detectable G6PD activity. Kupffer cell G6PD may play a role in liver defense by delivering NADPH to NADPH-dependent enzymes. G6PD enzyme-cytochemistry may be a useful tool for the study of Kupffer cell functions.     

Immunohistochemical localization of basic fibroblast growth factor in A431 human epidermoid carcinoma cells.

The intracellular location of basic fibroblast growth factor (bFGF) was determined in A431 human epidermoid carcinoma cells both on immunofluorescence and on immunoelectron microscopy using a monoclonal anti-bFGF antibody. The immunofluorescence was located in the cytoplasm in quiescent cells. Following the addition of FCS to the culture medium of quiescent sparse cells the growth factor was translocated to and accumulated in the nucleolus. Immunogold particles were dense near the ribosomes, but were not recognized in the cytoplasmic structures concerned with the usual secretory pathway such as the rough endoplasmic reticulum, the Golgi apparatus, and secretory granules. These results suggest that endogenous bFGF undergoes intracellular sorting and enters the nucleoli in A431 cells according to an extracellular growth signal.     

Immunohistochemical localization of basic fibroblast growth factor in A431 human epidermoid carcinoma cells

Summary The intracellular location of basic fibroblast growth factor (bFGF) was determined in A431 human epidermoid carcinoma cells both on immunofluorescence and on immunoelectron microscopy using a monoclonal anti-bFGF antibody. The immunofluorescence was located in the cytoplasm in quiescent cells. Following the addition of FCS to the culture medium of quiescent sparse cells the growth factor was translocated to and accumulated in the nucleolus. Immunogold particles were dense near the ribosomes, but were not recognized in the cytoplasmic structures concerned with the usual secretory pathway such as the rough endoplasmic reticulum, the Golgi apparatus, and secretory granules. These results suggest that endogenous bFGF undergoes intracellular sorting and enters the nucleoli in A431 cells according to an extracellular growth signal.     

Cytochemical observations on glucose-6-phosphatase, glucosan phosphorylase, glucose-6-phosphate dehydrogenase, and -glycerophosphate dehydrogenase in chick liver cell cultures infected with Trichomonas vaginalis

Cytochemical reactions specific for glucose-6-phosphatase, glucosan phosphorylase, glucose-6-phosphate dehydrogenase, and α-glycero-phosphate dehydrogenase were observed in the epithelial cells and macrophages of chick liver cell cultures; α-glycerophosphate dehydrogenase activity was observed also in the fibroblasts. Distribution of three of the enzymes was limited to the cytoplasm, their activity being localized primarily in cytoplasmic inclusions. Weak staining of the nuclei and strong staining of the nucleoli occurred in addition to the cytoplasmic reaction in cells treated for glucose-6-phosphatase. In cell cultures inoculated with Trichomonas vaginalis, the activity of three of the enzymes decreased progressively in the course of infection, but that of α-glycerophosphate dehydrogenase increased.     

Cytochemical Observations on Malic Dehydrogenase,Lipase, Nonspecific Esterase and Monoamine Oxidase in Chick Liver Cell Cultures Infected with Trichomonas vaginalis*

SYNOPSIS. In trypsin-dispersed chick liver cell cultures malic dehydrogenase activity is localized in granules distributed thruout the cytoplasm of fibroblasts, epithelial cells, and macrophages. A progressive increase of the enzymic activity in all cell culture elements, except for phagocytes whose external or internal surfaces remain in direct contact with the parasites, accompanies infection of the cultures with a relatively pathogenic strain of Trichomonas vaginalis. In such phagocytes most staining for malic dehydrogenase is lost. Epithelial cells and parasite-free macrophages in experimental cultures also have a diffuse cytoplasmic reaction. No lipase activity is present in fibroblasts, but epithelial cells and macrophages in chick liver cell cultures contain numerous reactive granules. A strong diffuse cytoplasmic reaction is found in the epithelial cells and a weaker one in the control phagocytes. In cultures infected with T. vaginalis the enzyme is lost progressively from the epithelium and from those macrophages which have engulfed the parasites or whose external surfaces are invested with the flagellates; however, no significant changes in lipase activity can be found in parasite-free experimental phagocytes. In all cell types found in chick liver cultures, the reaction for nonspecific esterase is localized in cytoplasmic inclusions of varying size, some of which tend to accumulate along the cell membranes and around the nuclei. In addition, a weak diffuse cytoplasmic reaction is seen in the epithelial cells. Most cells in cultures infected with T. vaginalis have a significant increase in esterase activity, the exception being the macrophages which contain parasites within their cytoplasm or those with flagellates applied closely to their external surfaces. Only a few specifically stained granules are retained by such phagocytes. Monoamine oxidase activity is limited to fine granules dispersed in the cytoplasm of fibroblasts, epithelial cells, and macrophages of control cultures. Infection of chick liver cultures with T. vaginalis results in lowered enzyme activity in non-phagocytic cells as well as in macrophages with engulfed flagellates and in those whose external surface are invested with the parasites. The number of reactive inclusions appears to increase in trichomonad-free phagocytes of experimental cultures.     

钐在小鼠肝脏细胞中的动态观察

It is generally considered that the rare earth compounds are plasma membrane-impermeable, thus affecting the cells only on their surface. Recently, we found that after repeated injections to mice of large dose of samarium trichloride, a soluble compound of rare earth, samarium aggregates appeared in Kupffer cells and hepatocytes of liver. In this study, we aimed at observing the route by which samarium enters the liver cells and the process of the formation of samarium aggregates. Samarium trichloride was given to Swiss mice at one dose of 70 mg/kg intravenously. Thereafter, at different intervals from 15 min to 48 h after the injection, the samarium in liver was traced dynamically by electron microscopy and X ray microanalysis. From 15 min to 2 h both Kupffer cells and hepatocytes endocytosed samarium-containing particles and formed phagosomes, in which the ingested particles were progressively concentrated. Besides, the small phagosomes fused with each other. Phagocytosis was especially active in Kupffer cells. During the 4 h to 24 h many Kupffer cells were degenerated and broken. In hepatocytes the phagosomes gathered mostly around the bile canaliculi. Groups of highly electron-dense particles were found in the lumen of bile canaliculi, implying the excretion of samarium by bile. At the 48 h, the samarium-containing phagosomies were found still in both kinds of cells in the liver.     

The hemocytes of Panstrongylus megistus (Hemiptera: Reduviidae)

Five hemocyte types were identified in the hemolymph of Panstrongylus megistus by phase contrast and common light microscopy using some histochemical methods. These are: Prohemocytes, small cells presenting a great nucleus/cytoplasm ratio; Plasmatocytes, the most numerous hemocytes, are polymorphic cells mainly characterized by a large amount of lysosomes; Granulocytes, hemocytes very similar to plasmatocytes which contain cytoplasmic granules and are especially rich in polysaccharides; Oenocytoids, cells presenting a small nucleus and a thick cytoplasm; they show many small round vacuoles when observed in Giemsa smears and many cytoplasmic granules under phase microscopy; Adipohemocytes, very large hemocytes, presenting many fat droplet inclusions which could correspond to free fat bodies which entered the hemolymph. Only prohemocytes and plasmatocytes can be clearly classified; all the other hemocyte types have a more ambiguous classification.     

An ultrastructural and histochemical study of oogenesis in the trichostrongylid nematode Heligmosomoides polygyrus

Oogenesis in trichostrongylids has been examined for the first time in a light and electron microscopic investigation of Heligmosomoides polygyrus. The female reproductive tract is a single straight tube containing small oogonia (6 micron in diameter), which are arranged in a rosette pattern around a central rachis at the anterior end of the tract. Developing oocytes separate from the rachis and pass posteriorly in single file down the growth zone. Oocytes increase rapidly in volume due to the accumulation of cytoplasmic inclusion granules. These granules are of 3 types. Type 1 granules are amorphous and probably consist primarily of lipoprotein. Type 2 granules are large lipid inclusions and type 3 granules are electron-dense lipoprotein yolk bodies, which are probably used for energy reserves in the developing embryo. Histochemical studies show a more intense reaction for DNA in the nuclei of oogonia than in the nuclei of oocytes. There is a strong reaction for RNA in the nucleoli and in the cytoplasm of oogonia and oocytes. Ultrastructural studies indicate that this RNA is probably in the form of rRNA in the abundant ribosomes. Mature oocytes are cylindrical (60 X 70 micron), have a distinct nucleus with nuclear pores, and the cytoplasm is filled with inclusion granules and ribosomes but contains only small amounts of glycogen. Prior to fertilization the plasma membrane of oocytes acquires a flocculent coat. These oocytes contain 6 distinct bivalent chromosomes in diakinesis. Thus the major changes that occur in developing germ cells are 2-fold: nuclear changes that prepare the chromosomes for fertilization by initiating reduction division, and cytoplasmic changes that involve the synthesis and storage of inclusion granules.     

不同生殖期鳜肝脏超微结构变化的观察

应用透射电镜对生殖季节与非生殖季节鳜肝脏超微结构的变化进行了观察。鳜肝细胞含有单个卵圆形的核,核仁清楚;细胞质内含有粗面内质网、线粒体、糖原颗粒和脂滴等细胞器和内含物。胆小管由相邻的数个肝细胞质膜凹陷围成,而肝血窦则由内皮细胞的胞质构成。还发现了贮脂细胞、枯否氏细胞和成纤维细胞。胆小管腔和窦周隙内浸润许多由肝细胞发出的微绒毛结构。鳜肝细胞的超微结构在产卵前后呈现明显变化：产卵前的肝细胞内富含线粒体、糖原颗粒和脂滴,粗面内质网发达;而产卵后的肝细胞内核仁发生迁移,部分细胞核囊泡化,糖原颗粒和脂滴排空,少数肝细胞具双核结构。非生殖期多数肝细胞核含有双核仁结构,胞质内溶酶体数量增多。     

An in vivo role of Mrp2 in the rat hepatocytes by immunocytochemistry for amoxicillin using the transporter-deficient EHBR

An in vivo role of the multidrug resistant-associated protein (Mrp2) in rat hepatocytes was examined by immunocytochemistry (ICC) for amoxicillin (AMPC) by the use of the transporter-deficient Eisai hyperbilirubinemic rats (EHBR). The ICC revealed that in the liver of EHBR at 3-h post-administration, amoxicillin accumulated in the cytoplasmic pools and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries. However, no amoxicillin was observed on the surface of the lumina ranging from the bile capillaries to the interlobular bile ducts. The drug persisted at least for 6-h after administration. In contrast, in the control rat liver at 3-h post-administration, AMPC-adsorption occurred on such luminal surface, while AMPC accumulated to a less level in both the cytoplasm and nuclei of the hepatocytes. The drug completely disappeared in the hepatocytes at 6-h post-administration. These results strongly suggest that AMPC taken up into the cytoplasm of the hepatocytes excretes via Mrp2 into the bile flow. Furthermore, electron microscopy demonstrated that the lower electron density areas in large sizes, corresponding to the cytoplasmic pools in ICC for AMPC, occurred in the cytoplasm peripheral to the nuclei of the hepatocytes in EHBR at 3-h post-administration, and then disappeared 24 h after administration.