Electron microscopic and morphometric studies of the main kidney segment of male and female rats following castration and testosterone substitution

The effect of testosterone on the 3 segments of the renal proximal tubule (S1, S2, S3) of male and female rats was studied by electronmicroscopic and morphometric methods. Only light, granulated and dark lysosomes as well as microbodies (peroxisomes) and dictyosomes (Golgi zones) were investigated. After castration the area density of light lysosomes in the S1 segment increases in males whereas it decreases in females; therefore the sex different pattern of light lysosomes, that is to be seen in normal animals, is reversed. The absolute size and number of light giant lysosomes is also elevated in castrated males in comparison to normal animals as well as to animals substituted by testosterone. - Dark lysosomes of the S1 segments are more numerous in castrated females and less numerous in castrated males than in normal animals. - The distinct sex difference in dark lysosomes of the S2 segment which is demonstrable in normal animals disappears after castration the area density of dark lysosomes increasing in castrated females and decreasing in castrated males. The three species of lysosomes in the S1 segments show no longer a sex difference after substitution with testosterone: substituted males develop the same pattern as normal animals and substituted females are almost comparable with normal males. However, the sex difference in dark lysosomes of the S2 segment is more pronounced after testosterone treatment. - The characteristic pattern of light lysosomes in the S1 and S2 segments as well as the change of the sex different lysosomal pattern after castration and substitution with testosterone, respectively - especially in S1 - seem to be caused by testosterone which results in an inhibition of resorption. Only after castration a sex difference appears in dark lysosomes of the S3 segment (males show more dark lysosomes than females). This sex difference is reversed by testosterone treatment. There are more numerous lysosomes with an non-homogeneous matrix in both sexes after castration which are seldom to be seen in normal and substituted animals. The area density of microbodies shows sex differences in all 3 segments of normal animals. While no significant changes in S1 and S2 are to be seen after castration and substitution, there is a pronounced decrease of the area density of microbodies in S3 of males after castration, so that no sex differences are then available.(ABSTRACT TRUNCATED AT 400 WORDS)     

MICROBODIES IN EXPERIMENTALLY ALTERED CELLS

A rapid and sustained increase in the number of microbodies in liver and kidney cells can be induced in male rats by ethyl chlorophenoxyisobutyrate (CPIB), a hypolipidemic drug. This phenomenon permits investigation of several aspects of microbody behavior in experimental conditions. Reversal experiments demonstrate that liver cells revert to normal between 2 and 3 weeks after withdrawal of CPIB and that one of the mechanisms for removal of excess microbodies is their incorporation into structures indistinguishable from lysosomes. In a state of rapid cell division, such as that present during liver regeneration, microbody proliferation apparently occupies a high biological priority. In necrotic or degenerating cells microbody structure remains relatively normal. The increase in microbodies induced by CPIB is inhibited by chloramphenicol. No increase in microbodies occurred in female rats or in chickens, guinea pigs, or rabbits at the dosage used (0.25% in diet). No changes in microbodies were seen in monkey liver. Catalase activity was generally parallel to the numerical response in microbodies. Additional observations suggest that the microbody response to CPIB is not related to hepatomegaly induced by this agent but may be related to the hypolipidemic effect of CPIB, though hypolipidemia per se is not a specific or sufficient cause of microbody proliferation.     

Sex-dependent changes in the rat kidney after hypophysectomy

Summary Renal changes following hypophysectomy are investigated. Particular attention is given to sex differences in the ultrastructure of proximal tubule cells and in protein excretion.Regardless of gender, hypophysectomy is followed by an increase in urine volume. However, there is a concomitant reduction of proteinuria, which is much more pronounced in males than in females. Partial hypophysectomy with anterior pituitary function preserved also leads to increased urine excretion, but does not alter proteinuria.In both sexes there is a reduction of the tubule circumference, which again is more pronounced in males thereby moderating the sex difference. The proximal tubule cells display a segment- and sex-independent reduction in surface area, a decrease of Golgi areas and reduction of ribosomes. Mitochondrial changes (condensation of cristae) selectively affect the S₃ segment.The changes in the lysosomes and microbodies are segment- and sexdependent. The volume density of microbodies in the S₃ segment increases considerably, particularly in females. The volume density of lysosomes undergoes an increase in the S₁ cells of the males and a decrease in the S₂ cells. In the females the volume density of these organelles shows little change in these tubule segments; a sex-dependent difference is not longer apparent in the S₁ and S₂ segments. By contrast, in S₃, there is an increase in the volume density of lysosomes in both sexes. The present study confirms a connection between the morphology of lysosomes in the proximal tubule and proteinuria. The findings also point to a possible involvement of male sex hormones in the reabsorption of protein in the renal proximal tubule.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Effects of endogenous and exogenous glucocorticoids on liver differentiation

The effects of maternal bilateral adrenalectomy on day 1 of gestation and betamethasone treatment on fetal liver development were compared, in terms of biochemical and morphological parameters. For fetuses 20 days old (E20), absence of maternal glucocorticoids during gestation caused an increase in the number of nuclei in whole livers, and a significantly decrease of both body weight and protein content per nucleus, in comparison with the control group (C). Betamethasone injection on days 15, 16 and 17 of gestation into adrenalectomized pregnant rats (ADX + BET) did not completely prevent these effects. The electron microscopic analysis of the ADX fetal liver (E20) showed some hepatocyte lesions such as loss of cytoplasmic organelles, increase in hematopoietic cell number as well as a lower cellular maturation in comparison with the control group. The fetal liver from ADX + BET mothers 20 days after gestation displayed a noticeable involution of the hematopoietic component in spite of its relatively immature stage. However, there was no significant change in the degree of fetal hepatocyte lesions. Therefore, supply of maternal glucocorticoids from the beginning of gestation is essential for maintenance of the integral structure of the rat fetal hepatic parenchyma, for the correct maturation of the blood strains and for the beginning of involution of the hematopoietic tissue at the end of gestation.     

Effects of clofibrate (alpha-p-chlorophenoxyisobutyryl-ethyl-ester) on male rat liver

In male rats, fed 0.5% clofibrate in their diet for 8 days and 21 days, the ultrastructural morphometric alterations of the hepatocytes were evaluated and compared with the biochemical data. The morphologic alterations of the microbodies were particularly related to the changes of the catalase activity of the liver homogenates. The results showed a marked hypertrophy of the liver and an increase in the volume of the individual hepatocyte. The numerical density and, even more pronounced, the volume density of the microbodies increased excessively during the treatment. The numerical density of the mitochondria decreased markedly after 21 days of administration. The surface of the rough endoplasmic reticulum showed a significant decrease, whereas the surface of the smooth endoplasmic reticulum showed a hypertrophy. The catalase activity of the liver homogenates increased 2-fold after 8 days and remained at this new steady-state after 21 days of treatment. The results suggest that the enzyme content of the microbodies changed after treatment with clofibrate, and support the suggestion that clofibrate may induce the synthesis of a yet unidentified peroxisomal protein.     

Chronic stress and its effects on adrenal cortex apoptosis in pregnant rats

The model of chronic intermittent stress by immobilization during pregnancy may produce alterations in the mechanisms that maintain adrenal gland homeostasis. In earlier investigations using this model, significant variations in plasma prolactin and corticosterone levels, and adrenal gland weights were observed. We hypothesized that chronic stress causes changes in apoptosis in the adrenal glands of pregnant rats. We identified and quantified apoptotic cells in the adrenal cortex and examined their ultrastructural characteristics using transmission electron microscopy. Adrenal glands of pregnant rats at gestation days 12, 17 and 21 were studied for control and experimental (stressed) rats. Immunolabelling techniques, stereological analysis and image quantification of adrenal gland sections were combined to determine differences in apoptosis in the different cell populations of the adrenal cortex. The apoptotic index of the experimental rats showed a significant reduction at gestation day 17, while at days 12 and 21 there were no differences from controls. Moreover, the apoptotic index of the reticular zones in control and experimental animals showed a significant increase compared to the glomerular and fascicular zones at the three gestation times studied. Chronic stress by immobilization reduced the caspase-dependent apoptotic index at gestation day 17, which may be related to variations in plasma concentrations of estrogens and prolactin.     

Regulation of uterine 5 alpha-reductase type 1 in mice.

A null mutation in the murine gene encoding steroid 5 alpha-reductase type 1 (5 alpha R1) leads to failure of normal parturition at term. This observation, together with the finding that mRNA levels of uterine 5 alpha R1 increase significantly at term in normal pregnant animals, indicates that 5 alpha R1 plays an important role in murine parturition. The current studies were conducted to elucidate the regulation of 5 alpha R1 in uterine tissues of nonpregnant and pregnant animals. Nonpregnant, ovariectomized ICR mice were treated with vehicle (control), 17 beta-estradiol (E(2)), progesterone (P(4) ), or E(2)+P(4) for 3 days. Thereafter, uterine tissues were obtained for histology, quantification of 5 alpha R1 specific activity, and Northern blot analysis of 5 alpha R1 mRNA expression. The 5 alpha R1 enzyme activity was significantly increased in animals treated with E(2)+P(4). However, activity was much less in uterine tissues from E(2)+P(4)-treated animals than in uterine tissues from pregnant animals near term. To evaluate further the regulation of 5 alpha R1 during gestation, mice underwent unilateral tubal ligation before timed matings. The 5 alpha R1 activity increased eightfold in uterine tissues from the fetal horn from Gestational Days 12 to 18. This temporal pattern in 5 alpha R1 activity paralleled marked increases in uterine diameter. Taken together, these studies indicate that expression of 5 alpha R1 is regulated by E(2)+P(4) in uterine tissues. Whereas E(2) alone is insufficient to induce enzyme activity, E(2) may be required to increase P(4) receptors and, thereby, mediate the effects of P(4) on 5 alpha R1 gene expression. Further increases in enzyme activity during late gestation are mediated by fetal occupancy, possibly through stretch-induced increases in endometrial growth. Thus, like other genes involved in parturition, expression of 5 alpha R1 is regulated by both hormonal and fetal-derived signaling pathways.     

Morphogenesis of chicken liver: identification of localized growth zones and the role of beta-catenin/Wnt in size regulation

During development and regeneration, new cells are added and incorporated to the liver parenchyma. Regulation of this process contributes to the final size and shape of the particular organs, including the liver. We identified the distribution of liver growth zones using an embryonic chicken model because of its accessibility to experimentation. Hepatocyte precursors were first generated all over the primordia surrounding the vitelline blood vessel at embryonic day 2 (E2), then became limited to the peripheral growth zones around E6. Differentiating daughter cells of the peripheral hepatocyte precursors were shown by DiI microinjection to be laid inward and were subsequently organized to form the hepatic architecture. At E8, hepatocyte precursor cells were further restricted to limited segments of the periphery, called localized growth zones (LoGZ). Adhesion and signaling molecules in the growth zone were studied. Among them, beta-catenin and Wnt 3a were highly enriched. We overexpressed constitutively active beta-catenin using replication competent avian sarcoma (RCAS) virus. Liver size increased about 3-fold with an expanded hepatocyte precursor cell population. In addition, blocking beta-catenin activity by either overexpression of dominant-negative LEF1 or overexpression of a secreted Wnt inhibitor Dickkopf (DKK) resulted in decreased liver size with altered liver shape. Our data suggest that (1) the duration of active growth zone activity modulates the size of the liver; (2) a shift in the position of the localized growth zone helps to shape the liver; and (3) beta-catenin/Wnt are involved in regulating growth zone activities during liver development.     

Subcellular localization of glycogen-UDP-glucosyltransferase in the hepatocytes of intact and fasting rats

Electron microscope cytochemical techniques by Takeuchi et al. were used to study the localization of glycogen-UDP-glucosyl-transferase (GUDPG) in hepatocytes of intact and fasting rats. Glycogen was found in the cytoplasm, nuclei, mitochondria, microbodies and lysosomes of the hepatocytes of intact and fasting animals after special cytochemical procedures for determination of GUDPG.     

Ultrastructural and Cytochemical Evidence for the Presence of Peroxisomes in the Generative Cell of Magnolia x soulangeana Pollen Grain

The generative cell (GC) development during three sequentialstages of Magnolia x soulangeana pollen grain maturation wasinvestigated by light and electron microscopy. Plastids werenot identified in this cell but mitochondria, Golgi bodies andvesicles as well as rough endoplasmic reticulum profiles werealways present. Microtubules were also present, their numberincreasing and their disposition varying during GC maturation.The most conspicuous components of the GC cytoplasm were themicrobodies. The latter were few in number in the newly formedGC, and the appearance of their matrix was different from laterdevelopmental stages. A clear microbodial proliferation occurredin the GC during an intermediate stage of pollen maturation.Then, the microbody matrix was either fibrillar to granularas in the vegetative cell microbodies or very dense and compact.The polymorphism and size range and the frequent aggregationof these organelles in one or more clusters were also noteworthy.Tilting of semithin sections as well as the analysis of serialsections suggested that a number or enlarged and irregularlyshaped microbodies co-exist with smaller and more sphericalones, the latter probably originating by budding. In the GCof the mature pollen the microbody-like organelles were in generalmore uniform both in shape and size. The cytochemical test ofDAB was positive in the microbodies of both the pollen cells,thus demonstrating their peroxisomic nature. The function ofthe microbodies in the GC is not clear. In this cell, a fewlipid droplets only exist during the first developmental stageand the microbodies were apparently unrelated to any other organelle.Possibly, these are unspecialized microbodies which are paternallytransmitted, but it is not excluded that, temporarily, theymay play some special role during GC maturation.Copyright 1994,1999 Academic Press Peroxisomes, generative cell, pollen maturation, Magnolia x soulangeana Soul.-Bod     

Quantitative analysis of immunogold labelling for ferritin in liver from control and iron-overloaded rats

Summary The distribution of ferritin antigenicity in control and iron-loaded rat hepatocytes was investigated with an immunogold-ferritin antibody technique. Antibody to horse spleen ferritin showed immunoreactivity as determined by dot blotting with immunogold/silver staining with purified rat liver ferritin but not with rat haemosiderin. The initial site of ferritin degradation was studied by analysing the density of gold labelling in the cytosol and lysosomes in combination with pre-embedding acid phosphatase cytochemistry.Immunoreactive ferritin was present in the cytosol, cytosolic clusters and lysosomes of normal hepatocytes. After iron-loading, the labelling density increased over tenfold in parenchymal cell cytosol with a smaller increase in Kupffer cells. Ferritin clusters contained substantially more immunoreactive ferritin than equivalent areas of lysosomes or cytosol. Analysis of the labelling density in hepatocyte lysosomes showed that, despite a striking increase in iron content, one-quarter of the lysosomes showed less immunolabelled ferritin than the cytosol. The existence of a wide range of ferritin labelling densities in the lysosomes with a large proportion unlabelled suggests that the ferritin protein shell is not degraded at a significant rate either in the cytosol or in clusters but only after incorporation into lysosomes.     

Changes with Development in the Expression of Cathepsin E in the Fetal Rat Stomach

The changes with development in the expression of cathepsin E in the fetal rat stomach were examined immunochemically and immunohistochemically. The activity of acid proteinase in fetal gastric extracts increased dramatically during late gestational stages, rising from 0.017 units per mg of protein on day 15 of gestation to 0.591 units per mg of protein on day 21 of gestation. Electrophoretic analysis, combined with immunological tests, showed that the increase was due exclusively to increases in the activity of the monomeric and dimeric forms of cathepsin E, while SDS-PAGE-immunoblot analysis revealed that both forms are present as a 43-kDa proenzyme. Immunohistochemically, cathepsin E was localized in the cytoplasm of all proliferating epithelial cells of pars glandularis on day 16 of gestation or later. As revealed by conventional histological methods, surface mucous cells and parietal cells appeared for the first time in specimens on day 19 of gestation, and all of these cells were immunopositive for cathepsin E. The present study further indicated that cathepsin E is the predominant aspartic proteinase in the stomach of young rats, until pepsinogen C appears. Based on these results, possible roles of gastric cathepsin E are discussed.     

Partially protected areas as a management tool on inshore reefs

Partially Protected Areas (PPAs) are a widely-used management tool, yet comparatively little is known about their effectiveness compared to more commonly studied No-Take Marine Reserves (NTMRs). Here, we examine the efficacy of two kinds of PPAs (with and without spearfishing) within the Great Barrier Reef Marine Park (GBRMP) that are subject to a range of fishing limitations, and assess their utility as a marine park zoning and fisheries management tool. Fish abundance, size, and habitat composition were compared inside PPAs and NTMRs on inshore reefs of the central GBR. Fish abundances were lower inside PPAs relative to adjacent NTMRs for primary fishing targets, with no detectable effects for secondary targets and non-targets, or for species richness. Fish assemblages differed amongst zones, but these variations were minor compared to regional variations in species composition. Partially Protected Areas supported 46%–69% of the relative abundance of total primary targets compared to adjacent NTMRs, with no evident increase in abundance in zones where spearfishing was prohibited. There were no reductions in the size of two key target species: coral trout (Plectropomus spp.) and stripey snapper (Lutjanus carponotatus) inside PPAs, and only stripey snapper had significant reductions in abundance inside PPAs compared to NTMRS. Habitat and biophysical characteristics (especially topographic complexity) were strong drivers of fish abundance, but the relative influence of zone was greater for target species compared to non-targets. This study provides novel data on PPAs and highlights their utility as a spatial management tool in contributing to conservation and fisheries management goals.

     

Development of Microbodies in the yeast Kloeckera growing on methanol.

A number of microbodies appear regularly in methanol-grown yeast cells, but rarely in ethanol- or glucose-grown cells. When one of representative methanol-utilizing yeasts, Kloeckera sp.no. 2201 (also known as Candida bodinii), was cultured on glucose and then transferred into a methanol medium, microbodies of small size could be observed in 2-h old cells. The number of microbodies per sectioned cell reached five to six after 4 h of cultivation. Though the number of microbodies did not change during prolonged cultivation, their size became larger with the passage of cultivation time. The activities of catalase and alcohol oxidase were confirmed in the particulate fractions throughout the cultivation period, whereas the activities of formaldehyde dehydrogenase and formate dehydrogenase were not detected in the particles. The activity of isocitrate lyase was detected in the particulate fractions only at the early growth phase.     

Subcellular localization of acyl coenzyme A: dihydroxyacetone phosphate acyltransferase in rat liver peroxisomes (microbodies).

Upon differential centrifugation of rat liver homogenate, the enzyme acyl-CoA:dihydroxyacetone-phosphate acyltransferase (EC 2.3.1.42) was found to be localized in the light mitochondrial (L) fraction which is enriched with lysosomes and peroxisomes. Peroxisomes were separated from lysosomes in a density gradient centrifugation using rats which were injected with Triton WR 1339. By comparing the enzyme distribution with the distribution of different marker enzymes, it was concluded that dihydroxyacetone phosphate acyltransferase is primarily localized in rat liver peroxisomes (microbodies). Similarly, the enzyme acyl dihydroxyacetone-phosphate:NADPH oxidoreductase (EC 1.1.1.101) was shown to be enriched in the peroxisomal fraction, although a portion of this reductase is also present in the microsomal fraction.     

Lysosome changes in exponentially growing, synchronized and differentiating L-cell cultures

Using supravital fluorescent staining of lysosomes with Euchrysine 3R, the morphology of these organelles was studied in L cells examined from cultures being at different growth phases in the course of cell cycle and after adipocyte conversion of L cells due to the 60% bovine serum administration. As cells were passing from the lag-phase to the stationary phase of culture growth, the number of lysosomes was seen to increase. The appearance of large lysosomes is characteristic of cells in confluent and senescent cultures. During G1-period, lysosomes are often confined to the perinuclear area of L-cells, to be extended later during S and G2-periods. In dividing cells, these are commonly seen scattered throughout the cell periphery, around the mitotic spindle. In cells undergoing differentiation, within 4-7 days the seeding in the medium supplemented with 60% bovine serum, the number of lysosomes became augmented to be gradually reduced during the next 10-15 days, concomittantly with the accumulation of lipid drops in the cell cytoplasm. The activity of the Golgi complex and the intensity of autophagy are discussed as possible regulation points of lysosome formation during the cell growth.     

Ultrastructure of methanotrophic yeasts.

The cellular structure of two yeast strains capable of growth on methane was investigated by electron microscopy. Microbodies were observed in cells of Sporobolomyces roseus strain Y and Rhodotorula glutinis strain CY when grown on methane but rarely when grown on glucose. The size of the microbodies and the number observed per cell in a thin section did not increase with culture age. No crystalline organization was observed within these organelles. Similar microbodies were also observed in cells of R. glutinis CY grown on hexadecane. The plasma membranes of both methane and hexadecane-grown cells exhibited increased invagination compared to that of glucose-grown cells. Catalase activity was detected in the microbodies of alkane-grown cells by using 3,3'-diaminobenzidine as a cytochemical stain. The data presented suggest that microbodies, and the catalase contained within them, play a role in eucaryotic methane metabolism.     

The nucleoids of rat liver cell microbodies. Fine structure and enzymes

The nucleoids of microbodies of rat liver cells were isolated in a highly homogeneous and pure state, by treating the microbody-rich fraction, prepared from 10% polyvinylpyrrolidone-0.25 M sucrose homogenate, with Triton X-100. Three treatments with 0.1% detergent were enough to render the nucleoids free from contamination with mitochondria, microsomes, lysosomes, and intact microbodies. Electron microscopically, the nucleoids were found to consist of parallel bundles of highly dense hollow tubules, the outer and inner diameters of which are approximately 150 and 50 A, respectively. Ten tubules are arranged around a longitudinal space 190 x 200 A in width. The nucleoids thus show a honeycomb appearance in the cross-plane and a parallel-packed structure in the longitudinal plane. Biochemically, the nucleoids were found to bear only urate oxidase among probably microbody-enzymes, and they might be the only cytoplasmic particles of rat liver cells in which the enzyme locates. Urate oxidase activity, on a unit protein basis, of the nucleoid preparation is approximately 380 times as high as that of the whole homogenate, and is almost comparable with that of a commercial type I enzyme preparation. No enzymes of mitochondrial, microsomal, and lysosomal origins were detected in the nucleoids. The fine structure of the nucleoids is described in detail, and a probable schematic diagram is presented.     

CYTOCHEMICAL AND DEVELOPMENTAL CHANGES IN MICROBODIES (GLYOXYSOMES) AND RELATED ORGANELLES OF CASTOR BEAN ENDOSPERM

Structural changes in endosperm cells of germinating castor beans were examined and complemented with a cytochemical analysis of staining with diaminobenzidine (DAB). Deposition of oxidized DAB occurred only in microbodies due to the presence of catalase, and in cell walls associated with peroxidase activity. Seedling development paralleled the disappearance of spherosomes (lipid bodies) and matrix of aleurone grains in endosperm cells. 6 to 7 days after germination, a cross-section through the endosperm contained cells in all stages of development and senescence beginning at the seed coat and progressing inward to the cotyledons. Part of this aging process involved vacuole formation by fusion of aleurone grain membranes. This coincided with an increase in microbodies (glyoxsomes), mitochondria, plastids with an elaborate tubular network, and the formation of a new protein body referred to as a dilated cisterna, which is structurally and biochemically distinct from microbodies although both apparently develop from rough endoplasmic reticulum (ER). In vacuolate cells microbodies are the most numerous organelle and are intimately associated with spherosomes and dilated cisternae. This phenomenon is discussed in relation to the biochemical activities of these organelles. Turnover of microbodies involves sequestration into autophagic vacuoles as intact organelles which still retain catalase activity. Crystalloids present in microbodies develop by condensation of matrix protein and are the principal site of catalase formerly in the matrix.