Studies on the toxic effect of cadmium in the rat. I. Testicular changes induced by a single subcutaneous injection of cadmium chloride.

Adult male rats of Wistar strain were subcutaneously injected with a single dose of 0.025 mM CdCl2/kg body weight. The autopsies were performed 2 and 12 weeks post cadmium injection. Experimental animals showed a normal gain of body weight while a marked lowering of the testes weight was observed. The lowering of testes weight of cadmium treated rats occured mainly due to the necrosis of seminiferous tubules, volume of which is markedly lower if compared to intact control rats. After 2 weeks of experiment a marked enlargement of the interstitial gland of the testis occured while after 12 weeks the gland is markedly lower if compared to control rats and to rats studied 2 weeks post cadmium injection. Histochemical reactions for lactic, succinic, alpha-glycerophosphate and 3beta-hydroxy steroid dehydrogenases, NADH tetrazolium reductase, acid phosphatase and nonspecific esterases showed for irreversible necrotic changes of seminiferous tubules of cadmium treated rats while the damage to interstitial gland is only temporary.     

Histochemical localization of enzyme activity in the kidneys of male marmosets (Callithrix jacchus and Callithrix penicillata).

The distribution of several hydrolases and oxidoreductases was studied in the renal parenchyma of adult male marmosets (Callithrix jacchus and Callithrix penicillata). The oxidative enzymes showed a high reactivity in the proximal and distal tubules, whereas the hydrolases reacted strongly in the proximal tubules but only weakly or not at all in the thick limb of Henle's loop, distal tubules and collecting ducts. The NAD-dependent enzymes (except alpha-GPDH) showed a stronger reactivity in the proximal tubules, while the NADP-dependent ones were more reactive in the thick limb of Henle's loop and distal convoluted tubules. Two groups of interstitial cells were found in the medulla. A first group inside the outer medulla, showing cells rich in acid phosphatase and nonspecific esterases and a second group, close to the papilla, reactive to a certain number of oxidative enzymes. A different reactivity in cells of the distal convoluted tubules, thick limb of Henle's loops and collecting ducts (dark cells) was seen in the case of some enzymes like nonspecific esterase, alpha-GPDH and SDH.     

Nonspecific esterase and alpha-glycerolphosphate dehydrogenase activity and the fat content in the regenerating chicken liver]

The data on histochemical study of changes in the activity of esterases, alpha-glycerophosphate dehydrogenase and the fat content in the chick regenerating liver are presented. The accumulation of the fat in hepatocytes during the early periods of regeneration (1--5th day of the experiment) is partially conditioned by the reduction of the activity of nonspecific esterases. The maximal liver steatosis is accompanied also by an increase in the activity of alpha-glycerophosphate dehydrogenase which indicates intensification of glycolysis and synthesis of triacylglycerines (liver spare lipids) during regeneration.     

Histochemical investigation of the folliculogenesis in the ovary of the Mongolian gerbil (Meriones unguiculatus)

The ovaries of 70 mature Mongolian gerbils (Meriones unguiculatus) were investigated morphologically and enzyme histochemistry for the appearance of acid phosphatase, non-specific esterases, succinate dehydrogenase and thiamine pyrophosphatase. In the oocyte two particular enzyme active zones exist depending on the state of development. In young oocytes acid phosphatase, succinate dehydrogenase and thiamine pyrophosphatase can be found only in the perinuclear zone. From the late secondary follicle on, activity of acid phosphatase, succinate dehydrogenase and non-specific esterases can be detected only in a peripheral area of cytoplasm, whereas thiamine pyrophosphatase is present in the entire ooplasm. In the follicular epithelium a different pattern of enzyme distribution suggests a functional differentiation of the epithelial cells during folliculogenesis.     

Histochemical investigation of the folliculogenesis in the ovary of the Mongolian gerbil (Meriones unguiculatus)

Summary The ovaries of 70 mature Mongolian gerbils (Meriones unguiculatus) were investigated morphologically and enzyme histochemically for the appearance of acid phosphatase, non-specific esterases, succinate dehydrogenase and thiamine pyrophosphatase. In the oocyte two particular enzyme active zones exist depending on the state of development. In young oocytes acid phosphatase, succinate dehydrogenase and thiamine pyrophosphatase can be found only in the perinuclear zone. From the late secondary follicle on, activity of acid phosphatase, succinate dehydrogenase and non-specific esterases can be detected only in a peripheral area of cytoplasm, whereas thiamine pyrophosphatase is present in the entire ooplasm.In the follicular epithelium a different pattern of enzyme distribution suggests a functional differentiation of the epithelial cells during folliculogenesis.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Histopathology of streptozotocin-induced diabetic DBA/2N and CD-1 mice

Histopathological examinations were carried out on female DBA/2N and CD-1 mice which were autopsied 4 and 12 weeks after six daily intraperitoneal injections of streptozotocin (SZ). Histopathological changes related to SZ treatment were found in the pancreas, liver and kidneys. Little difference was observed between the two strains in the histological changes of the pancreas (a decrease in size of the islets, and degranulation and a decrease in the number of B cells) and liver (hypertrophy of hepatocytes and cytoplasmic invagination into hepatocyte nuclei). With regard to the changes in the kidneys, DBA/2N mice showed characteristic inclusions positive to periodic acid-Schiff reagent in the distal tubule epithelial cells, while CD-1 mice showed remarkable luminal dilatation and epithelial cell deformation of distal tubules. SZ-induced diabetes had no influence on the development of spontaneous cardiovascular lesions in DBA/2N mice under the present experimental conditions.     

Age-related changes in the seminal vesicles of a Brazilian (Nelore) zebu.

Age changes in the structure of the seminal vesicles and in the rate of production of fructose and citric acid have been studied in a Brazilian (Nelore) zebu, from the fetal period to 36 months of age. At 3 and 6 months, the microscopic anatomy of the gland resembled that of the fetus; the tubules of the seminal vesicles had a reduced diameter and a low epithelial layer; only a few presented traces of secretion, and tissue contents of fructose and citric acid were accordingly low. At 12 months, the tubules were more ramified and had a larger diameter. In the 18-month-old animals the seminal vesicles presented substantial modifications; the tubules were large, with irregular lumina and surrounded by narrow stroma, the epithelial layer was higher than that of previous stages and its columnar cells had nuclei located basally. Tissue levels of fructose increased rapidly between 12 and 18 months. At 24 months, the seminal vesicles had reached the adult condition characterized by intense proliferation of tubules with irregular lumina and abundant secretory material. Numerous dark columnar cells were found in the epithelium. Seminal vesicles of Nelore zebus contain less fructose and citric acid than those of taurine bulls of comparable age.     

Characterization of nonspecific esterase activity in macrophages and intestinal epithelium of the rat.

We determined the histochemical characteristics of nonspecific esterase in different populations of rat macrophages. The cells included alveolar and peritoneal macrophages recovered by lavage and a mixed cell population obtained by collagenase digestion of the small intestine. The histochemically localized enzyme activity of alveolar and peritoneal macrophages was cytoplasmic, diffuse, and inhibited by sodium fluoride. Both populations were effectively stained using alpha-naphthyl acetate and alpha-naphthyl butyrate as the esterase substrate. When the intestinal cells were examined for activity, a greater percentage of cells showed positive nonspecific esterase than would be predicted by differential counts for macrophages on the basis of morphological criteria. We confirmed, using cell smears and tissue sections, that rat intestinal epithelial cells, a prominent component of the isolated cell population, possessed esterases that react similarly to macrophage esterases with histochemical procedures.     

Age and the course of nephrotoxic nephritis in rats]

In experiments on two groups of mongrel rats (4 weeks old and 4 months old) with induced nephrotoxic nephritis it was revealed that in comparison with adult rats the course of nephritis in ratlings was characterized by lesser proteinuria, selective in nature, by lesser reducticn of endogenous creatinine clearance and diuresis. The acido- and ammo-niogenesis decreased in ratlings and adult rats to the same extent. Morphological changes in the kidneys of ratlings were less pronounced than in adult animals, and were mostly localized in the convoluted tubules. The level of DNA-synthetic activity of the epithelial nuclei of the glomeruli prevailed over this index of the convoluted tubules epithelium. The weight index of the kidneys increased less in ratlings with nephritis than in adult rats. beta-lipoproteinemia in ratlings increased 8 times. Normalization of the urine and blood indices occurred more rapidly in ratlings than in adult rats.     

Correlative histochemical studies on preneoplastic and neoplastic lesions in the kidney of rats treated with nitrosamines

Renal tubular lesions induced in male rats by two different carcinogens, N-nitrosomorpholine (NNM) and N-ethyl-N-hydroxyethylnitrosamine (EHEN), using a limited exposure "stop" protocol were investigated histochemically to demonstrate phenotypic cellular changes. The parameters measured included basophilia, glycogen content and the activity of the enzymes glucose-6-phosphatase (G6PASE), glycogen synthetase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase (SDH), alkaline phosphatase (ALP), acid phosphatase (ACP) and gamma-glutamyl transpeptidase (gamma-GT). The lesions observed were predominantly of either basophilic or oncocytic types. In each case, tubular lesions (altered tubules) appeared to give rise to epithelial tumors (epitheliomas) with the same cellular phenotype. Basophilic tubules and epitheliomas proved to be strongly positive for GAPDH and G6PDH while demonstrating a reduction or loss of G6PASE, ALP, ACP, gamma-GT, and SDH compared with controls and the surrounding proximal or distal tubules. In addition, large basophilic epitheliomas demonstrated an increase in both SYN and PHO activities. In contrast, most oncocytic tubules and oncocytomas characterized by abundant densely granular cytoplasm showed a reduction in the activity of G6PDH, but were intensely positive for SDH. However, a few oncocytic lesions demonstrated a decrease in both SDH and G6PDH activity. Rarely, decreased SDH and elevated G6PDH activities were observed in altered tubules resembling oncocytic tubules. It remains to be clarified whether these tubules represent a variation of the oncocytic lesions or, perhaps, another type of tubular lesion. The results indicate that basophilic and oncocytic epithelial tumors differ in their cytochemical pattern and histogenesis. In line with earlier suggestions, the basophilic tumors apparently originate from the proximal renal tubules, while the oncocytomas develop from the distal parts of the nephron. The basophilic tumors are characterized by an increased pentose phosphate pathway and glycolysis, with a corresponding reduction in mitochondrial respiration. However, the majority of the oncocytomas show an increased activity of the mitochondrial enzyme SDH, and a marked decrease in the activity of the key enzyme of the pentose phosphate pathway.     

Monocyte nonspecific esterase. Enzymologic characterization of a neutral serine esterase associated with myeloid cells

Monocytes contain a characteristic, prominent set of membrane-bound nonspecific esterases with a slightly acid isoelectric point. These esterases are also detected at modest levels in some granulocyte preparations. They are not apparent in lymphocytes. Among 18 fresh myeloid leukemias and myeloid leukemia cell lines, those of subtypes M4 (myelomonocytic) and M5 (monocytic) were strongly positive; some of subtypes M1-M3 (granulocytic) were moderately positive. The esterases were not detected among 32 fresh lymphoid leukemias and lymphoid leukemia and lymphoblast cell lines. The membrane-bound monocyte esterases, solubilized by treatment of monocyte preparations with nonionic detergent, were resolved by ion-exchange chromatography. The monocyte species account for 80-95% of the total nonspecific esterase activity of monocytes. The resolved enzymes behave as neutral serine carboxyl esterases and are highly sensitive to inhibition by diisopropylfluorophosphate (DFP) and also by sodium fluoride. Similar analysis of a lymphocyte preparation yielded no detectable monocyte esterases, but yielded numerous other forms which were generally resistant to inhibition by DFP and NaF. These nonspecific esterases are also present at background levels in monocytes. The resolution and characterization of the membrane-bound serine esterases from monocytes demonstrates the basis for the well-known cytochemistry of monocytes. The results are also crucial to the development of an immunologic surface marker test for myeloid cells and the study of monocyte membrane physiology.     

Effect of diabetes mellitus induced by streptozotocin on renal Superoxide dismutases in the rat

Two forms of superoxide dismutase, CuZn-SOD and MnSOD, have been investigated in the kidneys of streptozotocin-induced diabetic rats using both radio-immunoassay and immunoenzyme staining. The rats were killed 2, 8 and 12 weeks after the induction of diabetes mellitus and the kidneys excised. Two weeks after the induction of diabetes, the kidneys were hypertrophied because of the proliferation of renal tubular epithelium. However, the total CuZnSOD content of the kidneys did not increase and, because of the epithelial proliferation, the CuZnSOD concentration in each proximal tubular cell was decreased. Armanni-Ebstein lesions were found in the distal tubules 8 and 12 weeks after the induction of diabetes. The cells in these lesions were intensely stained for CuZnSOD, suggesting an adaptive response to the enhanced oxidative stress. The MnSOD staining in the thick ascending limbs of Henle's loops was enhanced in the diabetic kidneys, while that in the cortical tubules was unaltered. MnSOD was assumed to increase in response to hypermetabolism associated with the proliferation of renal tubules. This was most marked in the cells which were rich in mitochondria, again suggesting an adaptive response to enhanced oxidative stress induced by diabetes mellitus. The glomeruli of both the diabetic and control groups were not stained for SODs, and no significant microscopic change was found even 12 weeks after the induction of diabetes mellitus.     

Reinitiation of spermatogenesis by exogenous gonadotropins in a seasonal breeder, the woodchuck (Marmota monax), during gonadal inactivity.

The present study was undertaken (1) to document structural and functional changes in the testes of seasonally breeding woodchuck during active and inactive states of spermatogenesis and (2) to evaluate the ability of exogenous gonadotropins to reinitiate spermatogenesis outside the breeding season. During seasonal gonadal inactivity, there were significant (P less than 0.05) reductions in volumes of several testicular features (testis, seminiferous tubules, tubular lumen, interstitial tissue, individual Leydig cells, Leydig cell nuclei, and Leydig cell cytoplasm) as compared with gonadally active animals. The diameter of the seminiferous tubules was decreased by 26%, and Leydig cell numbers also declined in the regressed testes. These changes were accompanied by a decline in testosterone (T) levels in both plasma and testis, and reduction in epithelial height of accessory reproductive organs. A hormonal regimen was developed that would reinitiate spermatogenesis in captive, sexually quiescent woodchucks. A combination of PMSG and hCG markedly stimulated testicular growth and function and restored spermatogenesis qualitatively. Quantitatively normal spermatogenesis was restored in 2 of 6 treated males. Morphometric analyses revealed substantial increases in seminiferous tubular diameter and in the volume of seminiferous tubules, tubular lumen, total Leydig cells, and individual Leydig cells in the hormone-treated animals. These increased values corresponded to 99, 75, 68, 51, and 200%, respectively, of the values measured in naturally active woodchucks. Leydig cell numbers, however, remained unchanged and approximated only 31% of the number found in naturally active testes. Hormonal stimulation also resulted in a significant rise in serum T as well as in the total content of testicular T, and a marked increase in epithelial height in various accessory reproductive glands. The most effective hormonal protocol for stimulating spermatogenesis was treatment with 12.5 IU of PMSG twice a week for 4 weeks followed by 12.5 IU of PMSG + 25 IU of hCG twice a week for 4 weeks.     

Observations on the histology and histochemistry of the oesophagus of the perch, Perca fluviatilis L.

The anatomy and enzyme histœhemistry of the œsophagus of a freshwater teleost perch ( Perca fluviatilis L.) has been studied. The mucosa is composed of an undifferentiated layer of basal epithelial cells, mucous cells and surface epithelial cells. The submucosa is compact and contains blood vessels, nerves and bundles of striated longitudinal muscles. The outer circular muscular layer contains both striated and smooth muscle fibres. Electron microscopy shows that the surface epithelial cells are degenerative and that they surround and support pores of the mucous cells. Undifferentiated epithelial cells undergo cytoplasmic changes and eventually become surface epithelial cells. Mucous cells arise from the undifferentiated epithelial cells and their droplets contain acid and neutral mucosubstances. Alkaline phosphatase, non-specific carboxylic esterases and aminopeptidase activity is absent in the mucosa. However, acid phosphatase activity is localized in the surface epithelial cells.     

大鼠颌下腺促性腺激素释放激素及其mRNA的免疫组织化学与原位杂交研究

用免疫组织化学及原位杂交法,研究了促性腺激素释放激素及其ｍＲＮＡ在大鼠颌下腺的分布。结果显示,大鼠颌下腺的浆液性腺泡的上皮细胞,各级导管的上皮细胞及副交感神经节细胞均呈促性腺激素释放激素免疫反应阳性,阳性反应物质分布在胞质,胞核呈阴性反应。颌下腺的浆液性腺泡上皮细胞,各级导管上皮细胞同样被检测到很强的促性腺激素释放激素ｍＲＮＡ杂交信号。以上结果提示,大鼠颌下腺能自身合成促性腺激素释放激素,促性腺激素释放激素对消化功能可能有重要调节作用。     

Histochemical studies on the localization of oxidative and dephosphorylating enzymes and esterases in the peritoneal mesothelial cells

Summary Histochemical localization of various groups of oxidative and dephosphorylating enzymes and esterases has been made in the peritoneal mesothelial cells. These cells were strongly positive for lactic dehydrogenase and succinic dehydrogenase, moderately positive for cytochrome oxidase, simple esterase, acid phosphatase, 5nucleotidase and nonspecific cholinesterase, and negative for monoamine oxidase and specific cholinesterase. They showed moderate positive activity for alkaline phosphatase, localized in the microvilli of these cells. They contain scanty amount of thiamine pyrophosphatase-positive Golgi complex and showed diffuse thiamine pyrophosphatase-positive activity in the cell cytoplasm. Adenosine triphosphatase-positive spaces found in between the adjacent cells and at the bases of the individual cells have been demonstrated. These results are discussed in view of the great absorptive power of the peritoneal mesothelium.     

2型糖尿病大鼠模型的肾脏和动脉血管并发症

目的制备发病过程类似人类2型糖尿病的动物模型,并观察其肾脏和主动脉的病变特点。方法8周龄SD大鼠高脂、高糖饮食一个月后给予小剂量STZ腹腔注射建立2型糖尿病模型,于成模后4周及8周观察血管功能指标和肾脏功能指标,并对肾脏和血管的病理改变进行观察。结果模型组于成模后4周及8周出现高血糖、高血脂、胰岛素抵抗、肾功能改变和血管功能改变。肾脏光镜下见肾小球内皮及系膜细胞增生;肾小管水肿,管腔内有大量蛋白管型和细胞管型;肾盂区有大量淋巴细胞浸润。动脉血管电镜下见内皮细胞局部损伤严重;内皮细胞与内弹力板连接处空隙增加等。结论用高脂高糖饮食加小剂量STZ腹腔注射可成功制备2型糖尿病大鼠模型,成模后4周及8周后观察肾脏及大血管相继出现病变,是2型糖尿病血管病变研究的理想模型。     

Morphometrical analysis of an ultrahistochemical demonstration of nonspecific esterases in hepatocytes of mice after fructose overload.

Changes in ultrastructural distribution patterns of nonspecific esterases (E.C. 3.1.19) are described quantitatively by means of morphometry. Esterases were demonstrated with O-acetyl-8-hydroxyquinoline (Q-O-2) and S-acetyl-8-mercaptoquinoline in livers of normally and exclusively fructose-fed mice. Conditions are discussed, under which the quantification of the ultrastructural products of enzyme histochemical reactions may be possible. Smooth endoplasmic reticulum and rough endoplasmic reticulum exhibit no alteration in enzyme distribution with both substrates since the enzyme-occupied proportion of each compartment remains the same despite an overall decrease of both compartments. Likewise an increase of O-acetyl-8-hydroxyquinoline-esterase at fat droplets corresponds to the increase in total surface of the fat. S-acetyl-8-mercaptoquinoline-positive fat surface however reveals an increase far beyond that of the total fat surface. The results support the hypothesis that a variety of esterases with different substrate spectra are present at the subcellular level in different cell compartments.     

Apoptosis and cell desquamation in repair process of ischemic tubular necrosis

To elucidate the role of apoptosis and cell desquamation in the repair phase of acute tubular necrosis, morphological findings after 60 min ischaemia were investigated in rats. A morphometric analysis of the cell proliferation and of the epithelial cellularity of reconstructing tubules was performed. The kinetics of apoptosis and cell desquamation were also examined. Ischaemia and reperfusion injury resulted in widespread necrosis of tubules at day 1. Subsequently, a regenerative epithelial hyperplasia took place in the aarly stage. The most marked increase in cellularlity in the damaged tubules was on day 6, when the tubules became lined by hyperplastic epithelial cells with papillary clusters. The number of papillary clusters decrease up to day 8, and during this period many desquamated cells from the clusters were observed in the tubular lumen. In the later stage, hyperplastic epithelial cells were reduced to their original cellularity and during this period the number of apoptotic cells obviously increased, while the damaged tubules were reconstructed. We conclude that epithelial over-production occurs in the early phase after tubular necrosis, and excess hyperplastic epithelial cells regress during the repair process by cell desquamation and apoptosis, both of which are essential for the recovery of the original tubular structure.     

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.