Smooth-surfaced Endoplasmic Reticulum Research Articles

The effects of the anti-Müllerian hormone on folliculogenesis in rats: light and electron microscopic evaluation

ABSTRACT In this study, we evaluated the effects of anti-Müllerian hormone on follicle development and oocyte quality with light and electron microscopy. Twenty-four adult female rats were divided into four groups. After estrous cycle synchronization, on the first day, control group rats were injected with 0.5 ml saline, 2nd, 3rd, and 4th groups were injected 1 µgr, 2 µgr, and 5 µgr anti-Müllerian hormone, respectively. On the third day, intracardiac blood samples were taken for follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone serum level measurements. Ovaries were obtained for light and electron microscopic examinations. Secondary (antral) follicles were decreased while atretic follicles were increased in number parallel with an increased dose of anti-Müllerian hormone injection. Atresia of the follicles was demonstrated with apoptosis of granulosa cells characterized by apoptotic bodies and with paraptosis characterized by the vacuole formation in the cytoplasm, enlargement of granular endoplasmic reticulum cisternae and perinuclear cisternae in granulosa cells. Premature luteinization characterized by increased lipid droplets, mitochondria with tubular cristae, and smooth-surfaced endoplasmic reticulum in the cytoplasm of granulosa cells were detected in some growing follicles. In the anti-Müllerian hormone injected experimental groups, cystic follicles characterized by a large antrum, attenuated granulosa cell layer, and flattened granulosa cells that face the antrum were observed. Corpus luteum and stroma were similar in all groups. It was concluded that increasing doses of anti-Müllerian hormone caused increased atresia in developing follicles, premature luteinization of granulosa cells in some follicles, and cystic follicle formation in the further developing follicles.

Ultrastructural differences in the endoplasmic reticulum among various pituitary tumor types

Our aim was to reveal ultrastructural differences in the endoplasmic reticulum (ER) among surgically removed human pituitary tumors. More than 2000 pituitary tumors were studied by electron microscopy. In growth hormone (GH) adenomas, the ER is well developed and consists mainly of rough surfaced endoplasmic reticulum (RER) and few ER membranes without ribosomes, called smooth surfaced endoplasmic reticulum (SER). In the sparsely granulated GH adenomas, the ER is less prominent and is seen in fibrous bodies containing keratin immunopositive microfilaments. In prolactin (PRL) adenomas, the RER is conspicuous, forming concentric whorls. In adenocorticotropin (ACTH) and thyroid stimulating hormone (TSH) adenomas, the ER and consists of RER and to a lesser extent, SER. In gonadotroph adenomas and null cell adenomas, the ER is poorly developed. In GH adenomas exposed to somatostatin analogs and in prolactin adenomas exposed to dopamine agonists, regression of ER is noted. In pituitary carcinomas, the ER is recognizable but it is less extensive than in the benign neoplasms. In conclusion, the ultrastructural investigation of ER provides valuable information on the cytogenesis and cellular composition of pituitary tumors. There is also a mild correlation between endocrine activity and extent of ER. However, no correlation exists between invasive and non‐invasive tumors and growth potential. Pituitary carcinomas cannot be distinguished from benign adenomas based on the ultrastructural features of ER.

Effect of formaldehyde inhalation on rat livers: A light and electron microscopic study

It is well known that formaldehyde (FA) is cytotoxic and potentially carcinogenic. Although the individual effects of this reactant on cells has been investigated, the cytotoxicity exerted by the coexistence of FA is poorly understood. The aim of this study was to investigate the effects of FA on the liver in rats, by light and electron microscopic level. We used 18 Wistar albino rats divided into three groups, exposed to 0 (control), 19.7 ppm FA gas for a total of 4 weeks, 8 h/day, 5 days a week (subacute) and 20.3 parts per million (ppm) FA gas for a total of 13 weeks, 8 h/day, 5 days a week (subchronic). After the completion of the exposure period, they were sacrificed by decapitation and their liver tissue samples were taken in order to be processed for light and electron microscopic studies. Light microscopic evaluation of liver tissue samples of FA-exposed rats revealed enlarged sinusoids filled with blood and mononuclear cell infiltration in the portal areas and around the central veins. In addition, some of the hepatocytes showed loss of cytoplasm, and some had a hyperchromatic nucleus. The cells of FA-exposed livers, on the other hand, showed an electron-lucent ground-cytoplasm and a hypertrophy of the smooth-surfaced endoplasmic reticulum. In conclusion, we observed that exposure FA caused diverse histopathological changes indicating the destruction in the liver tissue and this destruction has direct relationship with the length of the exposure period.

The inositol 1,4,5-trisphosphate receptor.

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger that releases Ca2+ from its intracellular stores. The InsP3 receptor has been purified and its cDNA has been cloned. We have found that the InsP3 receptor is identical to P400 protein, first identified as a protein enriched in cerebellar Purkinje cells. We have generated an L-fibroblast cell transfectant that produces cDNA-derived InsP3 receptors. The protein displays high affinity and specificity for InsP3. InsP3 induces greater Ca2+ release from membrane vesicles from transfected cells than from those from control L-fibroblasts. After incorporation of the purified InsP3 receptor into lipid bilayers InsP3-induced Ca2+ currents were demonstrated. These results suggest that the InsP3 receptor is involved in physiological Ca2+ release. Immunogold labelling using monoclonal antibodies against the receptor showed that it is highly concentrated on the smooth-surfaced endoplasmic reticulum and slightly on the outer nuclear membrane and rough endoplasmic reticulum; no labelling of Golgi apparatus, mitochondria and plasmalemma was seen. Cross-linking experiments showed that the receptor forms a homotetramer. The approximately 650 N-terminal amino acids are highly conserved between mouse and Drosophila, and this region contains the critical sequences for InsP3 binding. We have investigated the heterogeneity of the InsP3 receptor using the polymerase chain reaction and have found novel subtypes of the mouse InsP3 receptor that are expressed in a tissue-specific and developmentally specific manner.

Cell Density and Growth-dependent Down-regulation of Both Intracellular Calcium Responses to Agonist Stimuli and Expression of Smooth-surfaced Endoplasmic Reticulum in MC3T3-E1 Osteoblast-like Cells

A two-dimensional intracellular Ca(2+) ([Ca(2+)](i)) imaging system was used to examine the relationship between [Ca(2+)](i) handling and the proliferation of MC3T3-E1 osteoblast-like cells. The resting [Ca(2+)](i) level in densely cultured cells was 1.5 times higher than the [Ca(2+)](i) level in sparsely cultured cells or in other cell types (mouse fibroblasts, rat vascular smooth muscle cells, and bovine endothelial cells). A high resting [Ca(2+)](i) level may be specific for MC3T3-E1 cells. MC3T3-E1 cells were stimulated with ATP (10 microM), caffeine (10 mM), thapsigargin (1 microM), or ionomycin (10 microM), and the effect on the [Ca(2+)](i) level of MC3T3-E1 cells was studied. The percentage of responding cells and the degree of [Ca(2+)](i) elevation were high in the sparsely cultured cells and low in densely cultured cells. The rank order for the percentage of responding cells and magnitude of the Ca(2+) response to the stimuli was ionomycin > thapsigargin = ATP > caffeine and suggests the existence of differences among the various [Ca(2+)](i) channels. All Ca(2+) responses in the sparsely cultured MC3T3-E1 cells, unlike in other cell types, disappeared after the cells reached confluence. Heptanol treatment of densely cultured cells restored the Ca(2+) response, suggesting that cell-cell contact is involved with the confluence-dependent disappearance of the Ca(2+) response. Immunohistological analysis of type 1 inositol trisphosphate receptors and electron microscopy showed distinct expression of inositol trisphosphate receptor proteins and smooth-surfaced endoplasmic reticulum in sparsely cultured cells but reduced levels in densely cultured cells. These results indicate that the underlying basis of confluence-dependent [Ca(2+)](i) regulation is down-regulation of smooth-surfaced endoplasmic reticulum by cell-cell contacts.

Fine structure of the oxynticopeptic cells in the gastric glands of the ruin lizard, Podarcis sicula campestris De Betta, 1857.

The results of an ultrastructural investigation of the gastric glands of the ruin lizard are reported. In this reptile the stomach can be divided into a larger fundus and a smaller pars pilorica. Fundic glands are characterized by three main kinds of cells: mucous, endocrine, and oxynticopeptic; the latter were not observed in the pyloric glands. The morphological features of the oxynticopeptic cells change from the proximal to the distal region of the fundic mucosa. In the proximal region, numerous electron-dense secretory granules, a well-developed granular endoplasmic reticulum, an evident Golgi complex, and a reduced system of smooth-surfaced vesicles and tubules in the apical cytoplasm characterize these cells. In the distal fundic region, oxynticopeptic cells possessed numerous mitochondria and a well-developed smooth-surfaced endoplasmic reticulum, but secretory granules were rare. These data suggest the existence of a gradient in the production of proteolytic enzymes, and perhaps also of hydrochloric acid, along the oral-aboral axis of the stomach. The results are discussed with regard to the evolution of the gastric glands and of the digestive mechanism in vertebrates.

Immunolocalization of mitsugumin29 in developing skeletal muscle and effects of the protein expressed in amphibian embryonic cells.

The temporal appearance and subcellular distribution of mitsugumin29 (MG29), a 29-kDa transmembrane protein isolated from the triad junction in skeletal muscle, were examined by immunohistochemistry during the development of rabbit skeletal muscle. MG29 appeared in the sarcoplasmic reticulum (SR) in muscle cells at fetal day 15 before the onset of transverse tubule (T tubule) formation. In muscle cells at fetal day 27, in which T tubule and triad formation is ongoing, both SR and triad were labeled for MG29. In muscle cells at newborn 1 day, the labeling of the SR had become weak and the triads were well developed and clearly labeled for MG29. Specific and clear labeling for MG29 was restricted to the triads in adult skeletal muscle cells. When MG29 was expressed in amphibian embryonic cells by injection of the cRNA, a large quantity of tubular smooth-surfaced endoplasmic reticulum (sER) was formed in the cytoplasm. The tubular sER was 20-40 nm in diameter and appeared straight or reticular in shape. The tubular sER was formed by the fusion of coated vesicles [budded off from the rough-surfaced endoplasmic reticulum (rER)] and vacuoles of rER origin. The present results suggest that MG29 may play important roles both in the formation of the SR and the construction of the triads during the early development of skeletal muscle cells.

Calcium-containing, smooth-surfaced endoplasmic reticulum and vacuoles in cells of the blastopore-forming region during gastrulation of the newt, Cynops pyrrhogaster.

Ultrastructural changes in cells of the blastopore-forming region (BFR) were examined during gastrulation of the newt, Cynops pyrrhogaster. Smooth-surfaced endoplasmic reticulum (sER), tubular in shape and with two types of vacuole, empty vacuoles and multilamellar vacuoles, appeared in cells of the BFR at the beginning of formation of the blastopore. The extent of the tubular sER increased during formation of the blastopore. With the deepening of the blastoporal groove, the tubular sER and multilamellar vacuoles disappeared in cells of the BFR. The empty vacuoles increased in number and appeared throughout the cytoplasm of the cells in the BFR and the marginal zone as formation of the archenteron progressed. Many large empty vacuoles were closely associated with deformed lipid droplets. Cytochemistry and X-ray microanalysis revealed the accumulation of calcium in the tubular sER and in some of the empty vacuoles. These results suggest important roles for the calcium-containing tubular sER in the regulation of the intracellular concentration of calcium during formation of the blastopore, as well as a role for empty vacuoles in the dissolution and consumption of lipid droplets during early gastrulation.

Carbamylcholine-induced morphological changes and spatial dynamics of [Ca2+]c in Harderian glands of guinea pigs: calcium-dependent lipid secretion and contraction of myoepithelial cells.

To determine whether lipid-secreting cells have cytosolic Ca2+ concentration ([Ca2+]c)-related secretory mechanisms, morphological changes and intracellular calcium dynamics of Harderian glands of guinea pigs stimulated by secretagogue were studied by electron microscopy and Fura-2/AM digital image analysis. Control glandular cells contained large lipid vacuoles that were bordered by multi-layered membranes. Rough-surfaced endoplasmic reticulum, mitochondria, and smooth-surfaced endoplasmic reticulum may be involved in lipid vacuole formation. Myoepithelial cells surrounded alveoli. After carbamylcholine (CCh, 10(-6), 10(-5), and 10(-3) M) stimulation, lipid materials within the membranous structures were frequently discharged by an exocytotic mechanism. Conspicuous deformation of glandular cells caused by vigorous contraction of myoepithelial cells was observed in isolated alveoli after 10(-6) M CCh stimulation, whereas the deformities of glandular tissues perfused via vessels were small even after 10(-3) M CCh stimulation. Connective tissue between glandular alveoli inhibited unbridled myoepithelial-cell contraction. Fura-2/AM digital imaging analysis revealed that CCh stimulation caused an increase in [Ca2+]c in isolated alveoli. The morphological reactions and changes in [Ca2+]c were prevented by atropine. When extracellular calcium ions were absent, enhanced extrusion of lipid vacuoles, myoepithelial-cell contraction, and a rise in [Ca2+]c after CCh stimulation were not observed. Nicotine and catecholamines had no effect on the secretion or on the dynamics of [Ca2+]c. It can be concluded that acetylcholine elicits exocytosis in glandular cells and contraction of the myoepithelial cells of Harderian glands, accompanied by an increase in [Ca2+]c. The dynamics of [Ca2+]c of the gland alveoli are mostly dependent on extracellular Ca2+.

Distribution of taurine-like immunoreactivity in the mouse liver during ontogeny and after carbon tetrachloride or phenobarbital intoxication.

The ontogenic pattern of development of taurine-like immunoreactivity (TLI) was studied in the mouse liver. The effect on adult mice of carbon tetrachloride or phenobarbital treatment was also examined. Light-microscopically, granules of TLI were first found in the liver from 17-day-old embryos, diffusely distributed throughout the lobules. These positive granules increased with age, were most numerous in the two-week-old mouse, and were notably decreased in the central region of some lobules in the three-week-old mouse. In mature mice, hepatocytes containing TLI-positive granules were distributed unevenly in each liver lobule, and were located predominantly in the peripheral region. Electron-microscopically, TLI was observed in small vesicles in the cytoplasm of hepatocytes and was found mainly in the cisternal lumen of smooth-surfaced endoplasmic reticulum. Some taurine-positive vesicles surrounding the reticulum seemed to associate with the protoplasm. Similar positive vesicles were often located near the bile canaliculi. In carbon tetrachloride-intoxicated mature mice, TLI was no longer limited to the peripheral region of lobules; hepatocytes situated in the central region of lobules also contained intense TLI. In mice injected with a small and repeated dose of phenobarbital, the distribution pattern of TLI was similar to that in the untreated group. However, in mice injected with a large dose of phenobarbital, TLI was markedly increased, especially in the central region of lobules. The results demonstrate that the distribution pattern of TLI in mouse liver changes during development, and that the pattern in mature mice is affected by intoxication with carbon tetrachloride or a toxic dose of phenobarbital.

Tridimensional organization of Purkinje neuron cisternal stacks, a specialized endoplasmic reticulum subcompartment rich in inositol 1,4,5-trisphosphate receptors.

Stacks of regularly spaced, flat, smooth-surfaced endoplasmic reticulum cisternae frequently observed in both the cell body and dendrites of cerebellar Purkinje neurons, were previously shown by immunocytochemistry to be highly enriched in receptors for the second messenger, inositol 1,4,5-trisphosphate. Morphometric analyses have been carried out on randomly selected thin section images of rat Purkinje neurons to reveal the tridimensional organization of these structures. Individual stacked cisternae (on the average approximately 3.5 per stack) were shown to be separated from each other by a 23.5 nm space occupied by perpendicular bridges, approximately 20 nm in diameter, most probably composed by two apposed receptor homotetramer molecules, inserted into the parallel membranes in their hydrophobic domains. In the stacked membranes the density of the bridges was approximately 500 microns -2, corresponding to approximately 15% of the surface area. The lateral distribution of bridges was not random, but revealed regular distances that might correspond to unoccupied receptor slots. In each stack, the external cisternae were often in direct lumenal continuity with conventional elements of the endoplasmic reticulum, whereas the internal cisternae were not. Since continuities between stacked cisternae were never observed, the results indicate that the internal cisternae are at least transitorily discrete, i.e. they are not in permanent lumenal continuity with the rest of the endoplasmic reticulum. To our knowledge this is the first demonstration of a physical subcompartmentalization of the latter endomembrane system in a non-mitotic cells. A model for the biogenesis of cisternal stacks, based on the head-to-head binding and lateral interaction of the inositol 1,4,5-trisphosphate receptor molecules in the plane of the interacting membranes, is proposed and critically discussed.

Hepatocyte fine structure during maturation and senescence

Aging is accompanied by a myriad of changes in cell structure, function, and composition. The fact that much of the information concerning age-related alterations in cellular morphology is qualitative precludes meaningful correlations with biochemical changes in order to enhance data interpretation. The mammalian liver has been subjected to both qualitative and quantitative evaluations of hepatocyte structure as a function of aging, i.e., development, maturation, and senescence. Although these data are characterized by considerable variability and, in some instances, blatant contradictions, there exists sufficient agreement in several parameters to permit a consensus in the inbred rat model. Certainly the volume of individual hepatocytes increases with age, and many of the organelle compartments reflect this change. While old rats exhibit a high incidence of polyploidy, there is no definitive evidence to demonstrate a concomitant increase in the binuclear hepatocyte index. Several specific hepatocellular organelles undergo changes in their relative volume or surface area that appear to correlate with functional alterations. The volume density of the lysosomal compartment enlarges significantly during senescence and is accompanied by increased activities of several constituent hydrolases. The hepatic concentration of smooth-surfaced endoplasmic reticulum declines markedly with aging, as does the yield of liver microsomes and the activities of several microsomal enzymes, e.g., mono-oxygenases and glucose-6-phosphatase. However, the responses of the majority of hepatocyte organelles to aging is varied and inconsistent based on the limited data currently available.

FIFTY TWO-WEEK CHRONIC ORAL TOXICITY STUDY OF MOFEZOLAC (N-22) IN RATS

Fifty two-week oral toxicity study of mofezolac (N-22), a new developed analgesic and anti-inflammatory agent, was carried out in Wistar rats with dose levels of 5, 20, 60 and 120 mg/kg/day, and the 5-week withdrawal was followed for recovery study. Hematuria, blanching of the skin and suppression of body weight gain were observed in females given 120 mg/kg, and 9 of these prostrated and died from week 20 to week 52, or were euthanized when moribund. These symptoms were not seen in males at any dose levels. In 120 mg/kg group, increased positive cases of fecal occult blood were observed during the administration period, and the pathological examination revealed gastrointestinal lesions such as erosion, ulcer, hemorrhage and mucosal regeneration in the small intestine. Renal disorder was also involved mainly in females given 120 mg/kg, as shown by increase in urine volume with declined osmotic pressure and specific gravity, serum urea nitrogen, creatinine, inorganic phosphorus, and other related parameters. In addition to enlargement, rough surface and scar formation, dilated tubular lumen and papillary ducts of the kidney were observed as a main lesion. Incidental findings with those disorders, observed mainly in females given 120 mg/kg, included increase in leucocytes with high neutrophils ratio, and enlargement of the spleen, adrenals and mesenteric lymph node. There were some of the similar gastrointestinal and renal changes mainly in females given 60 mg/kg. Anemic findings were noted in both sexes of 120 mg/kg and in females of 60 mg/kg group, and mainly females given 120 mg/kg showed increase in platelets, reticulocytes and fibrinogen, shortening of blood coagulation time, as well as extramedullary or accented hematopoiesis of the liver, spleen and bone marrow. Other serum biochemical changes observed mainly in females given 120 mg/kg were decrease or decreasing trend in total protein, A/G ratio, and transaminase activity. Fine structure of the liver from females given 20 mg/kg or more revealed cisternal dilatation of smooth-surfaced endoplasmic reticulum of hepatocytes, and the increased liver weight was observed in females given 120 mg/kg. Accordingly, non-effective dose level of N-22 in 52-week chronic toxicity study was estimated to be 20 mg/kg for males and 5 mg/kg for females.

Reversible neuronal damage in hippocampal pyramidal cells with triethyllead: the role of astrocytes.

A single dose (19 mg kg-1) of triethyllead given to weanling rats produces necrosis in a small number of hippocampal pyramidal (CA3) and hilar neurons with reversible changes in the remaining neurons of this region. The sequence of events has been studied by light and electron microscopy over a period from 12 h to 14 days after dosing. Early changes resemble those previously described for trimethyltin, with the formation of characteristic tubulo-vesicular dense bodies by 12 h accompanied by vacuolation of Golgi and smooth surfaced endoplasmic reticulum (SER) elements which became generalized by 24 h. Large numbers of secondary dense bodies, formed from tubulo-vesicular dense bodies as well as from autophagosomes, were present by 48 h, whilst very little rough surfaced endoplasmic reticulum (RER) and few polyribosomes remained and vacuolation was much reduced. In those animals which did not die from seizures, the majority of hippocampal pyramidal cells were able to recover from these changes with astrocytes playing a significant role in the elimination of the dense bodies. This involved astrocytes inserting processes into the neuronal perikaryon from where the secondary dense bodies were selectively transferred into the astrocyte cytoplasm. This activity was first seen at 48 h, reached a peak at 4 days, when most CA3 neurons contained one or more astroglial intrusions and subsided soon after. The surviving neurons returned to apparent normality over the period from 3 to 7 days with a gradual return of polyribosomes. Golgi elements and RER.

Effect of Osmolarity of the Fixative on the Ultrastructure of Preimplantation Rabbit Embryos

In an attempt to clarify whether or not glutaraldehyde molecules contribute to the effective osmotic pressure of the fixative solution the ultrastructure of noncultured and in vitro-cultured day 1, 3 and 4 rabbit embryos was evaluated. Total osmolarity of the fixative solution (200-800 mosm) was only varied by changing the aldehyde concentration, whereas the vehicle osmolarity (145 mosm) remained unchanged. Optimum preservation in all embryonic stages was obtained when total osmolarity of the fixative solution was 285-340 mosm. Higher (480-800 mosm) or lower (250 mosm) osmolarities of the fixative solution led to alterations mainly in mitochondria and smooth-surfaced endoplasmic reticulum. Shrinkage of cells and condensation of the cytoplasm occurred only occasionally. Compared with early cleavage stage embryos blastocysts were generally more susceptible to hyperton and hypoton fixative solutions. In vitro culture for 24 h per se did not have any influence on the fixation.

Nerve regeneration in the dorsal funiculus of the rat spinal cord: A light and electron microscopic study.

In an attempt to identify regenerating axons in the central nervous system, a partial transection of the dorsal funiculus in the rat spinal cord was carried out with a pair of microdissection scissors, and a nylon thread loop was inserted into the lesion to demarcate the severed tissue. Nerve regeneration through the demarcated lesion was observed 4-20 days after the operation by light and electron microscopy. In the early stage, many naked axons appeared from the caudal part of the lesion, and some of these further extended into the demarcated space. They contained an accumulation of mitochondria, smooth-surfaced endoplasmic reticulum and vesicles in the axoplasm; this axoplasmic feature indicated that they were regenerating axons. They gradually increased in number, and took highly irregular courses exhibiting various fluctuations in diameter throughout their lengths. Immature Schwann cells as well as glial cells including oligodendrocytes and astrocytes appeared in close association with these regenerating axons. Oligodendrocytes eventually formed thin myelin sheaths. On the other hand, naked axons were present deflecting outside the thread loop; they showed no axoplasmic characteristics as described above. These axons could be regarded as uninjured ones merely undergoing demyelination due to the surgery. Thus, regenerating axons were clearly distinguished from merely demyelinated ones, and some of them were shown to grow through the traumatic lesion in the dorsal funiculus of the rat spinal cord.

Relationship between zonal distribution of microsomal cytochrome P-450s (P-450(17)alpha,lyase and P-450C21) and steroidogenic activities in guinea-pig adrenal cortex.

In an attempt to elucidate the functional role of the three zones of the adrenal cortex, the localization of microsomal cytochrome P-450s (P450(17)alpha,lyase and P-450C21) and their relation to steroidogenesis in the guinea-pig adrenal cortex were studied. The intracellular localization and zonal distribution of P-450(17)alpha,lyase and P-450C21 were examined by the direct peroxidase-labelled antibody technique. The cytochromes were localized immunocytochemically on the smooth-surfaced endoplasmic reticulum. P-450(17)alpha,lyase was found to be distributed in the zona fasciculata, especially in the externa, and less in the zona reticularis. The zona glomerulosa was negative for immunohistochemical staining for P-450(17)alpha,lyase. In contrast, P-450C21 was distributed in all three zones in the adrenal cortex. The cytochrome P-450 contents and their steroidogenic activities were determined with microsomes prepared from the outer zone (zonae glomerulosa and fasciculata) and from the inner zone (zona reticularis) of guinea-pig adrenals. Using an enzyme-linked immunosorbent assay, the ratio of P-450C21 content in the inner zone to that in the outer zone microsomes was estimated to be 1.24. 21-Hydroxylase activity was higher in the inner zone microsomes, which was in good agreement with the relative P-450C21 contents. The ratio of P-450(17)alpha,lyase content in the inner zone to that in the outer zone microsomes was estimated to be 0.75. 17 alpha-Hydroxylase and C17-20-lyase activities were five- to sixfold greater in the outer than in the inner zone microsomes, which could not be explained by the P-450(17)alpha,lyase contents in the two zones.(ABSTRACT TRUNCATED AT 250 WORDS)

THE SEQUENTIAL CHANGE OF A NAPHTHALENE-INDUCED BRONCHIOLAR DAMAGE AND OF PULMONARY CYTOCHROME P-450 IN MICE

Male ICR mice were exposed to 2 mmol/Kg body weight of naphthalene by single intraperitoneal injection. Treated mice were killed consecutively from 2 hours to 14 days after treatment. Tissues were examined for alterations of the bronchiolar epithelial cells by light and electron microscopes, and for amount of pulmonary cytochrome P-450 by ascorbate PES method. Morphologically, naphthalene caused a remarkable increase in smooth-surfaced endoplasmic reticulum of the Clara cells within 4 hours after treatment and the subsequent lesions including the exfoliated ciliated cells. Foamy cell-like Clara cells were observed in the bronchiolar lumen from 6 to 24 hours followed by a pronounced increase in number of flat ciliated cells covering the denuded surface of the bronchiolar tree 48 hours after treatment. These lesions wer almost completely recovered 7 days after treatment. The amount of pulmonary cytochrome P-450 increased promptly and reached up to the maximum level 4 hours after treatment, paralleling with the remarkable increase in smooth surfaced endoplasmic reticulum of the Clara cells. Thereafter, cytochrome P-450 showed a decrease in amount simultaneously with degeneration and desquamation of the Clara cells and reduced by 40% of control amount 24 hours after treatment. There results seem to show that the activity of cytochrome P-450 is closely related with smooth surfaced endoplasmic reticulum of the Clara cells, and naphthalene-induced pulmonary damage is mediated by cytochrome P-450.

Multiple Bilateral Apocrine Cystadenomas of the Lower Eyelids: Light and Electron Microscopic Studies

An 88-year-old man presented with 13 apocrine cystadenomas of both lower eyelids of at least 10 years' duration. The cysts were remarkable for possessing a superior dome of either creamy or yellowish material (probably reflecting the lipid-rich decapitation secretion), surmounting a clearer fluid inferiorly, as well as for being restricted to the eyelid margins where the glands of Moll are located. Light microscopy disclosed a double-layered cuboidal cellular lining, which focally became more plump or hobnailed, exhibited apical decapitation secretion, and was occasionally thrown into papillary folds. Electron microscopy demonstrated cellular debris and segments of interrupted plasmalemmas within the cystic cavity, intercellular widenings constituting canaliculi, cytoplasmic election-dense secretory granules, flocculent mucous vacuoles, and swirls of smooth-surfaced endoplasmic reticulum. Basal cells were focally interspersed between the secretory cells and the basement membrane, and occasionally manifested spotty myofilamentary differentiation. These findings are supportive of an origin of the lesions from the secretory rather than the ductal portion of the glands of Moll. They corroborate conclusions drawn from dermatologic investigations that the cysts are an essentially proliferative process as opposed to the result of passive ductal dilatation.

Progesterone and relaxin secretion in relation to the ultrastructure of human luteal cells in culture: Effects of human chorionic gonadotropin

This report describes the first study to correlate the ultrastructure of long-term monolayer cultures of human luteal cells with their secretion of relaxin and progesterone under basal and human chorionic gonadotropin-stimulated conditions. In culture from 14 to 28 days, cells from both corpora lutea of the menstrual cycle and corpora lutea of pregnancy took on characteristics of granulosa luteal cells, particularly after exposure to human chorionic gonadotropin. Relaxin was detectable in the luteal cell cultures only at early time points. The effect of human chorionic gonadotropin on media relaxin levels differed in cells of the cycle and cells of pregnancy. In the cells of the cycle, relaxin was detectable only on day 2 and was decreased by human chorionic gonadotropin (p < 0.01). In cells of pregnancy, relaxin was detectable for the first 4 days of culture and was not affected by human chorionic gonadotropin. Progesterone was detectable in all the luteal cell cultures and was enhanced by human chorionic gonadotropin (10 and 50 IU/ml) after 24 days of exposure. At earlier time points in both the luteal cells of the cycle and the luteal cells of pregnancy, the human chorionic gonadotropin-induced increases in progesterone levels were not as consistent. However, in all cases of progesterone enhancement, smooth-surfaced endoplasmic reticulum was increased in the human chorionic gonadotropin-treated cells compared with corresponding controls, consistent with more active steroid production. In addition, gap junctions, considered to be responsive to trophic hormones, were increased in the treated cells. In conclusion, this long-term monolayer culture of human luteal cells, as monitored by ultrastructural and hormonal changes, retained the differentiated function of progesterone secretion and exhibited responsiveness to human chorionic gonadotropin. Therefore, morphologic and functional aspects of progesterone secretion may be investigated more closely with use of this long-term luteal cell culture system.