Cytoplasmic Volume Densities Research Articles

Time-dependent effects of maternal continuous propranolol on fetal lung development in rats.

Previous studies have demonstrated a role for the beta-adrenergic system in the maturation of the fetal alveolar epithelium. Chronic blockade of beta-adrenergic binding sites has been shown to adversely effect physiologic and biochemical indices of fetal lung maturation. In the present study timed-pregnant female Sprague-Dawley rats were treated with a continuous 0.5 mg/hr dose of propranolol HCl, or saline, via an osmotic pump. The treatment periods were days 18-21, or 20-23 of gestation. Fetal body weights were obtained, and the morphology of the fetal lungs studied by light and electron microscopy. Cytoplasmic volume densities of lamellar inclusion bodies and glycogen within developing type II alveolar epithelial cells were also determined. In addition, total phospholipids (as phosphorus) and glycogen content were determined biochemically. The fetuses from females treated from day 20-23 demonstrated no differences between saline-treated and propranolol-treated groups, in either fetal weight or the morphologic appearance of the developing lung. In contrast, the fetuses from mothers treated from day 18-21 with propranolol were significantly smaller, and their lungs appeared less mature than saline-treated counterparts. The glycogen content of developing type II alveolar epithelial cells was significantly more abundant (as judged by stereologic and biochemical analyses) in the propranolol-treated fetuses. In addition, total phospholipids were decreased in the propranolol-treated 21-day fetuses. The results of the present study suggest that the development of the alveolar epithelium is sensitive to continuous beta-adrenergic blockade by propranolol during a critical time late in gestation.

Short-term morphologic and functional effects of bromocriptine on pituitary prolactin cell adenomas in vitro.

Bromocriptine, a dopamine agonist, rapidly reduces profactin (PRL) blood levels in vivo; it also causes a marked reduction in tumor size in patients with lactotroph adenomas within 2 weeks of initiation of therapy. The effects are direct, as shown in vitro, and are thought to be due to inhibition of gene transcription that causes alteration in tumor cell morphology. However, the morphologic changes that account for a functional response within hours of administration have not been elucidated. We report the functional and morphologic changes in 7 pituitary lactotroph adenomas studied in vitro after 2, 24, and 48 hours of incubation with bromocriptine. In 2-hour incubations, PRL release rapidly fell to 46% to 74% of control; morphologic analysis revealed an increase in the cytoplasmic volume density (CVD) of secretory granules. After 24 hours of treatment, PRL release was maximally suppressed at 18% to 32 % of control; at 24 hours, the CVDs of secretory granules and lysosomes were increased, and they remained higher than in control cells at 48 hours. The CVD of rough endoplasmic reticulum was reduced at 2 hours in some tumors, but that of Golgi complexes was reduced only at 48 hours. Cell size was altered only to slightly below 80% of control and only after 48 hours of exposure in 2 or 3 tumors. This study indicates that inhibition of PRL secretion occurs within 2 hours of exposure to bromocriptine; the initial morphologic correlates are an increase in the CVD of secretory granules, suggesting inhibition of hormone release, and a decrease in rough endoplasmic reticulum, suggesting reduction of hormone synthesis. Lysosomal accumulation occurs within 24 hours, most likely reflecting degradation of the stored hormone. Subsequently, Golgi complexes involute as the next step in reduced hormone synthesis occurs, and cell shrinkage occurs relatively later. These findings correlate well with the rapid reduction in PRL blood levels and the slower tumor shrinkage documented clinically in patients with PRL cell adenomas.

Reverse Hemolytic Plaque Assay: Electron Microscopic Observation of Plaque-Forming Single Adeno Cells in GH Producing Adenomas

In this study, 3 GH-producing adenomas were studied as materials to clarify the usefulness of electron microscopic investigation of plaque-forming single adenoma cells, and ultrastructural morphometric comparisons were made to determine whether some differences exist at the cytoplasmic organelle level between small plaque-forming cells and large plaque-forming cells. In two cases, the relative cytoplasmic volume density of Golgi apparatus and lysosomes were significantly greater and the diameter of seretory granules were significantly smaller in large plaque-forming cells compared to small plaque-forming cells. Moreover, in one of them, not only the diameter but also the cytoplasmic volume density of secretory granules were significantly smaller in large plaque-forming cells. These findings suggest a greater secretory granule synthesis and internal granule destruction, and faster granules secretion at an early stage of formation in large plaque-forming cells compared with small plaque-forming cells. It became apparent through this study that electron microscopic investigation of plaque-forming single cells is practically useful since plasma membrane and cytoplasmic organelles were preserved well after processing for EM in most plaque-forming cells.

Effects of somatostatin on somatotroph adenomas of the human pituitary: An in vitro functional and morphological study.

The effects of somatostatin or the somatostatin analog SMS 201-995 were studied on 4 densely granulated somatotroph adenomas and 4 sparsely granulated somatotroph adenomas in vitro. Release of growth hormone (GH) into culture media during incubation with somatostatin or SMS 201 -995 were measured by radioimmunoassay, and light-microscopical and ultrastructural morphometric parameters were compared with those of cultured control somatotroph adenoma cells of the same tumor. In all tumors except for 1 densely granulated somatotroph adenoma, somatostatin or SMS 201-995 decreased GH release into culture media in 24- and 2-hour incubations. After 48-hour incubation with somatostatin or SMS 201-995, there was no change in cell size or secretory granule diameter. One densely granulated adenoma showed decreased cytoplasmic volume density (CVD) of Golgi apparatus and secretory granules, and a sparsely granulated adenoma had reduced CVD of endoplasmic reticulum. All the tumors that responded with decreased GH release exhibited increased CVD of lysosomes after incubation with somatostatin or SMS 201-995. These results indicate that both densely and sparsely granulated somatotroph adenomas respond to somatostatin inhibition and, furthermore, that inhibition of hormone release is associated with accumulation of lysosomes, suggesting lysosomal degradation of stored hormone.

Human Pituitary Corticotroph Adenomas in vitro: Morphologic and Functional Responses to Corticotropin-Releasing Hormone and Cortisol

We examined the direct effects of corticotropin-releasing hormone (CRH) and cortisol on the morphology of cells from 6 functioning human pituitary corticotroph adenomas in culture using both light and electron microscopic morphometry and correlated the structural changes with alterations in adrenocorticotropin (ACTH) release in each case. During incubations lasting 2 or 24 h, ACTH release was increased by CRH and reduced by cortisol. After incubations lasting from 2 to 72 h, light microscopic morphometric analysis showed no significant differences in cell size, nuclear area, cytoplasmic area or nuclear/cytoplasmic ratio between treated and control adenoma cells. Ultrastructural morphometry documented increased cytoplasmic volume density (CVD) of rough endoplasmic reticulum and/or Golgi apparatus and reduced CVD of secretory granules in cells incubated with CRH. There was no consistent change in CVD of endoplasmic reticulum, Golgi apparatus or secretory granules in adenoma cells incubated with cortisol, but in all tumors there were marked filament accumulations indicating a direct effect of cortisol on adenomatous corticotrophs. The changes were similar after 2- and 72-hour exposures. These results indicate that (1) some adenomatous corticotrophs can respond to CRH and cortisol; (2) the morphologic changes observed in cells treated with CRH correlate with increased ACTH release, and (3) accumulation of filaments is the direct effect of cortisol and is associated with reduced ACTH release.

Correlation study of serum prolactin levels, pituitary weights, number and ultrastructure of pituitary lactotrophs in female long-evans rats

Twenty-five female, 23-month-old Long-Evans rats were sacrificed for investigation of correlations between serum prolactin levels, pituitary weights, numbers and ultrastructural features of pituitary lactotrophs. Serum prolactin levels measured by radioimmunoassay varied from 7.1 ng/ml to 1,534.0 ng/ml. Pituitary weights ranged from 16.0 mg to 255.0 mg. Serum prolactin levels were increased when pituitary weights were high; statistical analysis revealed a correlation between the two. No significant correlation was evident between serum prolactin levels and lactotroph percentage and ultrastructure. Light microscopy revealed a large number of lactotrophs in almost every pituitary. Values for nuclear, cell and cytoplasmic areas, nuclear/cytoplasmic ratios and nucleolar diameters remained similar in the lactotrophs of all animals. Electron microscopy disclosed no lactotroph damage or vascular injury. Ultrastructural morphometry of lactotrophs in the pituitaries studied showed no major differences in cytoplasmic volume densities of endoplasmic reticulum, Golgi complex, mitochondria, secretory granules and lysosomes as well as secretory granule diameters. It is reasonable to conclude that elevations of serum prolactin levels are due to an increase in pituitary mass, resulting from accumulation of lactotrophs. Thus hyperprolactinemia appears to be secondary to an increase in the number of lactotrophs and not hyperactivity of individual cells.

Effects of growth hormone-releasing hormone (GHRH) on densely granulated somatotroph adenomas and sparsely granulated somatotroph adenomas in vitro: a morphological and functional investigation

The effects of growth hormone-releasing hormone (GHRH) were studied on densely granulated somatotroph adenoma cells and sparsely granulated somatotroph adenoma cells in culture by measuring release of growth hormone (GH) as well as ultrastructural morphometrical parameters and comparing them with those of control adenoma cells. Both types of adenoma cells cultured with GHRH showed similar increases of GH release into culture media and exhibited similar increases in cytoplasmic volume densities (CVD) of endoplasmic reticulum and Golgi apparatus and decreases in CVD of secretory granules and secretory granule diameter. These results indicate that (1) both types of somatotroph adenoma cells react similarly to GHRH stimulation, despite their morphologic differences, and (2) GHRH stimulates GH synthesis as well as GH release by somatotroph adenoma cells.

Morphometric quantification of mitochondria in the two steroidogenic ovine luteal cell types.

Progesterone secretion is regulated by different mechanisms in large and small steroidogenic ovine luteal cells. Large cells secrete approximately 7-fold more progesterone in an unstimulated state than small cells. Since cholesterol side-chain cleavage, which is catalyzed by an inner mitochondrial membrane enzyme complex, is a major rate-limiting step in progesterone synthesis, mitochondrial components were quantified in the two steroidogenic cell types throughout the estrous cycle. Corpora lutea collected on Days 4 (n = 4), 8 (n = 4), 12 (n = 5), and 16 (n = 6) of the estrous cycle were prepared for electron microscopy. Volume densities of cell types within corpora lutea and mitochondrial densities within cell types were estimated by point-counting; nuclear and cytoplasmic volume densities were estimated by planimetric analysis. A total of 570 micrographs (magnification 5300 X) were analyzed. Large cell volume density was unchanged during the cycle (35 +/- 1%) while small cell volume density increased (p less than 0.05) from 13 +/- 1% on Day 4 to 20 +/- 3% on Day 12. Large cell mitochondrial volume density increased (p less than 0.05) from 13 +/- 1% on Day 4 to 23 +/- 1% on Day 16 accompanied by an increase in cytoplasmic volume density such that nuclear to cytoplasmic ratio increased (p less than 0.05) from 1:14 to 1:34 between Days 4 and 16. Small cell mitochondrial volume density increased from 11 +/- 1% on Day 4 to 14 +/- 1% (p less than 0.05) for the rest of the cycle while the nuclear to cytoplasmic ratio remained at 1:14.(ABSTRACT TRUNCATED AT 250 WORDS)

Volume fraction and ultrastructure of age pigment in the saccular epithelium of old mice

Age pigment in the sensory and supporting cells was a prominent characteristic distinguishing old saccules from young. However, the pigment was not distributed uniformly throughout the sensory epithelium but displayed cell-specific patterns of accumulation. The highest cytoplasmic volume density was in the old supporting cells followed by type I hair cells and then type II hair cells. The most common form of pigmented inclusion seen in old type I hair cells was a cluster of granules resembling melanin. This form was never seen in type II hair cells or supporting cells where a form containing a lipid-like droplet was prevalent. The differences in the amount of age pigment and the forms accumulated probably reflects metabolic differences between the three cell types.

In vitro studies of human pituitary adenomas

The application of morphologic and tissue culture techniques to the study of human pituitary adenomas allows further investigation of structure-function correlations. Using these methods, we have documented morphometric differences between densely and sparsely granulated somatotroph adenomas but the release of growth hormone in vitro and responses to adenohypophysial hormones/drugs do not correlate with tumor type. The morphologic and functional alterations in somatotroph adenomas exposed to SMS 201-995 appear to be reversible in vitro. Incubation of lactotroph adenomas with bromocriptine for 3 days directly reduces tumor cell size, cytoplasmic volume and cytoplasmic volume densities of endoplasmic reticulum and Golgi regions; these changes are similar to the effects of longterm bromocriptine therapy in vivo. Tissue culture studies of gonadotroph adenomas of men confirm that gonadotropin-releasing hormone (GnRH) stimulates gonadotropin release by tumor cells and yields morphologic evidence of increased hormone synthesis whereas these tumors have variable sensitivity to gonadal steroids; structural changes in tumor cells correlate with hormone release after stimulation, suggesting that morphologic parameters may reflect the hormonal milieu of these adenomas. Null cell adenomas and oncocytomas release small quantities of glycoprotein hormones, predominantly gonadotropins in vitro and there are no functional differences between these 2 tumor types; gonadotropin release responds to GnRH stimulation and, paradoxically, to other adenohypophysiotropic hormones, but such stimulation does not result in secretion of other adenohypophysial hormones by these tumors.

Effect of bromocriptine on secretion and morphology of human prolactin cell adenomas in vitro.

The direct effect of short-term bromocriptine administration on human prolactin cell adenomas in vitro was studied by light and electron microscopy and correlated with the effect on hormone release. The light microscopic and, for the first time, ultrastructural characteristics of adenomatous prolactin cells in control and treated cultures were analyzed morphometrically and the changes induced by bromocriptine were quantified. Following 72 h treatment which lowered levels of prolactin release, there was a marked reduction in cell size, cytoplasmic volume and cytoplasmic volume densities of endoplasmic reticulum and Golgi apparatus. In contrast, the percentage of cytoplasm occupied by lysosomes increased. There was no consistent change in number and diameter of secretory granules. These findings indicate that prolactin cell involution is a direct effect of bromocriptine and suggest that reduction in endoplasmic reticulum and Golgi complex is largely responsible for the decreased cell size. Lysosomal degradation of cytoplasmic components may play a role in this process.

Stereological analysis of coated pits and secondary lysosomes in cultured human glial and glioma cells.

The frequency, distribution and size of coated pits along the upper and lower cell surfaces, and the cytoplasmic volume density of secondary lysosomes were studied by ultrastructural stereological methods in sparse and post-confluent cell cultures of a human normal glial line and a human malignant glioma line. Neither the frequency of coated pits nor the volume density of secondary lysosomes showed any statistically significant changes between the normal glial cells at 2 and 14 days after subcultivation. The numerical surface density of the coated pits was significantly higher along the lower cell surface than on the upper free surface. The coated pits on the malignant glioma cells at 14 days were far more frequent than at 2 days after subcultivation, or than on the sparse and post-confluent glial cells. The surface density of the coated pits was higher along the upper free cell surface than on the lower surface of the glioma cells, in contrast to the glial cells. The volume density of lysosomes was significantly higher in the post-confluent glioma cells than in the sparse cells. The mean diameter of the coated pit openings on cells at 2 days after subcultivation was significantly larger than at 14 days, both in the glial and glioma cell cultures. The observed differences in frequency and distribution of coated pits between the glial and glioma cells may reflect differences in receptor-mediated endocytosis and be related to the different growth characteristics of the normal and malignant cells. The greatly increased load of secondary lysosomes in post-confluent cultures of the glioma cells, in contrast to the glial cells, may be due to a higher rate of autophagocytosis in the malignant cells. The larger size of the coated pit openings on isolated than on post-confluent cells may reflect differences in surface properties between sparse and dense cells.

Muscle ultrastructural characteristics of elite powerlifters and bodybuilders

Muscle ultrastructure of a group of subjects possessing extreme hypertrophy was compared with that of a control group which had undergone 6 months of heavy resistance training. Two needle biopsies were taken from triceps brachii of two international calibre powerlifters and five elite bodybuilders. In addition, samples were taken from five healthy volunteers before and after 6 months of training of the elbow extensors. One biopsy was prepared for electron microscopy and analyzed stereologically, and the other was stained for myosin ATPase activity and photographed under the light microscope. Despite large differences in elbow extension strength and arm girth there was no significant difference in fibre areas or percentages of fibre types between the elite group and the trained controls. This suggests that the elite group possessed a greater total number of muscle fibres than the controls did. Mitochondrial volume density of the elite group was similar to that of the control group following training but significantly less (p less than 0.05) than the pretraining control measurements. Myofibrillar volume density was significantly lower and cytoplasmic volume density significantly higher in the elite group than in the trained controls. There was a considerably higher incidence of structural abnormalities including central nuclei and atrophied fibres in the elite group than in the control group, which might possibly have been associated with the use of anabolic steroids by the elite group.