Nexuses between the smooth muscle cells of the guinea-pig ileum

The circular musculature of the guinea-pig ileum has been studied by freeze-fracture to analyze quantitatively the gap junctions (nexuses) between its smooth muscle cells. The average cell surface area and cell volume are 5,074 micron 2 and 3,260 micron 3. The packing density of nexuses is 48/1,000 micron 2 of cell surface or approximately 244/muscle cell. Nexuses range in area from less than 0.1 to approximately 1.5 micron 2 and they occupy 0.212% of the cell surface. The average packing density of intramembrane particles or pits in nexuses is approximately 7,200/micron 2 of nexal surface, indicating that there may be approximately 77,000 intercellular channels in the full complement of nexuses of one muscle cell.     

Limitation of substratum size alters cytoskeletal organization and behaviour of Swiss 3T3 fibroblasts

Cell spreading and adhesion formation in Swiss 3T3 cells was studied on circular adhesive islands of size 400-500 microns 2 made by evaporating palladium through a mask onto an underlying non-adhesive surface. Cell spreading was limited since focal contacts were restricted to the palladium. On islands less than 2000 microns 2, focal contacts and actin bundles were arranged at the cell periphery. On islands less than 1000 microns 2, the size and number of focal contacts were reduced. Focal contacts may be important regulators of proliferation, but they do not seem to form a deterministic link between substratum contact and proliferative stimulus.     

The ultrastructure of the cardiac Purkinje strand in the dog: a morphometric analysis

Purkinje strands from both ventricles of adult mongrel dogs were excised, and electrical properties were studied by the voltage-clamp technique. The strands were then examined with light and electron microscopy and structural properties were analysed by morphometric techniques. The canine Purkinje strand contains (by volume) about 28% myocyte and 55% dense outer connective tissue. The remainder of the volume is taken up by the inner shell of loosely packed connective tissue within 10 microns of a myocyte membrane. These volume fractions vary considerably from one strand to another. Clefts less than 10 microns wide occupy 18% of the myocyte volume and clefts less than 1 micron wide occupy 1%. The membrane surface area of the myocytes can be divided into three categories by reference to the size of the adjacent cleft. About 47.8% of the membrane surface area faces clefts wider than 1 micron, another 22.2% faces clefts between 0.1 and 1 micron wide, and the final 30% faces clefts less than 0.1 micron wide. The surface area facing the narrowest clefts (less than 0.1 micron wide) is divided between nexuses 3%, desmosomes 10%, and unspecialized membrane 17% (each figure is expressed as a percentage of the total surface area of myocyte membrane). The canine Purkinje strand has a more favourable anatomy than the sheep Purkinje strand for most physiological experiments. We expect that the complicating effects of series resistance and change in the concentration of extracellular ions will be much smaller than in sheep strands, but still not negligible.     

Size distribution and hormonal responsiveness of dispersed rabbit luteal cells during pseudopregnancy

Due to the evidence for two distinct steroidogenic cell types in corpora lutea of large domestic animals, cells of the rabbit corpus luteum were characterized with respect to cell diameters, relative abundance, steroidogenic capacity and responsiveness to hormones. Pseudopregnancy was induced in New Zealand rabbits by injection of 30-160 IU pregnant mare's serum gonadotropin (PMSG) followed in 2-4 days by an i.m. injection of 20-35 micrograms gonadotropin-releasing hormone (GnRH). Corpora lutea were obtained 2, 5 and 9 days after injection of GnRH and dissociated into single cell suspensions. Suspended steroidogenic cells were incubated (2 h, 37 degrees C) in medium 199 alone or in medium containing ovine luteinizing hormone (oLH) (100 ng/ml), or isoproterenol (100 microM). Media were collected and assayed for progesterone content. Secretion of progesterone (means +/- SE, n = 4) was stimulated (p less than 0.05) by oLH on each day: Day 2 = 1.7 +/- 0.2-fold; Day 5 = 3.5 +/- 0.4-fold; and Day 9 = 3.1 +/- 0.6-fold stimulation above controls. Isoproterenol also stimulated (p less than 0.05) secretion of progesterone by suspended luteal cells on Days 2 and 9. Microscopic examination of cell suspensions stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity provided identification of cells with steroidogenic capacity. The diameters (means +/- SE) for steroidogenic cells increased (p less than 0.05) from Days 2 to 9 (Day 2 = 15.2 +/- 0.2 micron; Day 5 = 22.4 +/- 0.4 micron; Day 9 = 28.3 +/- 1.6 micron). The large cell to small cell ratio increased from 0.01 on Day 2 to 2.03 on Day 9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphometric analysis of Leydig cells in the normal rat testis

Leydig cells are thought to be the source of most, if not all, the testosterone produced by the testis. The goal of this study was to obtain quantitative information about rat Leydig cells and their organelles that might be correlated with pertinent physiological and biochemical data available either now or in the future. Morphometric analysis of Leydig cells in mature normal rats was carried out on tissue fixed by perfusion with buffered glutaraldehyde, and embedded in glycol methacrylate for light microscopy and in Epon for electron microscopy. In a whole testis, 82.4% of the volume was occupied by seminiferous tubules, 15.7% by the interstitial tissue, and 1.9% by the capsule. Leydig cells constituted 2.7% of testicular volume. Each cubic centimeter (contained approximatelyy 1 g) of rat testis contained about 22 million Leydig cells. An average Leydig cell had a volume of 1,210 micron3 and its plasma membrane had a surface area of 1,520 micron2. The smooth endoplasmic reticulum (SER), the most prominent organelle in Leydig cells and a major site of steroidogenic enzymes, had a surface area of approximately 10,500 micron2/cell, which is 6.9 times that of the plasma membrane and is 60% of the total membrane area of the cell. The total surface area of Leydig SER per cubic centimeter of testis tissue is approximately 2,300 cm2 or 0.23 m2. There were 3.0 mg of Leydig mitochondria in 1 g of testis tissue. The average Leydig cell contained approximately 622 mitochondria, measuring on the average 0.35 micron in diameter and 2.40 micron in length. The mitochondrial inner membrane (including cristae), another important site of steroidogenic enzymes, had a surface area of 2,920 micron2/cell, which is 1.9 times that of the plasma membrane. There were 644 cm2 of inner mitochondrial membrane/cm3 of testis tissue. These morphometric results can be correlated with published data on the rate of testosterone secretion to show that an average Leydig cell secretes approximately 0.44 pg of testosterone/d or 10,600 molecules of testosterone/s. The rate of testosterone production by each square centimeter of SER is 4.2 ng/d or 101 million molecules/s: the corresponding rate for each square centimeter of mitochondrial inner membrane is 15 ng testosterone/d or 362 million molecules/s.     

Very small fat cells. II. Initial observations on basal and hormone-stimulated metabolism

Very small fat cells (less than 35 micron diameter) and normal large fat cells (greater than 40 micron diameter) were isolated from adult Fischer 344 rat epididymal adipose depots. These very small fat cell preparations were free from normal, large fat cells (40-130 micron diameter) and stromal-vascular elements. Examination by electron microscopy and lipid analysis showed a similarity in overall organization and composition to normal, large fat cells. Incubations with [U-14C]glucose showed that the very small fat cell preparations oxidized glucose in proportion to both cell number and time. These preparations also responded to insulin, increasing [U-14C]glucose oxidation in a manner similar to normal large fat cell preparations (i.e., 2- to 4-fold increases in CO2 production with insulin stimulation). The very small fat cells also incorporated radiolabeled glucose into lipids; but, unlike normal large fat cells, insulin failed to stimulate this process. Glycerol release from very small fat cells was stimulated by lipolytic hormones in a manner similar to these responses in co-isolated large fat cells.     

Temperature effects on osmotic fragility, and the erythrocyte membrane

Results are reported on the temperature-dependence of intact-cell surface area, isotonic volume, hemolytic volume, and ghost steady-state surface area and volume, using several techniques of resistive pulse spectroscopy. Temperature was found not to alter the intact cell surface area permanently: the area remains constant at 130 +/- 1 micron 2, at temperatures ranging from 0 to 40 degrees C. Temperature does alter the steady-state volume of the cells, with a colder temperature inducing swelling by about 0.29 micron 3/deg. C. Such a temperature-induced volume change is sufficient to explain only approximately half of the fragility differences which result from temperature changes. The remainder was found to result from higher temperatures enabling a substantial transient increase in surface area of intact cells (up to at least 14% of 40 degrees C), with a corresponding increase in the cell's hemolytic volume (up to 21%). The hemolytic volume apparently increases linearly with temperature, since steady-state ghost volumes are found to increase linearly with the temperature at which the ghosts were produced. In the steady state (at high temperature), the membranes of electrically-impermeable resealed ghosts can remain extended by more than 10%, compared with membranes of the corresponding unhemolyzed, intact red cells.     

Electrical coupling and uncoupling of exocrine acinar cells

The electrical communication network in the mouse pancreatic acinar tissue has been investigated using simultaneous intracellular recording with two separate microelectrodes and direct microscopical control of the localizations of the microelectrode tips. All cells within one acinus were electrically coupled, and the coupling coefficient (the electrotonic potential change in a cell neighboring to the cell into which current is injected [V2] divided by the electrotonic potential change in the cell of current injection [V1]) between two cells near each other (less than 50 micron) was always close to 1. Cells farther apart (50-100 micron) were, in some cases, coupled; in other cases, there was no coupling at all. Coupling coefficients varied between 0 and 1. There was rarely electrical coupling over distances of more than 110 micron. Using microiontophoretic acetylcholine (ACh) application, it was possible to evoke almost complete electrical uncoupling of two previously coupled pancreatic or lacrimal acinar cells from different acini or within one acinus. The effects were fully and quickly reversible. While the ACh-evoked uncoupling in the pancreas was associated with membrane depolarization, ACh caused hyperpolarization in the lacrimal acinar cells. The uncoupling was associated with a very marked reduction in electrical time constant, indicating a reduction in input capacitance (effective surface cell membrane area). The concentrations of stimulants needed to evoke reduction in pancreatic cell-to-cell coupling were 1 micron for ACh, 0.14 nM for caerulein, and 3 nM for bombesin. These concentrations are smaller than those required to evoke maximal enzyme secretion.     

Regeneration of changes produced in rabbit tracheal epithelium by saline lavage of the airways

The authors studied the course of the repair of changes induced in the rabbit tracheal epithelium by saline lavage of the airways. The tracheal epithelium was examined 2, 24, 48 and 72 hours after treatment. Saline lavage stimulated the goblet cells to instantaneous discharge of their secretion. 2 hours after treatment 98 +/- 3% of the goblet cells were completely exhausted and had degenerated. Repair of the changes began 24 hours after lavage and was associated with massive differentiation of new goblet cells resulting in hyperplasia of the mucus-secreting elements with formation of endoepithelial mucous glands. The most pronounced injury to the ciliated cells was apparent 2 hours after lavage, then the degree of alteration of these cells gradually decreased. Saline lavage markedly impaired the ciliary border. The mean number of kinocilia per micron2 fell to 1.5 +/- 0.3. In subsequent phases the number of kinocilia rose gradually to 7.5 +/- 0.5/micron2. This value was still significantly lower (P less than 0.005) compared with controls. The first signs of impairment of the self-cleaning ability of the epithelium were recorded 2 hours after lavage. The most pronounced disturbances of the mucus flow were observed after 24 hours. At the end of the experimental period small clumps of condensed mucus and rather numerous bacteria were still present in the area of the ciliary border.     

The insulin-stimulated cell surface presentation of low density lipoprotein receptor-related protein in 3T3-L1 adipocytes is sensitive to phosphatidylinositide 3-kinase inhibition

The regulation of low density lipoprotein receptor-related protein (LRP) activity by insulin was studied using 3T3-L1 adipocytes. The LRP mRNA and protein expression were independent of differentiation state of the cells and of insulin treatment. In differentiated cells, insulin treatment acutely stimulated the cell surface presentation of LRP (approximately 2-fold) as evidenced by methylamine-activated alpha(2)-macroglobulin binding and by biotinylation of cell surface LRP. The increased cell surface presentation was accompanied by a 39% decrease in LRP level in the low density microsomes. The magnitude of insulin-stimulated cell surface presentation of LRP was similar to that of transferrin receptor but was much less than that of GLUT4. Both the increases in LRP and GLUT4 cell surface presentation upon insulin treatment were abolished by inhibition of phosphatidylinositide 3-kinase. The increased cell surface presentation of LRP was associated with proportionally increased endocytic activity, and the internalization rate constant (K(e)) was not decreased by insulin treatment. Thus, insulin treatment most likely stimulates recycling of LRP from an endosomal pool to the plasma membrane, which is regulated in a phosphatidylinositide 3-kinase-dependent manner in 3T3-L1 adipocytes.     

Reconstruction of a type-B configuration monkey Sertoli cell: size, shape, and configurational and specialized cell-to-cell relationships

A model of a stage-V monkey Sertoli cell was reconstructed from electron micrographs taken of semiserial sections. The configuration (type-B) was one in which spermatids were positioned near the lumen, their heads occupying shallow cylindrical recesses at the apical portion of the Sertoli cell. The cell volume was calculated to be 4,100 micron3, the surface area 2,400.68 micron2, and the surface-to-volume ratio 0.58:1. The reconstructed cell extended from the basal lamina to the tubular lumen and was generally of the tall columnar type although its surface contour was highly irregular. The dimensions of the cell [centripetal (68.46 micron), circumferential (18.40 micron), and longitudinal (21.63 micron)] were determined and cell surfaces designated. Relative and absolute surface areas of the reconstructed cell which faced other Sertoli cells, germ cells, basal lamina, and tubular lumen were calculated. Junctions and surface specializations were enumerated, catalogued, and depicted on diagrams of the cell surface. Where appropriate, type-A rat and type-B monkey Sertoli cells were compared and discussed. Morphometry was utilized to analyze the relative surface areas of germ cells adjoining the reconstructed cell to determine the percentage of their surface facing cellular and acellular elements, and these data were compared to data obtained for the rat.     

Gliding movements in Myxococcus xanthus.

Prokaryotic gliding motility is described as the movement of a cell on a solid surface in the direction of the cell's long axis, but its mechanics are unknown. To investigate the basis of gliding, movements of individual Myxococcus xanthus cells were monitored by employing a video microscopy method by which displacements as small as 0.03 micron could be detected and speeds as low as 1 micron/min could be resolved. Single cells were observed to glide with speeds varying between 1 and 20 microns/min. We found that speed variation was due to differences in distance between the moving cell and the nearest cell. Cells separated by less than one cell diameter (0.5 micron) moved with an average speed of 5.0 micron/min, whereas cells separated by more than 0.5 micron glided with an average speed of 3.8 microns/min. The power to glide was found to be carried separately at both ends of a cell.     

Endgoenous protein kinase in outer plasma membrane of cultured 3T3 cells. Nature of the membrane-bound substrate and effect of cell density, serum addition, and oncogenic transformation.

Endogenous protein kinase activity was detected in the outer plasma membrane of 373 and SV40 transformed 3T3 cells. When intact cells were incubated with [gamma-32P]ATP, there was a transfer of [32P]phosphate into an acid-insoluble product. The reaction was: (a) linear as a function of time (up to 30 min), (b) proportional to the number of cells present and (c) dependent on temperature and Mg2+ concentration. The acid-insoluble product was susceptible to pronase but not RNase or DNase. More specifically, phosphomonoester bonds to serine and threonine were identified. There was less than 3% hydrolysis of the [gamma-32P]ATP during the reaction; moreover, free [32P]phosphate failed to substitute for the ATP. The reaction product was located on the cell surface, as evidenced by the fact that it could be removed by mild trypsin treatment of intact 3T3 cells. Further evidence for the surface location of the kinase was shown by its activity in phosphorlating exogenous substrate, histone, and phosvitin. The level of phosphorylation increased by 2- to 4-fold prior to the start of S phase when quiescent 3T3 cells were stimulated to reinitiate growth by the addition of serum. The SV40 3T3 cells had from 5- to 10-fold more activity per cell than the quiescent 3T3 cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioautography show at least 25 phosphorylated proteins; the surface label pattern of 3T3 cells differs from that of SV40-transformed 3T3 cells.     

Stimulation of murine T cell subsets with anti-CD3 antibody. Age-related defects in the expression of early activation molecules

Splenocytes from young (3 to 4 mo) and aged (24 to 26 mo) C57BL/6 mice were stimulated with anti-CD3 epsilon mAb in vitro. At the time of peak DNA synthesis (day 2), cells from aged mice incorporated congruent to 60% less [3H]TdR than cells from young mice. This age-related defect was not attributable to gross differences in anti-CD3 does optima, response kinetics, accessory cell function, numbers of T cells cultured, CD4+:CD8+ cell ratios or surface levels of CD3 epsilon molecules. In an attempt to analyze pre-S phase events in these responses, we monitored CD4+ and CD8+ cells in splenocyte cultures for the time-dependent expression of three T cell activation markers: RL388 Ag and IL-2R and transferrin R. Parallel analyses of mean T cell size and cell cycle phase distributions were performed. Non-activated T cells from both age groups similarly expressed moderate levels of RL388 Ag, low levels of transferrin R, and undetectable levels of IL-2R. Analysis of stimulated T cells revealed, in both age groups: 1) detectable increases in expression of all three markers by 6 h of culture, and continued increases associated with blastogenesis and G1 phase transit and 2) a preferential stimulation of the CD8+ subset to a state of high level marker expression. Age group comparisons of activation marker expression over time suggested that the age-related defect reflects proportionally smaller fractions of CD4+ and CD8+ cells that respond normally, rather than a general defect in all T cells or a subset-specific defect. Finally, we found that supernatants from aged donor cell cultures stimulated with anti-CD3 contained less Il-2 than those of young controls. Addition of an IL-2 containing supernatant to aged donor cell cultures increased, but did not restore, the S phase response on day 2; however, the response on day 3 was comparable to the peak (day 2) response of young controls. These data suggest that exogenous IL-2 can improve the aged response, perhaps by expanding the fraction of normally reactive T cells.     

Proliferation and differentiation sequence of osteoblast histogenesis under physiological conditions in rat periodontal ligament

To define the mechanism of osteoblast histogenesis, nuclear morphometry was utilized as a marker for precursor cell differentiation. One hour after 3H-thymidine injection, groups of 7-week-old rats were killed at hourly intervals over one complete 24-hr photoperiod (LD 12:12). S-phase and mitosis were assessed in autoradiographs of 3-micron sections of molar periodontal ligament (PDL) adjacent to a physiological bone-forming surface. Labeled nuclei were divided into four categories according to morphometry of nuclear size: A (40-79 micron3), B (80-119 micron3), C (120-169 micron3), and D (greater than or equal to 170 micron3) cells. C and D cells synthesize DNA during the light and divide in the following dark phase; the rhythm for A cells is the opposite. B cells demonstrated no preference and were subsequently determined to be nonosteogenic. Compared to A cells the S-phase photoperiod of C and D cells (combined) is approximately a one-to-one reciprocal relationship, suggesting two proliferating progenitors in series. Based on arrest points in the histogenesis sequence, five compartments are defined: 1) A cells, less differentiated, self-perpetuating precursors; 2) A' cells, committed osteoprogenitors; 3) C cells, G1 stage preosteoblasts; 4) D cells, G2 stage preosteoblasts; and 5) Ob cells, morphologically distinct osteoblasts. Minimal elapsed time for the A----A'----C----D----Ob sequence is about 60 hr (five alternating dark/light cycles). A stress/strain-mediated increase in nuclear volume (A'----C) is an important, rate-limiting step in osteoblast differentiation.     

Rapid redistribution of clathrin onto macrophage plasma membranes in response to Fc receptor-ligand interaction during frustrated phagocytosis

We have observed increases in assembled clathrin on the plasma membrane during "frustrated phagocytosis," the spreading of macrophages on immobilized immune complexes. Resident macrophages freshly harvested from the peritoneal cavity of mice and attached to bovine serum albumin (BSA)-anti-BSA-coated surfaces at 4 degrees C had almost no clathrin basketworks on their adherent plasma membrane (less than 0.01 coated patch/micron 2), as observed by immunofluorescence, immunoperoxidase, and platinum-carbon replica techniques, although abundant assembled clathrin was observed in the perinuclear Golgi region. When the cells were warmed to 37 degrees C they started to spread by 4 min and reached their maximum extent by 20 min. Spreading preceded clathrin assembly at the plasma membrane. Clathrin-coated patches were first observed on the adherent plasma membrane at 6 min. Between 12 and 20 min assembled clathrin coats appeared on both adherent and nonadherent plasma membranes with a concomitant decrease in identifiable clathrin in the perinuclear region. A new steady state emerged by 2 h, as perinuclear clathrin began to reappear. At 20 min at 37 degrees C the adherent plasma membranes of macrophages spreading on BSA alone had 0.9 coated patch/micron 2, whereas in cells spread on immune complex-coated surfaces, the clathrin patches increased, dependent on ligand concentration, to a maximum of 2.1 coated patches/micron 2. Because frustrated phagocytosis of immune complex-coated surfaces at 37 degrees C increased the area of adherent plasma membrane, the total area coated by clathrin basket-works increased 5-fold (28 micron 2/cell) as compared with cells plated on BSA alone (5.6 micron 2/cell) and 200-fold as compared with cells adhering to immune complexes at 4 degrees C. We then determined that macrophages cultured on BSA-coated coverslips for 24 h already have abundant surface clathrin. When immune complexes were formed by the addition of anti-BSA IgG to already spread macrophages cultured on BSA-coated coverslips for 24 h, clathrin assembled at the sites of ligand-receptor interaction even at 4 degrees C, before spreading, and a 2.6-fold increase in assembled clathrin was observed on the adherent plasma membrane of cells on immune complexes as compared with cells on BSA alone. Clathrin was reversibly redistributed to the Golgi region, returning to the steady state by 2 h.(ABSTRACT TRUNCATED AT 400 WORDS)     

On the mechanism of rapid plasma membrane and chloroplast envelope expansion in Dunaliella salina exposed to hypoosmotic shock

Dunaliella salina cells rapidly diluted from their normal 1.71 M NaCl-containing growth medium into medium containing 0.86 M NaCl swelled within 2--4 min to an average volume 1.76 X larger and a surface area 1.53 X larger than found in control cells. Morphometric analysis of thin section electron micrographs revealed that certain organelles, including the chloroplast, nucleus, and some types of vacuoles, also expanded in surface area as much or more than did the entire cell. It is likely that glycerol, the most important osmotically active intracellular solute, was present in high concentration within these organelles as well as in the cytoplasm itself. Thin section and freeze-fracture electron microscopy were utilized to trace the origin of membrane material whose addition permitted the large increase in plasma membrane surface area and the equally large growth of the chloroplast outer envelope. The findings indicated that the plasma membrane's expansion resulted from its selective fusion with numerous small (less than or equal to 0.25 micron diam) vesicles prevalent throughout the cytoplasm. In contrast, new membrane added to the chloroplast outer envelope was drawn from an entirely different source, namely, elements of the endoplasmic reticulum.     

Patch-clamp measurements reveal multimodal distribution of granule sizes in rat mast cells

Using patch-clamp techniques, we have followed the attributes of the secretory granules of peritoneal mast cells obtained from rats of different ages. The granule attributes were determined by following the step increases in the cell surface membrane area caused by the exocytosis of the granules in GTP gamma S stimulated mast cells. Our data show that the amount of granule membrane available for exocytosis depends exponentially on the weight (age) of the donor rat, reaching a maximum at approximately 300 g. The data are consistent with an exponential growth in the number of granules contained by mast cells of maturing animals. Histograms of the sizes of the step increases in surface area caused by exocytosis of the granules showed at least four equally spaced peaks of similar variance where the position of the first peak and the spacing between peaks averaged 1.3 +/- 0.4 micron2. In all cells recorded, no more than seven peaks could be found, the higher order peaks having a lower probability of occurrence. The distribution of granule sizes did not change measurably between young and adult animals. This study suggests that at least two separate steps may determine the size of a secretory granule: granule to granule fusion that may account for the subunit composition of granule sizes and traffic of microvesicles through the maturing granules that may account for the variance observed in the granule sizes. This study also demonstrates a novel way to study granulo-genesis in living cells.     

Lysosomal interconnections and horseradish peroxidase compartmentalization in three-dimensionally reconstructed bovine alveolar macrophages

Following a 1-h incubation of bovine alveolar macrophages in 1 to 2 mg/ml exogenous horseradish peroxidase (HRP), ultrathin sections revealed vacuolar interconnections among both labeled and unlabeled vacuoles constituting the lysosomal compartment. Four entire cells and their vacuolar components were subsequently computer resconstructed from serial transmission electron micrographs and measured using a morphometric technique. HRP-labeled and unlabeled vacuoles ranged in size from 0.5 micron to greater than or equal to 4.0 microns in diameter and occupied up to 25% of the cytoplasmic volume. HRP-containing vacuoles were distributed throughout each cell in a clumped distribution (P less than 0.05) and occupied up to 75% of the total vacuole compartment. Up to 60% of all vacuoles were interconnected through a series of openings formed by membrane fusions (average pore diameter 0.42 micron), which resulted in a labyrinth of vacuoles comprising up to 55% of the total volume of the lysosomal compartment. The area of open interconnections resulting from vacuolar fusions represented less than 1% of the total surface area of the lysosomal membrane. Rotation of a three-dimensionally reconstructed macrophage about the Y-axis revealed an interconnected vacuolar network of 75 fused vacuoles in a chain up to 21 microns in length. We have demonstrated that HRP-labeled vacuoles interconnect with each other as well as with preexisting unlabeled vacuoles. As a result of such interconnections, individual vacuoles become contributing members of a large, continuous, lysosomal compartment in bovine alveolar macrophages.     

Steroidogenic capacity and ultrastructural morphology of cultured ovine luteal cells

Corpora lutea were surgically collected from superovulated ewes 36 h post-injection of human chorionic gonadotropin (hCG) (Day 2), dissociated (0.2% collagenase), plated, and maintained in culture Days 2-10 in Medium 199 supplemented with 5% calf serum. Accumulation of progesterone in the cultures did not decrease (p greater than 0.05) from Day 3 (17.5 +/- 5.1 nmol/10(6) cells) to Day 10 (4.8 +/- 1.7 nmol/10(6) cells). Calf serum (5%) in the medium supported greater (p less than 0.05) progesterone production than fetal calf serum (5%) or medium without added serum. Steroidogenic cells did not increase (Days 2-10) in numbers, but increased (p less than 0.01) in mean cell diameter (Day 2, 11.7 +/- 0.4 micron; Day 10, 24.5 +/- 1.6 micron). Steroidogenic capacity on Day 10 of cells cultured Days 2-10 (in vitro) was not different (p greater than 0.05) from that of cells collected from the ovary on Day 10 (in vivo); however, steroidogenic cells recovered from plates had greater (p less than 0.01) mean cell diameters (24.5 +/- 1.6 micron, in vitro, compared to 15.2 +/- 1.0 micron, in vivo). Transmission electron microscopy revealed that cultured cells (Days 5, 10) possessed less smooth endoplasmic reticulum but more lipid droplet inclusions, ribosomes, and rough endoplasmic reticulum than cells obtained in situ (Day 10). Electron-dense secretory granules were rarely seen. Although subcellular morphology of ovine luteal cells in culture was altered, these changes did not appear to significantly affect the ability of these cells to produce progesterone.