Ultrastructural studies of human basophils and mast cells.

Ultrastructural studies of human mast cells (HMCs) and basophils (HBs) are reviewed. Sources of HMCs include biopsies of tissue sites and in situ study of excised diseased organs; isolated, partially purified samples from excised organs; and growth-factor-stimulated mast cells that develop de novo in cultures of cord blood cells. Sources of HBs for study include partially purified peripheral blood basophils, basophils in tissue biopsies, and specific growth factor-stimulated basophils arising de novo from cord blood cells. The ultrastructural studies reviewed deal with identity, secretion, vesicles, recovery, and synthesis issues related to the biology of these similar cells.     

Ultrastructural immunocytochemical distribution of tubulin in cultured cells treated with microtubule inhibitors.

Microtubules in tissue cultured cells are stained immunocytochemically with the PAP-method using a purified antitubulin antibody. Treatment of the cells with microtubule inhibitors (colchicine, nocodazole, vinblastine) results in the disappearance of microtubules. The diffuse cytoplasmic staining is strongly increased in the cells by colchicine and nocodazole. Vinblastine produces paracrystalline aggregates that are strongly stained and macrotubules that are unstained. The diffuse staining is much less in vinblastine-treated cells. The bundles of intermediate filaments that are induced by all microtubule inhibitors do not bind the antitubulin antibody.     

Ultrastructural immunoperoxidase investigations of HCG binding to isolated testicular intertubular cells.

The binding and processing of human chorionic gonadotrophin (HCG) on isolated rat testicular intertubular cells was investigated using an ultrastructural immunoperoxidase technique. Following incubation of isolated cells with HCG at a range of temperatures and time intervals, cells were fixed and the presence of HCG demonstrated using monoclonal and polyclonal antibodies to HCG and its alpha and beta subunits. Results indicated that HCG binds to the surface of the Leydig cells where it is rapidly split into its component subunits. Internalization of the HCG molecule was not demonstrated. The testicular macrophages also bound HCG. In contrast to the Leydig cells they were able to internalize the molecule into lysosomes in the cytoplasm. The complete molecule and the alpha and beta subunits were found within the cells. The study suggests that results of previous studies using mixed populations of isolated cells may be due to the combined effects of the different mechanisms present in the two cell types. It is postulated that Leydig cells bind HCG on the surface and cyclic AMP is activated prior to breakdown and loss of the hormone from the surface of the cells. It is postulated that the macrophages play a role in the breakdown of excess trophic hormone and act in concert with the Leydig cells in the local control of testicular function.     

Herpesvirus saimiri antigens and virus recovery from cultured cells and antibody levels and virus isolations from squirrel monkeys

In cultures of vero cells infected with a low multiplicity of Herpesvirus saimiri (HVS), the development of internal antigen, as demonstrated by indirect immunofluorescence, was detected early and preceded the release of infectious virus. Membrane antigen, determined on unfixed cells, developed shortly thereafter. A positive correlation was found between the percentage of antigen-positive cells and the amount of recoverable infectious virus in cocultivation cultures of cells of a low virus-producing line of HVS-induced tumor and vero cells. Cytosine arabinoside (araC) prevented the development of infectious HVS, typical internal antigen, and membrane antigen when vero cells were treated with the drug two hours after virus inoculation. Another type of internal antigen appearing predominantly in the nucleus was demonstrable in infected but araC-treated vero cells. The presence of antibody to this antigen was rare in naturally-infected squirrel monkeys. Colony-housed squirrel monkeys showed a high frequency of antibody to HVS internal antigen produced in infected vero cells in the absence of araC, and evidence for seroconversion in some of these monkeys was obtained. Virus was isolated from lymphocytes of six of seven squirrel monkeys tested.     

Collagen biosynthesis in cultured rat testicular Sertoli and peritubular myoid cells.

The incorporation of 3H-proline into protein was regarded as a measure of total protein synthesis and the incorporation into hydroxyproline as indicative of collagen synthesis. Relative collagen synthesis (expressed as percent of total protein synthesized) by Sertoli and peritubular myoid cells cultured from 20-22 day old rat testis was estimated. In both secreted and cellular pools, relative collagen synthesis by Sertoli cells was significantly greater than by peritubular myoid cells. Coculture of Sertoli and myoid cells resulted in a significant increase in relative collagen synthesis when compared to monocultures of each cell type. Addition of serum to peritubular myoid cells resulted in a stronger stimulation of relative collagen production. Sertoli cell extracellular matrix inhibited relative collagen synthesis by peritubular myoid cells in the presence or absence of serum. Radioactivity into hydroxyproline as corrected per cellular DNA also showed similar results. Immunolocalization studies confirmed that both cell types synthesize type I and type IV collagens. These results indicate that stimulation of collagen synthesis observed in Sertoli-myoid cell cocultures is due to humoral interactions, rather than extracellular matrix, and Sertoli cell extracellular matrix regulates serum-induced increase in collagen synthesis by peritubular myoid cells.     

Evaluation of culture systems for attachment and proliferation of epithelial cells cultured from ovine semen

Different culture systems were evaluated for their ability to support attachment and proliferation of the somatic cells obtained from ovine semen. Ejaculates (n = 14) were collected from eight rams representing three breeds, Dorper, Suffolk and Hampshire. All samples were processed immediately and somatic cells were obtained from 11 of the 14 ejaculates. These cells had classic epithelial morphology and expressed cytokeratin, indicating they were of epithelial origin. Cells from four rams with the greatest growth rates were used for subsequent studies. Cells were cultured in four different media for 5 days and total numbers of attached cells vs. total numbers of seeded cells were counted and compared each day. Four media were evaluated: (1) a supplemented medium composed of DMEM/F12, 10% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, 30 μg/ml bovine pituitary extract, 5 μg/ml insulin, 10 ng/ml cholera toxin, and 50 μg/ml gentamycin; (2) sheep fetal fibroblast (SFF)-conditioned medium; (3) swiss 3T3 fibroblast-conditioned medium; and (4) basic medium composed of DMEM/F12, 10% FBS, and 50 μg/ml gentamycin. Cell proliferation was greater in the supplemented medium, SFF-conditioned medium, and 3T3 fibroblast-conditioned medium compared to the basic medium by day 2 of culture (p < 0.05, n = 24), and greater in supplemented medium compared to the SFF-conditioned medium and 3T3 fibroblast-conditioned medium by day 4 of culture (p < 0.05, n = 24). Three different surfaces: (1) Matrigel basement membrane matrix-coated plastic; (2) collagen I-coated plastic; and (3) uncoated plastic were evaluated for their ability to support proliferation and attachment of the cells obtained from semen. Cell proliferation was greater when cells were cultured on the Matrigel-coated compared to the collagen I-coated and uncoated plastic by day 2 of culture (p < 0.05, n = 16). Cell attachment was greater when cells were plated on the Matrigel-coated and collagen I-coated plastic compared to the uncoated plastic (p < 0.05, n = 16). These studies describe an effective system for the culture and proliferation of epithelial cells obtained from ovine semen samples. The system may increase the likelihood of obtaining cells from frozen semen, which could be used for cloning to recover animals of genetic value in which semen is the only material that is available.     

Ultrastructural localization of acetylcholinesterase in cultured cells. II. Cycloheximide-treated embryo muscle.

The acetylcholinesterase activity (AChE) of cultured chick embryo muscle fibers that remains after the cells have been treated with the protein synthesis inhibitor cycloheximide was examined with cytochemical stains and the electron microscope. AChE activity that decreased rapidly after addition of the inhibitor was associated with enzyme within the cells, and AChE activity that was relatively insensitive to the inhibitor was associated with AChE outside of the cells. The results support the view that there are at least two fractions of AChE in developing muscle fibers, one intracellular and labile, the other extracelullar and stable.     

Ultrastructural and histochemical studies on guard cells

Serial thick sections of guard cells from Vicia faba L., Nicotiana tabacum L., Allium cepa L., Zea mays L. and Beta vulgaris L. were obtained systematically (600–800 nm) and viewed with the transmission electron microscope in an effort to demonstrate the presence or absence of a symplastic transport pathway within the stomatal complex. Eight to ten stomata from each species were examined, and no continuous plasmodesmata were found connecting guard cells to sister guard cells or to adjacent epidermal or subsidiary cells. Continuous plasmodesmata were observed in immature guard cells, but were sealed (truncated) during the development of the mature cell wall. Histochemical stains, phosphotungstic acid and silver methenamine, were used to demonstrate differentiation within the mature guard-cell wall. The structural differentiation of the stomatal apoplastic region is discussed in relation to fanctional specialization. Plasma-membrane elaborations or plasmalemmasomes were identified in the guard cells of Zea, and it is suggested that these structures may function in ion transport.Abbreviations PTA-HCl phosphotungstic acid and hydrochloric acid - SM silver methenamine - UA-LC uranyl acetate and lead citrate     

The fertilising ability of spermatogenic cells derived from cultured mouse immature testicular tissue

There are many reports about the in vitro culture of spermatogenic cells, but no-one has succeeded in inducing the differentiation from spermatogonia to intact sperm. Also the study of in vitro testicular tissue culture has hardly advanced. We studied the culture of mouse immature testicular tissue derived from 5-day-old mice. We aimed to achieve the differentiation of spermatogenic cells in order to observe spermatogenesis in testicular tissue in vitro. We also froze mature testicular tissue and immature testicular tissue cultured for 2 weeks. Furthermore, spermatogenic cells differentiated by culturing were injected into metaphase II oocytes to determine whether these differentiated cells and frozen-thawed testicular tissue have fertilising and developmental ability. Under the culture conditions employed, secondary spermatocytes and a few round spermatids differentiated from spermatogonia were observed in the immature testicular tissue cultured for 2 weeks. When spermatogenic cells derived from cultured immature testicular tissue, cultured frozen immature testicular tissue and frozen-thawed mature testicular tissue were injected into ooplasm, the oocytes were fertilised and fertilised oocytes developed to the 8-cell stage. We suggest that spermatogenic cells derived from cultured immature testicular tissue have fertilising and developmental abilities equivalent to that of sperm. Also these abilities of spermatogenic cells obtained from cultured frozen immature testicular tissue and frozen-thawed mature testicular tissue were better than those of the same cells before freezing.     

Patterns of musculoskeletal growth and dimensional changes associated with selection and developmental plasticity in domestic and wild strain turkeys

Domestication is a type of experimental evolution in which humans have artificially selected for specific desired traits. Selected strain animals can be utilized to identify correlated responses by comparing them to the wild strain. In particular, domestic turkeys have been selected for increased body mass and high‐growth rate, most significantly over the past 60 years. Yet it remains unclear how artificial selection has affected the morphology and evolution of the musculoskeletal system as a whole. Here, we compare growth rate over 21 weeks, hind limb bone scaling across ontogeny via in vivo CT scanning, and muscle proportions in wild and domestic turkeys to identify differences in structural scaling and the potential contributions of selection and developmental plasticity to whole‐organism morphology. The domestic turkeys grew at a higher rate (0.14 kg/day vs. 0.05 kg/day) and reached over 3 times the body mass of wild birds. Comparing the proportional muscle masses in adult turkeys, only the trunk had a greater mass ratio in the domestic turkey, driven solely by M. pectoralis (2.8 times larger). The proportional increase in only breast meat and no other muscles highlights the surgical precision attainable with artificial selection. The domestic turkey femur and tibiotarsus displayed increases in polar moment of area, apparently maintaining torsional strength as body mass increased. The lack of dimensional change in the more vertically held tarsometatarsus is consistent with the pattern expected due to developmental plasticity. These results from the domestic turkey emphasize that there are morphological limits to preserving the balance between growth and function, and varying rates of trait evolution can further complicate this equilibrium.     

Ultrastructural localization of acetylcholinesterase in cultured cells. III. DFP treated embryo muscle

Brief treatment with 10(-4)M diisopropylfluorophosphate (DEP) irreversibly inactivates acetylcholinesterase (E.C.3.1.1.7; acetylcholine hydrolase) (AChE) activity in 10 day old chick embryonic muscle cultures. Electron microscopic cytochemistry was employed to follow the distribution of new AChE during recovery from DEP treatment. In normal 10 day cultures of embryo pectoralis muscles AChE is localized in the nuclear envelope, perinuclear sarcoplasm, sarcotubular system, subsurface vesicles and bound outside the cells. Immediately after DFP treatment AChE activity is absent in large myotubes. Within 15 min, activity is randomly present in small amounts in the sarcotubular system and nuclear envelope. There is a dramatic increase in activitv in the nuclear envelope during the 1st hr of recovery, and connections between the nuclear envelope and sarcotubular system are often seen. The next few hr of recovery show increased AChE activity. By 4 hr activity approaches that of controls. Six to 8 hr after treatment, AChE activity can be detected spectrophotometrically in the medium and can be seen bound outside the cells with the electron microscope. The spatial and temporal patterns of AChE activity demonstrate that the recovery of AChE and its mobilization and release from DFP-treated cells are not governed solely by the levels attained by the enzyme in the cultured embryo muscle.     

R1881 regulation of steroidogenesis in cultured testicular cells

The influence of a synthetic androgen R1881 upon hCG stimulated steroidogenesis in cultured rat testicular cells was investigated. Testicular cells were cultured for 8 days in medium alone and thereafter reincubated for 48 h with appropriate treatments before the collection of media for steroid RIA. Addition of R1881 (10(-6) M) resulted in an overall decrease of hCG (0.3-10 ng/ml) stimulated pregnenolone and progesterone production by cultured cells. The conversion of exogenous steroids of the delta 4 pathway (progesterone,17 alpha-OH-P and delta 4-A) was also studied in cultures supplemented with cyanoketone (10(-5) M) and/or spironolactone (10(-5) M) to prevent endogenous testosterone production. R1881 inhibited progesterone and 17 alpha-OH-P conversion to testosterone (T) and was ineffective when delta 4-A served as precursor for T biosynthesis. The inhibitory effect of R1881 upon Testosterone production was prevented by concomitant treatment with CPA. These observations suggest that R1881 decreases the hCG stimulated testosterone production via inhibition of CSCCE,3 beta-HSD,C17-20 Lyase and likely 17 alpha-Hydroxylase, whereas no effect on 17 beta-HSD could be observed.     

Polyribosomes associated with microfilaments in cultured lens cells

Epithelial hamster lens cells, transformed by SV40 can be grown in suspension culture. Triton X-100 extraction of these cells grown under conditions when ribosome run off is blocked releases about 40% of the total amount of polyribosomes, designated as free- and loosely-bound polyribosomes. The Triton ghosts retain the remaining polysomal population which can be released by a combined treatment with deoxycholate and Nonidet P 40. Electron microscopic examination of the ghosts reveals microfilament-associated ribosome clusters next to a fraction of polysomes still attached to membranes. Preincubation of the cells with cytochalasin D prior to polyribosome isolation enables us to discriminate between these two latter polysome populations. The experiments indicate that about 25% of the polyribosomes are attached to microfilaments, while the remaining 35% are tightly bound to the membranes of the endoplasmic reticulum. When the different polyribosome classes were translated in a reticulocyte lysate, no significant differences could be observed in the patterns of the newly synthesized polypeptides. In all cases actin was one of the major products synthesized de novo.     

Ultrastructural characterization of the interphase nucleus of Gonyostomum semen.

The interphase nucleus of the chloromonadophycean alga, Gonyostomum semen (Ehrenberg) Diesing, has a highly distinctive appearance. Interphase chromatin is readily distinguishable in both light and electron microscope preparations. It extends throughout the neucleus and frequently makes contact with the nucleoli and with the nuclear envelope. Among the chromatin filaments are large numbers of 35-46 nm diameter granules which occur singly or in clusters. The nucleoli are characteristically located in the posterior half of the nucleus and are composed of granular and non-granular components. Nuclear pores occur in slight depressions of the nuclear surface; their lumen has a diameter of approximately 75 nm and contains electron-dense material. The chromatin and the large numbers of nuclear granules are unusual and warrant further investigation.     

Ultrastructural studies of C1300 neuroblastoma cell interaction with syngeneic spleen lymphoid cells.

Sensitized and unsensitized spleen lymphoid cells from A/J mice were induced to form rosettes with cells of clone NB6R of syngeneic C1300 neuroblastoma cells. Light and transmission electron microscopy were applied in combination with ⁵¹Cr release experiments to follow the time course of reaction after rosette formation. With unsensitized lymphoid cells, rosettes formed but target cell morphology in general remained unchanged. With sensitized lymphoid cells a progressive series of morphological changes in the target cells was seen, initially in the mitochondria and, later, when specific ⁵¹Cr release became significant, in the formation of large surface blebs and protrusions. Our data also show another phenomenon occasionally following rosette formation. Lymphocytes were seen within the target cell; these either apparently transformed to lymphoblasts and killed the target cell from the inside or alternatively were destroyed by the host cell and their material was reutilized.