The ultrastructure of mammary explants from virgin ovariectomized goats during hormone-induced lactogenesis

The effect of insulin (I), cortisol (F) and prolactin (P) on the ultrastructural morphology of epithelial cells of cultured mammary explants from virgin ovariectomized (OV-X) goats were studied. The epithelial cells showed little structural organization and were devoid of fat droplets and secretory protein granules at zero time of culture. The cytoplasm contained few profiles of smooth and rough endoplasmic reticulum and the Golgi apparatus was rudimentary. After being cultured in Waymouth's medium without added hormones the epithelial cells were indistinguishable from epithelial cells of uncultured explants. The addition of I induced changes mainly in the appearance of nucleoli. The nucleoli were enlarged and fibrillogranular areas with light spaces were observed. The most obvious cytological changes of epithelial cells of explants cultured in the presence of I and F are translocation of the nucleus into the basal cytoplasm, increase of rough endoplasmic reticulum, an increase in the size of the Golgi apparatus, presence of one or two lipid droplets and in some cells vacuoles with protein granules were present. Mitochondria were more abundant. The epithelial cells of explants cultured in the presence of I, F and P were characterized by the polarization of organelles within the cytoplasm and by the formation and release of protein granules and small and large fat droplets. The cell nucleus was in the basal cytoplasm, the Golgi apparatus was supranuclear. The rough endoplasmic reticulum was extensively developed and formed large sacs. Golgi vacuoles contained protein granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Casein accumulation in distended rough endoplasmic reticulum of collagen gel-cultivated mouse mammary epithelia

Mouse mammary epithelial cells cultivated on collagen gels synthesize and secrete casein in a hormone-dependent manner. Fine-structure electron microscopy of secretory cultures revealed numerous cytoplasmic structures surrounded by membrane that is studded with ribosomes. The structures appear to be distended rough endoplasmic reticulum (RER). Electron microscope protein A-colloidal gold immunolocalization showed casein antiserum-specific deposition of gold particles over the RER cytoplasmic vesicles in cells provided insulin, prolactin, and hydrocortisone (IPF). Nonimmune antiserum showed no gold particle deposition over these cytoplasmic structures. Epithelia provided only insulin showed no such cytoplasmic vesicles nor any specific deposition of gold particles. Immunoblot analysis of cell lysate and culture medium showed casein only in IPF-treated cultures. It appears that the casein secretory pathway in collagen gel cultured mammary epithelia is blocked at the step that fuses RER vesicles to Golgi membrane. The data raise questions regarding the processing and maturation of casein and the mechanism of casein secretion in these cultures.     

Ultrastructural studies on prolactin and growth hormone cells in Anguilla pituitaries in long term cultures

Summary Eel hemi-pituitaries were cultured in vitro on high or low sodium media, previously shown to affect differentially prolactin and growth hormone release. After 6 days culture, there were marked differences in the ultrastructure of both prolactin and growth hormone cells from the two groups. Morphometric data on the prolactin cells from SW-adapted eels showed a greater abundance of RER and paucity of secretory granules in cells from the low sodium medium. The size of the Golgi apparatus and the number of exocytosed secretory granules did not differ markedly between experimental groups, in contrast to previous findings on short-term cultures. Differences in the profile diameters of secretory granules are recorded between the experimental groups and the pattern differs markedly from that previously recorded for short-term cultures. The growth hormone cells from low sodium media were characterised by abundant, vesiculated RER, a prominent Golgi apparatus (in SW-adapted animals) and relatively few secretory granules. The activity of these growth hormone cells is in marked contrast to previous findings relating to short-term cultures. The shape and size of the non-granulated (stellate) cells of the RPD was again affected by the osmotic pressure of the medium.I should like to thank Mr. P.F. Hire for his photographic assistance     

Ultrastructure of growing oocytes and accessory cells in Austrognathia (Gnathostomulida, bursovaginoidea)

The ovary of Austrognathia cf. riedli consists of 4-6 oocytes which are located in the mid-body region between the epidermis and the gut epithelium. The ovary is not enveloped by a tunica and each growing oocyte is surrounded by one or more accessory cells, the function of which is hypothesized in this study. Oogenesis is not synchronous and can be subdivided into a previtellogenic phase and a vitellogenic phase. Previtellogenic oocytes undergo a number of cell differentiations which consist mainly of an increase in size of the nucleus and nucleolus and the appearance in the cytoplasm of chromatoid bodies, annulate lamellac and short cisternae of rough endoplasmic reticulum (RER). Vitellogenic oocytes are characterized by the increase of RER, the appearance of numerous Golgi complexes and the accumulation of electron-dense globules, glycogen and lipid droplets. The electron-dense globules have been interpreted as yolk on the basis of both their localization and composition. Yolk synthesis occurs mainly by an endogenous mechanism and, to a lesser extent, by micropinocytosis. No shell-granules have been identified in the oocytes. The present ultrastructural findings are discussed in comparison with those from other lower metazoans.     

Mast cells of lymph hearts during ontogenesis of frogs Rana temporaria

Electron microscopic observations of the lymph hearts of tadpoles and yearling frogs of Rana temporaria showed that mast cells (MCs) were present not only between muscle fibers (population of resident MCs), but in the cavities of lymph heart (population of circulating MCs), too. There were some differences in the ultrastructure of the resident MCs at each studied stage of larval development. The first recognizable MCs were revealed in the lymph hearts at premetamorphosis (stages 39-41). MCs presented as mononuclear relatively small and slightly elongated cells with a few immature secretory granules and numerous free ribosomes, polysomes and short cisternae of rough endoplasmic reticulum (RER) in the cytoplasm. Chromatin of their nuclei was poorly condensed; the Golgi apparatus was moderately developed. At pro-metamorphosis (stages 44-45), we revealed MCs at different levels of their differentiation. Some MCs demonstrated an active process of granulogenesis in their cytoplasm. Among densely packed cytoplasmic organelles, immature secretory granules were closely associated with cisternae of RER and free ribosomes. Other MCs appeared as more differentiated cells. They were characterized by a predominantly heterochromatic nuclei and cytoplasm filled with polymorphic and heterogeneous granules. MCs also showed a reduction in the number of free ribosomes and cisternae of RER in the cytoplasm. On the contrary, the Golgi apparatus was well developed. Stacks of Golgi cisternae, detaching vacuoles, and progranules occupied the perinuclear region. The majority of the outlines above ultrastructural features of differentiated MCs were typical for MCs of yearling frogs. At metamorphic climax (stages 52-53), MCs often tightly contacted with macrophages. We did not reveal apoptotic MCs. However, some MCs exhibited morphological features typical for programmed necrosis-like death, which was characterized by mitochondria swelling, dilatation of cisternae of RER and nuclear envelope, plasma membrane rupture and subsequent loss of intracellular contents. Electron microscopical immunocytochemistry revealed the localization of atrial natriuretic peptide (ANP), substance S (SP) and heat shock protein (Hsp70) in the secretory granules of the resident and circulating MCs at different stages of tadpole development and in yearling frogs.     

Virus-neuron interactions in the mouse brain infected with Japanese encephalitis virus

The virus-host interactions between Japanese encephalitis (JE) virus and mouse brain neurons were analyzed by electron microscopy. JE virus replicated exclusively in the rough endoplasmic reticulum (RER) of neurons. In the early phase of infection, the perikaryon of infected neurons had relatively normal-looking lamellar RER whose cisternae showed focal dilations containing progeny virions and characteristic endoplasmic reticulum (ER) vesicles. The reticular RER, consisted of rows of ribosomes surrounding irregular-shaped, membrane-unbounded cisternae and resembled that observed in JE-virus-infected PC12 cells, were also seen adjacent to the lamellar RER. The appearance of the reticular RER indicated that RER morphogenesis occurred in infected neurons in association with the viral replication. The fine network of Golgi apparatus was extensively obliterated by fragmentation and dissolution of the Golgi membranes and their replacement by the electron-lucent material. As the infection progressed, the lamellar RER was increasingly replaced by the hypertrophic RER which had diffusely dilated cisternae containing multiple progeny virions and ER vesicles. The Golgi apparatus, at this stage, was seen as coarse, localized Golgi complexes near the hypertrophic RER. In the later phase of infection, RER of infected neurons showed a degenerative change, with the cystically dilated cisternae being filled with ER vesicles and virions. Small, localized Golgi complexes frequently showed vesiculation, vacuolation, and dispersion. The present study, therefore, indicated that during the viral replication the normal lamellar RER which synthesized neuronal secretory and membrane proteins was replaced by the hypertrophic RER which synthesized the viral proteins. The hypertrophic RER eventually degenerated into cystic RER whose cisternae were filled with viral products. The constant degenerative change which occurred in the Golgi apparatus during the viral replication suggested that some of the viral proteins transported from RER to the Golgi apparatus were harmful to the Golgi apparatus and that increasing damage to the Golgi apparatus during the viral replication played the principal role in the pathogenesis of JE-virus-infected neurons in the central nervous system.     

An electron microscopical study of epididymal epithelium of rats treated with gonadotropic hormone

The ultrastructural modifications of the epithelial cells of rat corpus epididymis stimulated with gonadotropic hormone were studied. The structural variety of the cells depending on functional conditions becomes more prominent 6 h after the injection of gonadotropic hormone. Light large cells have one or often two nucleus-containing bing nucleoli, in their cytoplasm there are numerous vesicles, a well-developed Golgi apparatus, other organelles and lysosomal bodies. Some other cells are filled with many large vacuoles of different density, dense bodies and vesicles. Cells of another type which are in the majority show an unusually active structure reflecting the function of synthesis. The more prominent nucleolus is associated to clumps of chromatin. Their apical cytoplasm is filled by a structure related to absorption. The whole remaining part of their cytoplasm is covered with a very extensive Golgi apparatus and a very well developed granular endoplasmic reticulum. The extremely enlarged cisternae of this reticulum were found to be very closely applied to the basal cell membrane. There is a flocculent material inside the cisternae. Similar material is observed in the extracellular medium under the basal membrane. The epithelium seems normal 10 h after the injection of hormone, but large light cells make up the majority of them.     

Comparison of collagen gels and mammary extracellular matrix as substrata for study of terminal differentiation in rabbit mammary epithelial cells

Mammary epithelial cells were prepared by collagenase digestion of tissue from mid-pregnant rabbits and cultured for up to 6 days on either collagen gels or an extracellular matrix prepared from the same tissue. The behaviour of the cells in serum-supplemented medium containing combinations of insulin, prolactin, hydrocortisone, estradiol and progesterone were monitored by measuring rates of casein synthesis, lactose synthesis, DNA synthesis and protein degradation. After 6 days, epithelial cells on floating collagen gels showed substantial increases in casein synthesis and DNA synthesis over freshly-prepared cells, following a decline during the first 3 days when the collagen gels are contracting. The optimum hormone combination for casein synthesis was insulin + prolactin + hydrocortisone, whereas for optimum DNA synthesis the additional presence of estradiol and progesterone was required. Cells on extracellular matrix showed increased rates of both casein synthesis and DNA synthesis by day 6 in the presence of insulin + prolactin + hydrocortisone, with additional estradiol + progesterone having an inhibitory effect. Whereas on day 2 rates of intracellular protein degradation were generally lower in cells on extracellular matrix, by day 6 rates of protein degradation were lowest in cells cultured on collagen gels with insulin + prolactin + hydrocortisone. In all cases, rates of lactose synthesis fell to low levels as the culture proceeded. Pulse-chase labelling of freshly-prepared cells with [32P]orthophosphate in medium containing serum and insulin + prolactin + hydrocortisone demonstrated that newly-synthesized casein was degraded during its passage through the epithelial cell. The influences of the collagen gels and extracellular matrix and of the hormone combinations on epithelial cell differentiation and secretory activity are discussed.     

Polyamine requirement for prolactin action on macromolecular synthesis in the MCF-7 human mammary epithelial cell line

The actions of insulin, hydrocortisone, prolactin and growth hormone on the synthesis of macromolecules in MCF-7 cells was determined in a serum-free defined medium. The inclusion of the polyamine spermidine in the medium was shown to enhance the insulin stimulation of the rate of [3H]uridine incorporation into RNA in a manner similar to that demonstrated for hydrocortisone. Spermidine, in addition to insulin and hydrocortisone, was also essential for prolactin to manifest a stimulation of the rate of [3H]uridine incorporation; this effect of spermidine was optimal with spermidine concentrations between 1 and 5 mM. Prolactin also stimulated the rate of [3H]leucine incorporation into total cellular protein and into an isoelectrically precipitable (pH 4.6) phosphoprotein fraction. The actions of prolactin on total protein and phosphoprotein synthesis were only expressed if spermidine, in addition to insulin and hydrocortisone, was contained in the culture medium. All of the prolactin responses were observed employing physiological concentrations of prolactin. Specificity of the prolactin responses was established by demonstrating that porcine growth hormone had no effects on RNA or phosphoprotein synthesis in the MCF-7 cells.     

The monolayer–spheres–monolayer cycle: Ultrastructural changes in stem cells

Stem cell sphere formation is applied as a preconditioning treatment prior to transplantation. Here, stem cells (SC) isolated from human subepicardial adipose tissue were analyzed at different stages of the monolayer–spheres–monolayer cycle by transmission electron microscopy. Spheres obtained on a non-adherent surface or through hanging drops gave similar results. At the first two or three passages (stage 1), isolated SC displayed an embryonal cell-like ultrastructure. With increased passage number (stage 2), SC became larger and more electron-dense with a multilobed nucleus, well-developed rough endoplasmic reticulum (RER), well-developed Golgi apparatus, and numerous vacuoles. At 2 h after sphere induction (stage 3), SC gathered into clusters and formed desmosome-like intercellular contacts. Their nuclei possessed a large loose fibrillo-granular nucleolus, the cytoplasm was tightly packed with disintegrated cisternae of RER, and Golgi apparatus was not identified. Twenty-four hours afterward (stage 4), SC in well-formed spheres exhibited a large dense nucleolus and poorly developed Golgi apparatus and RER. One day after sphere dissociation (stage 5), SC looked like embryonal cells and were morphologically similar to the cells of the first stage except for the presence of a large nucleolus and several Golgi complexes. At 48 h after sphere dissociation (stage 6), SC became electron-dense and resembled SC of the second stage, bearing irregular nuclei and cytoplasm that was rich in RER. We interpret these results as SC senescence with increasing passage number after isolation from the tissue or 1 day after sphere dissociation and rejuvenation of the SC just after sphere dissociation. Further research is needed to determine the genetic, biochemical, and physiological parameters of the SC corresponding to morphologically defined distinct stages in order to provide high quality cellular material for cell therapy.     

Structural and ultrastructural differentiation of the thyroid gland during embryogenesis in the grass snake Natrix natrix L. (Lepidosauria, Serpentes)

The differentiation of the thyroid primordium of reptilian species is poorly understood. The present study reports on structural and ultrastructural studies of the developing thyroid gland in embryos of the grass snake Natrix natrix L. At the time of oviposition, the thyroid primordium occupied its final position in the embryos. Throughout developmental stages I-IV, the undifferentiated thyroid primordium contained cellular cords, and the plasma membranes of adjacent cells formed junctional complexes. Subsequently, the first follicular lumens started to form. The follicular lumens were of intracellular origin, as in other vertebrate species, but the mechanism of their formation is as yet unclear. At developmental stages V-VI, the thyroid anlage was composed of small follicles with lumens and cellular cords. Cells of the thyroid primordium divided, and follicles were filled with a granular substance. At developmental stage VI, the cells surrounding the follicular lumen were polarized, the apical cytoplasm contained dark granules and the Golgi complex and the rough endoplasmic reticulum (RER) developed gradually. Resorption of the colloid began at developmental stage VIII. At the end of this stage, the embryonic thyroid gland was surrounded by a definitive capsule. During developmental stages IX-X, the follicular cells contained granules and vesicles of different sizes and electron densities and a well-developed Golgi apparatus and RER. At developmental stage XI, most follicles were outlined by squamous epithelial cells and presented wide lumens filled with a light colloid. The Golgi complex and RER showed changes in their morphology indicating a decrease in the activity of the thyroid gland. At developmental stage XII, the activity of the embryonic thyroid gradually increased, and at the time of hatching, it exhibited the features of a fully active gland.     

Effects of insulin, hydrocortisone and prolactin on cell morphology, growth and survival of mammary organoids from mid-pregnant mice cultured on collagen gels.

Mammary epithelial organoids consisting of groups of lobular-alveolar acini were prepared from mid-pregnant mice and cultured for 24, 48, 96 and 192 hr on attached collagen gels in the presence of combinations of insulin, hydrocortisone and prolactin. The organoids rapidly attached to the gels and with all the combinations of hormones used colonies of cells spread out as a monolayer from the organoids within 48 hr. Although colony formation continued for up to 192 hr in culture, the maintenance of parental organoid structure after 96 and 192 hr was strongly favoured when hydrocortisone was present in the culture medium. The presence of hydrocortisone produced a dose-dependent increase in the amount of organoid DNA associated with the collagen substratum but decreased the rate of DNA synthesis by the organoids, as measured by the incorporation of labelled thymidine into DNA, in a dose-dependent manner under these conditions. The results suggest that the presence of hydrocortisone minimised the loss of cells from the collagen matrix in these cultures.     

Immunocytochemical localization of Mullerian inhibiting substance in the rough endoplasmic reticulum and Golgi apparatus in Sertoli cells of the neonatal calf testis using a monoclonal antibody

Mullerian Inhibiting Substance (MIS) has been localized in the Sertoli cells of the neonatal calf testis using preembedding immunoperoxidase techniques and a monoclonal antibody which almost completely blocks the biological activity of MIS. Both the peroxidase-labeled antibody method using a peroxidase-conjugated F(ab')2 fragment of IgG as a second antibody and the unlabeled antibody peroxidase-antiperoxidase (PAP) method using Fab fragments of the PAP complex were employed. With both methods, MIS was demonstrated within the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus. In the Golgi, MIS was concentrated in the transmost cisternae especially at their peripheral expansions. This study indicates that MIS is synthesized in the RER and transported to the Golgi apparatus, presumably for glycosidation, before secretion from Golgi derived vacuoles.     

Chemical inducers of differentiation,dimethylsulfoxide, butyric acid,and dimethylthiourea,induce selective ultrastructural patterns in B16 melanoma cells

The morphology and ultrastructure of B16 melanoma cells was examined after treatment of the cells with the chemical inducers of differentiation dimethylsulfoxide (DMSO), butyric acid, and dimethylthiourea (DMTU). The treated B16 melanoma cells seemed to be enlarged and more flattened, and to possess dendrite-like structures as revealed by scanning electron microscopy. The main ultrastructural features, depicted by transmission electron microscopy in DMSO-treated B16 cells were: a marked increase in melanin granules, migration of the melanin granules to the dendrites, and appearance of melanosome aggregates. Butyric acid did not induce melanin biosynthesis; however, it stimulated rough endoplasmic reticulum (RER) formation all over the cytoplasm. The DMTU-treated cells also showed a well developed RER accompanied by early stages of melanosomes and melanin granules. The increase in the endoplasmic reticulum was also reflected by enhancement of NADPH cytochrome c reductase activity, an enzymatic marker of the endoplasmic reticulum. The mitochondria in the DMTU-treated cells were swollen with disrupted cristae. The results indicate that DMSO, butyric acid, and DMTU induce different ultrastructural patterns in B16 melanoma cells. These findings correlate with the biochemical alterations induced in melanoma cells by these agents.     

Immunolocalization of the oligosaccharide trimming enzyme glucosidase II

We used immunoelectron microscopy to localize glucosidase II in pig hepatocytes. The enzyme trims the two inner alpha 1,3-linked glucoses from N-linked oligosaccharide precursor chains of glycoproteins. Immunoreactive enzyme was concentrated in rough (RER) and smooth (SER) endoplasmic reticulum but not detectable in Golgi apparatus cisternae. Transitional elements of RER and smooth membraned structures close to Golgi apparatus cisternae contained labeling for glucosidase II. Specific labeling was also found in autophagosomes. These results indicate strongly that glucosidase II acts on glycoproteins before their transport to, and processing in Golgi apparatus cisternae, and suggest that an important transitional region for glucosidase II exists between RER and Golgi apparatus cisternae. Degradation in autophagolysosomes could form a normal catabolic pathway for glucosidase II.     

Maintenance of Golgi structure and function depends on the integrity of ER export.

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems.     

Insulin and prolactin synergize to induce translation of human serum albumin in the mammary gland of transgenic mice

A dramatic uncoupling of the expression of chimaeric -lactoglobulin (BLG)/human serum albumin (HSA) gene constructs at the RNA and protein levels was observed in cultured mammary explants of virgin transgenic mice. Upon explantation, both HSA RNA and protein were expressed at high levels. However, when the explants were grown in hormone-free medium, HSA RNA continued to accumulate, whereas the synthesis of the corresponding protein was dependent on the presence of insulin and prolactin with a minor contribution of hydrocortisone. The untranslated HSA RNA was indistinguishable from its translatable counterpart in its mobility on agarose gels, was transported normally from the nucleus to the cytoplasm and was translated efficiently in rabbit reticulocyte lysate. In the presence of cycloheximide, HSA RNA rapidly disappeared, suggesting a dependency on ongoing protein synthesis. Its estimated half-life of 5--6 h in hormone-free medium increased significantly in the presence o f insulin, hydrocortisone and prolactin and was comparable to that of -casein RNA. The uncoupling of the expression of the BLG/HSA transgenes at the RNA and protein levels was also confirmed by in situ hybridization and immunohystochemistry on sections from virgin mammary explants. HSA synthesis was initiated within 13 h of the addition of insulin and prolactin in explants that had accumulated untranslated HSA RNA and was fourfold higher than that observed with insulin alone. Addition of hydrocortisone contributed to an additional 20% in HSA synthesis. We believe this is the first demonstration of translational control of exogenous milk protein gene expression in the mammary gland of transgenic animals     

Disproportional immunostaining patterns of two secretory proteins in guinea pig and rat exocrine pancreatic cells. An immunoferritin and fluorescence study

Amylase (Am) and chymotrypsinogen (Chtg) were demonstrated in rat and guinea pig exocrine pancreatic cells by immunofluorescence and immunoferritin cytochemistry on thin and ultrathin frozen sections. We describe two observations indicating that Am and Chtg may behave differently in the pre-Golgi phase of their intracellular transport. Firstly, aggregates of material within the RER cisternae of the guinea pig (so-called intracisternal granules) reacted strongly with anti-Chtg, but showed no affinity for anti-Am. Secondly, in both rat and guinea pig, the increase in labeling intensity from cytoplasm (RER) to secretory granules was larger for Chtg than for Am. We hypothesize that the two proteins do not travel in-parallel towards the Golgi complex. Compared with Chtg, Am would lag behind in the RER cisternae.