Ultrastructure and lectin cytochemistry of secretory cells in lingual glands of the Japanese quail (Coturnix coturnix japonica).

In the present study, as continuation of our previous research, Japanese quail (Coturnix coturnix japonica) lingual glands were investigated by means of transmission electron microscopy (TEM) to understand the cytoarchitecture and the subcellular sugar distribution within the different secretory structures. Indeed, glycosidic residues were visualized by applying an indirect technique of binding and the terminal sialoglycoconjugate sequences were characterized by employing sialidase digestion combined with lectin affinity. The ultrastructural analysis revealed an unusual cytoarchitecture of the caudal portion of anterior lingual gland that was composed of both secretory cells, filled with granules, and non-secretory cells, filled with mitochondria. Conversely, the posterior lingual gland was composed of secretory units of lingual glands only containing mucous cells filled with secretory granules with a variable morphology, including bipartite features characterized by an electron-lucent matrix and one or more electron-dense areas. Actual findings further supported that the quail lingual glands produce sialoglycoconjugates characterized by a heterogeneous composition. In conclusion, the cytological characteristics and the carbohydrate composition of quail lingual glands suggest that, analogously to mammal salivary glands, avian lingual glands could also be involved in several functions that can be correlated with the occurrence of sialic acids.

Involvement of p53 in phthalate effects on mouse and rat osteoblasts.

The role of two estrogen-mimicking compounds in regulating osteoblast activities were examined. Previously, our attention was focused on benzyl butyl phthalate (BBP) and di-n-butyl phthalate (DBP) since previous works showed that they enter the cytoplasm, bioaccumulate, modify actin cytoarchitecture and exert mitogenic effects involving microfilament disruption, and nuclear actin and lamin A regulation in Py1a rat osteoblasts. In this study we showed that BBP and DBP cause DNA base lesions both in MT3T3-E1 osteoblasts and in mouse primary calvarial osteoblasts (COBs). In addition, treatment with the above effectors caused an increase of p53 and phospho-p53 (ser-15 and ser-20) as well as an increase of apoptotic proteins with consequent decrease of cell viability. Moreover, treatment with phthalates did not modified p53 and phospho-p53 expression in Py1a rat osteoblasts. It is of relevance that in p53 knockdown mouse osteoblasts a proliferative effect of phthalates, similar to that observed in rat Py1a osteoblasts, was found. In conclusion, our data demonstrated that phthalates induce osteoblast apoptosis, which is, at least in part, mediated by p53 activation, suggesting that the proliferative effects could be due to p53 missing activation or p53 mutation.

Phthalate esters immunolocalized in the gastrointestinal tract of shi drum Umbrina cirrosa (L.) and rainbow trout, Oncorhynchus mykiss (W.).

The occurrence of phthalate esters in freshwater and marine aquacultural species like rainbow trout Oncorhynchus mykiss and shi drum Umbrina cirrosa, respectively, were determined by immunohistochemical approach. The results showed a similar distribution in the gastrointestinal tract of both species. In particular, intense immunoreactivity was found at gastric gland level. In the intestinal tract, goblet cells failed to stain, whereas enterocytes showed the highest binding of phthalates restricted to the apical cytoplasm. This distribution of phthalate esters at gastric gland and enterocyte level may have implications for the physiology of the digestive process and intestinal biotransformation. Phthalates are confirmed to be widely diffused contaminants, absorbed via the alimentary canal; thus a multidisciplinary approach could be useful to examine sea and freshwater environments.

Lectin cytochemistry on developing rat submandibular gland primary cultures.

Lectin cytochemistry was performed in vitro on primary cultures from the rat submandibular gland. For this purpose, prepubertal rats (17, 27, 33 days old) of both sexes were used. Several types of medium supplements were tested and it was found that cells survived until 15 days in presence of all medium supplements and extracellular matrix gel. The binding patterns of all FITC/TRITC-labeled lectins, with and without prior sialidase digestion and deacetylation, were analyzed in a confocal laser scanning microscope. In particular, the occurrence of C4 acetylated sialic acid linked to beta-galactose at day 27 and the presence of fucose residues at day 33 indicated that lectin probes applied to cultured cells give results similar to those obtained in intact tissues and can be used as markers of growth and differentiation.

Occurrence of beta-endorphin binding sites in the pituitary of the frog Rana esculenta: effect of beta-endorphin on luteinizing hormone secretion.

The possible effect of proopiomelanocortin-derived peptide, beta-endorphin on frog gonadotrope cells was investigated. Binding and internalization of beta-endorphin to pituitary pars distalis cultured cells were visualized by immunofluorescence and analyzed by means of confocal laser scanning microscopy. Using biotinylated endorphin, the time-course of beta-binding showed that this opioid was internalized through receptor-mediated endocytosis, the mechanism in which actin and clathrin were involved; then, the lysosomal degradation program occurred at later stages. The beta-endorphin binding was well antagonized by Naloxone, the opiate receptor antagonist, and up-regulated since more rapid response was obtained in the previously primed cells. The double immunostaining reaction for beta-endorphin and LH beta-subunit revealed that half the beta-endorphin labeled cell population was positively immunostained for LH beta-subunit, and beta-endorphin was able to induce an increasing trend of LH secretion in cultured pars distalis cells. Therefore, it seems that beta-endorphin acts directly on pituitary pars distalis and influences gonadotropin secretion through the interaction with its own receptor.

In situ visualization of o-phthalate esters in gastrointestinal tract of the frog Rana esculenta.

The regional distribution and relative occurrence of phthalates were studied immunohistochemically by confocal laser scanning microscopy in the alimentary tract of the green frog, Rana esculenta, using an antibody against o-phthalate esters. Many positive sites indicating the basal presence of phthalate esters were identified. The immunoreactive cells were located in the gastric glands of the stomach and in the intestinal epithelium regions with variable frequencies. The regional distribution of phathalate-accumulating cells resembled that of fish and demonstrated that these endocrine disruptors not only enter via the alimentary canal, but also bioaccumulate inside cells specialized in secretion as well as absorption functions.

Effects of phthalate esters on actin cytoskeleton of Py1a rat osteoblasts.

We evaluated, by confocal laser scanning microscopy, the actin cytoskeleton of immortalized rat Py1a osteoblasts treated with phthalate esters (butyl benzyl phthalate, BBP and dibutyl phthalate, DBP), endocrine disruptors with estrogenic activity. We observed some peculiar modifications of actin cytoskeleton and cells changing from a spindle shape to a rounded form. In particular, F-actin formed thick bundles around the cell membrane but only a weak labeling was observed in rounded cells. Also influence on apoptosis and short-term effects on FGF-2 were studied. It was found that BBP and DBP exert their action in a similar way, act in a transient manner and do not induce apoptosis.

Phthalate esters influence FGF-2 translocation in Py1a rat osteoblasts.

Exposure of the Py1a rat osteoblastic cells to butyl benzyl phthalate (BBP) and dibutyl phthalate (DBP) showed that these endocrine disrupting chemicals (EDC) strongly and reversibly affect the cytoplasmic fibroblast growth factor-2 (FGF-2) translocation into the nucleus in a dose-dependent and time-related manner. Stimulation of cells with high concentrations of BBP or DBP for short timing gave results comparable to those of cells treated with low concentrations for long timing. By confocal laser scanning microscope (CLSM) analysis it was found that the first relevant effect resulted in an accumulation of FGF-2 near the nuclear envelope, sometimes in the shape of clusters; the growth factor was then translocated into the nucleus and, finally, after long periods of exposure, the basal nuclear and cytoplasmic binding, typical of unstimulated cells, was re-established. In addition it was found that phthalate esters did not affect the FGF receptor 2 (FGFR-2) but decreased Con A binding indicating a possible inhibition of collagen fiber assembly. The different concentrations and timing of exposure of BBP and DBP affected the FGF-2 modulation in a similar way. Noticeable cumulative effects of BBP and DBP were not observed.

Nuclear and cytoplasmic lectin receptor sites in rat Py1a osteoblasts.

The intracellular distribution of lectin receptor sites was studied in the rat Pyla osteoblasts using immunofluorescence at the confocal microscopy level. This immortalized cell line was found to represent a satisfactory model to study the occurrence and distribution of sugar moieties. Our data showed distinct affinity patterns of lectins recognizing different terminal or internal sugar residues. For some lectins, the binding patterns appeared to be cell cycle-independent, whereas for PNA the cell cycle greatly influenced the nuclear binding. By combining lectin affinity with sialidase degradation and alcoholic saponification the sialic acid acceptor sugars and derivatives were also visualized. In particular, glycoconjugates with sialic acids linked to beta-galactose, and mainly C4 acetylated, were located in the cytoplasm, while glycoconjugates characterized by sialic acids linked to alpha-N-acetylgalactosamine, and devoid of acetyl groups at C4, were almost exclusively found in the nucleus. The comparison of lectin affinities, with and without prior glycosidase digestions, allowed us to gain further insight into the chemical composition of glycoconjugates that act as the lectin receptor sites that appeared to belong to O- and N-linked glycoconjugates. The use of additional enzymatic treatments were useful to better establish the localization of nuclear receptor sites and results were compared with previous studies about endogenous and exogenous lectins in an attempt to reconcile the association of lectins and sugars within the nucleus and their possible involvement in modulation of cell proliferation and/or response to chemical signals. The above digestions also provided information about the cytoplasmic binding patterns.

Confocal evaluation of native and induced lectin binding contributes to discriminate between lingual gland glycocomponents in quail.

A confocal analysis was performed on the quail (Coturnix coturnix japonica) lingual salivary glands where the carbohydrate chains were studied by lectin histochemistry. For this purpose, appropriate FITC- and TRITC-conjugates were used for double binding also accomplished with sialidase digestion. The glycosidic components of the quail lingual salivary glands were found to be heterogeneously distributed on the different secretory structures as well as on the single secretory elements of each adenomere. The rostral portion of the anterior lingual gland was found to only secrete neutral glycocomponents, characterized by terminal beta-galactose, N-acetylgalactosamine and fucose residues in contrast to the caudal portion that was shown to be extremely heterogeneous and to produce sialylated glycoconjugates characterized by the terminal sequences sialic acid-beta-galactose-N-acetylgalactosamine, sialic acid-beta-galactose-N-acetylglucosamine, and sialic acid-alpha-N-acetylgalactosamine partly codistributed within secretory adenomeres. The posterior lingual gland was observed to be the major contributor to the secretion of salivary mucins containing sialoglycoconjugates with terminal sialic acid residues linked to beta-galactose-N-acetylgalactosamine or alpha-N-acetylgalactosamine often located in distinct secretory elements.

Double-sided staining with a gold probe and silver enhancement to detect alpha-amylase and sugar moieties in the mouse salivary glands.

In the present study we report the development of an ultrastructural electron microscopic double-sided staining technique that, using gold probes of 10 nm and enhancement of the gold signal by silver amplification, allows the demonstration of two antigenic sites on the same section. The labeling was carried out in the following manner: one face of uncoated floating grids was incubated with an antibody directed to alpha-amylase, followed by a secondary gold-labeled antibody, amplification of gold particles, drying and carbon coating; subsequently, the reverse face of the same grid, was processed for lectin cytochemistry, with and without sialidase digestion, and it was incubated with HRP-conjugated lectins, anti-HRP antibody and protein-A gold. Also the reverse sequence of steps and amplification of gold signal after the first or second labeling were experimented. The resultant small and large particles revealed different distributional patterns of antigenic sites on the opposite faces of the same tissue section. The transparency of the resin-embedded ultrathin sections in the electron beam allowed the simultaneous visualization of the gold probes of different sizes present on the two faces. The analysis of immunolabeling revealed that the alpha-amylase is chiefly secreted by the parotid and submandibular glands. The application of this double-sided staining technique also indicated that, when present in glycosylated form, the alpha-amylase enzyme does not contain sialic acid in the submandibular and sublingual glands; conversely, its location on the electron-dense areas of target granules in the parotid acinar cells seems to suggest that a sialylated isoenzymatic form can occur within these granule regions where sialic, acid linked to beta-galactose, was found to be located.

Sex-related expression of sialic acid acceptor sugars in the mouse submandibular gland. Simultaneous visualization by confocal laser scanning microscopy.

The novel combination of sialidase digestion with simultaneous PNA and DBA binding yielded marked differences on sialoglycoconjugate occurrence and distribution in the mouse submandibular gland acinar cells of the two sexes. Striking differences in the structure of terminal disaccharides within stored secretory sialoglycoconjugates were also found. High content of sialic acid, characterized by the terminal sequence sialic acid-alpha-N-acetylgalactosamine, was established to only occur in the male acini where secretory cells appeared to be differently stained; indeed, some cells exhibited codistribution of sialic acid-alpha-N-acetylgalactosamine and sialic acid-beta-galactose terminal disaccharides, whereas other ones exclusively contained one of the two kinds of terminal sequences. In the female acinar cells, the secretory products were found to be almost exclusively composed by glycoconjugates having sialic acid subtended to beta-galactose without appreciable differences between acinar cells. Our finding of such extensive differences in the acinar cells of male and female mice adds new insights into the submandibular gland sexual dimorphism, commonly attributed to the androgen responsiveness of the granular convoluted tubule portion of the gland.

Parathyroid hormone regulates the expression of fibroblast growth factor-2 mRNA and fibroblast growth factor receptor mRNA in osteoblastic cells.

We examined the effect of parathyroid hormone (PTH) on basic fibroblast growth factor-2 (FGF-2) and FGF receptor (FGFR) expression in osteoblastic MC3T3-E1 cells and in neonatal mouse calvariae. Treatment of MC3T3-E1 cells with PTH(1-34) (10-8M) or forskolin (FSK; 10-5M) transiently increased a 7 kb FGF-2 transcript with a peak at 2 h. The PTH increase in FGF-2 mRNA was maintained in the presence of cycloheximide. PTH also increased FGFR-1 mRNA at 2 h and transiently increased FGFR-2 mRNA at 1 h. FGFR-3 and FGFR-4 mRNA transcripts were not detected in MC3T3-E1 cells. In cells transiently transfected with an 1800-bp FGF-2 promoter-luciferase reporter, PTH and FSK increased luciferase activity at 2 h and 4 h. Immunohistochemistry showed that PTH and FSK increased FGF-2 protein labeling in the nuclei of MC3T3-E1 cells. PTH also increased FGF-2 mRNA, and FGFR-1 and FGFR-2 mRNA levels within 30 minutes in neonatal mouse calvarial organ cultures. We conclude that PTH and cAMP stimulate FGF-2 mRNA abundance in part through a transcriptional mechanism. PTH also regulated FGFR gene expression. We hypothesize that some effects of PTH on bone remodeling may be mediated by regulation of FGF-2 and FGFR expression in osteoblastic cells.

Prostaglandins regulate the expression of fibroblast growth factor-2 in bone.

We examined the effect of PGs, particularly PGF2alpha, on basic fibroblast growth factor-2 (FGF-2) messenger RNA (mRNA) and protein in the rat osteoblastic cell line Py1a and in fetal rat calvariae. Py1a cells expressed multiple FGF-2 mRNA transcripts. PGF2alpha dose-dependently increased the 6-kb transcript at 6 h. The selective PGF2alpha agonist, fluprostenol (Flup), was more potent than PGF2alpha. Phorbol myristate acetate (10(-6) M) also increased a 6-kb mRNA at 6 h. By immunofluorescence microscopy, Flup increased perinuclear staining for FGF-2 protein at 6 h and nuclear labeling at 24 h. Immunogold labeling of calvariae revealed that treatment with Flup for 3 h caused a transition of FGF expression from matrix to cells and an increase in cytoplasmic labeling for FGF-2 protein in periosteal cells and in osteoblasts. After treatment with Flup for 24 h, nuclear labeling was marked in periosteal cells and in osteoblasts, and a further increase in cytoplasmic labeling for FGF-2 was noted in osteocytes, periosteal cells, and osteoblasts. We conclude that PGs can increase FGF-2 mRNA and protein in bone cells. Because the effect of Flup was mimicked by phorbol myristate acetate, we hypothesize that PGs' regulation of FGF-2 is mediated by a PGF2alpha-selective receptor acting through protein kinase C. Hence, effects of PGs on bone remodeling may be mediated, in part, by endogenous FGF-2.

Multihormonal control of vitellogenin mRNA expression in the liver of frog, Rana esculenta.

In Rana esculenta in an in vitro system, hepatic vitellogenin synthesis can be induced by growth hormone in both sexes. In this study: (1) the ability of this hormone to induce transcription of the VTG gene was determined, and (2) this ability was compared with that of estradiol-17 beta. The results indicate that growth hormone stimulates VTG mRNA transcription both in vivo and in vitro, in both sexes. The levels of mRNA are related to protein levels in the medium. In addition, seasonal variation occurs in the VTG gene transcription under growth hormone and estradiol-17 beta; indeed the more active inducer was growth hormone during the reproductive period and estradiol-17 beta during the preproductive phase.