Ultrastructural investigation of a salivary gland in a centipede: structure and origin of the maxilla I-gland of Scutigera coleoptrata (Chilopoda, Notostigmophora)

The maxilla I-gland of Scutigera coleoptrata was investigated using light and electron microscopy methods. This is the first ultrastructural investigation of a salivary gland in Chilopoda. The paired gland opens via the hypopharynx into the foregut and extends up to the third trunk segment. The gland is of irregular shape and consists of numerous acini consisting of several gland units. The secretion is released into an arborescent duct system. Each acinus consists of multiple of glandular units. The units are composed of three cell types: secretory cells, a single intermediary cell, and canal cells. The pear-shaped secretory cell is invaginated distally, forming an extracellular reservoir lined with microvilli, into which the secretion is released. The intermediary cell forms a conducting canal and connects the secretory cell with the canal cell. Proximally, the intermediary cell bears microvilli, whereas the distal part is covered with a distinct cuticle. The cuticle is a continuation of the cuticle of the canal cells. This investigation shows that the structure of the glandular units of the salivary maxilla I-gland is comparable to that of the glandular units of epidermal glands. Thus, it is likely that in Chilopoda salivary glands and epidermal glands share the same ground pattern. It is likely that in compound acinar glands a multiplication of secretory and duct cells has taken place, whereas the number of intermediary cells remains constant. The increase in the number of salivary acini leads to a shifting of the secretory elements away from the epidermis, deep into the head. Comparative investigations of the different head glands provide important characters for the reconstruction of myriapod phylogeny and the relationships of Myriapoda and Hexapoda.     

Freeze-fracture,ultrastructural and autoradiographic analysis of the ventral prostate glands in castrated mice

Summary Ultrastructure of the ventral prostate glands was studied in mice castrated for 1 through 60 days and for 11 and 17 months and in age-matched normals. We have described freeze-fracture and ultrastructural characteristics of acinar epithelial cells in addition to the patterns of thymidine incorporation in the cells of castrates and normal animals. Our study has shown a biphasic pattern of prostatic involution in the long-term castrated mice. In castrates the initial atrophy of prostate glands occurred by sloughing of the apical portions of columnar cells, autophagia of the cytoplasmic organelles as well as by occasional sloughing of the individual cells into the acinar lumen. Concurrent with the initial atrophy, the glands and stroma were infiltrated by neutrophils and lymphocytes. The cell loss by sloughing and leucocyte infiltration of glands became infrequent in 7- to 21-day castrates. However, the cell loss by sloughing increased secondarily in mice castrated for 21 to 37 days along with the increased leucocyte infiltration of the glands. The cell loss became minimal in castrates of 60 days and beyond. Our evidence suggests that the cell loss by sloughing was an active process in the involution of prostate glands which also showed differential sensitivity to castration stimuli in mice.This research was supported by Medical Research Service of Veterans Administration     

Fine structural studies of rat seminal vesicle in castrated and intact animals following estrogen treatment.

The effect of estradiol and/or testosterone upon secretion by seminal vesicle in castrated and intact rats was assessed in young adult Sprague-Dawley rats, using light microscopy (LM), transmission (TEM) and scanning (SEM)electron microscopy. Hormones were injected daily for ten days beginning ten days after castrations were performed. The normal rat seminal vesicle, as revealed by SEM, was characterized by a large saccular lumen with highly folded walls. Cell surfaces were covered with microvilli, or occasionally displayed a protruding, ruffled surface, sparsely covered with short microvilli. Cytology was normal in testosterone-treated animals. Estradiol treatment of castrated animals stimulated secretion by seminal vesicle epithelial cells as evidenced by the presence of normal secretory bodies, the presence of RER, and moderately hypertrophied Golgi complexes. These glands were not heavier than were glands from castrated, untreated animals, although the epithelial cells were significantly taller. Secretion was maintained in intact animals treated with estradiol, although glands were smaller and epithelial height was reduced. Estradiol and testosterone treatment in combination did not appear to have an additive effect on secretion, weight of the gland, or epithelial height. The following results support the hypothesis that estrogen-induced prolactin synthesis and release may be involved in the mechanism by which estradiol effected stimulation of seminal vesicle epithelium. Prolactin-treated, castrated animals exhibited focal areas of stimulated epithelium. In hypophysectomized animals (untreated controls), the seminal vesicle epithelium retained some secretory bodies and secretory fluid in the glandular lumen; epithelial height was taller than that in castrated controls. Estrogen treatment reduced the epithelial height to that of castrated controls; there was no evidence of secretion. This suggests that in the absence of anterior pituitary hormones, including prolactin, the stimulatory effect of estradiol on seminal vesicle epithelium was nullified. In adrenalectomized/castrated animals, estradiol treatment stimulated secretion in seminal vesicle epithelium just as in non-adrenalectomized/castrated animals. This indicates that the adrenal gland plays a non-essential role in the action of estrogen on seminal vesicle epithelium.     

Antigenic homogeneity of male Müllerian gland (MG) secretory proteins of a caecilian amphibian with secretory proteins of the mammalian prostate gland and seminal vesicles: evidence for role of the caecilian MG as a male accessory reproductive gland

Whereas in all other vertebrates the Müllerian ducts of genetic males are aborted during development, under the influence of Müllerian-inhibiting substance, in the caecilian amphibians they are retained as a pair of functional glands. It has long been speculated that the Müllerian gland might be the male accessory reproductive gland but there has been no direct evidence to this effect. The present study was undertaken to determine whether the caecilian Müllerian gland secretory proteins would bear antigenic similarity to secretory proteins of the prostate gland and/or the seminal vesicles of a mammal. The secretory proteins of the Müllerian gland of Ichthyophis tricolor were evaluated for cross-reactivity with antisera raised against rat ventral prostate and seminal vesicle secretory proteins, adopting SDS-PAGE, two-dimensional electrophoresis and immunoblot techniques. Indeed there was a cross-reaction of five Müllerian gland secretory protein fractions with prostatic protein antiserum and of three with seminal vesicle protein antiserum. A potential homology exists because in mammals the middle group of the prostate primordia is derived from a diverticulum of the Müllerian duct. Thus this study, by providing evidence for expression of prostatic and seminal vesicle proteins in the Müllerian gland, substantiates the point that in caecilians the Müllerian glands are the male accessory reproductive glands.     

Anatomy of the major lacrimal gland of rhesus monkeys (Macaca mulatta)

The major lacrimal gland of rhesus monkeys is impalpable within the fatty connective tissue of the upper lateral quadrant of the orbit. Acini of the lacrimal glands are composed of both sparsely and heavily granulated cells that histochemically resemble serous acinar cells of the submandibular salivary gland. The cytoplasmic granules are strongly periodic acid-Schiff (PAS)-positive, and some are also stained by alcian blue for acidic mucosubstances. The lacrimal gland has a simple duct system of intralobular ducts and interlobular excretory ducts. Lymphocytes and plasma cells are common in the periductal stroma. Major lacrimal glands of rhesus monkeys are suitable for comparative and correlative studies of lacrimal and salivary diseases and radiation responses.     

Ultrastructural localization of WGA,RCA I,LFA and SBA binding sites in the seven-day-old mouse embryo

Summary Transglutaminases are Ca²⁺-dependent intra-and extracellular enzymes catalyzing the cross-linking between proteins and/or polyamines, thereby eliciting divergent physiological effects such as fibrin clot stabilization or hair follicle cross-linking. A secretory transglutaminase (EC 2.3.2.13) was isolated from the coagulating gland of the rat. The protein is highly glycosylated. A fraction purified to homogeneity was used as an antigen to raise polyclonal antibodies in rabbits. These antibodies were used to identify the secretion sites of the protein within the male accessory sex glands as well as to study the immunological relationships of the respective antigen within different organs of different species. In the rat, the coagulating gland and likewise the dorsal prostate gave a positive immunoreaction. In the guinea pig, a closely related protein was detected in the anterior prostate. No cross-reactivity was found with membrane-bound transglutaminase from liver, erythrocytes or blood clotting factor XIIIa. The intraluminal secretion of the aforementioned glands was only weakly stained. No secretory granules were observed in the glandular epithelium but instead bleb-like structures reminiscent of apocrine secretion. A slight background stain of the epithelium remained even in castrated animals where secretion is largely suppressed. The background stain is attributed to a tissue-type, membrane-bound, non-secretory transglutaminase that is not androgen dependent, but instead synthesized only after androgen deprivation.     

Fertilization-promoting peptide in reproductive tissues and semen of the male marmoset (Callithrix jacchus)

Fertilization-promoting peptide (FPP) is present in the prostate gland and semen of some mammals, and has been shown to enhance the fertilizing ability of both epididymal mouse and ejaculated human spermatozoa. The novel peptide may prove of importance for the treatment of some cases of male infertility, and a suitable animal model would be useful to test this hypothesis. To this end, we examined reproductive tissues and semen of the male marmoset for the presence of FPP. Peptides were extracted from seminal plasma, testes, prostate, and bulbourethral glands of intact and castrated male marmosets. The peptides were identified by ion-exchange chromatography followed by radioimmunoassay. The mean concentration of FPP immunoreactivity in semen from intact males was 58.7 nM (SE ± 9.9 nM, n = 10), and anion-exchange chromatography revealed FPP as the only immunoreactive peptide present. Analysis of tissues revealed that FPP in semen was likely to be derived from the prostate gland, which contained this peptide as the major source of immunoreactivity (10.86 pmol/gland; SE ± 4.39 pmol/gland, n = 4). Only low concentrations of FPP were detectable in the bulbourethral glands, and the peptide was undetectable in the testis. Surprisingly, FPP was readily detectable in the seminal plasma from one castrated marmoset and was present in the prostate gland from 3 castrates at levels which did not differ significantly from those in intact animals (5.47 pmol/gland, SE ± 1.64 pmol/gland, n = 3). Plasma testosterone measurements indicated that residual circulatory androgens remained after castration, which may be consistent both with the maintenance of mating behavior and the presence of prostatic FPP. We conclude that FPP is present within the prostate gland and seminal plasma of the marmoset at concentrations consistent with a role in male fertility in this species. Mol. Reprod. Dev. 47:113–119, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of lacrimal gland carbonic anhydrase VI.

We have previously demonstrated by immunohistochemistry the presence of secreted carbonic anhydrase (CA VI) in the acinar cells of the rat lacrimal glands. In this study we purified the sheep lacrimal gland CA VI to homogeneity and demonstrated by Western analysis that it has the same apparent subunit molecular weight (45 kD) as the enzyme isolated from saliva. RT-PCR analysis showed that CA VI mRNA from the lacrimal gland was identical to that of the parotid gland CA VI mRNA. An RIA specific for sheep CA VI showed the lacrimal gland tissue concentration of the enzyme to be 4.20 +/- 2.60 ng/mg protein, or about 1/7000 of the level found in the parotid gland. Immunohistochemistry (IHC) and in situ hybridization (ISH) showed that lacrimal acinar cells expressed both immunoreactivity and mRNA for CA VI. Moreover, CA VI immunoreactivity was occasionally observed in the lumen of the ducts. Unlike the parotid gland, in which all acinar cells expressed CA VI immunoreactivity and mRNA, only some of the acinar cells of the lacrimal gland showed expression. These results indicate that the lacrimal gland synthesizes and secretes a very small amount of salivary CA VI. In tear fluid, CA VI is presumed to have a role in the maintenance of acid/base balance on the surface of the eye, akin to its role in the oral cavity.     

Androgens affect the processing of secretory protein precursors in the guinea pig seminal vesicle. I. Evidence for androgen-regulated proteolytic processing

The guinea pig seminal vesicle epithelium synthesizes and secretes four major secretory proteins (SVP-1-4). Previous work has established that these four proteins are cleaved from two primary translation products in a complex series of protein processing reactions. The present studies suggest that these protein processing reactions are regulated by androgens. In vitro labeling of seminal vesicle proteins revealed significant differences in the patterns of secretory protein intermediates produced by tissue from intact and castrated animals. Seminal vesicle tissue explants from castrated animals secreted a subset of the processing intermediates secreted by tissue from intact animals. The changes in the patterns of secretory protein intermediates became more pronounced with increasing time after castration, and were fully reversible by treatment of castrated animals with testosterone, suggesting that androgens were affecting the processing or secretion of secretory protein precursors. Amino-terminal protein sequencing of secretory protein processing intermediates that accumulate in the seminal vesicle lumen after castration suggests that the guinea pig seminal vesicle contains an androgen-regulated proteolytic processing activity.     

Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.     

Quantitative morphology and carbohydrate histochemistry of the mouse submandibular gland following prepubertal castration.

The endocrinologic basis for morphological and biochemical sex differences in the mouse submandibular gland have not been clarified. Previous studies have emphasized the maintenance of glandular differences in adult animals, rather than considering the factors responsible for their developmental etiology. Male CD-1 mice were castrated at intervals between 10 and 50 days of age and killed at 100 days. The quantitative development of granular tubules and the carbohydrate histochemistry of the submandibular glands were compared to untreated males and females. The area of granular tubules increased with age at castration. Nested analysis of variance indicated significant differences among treatments and among sections within individual glands. No group of castrated males had a greater development of tubules than untreated females. Carbohydrate histochemistry demonstrated an increase in carboxylated mucosubstances in the acinar cells and granular tubule cells of castrated animals.     

The secretory activity of rat nasal glands and the effect of cholinergic drugs

Summary The secretory behaviour of rat nasal glands, under normal conditions and after the application of cholinergic drugs, has been studied using morphological and radiobiochemical techniques.Autoradiography and electrophoresis provide evidence for the selective incorporation of ³H-arginine into the glycoprotein containing fraction of the nasal glandular secretion. Radiobiochemical experiments show that labelled arginine is rapidly incorporated into the acinar cells of unstimulated glands, although it takes approximately 4 h before the labelled secretory proteins leave the cells. The secretion of proteins is stimulated by the parasympathetic agonist pilocarpine, whose main action is to promote discharge. Histological sections show a depletion of secretory granules after pilocarpine treatment. The cholinergic antagonist atropine inhibits the secretion; the acinar cells are completely filled with secretory granules following this treatment. The time course of the events following atropine administration suggests that there is no feed-back system controlling glycoprotein synthesis.The techniques employed here therefore appear to be useful for studying the effects of drugs that interfere with the secretory activity of the nasal glands.     

Characterization of immortalized rabbit lacrimal gland epithelial cells

Summary To establish an immortalized lacrimal gland epithelial cell line, the orbital lacrimal glands of normal New Zealand White rabbits were multiply injected with an immortalizing amphotropic retroviral vector (LXSN16E6E7) containing the E6 and E7 genes of human papillomavirus type 16. Lacrimal glands were removed after 2 d and acinar epithelial cells were isolated and cultured on Matrigel-coated 60 mm² plates containing DMEM-F12 supplemented with 5% Nu-serum V. Transformed cells were selected in G418 sulfate for 7 d and passaged. Morphology of the immortalized cells was similar to that described for normal acinar cells both in vivo and in vitro, with rough endoplasmic reticulum and secretory granules. These characteristics remained unchanged and the cells continued to exhibit typical polygonal epithelioid structure. The cells have been maintained in culture for 14 mo. and have gone through 58 passages without loss of proliferation or epithelial cell characteristics. Immunohistochemistry and Western blots showed positive reactivity to secretory component, transferrin, and transferrin receptor, which are typical proteins found in the lacrimal gland. Functional analysis by stimulation with a cholinergic agonist, carbachol (100 μM), resulted in a significant release of protein. This is the first report of an immortalized rabbit lacrimal epithelial cell. These cells will provide a valuable tool for the molecular analysis of lacrimal gland epithelial cell functions.     

Urethral glands of the male mouse contain secretory component and immunoglobulin A plasma cells and are targets of testosterone.

The occurrence and possible functions of mucosal immunity in the male urogenital tract have not been extensively investigated. In this study we used immunolabeling to localize secretory component (SC) and immunoglobulin (Ig) A in the urogenital tract of the male mouse. SC was located in the ventral prostate, while SC and IgA plasma cells were both detected in the urethral glands in the pelvic and bulbous portions of the urethra. SC and IgA were not observed elsewhere in the urogenital tract. We also examined the ventral prostate and urethral glands of sham-castrated, oil-treated castrated, and testosterone-treated castrated mice. There was a striking reduction in the size of the ventral prostate and urethral glands in oil-treated castrates compared to the other two groups, based on gross and histological morphology. Morphometric analysis showed that the cell and nuclear sizes of the urethral gland acinar cells were reduced after castration and restored to normal size by testosterone treatment. Androgen receptors (AR) were localized in the nuclei of urethral gland cells by immunocytochemistry using anti-AR antibodies. Labeling of SC and IgA plasma cells was similar in the urethral glands and ventral prostates of sham- and testosterone-treated castrates, but was reduced or absent at these sites in oil-treated castrates. These studies show that the ventral prostate and urethral glands may be sites for secretory immunity in the male murine urogenital tract, and that the urethral glands are targets for testosterone.     

Isoprenaline-induced cell proliferation in mouse salivary glands: the effect of castration

The proliferative response to isoprenaline in the submaxillary and parotid glands of the Balb/c mouse has been studied in the intact male and female, and also in the male castrated one month prior to stimulation. The hyperplastic response of the acinar cells has been monitored by serial measurements of the flash tritiated thymidine labelling index and the mitotic index. Castration caused the atrophy of the granular ducts in the submaxillary gland, and therefore an increased predominance of the acini. At one month after castration the acini occupied an area almost 1.5-fold greater than that of the granular ducts, but this was not as great as in the intact female gland where acini occupied twice the area of the granular ducts. Hyperplasia was induced by a single injection of isoprenaline (0.3 mM/kg body weight). The response of the submaxillary gland in the intact male and intact female was very similar, DNA synthesis commencing 21-24 h after stimulation and mitotic activity first noted after 33-36 h. On the other hand, in the submaxillary gland of the castrated male, DNA synthesis began after only 18-21 h and mitotic activity after only 27-30 h. A metaphase arrest experiment with vincristine confirmed the more prompt response in the castrated animals; between 33-36 h after isoprenaline injection, the rate of entry of cells into mitosis was 4 cells/100 cells/h in the castrated group but only 0.4 cells/100 cells/h in the intact males. Thus castration appears to bestow a unique state of responsiveness upon the submaxillary gland to isoprenaline stimulation. The mechanisms underlying this change are not yet understood, for it is paradoxical that atrophy of a structural component rich in specific protein growth factors can alter the format of isoprenaline-induced hyperplasia in acinar cells that produce secretory glycoproteins.     

Intravesicular Fas localization in epithelial cells of castrated rat prostate glands

Androgenic steroids regulate the development and size of mammalian prostate epithelial cells. To evaluate the relationship between Fas-Fas ligand system and apoptosis in prostate epithelial cells of the castrated rats, we have examined immunocytochemical localization of Fas antigen in the castrated rat prostate glands at a series of different times. We used a rabbit polyclonal anti-Fas antibody with a streptavidin-biotin method and confocal laser scanning method or an immunogold method. Fas immunolocalization was examined in ventral lobes of prostate glands taken from intact or castrated adult male Wistar rats on day 1, 2, 3, 4 and 5 by light or electron microscopy. At a light microscopic level, the castrated prostate epithelial cells showed mostly Fas immunolocalization in their apical parts of cytoplasm on day 2 after the castration. In addition, their extent of the Fas expression was expanded throughout the cytoplasm in proportion to the androgen ablation periods, and later the Fas expression was detected at luminar or basolateral sides of the epithelial cells. Both immunogold labeling with ultrathin sections and immunoperoxidase technique with cryostat sections demonstrated that Fas was localized mainly in secretory granules of the castrated prostate epithelial cells and some parts of their cell membranes at later stages. Our immunocytochemical findings showed that Fas expression was time-dependently induced in most of the prostatic epithelial cells after castration of rats. The rate of Fas-expressing epithelial cells was too high and inconsistent with the previously reported rate of TUNEL-positive ones. The membrane-associated Fas may have little effect on the apoptosis in the present case, bacause a lot of soluble Fas was secreted from the prostatic epithelial cells. A further study is needed to clarify some significance of the secretory Fas in the prostatic epithelium after the rat castration.     

The human submandibular gland: immunohistochemical analysis of SNAREs and cytoskeletal proteins

Submandibular acinar glands secrete numerous proteins such as digestive enzymes and defense proteins on the basis of the exocrine secretion mode. Exocytosis is a complex process, including a soluble NSF attachment protein receptor (SNARE)-mediated membrane fusion of vesicles and target membrane and the additional activation of cytoskeletal proteins. Relevant data are available predominantly for animal salivary glands, especially of the rat parotid acinar cells. The authors investigated the secretory molecular machinery of acinar (serous) cells in the human submandibular gland by immunohistochemistry and immunofluorescence and found diverse proteins associated with exocytosis for the first time. SNAP-23, syntaxin-2, syntaxin-4, and VAMP-2 were localized at the luminal plasma membrane; syntaxin-2 and septin-2 were expressed in vesicles in the cytoplasm. Double staining of syntaxin-2 and septin-2 revealed a colocalization on the same vesicles. Lactoferrin and α-amylase served as a marker for secretory vesicles and were labeled positively together with syntaxin-2 and septin-2 in double-staining procedures. Cytoskeletal components such as actin, myosin II, cofilin, and profilin are concentrated at the apical plasma membrane of acinar submandibular glands. These observations complement the understanding of the complex exocytosis mechanisms.     

Early changes in the dog prostate after castration. An ultrastructural study

Using electron microscopic techniques the prostate glands of male Beagle dogs were studied 3 days after castration. At this time marked differences in the extent of alterations of the glandular epithelium were observed: Whereas several acini showed only minor changes with reduction of epithelial height and diminution of secretory granules, many acini were severely affected with pronounced alteration of cellular structure and accumulation of large lipid droplets. A constant feature was the stimulation of the basal cells of the grandular epithelium. Additionally, in some areas of the gland aggregations of stimulated basal cells forming an acinus-like structure with a slit-like lumen were found. Our study shows that castration leads to marked alterations of prostatic epithelium within a short time. Androgen deprivation causes regressive changes of secretory epithelial cells, but clearly stimulates the basal cell population.