Electron microscopic identification of the histamine-storing argyrophil (enterochromaffin-like) cells in the rat stomach

Summary In the oxyntic gland area of the rat stomach the histamine-containing epithelial cells (also referred to as enterochromaffin-like cells because of their morphologic similarity with the 5-hydroxytryptamine-storing enterochromaffin cells) constitute the system of argyrophil cells in this area as previously shown by the combined use of fluorescence and light microscopic techniques. By performing the argyrophil staining reaction directly on ultra-thin sections it could be demonstrated in the electron microscope that the argyrophil cells have features suggesting that they are endocrine. Based on the ultrastructure of their secretory granules at least two such endocrine cell systems—both argyrophil—could be recognized in the oxyntic glands. The silver deposits were accumulated over the secretory granules of both these cell systems.It is well known that after injection of 1-3,4-dihydroxyphenylalanine, the histamine-storing (enterochromaffin-like) cells of the oxyntic glands store also dopamine. Under these conditions the enterochromaffin-like cells stain argentaffin, which has been shown at the light microscopic level. Also this reaction could be performed directly on ultra-thin sections. By electron microscopy it was then established that the two endocrine cell systems of the oxyntic gland area stained argentaffin upon treatment with 1-3,4-dihydroxyphenylalanine, and that the staining was confined to the secretory granules.The results clearly show that the enterochromaffin-like cells of the rat oxyntic gland area (which is devoid of 5-hydroxytryptamine-containing enterochromaffin cells) are identical with cells characterized as endocrine by ultrastructural criteria, and that gastric non-mast-cell histamine occurs in at least two separate systems of enterochromaffin-like cells.     

Androgen regulation of the cellular Distribution of the true tissue kallikrein mK1 in the submandibular gland of the mouse.

The kallikrein gene family encodes for at least four different proteases in the mouse submandibular gland (SMG): mK1 (true tissue kallikrein), mK9, mK13, and mK22. These enzymes and many other biologically active proteins are synthesized by the granular convoluted tubule (GCT), a specialized segment of the SMG duct system. The GCT is under multihormonal regulation by androgens, thyroid hormones, and adrenocortical hormones. Androgens suppress synthesis of mK1 in the SMG but enhance expression of the other three kallikreins. We prepared an antibody with limited immunoreactivity for mK1 and used it to examine the effects of androgen status on the distribution of this isozyme in the SMGs of developing and mature mice by immunoperoxidase staining for the light microscope and immunogold labeling for the electron microscope. In prepubertal mice, every immature GCT cell contains mK1, confined to an accumulation of small granules in the subluminal cytoplasm. In mature mice, not every GCT cell contains mK1, and in those cells that do there is considerable intergranular variation in the intensity of staining for mK1. GCT cells containing mK1 are much more abundant in the glands of females than of males, resulting in a peculiar sexually dimorphic mosaic distribution of this isozyme in the mature SMG. Castration of adult males increases the number of GCT cells expressing mK1. Administration of androgen to intact or castrated males or to intact females reduces the number of cells staining for mK1. In all cases, immunogold labeling for mK1 is confined to secretory granules. No fine structural differences were noted between cells that were positively or negatively stained for mK1. Therefore, although GCT cells appear to be composed of a uniform population of cells on the basis of morphology alone, they are not homogeneous in their content of secretory proteins. These results indicate that androgen regulation of GCT cells is more complex than has been appreciated to date.     

Ultrastructural localization of prolactin,growth hormone and luteinizing hormone by immunocytochemical techniques in the bovine pituitary

Summary The technique of ultrastructural immunocytochemistry involving the unlabeled antibody and the soluble peroxidase-antiperoxidase complex was used to identify and describe the prolactin (P) cells, somatotropic (STH) cells and luteinizing hormone (LH) cells in the bovine anterior pituitary gland. This method was used to localize the three hormones at the electron microscopic level. Staining of varying intensity was found on the secretory granules and on the small granules and vesicles within the Golgi complex. No stain was found in nuclei, on mitochondria or in the endoplasmic reticulum.     

On argyrophil reactions of endocrine cells in the human fetal pancreas

Summary The endocrine cells in the pancreas of five human fetuses with gestational ages of 18–20 weeks were examined by light and electron microscopy with special regard to argyrophil reactions. B-cells and typical A and D-cells were easily identified electron microscopically on the basis of their typical secretory granules. In the Grimelius argyrophil silver stain, a concentration of silver grains over the less electron dense peripheral mantle of the A-cell secretory granules was observed by electron microscopy. In the Hellerström and Hellman modification of the argyrophil Davenport alcoholic silver stain, silver grains were concentrated over the internal structures of the D-cell secretory granules. With this stain an accumulation of silver grains was also seen at the surface of the A-cell secretory granules. The argyrophil reaction of the A-granules was less pronounced than in the D-cells. In addition to B-cells and A- and D-cells, two other types of endocrine cell were observed by electron microscopy. These cells were argyrophil with the silver impregnation method of Grimelius. The electron microscopic findings at least partly explain the frequent overlapping between the two staining methods observed at the light microscope level.This study was supported by the Swedish Medical Research Council (Project No. 102)     

Ultrastructure and complex carbohydrate ultracytochemistry of the cat parotid gland

The structure and glycoconjugate content of the cat parotid gland were analyzed at electron microscopic level by applying morphological techniques and three ultrastructural histochemical methods - HID-TCH-SP, LID-TCH-SP and PA-TCH-SP. This gland appeared as a typical salivary gland composed of acinar secretory cells, intercalated ducts, striated ducts and excretory ducts. The most common configuration of secretory granules consisted of a dense core surrounded by a variable electron-lucent halo. All ductal segments were characterized by the presence of different cell populations and small apical granules greatly different from those localized in the acinar cells. By using HID-TCH-SP we were able to demonstrate that in a few acinar cells there are sulphated sites, whereas PA-TCH-SP staining revealed the presence of vic-glycol radicals in all acinar cells preferentially located on the halo of secretory granules.     

The influence of fixation on the carbohydrate cytochemistry of rat salivary gland secretory granules

Synopsis A series of studies was performed to assess the optimum fixation conditions for staining of carbohydrate-containing constituents of rat salivary gland secretory granules. In the parotid and submandibular salivary glands of the rat, the reactivity of secretory granules, at both the light and electron microscopic level, with routine stains and with cytochemical reagents was highly dependent upon the nature of the fixative employed. At the light microscopic level, secretory granules in rat parotid gland were periodic acid-Schiff (PAS) positive if fixed with buffered formalin fixatives. However, if the gland was fixed with lipid-solvent-containing fixatives, or with formalin at a very acid pH (as in Bouin's fixative), the PAS reactivity of the granules was lost. In the submandibular gland of rats, the acinar cells and granular tubules behaved similarly after such fixation in terms of their PAS reactivity, particularly in males; acinar cells of the female submandibular gland stained only lightly with PAS. At the fine structural level, the morphology of secretory granule constituents depended on the buffer used (cacodylate, phosphate or collidine) and on whether or not tissue was post-osmicated. Post-osmication considerably reduced the reaction of secretory granule components with stains for carbohydrates.The experimental evidence indicated that the carbohydrate-containing components of both parotid and submandibular gland secretory granules were not typical long-chain neutral or acidic mucins, but were rather glycolipids or lipophilic glycoproteins that were solubilized by lipid solvents or at acidic pH and were lost or destroyed in the presence of strong oxidants.     

Immunocytochemical localization of nerve growth factor (NGF) in the submandibular gland of adult mice by light and electron microscopy

Summary Nerve growth factor (NGF) was localized in the submandibular gland of adult male mice by a direct immunocytochemical method using highly purified antibodies against NGF coupled to horseradish peroxidase. In light microscopic sections the reaction product was entirely confined to the cells of the secretory tubules. The acinar part of the gland was free of reaction product. This finding was confirmed by electron microscopy. Within the cells NGF was localized exclusively in the apical secretory granules.No reaction was observed in the rough endoplasmic reticulum, the Golgi region or in the granules of the basal part of the cells. This observation favours the assumption that NGF is derived from a precursor molecule and that the precursor is transformed into immunologically active NGF within the secretory granules during their transport from the basal to the apical part of the tubular cells. Stimulation of the submandibular gland with carbachol (2 mg/kg) led to a massive release of the content of the secretory granules, including NGF, into the salivary duct.We wish to thank Dr. C. Rufener, Geneva, for communicating to us a new method of antibodyperoxidase coupling and Mrs. M. Durand-Wenger for her excellent technical assistance. This study was supported by the Swiss National Foundation for Scientific Research (Grant Nr. 3.432.74)     

Light and electron microscopic localization of ACTH and pro-opiomelanocortin-derived peptides in human developmental and neoplastic cells

Oncofetal aspects of ACTH and pro-opiomelanocortin (POMC)-derived peptides were studied immunohistochemically at the light and electron microscopic level in human fetal pituitary glands, pituitary adenomas, and small-cell carcinoma of the lung. ACTH, beta-endorphin, and gamma-MSH were localized in the same cells of both fetal and adult pituitary, as well as in the above-mentioned neoplastic tissues. However, alpha-MSH was observed only in the early fetal pituitary, its concentration decreasing with advancing gestational age. The adult pituitary contained only a few alpha-MSH-positive cells. By immunoelectron microscopy, ACTH in the adult pituitary was localized exclusively in the secretory granules. In fetal pituitary at 9 weeks' gestation, ACTH was localized in the perinuclear spaces (PNS), cisternae of rough endoplasmic reticulum (RER), Golgi saccules, and secretory granules. The staining pattern of ACTH in these organelles varied from cell to cell. In fetal pituitaries of greater gestational ages, ACTH was localized in secretory granules. The pituitary adenomas mimicked the staining characteristics of the adult pituitary, i.e., negative or only very occasional alpha-MSH staining and localization of ACTH in the secretory granules. The ectopic ACTH-producing tumors showed a staining pattern similar to that of the early fetal pituitary, i.e., positive staining for alpha-MSH and the presence of ACTH in PNS and cisternae of RER.     

Coexistence of gland mucous cell-type mucin and lysozyme in gastric gland mucous cells

Class III mucin, identified by paradoxical concanavalin A staining, is confined to gastric gland mucous cells and is an essential component of the gastric surface mucous gel layer. The pretreatment required has hampered the application of this method to electron microscopic studies. Antibody HIK1083 reacts selectively with class III mucins. The present study was undertaken to explore, electron microscopically, the immunoreactivity of the human stomach to HIK1083. We examined normal mucosa from resected human stomachs (five cases; formalin-fixed, paraffin-embedded) and gastric biopsy specimens from patients with early gastric cancer [nine cases; glutaraldehyde- and osmium-fixed, epoxy-embedded (seven cases) and half-strength Karnovsky’s solution-fixed, Lowicryl K4M-embedded (two cases)]. Immunostaining with HIK1083 and anti-lysozyme antibody was examined under light and electron microscopes. Gland mucous cells were labeled with HIK1083, and lysozyme was detected in some gland mucous cells and surface mucous cells. Electron microscopically, the secretory granules of gland mucous cells contained a single electron-dense core. HIK1083-positive mucins and lysozyme coexisted in the secretory granules of gastric gland mucous cells. HIK1083-reactive mucins and lysozyme were distributed in the matrix and in the dense core of these secretory granules, respectively. HIK1083 can be used for electron immunohistochemistry. Accepted: 1 December 1999     

Developmental and androgenic regulation of the immunocytochemical distribution of mK1, a true tissue kallikrein, in the granular convoluted tubule of the mouse submandibular gland.

The action of androgens on the immunocytochemical distribution of mK1, a true tissue kallikrein, was examined in the submandibular gland (SMG) of developing and adult mice by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. In both sexes at 3 weeks of age, essentially all of the immature granular convoluted tubule (GCT) cells were uniformly immunostained. At 4 weeks of age (the onset of puberty), morphological differences between the two sexes appeared in the GCTs, in which some cells became immunonegative. Thereafter, the immunonegative GCT cells became more abundant in the SMG of males than of females and considerable intercellular variation in staining intensity for mK1 was seen, especially in males. A few slender GCT cells with strong immunoreactivity appeared in GCT segments only in males. Castration of males resulted in an increase in the number of immunopositive GCT cells, whereas administration of dihydrotestosterone (DHT) decreased the number of immunopositive GCT cells in the SMGs of both sexes. Slender GCT cells immunoreactive for mK1 were seen in females treated with DHT for 6 days. However, there were no immunostained slender GCT cells in female SMGs after injection of DHT for 2 weeks. Immunoelectron microscopy disclosed this type of cell in male SMGs, which closely resembles immature GCT cells of prepubertal mice, with a few small secretory granules uniformly labeled with gold particles, a sparse Golgi apparatus and RER, and basal infoldings. In mature male SMGs and in SMGs of DHT-treated females and castrated males, typical GCT cells had a well-developed Golgi apparatus and a net-like RER but few to no basal infoldings, whereas in the female gland equivalent cells had moderately developed RER and some basal infoldings. These results suggest that mK1 is one of the enzymes characteristically present in immature GCT cells and that its synthesis is inhibited in part by androgens, resulting in decreased numbers of immunopositive cells.     

Electron microscopic classification of amine-producing endocrine cells by selective staining of ultra-thin sections

Summary The argyrophil, argentaffin and chromaffin reactions were performed directly on ultra-thin sections for examination in the electron microscope. Glutaraldehyde fixation was appropriate for the argentaffin and chromaffin reactions; additional fixation with osmium tetroxide, however, caused impairment of these reactions. Fixation with formaldehyde, but not with glutaraldehyde, was adequate for the argyrophil reaction; post-fixation with osmium tetroxide did not affect this staining. At the light microscopic level the staining reactions were correlated with fluorescence histochemistry according to the method of Falck and Hillarp. The techniques described were used to study certain amine-producing endocrine cell systems: adrenal medullary cells and thyroid parafollicular cells of the mouse, gastric endocrine cells from the oxyntic gland area of the mouse, rat and rabbit. All these cells stained argyrophil. The adrenal medullary cells and one cell type in the oxyntic gland area of the rabbit were strongly argentaffin and chromaffin. The remainder of the cells were non-argentaffin and non-chromaffin but could be induced to give an argentaffin (and chromaffin) reaction after injection of the animals with l-3,4-dihydroxyphenylalanine or l-5-hydroxytryptophan, a treatment which is known to result in the accumulation of the highly reducing dopamine and 5-hydroxytryptamine, respectively, in these endocrine cells. Without exception the precipitates formed in all the staining reactions accumulated selectively over the secretory granules of the cells.The techniques described permit differential staining of consecutive ultra-thin sections for electron microscopic characterization of one and the same cell. They will provide information necessary for correlative studies of the stainable cells at the light and electron microscopic levels.     

Thyroxine accelerates the differentiation of granular convoluted tubule cells and the appearance of epidermal growth factor in the submandibular gland of the neonatal mouse

Summary The effects of the administration of thyroxine (T4) on the postnatal cytodifferentiation of granular convoluted tubule (GCT) cells of the submandibular gland (SMG) of Lewiss-Webster mice were studied by light and electron microscopy. From birth, mice of both sexes were injected daily with T4 (sc 0.4 g/g BW) and were sacrificed 24 h after the last injection at 7, 9, 11, 14 and 21 days of age. Control mice received vehicle only. In control mice, granulated striated duct (SD) cells were first detected at 9 days and 7 days of age by light- and electron microscopy, respectively. Furthermore, a few scattered granulated SD cells were observed by light microscopy as early as day 7 in T4-treated mice of both sexes. At 21 days of age, in mice given T4, GCT cells were larger and more numerous and the Golgi apparatus, rough endoplasmic reticulum, and secretion granules were more abundant. In control mice, immunocytochemical staining for epidermal growth factor-(EGF) was first detectable at day 21 at the light- and electron-microscopic levels. However, positively stained cells were first observed in T4-treated mice by light- and electron-microscopic immunocytochemistry at 14 and 11 days of age, respectively. Moreover, in the 21-day-old T4-treated mice, the number of immunoreactive GCT cells, as well as the intensity of the staining per cell, was markedly increased as compared to controls. EGF immunostaining was restricted to GCT cells, and by immuno-electron-microscopy was only seen in apical secretory granules in granulated SD cells and GCT cells. There were no sex differences in the differentiation of the duct system under any conditions. It is concluded that T4 stimulates the biosynthesis of EGF by an acceleration of the differentiation of the GCT precursor cells to mature cells.Supported in part by grant no. MT-5730 from the Medical Research Council of CanadaHolder of a fellowship from the Medical Research Council of CanadaScholar of the Fonds de la Recherche en Santé du Québec     

Ultrastructural visualization of galactosyl residues in various alimentary epithelial cells with the peanut lectin-horseradish peroxidase procedure

Summary A conjugate of peanut lectin with horseradish peroxidase (PL-HRP) has been employed for ultrastructural localization of glycoprotein with presumed terminal galactose residues in mouse alimentary epithelial cells. The PL-HRP conjugate imparted electron opacity in sites that stain at the light microscopic level, as for example, Golgi cisternae in surface epithelial cells of the stomach and in superficial and deep crypt cells and goblet cells of the large intestine. Ultrastructural staining revealed that Golgi cisternae intermediate between the trans and cis faces stained selectively in these sites. Secretion stored in secretory granules or Golgi vesicles in the cells lacked affinity for PL-HRP conjugate, however. Selective staining of intermediate Golgi cisternae in cells with unreactive secretory product is interpreted as indicating the site of galactosyl transferase activity and a location where glactose occurs transitorily as the terminal sugar in the glycoprotein side chains. The luminal aspect of the surface epithelial cells in the stomach and columnar cells in the colon also stained, but with some variability. Staining of these surfaces was considered possibly attributable to PL affinity of some of the secretory glycoprotein which, after absorbing to the cell surface, lost terminal sialic acid through action of luminal enzyme. PL-HRP conjugate stained granules in pancreatic zymogen cells near the block surface but not in other cells, presumably because of limited penetration of reagent. Secretion on the surface of pancreatic acinar cells or in the lumen also exhibited affinity for PL-HRP complex as did the luminal surface of gastric chief cells. Staining of secretion in the pancreatic zymogen cells and gastric chief cells for galactose appeared inconsistent with lack of evidence for presence of glycoprotein in these sites which failed to stain with the periodic acid-Schiff or periodic acid-thiocarbohydrazide-silver proteinate methods for demonstrating glycoprotein at the light and electron microscopic levels. This discrepancy points to possible selective binding of PL-HRP conjugate to a moiety other than terminal galactose of glycoprotein in a few histologic sites. These results demonstrate the applicability of the PL-HRP technique at the ultrastructural level and provide information concerning the chemical structure of epithelial cell glycoproteins and their biosynthesis.     

Localization of glucagon-like peptide (GLP) immunoreactants in human gut and pancreas using light and electron microscopic immunocytochemistry

The distribution of peptide immunoreactivities predicted from the sequence of the human preproglucagon gene in enteroglucagon (EG; glicentin-like immunoreactant-containing) cells of the human gut and A cells of the pancreas has been determined by light and electron microscopic immunocytochemistry. At light microscopy the application of peroxidase-antiperoxidase and immunogold-silver staining methods has revealed that glucagon-like peptide (GLP-1 and GLP-2) immunoreactivities coexist with a glicentin-related immunodeterminant in human colorectal EG cells and pancreatic A cells. Using single and double colloidal gold probe electron immunocytochemistry, we have been able to show the coexistence of glicentin, GLP-1, and GLP-2 immunoreactivities within single EG cell secretory granules. No morphologic segregation of the proglucagon immunoreactants was observed in EG cells of the colonic mucosa. In pancreatic A cells we have localized GLP-1, GLP-2, and glucagon-[16-29] immunoreactivities solely to the electron-dense core of the secretory granules, whereas glicentin-related immunoreactivity was restricted to the electron-lucent halo. The results obtained in the present study have shown that the peptide immunoreactivities predicted from cDNA sequencing of the human preproglucagon gene are indeed expressed in colorectal EG and pancreatic A cells. The topographical segregation of immunoreactivities in the A cell secretory granule shows that antigenic determinants derived from the C-terminal portion of proglucagon are stored with glucagon in the core of the secretory granule.     

Immunocytochemical localization and lectin-binding properties of the 22 kDa secretory protein from rat ventral prostate

The distribution of the 22 kDa secretory protein from rat ventral prostate was studied by light and electron microscopic immunocytochemistry. An anti-22 kDa protein antiserum was raised in rabbits and its specificity was tested by Western blotting. With the immunofluorescence technique, the 22 kDa protein was detected in the luminal secretions and intracellular apical granules of the ventral prostate. No reaction was observed in the seminal vesicle or dorsolateral prostate. After castration, no intracellular immunoreactivity was detected in ventral prostate, although positively labeled secretory material was retained within the acinar lumen. Restoration of normal intracellular staining pattern was incomplete after 5 daily testosterone injections. At the ultrastructural level, labeling was confined to apical secretory granules and condensing vacuoles. The 22 kDa protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose was shown to bind intensely to wheat germ agglutinin (WGA) but only faintly to Concanavalin A. This protein was thus demonstrated to contain N-acetylglucosamine residues. Accordingly, on tissue sections, WGA reacted intensely with condensing vacuoles and secretory granules.     

Intracellular localization of Prostatic Binding Protein (PBP) in rat prostate by light and electron microscopic immunocytochemistry

Extra- and intracellular distribution of Prostatic Binding Protein (PBP) was studied in the different genital organs of the male rat by immunocytochemistry at the light and electron microscopic levels. PBP was extracted from cytosols of rat ventral prostate and used for immunization of rabbits. The specificity of the antiserum raised was tested by "western blotting" and immunoelectrophoresis. From the different fixatives tested for optimal structural and antigenic preservation of the ventral prostate a mixture containing 2.5% paraformaldehyde, 0.5% glutaraldehyde and 0.5% CaCl2 in cacodylate buffer, 0.05 M, pH 7.3 was selected. Using the immunofluorescence technique and the unlabeled antibody enzyme method PBP-immunoreactivity was detected at the light microscopic level in the luminal secretions of the ventral prostate. No reaction was observed with the seminal vesicle, the coagulating gland, the dorsal and lateral prostates, the epididymis and the testis. Intracellular secretory granules reacting with PBP antiserum were exclusively found in the secretory cells of the ventral prostate. Insufficiently fixed cells showed a diffuse generalized reaction of the cytoplasm indicating a leakage of the antigen from the secretory granules. Such artifacts were common in tissue sections processed with the preembedding-staining procedure. At the ultrastructural level therefore mostly the postembedding staining method was performed using both the unlabeled antibody enzyme method and the ferritin-labeled immunoglobulin technique in osmicated, Epon-embedded tissue. Labeling with either method was intense in the secretory granules and the condensing vacuoles, while the labeling density of the rough endoplasmic reticulum and the Golgi cisternae was in the background range. Castration experiments showed that secretory material displaying PBP immunoreactivity was retained within the acinar lumen of the gland for several days after castration, but was absent from most secretory cells already by four days after castration. Immunocytochemistry of PBP therefore is a very sensitive method for analysing the secretory activity and its androgen dependence of the prostate of the rat.     

Application of silver stains to gastric endocrine cells in plastic sections

Summary A simultaneous light and electron microscopic study of mouse gastric mucosa was made to determine whether the silver nitrate methenamine stain of Duk-Ho Lee could be used to stain gastric endocrine-like cells in plastic embedded tissue. Examination of consecutive thick and thin sections showed that this stain blackened the granules of the predominant type of endocrine-like cell present. Blackening of the granules with silver occured in tissue fixed in osmium tetroxide solution with or without dichromate salt or in tissue fixed in glutaraldehyde then treated with osmium. The intensity of staining was deepest in the osmium-dichromate fixed tissue, but the glutaraldehyde-osmium procedure gave less interference from diffuse silver impregnation and better preservation of detail for electron microscopy.     

Ordered flow of secretion from accessory glands to specific layers of the spermatophore of mealworm beetles: demonstration with a monoclonal antibody

Monoclonal antibodies were produced against the secretory product of the bean-shaped accessory gland (BAG) of male mealworm beetles (Tenebrio molitor). Antibodies from one clone (PL 6.3) recognized a 9,600 dalton protein with a pI of 6.6 which was found in homogenates of the BAG. The PL 6.3 antigen was first detected on Western blots of BAG proteins from 2-day adults, and amounts increased for the next 6 days until reproductive maturation was achieved. The antibody also recognized a polypeptide with a molecular weight (mw) of about 5,000 daltons which we believe to be derived from the larger 9,600 dalton antigen. There are eight types of secretory cells in the BAG. By using light microscopic immunohistochemistry, we localized the antigens recognized by PL 6.3 in cell type 7 (intense staining) and cell type 5 (weak staining). Results from electron microscopic immunocytochemistry showed that antigen PL 6.3 was concentrated in the secretory granules characteristic of each of these two cell types and was absent in all other cell types. PL 6.3 antigens were traced from the BAG into its secretory product and then into the prespermatophoric mass in the ejaculatory duct. The antigen was not randomly mixed with other secretory products of the accessory glands. As it flowed from the BAG and into the ejaculatory duct, it remained in a coherent, precisely localized mass. Within the definitive spermatophore, the PL 6.3 antigen was concentrated in discrete layers of material that line the lumen.     

Immunocytochemical study of the GH cells in the anterior pituitary gland of human fetus II. Anencephalic fetus

In order to elucidate the effects of hypothalamic regulation on the morphology of GH cells, light and electron microscopic immunocytochemical examinations were carried out comparing GH cells in the anterior pituitary gland of anencephalic fetus with those of normal fetuses. Three types of GH cells were identified in the anterior pituitary gland of anencephalic fetus as well as in the normal fetus. Type-I is a small, round cell containing a few small secretory granules. Type-III is a large, polygonal cell with numerous large secretory granules. Type-II is a polygonal cell with medium-sized secretory granules. The Type-II GH cell was predominant in both anencephalic and normal fetuses. The most striking difference between anencephalic and normal fetuses was the presence of atypical forms of the Type II cell. These were polygonal cells containing secretory granules, which were either immunopositive or immunonegative to anti-human GH (anti-hGH) serum. Furthermore, two other types of GH cells were identified. The somatomammotroph (SM cell) contained GH and PRL in different granules within the same cell. Also, a different type of the GH cell was noted containing two varieties of secretory granules; one was immunolabeled only with anti-hGH and the other was not immunolabeled to either anti-hGH or anti-human PRL (anti-hPRL). From these results, we suggest that an absence of hypothalamic regulation in the anencehpalic does not seriously modify GH cell morphology but induces an altered GH storage pattern in some of the cells.     

Immunocytochemical localization of amylase in the parotid gland of developing and adult rats

An antiserum against purified rat parotid amylase was used to localize the protein in parotid glands of developing and adult rats. The unlabeled antibody peroxidase-antiperoxidase method and the protein A-gold colloid technique were used at the light and electron microscope levels, respectively. Immunoreactive amylase was detected in a few scattered cells in the glands of 2-day-old rats. During the following days the number of cells stained immunocytochemically for amylase increased rapidly; at 15 days of age all acinar cells revealed amylase, but the intensity of immunostaining varied from cell to cell. Electron microscopically, amylase was localized in the secretory granules, and by using a more concentrated antiserum, in the rough endoplasmic reticulum and Golgi complex. At early stages of development the acinar cells contained fewer and smaller secretory granules than in adult animals; the gold particles indicative of amylase were randomly distributed over the secretory granules. In the glands of adult rats, amylase was distributed inhomogeneously within the secretory granules. In the majority of secretory granules gold colloid particles were located over the electron-dense portions of the granules. However, secretory granules in which an amylase-rich shell surrounded an amylase-poor or amylase-negative "core" were not infrequent.