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.     

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.     

Estrogen-mediated exocytosis in the glandular epithelium of prostates in castrated and hypophysectomized dogs

Summary Glandular cells in the prostate of the intact, adult dog contain numerous, large secretory granules that are released by exocytosis. Following hypophysectomy or castration, the glandular epithelium atrophies and the secretory granules degenerate and eventually disappear. Pharmacologic doses of estradiol-17 17-cyclopentylpropionate cause the regressed glandular cells to synthesize a new population of smaller granules that are also released by exocytosis, even though estrogen is known to inhibit fluid secretion by the canine prostate. Thus, the mechanisms involved in prostatic synthesis and exocytosis of secretory granules are independent of those regulating fluid secretion and are operative in the absence of androgen or pituitary hormones.     

Epithelial-stromal transition of MMP-7 immunolocalization in the rat ventral prostate following bilateral orchiectomy

Epithelial cells from involuting rat ventral prostate (VP) express Matrilysin (MMP-7) mRNA. Herein, we investigated by immunohistochemistry the MMP-7 protein location and its association with tissue changes following castration in the VP. Normal and castrated adult male Wistar rats were sacrificed at different times after surgery. VP was examined by immunocytochemistry and immunoprecipitation. Castration promoted a shrinking of prostate ducts with an extensive stromal remodeling. In the VP from normal rats, MMP-7 immunoreactivity was found in epithelial secretory granules. Three days after castration, immunostaining for MMP-7 was found in both the epithelial secretory granules and in the stroma just below the epithelium, mainly at the distal ductal tips. At seven and 21 days after castration, the immunostaining for MMP-7 was found only in the stromal space. Immunoprecipitation confirmed the specificity of the primary antibody by rescuing a pro-enzyme form (28kDa) in the prostate extracts. The present results suggest that MMP-7 participates in the epithelial-stromal interface remodeling of the ventral prostate during the involution achieved by castration, probably in the degradation of components of the epithelial basement membrane.     

Immunocytochemical and morphometrical study of thyrotropic cells of Rana ridibunda

Summary Indirect immunoflorescence and PAP techniques for light microscopy as well as the immunogold complex technique for electron microscopy were used to localize and identify thyrotropic (TSH) producing cells in the pars distalis of Rana ridibunda. A double immunostaining procedure was used to distinguish TSH cells from other glycoprotein hormone producing cells. Rabbit anti-human--TSH was used as the primary antiserum and revealed a basophil, PAS and alcian blue positive cell type in the ventro-central zone of the gland. Under the electron microscope, TSH cells show irregular morphology, polymorphic secretory granules with diameters ranging between 120 and 375 nm and poor development of the endoplasmic reticulum and Golgi complex; they are usually polarized towards capillaries. Ultrastructural morphometry (point-counting method) was used to evaluate stereological parameters of rough endoplasmic reticulum, Golgi complex, secretory granules and mitochondria.This work has been supported by grant 2184-83 from the Comisión Asesora (CAICYT) of Spain     

Unique features of secretory granules observed in the pituitary growth hormone-secreting (GH) cells of the musk shrew (Suncus murinus L.)

Summary Unique rod-shaped secretory granules were observed among oval or spherical secretory granules in GH cells of the anterior pituitary gland of musk shrew using the protein A-gold procedure combined with electron microscopy. The rod-shaped and spherical secretory granules were both immunoreactive by the immuno-gold method using antiserum to sheep GH. The rod-shaped secretory granules, which seem to be formed directly from the Golgi vesicles, extend from several hundred to several thousand nm in length. They often touch each other and fuse. The spherical secretory granules are also unique in that they may also fuse with loss of dense contents to leave empty circular membrane profiles in the cytoplasm. Both the rodshaped and spherical secretory granules are secreted from the cell by exocytosis.     

Regulation of gene expression in rat prostate by androgen and beta-adrenergic receptor pathways

Denervation of rat ventral prostate has been accomplished by excising prostatic tissue fragments and implanting them under the renal capsules of intact syngeneic rats. This resulted in a substantial reduction of expression of a major organ-specific secretory protein, prostatic binding protein (PBP). The depressed level of PBP and its subunits and mRNAs could be restored, however, to as much as 80% of control levels by the administration of a pharmacological dose of exogenous androgen, testosterone propionate (TP), and/or a beta-adrenergic agonist, isoproterenol (ISO). Furthermore, compared to ascorbate-treated controls, TP and ISO increased the synthesis of total cellular protein and PBP by the prostatic renal implants. TP and/or ISO also remodelled the luminal epithelial structure and elevated secretory functions. ISO alone had no effect, however, in castrated animals, indicating that androgen plays a dominant role in the restoration of tissue PBP content. Concomitant to increased PBP content and remodelling of prostatic histomorphology, androgen was also found to raise the depressed levels of beta 2-adrenergic and androgen receptors in the prostatic isografts maintained in intact hosts. In contrast, although an established rat prostatic epithelial cell line (NbE-1) contains high affinity androgen receptor, androgen failed to restore beta-adrenergic receptor as well as PBP content in this cultured cell line. These results, taken together, suggest that a tight coupling between androgen receptor and beta 2-adrenergic receptor pathways may be a prerequisite for PBP expression and functional differentiation in the rat ventral prostate gland.     

Distribution of nerve growth factor in the submandibular gland of the male and female mouse

Summary Nerve growth factor (NGF) was localized in the mouse submandibular gland by means of indirect immunofluorescence applied to 0.5 mthick sections of freeze-dried, plastic-embedded tissue. The antibody to NGF (I_gG-fraction) was raised in rabbits immunized with pure 2.5 S NGF from submandibular glands of adult male mice.In the male gland anti-NGF bound selectively to the secretory granules was present in the cells of the granular ducts. Immunoreactive granules extended from the perinuclear region toward the apical pole. In the female gland immunoreactive cells and granules were considerably less abundant than in males. Immunofluorescence was confined to individual secretory cells located in the wall of the granular striated duct.In the present study no support was found for the hypothesis suggesting that immunoreactive NGF is formed within the secretory granules during their transport from the perinuclear region to the apical pole.     

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.     

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.     

Prolonged lifetime of the 410-nm intermediate of bacteriorhodopsin in the presence of guanidine hydrochloride.

Prostatic binding protein (PBP) is a quantitatively important steroid-binding protein present in rat ventral prostate. Electrophoresis on SDS-containing polyacrylamide gels shows that PBP is composed of two subunits, F and S having molecular weights of 16,000 and 18,000. Upon reduction these subunits dissociate further into smaller components. Translation of mRNA from rat ventral prostate in a wheat germ cell-free system or in $\text{Xenopus}$ oocytes results in the formation of polypeptides immunoprecipitable with an anti-PBP antiserum. However, as opposed to the wheat germ system, only the oocytes synthesize polypeptides, that are electrophoretically identical to those of native cytosolic PBP.     

Ultrastructure of the rat posterior pituitary gland and evidence of hormone release by exocytosis as revealed by freeze-fracturing

Summary Posterior pituitary glands from normal rats, and rats which had been deprived of water for varying periods, were examined by the freeze-fracture method. This technique reveals large areas of the nerve cell membrane. Images consistent with exocytosis as the mechanism of release of the neurohypophysial hormones were observed. These modifications were most numerous after the rat had been starved of water for 2 days.In normal rats, the large number of neurosecretory granules within the nerve fibres caused a bulging of the nerve cell membrane. The bulges disappeared 2 days after removal of drinking water. Regions of the membrane displaying bulges were characterised by the absence of the typical membrane-associated particles.It is postulated that the close proximity of the neurosecretory granules to the nerve cell membrane may result in rapid fusion of the neurosecretory granules on stimulation of the gland. The change in properties of the nerve cell membrane overlying the neurosecretory granules, as suggested by the loss of membrane-associated particles, may represent a change in the structure of the membrane to a form which is more favourable for fusion.This work was supported in part by a grant from the New Zealand Medical Research Council.     

Ultrastructure of STH cells of the pars distalis of castrated male mice after hepatectomy

Summary An electron microscopic study aimed at differentiation between castration gonadotrophs and posthepatectomy STH cells was performed on the pars distalis of the pituitary of castrated male mice, after partial hepatectomy. The ultrastructural features observed permit the distinction of both cell types. In the present experiments some remarkable ultrastructural changes, other than those described in a previous report, have been found in STH cells of hepatectomized mice with or without previous castration. Most of them contained masses of heterogeneous electron density, suggesting fusion of granules. These masses and some secretion granules were observed close to the plasma membrane, apparently in the process of discharging material into the pericapillary space. Granular extrusion was more frequent than normally. An increased number of lysosomes, probably related to the digestion of overproduced secretion material, was evident. The appearance of concentric lamellar formations might be related to an increase in cell movements. STH cells with severe cytoplasmic damage were also found, indicating an increased rate of cell loss.Work carried out with the financial assistance by a grant from the Comisión de Investigación Científica de la Universidad Nacional de La Plata. Thanks are due to the members of the Technical Staff of the Institute for their technical assistance.     

Oxidative stress signalling in the apoptosis of Jurkat T-lymphocytes

Within the first 24 h after castration of an adult male rat, the vascular system of the ventral prostate gland undergoes a degenerative process that drastically reduces blood flow to the tissue. Since the vascular degeneration precedes the loss of the prostatic epithelium (by apoptosis), we have proposed that the onset of epithelial cell apoptosis in this tissue is caused by an ischemic/hypoxic environment resulting from the loss of blood flow. In order to further evaluate the extent to which ischemia/hypoxia might be a factor in apoptosis of the prostate epithelium after castration, we analyzed for biomarkers of cellular hypoxia in rat ventral prostates during the first 3 days following castration. Ventral prostate tissues removed from hypoxyprobe-1-treated adult male rats (uncastrated controls; surgically castrated for 24, 48 or 72 h, or sham-castrated for equivalent times) were directly analyzed for evidence of hypoxia by in situ immunohistochemical evaluation of hypoxyprobe-1 adduct formation in the prostate cells. Protein extracts from these tissues were also tested for expression of the 120 kDa hypoxia-inducible factor-1-alpha (HIF-1-alpha) protein as well as for expression of mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins using a Western blot assay. The tyrosine phosphorylation status of the latter signaling molecules was also evaluated by Western blotting using anti-tyrosine phosphate antibodies. Our results showed that epithelial cells of the rat ventral prostate stained positively for hypoxyprobe-1 adducts at all times after castration, whereas cells in control tissues were unstained by this procedure. In addition, the prostatic expression of HIF-1-alpha protein was increased approximately 20-fold at 48 h after castration compared to control tissues. Finally, although prostatic MAPK and JNK protein expression was unaltered during the early period after castration, phosphorylation of the JUN kinase protein was significantly elevated, indicating that this stress-activated cellular signaling pathway becomes more active subsequent to castration. These results support our proposal that early castration-induced degeneration and constriction of the vascular system of the rat ventral prostate gland leads to reduced oxygenation of prostatic epithelial cells and the activation of hypoxic cellular signaling in these cells through upregulation of HIF-1-alpha expression and stimulation of the JUN kinase signaling pathway.     

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.     

Cytochemistry and biochemistry of acid phosphatases

Summary Acid phosphatases of the rat ventral prostate were studied cytochemically using different substrates. The results were compared to findings on isoelectric focussing gels stained for acid phosphatase activity. This is a highly specific and reproducible method which allows the distinction between secretory androgen-dependent and lysosomal acid phosphatases. Activity of lysosomal acid phosphatase was increased after castration, while the activity of the secretory enzyme gradually decreased after androgen deprivation. None of the substrates tested was selectively hydrolyzed by either secretory or lysosomal acid phosphatase. Phenylphosphate, creatine phosphate and choline phosphate were found to be inappropriate substrates for histochemical purposes, however, reproducible results were obtained with -naphthylphosphate, -glycerophosphate and p-nitrophenylphosphate. The method of isoelectric focussing (pH range 4.0–8.0) of enzymes with subsequent histochemical staining demonstrated lysosomal enzymes at pH 7.9 and 8.2 respectively. Small amounts of identical enzymes were found in liver, kidney, blood or epididymis. Secretory acid phosphatases were focussed at pH 5.5, 5.6, 5.65 and 7.15. Similar enzymes have been identified in epididymis, kidney, liver and pancreas. These results indicate that 1) at present no specific substrate for prostatic secretory or lysosomal acid phosphatases is available and 2) that no prostate-specific prostatic acid phosphatase (PAP) exists in the rat ventral prostate.Supported by the Deutsche Forschungsgemeinschaft (Au 48/6)     

Fine structural observations on magnum mucosa in quail and hen oviducts

Summary Previous investigators have reported that albuminous material in the albumin-secreting (tubular gland) cells of the magnum of hen oviduct accumulates in the ergastoplasmic cisternae and is released directly into the glandular lumen without being first concentrated into secretory granules in the Golgi region (Zeigel and Dalton, 1962). Present fine structural studies on the tubular gland cells in oviducts from actively laying wild-type Japanese quail and in an oviduct from an actively laying hen indicate that the Golgi apparatus is directly involved in the formation of secretory granules. At least three types of granules can be observed in the tubular gland cells at various times, and all types seem to be associated initially with the Golgi apparatus.In actively laying quail, the distribution of electron opaque, intermediate, and light granules within the superficial and deep regions of the glandular epithelium varies, depending on the presence of an egg in a particular region of the oviduct. Secretion of the product is merocrine, involving fusion of granule membranes with the plasmalemma of the cell surface.Granules first appear in the tubular gland cells of quail oviducts at about 4 1/2 weeks of age. The granules are of the electron opaque type and probably represent secretion in concentrated storage form. At this age, a few of the tubular gland cells exhibit distended ergastoplasmic cisternae containing material of low electron density. The appearance of these light cells, which occur with greater frequency in oviducts from older quail, probably reflects an increased level of secretory activity initiated by changes in hormonal levels. From 5 weeks of age on, intermediate and light (less concentrated) granules, as well as dark granules, are present.Supported by the National and Medical Research Councils of Canada.     

The fate of prorenin during granulopoiesis in epithelioid cells

Summary Comparative immunocytochemical experiments with antisera directed against renin and three synthetical peptides (Pro 1, Pro 2 A and Pro 3) covering almost the entire span of human renin prosegment were performed on human kidney tissue. With anti-Pro 1, i.e. the antiserum which recognizes the NH₂ terminus of human prorenin, no clear immunolabeling of juxtaglomerular epithelioid cell secretory granules could be obtained. It is therefore concluded that the corresponding portion of human prorenin may be cleaved off in the Golgi complex.After application of anti-Pro 3, the antiserum which recognizes the COOH terminus of the prosegment, only the juvenile secretory granules of epithelioid cells were consistently labeled, whereas, in contrast, some of the intermediate and most of the mature secretory granules were anti-Pro 3-negative. As the immunoreactivity of mature renin increased remarkably from protogranules to mature secretory granules, it is suggested that the cleavage of the COOH terminus of the prosegment, i.e. the activation of renin, takes place in juvenile and intermediate granules during condensation of the enzyme.The immunoreactivity of Pro 2A, corresponding to the middle portion of the prosegment, disappeared in a some-what earlier stage of granulopoiesis than that of Pro 3. It is therefore concluded that the corresponding segmental cleavage, the result of which is a truncated version of intact prorenin, occurs in the protogranules of epithelioid cells.The data presented are consistent with the assumption that the secretion of active renin takes place by the exocytosis of mature secretory granules, while the secretion of inactive renin, which is a truncated version of intact prorenin, is mediated by the exocytosis of juvenile and intermediate granules.These studies were supported by the German Research Foundation within the Forschergruppe Niere/Heidelberg