Immunocytochemical localization of amylase and chymotrypsinogen in the exocrine pancreatic cell with special attention to the Golgi complex

Affinity-purified, monospecific rabbit antibodies against rat pancreatic alpha-amylase and bovine pancreatic alpha-chymotrypsinogen were used for immunoferritin observations of ultrathin frozen sections of mildly fixed exocrine pancreatic tissue from secretion-stimulated (pilocarpine) rats and from overnight-fasted rats and guinea pigs. The labeling patterns for both antibodies were qualitatively alike: Labeling occurred in (a) the cisternae of the rough endoplasmic reticulum (RER) including the perinuclear cisterna, in (b) the peripheral area between the RER and cis-Golgi face, and (c) all Golgi cisternae, condensing vacuoles, and secretory granules. Labeling of cytoplasmic matrix was negligible. Structures that appeared to correspond to rigid lamellae were unlabeled. Differences in labeling intensities indicated that concentration of the zymogens starts at the boundary of the RER and cis-side of the Golgi complex. These data support the view that the Golgi cisternae are involved in protein processing in both stimulated and unstimulated cells and that Golgi cisternae and condensing vacuoles constitute a functional unit.     

Condensation-sorting events in the rough endoplasmic reticulum of exocrine pancreatic cells

In guinea pig exocrine pancreatic cells intracisternal granules (ICGs) occur at a low frequency within the lumen of the RER. By starving and refeeding guinea pigs or injecting them in CoCl2 solution, the number of these granules is greatly increased. We show here that ICGs contain the complete set of secreted pancreatic digestive enzymes and proenzymes. Two other soluble proteins in the lumen of the RER, GRP 78/BiP and protein disulphide isomerase (PDI), are specifically excluded from ICGs. The formation of ICGs, which occurs without acidification of the RER cisternae, is therefore a sorting event involving the cocondensation of a complete set of secretory enzymes and proenzymes, which for brevity we refer to collectively as the zymogens. With the exception of approximately 50% of the RNase, the zymogens in ICGs are covalently cross-linked by intermolecular disulphide bonds. The synthesis of all three resident ER cisternal proteins--PDI, GRP 78/BiP, and GRP 94--with the carboxy-terminal sequence KDEL, is induced in response to the accumulation of massive amounts of misfolded secretory protein in the ICGs in the lumen of the RER. After injection of rats with large doses of parachlorophenylalanine-methylester, crystals form in the lumen of the RER. We show that these crystals appear to be a lattice of amylase with the other zymogens incorporated between the layers. Both GRP 78/BiP and PDI are excluded from these crystals. The formation of these amylase crystals within the RER and the inclusion of other zymogens is, therefore, also a sorting event. These data establish that in exocrine pancreatic cells zymogens can cocondense in the RER into either amorphous aggregates or crystals that exclude other soluble RER proteins. This demonstrates that cocondensation is a mechanism capable of sorting zymogens within the secretory pathway.     

Immunolocalization of the oligosaccharide trimming enzyme glucosidase II

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

Ultrastructural immunocytochemical localization of cyclic AMP-dependent protein kinase regulatory subunits in rat parotid acinar cells

Polyclonal antibodies to types I and II regulatory (R) subunits of cyclic AMP-dependent protein kinase (cA-PK) were utilized in a post-embedding immunogold-labeling procedure to localize these proteins in rat parotid acinar cells. Both RI and RII were present in the nuclei, cytoplasm, rough endoplasmic reticulum (RER), Golgi apparatus, and secretory granules. In the nuclei, gold particles were mainly associated with the heterochromatin. In the cytoplasm, the label was principally found in areas of RER. Most gold particles were located between adjacent RER cisternae or over their membranes and attached ribosomes; occasional particles were also present over the cisternal spaces. Labeling of the Golgi apparatus was significantly greater than background, although it was slightly lower than that over the RER cisternae. In secretory granules, gold particles were present over the granule content; no preferential localization to the granule membrane was observed. Morphometric analysis revealed equivalent labeling intensities for RI and RII in the cytoplasm-RER compartment. Labeling intensities for RII in the nuclei and secretory granules were about 50% greater than in the cytoplasm-RER, and 3 to 4-fold greater than values for RI in these two compartments. Electrophoresis and autoradiography of the postnuclear parotid-tissue fraction, the contents of purified secretory granules and saliva collected from the main excretory duct, after photoaffinity labeling with [32P]-8-azido-cyclic AMP, revealed the presence of R subunits. Predominantly RII was present in the granule contents and saliva, while both RII and RI were present in the cell extracts. Additionally, R subunits were purified from saliva by affinity chromatography on agarose-hexane-cyclic AMP. These findings confirm the localization of cA-PK in parotid cell nuclei and establish the acinar secretory granules as the source of the cyclic AMP-binding proteins in saliva.     

Is Lysolecithin an In Vivo Constituent of Chromaffin Granules?

Abstract: It was recently claimed that lysolecithin (lysophosphatidylcholine) in chromaffin granules is a postmortem artefact. We have, therefore, determined catecholamine/lysolecithin ratios in adrenal tissues and isolated chromaffin granules. In rat adrenals and bovine medulla the ratios in both tissues and granules were similar. This indicates that even in rapidly frozen rat adrenal glands, sufficient lysolecithin is present in the total tissue to account for its presence in isolated organelles. Owing to the high cortexhedulla ratios such studies cannot be performed with guinea pig or rabbit adrenals. However, isolated chromafh granules from guinea pig, in contrast to a previous study, do contain lysolecithin. We conclude that lysolecithin is an in vivo constituent of chromaffin granules of all species so far investigated.     

Mast cells of lymph hearts during ontogenesis of frogs Rana temporaria

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

Relationship between the golgi apparatus, gerl, and secretory granules in acinar cells of the rat exorbital lacrimal gland

The method of secretory granuleformation in the acinar cells of the rat exorbital lacrimal gland was studied by electron microscope morphological and cytochemical techniques. Immature secretory granules at the inner face of the Golgi apparatus were frequently attached to a narrow cisternal structure similar to GERL as described in neurons by Novikoff et al. (Novikoff, P. M., A. B. Novikoff, N. Quintana, and J.-J. Hauw. 1971. J. Cell Bio. 50:859-886). In the lacrimal gland. GERL was located adjacent to the inner Golgi saccule, or separated from it by a variable distance. Portions of GERL were often closely paralleled by modified cisternae of rough endoplasmic reticulum (RER), which lacked ribosomes on the surface adjacent to GERL. Diaminobenzidine reaction product of the secretory enzyme peroxidase was localized in the cisternae of the nuclear envelope, RER, peripheral Golgi vesicles, Golgi saccules, and immature and mature secretory granules. GERL was usually free of peroxidase reaction product or contained only a small amount. Thiamine pyrophosphatase reaction product was present in two to four inner Golgi saccules; occasionally, the innermost saccule was dilated and fenestrated, and contained less reaction product than the next adjacent saccule. Acid phosphatase (AcPase) reaction product was present in GERL, immature granules, and, rarely, in the innermost saccule, but not in the rest of the Golgi saccules. Thick sections of AcPase preparations viewed at 100 kV revealed that GERL consisted of cisternal, and fenestrated or tublular portions. The immature granules were attached to GERL by multiple connections to the tublular portions. These results suggest that, in the rat exorbital lacrimal gland, the Golgi saccules participate in the transport of secretory proteins, and that GERL is involved in the formation of secretory granules.     

Immunogold determination of amylase concentrations in pancreatic subcellular compartments

We used the immunogold method on ultrathin cryosections to measure intracellular amylase (Am) concentrations in subcellular compartments of rat exocrine pancreatic cells. Previously, the quantitation procedure was characterized in a model system consisting of Am dispersed at known concentration in a matrix of gelatin. Variations in labeling efficiency, due to differences in matrix density, were equalized by embedding in 30% polyacrylamide (PAA). Here we applied these model conditions to rat pancreas and established intracellular Am concentrations [Am]. Specimen blocks were composed of tissue and a reference layer of gelatin mixed with a known Am concentration ([Am]r), both fixed in glutaraldehyde. Cryosections of the PAA embedded blocks were immunogold labeled for Am. The labeling density was measured in the reference layer (LDr) and in structures in exocrine cells that were involved in Am synthesis and transport (LDs). In each of these structures the Am concentration ([Am]s) was calculated from: [Am]s = [Am]r. LDs/LDr In this way we measured average concentrations ranging from 63 mg/ml in rough endoplasmic reticulum to 261 mg/ml in secretory granules. Concentration of Am appeared to occur mainly in the most cis- and the most trans-Golgi cisternae. To check whether sterical hindrance was an inherent bias to the [Am] measurements in compartments that contained high concentrations of the enzyme, the labeling efficiency for Am in intact isolated secretory granules in gelatin and embedded in PAA, was compared with the efficiency when the granules were lysed and approximately 50 times diluted in gelatin before PAA embedment. It appeared that Am was detected with similar efficiency under both conditions. This demonstrated that sterical hindrance did not cause errors in the measurements of cellular Am concentrations.     

Subcellular distribution of secretogranins I and II in GH3 rat tumoral prolactin (PRL) cells as revealed by electron microscopic immunocytochemistry

The GH3 rat pituitary cell line which secretes prolactin (PRL) is characterized by the paucity and small size of secretory granules. We looked for the presence, in these cells and in normal PRL cells, of two acidic tyrosine-sulfated proteins which are widely distributed in dense-core secretory granules of endocrine and neuronal cells, secretogranins I and II, using immunofluorescence and electron microscope immunoperoxidase techniques. Both secretogranins were detected in secretory granules of GH3 cells and of normal cells. Moreover, with our pre-embedding approach, secretogranins were localized within some RER cisternae and within all sacules of the Golgi stacks in both PRL cell models. A few small vesicles, large dilated vacuolar or multivesicular structures, and some lysosome-like structures were also immunoreactive. Double localization of secretogranins and PRL performed on GH3 cells by immunofluorescence indicated that all cells contained secretogranins I and II, whereas only 50-70% of the cells contained PRL. Moreover, in the case of hormone treatment known to increase the number of secretory granules, most if not all mature secretory granules were immunoreactive for secretogranins, whereas in certain cells some of the granules were apparently not immunoreactive for PRL. These immunocytochemical observations show that GH3 cells, which under normal conditions form only a small number of secretory granules, produce secretogranins and package them into these granules.     

A lysophospholipase specific for exocrine pancreatic cells is stored in zymogen granules and secreted into pancreatic juice

We separated by two-dimensional (2D) gel electrophoresis the content of isolated rat zymogen granules and from the gel excised a protein of apparent MW 77,500 and an isoelectric point of about 4.7. A rabbit antiserum against this previously uncharacterized rat zymogen granule protein recognized two cDNA clones in a rat pancreas expression library. The cDNA inserts of these two clones had sequences showing perfect homology to the published cDNA sequence of rat pancreatic lysophospholipase. The antiserum recognized only a single protein, lysophospholipase, on one and two-dimensional immunoblots of rat pancreas homogenates and isolated zymogen granules. The antiserum did not react with any protein in homogenates of rat liver, spleen, adrenal, parotid, and prostate tissue. The zymogen granule protein of the guinea pig, previously identified as Lipase 1, was recognized specifically by the antiserum against rat lysophospholipase. This guinea pig protein can now be regarded as lysophospholipase. The same protein was demonstrated in the transformed rat acinar cell line AR4-2J, where both the rate of total enzyme synthesized and the amount of mRNA increased following treatment with dexamethasone. Immunogold labeling established that pancreatic lysophospholipase is restricted exclusively to exocrine cells where it occurs only in compartments of the exocytotic pathway. It could also be detected in pancreatic juice in the ducts of the tissue. Finally, we have shown that lysophospholipase is not related to the zymogen granule membrane protein GP2. This work establishes that lysophospholipase is a normal member of the set of soluble enzymes and proenzymes that are stored in zymogen granules and secreted into pancreatic juice.     

Tumor-basophil interactions in vitro--a scanning and transmission electron microscopic study.

Purified guinea pig basophils, or basophils either specifically degranulated with antigen or nonspecifically degranulated with lectin, were cultured with guinea pig line 1 hepatoma cells for 1 to 24 hr and studied ultrastructurally. As early as 1 hr of culture, degranulated or nongranulated basophils and tumor cells formed close contacts by mutually intertwined elongated cell processes and also in cultures containing degranulated basophils, extruded membrane-free basophil cytoplasmic granules became firmly attached to tumor cells. At later intervals, some tumor cells cultured with basophils exhibited cytostatic and cytopathic changes, including dense mitochondria, centralization of organelles, dilated perinuclear and rough endoplasmic cisternae, cell swelling and cytoplasmic lucency, disrupted cytoplasmic organelle and plasma membranes, nuclear pyknosis and fragmentation. Some tumor cell specialized surface attachments were either disrupted or damaged at points of basophil or basophil granule adhesion. Tumor damage was most extensive in cultures containing degranulated basophils, although only a minority of tumor cells (less than 10%) was affected. Tumor injury was seen much less frequently in the presence of nondegranulated basophils, and was absent in control cultures of tumor alone. The occasional viable tumor cells that phagocytosed basophil granules were apparently unharmed, suggesting that internalization of basophil granules by tumor cells is not cytotoxic.     

A post-embedding immunoelectron-microscopic demonstration of apolipoprotein-B-containing lipoprotein particles in hepatocytes of fetal rats

We used the protein-A gold technique to demonstrate the presence of apolipoprotein-B in ultrathin sections of fetal rat liver tissue. It was possible to show for the first time that the electron-dense, osmiophilic particles with diameters of 20-40 nm located within the RER cisternae and Golgi complexes of fetal rat hepatocytes contain apolipoprotein-B components and therefore are lipoproteins. After specific labelling an accumulation of gold label was observed on the RER cisternae, Golgi cisternae and the Golgi-associated secretory vesicles of hepatocytes. The specificity of this labelling pattern was assessed by comparison with cytochemical controls. Our qualitative findings were confirmed by a quantitative analysis of the mean labelling intensity (mean number of gold particles per square micron of the surface area of a particular cellular compartment) on the RER, Golgi complexes, mitochondria, nuclei and the remaining cytoplasm of hepatocytes. It is concluded that the hepatocytes of fetal rats are capable of forming apolipoprotein-B-containing lipoprotein particles. With respect to the size-distribution pattern of the observed intra-hepatic lipoprotein particles, we suggest that the hepatocytes of fetal rats produce lipoproteins of the low- and very low-density-lipoprotein type.     

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.     

Comparative ultrastructure of Clara cells in neonatal and older cattle

Calf lungs were fixed with glutaraldehyde and examined by scanning (SEM) and transmission (TEM) electron microscopy to compare the ultrastructure of Clara cells in terminal bronchioles of neonatal calves and older cattle. In the neonatal calf, SEM revealed numerous smooth-surfaced Clara cells protruding above a similar number of ciliated cells, whereas in older animals the surface of Clara cells was lobulated. Thin sections examined by TEM revealed numerous cuboidal to columnar Clara cells with indented nuclei and a pale cytoplasm filled with faintly granular glycogen in the neonatal calf. Some cells were characterized by apical dense and/or pale membrane-bound granules or secretory droplets. Many cells had an apical tubular network of cisternae that were partly smooth and partly decorated with ribosomes. Ultrastructural comparison of Clara cells in a 2-day-old calf with those of 14- and 19-day-old, 4- and 5. 5-month-old, and 3.5-year-old cattle revealed a striking reduction in the amount of glycogen per cell after 14 days. The number of cells with apical granules was small at all ages, and the density of the secretory granules varied greatly in different cells. A variable amount of smooth endoplasmic reticulum (SER) was present but was less prominent than cisternae of ribosomal endoplasmic reticulum (RER). In older cattle, the limited amount of SER compared to the RER and secretory granules suggests that bovine Clara cells are more likely to be secretory than detoxifying.     

Paraspermatogenesis in Littoraria (Palustorina) articulata, with reference to other Littorinidae (Littorinoidea, Caenogastropoda)

Abstract. The ultrastructure of paraspermatogenesis is examined in the littorinid subfamily Littorininae, with special emphasis on Littoraria (Palustorina) articulata (PHILIPPI 1846). In particular the study focuses on the fate of the nucleus and origin of the rod bodies during parasperm development. Parasperm of the Littorininae are rounded or oblong cells, which undergo an abortive meiosis and eliminate part of the nucleus but often retain a nuclear remnant. The cytoplasm is filled with numerous spherical vesicles in all Littorininae, but in Littoraria (and in certain species of Nodilittorina, Tectarius and Cenchritis) dense 'rod-bodies' also occur. Littoraria (Palustorina) are unique in possessing a flagellum-like structure termed the 'pseudotrich", which lacks an axoneme but contains microtubules during its development. Paraspermatogonia differ from euspermatogonia in the structure of the nucleus and in the extensive rough endoplasmic reticulum (RER) and swollen cytoplasm. Two types of secretions develop in Littoraria : (1) numerous, spherical granules (composed of putative glycoprotein, also seen in other Littorininae) and (2) rhomboid granules (composition uncertain but reacting positively to RNA stains; these granules arising within RER cisternae close to the nucleus). As the rhomboid granules fuse to form the larger, rod-bodies (polygonal in cross section), the RER membrane enclosing the rod-bodies becomes confluent with the outer nuclear membrane, thereby forming a common compartment. Results of this study clearly show that the rod-bodies are secretions of the RER cisternae and not, as claimed in some light microscopic accounts, the product of fusion of eusperm nuclei which have entered the parasperm cytoplasm (either by active eusperm penetration or by phagocytosis). Developmental characteristics of littorinid parasperm show differences between species and may, in some cases, provide characters diagnostic of subgenera.     

Different tolerance to hypothermia and rewarming of isolated rat and guinea pig hearts.

We examined the effect of hypothermia and rewarming on myocardial function and calcium control in Langendorff-perfused hearts from rat and guinea pig. Both rat and guinea pig hearts demonstrated a rise in myocardial calcium ([Ca]total) in response to hypothermic perfusion (40 min, 10 degrees C), which was accompanied by an increase in left ventricular end diastolic pressure (LVEDP). The elevation in [Ca]total was severalfold higher in guinea pig than in rat hearts, reaching 12.9 +/- 0.8 and 3.1 +/- 0.6 micromol.g dry wt-1, respectively. The rise in LVEDP, however, was comparable in the two species: 62.5 +/- 2.5 (guinea pig) and 52.5 +/- 5.1 mm Hg (rat). Following rewarming, [Ca]total remained elevated in guinea pig, whereas a moderate decline in [Ca]total was observed in the rat (13.6 +/- 1.9 and 2.2 +/- 0.3 micromol.g dry wt-1, respectively). Posthypothermic values of LVEDP were also significantly higher in guinea pig compared to rat hearts (42.5 +/- 6.8 vs 20.5 +/- 5.1 mm Hg, P < 0.027). Furthermore, whereas rat hearts demonstrated a 78 +/- 7% recovery of left ventricular developed pressure, there was only a 15 +/- 7% recovery in guinea pig hearts. Measurements of tissue levels of high energy phosphates and glycogen utilization indicated a higher metabolic requirement in guinea pig than in rat hearts in order to oppose the hypothermia-induced calcium load. Thus, we conclude that isolated guinea pig hearts are more sensitive to a hypothermic insult than rat hearts.     

棉花花粉水合和萌发时超微结构的变化：着重论述内质网和被膜小泡

棉花(Gossypium hirsutum L.)花粉在授粉后水合至萌发时期的营养细胞中贮藏的大量淀粉粒和脂体被动用。超微结构的观察表明,首先是造粉质体中的淀粉粒降解,尔后是脂体。在花粉水合至萌发时期,营养细胞中内质网和高尔基体十分活跃,并含丰富的被膜小泡。内质网的构型发生明显的变化:花粉刚水合时内质网潴泡高度扩张,不同程度扩张的内质网潴泡连续成网状并折迭形成许多囊袋状结构单位,其中包含造粉质体、脂体和被膜小泡群;其后,内质网潴泡形成的囊袋状结构消失,变为分支互通的网状结构;至萌发时,内质网潴泡略为扩张,有些连续成简单的网状,有些呈游离的囊泡状。被膜小泡始终是成群地分布,并与脂体联结,当脂体降解时一些被膜小泡与之融合。根据棉花花粉在水合至萌发时期,营养细胞质中存在独特形态的内质网系统和含丰富的被膜小泡,它们的动态行为及与淀粉和脂体的转化和降解之间的密切关系,讨论了这两种细胞器可能的功能。     

Ultrastructural and cytochemical studies of the effects of prolactin on the lateral prostate and the seminal vesicle of the castrated guinea pig

Summary Administration of ovine prolactin to castrated guinea pigs for 2 weeks induced hypertrophy of secretory cells in the lateral prostate when compared with the castrated controls. This was accompanied by an apparent increase in the number of profiles of granular endoplasmic reticulum and well developed Golgi complexes with dilated cisternae. An increase in the number of low-contrast electron-dense secretory granules was observed 4 weeks after prolactin treatment. In the seminal vesicle, dilatation and degranulation of granular endoplasmic reticulum and an apparent decrease in the number of secretory granules were observed 4 weeks after prolactin administration. Following castration and 2 weeks after prolactin treatment, thiamine pyrophosphatase (TPPase)-reaction product was mainly confined to 1–2 trans cisternae of the Golgi complexes in secretory cells of the lateral prostate and the seminal vesicle. In both glands, a reduction of TPPase activity was observed 2 weeks following prolactin administration, and the reaction product was totally absent after prolonged treatment for 4 weeks. The present study has provided morphological evidence that prolactin is capable of stimulating the secretory function of the lateral prostate while exerting some inhibitory effects on the seminal vesicle of the castrated guinea pig. In both glands, TPPase activity, and hence the process of glycosylation was inhibited after prolactin administration. The results from radioimmunoassay indicated that the action of prolactin on these glands could be a direct effect and not mediated through testosterone.     

Use of immunoperoxidase and immunogold methods in studying prolactin secretion and application of immunogold labelling for pituitary hormones and neuropeptides

Two immunocytochemical methods, immunoperoxidase and immunogold (IG), were used in an attempt to study the dynamic process of prolactin release from stimulated rat pituitary mammotrophs. The immunogold method was also used to localize other pituitary hormones including growth hormone, follicle-stimulating hormone, luteinizing hormone, and the neuropeptides substance P, neuropeptide tyrosine, leu-enkephalin, and atrial natriuretic factor in peripheral nerves. Light-microscopic immunoperoxidase staining of prolactin revealed a unique distribution of immunoreactive mammotrophs. Two groups of cells were seen, one centrally located and one forming a narrow peripheral rim on the gland. The two groups were separated by a zone of nonimmunoreactive cells. In addition, the distribution of immunoperoxidase-stained material was not uniform in all mammotrophs. In some, prolactin immunoreactive material was clumped near the nucleus (in the Golgi cisternae); in others it was more diffused within the cytoplasm (but immediately surrounding the cisternae of rough endoplasmic reticulum). After stimulation of mammotrophs, via suckling, prolactin-immunoreactive material was visualized in extracellular spaces. With immunogold methods, prolactin labelling was seen mainly in secretory granules; but some labelling of Golgi cisternae and rough endoplasmic reticulum also occurred. Immunogold labelling revealed that material immunoreactive for leu-enkephalin and atrial natriuretic factor was present in nerve terminals in the rat paracervical ganglion. Material immunoreactive for substance P and neuropeptide tyrosine was present in nerve terminals in the guinea pig heart. Thus, in some situations the immunoperoxidase technique was useful and helped to visualize "grossly" the presence of specific antigens, but it was inadequate for fine ultrastructural localization of these antigens. The immunogold technique was excellent for precise localization of antigens and especially for the detection of colocalization of different antigens. This method can be used in very different structures, such as the adenohypophysis and peripheral nervous tissue, without any modification except for the nature of the antibodies.     

Synthesis and intracellular transport of proteins in the exocrine pancreas of the frog (Rana esculenta)

Summary Frog pancreatic tissue was pulse-labelled in vitro with ³H-leucine and protein transport was studied in exocrine cells by electron microscope autoradiography. The proteins appeared to be synthesized in the RER and transported to the secretory granules along a similar route and with the same velocity as previously described under in vitro conditions.Evidence was obtained for the involvement of the vesicular and tubular elements at the periphery of the Golgi system in transferring protein from the RER to the Golgi cisternae.Kinetics of the release of newly synthesized proteins from the RER and their appearance in the condensing vacuoles are discussed and related to results reported from other tissues.The transport velocity in this poikilothermic system was studied in relation to the incubation temperature and compared with results reported from its mammalian counterpart. At temperatures between 20 and 30° C intracellular protein transport occurs faster in the frog than in the Guinea pig pancreas. At higher temperature the transport process was severely disturbed in the frog.