Immunocytochemical demonstration of a lipid droplet-specific capsule in cultured Leydig cells of the golden hamsters

In this report, we provide direct evidence for the presence of a lipid droplet-associated capsule in hamster steroidogenic Leydig cells by using a monoclonal antibody A2. Leydig cells are characterized by containing many lipid droplets and having 3β-hydroxysteroid dehydrogenase activity. Immunofluorescence staining with this antibody demonstrated a rim or capsule surrounding the lipid droplets in Leydig cells, a pattern not seen with anti-vimentin antibody. Immunogold labelling confirmed ultrastructurally that antibody binding was distributed on the lipid droplet surface. In order to investigate the possible function of the capsule, we examined the morphological changes induced in the capsule following stimulation with LH or dibutyryl cAMP; the fluorescent intensity of the capsule was seen to gradually decrease, accompanied by a decrease in number and size of lipid droplets, and the response to both reagents was time- and concentration-dependent. We thus conclude that hormonal stimulation resulting in the detachment of certain capsular proteins from the surface of lipid droplets is mediated via the cAMP signaling pathway and may allow cholesterol ester hydrolytic enzyme direct access to its substrate in the lipid droplet. © 1996 Wiley-Liss, Inc.     

Immunocytochemical localization of substance P in mammalian intestine

Summary In mammalian intestine immunoreactive Substance P is localized not only in the plexuses of Auerbach and Meissner, as could be anticipated, but also in a number of basally situated, often basigranular, endocrine cells which have been identified tentatively as enterochromaffin.The presence of a neurohormone in cells of this type confirms their close association with the nervous system, noted by Masson (1924), and suggests that their postulated origin from the nervous system (Danisch, 1924) may well be correct.     

Immunocytochemical demonstration of ecto-galactosyltransferase in absorptive intestinal cells

Galactosyltransferase immunoreactive sites were localized in human duodenal enterocytes by the protein A-gold technique on thin sections from low temperature Lowicryl K4M embedded biopsy specimens. Antigenic sites detected with affinity-purified, monospecific antibodies were found at the plasma membrane of absorptive enterocytes with the most intense labeling appearing along the brush border membrane. The lateral plasma membrane exhibited a lower degree of labeling at the level of the junctional complexes but the membrane interdigitations were intensely labeled. The labeling intensity decreased progressively towards the basal part of the enterocytes and reached the lowest degree along the basal plasma membrane. Quantitative evaluation of the distribution of gold-particle label proved its preferential orientation to the outer surface of the plasma membrane. In addition to this membrane-associated labeling, the glycocalyx extending from the microvillus tips was heavily labeled. Occasionally, cells without plasma membrane labeling were found adjacent to positive cells. The demonstration of ecto-galactosyltransferase on membranes other than Golgi membranes precludes its general use as a marker for Golgi membrane fractions. The possible function of galactosyltransferase on a luminal plasma membrane is unclear at present, but a role in adhesion appears possible on the basolateral plasma membrane.     

Immunocytochemical demonstration of 5-hydroxytryptamine in gastrointestinal endocrine cells

Symopsis We have been able to demonstrate 5-hydroxytryptamine in the enterochromaffin cells of the mammalian gastrointestinal tract, using a highly specific antiserum. Conventional histochemical techniques for identifying amines as cell markers can thus be replaced by more reliable and sensitive immunocytochemical methods. This has been facilitated by the use ofp-benzoquinone as fixative which has been shown to preserve the localization and antigenicity of amines, as well as peptides.     

Immunocytochemical demonstration of carbonic anhydrase in human epithelial cells

Human tissues obtained early postmortem were immunostained to demonstrate carbonic anhydrase (CA) and, in some instances, to differentiate CA I and CA II, employing an immunoglobulin-peroxidase bridge method. Optimal immunostaining was obtained in tissues fixed a few hours in Carnoy's fluid or a buffered HgCl2 solution. Specimens fixed 1/2 to 2 hr with buffered formalin or Bouin's fluid stained less well but better than those fixed 24 hr with formalin. In tracheobronchial glands, serous acini and demilunes exhibited intense immunoreactivity demonstrative of the isozyme CA II. In kidney, all cells of the distal convoluted tubules were strongly positive for CA and cortical collecting tubule cells stained strongly but with some variability among individual cells. Cells in medullary collecting tubules ranged from intensely to negligibly reactive. Proximal convoluted tubules and thick ascending limbs showed moderate to light, uniform staining, but the thin limbs of the loop of Henle were negative. Renal cell immunoreactivity occurred only with antiserum to CA II. Seromucous acini in submandibular glands stained strongly and selectively for CA. Ducts in liver and pancreas showed strong selective immunostaining. The most superficial columnar cells lining the main lumen of the colon and appendix displayed strong reactivity, as did columnar cells lining the gall bladder.     

Immunocytochemical localization of substance P neurokinin-1 receptors in rat gingival tissue

The distributions of substance P (SP) and the neurokinin-1 receptor (NK1-R), the receptor preferentially activated by SP, were examined in rat gingiva by immunocytochemical methods with light and electron microscopy. SP-immunoreactive nerve fibers were located preferentially in the junctional epithelium (JE) but few in the other oral and oral sulcular epithelia. NK1-R immunoreactivity was found in the endothelial cells (capillaries and postcapillary venules underlying the JE). NK1-R-labeled and -unlabeled unmyelinated nerve fibers were located close to the blood vessels and partially or completely covered by a Schwann cell sheath. In the JE, labeled naked axons without Schwann cell sheaths were observed. Neutrophils and macrophages in the connective tissue underlying the JE and in the JE were also labeled with NK1-R. Furthermore, NK1-R was found in the JE cells. Basically, immunoreaction products for NK1-R were found throughout various cells (endothelial cells, neutrophils, and JE cells) at invaginations of the plasma membrane and in vesicular and granular structures that are probably endosomes and are found close to both the plasma membrane and the nucleus. This is a first report, demonstrating the presence of NK1-R in the gingival tissue in the normal nonstimulated condition. Furthermore, it is thought that SP may modulate the permeability of blood vessels beneath the JE, the production of antimicrobial agents in neutrophils, and the proliferation and endocytotic ability of JE cells through NK1-R.     

Immunocytochemical study of substance P containing nerve terminals in rat spinal cord

Summary The subcellular localization of substance P (SP) in the dorsal horn of the rat spinal cord was studied using the unlabelled antibody procedure of Sternberger with different fixatives (4% paraformaldehyde alone or with varying amounts of glutaraldehyde), buffer systems for the immunohistochemical incubations, and the presence or absence of the detergent, Trition X-100. Hand-sliced tissues were compared with Vibratome sections, and showed adequate results which are described below. Labelled terminals of two types could be seen in all samples incubated with anti-SP sera. The two types of positive terminals can be described as those which contained mostly immunoreactive clear vesicles, and those which contained both immunoreactive clear and dense core vesicles. Brief fixation during pressure perfusion with increased concentrations of glutaraldehyde (up to 2%) improved the tissue preservation and, as a result, the intensity and definition of the SP immunoreaction products. The use of Tris or phosphate buffer for the immunohistochemical incubations maintained the intensity of staining in well-fixed tissues. However, Tris incubations contributed to a diffusion of immunoreaction products and increased the number of broken membranes in the labelled processes as well as those of myelin. These phenomena were not observed in phosphate buffer, which preserved the tissue better than Tris. Like Tris, pretreatment with the detergent Triton X-100 (TX) contributed further to the diffusion of the immunoreaction products, and increased the number of broken membranes. For example,without TX, the outer membrane and envelope of the mitochondria became intensely and clearly labelled when phosphate buffer was used for incubations;with TX pretreatment, the staining was far more diffuse, and the intensity of staining became reduced such that only the mitochondrial outer surface appeared somewhat immunopositive. Using phosphate buffer alone, we observed well-defined immunoreaction products around the microtubules of many-containing processes. This finding was less clear in other preparations, especially those pretreatedwith TX. We therefore submit that the conditions of tissue fixation and incubation may influence greatly the data amassed by the technique of immunocytochemistry. Results must be evaluated in view of the methods chosed for each immunocytochemical study. Optimal technical conditions encourage new morphological findings, as shown below concerning the circuitry of SP neurons in the dorsal horn.This work was initiated while Dr. K. Kakudo was a postdoctoral Fellow in Anatomic Pathology, Medical College of Georgia, from Osaka University Medical School, JapanPresently at the Department of Anatomy, Tulane Medical School, New Orleans, Louisiana, USAThe work, presented at the Annual Meeting of the 31st Annual Meeting of the Histochemical Society held in New Orleans, April 11–15, 1980, was partially supported by BRSG 10-16-04-3611-23, CCHD10-12-04-3600-00 (LLV) and the Department of Pathology (address reprint requests to LLV)     

A dense-cored filamentous body in Leydig cells of the golden hamster

Summary A unique cytoplasmic structure has been observed in Leydig cells of the golden hamster. It consists of a laminar core made up of electron dense material surrounded by a filamentous matrix of lower density, and is tentatively called a dense-cored filamentous body (DCFB). DCFBs vary in overall size and in configuration of the centrally disposed dense lamina. They are typically located in the vicinity of the centrosome and the Golgi complex. The body has no limiting membrane, and may be in contact with virtually every type of organelle. The DCFB is well developed in active Leydig cells, whereas it is small in the quiescent stage of the secretory cell. It is likely that the DCFB is a constant organelle in the hamster Leydig cell and may be involved in the physiological function of the Leydig cell, which remains to be specified.This work was supported in part by a grant from the National Science Council, the Republic of China (NSC-66B-0412-02-13)     

P2X receptors in mouse Leydig cells

ATP-activated currents were studied in Leydig cells of mice with the patch-clamp technique. Whole cell currents were rapidly activating and slowly desensitizing (55% decrement from the peak value on exposure to 100 µM ATP for 60 s), requiring 3 min of washout to recover 100% of the response. The concentration-response relationships for ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPS), and 2-methylthio-ATP (2-MeS-ATP) were described by the Hill equation with a concentration evoking 50% of maximal ATP response (K_d) of 44, 110, and 637 µM, respectively, and a Hill coefficient of 2. The order of efficacy of agonists was ATP ATPS > 2-MeS-ATP > 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). -Methylene-ATP (-MeATP), GTP, UTP, cAMP, and adenosine were ineffective. Suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) blocked the responses in a concentration-dependent manner. The ATP-activated currents were dependent on extracellular pH, being maximal at pH 6.5 and decreasing with both acidification and alkalinization (apparent dissociation constant (pK_a) of 5.9 and 7.4, respectively). The whole cell current-voltage relationship showed inward rectification and reversed near 0 mV. Experiments performed in bi-ionic conditions for measurement of reversal potentials showed that this channel is highly permeable to calcium [permeability (P)_Ca/P_Na = 5.32], but not to chloride (P_Cl/P_Na = 0.03) or N-methyl-D-glucamine (NMDG) (P_NMDG/P_Na = 0.09). Unitary currents recorded in outside-out patches had a chord conductance of 27 pS (between –90 and –50 mV) and were inward rectifying. The average current passing through the excised patch decreased with time [time constant () = 13 s], resembling desensitization of the macroscopic current. These findings indicate that the ATP receptor present in Leydig cells shows properties most similar to those of cloned homomeric P2X₂. patch clamp; single channels; ATP; desensitization     

Compensatory hypertrophy of golden hamster ovaries during early postnatal ontogenesis]

The right-side ovariectomy in the golden hamster females during the prepubertate period resulted in the compensatory hypertrophy of the rest paired organ. The morphological rearrangement of the hypertrophied ovary represented the shift in the quantitative distribution of follicles by the maturation stages towards the predominance of mature forms. The accelerated growth and differentiation along the path of normal postnatal ontogenesis led to the earlier ovulations in the hypertrophied ovary.     

Immunocytochemical demonstration of alpha-tubulin modification during axonal maturation in the cerebellar cortex

Previous light microscopic immunocytochemical studies using two monoclonal antibodies that recognise alpha-tubulin (YOL/34 and YL1/2) but differ in their isotypic specificity have shown that the unmyelinated parallel fiber axons in the cerebellar cortex are labeled with only one of the antibodies (YOL/34). We now show that at 10 d postnatally the parallel fibers are labeled with both antibodies, and that during development YL1/2 (but not YOL/34) immunoreactivity disappears progressively from parallel fibers in the lower regions of the molecular layer upwards towards the external germinal layer. By approximately 28 d postnatally, the differential staining pattern of parallel fibers by the antibodies is established throughout the molecular layer. The time course, light microscopic, and ultrastructural staining distribution corresponds to a progressive change in alpha-tubulin immunoreactivity as the parallel fibers form synaptic contacts. This modification of alpha-tubulin (which was not observed in Purkinje cell dendrites or Bergmann glia) may be related to the formation of a basic isotype of alpha-tubulin within parallel fiber axons at maturation.     

Morphology and function of human Leydig cells in vitro. Immunocytochemical and radioimmunological analyses

The aim of our study was to show whether the cells isolated from testes of patients underwent bilateral orchiectomy for prostatic cancer are able to grown in vitro, and if so, are functionally active. Immuncytochemistry was performed to show the functional status of human cultured cells. In detail, immunolocalization of luteinizing hormone receptors (LHR), mitochondria, and cytoskeletal elements was demonstrated. Moreover, radioimmunological assay was used to measure testosterone secretion by cultured Leydig cells. Using Nomarski interference contrast and fine immunofluorescence analysis the positive immunostaining for LHR was observed in almost all Leydig cells, however it was of various intensity in individual cells. Testosterone measurement revealed significant difference between testosterone secretion by hCG-stimulated and unstimulated Leydig cells (p<0.05). Moreover, testosterone levels were significantly higher in 24- and 48-hour-cultures than in those of 72 hrs (p<0.05). Morphological analysis of Leydig cells in culture revealed the presence of mononuclear and multinucleate cells. The latter cells occurred in both hCG-stimulated and unstimulated cultures. In Leydig cells labeled with a molecular marker MitoTtracker, an abundance of mitochondria and typical distribution of microtubules and microfilaments were observed irrespective of the number of nuclei within the cell, suggesting no functional differences between mono- and multinucleate human Leydig cells in vitro. Since the percentage of multinucleate cells was similar in both hCG-stimulated and unstimulated cultures (23.70% and 22.80%), respectively, the appearance of these cell population seems to be independent of hormonal stimulation.Key words: human Leydig cells, LH receptors, primary culture, hCG-stimulation, immunocytochemistry, testosterone secretion, multinucleate cells, multicolor staining.It is well established that testosterone biosynthesis depends on the existence of mature Leydig cells in the testicular interstitium. Human Leydig cells arise from mesenchymal cells or fibroblast-like precursor cells through a hormonally regulated differentiation process (Chemes, 1996). Production of testosterone in human and mammalian Leydig cells is dependent on LH stimulation in vivo and on LH/hCG stimulation in in vitro conditions; to respond to hormonal regulation the cells are equipped with functional receptors for LH (Amador and Bartke, 1987; Simpson et al., 1987; Mendis-Handagama et al.,1990; Cooke, 1996; Ramadoss et al., 2006). In man, the Δ⁵-metabolic pathway is the major pathway for the metabolism of pregnenolone to testosterone (Rommerts, 1990). According to Hammar and Petersson (1986) in human testis from young and elderly men with prostatic carcinoma also the 5-ene pathway is preferred. For optimal steroidogenic function a number of neuroendocrine and neuronal markers have been demonstrated in human Leydig cells in vivo by the group of Holstein (Middendorff et al., 1993; 1995). Moreover, production of testosterone in Leydig cells, requires the presence of functionally active enzymes acting within mitochondria and the smooth endoplasmic reticulum (Payne and O’Shaughnessy, 1996; for review see Haider, 2004).Recent studies have shown that Leydig cells become hypofunctional with age. In the rat, aged Leydig cells produce less testosterone than Leydig cells from young adult rats (Luo et al., 1996; for review Zirkin et al., 1997). A detailed characteristics of aged rat Leydig cells in vivo, including reduced testosterone biosynthesis and reduced cell volume has been described by Ewing and Zirkin (1983). Now, there is evidence from in vitro studies that reactive oxygen species can result in the inhibition of testosterone production in mouse Leydig cells by affecting steroidogenic enzymes (Stocco et al. 1993; Peltola et al., 1996; Cao et al., 2004).Considering human samples as a very rare and valuable biological material, the aim of this study was to show whether Leydig cells obtained from testes of elderly patients who underwent orchiecto-my for prostatic cancer are able to grown in vitro, and if so, are functionally active. For this purpose localization of luteinizing hormone receptors (LHR) and visualization of mitochondria and cytoskeletal elements in both hCG-stimulated and unstimulated Leydig cell cultures were performed, as well as testosterone secretion by cultured Leydig cells was measured. It is worth noting that the effect of LH and an involvement of cytoskeletal proteins in steroidogenesis of mouse Leydig cells in vitro have been demonstrated by our own (Bilinska, 1989) and mitochondria have been described as integrally involved in Leydig cell steroidogenesis (Bilinska 1994; Kotula-Balak et al., 2001).     

Immunocytochemical studies on prolactin cells in the adenohypophysis of the golden hamster

Mammotrophs or prolactin (PRL) cells were identified in the adenohypophysis of adult golden hamsters by immunocytochemical techniques with a polyclonal anti-PRL, that was proved to be specific to PRL by the dot immunoblotting test. Postembedding immunostaining was performed on Araldite thin sections by immunoperoxidase and immunogold methods. PRL cells were classified into three types according to the different size of the secretory granules. The Type A cells were usually small and angular or oval in shape, and had secretory granules ranging in diameter from 100-230 nm, and showed poorly developed organelles. The Type B and C cells were larger and round or ovoid in shape, contained larger granules, 230-280 nm and 280-570 nm, respectively, and displayed well developed organelles. Immunoreactive PRL cells in the male pituitaries were far less numerous than in the nonpregnant female glands, and were mostly of the Type A and B, whereas in the female the Type C and B cells predominated. In pregnant females, Type C cells became activated and increased in number, while the other two types decreased in proportion. In lactating females, Type A and B cells significantly increased in number at the expense of the Type C cells; meanwhile, the exocytosis of secretory granules was frequently found in all types of PRL cells. The present findings suggest that Type C and B PRL cells, especially the former, are potent in producing and releasing PRL and highly responsive to various physiological stimuli, while Type A cells are probably relatively inert in synthetic activity.     

Immunocytochemical demonstration of Langerhans' cells in smears and pellets of exfoliated cells from human exocervices

Immunocytochemical staining with OKT6 monoclonal antibody and S-100 protein antiserum was used to reveal Langerhans' cells in smears and/or pellets of exfoliated cells from uterine cervices (normal and with squamous carcinoma). Immunoreactive Langerhans' cells were found exclusively in smears and pellets of cervices with squamous carcinoma. Langerhans' cells, which appear as rounded cells, showed a peripheral ring of intense fluorescence with OKT6 antibody whereas the entire cell stained with S-100 protein antiserum. The presence of Langerhans' cells among cells exfoliated from exocervical squamous carcinoma can be explained by the increased density of these cells in tissue with neoplastic changes.     

Immunocytochemical identification of growth hormone cells in the adenohypophysis of the golden hamster

Somatotrophs or growth hormone (GH) cells in the adenohypophysis of golden hamsters were identified by immunocytochemical staining with polyclonal rabbit anti-human GH. They were oval or columnar in shape, and had secretory granules of two size ranges, 90-150 nm and 280-320 nm, which were present in the same cells; no subtypes of GH cells were observed. Secretory granules were located in the peripheral portion of the cytoplasm or concentrated at the vascular pole of the cell. Flattened cisternae of the rough endoplasmic reticulum in parallel array and a moderately developed Golgi apparatus were often found in the cytoplasm. No sex difference was noticed in the population ratio of GH cells. Immunocytochemical staining with anti-GH or anti-prolactin (PRL) antibodies on separate adjacent sections revealed that the GH and PRL were stored in two different cell types.     

Immunocytochemical studies of hamster oocyte activation

By indirect immunofluorescence, using rabbit anti-heparin-binding placental protein (HBPP) antiserum, we studied HBPP expression by physiologically and non-physiologically (microsurgically) activated hamster gametes. Whereas mature gametes (sperm, metaphase II oocytes) were negative, in vivo conceived preimplantation embryos, from pronuclear to two- and four-cell stages, were HBPP positive. No HBPP was demonstrated in the zona pellucida, but HBPP-dependent immunofluorescence was localized in the perivitelline space. Oocytes incubated with hyaluronidase demonstrated variable responses from negative to positive. (Diluent or sperm) microinjected oocytes were all activated and HBPP positive within 4 h after stimulation. Thus neither activation by microinjection nor HBPP expression required paternal gametes. These kinetics suggest that HBPP may be a cortical granule secretogogue which can be applied to monitor oocyte responses during in vitro manipulations.     

Immunocytochemical demonstration of kinetochores in human sperm heads

CREST sera have been used to identify kinetochores in mature mammalian sperm heads. It is necessary to decondense the sperm heads artificially to permit access of the reagents before the kinetochores can be demonstrated immunocytochemically. The distribution of kinetochores in the sperm heads appears to be random. These results show that the kinetochore antigen recognized by the CREST sera used here is retained during spermiogenesis and is passed on to the zygote at fertilization.