Immunohistochemical and cytochemical evidences for a possible localization of leucine aminopeptidase in the Merkel cell granule

Localization of leucine aminopeptidase (LAP) in the Merkel cell-axon complex was studied immunohistochemically and cytochemically in the labial tissues of the mouse, rat, dog, and monkey. Anti-LAP was obtained in rabbits by the injection of commercially supplied swine LAP which was confirmed as electrophoretically pure. The Merkel cells of the mouse, rat, and monkey were positively stained by treatment with anti-LAP but the Merkel cells of the dog were negative. When ultrathin sections of the hair follicle from the rat whisker pad, which contain an abundance of Merkel cells, were processed by immuno-peroxidase or by the immuno-gold method, the reaction products were predominantly deposited on the Merkel cells granules. Furthermore, an immuno-blot assay revealed that an extract of the hair follicles from murine whisker pads contained a molecule of relative molecular mass Mr = 60,000 which is similar in size to a subunit of swine LAP. Thus, it appears that Merkel cell granules of rodents and the monkey contain a protein which resembles lucine aminopeptidase.     

Neurotrophin-3 signaling in mammalian Merkel cell development.

Merkel cells are sensory cells of neural crest origin. Because little is known about the mechanisms that direct their differentiation, we have investigated the potential role of a candidate regulatory factor, neurotrophin-3 (NT-3). At embryonic day 16.5 (E 16.5), neither NT-3 nor its primary receptors, TrkC and p75NTR are expressed by Merkel cells in the murine whisker. At the time of birth, however, Merkel cells are immunoreactive for NT-3, TrkC and p75NTR. In TrkC null and NT-3 null mice, Merkel cells differentiate initially, but undergo apoptosis perinatally. These results show that NT-3 signaling is not required for the differentiation of Merkel cells, but that it is essential for their postnatal survival.     

Cell death by apoptosis during involution of the lactating breast in mice and rats

The role of cell death in involution of lactating breast was investigated in mice and rats by light and electron microscopy. Apoptosis, recognized by sharply demarcated compaction of chromatin against the nuclear envelope and by shrinkage and budding of the whole cell to form membrane-bounded apoptotic bodies, was responsible for major loss of cells in both species. In the mouse, rapid involution during the first 2 days was associated with shedding of large numbers of apoptotic bodies derived from alveolar epithelial cells into alveolar lumens. This was followed by more gradual regression, during which the bodies were mostly phagocytosed by macrophages within the epithelium. In the rat, glandular involution was a more gradual and uniform process, with shedding of apoptotic epithelial cells into alveolar lumens being much less conspicuous. Apoptosis of myoepithelial cells was observed in mice, the resulting apoptotic bodies being phagocytosed by intraepithelial macrophages, but was not detected in rats. Apoptosis of capillary endothelial cells caused rapid regression of the capillary beds in both mice and rats. Intraepithelial macrophages increased in number during involution, developed cytoplasmic lipofuscin pigment, and either remained within the epithelium or migrated to the interstitium and regional nodes. Cell loss by apoptosis has been demonstrated during involution and atrophy of a variety of other glands. It characteristically results in shrinkage of a tissue without disruption of its basic architecture.     

An ultrastructural study of meconium corpuscles in human foetal colon

Summary In human foetal colon meconium corpuscles were observed in the colonic epithelium during the stage of secondary lumina development and enlargement.Transmission electron microscopy of these specimens revealed inclusion bodies in the superficial and deeper layers of the epithelium. Many of the membrane-bounded inclusion bodies contained well-preserved organelles and some inclusions contained nuclear fragments. There was evidence of nuclear fragmentation with condensed chromatin arranged in crescentic caps. The ultrastructural observations are typical of apoptosis, a mode of cell death first described in 1972 by Kerr and colleagues.Thus, meconium corpuscles are apoptotic bodies found as a result of the deletion of healthy normal cells during the reshaping and development of organs.     

Caspase-3, TUNEL and ultrastructural studies of small follicles in adult human ovarian biopsies

BACKGROUND: The aim of this study was to investigate evidence for cell death by apoptosis in small unilaminar ovarian follicles of adult humans. METHODS: Cortical biopsies from 13 healthy donors were either frozen and protein extracted for western blots or fixed for immunohistochemistry (IH) to localize procaspase-3 and active-caspase-3, to detect DNA fragmentation in situ and undertake routine transmission electron microscopy (TEM). RESULTS: Blots identified the presence of the inactive pro-form of caspase-3, and IH localized this in all follicles studied. In contrast, the active form of caspase-3, a major effector of apoptosis, was only detected in large antral follicles that also had microscopic signs of atresia. Active caspase-3 was not detected in primordial (n = 87), primary (n = 8) or secondary follicles. The atretic follicles were also the only ovarian structures with positive evidence of DNA fragmentation after terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) treatment. Confocal microscopy showed dual labelling for both active caspase-3 and TUNEL in individual granulosa cells in large atretic follicles, but no such labelling was evident in any other follicles. No apoptotic bodies were seen by TEM in sections of 39 small follicles from seven patients. CONCLUSION: This study found evidence for TUNEL and active caspase-3 only in human ovarian antral follicles.     

Elimination of atretic follicles from the mouse ovary: a TEM and immunohistochemical study in mice

We examined numerous ovarian follicles from 32–35 d virgin mice by transmission electron microscopy and light microscopic immunohistochemistry. No macrophages were seen, but various stages of apoptotic granulosa cells were encountered. Presumably a granulosa cell or its debris in an advanced stage of apoptosis was destined to be phagocytosed by adjacent normal-looking granulosa cells. Other granulosa cells of normal appearance were seen in the region of the zona pellucida in contact with and apparently phagocytosing atrophic oocytes. Such granulosa cells were characterised by the presence of gap junctions with other cells and frequently contained annular gap junctions in the cytoplasm. To confirm the lack of involvement of macrophages in the process of follicular atresia and elimination, specially prepared ovarian sections were incubated with antimouse macrophage monoclonal antibodies (F4/80, Mac-1, Mac-2). None of the follicles examined showed positive immunoreactivity with these antibodies. Atretic follicles may shrink and eventually disappear from the ovary as a result of repeated apoptosis and phagocytosis by granulosa cells. There is no evidence for the presence or involvement of macrophages in the atretic follicles, at least in prereproductive mice as examined.     

Transmission electron microscopic analysis of malathion-induced cytotoxicity in granulosa cells of caprine antral follicles

Malathion, one of the most abundantly used organophosphate pesticides, has immoderate potency as a cytotoxic and genotoxic compound that induces toxicity in granulosa cells, resulting in its apoptosis. Thus, the present study aims to employ ultrastructural analysis for assessing the cytotoxicity of malathion at nanomolar concentrations (1 nM and 10 nM) in granulosa cells of caprine antral follicles at different exposure durations. Transmission electron microscopy revealed diminished cell–cell contact and cellular integrity, presence of crescent-shaped nucleus, chromatin condensation, and pyknosis with nuclear membrane folding, accumulation of lipid droplets with occurrence of cytoplasmic protrusions in granulosa cells treated with 1 nM malathion, whereas at 10 nM concentration, along with apoptotic attributes, prominent association of nucleus, endoplasmic reticulum, mitochondria and lipid droplets, nucleus invagination into lipid droplets, apical localization of lipid bodies, and occurrence of autophagic body were observed as compared to healthy granulosa cells in control with normal intact cellular integrity, well-developed cellular association, and doubled membrane nuclear lamina with homogenously dispersed chromatin surrounded by intact mitochondria with well-developed cristae. Thus, the results of ultrastructural analysis clearly suggest that nanomolar concentration of malathion induces apoptotic hallmarks within the granulosa cells of antral follicles that play a consequential role in increasing the incidence of follicular atresia, thereby affecting the overall fertility.     

Neural Cell Apoptosis Induced by Microwave Exposure Through Mitochondria-dependent Caspase-3 Pathway

To determine whether microwave (MW) radiation induces neural cell apoptosis, differentiated PC12 cells and Wistar rats were exposed to 2.856GHz for 5min and 15min, respectively, at an average power density of 30 mW/cm². JC-1 and TUNEL staining detected significant apoptotic events, such as the loss of mitochondria membrane potential and DNA fragmentation, respectively. Transmission electron microscopy and Hoechst staining were used to observe chromatin ultrastructure and apoptotic body formation. Annexin V-FITC/PI double staining was used to quantify the level of apoptosis. The expressions of Bax, Bcl-2, cytochrome c, cleaved caspase-3 and PARP were examined by immunoblotting or immunocytochemistry. Caspase-3 activity was measured using an enzyme-linked immunosorbent assay. The results showed chromatin condensation and apoptotic body formation in neural cells 6h after microwave exposure. Moreover, the mitochondria membrane potential decreased, DNA fragmentation increased, leading to an increase in the apoptotic cell percentage. Furthermore, the ratio of Bax/Bcl-2, expression of cytochrome c, cleaved caspase-3 and PARP all increased. In conclusion, microwave radiation induced neural cell apoptosis via the classical mitochondria-dependent caspase-3 pathway. This study may provide the experimental basis for further investigation of the mechanism of the neurological effects induced by microwave radiation.     

Genesis of renal tubular atrophy in experimental hydronephrosis in the rat. Role of apoptosis

A morphological study was undertaken to assess the role of cell deletion by apoptosis in experimental hydronephrosis. Male Sprague-Dawley rats (200 +/- 20 gm) were used. The left ureter was ligated or a sham operation was carried out. Animals were killed from 4 days to 12 weeks after operation. Two parallel studies were undertaken: one to demonstrate and quantitate specific morphological changes in the affected kidney using light and electron microscopy, and the other to measure changes in dry kidney weights. Renal tubular atrophy is an inevitable consequence of chronic occlusion of the ureter. As expected, the present study showed a progressive loss of tissue mass in the hydronephrotic kidney. This occurred from 1 week after permanent ureteric ligation, and was most rapid between 2 and 4 weeks. The tubular epithelium contained cells undergoing a distinct form of cell death termed apoptosis, characterized ultrastructurally in its early stage by the presence of rounded cells with condensed cytoplasm and condensed and marginated nuclear chromatin, and later by the presence of discrete membrane-bounded intact cellular fragments (apoptotic bodies), which were phagocytosed and digested by adjacent viable cells, or were shed into the tubular lumens. Numbers of apoptotic cells or clusters of apoptotic bodies were increased significantly in all animals with ureteric obstruction in comparison with controls. The greatest increases occurred at 2 and 4 weeks, when loss of renal mass was occurring rapidly. Diminished blood flow in hydronephrosis has been well-documented by others, and therefore our results are consistent with studies which have shown mild ischemia to be the cause of tissue atrophy involving apoptosis. We conclude that cell deletion by apoptosis plays an important role in the pathogenesis of renal tubular atrophy associated with hydronephrosis.     

Induction of apoptosis in a carp leucocyte cell line infected with turbot (Scophthalmus maximus L.) rhabdovirus

A rhabdovirus was observed from the diseased turbot (Scophthalmus maximus L.) with lethal syndrome. In this study, a carp leucocyte (CLC) cell line was used to investigate the infection process and cell death mechanism occurring during the virus infection. Strong cytopathogenic effect (CPE) and the morphological changes, such as extreme chromatin condensation, nucleus fragmentation, and apoptotic body formation, were observed under fluorescence microscopy after DAPI staining in the infected CLC cells. Transmission electron microscopy analysis showed cell shrinkage, plasma membrane blebbing, cytoplasm vacuolization, chromatin condensation, nuclear breakdown and formation of discrete apoptotic bodies. The bullet-shaped nucleocapsids were measured and ranged in size from 110 to 150 nm in length and 40 to 60 nm in diameter. And therefore the virus is called Scophthalmus maximus rhabdovirus (SMRV). Agarose gel electrophoresis analysis of the DNA extracted from infected cells showed typical DNA ladder in the course of SMRV infection. Flow cytometry analysis of SMRV infected CLC cells detected apoptotic peak in the virus infected CLC cells. Virus titre analysis and electron microscopic observation revealed that the virus replication fastigium was earlier than that of the apoptosis occurrence. No apoptosis was observed in the CLC infected with UV-inactivated SMRV. All these supported that SMRV infected CLC cells undergo apoptosis and the virus replication is necessary for apoptosis induction of CLC cells.     

The existence of Merkel cells in the lingual connective tissue of the Surinam caiman, Caiman crocodilus crocodilus (order Crocodilia)

The tongue of the Surinam caiman (a reptilian species) was studied by light microscopy including immunohistochemistry for protein gene product 9.5 (PGP 9.5), and transmission electron microscopy. The connective tissue immediately under taste buds housed a cluster of cells immunoreactive for PGP 9.5. These cells synapsed on nerves, and their cytoplasm contained characteristic granules of 90 nm in the mean diameter, glycogen particles, and bundles of intermediate filaments. In light of these ultrastructural features, they were identified as Merkel cells. The Merkel cells were also surrounded by Schwann cells. These findings indicate that the present Merkel cell-neurite-Schwann cell complex is comparable to the avian Merkel corpuscle. On the basis of the granule localization in the cytoplasm, the caiman Merkel cell was presumed to be involved in not only mechanoreception but also endocrine or paracrine functions.     

Different types of neural cell death in the cerebellum of the ataxia and male sterility (AMS) mutant mouse

To investigate the mechanisms(s) of age-dependent atrophy of the cerebellum of the ataxia and male sterility (AMS) mouse at young age, the morphological changes were evaluated and the nature of neural cell death was examined. Dying Purkinje cells lacked characters of classical apoptosis except for light microscopic morphology, but their death was considered to be autonomous death triggered by the direct effect of ams mutation, because of the acute and near-complete disappearance and particular change of the cytoplasm. In contrast, in the granular layer, typical apoptotic bodies were recognized by electron microscopy, and substantial numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end-labeling (TUNEL)-positive cells and activated caspase-3-positive cells were observed. Granule cell death was considered to be target-related apoptosis induced after post-synaptic Purkinje cell death, because the age-dependent changes in TUNEL-positive cell counts followed that of Purkinje cell loss and the peak value was still noted 1 week after total loss of Purkinje cells. These results indicate that both total and partial losses of Purkinje cells and granule cells, respectively, contributed to the atrophy of the AMS cerebellum. Furthermore, different types of neuronal death were recognized; the granule cell death was apoptotic while Purkinje cell death was different from that of classical apoptosis.     

Histochemical, ultrastructural, and biochemical evidences for Azadirachta indica-induced apoptosis in benzo(a)pyrene-induced murine forestomach tumors

The present study reports aqueous Azadirachta indica leaf extract (AAILE)-mediated induction of apoptosis in a murine forestomach tumorigenesis model. Histochemistry-based quantification of apoptosis revealed enhanced apoptotic index in the forestomach tumors of mice receiving AAILE along with benzo(a)pyrene (B(a)P). Transmission electron microscopy confirmed the presence of classical morphological features of apoptosis including chromatin condensation/marginalization, nuclear fragmentation, and formation of apoptotic bodies. Scanning electron microscopy showed surface modifications on the transformed squamous epithelial cells and certain mitotic cells among them over the forestomach tumors of mice receiving only B(a)P. In tumors of the mice receiving AAILE along with B (a)P, such mitotic cells were found to be absent; however, certain cells showing shrinkage and blebbings (characteristics of apoptosis) were observed. DNA fragmentation was observed to increase exclusively in the tumors of mice that received AAILE along with B(a)P. Lipid peroxidation (LPO) levels decreased in forestomach tissues of mice in all the groups studied when compared to control counterparts. However, levels of LPO were found to increase in the tumorous tissue of mice that received AAILE along with B(a)P when compared to mice receiving only B(a)P. Taken together, observations of the present study suggest that A. indica induces apoptosis in B(a)P-induced murine forestomach tumors.     

Apoptosis in the reduced enamel epithelium just after tooth emergence in rats

The reduced enamel epithelium transforms into a stratified squamous epithelium, i.e. a junctional epithelium, as the tooth erupts. In this study, we observed apoptosis in the reduced enamel epithelia of rats just after tooth eruption and before complete junctional epithelium formation, by the TUNEL method and electron microscopy. TUNEL-positive reactions were scattered in the reduced ameloblasts and in the external cells of the reduced enamel epithelium. Electron microscopic observation confirmed features of apoptosis, such as nuclei with chromatin condensation, cell shrinkage, and phagocytosis of apoptotic bodies by macro-phage-like cells and epithelial cells. These results suggest that apoptotic cell death is involved in the disappearance of reduced ameloblasts and the external cells of the reduced enamel epithelium during the formation of the junctional epithelium.     

Osteoblasts engulf apoptotic bodies during alveolar bone formation in the rat maxilla

During bone formation, as in other tissues and organs, intense cellular proliferation and differentiation are usually observed. It has been described that programmed cell death, i.e., apoptosis, takes place in the control of the cellular population by removing of the excessive and damaged cells. Although it is generally accepted that apoptotic bodies are engulfed by professional phagocytes, the neighboring cells can also take part in the removal of apoptotic bodies. In the present study, regions of initial alveolar bone formation of rat molars were examined with the aim to verify whether osteoblasts are capable of engulfing apoptotic bodies, such as professional phagocytes. Rats aged 11-19 days were sacrificed and the maxillary fragments containing the first molar were removed and immersed in the fixative solution. The specimens fixed in glutaraldehyde-formaldehyde were processed for light microscopy and transmission electron microscopy. For the detection of apoptosis, the specimens were fixed in formaldehyde, embedded in paraffin, and submitted to the TUNEL method. The results revealed round/ovoid structures containing dense bodies on the bone surface in close contact to osteoblasts and in conspicuous osteoblast vacuoles. These round/ovoid structures showed also positivity to the TUNEL method, indicating that bone cells on the bone surface are undergoing apoptosis. Ultrathin sections showed images of apoptotic bodies being engulfed by osteoblasts. Occasionally, the osteoblasts exhibited large vacuoles containing blocks of condensed chromatin and remnants of organelles. Thus, these images suggest that osteoblasts are able to engulf and degrade apoptotic bodies.     

The Influence of Different Cultivating Conditions on Polymorphonuclear Leukocyte Apoptotic Process In Vitro,II: Ultrastructural Characteristics of PMN Populations Incubated with Proteinase 3 Anti-neutrophil Autoantibodies

The present study shows the effects of proteinase 3 anti-neutrophil cytoplasmic autoantibodies (PR3 ANCA) on polymorphonuclear leukocytes (PMN) apoptotic processes in vitro. The results are part of a generalized morphological analysis of 3 identical experiments on the influence of different cultivating conditions on the apoptotic processes. As controls, the authors use the results on spontaneous PMN apoptosis (Guejes L, Zurgil N, Deutsch M, Gilburd B, Shoenfeld Y. Ultrastruct Pathol. 2003;27:23–32) and PMN populations incubated with normal human IgG. Interaction of PR3 ANCA with the target antigen proteinase 3 (PR3) is one of the crucial pathogenic factors in Wegener granulomatosis (systemic autoimmune vasculitis). Following 40 min and 12 h incubation, PMN populations were evaluated by light microscopy, transmission electron microscopy, and immunogold electron microscopy. Twelve-hour cultures, either control or incubated with PR3 ANCA, contained different cell forms ranging from normal cells to cells at the final stages of apoptosis. Neutrophils at the state of complete manifestation of apoptotic phenotype were analyzed and compared. Three morphologically distinct apoptotic cell lines were characteristic for all PMN populations studied, regardless of cultivating conditions. As in spontaneous apoptosis, these cell lines are code-named “first,” “second,” and “third.” The present study has shown, firstly, that in the presence of PR3 ANCA, all 3 apoptotic lines were modified or altered. Secondly, the modifications or alterations of apoptotic cell lines effected by PR3 ANCA are specific for each cell line: the “first” line is characterized by intensification and modification of activation; the “second” by vacuolized cell forms; and the “third” by pronounced lytic alterations of the nuclei, while the cytoplasm is fully identical to that of control cell lines.     

Role of apoptosis in copper deficiency-induced pancreatic involution in the rat.

Rats maintained on a copper-deficient diet supplemented with a copper-chelating agent, triethylenetetramine tetrahydrochloride, for 8 to 10 weeks show marked involution of pancreatic acinar tissue. The present study deals with the possible mechanism of pancreatic acinar cell involution during copper deficiency. Sequential light and electron microscopic observations during the copper-depletion regimen, suggest that apoptosis is the main cause of progressive loss of acinar cells. At 4 weeks of copper deficiency, the apoptotic index was 2 +/- 0.6/1,000 cells. By 6 weeks, the apoptotic index reached a maximum of 95 +/- 25/1,000. By 8 weeks, there was almost total loss of acinar cells. The earliest change of apoptosis was characterized by condensation and margination of chromatin against nuclear membrane. Subsequently, several apoptotic bodies displayed pyknotic nucleus and eosinophilic cytoplasmic condensation. Apoptotic bodies were extruded into the interstitium or phagocytosed by unaffected acinar cells. No associated pancreatic inflammation was present. These results indicate that apoptosis is the process involved in pancreatic involution caused by copper deficiency. The molecular mechanism(s) by which copper deficiency causes apoptosis remain unclear.     

Light microscopic immunoenzyme and electron microscopic immunogold cytochemistry reveal tachykinin immunoreactivity in Merkel cells of pig skin

Light microscopic (LM) immunoenzyme and electron microscopic (EM) immunogold cytochemistry were used to demonstrate the presence and subcellular distribution of tachykinin (substance P)-like immunoreactivity in Merkel cells of pig skin. Merkel cells of sinus hair follicles were strongly immunoreactive for tachykinins. In contrast, tachykinin-like immunoreactivity was absent from or very weak in epidermal Merkel cells while subepidermal and some intraepidermal nerve fibres were clearly immunopositive for tachykinins. Postembedding immunogold cytochemistry on the EM level revealed that tachykinin-like immunoreactivity was confined to the secretory granules in Merkel cells. Tachykinin immunoreactivity was clearly absent from the axon of the Merkel cell-axon complex. The selective presence of tachykinin immunoreactivity in secretory granules strongly suggests that tachykinins are synthesized in cutaneous Merkel cells, at least of pig. This is a further indication for the concept that the Merkel cell is a member of the diffuse neuroendocrine system. However, the functional role of tachykinins like substance P and neurokinin A and of other peptides present in Merkel cells remains enigmatic.     

顺铂诱导人胃癌细胞SGC7901和人肝癌细胞HepG2凋亡的细胞形态学变化的比较

目的观察顺铂诱导人胃腺癌细胞SGC7901和人肝癌细胞HepG2细胞凋亡的细胞形态学的异同并探讨其意义。方法用倒置显微镜、荧光显微镜、透射电镜观察细胞经顺铂处理后的形态学改变。采用流式细胞术研究CDDP对两株肿瘤细胞诱导凋亡中DNA含量的影响。结果经顺铂处理后,两株肿瘤细胞均表现出一些共同的凋亡形态学特征,如细胞及细胞核皱缩,微绒毛消失,细胞膜皱缩但完整,细胞内荧光强度增强。细胞超微结构变化如线粒体肿胀,胞质空泡化。但两者在细胞核形态方面表现出较大的差异;SGC7901表现为核染色质呈团块状或环状,聚集于核膜下;而HepG2主要表现为核碎裂、核膜崩解,核染色质呈梅花状散落在细胞中和一些细胞器通过细胞出芽裂解成由细胞膜包绕的凋亡小体并被周围细胞吞噬。在两者的流式细胞DNA的直方图上均检测到了亚二倍体峰。结论顺铂可诱导SGC7901和HepG2产生不同形态的细胞凋亡,其中SGC7901主要表现为核固缩,HepG2表现为核碎裂。这种差异具有重要的生理意义。     

Morphological analysis of synovial exsudate in rheumatoid arthritis. II. Transmission electron microscopy

Synovial exsudate cells from patients with rheumatoid arthritis were investigated by transmission electron microscopy. Most of them represented polymorphous leukocytes. They contained vacuoles full of material reminding of immune complexes (in addition to typical granules) and plentiful alpha and beta glycogen granules. Some lymphoid cells nuclei were alike to those of Sézary disease with frequent ring-shaped nucleoli and enlarged mitochondria. Variegated non-lymphoid mononuclear cells had lateralized nuclei with marginated chromatin, mitochondria with sparse crists, short membranes of reticulum, conspicuous cisterns in trans-Golgi area and lysosomes with myeline bodies as a sign of big capacity of phagocytosis. Importance of using transmission electron microscopy in analysis of synovial exudate was discussed.