Apoptosis in the myocardium of the adult dromedary camel: ultrastructural characterization.

Apoptosis is a highly regulated mode of cell death that occurs in the absence of inflammation. Light microscopic (LM) examination of the myocardium of apparently healthy camel did not reveal evidence of apoptosis in any samples; however, evidence of apoptosis was apparent by transmission electron microscopy (TEM). The most common apoptotic features observed by TEM included (1) an intact sarcolemma with some bleb formation; (2) nuclear chromatin condensation and margination with nucleolar disruption; (3) mitochondrial swelling and disorganization, accompanied by degeneration or hypercondensation of cristae; and (4) an intercalated disc region with a higher-than-normal mitochondrion/myofibril ratio, or surrounded from both sides by asymmetrically contracted sarcomeres. Apoptotic alterations were also noted among the endothelial cells lining the microvasculature of the myocardium. These alterations included (1) marked nuclear chromatin condensation and margination; (2) villous blebs on the adluminal plasmalemma, which projected into the lumen; (3) cytoplasmic vacuolation; (4) presence of intraluminal membrane-bounded vesicles; and (5) occasional pericapillary edema and accumulations of cellular debris. The results of this study indicate that myocardial apoptosis can occur in apparently healthy camels, in the absence of a clear-cut etiology.

A morphological and histological examination of the pan-tropical spotted dolphin (Stenella attenuata) and the spinner dolphin (Stenella longirostris) adrenal gland.

The morphology and histology of the cetacean adrenal gland are poorly understood. Therefore, this study examined 32 pairs of adrenal glands from 18 pan-tropical spotted dolphins (Stenella attenuata) and 14 spinner dolphins (Stenella longirostris). In both species, the cortex was pseudolobulated and contained a typical mammalian zonation. Medullary protrusions (0-3 per section) and a medullary band were identified in both species. For S. attenuata, no statistical differences were found in the cortex to medulla (CM) ratio or the percent cross-sectional area (PCA) of the adrenal glands compared with sex or sexual maturity. The mean CM ratio for S. attenuata was 2.34 and the PCA was 64.4% cortex, 29.4% medulla and 6.2%'other'. 'Other' indicates blood vessels, connective tissue and the gland capsule itself. For S. longirostris, there was no statistical difference in the CM ratio compared with sexual maturity. However, a statistical difference was found between the CM ratio and sex, suggesting sexual dimorphism (female CM ratio = 2.46 and males = 3.21). No statistical differences were found in the PCA of S. longirostris adrenal glands by sexual maturity. However, a statistical difference was found between the PCA by sex. Female S. longirostris adrenal glands consisted of 65.0% cortex, 27.3% medulla and 7.7% 'other', whereas male adrenal glands consisted of 71.7% cortex, 22.7% medulla and 5.6% 'other'.

Morphological changes in the Atlantic bottlenose dolphin (Tursiops truncatus) adrenal gland associated with chronic stress.

Beach-stranded Atlantic bottlenose dolphins (n=68) were categorized as either "acutely stressed" (if they died from net entanglement, boat strike, or acute infection; 31 animals) or "chronically stressed" (if they suffered from or died as a result of long-term disease or debilitating injury; 37 animals). No significant differences in mass between the right and left adrenal glands were found within each category. However, the average gland mass (AGM), based on the right and left glands together, was 5.2g for acutely stressed animals and 11.01 g for chronically stressed animals (P<0.001). Significant differences were also found, in terms of the ratio of cross-sectional areas of the cortex to medulla, between acutely stressed (ratio 1.22) and chronically stressed (ratio 1.63) animals (P=0.027). Adrenal glands of acutely stressed animals consisted of 48% cortex, 41% medulla, and 11% other tissue elements (connective tissue, blood vessels and gland capsule), whereas the corresponding figures for chronically stressed animals were 53%, 36%, and 11%. The mean estimated mass values for cortex, medulla and other tissue were, for acutely stressed animals, 2.36, 1.9, and 0.54, respectively, whereas for chronically stressed animals the corresponding figures were 6.06, 4.04, and 1.29 (P<0.001 for each of the three comparisons). Overstaining with haematoxylin (HEM) and immunohistochemical labelling (IHC) of the enzyme phenylethanolamine N-methyl transferase (which converts norepinephrine to epinephrine) were used to determine the percentage of epinephrine-producing cells in relation to the overall cross-sectional area of the adrenal gland. The percentage values in acutely as compared with chronically stressed dolphins were 6.7% and 15.93%, respectively (P=0.021). The results therefore suggest that in bottlenose dolphins chronic stress results in increases in (1) adrenal mass, (2) cortex to medulla ratio, and (3) epinephrine-producing cells within the medulla, giving rise to an increase in the thickness of the medullary band.

Ultrastructural analysis of the integument of a desert-adapted mammal, the one-humped camel (Camelus dromedarius).

In this study, we conducted a light microscopic and ultrastructural analysis of the integument of the one-humped camel (Camelus dromedarius). In general, the epidermal strata of the camel integument appeared typical of those found in non-desert mammals. Two cell populations were noted in the stratum basale: one with a flat, non-serrated base and the other with a highly serrated base. Typical fine structure was observed in keratinocytes of the stratum spinosum and stratum granulosum. The stratum corneum was six to 10 cells thick. Within the different strata, overall cell morphologies and the general distribution and relative abundance of cellular organelles appeared typical. Dermal features included the presence of myoepithelial cells surrounding apocrine tubular glands. Inter- or intracellular canaliculi within the secretory cells of the apocrine glands, reported to be present in certain other non-desert mammals, were not evident in the camel. Together, these data indicate that while the camel is clearly adapted for a desert lifestyle, these adaptations do not include significant specializations at the cellular or subcellular level in the integument.

Morphology and histology of the atlantic bottlenose dolphin (Tursiops truncatus) adrenal gland with emphasis on the medulla.

This study provides the first detailed description of the Atlantic bottlenose dolphin (Tursiops truncatus) adrenal gland with emphasis on the medulla. Thirty-one dolphins of varying age and sex were used in this study. No statistical differences were found between the right and left gland mass, however, the left was typically greater. Mean mass for the right and left adrenal glands were 4.99 +/- 0.513 and 5.36 +/- 0.558 g, respectively. No statistical differences were found between average gland mass and sexual maturity or sex. The average cortex/medulla ratio was 1.22 +/- 0.060 meaning approximately 48% is cortex, 41% is medulla, and 11% was categorized as other (i.e. blood vessels, connective tissue, etc.). The cortex contained pseudolobules and the typical zonation. A medullary band, consisting of highly basophilic staining cells was found at the periphery of the medulla. Projections of the medulla to the gland capsule were noted. Immunolabelling with polyclonal antibodies against the enzymes dopamine beta hydroxylase and phenylethanolamine N-methyl transferase indicated that noradrenaline producing cells are found throughout the medulla including the medullary band while adrenaline producing cells are only found within the medullary band. Transmission electron microscopy confirmed the presence of two distinct cell populations within the medullary band and a single cell population throughout the medulla.

Actin-related intercellular adhesion junctions in the germinal compartment of the testis in the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus).

The germinal epithelium of male vertebrates consists of Sertoli cells and spermatogenic cells. Intercellular junctions formed by Sertoli cells assume critical roles in the normal functions of this epithelium. While Sertoli cell junctions have been well characterized in mammals, similar junctions in nonmammalian vertebrates have received little attention. We examined the intercellular junctions found within the germinal epithelium of the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus). Ultrastructurally, Sertoli cells were seen to form filament-associated junctions in both species. Adjacent Sertoli cells formed microfilament-related junctions near their apices. Filaments of these junctions were arranged in loose networks and were not associated with cisterns of endoplasmic reticulum. In fixed, frozen sections of hagfish testis, similar areas labeled with rhodamine phalloidin, indicating the filament type is actin. In the lamprey, desmosomes were observed immediately below the microfilament-related junctions. In appearance and location, the Sertoli cell junctions observed in these species resembled those of the typical junctional complex of other epithelial cell types. No junctions were observed between Sertoli cells and elongating spermatids. In the hagfish, but not the lamprey, an additional zone of microfilaments occurred near the base of Sertoli cells in areas of association with the basal lamina. Our observations are consistent with the proposal that the unique forms of intercellular attachment found in the testes of higher vertebrates evolved from a typical epithelial form of intercellular junction.

Lingual ultrastructure of the long-finned pilot whale (Globicephala melas).

Microscopic studies on the cetacean tongue are limited and, to date, only a few ultrastructural reports on dolphins have been published. This report presents the initial description of the lingual ultrastructure of the long-finned pilot whale. The lingual integumental surface was smooth, lacking papillae, although flaking of outer stratum corneum cells could be observed at high resolution. The keratinocytes of the stratum spinosum of the epidermis resembled those of cetacean skin on other regions of the body. The similarities included the presence of cytoplasmic lipid droplets around the nuclei of stratutm spinosum cells, a lingual feature not seen in terrestrial mammals. Keratin intermediate filaments were numerous and occasionally formed aggregates of circular whorls. At cell surfaces, bundles of keratin intermediate filaments were frequently observed inserting into desmosomal plaques. Pigment granules were not evident and organelles were sparse. Stratum corneal cells contained nuclear remnants (parakeratosis) and small multivesicular bodies, and the corneal layer was approximately 18 cells thick. The nuclei of the stratum basale keratinocytes possessed exceptionally numerous and deep clefts. The dermis was nondistinctive. The skeletal muscle of the tongue was arranged in widely separated fasiculi containing small numbers of muscle fibres. Typical fine structure of skeletal muscle bands and tubular elements was observed by transmission electron microscopy.

Unique and multifunctional adhesion junctions in the testis: ectoplasmic specializations.

In this paper, we review the structure and function of a unique type of actin-related intercellular adhesion junctions in the testis. Based on their ultrastructure, the junctions are divided into five distinct domains. The currently identified molecular components of each domain are summarized. In addition, the architecture of the mammalian system is compared with that of non-mammalian vertebrates. Functionally, the junctions are related to the turnover of adhesion between Sertoli cells, to the attachment of spermatids to the seminiferous epithelium, and to sperm release. They also are part of the mechanism by which spermatids are moved through the epithelium. Evidence consistent with adhesion and motility related functions is discussed. Control, both of junction turnover and of microtubule-based transport, is identified as an important avenue for future research.

Microtubules in Xenopus oocytes are oriented with their minus-ends towards the cortex.

Despite lacking centrosomes, stage VI Xenopus oocytes contain extensive networks of cytoplasmic microtubules (MTs). To gain additional insight into the factors regulating MT organization during oogenesis, we have used electron microscopy and "hook decoration" to examine the distribution and orientation of MTs in Xenopus oocytes. A limited survey of two "undecorated" stage VI oocytes revealed 218 MTs in images covering approximately 2,500 microm(2), and indicated that the MT number density of the animal cytoplasm was greater than that of the vegetal cytoplasm. Examination of five "decorated" stage VI oocytes (three animal and five vegetal hemispheres) revealed 653 MTs. Of these, 76% could be scored as having exclusively counterclockwise (CCW) or clockwise (CW) hooks. In the animal hemispheres, 93% of the scored MTs exhibited CCW hooks when viewed from the direction of the cortex, indicating that most MTs were oriented with their minus-ends out. MT orientation appeared relatively uniform throughout the animal cytoplasm: more than 90% of the scored MTs in the cortical (90%), subcortical (96%), or perinuclear (98%) cytoplasm were oriented with their minus-ends out. In the vegetal hemispheres, approximately 80% of the scored MTs exhibited CCW hooks, and thus were oriented with their minus-ends out; 96% of the scored MTs in stage III oocytes were oriented minus-end out. These observations support a model in which the cortex plays a significant role in MT nucleation and organization in Xenopus oocytes, and have significant implications for the MT-dependent transport and localization of cytoplasmic organelles and RNAs during oogenesis.

Sperm nuclear basic proteins (SNBPs) of agnathans and chondrichthyans: variability and evolution of sperm proteins in fish.

We have characterized for the first time SNBPs from the hagfish Eptatratus stouti (Myxini) and the lamprey Lampetra tridentatus (Cephalaspidomorphi) and have found that histones are the major protein components of the sperm of these agnathans. We have also conducted a systematic analysis of SNBPs from different groups of chondrichthyan fishes, including the skate Raja rhina and seven species of sharks. Together with our previous data showing the sporadic nature of SNBP evolution in bony fish (Saperas, N., Ausio, J., Lloris, D. and Chiva, M. [1994] J. Mol. Evol. 39: 282-295), the present study provides a unique insight into the overall evolutionary complexity and variability of the nuclear sperm proteins of fishes. It would appear that despite the discontinuous evolution of these proteins, the macroevolutionary pattern of histone (H type) --> protamine-like (PL type) --> protamine (P type) has been conserved in fish evolution, as it has in the evolution of other Deuterostomes.

The perinuclear centriole-containing centrosome is not the major microtubule organizing center in Sertoli cells.

Microtubules are abundant in Sertoli cells and are predominantly arranged parallel to the long axis of the cell. In addition, the centrioles occur in a perinuclear position in the basal one third of the cell. In this study we investigate the importance of the perinuclear centriole-containing centrosome as a microtubule organizing center (MTOC) in Sertoli cells. In all experiments, rat testes were perfusion fixed and then processed for electron and/or fluorescence microscopy. For fluorescence microscopy, fragments or dissected pieces of seminiferous epithelium were labeled for tubulin and actin. The three-dimensional pattern of microtubules in Sertoli cells was determined using data collected with a confocal microscope and analyzed using the NIH-Image program (written by Wayne Rasband at the NIH). The detailed arrangement of microtubules around, and the number of microtubule ends associated with, the centrosome were determined from composite projections constructed from serial thin sections. The nucleating potential of the perinuclear centrosome was determined by perfusing testes for 6 h with 10 micrograms/ml nocodazole and then for up to 3 h with control buffer prior to fixation and analysis with confocal and standard fluorescence microscopy. Microtubules are not organized around a focal perinuclear site and few microtubule ends are associated with the centrosome. Moreover, in cells recovering from nocodazole treatment, microtubules first appear in apical (peripheral) processes. Our data indicate that the centriole-containing perinuclear centrosome is not a significant MTOC in Sertoli cells. Rather, microtubules are nucleated in peripheral regions and project basally. Based on the observations that microtubules appear to "cuff" the nucleus, intermediate filaments are concentrated around the nucleus, microtubules project into the perinuclear intermediate filament network, and microtubules and intermediate filaments are often coaligned, we suggest that microtubules are anchored into position at the base of the cell via linkages with the intermediate filament network. Our nucleation-anchorage model of microtubule organization in Sertoli cells may be applicable to other epithelial systems.

Lack of inter-species reactivity between antigens and antibodies is overcome by protease treatment of western blots.

The analysis of proteins by Western blotting is frequently limited by the inability of antibodies to recognize antigenic epitopes in proteins of different species. In the present study, we investigated the influence of mild protease digestion on the reactivity of nitrocellulose-blotted proteins and microscopic sections with antibodies produced against analogous proteins of different species. The proteins were partially purified, electrophoresed and blotted by standard procedures. They were then incubated with either trypsin or pepsin, at concentrations ranging from 0.5 to 80 micrograms/mL, for different time periods prior to reaction with the first antibody. After incubation with the second antibody, the bands were visualized by chemiluminescence. The following antibody/antigen pairs produced no signal by conventional methods but resulted in the appropriate bands following protease treatment: (i) monoclonal antibody (Mab) to human keratin #18/rat keratin; (ii) Mab to starfish extracellular matrix/mouse laminin; (iii) rabbit antiserum to human platelet myosin II/ratfish nonmuscle myosin II. In the latter system, protease treatment also revealed previously hidden epitopes in microscopic sections. Appropriate controls demonstrated that the antibodies retained their specificity after the protease treatment of the preparations. Optimal conditions varied and had to be defined for each protein under study.(ABSTRACT TRUNCATED AT 250 WORDS)

Filamentous actin detected in rat spermatozoa.

In this paper we report the positive staining of epididymal spermatozoa and testicular cells (late spermatids and spermatozoa) with fluorescent phallotoxins. Staining is most obvious with rhodamine phalloidin, but is also detectible with NBD-phallacidin. Specific fluorescence is emitted as a linear tract along the dorsal curvature of the head and as an inverted V-shaped structure in what appears to be the anterior aspect of the post-acrosomal region. We conclude that filamentous actin occurs in the heads of rat spermatozoa. Moreover, we speculate that this filamentous actin is concentrated in two regions of the perinuclear theca; in the subacrosomal space along the dorsal curvature of the nucleus, and in the post-acrosomal region in an area termed the ventral spur.

Ectoplasmic ("junctional") specializations in Sertoli cells of the rooster and turtle: evolutionary implications.

Ectoplasmic specializations are complex actin-containing structures found at certain sites of intercellular attachment in Sertoli cells. Current evidence indicates that these structures are a form of actin-associated adhesion junction. In the turtle (Pseudemys scripta) and rooster (Gallus domesticus) ectoplasmic specializations are known to occur adjacent to sites of attachment to elongate spermatids and are characterized by a layer of "loosely" cross-linked actin filaments that lies next to the plasma membrane. In the turtle, a cistern of endoplasmic reticulum is associated with the cytoplasmic face of the filament layer. We have found that, in fixed frozen sections of turtle and rooster testes, immunological probes for myosin II react with epithelial regions that also stain with probes for filamentous actin and that are known to be sites at which ectoplasmic specializations occur. Furthermore, when exposed to standard contraction buffers, the diameters of glycerinated ectoplasmic specializations of the turtle are statistically smaller than those of the same structures exposed to control buffers. We interpret these smaller diameters as being produced by the contraction of actin bundles within the ectoplasmic specializations. Our results indicate that ectoplasmic specializations in the rooster and turtle, unlike those in mammals, possess contractile properties. We speculate that ectoplasmic specializations in eutherian mammals may have evolved from actin-associated adhesion junctions in which the actin bundles were initially contractile and from which myosin II was secondarily lost.

Actin filaments associated with the basal Sertoli cell surface in the alligator and turtle.

We have studied, using electron microscopy, the basal cytoplasm of Sertoli cells in the alligator (Alligator mississippiensis) and the turtle (Pseudemys scripta). Filament bundles occur immediately adjacent to the basal plasma membrane. The diameter of these filaments is smaller than that of perinuclear intermediate filaments, but is similar to that of filaments present in peritubular cells. In the alligator, the filament bundles appear to form a branching network over the base of the cell and are related to cisternae of the endoplasmic reticulum. In the turtle. filaments occur either in distinct bundles within folds of the plasma membrane or as a more uniform layer over the base of the cell. In fixed frozen sections, labelled with rhodamine phalloidin, linear bands of fluorescence occur in regions which correspond to those containing the filament bundles observed by electron microscopy. We conclude that the basal filaments in alligator and turtle Sertoli cells are actin and speculate that these filaments may be involved in cell/substratum adhesion.

Evidence that vinculin is co-distributed with actin bundles in ectoplasmic ("junctional") specializations of mammalian Sertoli cells.

Ectoplasmic specializations of Sertoli cells are actin containing structures found at sites of attachment to spermatids and to neighboring Sertoli cells. We suspect that these cytoskeletal structures are a form of actin-associated adhesion junction. If this is true, then molecular components, such as vinculin, that characterize actin-associated adhesion junctions in general should be present in ectoplasmic specializations. In this paper we have used two approaches to verify the prediction that vinculin is a component of ectoplasmic specializations. First, we have used fluorescence microscopy to probe immunologically for vinculin in ectoplasmic specializations associated with spermatids of the ground squirrel. Second, we have used immunogold techniques to probe for vinculin in ectoplasmic specializations of rat testis. Our results indicate that the immunological probe for vinculin was reactive with ectoplasmic specializations. In single label fluorescence experiments, linear patterns obtained with the vinculin probe were similar to those obtained with probes for filamentous actin. In double label experiments, the vinculin probe was co-distributed with the actin probes. In immunogold studies, specific labelling with the probe for vinculin occurred in ectoplasmic specializations both at sites of attachment to spermatids and adjacent to basal Sertoli cell junctions. Moreover, gold particles were concentrated adjacent to filament bundles within each ectoplasmic specialization. Our results support the conclusion that vinculin is present in ectoplasmic specializations. Further, they indicate that vinculin is co-distributed with actin bundles within each ectoplasmic specialization.

Evidence for the presence of actin-associated intercellular adhesion junction between interstitial cells of Leydig in the ground squirrel testis.

Interstitial cells of Leydig characteristically occur in clusters around blood vessels. Often these clusters remain intact when interstitial tissues are mechanically separated from other components of the testis. The presence of strong intercellular attachments is most likely one of the factors responsible for maintaining the integrity of Leydig cell clusters. In many tissues, actin associated adhesion junctions commonly provide intercellular attachment. To determine if actin associated adhesion junctions are present between Leydig cells, we have used 1) immunofluorescence to probe for two components that characterize these junctions in other tissues and 2) electron microscopy to examine areas of intercellular contact for evidence of microfilament related adhesion junctions. Isolated clusters of unsectioned cells, which had been fixed and detergent extracted, were probed with the F-actin specific strains rhodamine phalloidin and NBD-phallacidin and with an affinity purified primary antibody raised against human platelet vinculin. In regions of intercellular contact, fluorescence staining with the actin probes was intense and appeared as a solid linear band. Similar regions also stained with the vinculin probe. In double label experiments, actin and vinculin probes were co-distributed at sites of intercellular contact. Zones of intercellular contact, apparently similar to those detected with fluorescence microscopy, were observed at the ultrastructural level. At these sites, subsurface filaments, interpreted by us as actin, formed a dense carpet adjacent to the plasma membrane on each side of the junction. These filaments appeared to be organized into networks rather than discrete bundles.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunofluorescence localization of vinculin in ectoplasmic ("junctional") specializations of rat Sertoli cells.

We have investigated, using indirect immunofluorescence techniques, the possibility that vinculin is a component of Sertoli cell ectoplasmic specializations. Affinity-purified polyclonal antibodies produced against human platelet vinculin were used to probe fixed frozen sections of rat testis. Specific fluorescence occurs in Sertoli cell regions adjacent to spermatids and to basally situated junctional complexes, sites at which ectoplasmic specializations are known to occur. Staining also occurs in Sertoli cell regions associated with tubulobulbar complexes. The antibody also labels focal contacts in cultured human dermal fibroblasts, apical junctional sites of rat epididymal epithelium, and dense plaques of smooth muscle. Our results are consistent with the prediction that vinculin is likely a component of ectoplasmic specializations and are also consistent with the hypothesis that these structures are a form of actin-associated adhesion complex.

Gastric and cardiac organoprotection by lidocaine.

The concept of cytoprotection has been applied to many tissues afforded protection by drugs or endogenous chemicals against organelle, cyto- or histopathologic damage. We review here the "organoprotection" by lidocaine in rats and dogs as appraised by in vitro, ex vivo, and in vivo experiments with the stomach and heart, and as revealed at organelle to organ functional levels. Gastric mucosal lesions induced by 80% ethanol with 100 mM HCl on the ex vivo rat stomach were significantly reduced by lidocaine (2.2-4.4 mg/kg bolus followed by 66-132 micrograms/kg/min i. v. infusion). In anesthetized dogs with gastric corporeal lesions induced by increased gastric intraluminal pressure (50 mm Hg, 2.5 hrs), lidocaine (2.2 mg/kg bolus plus 66 micrograms/kg/min infusion) significantly reduced lesion severity. In the isolated rat heart, reperfusion after a 60 min period of ischemia induced localized cardiac mitochondrial swelling and disruption in ventricular apices which was greatly reduced if hearts were pretreated (15 min perfusion with lidocaine). In intact rats subjected to hemorrhagic shock, lidocaine pretreatment also facilitated shock resuscitation and reduced ultrastructural damage. In these diverse experiments, lidocaine organoprotection was likely mediated in part through reduction of ischemia induced organelle membrane damage and through reduction of reperfusion-induced superoxide and other oxygen-derived free radical related damage.