Showing papers in "Anatomical Record-advances in Integrative Anatomy and Evolutionary Biology in 1981"

The origin of the epicardium and the embryonic myocardial circulation in the mouse

Abstract: The origin of the epicardium and the formation of the early blood vessels of the heart prior to the opening of the coronary arteries from the aorta have been studied in the 9–13.5 day post coitum (dpc) mouse embryo heart. The epicardium begins to appear by 9 dpc. The majority of the epicardial cells derive from the somatopleural investment of the septum transversum, from where they migrate, associated to form vesicles, to the dorsal aspect of the ventricles and atria. The epicardial cells then migrate over the lateral surfaces and the AV sulcus to the ventral aspect of the heart. In the subepicardial space around the sulcuses, the proliferating epithelial tissue is found, also in vesicular form, for a time. The ventrally migrating primordial epicardial tissue ensheaths lastly the truncus arteriosus, while the sinus venosus is coated with epicardium ab initio, where (and also in the SA sulcus) the epicardial cells derive in part from the cuboidal cells of the pleuroperitoneal canal and in part from the somatopleural cells. The early blood vessel formation follows in space and time the development of the epicardium. The first blood vessels appear by 10 dpc by the invagination of the endocardium into the early sinus muscle, and at the same time in the ventricular chamber by the encasing of the endocardium, as the trabeculae become consolidated into the myocardial walls. By this process sinusoids are formed, some of which penetrate through the myocardium and which, by rapid proliferation, form an interconnected subepicardial plexus. These capillaries proliferate ventrally in the wide subepicardial space, reaching the septating truncus, in which the aorta and pulmonary artery are developing. The definitive coronary artery openings appear by 13 dpc, allowing the high pressure blood from the aorta to flow into a preexisting vascular bed.

Sympathetic innervation of murine thymus and spleen: a comparative histofluorescence study.

Abstract: Fluorescence microscopy of thymus and spleen from four strains of mice (C3H and ICR controls, AKR spontaneously leukemic and NZB autoimmune) revealed varicose noradrenergic (NE) fibers in perivascular and parenchymal regions of both organs. Thymic innervation was largely perivascular, but isolated islands and strings of free NE fibers were noted among thymic parenchymal cells. A morphological proximity between NE fibers in the thymus and mast cells was noted in all strains studied, but was exceptionally prominent in the NZB thymus. Perivascular plexuses within the splenic white pulp sent single NE fibers between the surrounding lymphocytes. Catecholamines and histamine have been shown to modulate lymphocyte development and activity in vitro. The present study provides morphological evidence that both NE and histamine are available to lymphocytes in thymus and spleen, and thus provides morphological evidence for neural modulation of immune activity in vivo.

Methods for the isolation of intact epithelium from the mouse intestine

Abstract: Methods are described for the isolation of intact intestinal epithelium in several novel forms. The first method described is for application to segments of bowel that must be studied without killing the animal, e.g., a resected length of bowel. In this method, the isolated segment is everted onto a glass rod and incubated at 37°C in fresh 30 mM ethylenediaminetetraacetic acid (EDTA) in calcium-magnesium-free Hank's balanced salt solution (CMF) for either 15 or 20 minutes. At the end of the incubation, the epithelium is isolated by vibration into a tube of cold CMF. If it is incubated for 20 minutes, the entire epithelium is isolated in the form of single crypt-villus units. If it is incubated for 15 minutes, the epithelium is isolated in the form of small intact sheets, but not all of the epithelium is removed. The second method requires the death of the animal but yields better results. The anesthetized animal is perfused through the left ventricle with a fresh 37° solution of EDTA (0.01–30 mM) in CMF. Then the segment of gut is removed, everted onto a glass rod, and the epithelium isolated by vibration into cold CMF. The whole of the epithelium is isolated with this procedure and, with higher concentrations of EDTA, it is isolated in the form of large sheets (1–2 cm2). Structurally inact units could be obtained with either procedure. There was no contamination with underlying nonepithelial elements. The best results, as determined by morphology and viability, were obtained with the perfusion method using 30 mM EDTA. With this concentration, the whole procedure, from sacrifice of the animal to isolation of the epithelium, requires less than 4 minutes. Procedures are also described for the isolation of either pure intact crypts or of pure intact vill from small intestine. Each of the methods described could be used to isolate the epithelium from any region of the intestinal tract with equal ease. Some applications of the methods are also discussed.

A morphologic and morphometric analysis of fetal lung development in the sheep.

Abstract: In the sheep, fetal lung development proceeds to a later stage of maturity than in smaller laboratory animals. Of the four stages in pulmonary development recognizable in this species - embryologic, pseudoglandular, canalicular, and aveolar - the latter three are described in the present study using histologic, morphometric, and ultrastructural techniques. During the pseudoglandular stage, the major airways developed centrifugally. Cartilaginous, glandular, muscular, vascular, and neural elements were present in major airway walls from an early age. During the canalicular stage, volume expansion of the lung was accomplished by rapid growth of large terminal spaces. In the final stage, alveoli were formed following subdivision of the large terminal spaces by alveolar crests. The alveolar lining epithelium differentiated during the latter two stages producing a large increase in alveolar surface area, particularly during the alveolar stage; a large increase in pulmonary capillary surface area also accompanied alveolar development. Thus, just prior to birth, the fetal sheep lung has a well-developed air-way system and alveolar network, in preparation for postnatal gas exchange.

Membranous systems in the "chloride cell" of teleostean fish gill; their modifications in response to the salinity of the environment.

Abstract: The membranous systems of the "chloride cell" were studied in teleostean fish gills stained in ferrocyanide-reduced osmium (Karnovsky, '71). Three distinct systems were observed: (1) the tubular system, densely stained with ferrocyanide-reduced osmium, was made up of anastomosed tubules opening in the latero basal intercellular space; (2) the endoplasmic reticulum, faintly stained, and continuous with the nuclear envelope; (3) the vesiculotubular system, the staining of which was intermediate between those of both previously cited systems, was made up of vesicles and short tubules. These membranous systems underwent modifications according to the salinity of the exterior medium: (1) the tubular system formed a broad and loose network in fresh water adapted fishes; in salt water, the meshes of the network became small, tight, and regular thus increasing the cell surface area. (2) the endoplasmic reticulum, which in fresh water, consisted of dilated cisternae often studded with ribosomes; in salt water, it developed in a network of anastomosed smooth sheets interdigitated with the tubular system. (3) the vesiculotubular system seems to be also more developed in salt water than in fresh water-adapted fishes.

Anatomy of the nasal-pharyngeal airway of experimental animals

Abstract: Silicone rubber casts were prepared of the nasal, pharyngeal and laryngeal regions of two rats, a rhesus monkey, and three beagle dogs and one for each species selected for detailed measurements. Cross-sections of the casts were made and the area and perimeter of each section measured using an image analyzing computer. Considerable anatomical differences were found between the species. Some of the differences, such as the sharp bend in the nasopharynx of the monkey, could be related to normal posture. One of the main differences was the greater complexity of the turbinate region of the dog as compared to the corresponding area of the monkey.

The somatotopic organization of the cat trigeminal ganglion as determined by the horseradish peroxidase technique.

Abstract: The somatotopic organization of the cat trigeminal ganglion has been investigated in the present study by using the horseradish peroxidase (HRP) technique. In separate animals, the corneal, supraorbital, infraorbital, inferior alveolar, or mental branches of the trigeminal nerve have been transected and then soaked in concentrated solutions of HRP. Retrogradely labeled corneal and supraorbital neurons have been found, with extensive overlap between the two cell populations, in the anteromedial region of the trigeminal ganglion. Inferior alveolar and mental neurons have been found to possess similar distributions within the posterolateral part of the trigeminal ganglion. Infraorbital cells have been localized in a central position. The cell bodies of any given nerve are found in at least minimal numbers in all dorsoventral levels of the trigeminal ganglion. However, cell bodies of origin of the supraorbital nerve and the lateral branch of the infraorbital nerve, innervating more posterior or lateral areas of the head and face, are found in greater numbers dorsally. Conversely, cell bodies of origin of the medial branch of the infraorbital nerve, the inferior alveolar nerve, and the mental nerve, supplying more rostral or intraoral areas of the orofacial region, are present in greater numbers ventrally. In contrast, corneal neurons are distributed uniformly in the dorsoventral axis. The ophthalmic and maxillary regions of the trigeminal ganglion appear to be well segregated, whereas the maxillary and mandibular regions exhibit a somewhat greater degree of overlap. Cell bodies of corneal afferent neurons range from 20 to 50 μm in diameter, whereas those of supraorbital, infraorbital, inferior alveolar and mental neurons measure from 20 to 85 μm. It is concluded from the findings of the present work that much of the cat trigeminal ganglion is organized somatotopically in not only the mediolateral axis but also in the dorsoventral axis.

The development of enamel structure in rat incisors as compared to the teeth of monkey and man

Abstract: The rat incisor is an excellent model system in which to study amelogenesis. However, the information obtained has not been extrapolated to the human because of alleged structural differences between the teeth. The obvious differences include continuous eruption in rat incisors and an enamel rod pattern in rats which seemingly differs from the keyhole pattern of human enamel. A comprehensive analysis was made of those features of enamel structure considered fundamental to the understanding of its formation. This was done by applying the knowledge of amelogenesis obtained in rat incisors to the teeth of monkey and man. The following points of basic similarity were established between these species: (1) Interrod enamel is secreted first. It forms the side walls of cavities which are initially occupied by Tomes' processes. (2) The formation of interrod cavities is followed by deposition of enamel rods within these spaces. (3) The rods conform to the shape of the cavities and are secreted from one surface of Tomes' process. (4) At the initial site of rod deposition its enamel is continuous with the interrod enamel wall. (5) Growth of the rod compresses the process to one side of the cavity resulting in an arcade-shaped “space” between the rod and the remaining interrod walls. This study demonstrates that it is no longer necessary to postulate a keyhole structure for primate enamel, and it has established that a fundamental similarity exists in the basic structure and in the mode of formation of enamel in all three species.

Effects of varying chamber construction and embryo pre-incubation age on survival and growth of chick embryos in shell-less culture.

Abstract: Shell-less culture involves culturing chick embryos with associated yolk and albumen outside of the eggshell and shell membranes. The technique allows direct access to and continuous observation of cultured embryos almost to the time of hatching. The plastic wrap/culture tripod technique described in this paper allows normal embryonic growth and differentiation from 48 hours (in ovo pre-incubation age) through at least 10 days of total incubation. As the duration of in ovo pre-incubation is decreased below 36 hours, there is a concomitant increase in the percentage of grossly abnormal embryos associated with decreased survival and retarded growth and differentiation. Survival of embryos pre-incubated for 72 hours through 13 and 18 days of total incubation is greater than 80% and 40%, respectively. The wrap/tripod technique allows substantially better survival and more normal development of cultured embryos than does the petri dish technique of Auerbach et al. (1974). Embryonic growth in 7.8-cm-diameter chambers is significantly greater than in either larger (10.6 cm) or smaller (5.2 cm) diameter chambers. Some gas exchange through the culture chamber walls appears necessary for optimal embryonic survival and growth. Suspending egg contents in either Safeway or Handi-Wrap plastic wrap (both of intermediate permeability) in 7.8-cm tripods resulted in superior growth and/or survival compared to suspension in Silastic sheeting (high permeability), Saran Wrap (low permeability), glass dishes (nonpermeable), or glass dishes lined with an inner layer of Safeway wrap(nonpermeable).

Ultrastructural evidence indicating reorganization at the neuromuscular junction in the normal rat soleus muscle.

Abstract: The ultrastructural organization of 40 soleus neuromuscular junctions from ten normal young adult male and female Sprague-Dawley (SD)-derived rats (Charles River Breeders, CD-Crl:COBS (SD)BR) has been studied. A smaller sample of motor endplates from the gastrocnemius, diaphragm, and extensor digitorum longus muscles of these rats as well as from the soleus muscles of two adult Wistar (W) rats (Crl:COBS(WI)BR) was included. Widespread ultrastructural reorganization was evident at the soleus neuromuscular junction during the growth period from three to five months of age. A major characteristic of reorganization is the presence of junctional folds not associated with axonal terminals; such sites occur within a single endplate adjacent to areas with typical intact synaptic associations. Additional features possibly related to remodelling are: (1) spatial separation of axonal terminals from the myofiber, (2) intervention of Schwann cell cytoplasm between an axon terminal and myofiber, (3) aggregates of satellite cells, and (4) folded or multilayered basal lamina. These features are most pronounced in the soleus muscle but occur to varying degrees in the neuromuscular junctions of other muscles of SD-derived rats. Distinctive characteristics of the rat soleus postjunctional sarcoplasm include the widespread occurrence of myofibrillar components, abundant free and membrane-associated polysomes, and triads oriented in various planes. Away from such discrete sites, myofibers possess the usual highly oriented organization of myofibrils, T tubules, sarcoplasmic reticulum, and mitochondria. The soleus muscle is a postural muscle that responds directly to rising workload imposed by continuous body growth during young adulthood by steady myofiber hypertrophy and conversion of motor units (Kugelberg, '76). This changing structural-functional relationship may be reflected also by ultrastructural remodelling of the neuromuscular junctions reported here.

Shunting in renal microvasculature of the rat: a scanning electron microscopic study of corrosion casts.

Abstract: We reinvestigated the still controversial existence of arterial pathways by-passing glomeruli within kidneys of rats from weaning to more than 12 months old (i.e., body weight ranging from 39 g to 643 g). For this purpose, the arterial injection of microspheres 7.5 micrometer to 17 micrometer in diameter was combined to corrosion-replication of the arterial bed of a vasodilated perfused kidney preparation. This procedure allowed easy detection of arterial by-passes with light microscope and detailed observation with scanning electron microscope. Our results clearly demonstrate the existence of various categories of arterial by-passes throughout renal cortex regardless of age. Some of them had never been described before. These vascular by-passes were found with increased frequency from superficial to juxtamedullary cortex. In the latter area, frequency was not age-dependent, and approximately 10% (range 4-22%) of juxtamedullary glomeruli were involved. Data derived from previous microsphere studies would suggest that these structures are (partially) nonfunctioning in basal physiological conditions, but more information is needed to assess their possible functional role in the rat.

Role of microtubules in the organization of the Golgi complex and the secretion of collagen secretory granules by periodontal ligament fibroblasts.

Abstract: Fibroblasts of the periodontal ligament (PDL) of the mouse exhibit a high degree of cytoplasmic and functional polarity. This polarity is dependent upon an elaborate system of microtubules. The location of the microtubular arrays and the observed effects of colchicine administration suggest that microtubules play an important role in maintaining the organization of the Golgi complex and its functional relationship to the rough endoplasmic reticulum. Smooth-walled intermediate vesicles, apparently derived from the rough endoplasmic reticulum, are aligned along microtubules at the immature face of the Golgi apparatus, and mature secretory granules are closely related to microtubules at the mature face of the Golgi apparatus. In distal cell processes the granules are closely parallel to microtubules and on occasion bridge-like attachments from granules to microtubules were noted. This relationship of secretory granules to microtubules, the lack of granule storage, and the effects of colchicine on granule secretion suggests that microtubules are part of a mechanism for collagen granule translocation from the Golgi complex to the cell periphery.

Structure and function of the murine muscle–tendon junction

Abstract: The muscle-tendon junctions of the extensor carpi radialis longus and brevis muscles from adult Balb C Bailey/J mice have been examined tensiometrically and ultrastructurally following removal of cellular membrane and soluble cytoplasm by exposure to nonionic detergent. As judged by the ability of the extracted muscle to generate tension upon exposure to ATP and to transmit the generated tension to the tendon, detergent extraction leaves the muscle-tendon junction functionally intact. Electron microscopic analysis of the extracted muscle-tendon junctions reveals that the relationship between the terminal myofilaments and the lamina densa of the basal lamina is retained, despite the extensive extraction of the plasma membrane. Fine filaments (2-7 nm) are seen to connect the lamina densa with an electron-dense intracellular layer into which terminal actin filaments appear to insert. These fine filaments are considered to represent an important component of the structural linkage between myofilaments and connective tissue and hence to be a significant component of the tension transmitting mechanism. Their precise nature is not known, but some part of the filaments must pass through the hydrophobic compartment of the plasma membrane and thus must be a transmembrane component of considerable tensile strength. These studies suggest that detergent-extractable membrane lipids play no significant role in the transmission of tension at the muscle-tendon junction, and that fine filaments, probably protein, are responsible for transmitting tension from myofilaments, through the plasma membrane, to the lamina densa of the basal lamina.

Comparative morphometry of the upper bronchial tree in six mammalian species.

Abstract: The length, diameter, and angle of branching of all airways through the sixth level of branching below the trachea were measured on corrosion casts prepared from the lungs of two animals whose bronchial geometry has not previously been studied, namely the donkey and the rabbit. These measurements and morphometric data for the rat, hamster, dog, and human obtained from other sources were analyzed and compared. The case prepared from human lungs exhibited an airway geometry that was clearly distinct from that shown by the nonhuman species. The human upper bronchial tree was the most symmetrical with respect to airway diameter and angle of branching. In all species studied, airway length was the most irregular parameter. The reasons for differences in branching geometry are not clearly understood. However, when attempting to determine whether a particular species may be used as a model for man in inhalation toxicology, and in the subsequent interpretation of animal data, an appreciation of differences in airway morphometry is essential.

Olfactory receptor cell staining using horseradish peroxidase

Abstract: Iontophoretic injection of horseradish peroxidase into severed olfactory nerve fascicles has been used to stain salamander olfactory receptor cell somata, their associated nerves, and their axonal terminations in the glomerular layer of the olfactory bulb. This technique gives homogeneous, Golgi-like staining of individually identifiable receptor cells and has permitted preliminary mapping of the topographical relationship between the loci of receptors in the olfactory mucosa and sites of their termination in the glomerular layer in the olfactory bulb.

A histological study of the Harderian gland of mongolian gerbils, Meriones meridianus

Abstract: The histological structure of the gerbil Harderian gland was investigated by means of light and transmission electron microscopy. The single excretory duct of the gland is directly continuous with endpieces at the hilus and opens nasally and ventrally to the third eyelid. The excretory duct is accompanied by many acini of small serous glands around it. The gland is composed of tubuloalveoli (tubular alveoli) with wide lumina and is not divided into lobules. There is no branched duct system within the gland. The tubuloalveoli themselves convey the secretory materials to the hilus where the excretory duct begins. The alveolar epithelium is composed of only one type of glandular cell as well as myoepithelial cells. The glandular cells contain many clear secretory vacuoles containing lipids and well-developed tubular smooth endoplasmic reticulum. The secretory vacuoles are surrounded by a unit membrane and are secreted by exocytosis. The interstices of the gland contain two types of autonomic nerve varicosities and a number of melanocytes. The surface of the gland is covered with the endothelium of the orbital venous sinus.

The wolff‐parkinson‐white syndrome and its anatomical substrates

Abstract: The anatomical substrates that underlie the clinical syndromes of preexcitation are still incompletely understood. Regarding the Wolff-Parkinson-White syndrome, there is increasing evidence that early ventricular activation is caused by accessory atrioventricular connections. These are muscular connections existing outside the specialized conduction tissue axis, which are almost always composed of working myocardial fibers. Left-sided accessory atrioventricular connections have been identified with most frequency. They occur in the presence of a well-developed fibrous annulus, thus contradicting the hypothesis that faulty development of the annulus is a necessary accompaniment. For right-sided and septal connections, this developmental concept may still hold, since there is marked variability in the formation of the right-sided annulus fibrosus. Even within these settings, however, the topography of accessory atrioventricular connections may vary considerably. Usually the accessory connection will pass on the epicardial aspect of the annulus fibrosus, particularly when left-sided. The connections are closely related to the fibrous annulus, but in other instances they may be in a superficial location. Multiple connections can occur in one heart. These anatomical features are of paramount significance for successful surgical ablation of accessory atrioventricular connections.

A stereologic analysis of collagen phagocytosis by fibroblasts in three soft connective tissues with differing rates of collagen turnover.

Abstract: The purpose of this study was to assess the importance of the phagocytic mechanism of collagen resorption in the normal turnover and remodelling of soft connective tissues. Collagen phagocytosis by fibroblasts in rat skin, attached gingiva, and periodontal ligament was quantitated using the methodology of electron microscopic stereology. Periodontal ligament contained five and 15 times as much phagocytosed collagen as attached gingiva and skin respectively. Also, for each tissue examined, a positive correlation was observed between the amount of collagen phagocytosed and the known rate of mature collagen turnover.

The origin of the hippocampal commissure in the rat.

Abstract: The present study was designated to determine the origin of commissural axons in the hippocampus. One hippocampus of 94 rats was pressure injected with 40% horseradish peroxidase (Sigma VI), or with 2-4% wheat germ agglutinin HRP (E-Y Labs). Injections (0.001 to 0.1 microliter) were made through glass micropipettes with fitted plungers. Pipettes were positioned stereotaxically, and by electrophysiological monitoring through the injection syringe. An ipsilateral stimulating electrode activated CA3 and CA1 cells via Schaffer collaterals. Population potentials were monitored as the recording pipette was advanced from the cortical surface into the hippocampus. Wave forms of monosynaptically elicited field potentials provided an accurate indicator of its position. Following survival periods of 24 hours, the brains were processed according to the Mesulam method. Forty-micron sections were serially mounted and counterstained. Injection sites and filled cells were plotted manually on a standard set of coronal sections. Our results indicate that field CA1 receives input from contralateral subfields CA1a and c, as well as from all CA3 subfields. In addition, rostral CA1 injections resulted in labeling of cells in the contralateral subiculum and entorhinal cortex. Homotopic connections exist between subfields CA3a and b; it appears that a major input to CA3c is from the contralateral polymorph cells of the dentate hilus. Commissural input to the dentate granule cells appears to be the giant polymorph and CA3c cells of the contralateral dentate hilus. With respect to the question of homotopicity, our results suggest that commissural connections are predominantly homotopic in the mediolateral plane, although CA1 and CA3 injections also resulted in contralateral labeling of hippocampal cells caudal to the levels of injection.

Three dimensional arrangement of mitochondria and endoplasmic reticulum in the heart muscle fiber of the rat.

Abstract: The three-dimensional arrangement of mitochondria and endoplasmic reticulum was studied in thick sections of the heart left ventricle fixed in glutaraldehyde and impregnated with the Ur-Pb-Cu technique and in thin sections of glutaraldehyde-fixed tissue post-fixed in potassium ferrocyanide-reduced osmium. Squarish flattened mitochondria, approximately the size of a sarcomere, were arranged in longitudinal columns in the clefts between the myofibrils. At the periphery of the fiber, the endoplasmic reticulum took the appearance of a subsarcolemmal network of plate-like and tubular cisternae running parallel to the cell surface. Between the myofibrils, the ER network formed longitudinally oriented repetitive units whose structure varied according to their position in relation to the A- or I- bands of the myofibrils. In front of the A-band, the endoplasmic reticulum appeared as a single layered network of anastomotic tubules compressed between the adjacent myofibrils. In front of the I-band, it formed a multilayered network the three-dimensional arrangement of which was dependent upon the presence or absence of the T-tubule. In the absence of the T-tubule, the ER cisternae were loosely anastomosed and occasionally displayed bulbous terminal swellings. In the presence of T-tubules, tubular ER cisternae were seen running parallel on both sides of the T-tubules and were continuous with sheet-like cisternae sandwiched between the distended T-tubule and adjacent extremities of longitudinally arranged mitochondria. These tubular or flattened cisternae were connected to each other by numerous bridging cisternae around the T-tubules.

Autoradiographic demonstration of ipsilateral and contralateral sensory nerve endings in cat dentin, pulp, and periodontium

Abstract: In order to determine the location of sensory nerve endings in cat teeth, 3H-proline and 3H-leucine were injected into the left trigeminal ganglion of eight cats aged 6.5–10 months; 24 hours was allowed for axonal transport of radioactive protein to dental nerve endings, and the endings were then detected by autoradiography. The pulps of most ipsilateral (left) teeth contained some labeled axons. These axons ended in the odontoblastic layer and predentin of roots and crown; at the tip of the pulp horn of each cusp, nerve endings also extended as far as 150 μm into dentinal tubules. Labeled nerve endings were extremely rare in contralateral (right) teeth; only one tooth of 83 studied (eight cats) contained heavily labeled axons, and one other had faintly labeled axons. Both labeled contralateral teeth were central maxillary incisors. Their labeled axons were unbranched in the root and arborized in the crown to end among odontoblasts and many adjacent dentinal tubules. Labeled periodontal nerve endings were most numerous in the apical one-third of the ligament, with some endings extending as far as the gingiva. The nerve endings in the periodontal ligament were often clustered and appeared to end freely between the collagen bundles; their radioactivity varied in the same way as that of pulp nerves in the adjacent root.

Orderly particle arrays on the mitochondrial outer membrane in rapidly‐frozen sperm

Abstract: By deep-etching and rotary replication of unfixed, non-cryoprotected tissue frozen on a helium-cooled copper block, previously undemonstrable organellar surfaces and intramembranous structures can be examined. Among the more remarkable features of mammalian spermatozoa thus prepared are the highly ordered particulate arrays on the surface of the mitochondrial outer membrane. In the midpiece of the sperm, mitochondria curl around dense fibers and the axoneme. The surface of the mitochondrion that faces the plasmalemma carries closely packed rods in haphazard dispersement, composed of two to four 70-to 80-A particles, less than 20 A apart, while the concave aspect of the organelle contains rods in stepladder pattern. These ladders are parallel, with their particles in neighboring rungs apparently in register at a 40--45 degree angle relative to the mitochondrial axis. This organizational disparity between the convex and concave surfaces of the organelle not only affords evidence of a new mitochondrial substructure, but represents a type of topographical heterogeneity rarely found except within specialized areas of the plasma membrane. Other novel findings in the sperm cell include the observation of "lipid" tracts flanking intramembranous particle-strands in the plasmalemma of the cytoplasmic droplet, and a gridiron design on the cytoplasmic faces of the droplet's microcisternae, as well as both within and atop its plasma membrane--a motif consistent with the presence of exocytotic or endocytotic activity in this portion of the cell. Additional recent observations are the differing internal and external periodicities of axonemal microtubules and the subunit structure of rectangles on the tail surface overlying the intramembranous particles of the zipper.

Sequential events in the formation of collagen secretion granules with special reference to the development of segment‐long‐spacing‐like aggregates

Abstract: The formation and maturation of collagen secretion granules in periodontal ligament (PDL) fibroblasts of young male Balb-C mice were studied by electron microscopy and cytochemistry. The Golgi apparatus was composed of several dictyosomes, each consisting of a stack of five smooth-walled cisternae. Each cisterna was connected at both ends to a dilated saccule. The cisternae, with their associated pairs of saccules, underwent progressive maturational changes from the immature to mature face of each dictyosome. The most immature saccules (type 1) were large, spherical, and filled with loosely arranged short filamentous structures. These saccules were continuous with the first and second Golgi cisternae (counting from the immature side). Golgi saccules type 2 were ellipsoidal, associated with the third and fourth cisternae, and contained parallel, elongate filaments. The most mature saccules, type 3, were more rectangular and connected by a short fifth cisterna. They contained aggregated filamentous material in the form of eight segment-long-spacing (SLS)-like crystallites, one in the center and seven at the periphery, to form a basic secretory unit. As type 3 saccules matured, the interconnecting cisterna progressively shortened until the two saccules were nearly juxtaposed. At this time the shortened fifth cisterna split to give rise to two independent presecretory granules. By progressive condensation, presecretory granules matured into secretion granules that contained a densely packed SLS- like aggregate, within which individual crystallites were no longer discernable. Maturation of cisternae and saccules involved removal of membrane, apparently by the formation and detachment of coated vesicles. The staining reaction with silver methenamine and phosphotungstic acid increased over the procollagen as the saccules matured, indicating addition of carbohydrate moieties and possible crosslinkages. It is concluded that the formation and maturation of collagen secretion granules in PDL fibroblasts involves the packaging and further modification of eight SLS-like crystallites, which are secreted as a basic unit.

Tridimensional study of the deep cortex of the rat lymph node. III. Morphology of the deep cortex units

Abstract: Recently we reported that the deep cortex of the rat lymph node is made up of semi-rounded "units," some of which are partially fused into "complexes" We further found that each unit is centered on the opening(s) of an afferent lymphatic vessel, the topographical organization of the deep cortex of a node correlating with the distribution pattern of the opening(s) of its afferent lymphatic(s) The present study aims to clarify the morphology of the deep cortex unit, particularly with regard to its reticular framework, its lymphatic sinuses, as well as its network of postcapillary venules For that purpose, we analyzed rat nodes from various locations by way of tridimensional reconstruction The observations revealed that each unit is formed of a "center" and a "periphery," distinguishable from one another on the basis of their morphological features The center is nearly devoid of reticular fibers, whereas the periphery exhibits a dense framework of fibers Moreover, the periphery is the site of concentration of most postcapillary venules of a unit and contains lymphatic sinuses which, peculiarly, are often loaded with small lymphocytes While both regions are populated mainly by small lymphocytes, the periphery usually contains a lower concentration of these cells than the center The overall findings support the view that the center is a site of cellular retention and proliferation, whereas the periphery is a site of rapid lymphocyte migration in and out of the unit

Development of seminal vesicles and coagulating glands in neonatal mice. I. The morphogenetic effects of various hormonal conditions.

Abstract: Seminal vesicles (SV) and coagulating glands (CG) from neonatal mice 1- to 7-days old were observed in whole mount preparations. Untreated, normal SV developed elaborate epithelial branches beginning on day 3 with secondary branches appearing on day 6. Castration (C), estradiol treatment (E), and castration combined with estradiol treatment (C + E) inhibited the morphogenesis of the epithelial branches. Untreated CG formed solid epithelial stalks that developed lateral epithelial buds on day 3 which attained a complex morphological pattern by day 7. Treatment groups (C, E, and C + E) displayed a pattern of retarded growth with few epithelial buds appearing even at day 7. The effects of castration on both SV and CG were reversed by the addition of testosterone. Short term in vitro culture of 1-day-old SV and CG glands in control medium or medium supplemented with estradiol did not exhibit visible growth. Culture of SV and CG glands with testosterone or a piece of testis showed pronounced development.

Ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase), thiamine pyrophosphatase (TPPase), and cytidine monophosphatase (CMPase) in the Golgi apparatus of early spermatids of the rat.

Abstract: At the early steps 3-7 of spermiogenesis the hemispherical Golgi apparatus elaborates and is closely associated to the acrosomic system which grows at the surface of the spermatid's nucleus. It shows two distinct zones, a cortex made up of flattened saccules and related membranous tubules, and a medulla containing various types of vesicular profiles. The various components of the cortex of the Golgi apparatus were tested for their reactivity to three phosphatases. Nicotinamide adenine dinucleotide phosphatase activity (NADPase, Smith, 1980) was observed in the middle two to six saccules in the stack with a midsaccule being more reactive than the saccules above and below. A weak and spotty reaction was also noted in the remaining saccules on the trans-face of the stack and in the thick elements making up the GERL on the trans aspect of the stacks of saccules. Thiamine pyrophosphatase activity (TPPase, Novikoff and Goldfisher, 1961) was found in one or two saccules on the trans-face of the stacks but was absent from the other Golgi components. Cytidine monophosphatase activity (CMPase, Novikoff, 1967) was observed in the GERL, in vesicles of the medulla and in the developing acrosomic system. In the intersaccular regions of the cortex the branching membranous tubules showed the same reactivity for the phosphatases to that of the saccules to which they are connected. ER cisternae associated with the Golgi apparatus, anastomotic membranous tubules seen in the peripheral Golgi region, small vesicles, as well as the first saccule on the cis-face of the stacks were all negative for the three enzymes studied. These data indicated that in the cortex of the Golgi apparatus there were several distinct compartments that could be distinguished on the basis of structural and cytochemical features.

Effects of acid-stress on epidermal mucous cells of the brown bullhead Ictalurus nebulosus (LeSeur): a morphometric study.

Abstract: The effects of acid water on alcian blue-periodic acid Schiff's stainable mucosubstances of epidermis from the anterior abdominal wall of brown bullhead catfish, Ictalurus nebulosus, were studied. Standard morphometric techniques were used to determine mucous cell volume density (MCVv) and number density (MCNv) of control (pH 6.8-7.0) and acid-stressed (pH 5.7-6.15) fish. Mucous cell volume density was significantly increased (P less than 0.005) in fish which had been exposed to acid-stress for five days. Since increase in volume density can arise from either an increase in the size of individual mucous cells (hypertrophy), an increase in the number of mucous cell per unit volume (hyperplasia), or a combination of the two, average cell data were computed. No significant difference in cell diameter or cell volume between treated and control groups was seen. However, the number of mucous cells in acid-stressed fish was nearly twice (p less than 0.005) that of controls. These results demonstrated that acid-stress resulted in increased number but not volume of mucous cells (i.e., hyperplasia without hypertrophy).

Morphological changes in the mechanically unloaded myocardial cell.

Abstract: The role of stretch and/or tension in maintaining the structural integrity of the myocardial cell was investigated in 16 cats. Right ventricular papillary muscles were studied 1-28 days after transection of the chordae tendinae and compared to adjacent intact papillary muscles. A progressive atrophy, as shown by decreased mean cell cross-sectional area, occurred; by the 28th day mean cardiocyte area was only 28% of the control value. The earliest ultrastructural changes (one day after surgery) were primarily focal and included disorientation of contractile filaments and loss of Z-line substance. During the first week, vacuolation, loss of contractile filaments and infiltration of macrophages and fibroblasts were characteristic. By the second week a massive loss of contractile substance, disappearance of the sarcoplasmic reticulum, and a marked increase in connective tissue occurred. Leptomere structures, membrane alterations, and phagocytosis were the most typical changes between the second and fourth week. Hydroxyproline assays on papillary muscles unloaded for three days showed a 38% increase in connective tissue, indicating an early increase in connective tissue/muscle mass associated with mechanical unloading. It is concluded that the cardiocyte is extremely dependent upon mechanical loading, i.e., stretch and/or tension. Mechanical unloading (tenotomy) results in rapid and marked cellular degeneration which exceeds that observed in skeletal muscle following either disuse or denervation.

Change in intramembranous particle distribution in epididymal spermatozoa of the boar

Abstract: Membranes of boar spermatozoa from different regions of the epididymis and after ejaculation were studied by the freeze-fracture replica technique. The ordered pattern of the intramembranous particles of spermatozoan plasma membranes was different in the five arbitrary zones of the epididymis and in the semen. A distinctive ordered pattern was absent in zone 1, which is the proximal segment of the epididymis. In zone 2, paired parallel rows of the particles were present in the plasma membrane over the acrosomal region. This parallel arrangement was not present in zone 3 spermatozoa. Anterior to the posterior ring, cords formed by packed particles were apparent in zone 2 spermatozoa and reached their maximum prominence in zone 3, and persisted in zones 4 and 5 and in the semen. The plasma membrane over the marginal ridge of the acrosome had a hexagonal array of particles only in zones 4 and 5 spermatozoa. A similar pattern appeared on the post-acrosomal region of spermatozoa in zone 5 and in the semen. The plasma membrane of the middle piece had a rectilinear arrangement of the particles in zone 2 spermatozoa in which the migration of the cytoplasmic droplet was complete. Rudiments of the rectilinear arrangement persisted in spermatozoa in zones 4 and 5 and in the semen. These changes are discussed in relation to sperm maturation in the epididymis. The acrosomal membrane had a hexagonal arrangement of particles in the equatorial segment. The marginal ridge of the outer acrosomal membrane had parallel rows of intramembranous particles. The organization of the acrosomal membrane particles did not change during the epididymal passage of boar spermatozoa.

Histology of the canine urethra. I. Morphometry of the female urethra

Abstract: Urinary bladders and urethrae were collected from six adult and two juvenile female dogs. Five urethral regions and the neck and body of the bladder were sampled. Volume fractions for connective tissue including elastic fibers, smooth and striated muscle, and epithelium were obtained by projecting section images onto an array of points and computing the number of points overlying a tissue constituent per total points overlying the tissue section. Smooth muscle occupied approximately half the volume of the bladder wall, one-third the volume of the vesical neck, and one-fourth the volume of the proximal urethra. Striated muscle was present in the distal half of the urethra, where the total muscle coat occupied about one-third of the urethral wall volume. Smooth muscle was practically absent in the terminal urethra, where the striated urethralis muscle encircles urethra and vagina in common. Epithelial area and lumen perimeter were not significantly different along the length of the urethra except that urethral epithelium was significantly thicker adjacent to the vesical neck. In terms of histological proportions, the vesical neck was intermediate between the body of the bladder and the proximal urethra.