Showing papers in "Anatomical Record-advances in Integrative Anatomy and Evolutionary Biology in 1971"

Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action

Abstract: The properties of the inorganic dye ruthenium red are presented with emphasis upon its use for electron microscopy of cells and tissues. Although commercial ruthenium red often can be used directly, it always contains various impurities and by-products. One of these, termed ruthenium violet, can be isolated and is useful by itself. Absorption spectra of the ruthenium dyes and common impurities are given so that an assay is possible for any sample. Convenient fixative recipes containing ruthenium red or violet are provided together with constraints necessary for a reliable reaction to label extracellular acidic mucosubstances. Perfusion was not successful. The specificity of the ruthenium red reaction was evaluated by spot testing with 57 substances, and by titration with chemically defined pectins. The results indicate that ruthenium red, as a hexavalent cation, precipitates a large variety of polyanions by ionic interaction, and that its classical reaction with pectin is typical rather than specific. New data are presented regarding its reaction with phospholipids. For electron microscopy, a further reaction with OsO4 amplifies the feeble electron density, which is the counterpart of its intense optical labeling, to a practical level resulting in strong contrast. An hypothesis is presented for the mechanism underlying this intensification.

Satellite cells as the source of nuclei in muscles of growing rats.

Abstract: The source of the new nuclei appearing during the growth of muscle fibers was examined in the tibialis anterior muscle of young Sherman rats (14–17 days of age) using radioautography at various intervals after a single injection of a small, non-toxic dose of 3H-thymidine (2 μCi/g body weight). Two techniques were employed: (1) labeled nuclei were detected in 1 μ thick radioautographs examined in the light microscope, and identified by simultaneous electron microscope examination of an adjacent section. The nuclei were then classified either as “true” muscle nuclei (within the plasmalemma of the fibers) or as belonging to “satellite cells” (which are mononucleated cells with scanty cytoplasm wedged between plasmalemma and basement membrane). (2) Muscle fibers freed by collagenase digestion were radioautographed one hour after 3H-thymidine injection in order to determine the total number of labeled nuclei (true muscle nuclei plus those of satellite cells) per unit length of fiber. Certain nuclei within the basement membrane of muscle fibers are labeled one hour after 3H-thymidine and, therefore, synthesize DNA. The electron microscope demonstrates that these nuclei invariably belong to satellite cells, never to true muscle nuclei. Furthermore, the total number of labeled nuclei per unit length of fiber doubles between 1 and 24 hours; and, therefore, the labeled satellite cell nuclei undergo mitosis. Following mitosis, half of the daughters of satellite cells are incorporated into the fibers to become true muscle nuclei. The remaining half divides again later; and half of their daughter cells are incorporated. Thus, satellite cells in young rats divide repeatedly and function as a source of true muscle nuclei.

Ruthenium red and violet. II. Fine structural localization in animal tissues.

Abstract: The inorganic dye, ruthenium red, stains extracellular materials in animal tissues which probably are acidic mucopolysaccharides. It complements other techniques, its advantages being fine grain, high resolution and good contrast. Localization is shown in mouse and rat muscle, heart, lung and intestine, frog cartilage and cells scraped from oral epithelium of human beings. Attention is paid to collagen bundles, the cell/collagen interface and particularly the myotendinal junction, cartilage matrix and agar gel, desmosomes, intestinal microvilli, erythrocytes and vascular endothelium, nerve fibers and the T-system of striated muscle. Although ruthenium red generally is excluded by plasma membranes, it penetrates giving intracellular density, if the membrane is broken. Even when the cell membrane is intact, exceptions occur with selective staining of the T-tubules or the sarcoplasmic sacs depending upon the state of contraction of the muscle cell, and with intracellular staining of certain nuclei and epithelial cells. Ruthenium red stains intracellular lipid droplets revealing lamellae, and stains myelin forms grown from crude egg lecithin but cannot penetrate deeply. It is localized in extracellular materials which have an important mechanical function. Its exclusion by cell membranes permits tracing tortuous cellular invaginations and those exceptions to its exclusion invite a comparison of the localization of the dye with the function of the cell.

The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation.

Abstract: Whole mounted segments of seminiferous tubules from rat testes have been used to investigate the morphology and proliferative activity of the undifferentiated type A spermatogonial population. This has led to the formulation of a new model for spermatogonial stem cell renewal. Three groups of undifferentiated A spermatogonia were classified according to their topographical arrangements as isolated, paired, and aligned spermatogonia. It was proposed that the isolated (as well as a few paired) spermatogonia, which were always present throughout the seminiferous epithelium, are the functional stem cells and should therefore be designated as As. Through sporadic divisions, the As spermatogonia both maintain their own numbers and give rise to pairs of cells which are destined to eventually differentiate. The latter undergo several synchronous divisions in succession, thereby forming increasingly longer chains of aligned spermatogonia. The proliferation of these chains, primarily in stages I–V, leads to a gradual expansion in the size of the undifferentiated type A population. When the population attains its maximal size in stage V, mitotic activity among the aligned cells ceases, and all of these cells morphologically transform without further division into typical A1 spermatogonia. Subsequently, the cohort of A1 cells synchronously divides in stage IX to begin the long process of spermatogonial maturation. The isolated (and a few paired) cells, which do not undergo this transformation and remain quiescent during the stage IX peak of mitosis, form a residual stock of stem cells, that, during the course of another cycle, rebuild the population of aligned A spermatogonia. In this way, a continual supply of type A1 spermatogonia which will cyclically differentiate is insured.

Spermatogonial stem-cell renewal in the mouse†

Abstract: Type A spermatogonia in the mouse can be separated into five successive classes on the basis of nuclear morphology and stage of the cycle in which they occur. Enumeration of all types throughout the cycle of the seminiferous epithelium reveals that the As spermatogonia are the stem cells. They divide throughout the cycle and, especially at stages IX to I, form chains of cells which then give rise to spermatogonia A1 at stages II–VIII. The A1 cells divide in IX to form the A2, which divide in XI to form A3, and the A3 cells divide in I to form the class A4 spermatogonia. Spermatogonia A4 give rise only to the In type; there is no evidence for the formation of either As or A1 from A4 spermatogonia. Repeated injections of 3H-thymidine and tracing the history of labeled cells to 15 days after labeling supported the conclusion obtained from morphological and numerical data that As spermatogonia are the stem cells of the testis.

A phase and electron microscopic study of vasculogenesis and erythropoiesis in the yolk sac of the mouse.

Abstract: By seven days of gestation, the yolk sac of the mouse has a sheet of mesoderm adjacent to the basement membrane separating it from the endodermal epithelium. Localized proliferations of this mesoderm produces thickened cellular regions which transform into the angioblastic cords; all of these developmental cells are attached by tight junctions and desmosomes. By eight and onehalf days, lumina appear within the angioblastic cords; the peripheral cells become attenuated and form endothelial cells which will line the primitive vessels while the more central cells become the primitive erythroblasts of the blood island. The process of vasculogenesis and lumenization occurs between eight and onehalf and nine days of gestation and has been correlated with the reduction of cellular junctions between angioblasts and fixed primitive erythroblasts, a loss of the visceral basement membrane and the formation of wide intercellular channels between endodermal epithelial cells. The primitive erythroblasts comprising the blood islands have abundant polysomes, sparse rough endoplasmic reticulum and possess coated vesicles and ferritin aggregates in their cytoplasm and coated invaginations of their plasma membrane. By nine days of gestation, the primitive erythroblasts lose their attachments and become free in the vitelline vessels. Mitochondria of the primitive and free erythroblasts are slightly enlarged and have lighter matrices than angioblasts and mesodermal cells. By 10 to 11 days of gestation, as differentiation proceeds, coated vesicles and invaginations become more numerous and the developing erythroblasts gradually decrease in both cell and nuclear size. Concomitant with these changes is the decrease in the number and size of the mitochondria, a decrease in polysomal numbers and an increase in hemoglobin and cytoplasmic density.

Extracellular lining of bronchioles after perfusion-fixation of rat lungs for electron microscopy.

Abstract: Rat lungs were fixed by perfusion of fixatives through pulmonary vessels which resulted in good fixation of bronchioles. This technique allows the preservation of a hitherto not described extracellular lining of the bronchiolar surface, which by conventional fixation (immersion or instillation of the fixatives into the airways) is washed out. The cilia appear to be embedded in an extra-cellular, amorphous material. This material is often lined at its surface by a strongly osmiophilic layer. Tubular myelin figures can also be identified. Comparison with recent physiological and biochemical work, which establishes the presence of surface-active material in the airways, strengthens the conclusion that this layer is related to bronchiolar “surfactant” fixed in situ. A mixed origin of this material from the Clara cells and from the alveolar surface lining layer is suggested.

The development of the periodontium. A transplantation and autoradiographic study.

Abstract: First molar tooth germs were dissected from one-day-old mice; placed for one hour in McCoy's medium containing 10 μc tritiated thymidine and transplanted subcutaneously into young adult animals of the same strain. Seven, 14, 21 and 28 days after implantation the host animals were sacrificed and the transplants harvested. The transplants were then serially sectioned and autoradiographs prepared. Control sections were prepared of first molar tooth germs in situ, after dissection from the jaws and after labelling with tritiated thymidine. Forty-nine of the 115 transplanted tooth germs continued development with the formation of enamel, dentine, cement, periodontal ligament and bone. In some instances the transplanted tooth germs “erupted” through the skin with the establishment of an epithelial attachment. Examination of control sections showed that the transplants consisted of dental organ, dental papilla and a layer of ectomesenchymal cells continuous with the dental papilla and investing the dental organ. Examination of autoradiographs of the transplants showed labelling of cementoblasts and periodontal ligament fibroblasts, thereby establishing their origin from the ectomesenchymal cells investing the tooth germ.

Hair loss and cyst formation in hairless and rhino mutant mice

Abstract: Structural abnormalities begin in the integument of hairless (hrhr), hairless-rhino (hrhrrh), and rhino (hrrhhrrh) male and female mutant mice prior to the end of the first hair growth cycle. The pilary canals begin to widen and accumulate keratin and sebaceous material. During the first catagen phase the lower internal root sheath coalesces around the terminal part of the hair shaft and abnormal club hairs form. The lower part of the external root sheath fails to follow the ascending club hair and becomes stranded in the dermis. The abnormal club hairs move above their normal anchoring position and fall out of the follicle at the end of the first growth cycle. The formation of abnormal club hairs and the loss of hair probably are related to the mispositioning of the internal root sheath. Subsequent to the loss of hair there are changes in the sebaceous glands and adipose tissue, disorganization of the peripheral neurovascular system, and the formation of cysts that are associated with either the pilary canals or with epithelial units of disorganized hair follicles stranded in the dermis. Cysts arise from proliferation of epithelial tissue; sebaceous cells are not necessary for cyst formation. Comparisons among similar and dissimilar anomalies indicate that the degree of gene action on the integument is in the following order of increased severity: hrhr, hrhrrh, and hrrhhrrh.

A light and electron microscopic study of the nearly mature enamel of rat incisors.

Abstract: The structure of the organic matrix close to the mature enamel in 100 gm rat incisors was studied by light and electron microscopy using EDTA decalcified teeth. Under the light microscope, in 0.5 μ Epon sections, the enamel layer of cross sectioned upper incisors was about 60 μ thick. The inner enamel was about 40 μ thick and consisted of an initial enamel layer (4 μ) adjacent to the dentin in which no rod profiles were seen, and an inner layer proper which contained six to eight rows of oval-shaped rod profiles set in a homogeneous background. The profiles in any given row were inclined mesially or laterally and alternated in adjacent rows. The outer enamel was about 20 μ thick and consisted of an outer enamel proper and a 2–4 μ thick final enamel layer which smoothed out the enamel surface. The outer enamel proper contained smaller elliptical rod profiles in a more abundant background. These profiles were not arranged in rows and were oriented at right angles to the enamel surface. The final enamel layer contained no rod profiles and was lined on its outer surface by a PA-Schiff positive layer resembling a basement membrane. Under the electron microscope the matrix of rod profiles and interrod material could be distinguished. This consisted of aggregated tubular (and filamentous) subunits, 250 A in diameter, with empty space between them. Within the rod profiles the subunits ran parallel to the rod's long axis, whereas in the interrod material the subunits were again parallel but but running at right angles to the subunits of the rods. In addition to forming the material between the rods, the interrod material also formed the initial and final enamel layers.

Weight standards for organs from early human fetuses.

Abstract: One hundred and thirty-six unfixed fetuses below 500 gm of body weight were examined and the brain, liver, lung, kidney, heart, adrenal, thymus, spleen, and thyroid were weighed. The ratios of organ weight to body weight could be divided into four general patterns: (1) constant; liver, heart, brain, and thyroid (2) increasing; thymus, adrenal, and spleen (3) initially increasing and then reaching a plateau; kidney, and (4) increasing and then decreasing; lung. Graphs of the mean and 95% confidence levels along with the formulas used for derivation are given for each organ on the basis of body weight.

Neuroglial population in the spinal white matter of neonatal and early postnatal rats: an autoradiographic study of numbers of neuroglia and changes in their proliferative activity.

Abstract: The temporal pattern of increase in number of neuroglia in spinal cord white matter and the amount of multiplication in situ of these cells were studied in rats ranging from less than 12 hours following birth to 30 days of age. Autoradiographs of sections from animals killed four hours after injection of tritiated thymidine were studied; counts of the total neuroglial population and of the number of labeled neuroglia were made in the white matter of each half of the cord (hemisection). During the first two weeks following birth, the number of neuroglia per hemisection increased six times; thereafter, the population appeared to be stable. The increases in number of labeled neuroglia appeared to parallel the rise in total population, particularly during the period of greatest population increase, i.e., days 7 through 12. After 12 days, when the total population stabilized, there was a marked and rapid decrease in the number of labeled cells. The walls of the central canal were examined in view of the role this area plays in the initial development of the nervous system. In rats killed during the first 14 days postnatally, the dorsal and ventral walls were incomplete; however, numerous, unidentified cells adjacent to these walls were oriented as if entering the dorsal median septum and the ventral median sulcus. By 22 days the central canal area had more of its adult appearance.

The fine structure of the neck of mammalian spermatozoa.

Abstract: The study of the fine structural organization of the various components of the neck of mature spermatozoa of rabbit, monkey and man has demonstrated that the striated columns of the connecting piece implant on the proximal centriole, that the distal centriole does not disappear but persists albeit in a modified form and that the central tubules of the axoneme of the flagellum terminate at the lower vault of the proximal centriole. Of the two centrioles, the most plausible candidate for the role of basal body of the flagellum and center of the sperm motility appears to be the proximal centriole. This hypothesis is supported directly by the apparent continuity of this centriole with all the contractile elements of the flagellum, and indirectly by the consideration that the distal centriole cannot be a basal body in that its lumen is traversed throughout by a central pair of tubules. The orientation of the proximal centriole at an angle to the flagellum, a unique situation since basal bodies are normally oriented on the same axes of cilia and flagella, has been tentatively accounted for by the particular type of motility of the spermatozoon.

Dendrite bundles in lamina IX of cat spinal cord: a possible source for electrical interaction between motoneurons?

Abstract: Longitudinally-oriented bundles of closely-aligned motoneuron dendrites in Lamina IX of cat spinal cord were examined. These dendrites were interrelated in one of several ways: (1) They were separated by a space of several microns containing small myelinated and unmyelinated fibers, synaptic boutons, and glial processes; (2) Some dendrites were separated by a gap of 0.2–0.5 μ which contained only an attenuated astrocyte process; (3) The plasma membranes of the adjacent dendrites were occasionally found in direct apposition and in most of these instances there was no detectable specialization of the opposing membranes; (4) There were definite modifications along the appositional membranes. These zones were characterized by a gap of ∼ 180 A, and symmetrical, non-polarized aggregations of electron-opaque material were found along the cytoplasmic surfaces of the membranes. It is suggested that the spatial arrangement and appositional relations of these dendrites represent a morphological substrate for the weak electrical facilitation known to occur between motoneurons in the cat spinal cord.

Intercellular bridges between follicle cells and oocyte in the lizard, Anolis carolinensis.

Abstract: Certain cells in the follicular epithelium of lizard (Anolis caro-linensis) ovaries are connected to the oocyte by true intercellular bridges. Unlike other known intercellular bridges, these may form by secondary cell contact and membrane fusion rather than by incomplete cytokinesis. These intercellular bridges are abundant in previtellogenic tertiary follicles, and they contain glyco-gen, ribonucleoprotein particles, and a dense, fibro-granular material which may be exchanged between the two cells. Prior to the onset of yolk accumulation in the oocyte, the intercellular bridges and the pyriform cells from which they originated disappear from the follicular epithelium. The abundance of intercellular bridges in previtellogenic follicles suggests that they may be involved in the maintenance of dormant oocytes during periods of suspended reproductive activity and in the preparation of these oocytes for ensuing periods of intense metabolic activity associated with yolk accumulation.

An electromyographic study of reciprocal activity of muscles.

Abstract: An electromyographic study of elbow flexors and extensors was conducted using 20 adult human subjects to determine if cocontraction occurred during voluntary movement. The interplay of proprioceptive reflex influences arising in the primary and secondary endings of the muscle spindle and in the Golgi tendon organ provide the neurophysiological basis for cocontraction. Whether or not cocontraction occurs during a movement is dependent on the degree to which one proprioceptive influence predominates over the others. This in turn appears to be dependent on factors such as those described below. Incidence and degree of cocontraction was greater during extension than during flexion movements. This may be attributed to the influence of muscle spindle secondary endings. Cocontraction increased with increasing load. Proprioceptive reflexes arising in tendon organs may be involved in this phenomenon. No evidence was found to indicate that the incidence of cocontraction increased with increasing precision of movement. In general, cocontraction was less in skilled and strong subjects than in average subjects during all types of movements. Under the special circumstances of voluntarily attempted cocontraction, evidence of reciprocal inhibition of the antagonist appeared. Proprioceptive reflex activity from tendon organs and from muscle spindle secondary endings are implicated as primarily responsible for this inhibition.

Morphologic studies of human subcutaneous adipose tissue in vitro.

Abstract: A simple method for in vitro studies of human subcutaneous adipose tissue is described. Explants of adipose tissue have been maintained in vitro for 30 weeks in Parker medium 199. The morphology of cultured explants compared well with that of freshly excised specimens. In the absence of serum there was no outgrowth of fibroblast-like cells. When human serum was added to the medium at concentrations exceeding 5% there was a proliferation of fibroblast-like cells. This cell proliferation could still be obtained when serum was added after two weeks of culture in a serum free medium. The adipose cells were isolated with collagenase and the cell size determined. Explants with an initial mean cell size larger than about 95 μ showed a significant decrease in cell size during the incubation. This could not be attributed to a traumatic effect. Metabolic differences between large and small adipose cells was suggested as a possible reason.

The fine structure of the mouse adrenal cortex and the ultrastructural changes in the zona glomerulosa with low and high sodium diets.

Abstract: The ultrastructure of the normal mouse adrenal cortex and the zona glomerulosa as stimulated by sodium restriction and repressed by high salt intake is reported. The mitochondria are zone specific, the endoplasmic reticulum tubular and the endothelium fenestrated. An intimate relationship between elements of reticulum, mitochondria and lipid droplets is seen in all animals. The rough reticulum is poorly developed in all but the glomerulosa of salt-restricted animals. The Golgi apparatus, associated vesicles and surface microvilli are relatively more developed in the inner cortical zones and in the glomerulosa cells of stimulated animals, whereas surface coated pits and vesicles do not vary. Lysosome-like granules are more prominant in the control reticularis and in the glomerulosa of animals on high salt intake. In stimulated glomerulosa cells, there is an early depletion of lipid droplets and a transient increase in rough reticulum followed by a progressive increase in smooth reticulum and Golgi apparatus. At three weeks, the lipid droplets are restored. In repressed glomerulosal cells, there is atrophy of the cytoplasmic organelles while lipid droplets are increased in number and osmiophilia. An accompanying feature is the appearance of cytoplasmic β-glycogen. These observations are discussed as to their relation to adrenocortical steroidogenesis and secretion.

The fine structure of a lateral recess of the subarachnoid space in the rat

Abstract: This study reports an electron microscopic investigation to the spinal meninges of the rat as they exist between the dorsal and ventral nerve roots in upper thoracic and cervical levels of the vertebral column. Between these roots, at a point where they penetrate the spinal dura, the leptomeninges are characterized by a histological pattern comparable to that elsewhere comprising the subarachnoid angle. However, this region is modified and possesses a number of heretofore unknown histological features. The most noteworthy is a lateral recess of the subarachnoid space. The meninges located between the nerve roots do not form a definite lateral boundary for the subarachnoid space. Here the subarachnoid space opens into a lateral recess which extends peripherally between the dorsal and ventral nerve roots. Conspicuous amounts of cellular debris are collected within the lateral recess. Numerous free macrophages congregate here. The lateral recess may be a communicating pathway between the central and peripheral nervous systems by way of the endoneurium of the nerve trunk.

The fine structure of monkey and human spermatozoa

Abstract: The fine structure of spermatozoa of two species of monkey, Macaca mulatta and Macaca nemestrina, and man was studied following fixation of seminal fluids in buffered picric acid-formaldehyde (PAF). The various components of the sperm have been studied mostly in light of the behavior of this cell at the time of conjugation with the ovum. The possible equivalence between acrosome and lysosomes has been discrussed considering the origin, enzymatic activity, and mechanism of action of these two classes of organelles. The fine morphology of the post-acrosomal cap has been found to be comparable to that of a septate desmosome. We have introduced the hypothesis that the cap plays a role in establishing and maintaining adhesion between the spermatozoon and the ovum at the time of gamete conjugation. The formation of the scrolls of the nuclear membrance in the posterior region of the nucleus has been put into relation with the modifications of volume and shape of the spermatid nucleus. The function of the scrolls could be to increase the surface through which nucleocytoplasmic exchanges take place. The possibility that the presence of a voluminous cytoplasmic droplet in human spermatozoa is a sign of cellular immaturity has also been discussed.

A three-dimensional reconstruction of the rods in rat maxillary incisor enamel.

Abstract: A three-dimensio nal reconstruction of the rod profiles seen in inner and outer rat incisor enamel was made from serial 1 µ cross sections of a decalcified upper incisor. The enamel rod was found to be an elongated structure which travelled incisally relative to its origin and ran continuously from the dentino-enamel junction to the enamel surface. The rod was divisible into an inner and outer enamel portion. The inner enamel portion began at the dentino- enamel junction and travelled incisally for about 20 µ with either a mesial or lateral tilt towards the outer enamel. The outer enamel portion of the rod was straight and ran incisally for about 60 µ as it gradually approached the enamel surface. Inner enamel portions of rods were arranged in rows parallel to the cross sec- tional plane of the incisor. All the rods in each row were tilted either mesially or laterally such that individual rods of adjacent rows crossed each other at 90°. Outer enamel portions of rods were not arranged in rows but all passed incisally parallel to one another. In the persistently erupting incisor of the rat two basic enamel patterns have been described, the inner enamel which is laid down first and forms most of the enamel thickness, and the outer enamel which is thinner and laid down last (see review in the preceding article; Warshawsky, '71). Although several papers have presented morphological descriptions of the rat in- cisor enamel with the light and electron microscope (see Warshawsky, '71), the validity of speculations concerning the three-dimensional arrangement of enamel rods is limited in that these conclusions were always based on two-dimension al ob- servations. In this study a precise three-dimensional

Microvascular organization of the adult human testis.

Abstract: The microvascular architecture of the adult human testis was studied from autopsy material using microangiography. The intratesticular arterial vessels show peculiar coiling independent of the age of the subject. These arteries run in either a centripetal or a centrifugal direction and give rise to inter-tubular and peritubular capillary networks which are basically similar to those of the rodent testis. Venous drainage is directed either towards the surface of the testis or towards the rete testis. Some capillaries in the vicinity of the seminiferous tubules penetrate some of the layers of the tunica propria. The rete testis has a completely different and rather sparse microvascular architecture, and both the rete and the tunica albuginea receive blood from extratesticular sources as well as from the testis.

Ultrastructural changes in mouse yolk sac associated with the initiation of vitelline circulation.

Abstract: The endodermal cells of the visceral yolk sac have been studied from the seventh to eleventh day of gestation. The absorptive capacity of the cells is established by the seventh day as indicated by the presence of microvilli, coated invaginations and vacuoles, the apical canalicular system and abundance of absorptive droplets and vacuoles in these cells. Changes in cellular structure during the next three to four days include the development of the cisternal system of the rough endoplasmic reticulum, an increase in mitochondrial number and their localization near absorptive droplets and the formation of multiple Golgi complexes. Intercellular spaces form between the endodermal cells by eight to eight and one-half days and coincide with the disappearance of the basal lamina separating the endoderm and mesoderm (angioblastic cords); these changes correlate with the formation of the vitelline vessels. As the vitelline circulation becomes functional, the visceral basal lamina is re-established, the intercellular clefts decrease in prominence and the absorptive storage droplets and vacuoles decrease in size and number. The apical junctional complex of the endodermal cells forms a continuous barrier for the direct passage of material from the yolk sac cavity to the developing vitelline vessels as established by the use of ruthenium red. The absorption and intracellular storage of macromolecules in the visceral endodermal cells was traced through the intermicrovillous apical coated invaginations, coated vesicles, apical canaliculi and storage vacuoles using ferritin. Immunofluorescent studies indicate the presence of immunoglobulin (anti-mouse gamma globulin) in the cytoplasm of the visceral endodermal cells as early as 9 to 11 days. Several blast-like cells which also were observed in the vitelline vessels at 11 days exhibited positive fluorescence for immunoglobulin.

The relationship between the pubo-urethral ligaments and the urogenital diaphragm in the human female.

Abstract: The connective tissue supports of the human female urethra have been investigated in 14 cadavers and 20 fetuses. In all cases the urethra was found to be suspended from the pubic bone by bilaterally symmetrical anterior, posterior and intermediate pubo-urethral ligaments. The anterior and posterior ligaments were formed by reflections of the inferior and superior fascial layers of the urogenital diaphragm. The intermediate ligament represented a fusion of these fascial layers and no transverse perineal ligament was found. It is suggested that the term pubovesical ligament is a misnomer since this band of connective tissue passes from the pubic bone to the urethra and not to the bladder. It is thus analogous to the puboprostatic ligament of the male. An anatomical defect in the pubo-urethral ligaments might be a contributing factor to urinary stress incontinence in the female.

Ultrastructural aspects of the main excretory duct of rat submandibular gland.

Abstract: Recent data indicate active transport of Na+ and K+ by the main excretory duct (MED) of the rat submandibular gland. In view of these data submandibular MED's of four adult rats were investigated by transmission electron microscopy. Luminal surfaces of two MED's were examined with the scanning electron microscope. The duct is surrounded by connective tissue, blood vessels and neuron perikarya. Blood supply to the ductal epithelium consists of a plexus of sinusoidal capillaries. Three principal cell types compose the epithelium: light cells, dark cells, and basal cells. The basal cells resemble those of striated ducts except for the presence of numerous hemidesmosomes along basal plasma membranes of MED basal cells. Light cells show basal infoldings, and bulbous enlargements may occur at their distal aspects. Dark cells are narrow, electron dense cells with prominent microvilli at the luminal surface. Intercellular clefts penetrate MED epithelium from the lumen to a depth of one-third the epithelial thickness. Surfaces of the clefts are lined by dark and light cells. Intercellular tissue spaces are present at lateral boundaries of all cells and extend from the epithelial base to distal cytoplasmic levels.

Ruthenium red and violet. III. Fine structure of the plasma membrane and extraneous coats in amoebae (A. proteus and chaos chaos)

Abstract: Ruthenium violet, closely related to ruthenium red, supplements the ultrastructural knowledge of the plasma membrane complex. Amoebae throughout were handled individually with braking pipettes and were exposed to ruthenium violet alive, during fixation with acrolein and OSO4, or during dehydration. Ruthenium violet was less toxic than ruthenium red but still killed the amoebae. Conventional methods reveal a filamentous layer 2000 A thick, an amorphous layer 150 A thick, and a typical trilaminar plasma membrane (48 A center-to-center). Ruthenium violet binds to the plasma membrane, and to the extraneous coats revealing globules in the filamentous layer. The diameter of the globules decreased according to the stage of processing at which the amoebae first encountered ruthenium violet; they were 1200 A in diameter when amoebae were alive, 600 A in acrolein and 300 A in dehydration. The appearance of the filamentous layer varied when ruthenium violet was replaced by very pure ruthenium red or red containing ruthenium brown (typical of commercial ruthenium red). The globules could be demonstrated without using ruthenium dyes when amoebae were treated after fixation with uranyl acetate or phosphotungstic acid. The relationship of extraneous coats of amoebae is compared with the coats and laminae of animal tissue cells.

The uptake of tritiated thymidine by the dorsal epidermis of the fetal and newborn rat

Abstract: The dorsal epidermis of fetal and newborn rats was examined to determine the difference in ability of the basal cells to bind tritiated thymidine during different stages of epidermal morphogenesis. Five rats were examined for each time period from the eleventh day of gestation to the fifth day after birth. The number of labeled cells in 5000 basal cells was counted and expressed as a percentage. The labeling index is ∼ 10% from the eleventh to the fifteenth fetal day. It increases to ∼ 30% by the eighteenth day, decreases to ∼ 10% from the twenty-first day until the first postpartum day and drops to 5% or less from the second to fifth day. These changes in labeling index are accompanied by and apparently correlated with the normal differentiation of rat epidermis. The growth of the epidermis is continuous during the course of the study. Keratohyalin granules begin to form on the eighteenth day and by the twentieth day the first cornified cells appear. The s. corneum becomes progressively thicker each day thereafter. The s. Malpighii, on the other hand, decreases somewhat in thickness after birth. The labeling index curve represents a relationship between basal cell activity and control or influencing mechanisms inherent in the maturational system of skin. The increase and decrease are not related to growth alone, but appear to be related to differentiation.

Popliteus muscle in man.

Abstract: This paper describes the morphology of the popliteus muscle based on the dissection of 15 human cadaver's knees. The muscle is found to have three origins: the strongest from the lateral femoral condyle is already wellknown, but there is also an important band from the fibula and a firm attachment to the posterior horn of the lateral meniscus. The femoral and fibular origins form the arms of an oblique Y-shaped ligament, the base of which is formed by the capsule and the meniscal origins. This previously was described as a separate entity, the “arcuate ligament,” attached to the belly of the muscle, but it is not a separate ligament. Rather it is a condensation of the fibers of origin of the popliteus including those from the fibula.

Arterial supply of the cervical spinal cord (with special reference to the radicular arteries)

Abstract: Arterial supply of the cervical spinal cord has been discussed with special reference to the radicular arteries. Thirty-one human spinal cords have been studied with postmortem positive pressure injection techniques using coloured, and radio-opaque media. Observations concluded that radicular arteries were main sources of supply to spinal cord except at the highest segments (C1C2C3), where intracranial vertebral branches contribute. Average number of significant radicular arteries is two or three, in two-thirds of the specimens only one was present. These feeding radicular arteries usually enter into the spinal canal through the intervertebral foramina accompanying C4C5C6 nerve roots to join the anterior and posterior spinal arteries. Anterior, and posterior spinal arteries are probably of segmental origin, and there is only a sparse anastomosis between them. The common radicular artery divides into an anterior, and a posterior branch of which one predominates in size. Cervical radicular arteries may originate from subclavian branches other than vertebral, of these ascending cervical branch of thyrocervical trunk is most important. A terminal zone probably exists at highest thoracic segments where craniocervical, and thoracic radicular flows meet. The filling of the anterior spinal trunk in the cervical region depends on the availability of at least one major anterior radicular artery. Interruption of radicular supply may be precipitated by trauma, spondylosis and other lesions resulting into ischaemia, and myelopathy; the risk is greater if there is only one radicular artery which is involved.

The ultrastructure of the human adrenal medulla: with comparative studies of white rat.

Abstract: There is wide support for the concept that there are two types of secretory cells in adrenal medulla of various species. One cell is filled with electron dense bodies believed to contain norepinephrine; the other cell's lesser stained bodies contain epinephrine. This differential density may be seen by fixation in osmium tetroxide after glutaraldehyde. The two cell types have not been clearly shown in human tissues. Such a failure may be related to fixation. In consideration of this, fresh human adrenal was fixed in cold 1% glutaraldehyde, then 1% osmium tetroxide — a procedure known to give satisfactory differentiation in other species. A parallel study with rat tissue was then done. Electron microscopy revealed two types of cells in rats, while in man an obvious cell difference was not found. There is, however, a clear difference in human chromaffin granule density, which implies that the human cells have random numbers of both light and dark granules. The morphologic difference of the cell types in man may be less distinct than in the rat. Another possibility is that in cells with predominantly dark granules, there are co-mingled lighter granules with the coexistence of cells of opposite dominance. Both hormones then would occur in the same cell in varying proportions according to functional demand. It seems highly unlikely that two distinct cell types would have been overlooked with wide sampling, however, in a study of three adrenal glands, one cannot rule out their existence.