An unusual cylindrical body in interstitial cells of rat testis.

Testes of 6 rats were processed routinely for electron microscopy and, as an incidental finding, interstitial (Leydig) cells were found to contain bundles of unusual cylindrical bodies or macrotubules. These cytoplasmic structures were found in testes of only 2 of the 6 rats and varied in number from 2 to 346 per cell profile, usually in parallel array but with an irregular orientation in approximately 5% of the cells. The macrotubules were up to 11 microns long, showed a diameter of 130 nm with a wall of 18-20 nm and an inside diameter of about 94 nm. The wall appeared to be formed by two membranes 6-7 nm thick showing a unit membrane structure with a central electron-lucent space of 6 nm. In several instances, the wall membranes appeared corrugated, which may account for the apparent formation of the wall by a spiralling small tubule or tubules of 18 nm diameter. Several macrotubules showed continuity between their wall membranes and elements of endoplasmic reticulum and occasionally two adjacent macrotubules showed continuity at their ends in a U-form. Similar if not identical structures have been described previously in four studies of interstitial cells of the rat renal medulla, in one instance correlated with water-deprivation. They have been considered an alteration in the endoplasmic reticulum. Also, they have been reported in pig uterine glands in pregnancy. They may represent a cellular response to an unrecognized physiopathological state.

The use of lanthanum chloride as a marker for intercellular junctions in rat exocrine pancreas.

A simple technique of perfusion and immersion of tissue in fixative containing lanthanum chloride as an extracellular tracer is described. In addition to functioning as a tracer, the lanthanum chloride appears to enhance electron staining. In rat exocrine pancreas, intercellular spaces between exocrine and centroductular cells were outlined clearly be electron dense material and, at cellular interfaces, spot desmosomes, gap junctions, and tight junctions were demonstrated. The technique proved simple and effective and should prove useful in studies of epithelial and other tissues.

Postnatal development and differentiation of contractile cells within the rabbit testis.

This study has been determined the postnatal development and differentiation of smooth muscle cells within the rabbit testicular capsule and within the peritubular tissue surrounding seminiferous tubules. Smooth muscle cells within the tunica albuginea are not identifiable at birth by light microscopy but by electron microscopy myocytes in early stages of development may be shown to be present. It is not until 42 to 49 days postnatum that smooth muscle cells can be identified by light microscopy. Differentiation of smooth muscle cells within the capsule is completed by 128 days postnatum. At this time, the muscle is arranged in two organized layers, a superficial layer of longitudinally oriented cells and a deeper layer of circularly arranged cells. At birth, the peritubular tissue consists of two to four layers of undifferentiated cells and, during the first postnatal week, the tissue becomes more condensed and generally is arranged in two cellular layers. Cells of the inner layer contain small bundles of microfibrils whereas cells of the outer layer are fibroblast in nature. Differentiation of the peritubular tissue is completed by 112 days postnatum. At this stage, it consists of four layers, two acellular and two cellular. The inner cellular layer, composed of attentuated myoid cells, possesses a basal lamina on both surfaces and is surrounded by two delicate connective tissue lamellae. The myoid cells of the peritubular tissue thus achieve structural maturity at approximately the same time postnatally as do those within the testicular capsule, which corresponds to the time when spermatogenesis becomes established. The relative contributions of the myoid cells in the peritubular tissue and within the testicular capsule to the movement of non-motile spermatozoa out of the testis and the possible significance of the peritubular tissue as a component of the permeability barrier are discussed in relation to the present findings.

Postnatal development and differentiation of the secretory elements of the rabbit parotid and submandibular glands.

The development of the secretory elements of the parotid and submandibular glands has been examined in postnatal rabbits. In the submandibular gland, there are 2 principal types of secretory cells, serous and mucous. At birth, these occur in approximately equal proportions, and, by 28 days postnatal, the mucous elements greatly outnumber the serous ones. Initially, development of the parotid gland lags significantly behind that of the submandibular gland. Lobules, which are widely separated at birth, appear compact by 28 days postnatal, at which time also serous acinar cells contain an adult content of secretory granules. In both glands, secretory granules develop within component cells of intercalated ducts by 14 days postnatal. Although the glands appear virtually mature by 28 days postnatal, which corresponds approximately to the onset of weaning, minor structural changes occur over the following 3 postnatal weeks.

Changes in the erythrocytes of the developing opossum, Didelphia virginiana.

Changes in the erythrocyte population during postnatal development of the opossum have been studied. At birth, the red cells of the peripheral blood number less than 600,000/cmm and the characteristic erythrocyte is a large nucleated cell ('megaloblast'). These are rapidly replaced by smaller, nonnucleated cells. The results indicate that the yolk-sac period of blood formation is short and that the hepatic phase of hemopoiesis begins prior to birth. The data also suggest that megaloblasts in the opossum may be able to transform into megalocytes.

Morphological observations on the metanephros in the postnatal opossum, Didelphis virginiana.

The metanephros of the newborn opossum is very immature, consisting only of collecting tubules and a few immature nephrons. Development during the postnatal period can be divided into two distinct phases. The initial phase occurs during the first 60 days of postnatal life and is concerned with nephronogenesis and the differentiation of nephrons that have formed during this period. The second phase lasts through the remainder of the postnatal period and is concerned with further differentiation and growth of established nephrons. During this latter period the tubular portion of the nephron increases in length and the renal corpuscle increases in diameter. Ultrastructural observations suggest that metanephric nephrons are not functional during the first 4 days of postnatal life, while the mesonephros reaches the height of its development during this period: there may be some functional overlap between the mesonephros and metanephros during the latter part of the first week of postnatal life. The pattern of nephron induction and differentiation in the opossum is discussed.

Morphological observations on the mesonephros in the postnatal opossum, Didelphis virginiana.

The mesonephros of the opossum persists for 3-4 weeks into the postnatal period. Based on our observations of its structure, and the vital dye experiments of others, it appears that the opossum mesonephros is functional during the first 10 days of the postnatal period. The mesonephros of the newborn opossum consists of 35-45 nephron units which are structurally very similar to metanephric nephrons except that they lack a loop of Henle. By the end of the first postnatal week, regression which proceeds in a craniocaudal direction, is observed. By the end of the second week most nephrons show some signs of regression. The regressing mesonephric nephrons are replaced by connective tissue.

Postnatal development and differentiation of the opossum submandibular gland.

The postnatal development and differentiation of the submandibular salivary gland has been examined in sixteen groups of young opossums. At birth the glandular elements, dispersed in loose connective tissue, consist only of ducts that are immature in appearance and of irregular secretory end-pieces. Development occurs in two phases, the first from birth to approximately 31 days postnatum, and the second thereafter. During the first phase the ductular elements show separation into intercalated and intralobular ducts, and attain structural maturity. The larger ducts are concentrated centrally within each lobule and lie in a markedly vascular connective tissue. The secretory end-pieces, initially acinar in form, are lined by proacinar cells which exhibit intercellular canaliculi at the lateral cell membranes and a few dense granules in the apical cytoplasm. During the second phase of development extensive changes occur within the secretory end-pieces, which elongate to form a system of branching tubules. Component cells show an increased granular content, and those in the main body of the tubules differentiate into mucous cells. By 34 cm postnatum the proacinar cells in the bulbous endings of the tubules are replaced by special serous cells possessing intercellular canaliculi and secretory granules which are either electron-lucent or electron-dense. The sequence of changes that occur during postnatal development is discussed and related to possible functional activities. The early development of the ducts may be correlated with their role in homeostasis, while the later development of secretory tubules and the differentiation of secretory cell types may be related to the onset of weaning, and may possibly be induced by this major change in dietary habit.

Virus-like particles in the opossum submandibular gland.

Numerous inclusion bodies (virus-like particles) were observed in the lumina of the intercellular canaliculi, mucous tubules and intralobular ducts of the opossum submandibular gland. The particles are spherical in outline, show an electron dense core, and are surrounded by a peripheral membrane.

Development of the external muscle coats in the digestive tract of the opossum, Didelphis virginiana.

Development of the external musculature of the gastrointestinal tract has been studied in the stomach, small intestine and colon of the postnatal opossum. The muscle support is thin and poorly developed at birth, especially in the stomach and small intestine where only the inner layer is completely formed. The outer layer is discontinuous and formed by scattered myoblasts. The muscularis externa of the colon at birth is considerably thicker and both layers are present. Subsequent development of the muscularis externa consists of an early period of proliferative activity followed by hypertrophy. A low rate of mitotic activity continues throughout development and into the adult. Elements of the myenteric plexus are present at birth.

Postnatal development of the epidermis in a marsupial, Didelphis virginiana.

At birth the epidermis of the opossum is 43 micron thick and consists of a basal layer of columnar cells, an intermediate layer of fusiform cells, a layer of incompletely cornified cells and a single surface layer of cells (the periderm). The latter shows central nuclei and distinct cell boundaries. Adjacent surface cells. are contiguous and show extensive interdigitations of the lateral cell membranes. The periderm is lost during the first week of postnatal development. The epidermis attains its greatest thickness (58 micron) at the 4.0 cm stage (18 days postnatum), and this is due primarily to an increase in thickness of the spinous layer. After this the epidermis thins to 14 micron in the adult. The epidermis of the adult consists of a thin Malphighian layer and a desquamating cornified layer. Hair follicles begin to differentiate at the 2.5 cm stage (7 days postnatum). They continue to differentiate and develop while the epidermis is increasing, and then decreasing, in thickness. The young are fully furred prior to the time they first venture from the protection of the pouch.

General observations on the growth and development of the young pouch opossum, Didelphis virginiana.

A 3-year study of general growth and development revealed a uniform increase in body length of young pouch opossums during the first 10 weeks of life. Throughout this period, growth was linear and constant for all animals regardless of sex, litter size, or whether animals were obtained from first, second or third litters. Body weights were somewhat more variable, but there were no significant sex differences. Various aspects of external gross morphology are presented and discussed as they relate to growth of the young opossum.

The postnatal development of the alimentary canal in the opossum. III. Small intestine and colon.

The duodenum of the newborn opossum exhibits a patent lumen containing scattered elongate villi, whereas the distal segments of the small intestine are smaller in diameter and are filled with short immature villi. The muscularis externa through the small intestine consists of a single layer of myoblasts. Interposed between the intestinal lining epithelium and the muscularis externa is an extensive capillary bed that occupies a considerable proportion of the intestinal wall. Additional villi appear to form during the postnatal period as a result of evaginations of the epithelium, together with underlying connective tissue and vasculature, into the intestinal lumen. Intestinal glands are not observed until 8.5cm, and are shallow in depth even in the adult. The epithelium of the entire small intestine is modified for absorption until just prior to weaning. The principal intestinal lining cells show an extensive apical endocytic complex, large supranuclear vacuoles and numerous cytoplasmic inclusions. Intestinal epithelial cells of the colon also appear to be modified for absorption during the first two weeks after birth. Although goblet cells and Paneth cells are present during the suckling period, they do not comprise a significant population in the intestinal epithelium until after weaning. In contrast to the small intestine, goblet cells are numerous in the colon by the ninth postnatal day. The significance of macromolecular absorption and the possibility of passive immunity being transmitted in the opossum during suckling are discussed and related to similar events that occur in the slckling young of several eutherian species. The possible functional significance of two large membranes that develop in the lamina propria of the intestines after weaning also is discussed.

The postnatal development of the alimentary canal in the opossum. II. Stomach.

The postnatal development of the gastric mucosa in the opossum has been traced with the light, transmission and scanning electron microscopes. The formation of fovea and gastric glands occurs simultaneously during the postnatal period. During the first 60 postnatal days the developing gastric glands are composed of undifferentiated cells, parietal cells and scattered endocrine cells. Chief cells are not present until just before weaning (13 cm, i.e. ca. 75 days). Juvenile and adult animals show only a small population of chief cells, and these are confined to the bases of the gastric glands. The pH of stomach contents ranges from 6-0 to 6-5 until the time of appearance of solid food within the stomach, when it drops to 2-0-2-5. The surface cells lining the gastric lumen contain a considerable amount of what appears to be lipid during the first 3 weeks after birth, and this may indicate that the gastric mucosa is involved in the absorption of lipid during this period. The mode of lipid absorption appears to be different from that described for the intestinal tract of several other species.

The postnatal development of the alimentary canal in the opossum. I. Oesophagus.

The oesophageal epithelium of the newborn opossum generally is two to three cells in depth and in some regions appears pseudostratified. By the 9th postnatal day the epithelium shows two distinct strata. Ciliated cells and occasional goblet cells also are observed within the epithelium during this stage and in subsequent stages. Cilia persist in the oesophagus of the adult opossum, but are restricted to the depths of the transverse folds found in the distal part of the organ. The epithelium covering the transverse folds of the adult likewise has an immature appearance. By 4-5 cm (ca. 20 days), the epithelium has assumed a more mature appearance and is of greater depth. This and later stages show three basic strata: a germinal layer, a spinous layer and, adjacent to the lumen, a flattened layer of cells that retain their nuclei. The epithelium throughout the postnatal period and in the adult does not undergo complete keratinization. The oesophageal glands begin as outgrowths from the epithelium just prior to 4-5 cm (ca. 20 days). The glands continue their development throughout the remainder of the postnatal period. The secretory units of the oesophageal glands of the the major portion of the secretory elements, and a light, rounded cell type which is less numerous and which occupies the terminal portions of the secretory units. Secretory material of the former appears complex, consisting of both neutral and acid glycoproteins. The secretory product of the light cell type is unknown at present. Both cell types are encompassed by myoepithelial cells. The relationship of the mitotic sequences to the observations made by microscopic examination of the developing oesophagus is discussed.

Type II pulmonary epithelial cells of the newborn opossum lung.

The air chambers of the newborn opossum are lined by a respiratory epithelium which appears similar to that lining alveoli of other species. The type II pulmonary epithelial cell is cuboidal in shape, shows apical microvilli, several Golgi complexes, lipid droplets, and numerous cytosomes. The cytosomes contain osmiophilic lamellae and are similar in appearance to whorls of osmiophilic material lying free within the alveolar lumina and on the surface of the respiratory lining epithelium. In the newborn opossum lung, examination of the air chambers with the scanning electron microscope permits the observation of several type II pulmonary epithelial cells in a single field. The potential use of the air chamber of the newborn opossum as an experimental model is suggested.

The postnatal development of the liver in a marsupial, Didelphis virginiana. 2. Electron microscopy.

The postnatal development of the liver has been examined with the electron microscope in 15 groups of young opossums. At birth, hepatic cells show little organization and large islands of haemopoietic elements occur between clusters of hepatic cells. Hepatocytes appear small and contain an abundance of granular endoplasmic reticulum. The cytoplasm is also characterized by scattered lipid droplets of varying sizes. Canaliculi occasionally are found between hepatocytes. Only a few scattered lining cells are found in relation to the sinusoids and extensive areas appear to lack a lining endothelium. By the 9th postnatal day large accumulations of glycogen are present in the majority of hepatic cells and large areas of the sinusoids remain devoid of a lining endothelium. Glycogen and lipid droplets remain abundant in hepatocytes of the 15 day old opossum and later stages. By the 69th postnatal day, hepatocytes are relatively mature in appearance and the principal cytological features appear similar in this and subsequent stages. The cytoplasm is characterized by the presence of glycogen, granular endoplasmic reticulum and numerous small, spherical mitochondria. A heterogeneous population of hepatic cells was noted in the majority of stages examined. This ultrastructural study confirmed the tendency for haemopoietic cells to remain in discrete foci of like kind, both with regard to cell lineage and stage of maturation.

Postnatal development of the respiratory system of the opossum. II. Electron microscopy of the epithelium and pleura.

At birth, the opossum lung is remarkably primitive and consists of a system of branching airways that end in a number of terminal air chambers. From the newborn through the 10 cm stage of development the conducting portion of the lung predominates. The air chambers, which represent portions of the conducting system modified for respiration, are in a constant state of evolution since they are destined to become part of the expanding bronchial system. The airways are devoid of cilia and goblet cells at birth, and are lined by columnar epithelial cells which contain two types of cytoplasmic granules: an electron-dense form and a heterogeneous form. The latter exhibits an electron-dense core surrounded initially by a large halo of flocculent material. This type of granule is not seen beyond the 8 cm stage. The terminal air chambers of the newborn and later stages are lined type I and type II alveolocytes that appear identical to the alveolocytes lining alveoli in the adult. By the 2.5 cm stage, scattered cilia are present in the trachea and bronchi and bands of smooth muscle have differentiated in relation to bronchial epithelium and to proximal areas of the terminal chambers. Citiated cells are separated by ridges composed of light and dark cells which are without cilia and which contain scattered electron-dence granules. Throughout the postnatal period numerous alveolar macrophages and mast cells are noted in relation to the conducting system and pleura. Differentiation of the pleura also occurs during the postnatal period. In the newborn the pleura is simple squamous mesothelium. Later stages develop a thick connective tissue lamina between the pleural mesothelium and lung parenchyma. A large band of elastin is interposed between the mesothelium and underlying bundles of collagen.