Steroidogenic characteristics of the adrenal cortex of the mare studied by electron microscopy

The three concentric zones of the horse adrenal cortex (zonae glomerulosa, fasciculata and reticularis) showed marked interpenetration and exhibited a different relative development according to their position in the gland. Whereas the three cortical zones each had a specific histological structure, the ultrastructure of their cells showed a certain qualitative homogeneity. The differences corresponded essentially to the relative abundance of the constituents which are generally considered typical of steroidogeneous cells: mitochondria with vesicular cristae, smooth endoplasmic reticulum and lipid droplets. Their importance increased progressively from the zona glomerulosa to the zona reticularis. In this zone, the presence of gap and septate-like cell junctions, and mitochondria with vesicular cristae in close proximity to a smooth endoplasmic reticulum with numerous dilated tubules, suggested that steroidogenesis may be the most active. Ultrastructural findings were indicative of only quantitative differences between the steroidogenic capacities of the three zones of the mare adrenal cortex.     

Nuclear roles in the postconjugant development of the ciliated protozoan Euplotes aediculatus. 1. Evidence for sequential roles of the differentiating macronucleus in exconjugant development

Thin-section and freeze-fracture data retrieved from developing fetal adrenals of mouse, rabbit, and rat reveal that gap junctions which appear as cortical and medullary cells morphogenetically assort themselves. In the presumptive zona fasciculata and reticularis communicating junctions develop between neighboring cells just prior to the onset of steroidogenesis. In the rodents, gap junctions assemble in large, particle-free formation plaques 1 day before the proliferation of smooth endoplasmic reticulum and the vesiculation of mitochondria (morphological features which are indicative of steroidogenesis). Similar observations have been obtained in fetal rabbits, but here gap junctions apparently develop in the absence of formation plaques. In each species, gap junctions apparently develop 1 or 2 days prior to a surge in corticosteroid production, suggesting that the formation of communicating junctions during the early phases of adrenal cortical differentiation may provide an avenue to coordinate steroidogenesis in the developing fetal cortex.     

Fine structure of the corpus Luteum of the raccoon during pregnancy

Summary Ultrastructure of the granulosa lutein cells of the raccoon from throughout pregnancy has been described. The lutein cells often from epithelial cords which are separated by the connective tissues, capillaries and lymphatics. Based on the arrangements and modifications of the cytoplasmic organelles and inclusions, three types of lutein cells have been recognized. The type I lutein cells predominantly contain tubular, agranular endoplasmic reticulum, juxtanuclear Golgi complexes, a few round to rod-shaped mitochondria, some free ribosomes, and occasional lipid droplets. Occasionally the tubular cristae of mitochondria and tubular smooth endoplasmic reticulum appear contiguous. The type II cells contain abundant lace-like and/or stacked fenestrated endoplasmic reticulum cisternae that frequently form membranous whorls, some tubular, agranular endoplasmic reticulum, mitochondria, and lipid droplets. Mitochondria are usually small, but unusual large ones also occur. The small, rod-to round-shaped mitochondria usually have tubular cristae; but the large, oval, elongate, and cup shaped mitochondria possess tubular, lamellar, plate like, and whorl-like cristae. The plasma membranes of the cells are complexly elaborated and folded, especially when apposing each other. In favorable sections, strands of fenestrated cisternae appose the folds of the plasma membranes. In general, the amount of cytoplasmic organelles and inclusions vary greatly in the cells. The type III cells predominantly contain lipid droplets and sparse cytoplasmic organelles. The type I and II cells are found throughout pregnancy, but the type III cells are observed from mid gestation to term. The cytological features of type I and II cells suggest that they probably secrete most of the steroids, whereas the type III cells primarily store lipids.This research was supported by UPSHS grant AM-11376 and NIH contract 69-2136.     

Fine structure of the sensory epithelium of guinea-pig organ of Corti: Subsurface cisternae and lamellar bodies in the outer hair cells

The fine structure of subsurface cisternae and lamellar bodies in the outer hair cells of the guinea-pig organ of Corti was studied with thin sections and freeze-fracture replicas. Subsurface cisternae in the outer hair cells consist of multilayers along the lateral plasma membrane of the cell. The outermost layer is a flattened cistern in the upper part of the supranuclear region, but comprises a series of tubules in the lower part. Deeper layers are fenestrated cisternae in which disc-like areas are found in the upper part of the supranuclear region. Lamellar bodies consist of concentric layers of fenestrated cisternae and are located in the apical cytoplasm beneath the cuticular plate. They are continuous with the subsurface cisternae. In the supranuclear cytoplasm, bulges of the subsurface cisternae and the lamellar bodies are found. Dilated cisternae are also present. Some dilated cisternae contain many small vesicles, which display acid phosphatase activity. The dilated cisternae are considered as forms of the bulges undergoing transformation into multivesicular bodies. The possible role of the lamellar bodies, and the origin and fate of the subsurface cisternae are discussed.     

Gap junctions in the cardiac muscle cells of the lamprey

Summary Electron microscopy of both thin sections and freeze-fracture replicas has demonstrated the occurrence of gap junctions (nexuses) in the cardiac muscle cells of the lamprey. These gap junctions are identical in basic structure with those found in the mammalian heart. However, they are much smaller (less than 0.5 in diameter), and more irregularly distributed than the typical gap junction in the mammalian heart. These small gap junctions seem to provide a structural basis for the electrical coupling between cardiac muscle cells in the lower vertebrates.In addition, the well developed sarcoplasmic reticulum and subsurface cisternae, which contain an electron dense spheroidal cast, are frequently observed in the cardiac muscles of the lamprey.This work is supported by a research grant from the Ministry of Education, Japan     

PEROXISOMES IN INNER ADRENOCORTICAL CELLS OF FETAL AND ADULT GUINEA PIGS

Abundant membrane-bounded granules, 0.1–0.45 µm in diameter, occur among the elements of the smooth-surfaced endoplasmic reticulum in zona fasciculata and zona reticularis adrenocortical cells of guinea pigs. Acid phosphatase cannot be cytochemically demonstrated in them, and they are therefore distinct from lysosomes. Incubation in medium containing 3,3'-diaminobenzidine results in dense staining of the granules, identifying them as peroxisomes. These small peroxisomes increase in number as fetal adrenocortical cells differentiate, and they appear to arise from dilated regions of endoplasmic reticulum. They maintain interconnections with the smooth endoplasmic reticulum and with one another.     

Freeze-fracture analysis of phloem structure in plant tissue cultures. I. The sieve element reticulum

During the differentiation of phloem sieve elements, the endoplasmic reticulum undergoes unique modifications to form the sieve element reticulum (SER) which persists in mature, functioning sieve tubes. Cisternae of the SER lack ribosomes and are restricted to the periphery of the sieve element at late stages of development. Some of the SER is seen as single cisternae that are in close contact with the sieve element plasma membrane. Thin sections and freeze-fracture images of sieve elements formed in tissue cultures demonstrate that the SER consists of both single cisternae and regions of stacked cisternae at some stages of maturity. The unstacked regions of the SER are continuous with the cisternae of the stacked regions. In freeze-fracture images the single cisternae adjacent to the plasma membrane are seen to be fenestrated and the openings allow continuity between the plasma membrane and the cell lumen. It is concluded that the interface between the SER and the plasma membrane of the sieve element serves to allow membrane functions such as proton efflux, proton-sucrose cotransport and compensating movements of ions to occur in a microenvironment that is separated from the moving translocation stream in the sieve element lumen. Passage of water and translocated solutes from the plasma membrane or the SER/PM interface to the interior of the cell is enhanced by the openings in the fenestrated regions of the SER. It is suggested tha the SER may also play a role in channeling ATP from mitochondria associated with the SER to the proton-pumping ATPase in the plasma membrane and that the SER may function in the uptake and release of potassium ions in the sieve element.     

Ultrastructure of cortisol-secreting adrenal adenomata

Two adrenocortical adenomata causing Cushing's syndrome were examined by electron microscopy. Adenomatous cells were arranged in islets and contained ovoid nuclei with a prominent nucleoli. Mitochondria were polymorphic and displayed tubular and lamelliform cristae. Some cells were apparently devoid of lipid droplets and possessed an exceedingly well developed SER, the tubules of which were intermingled with small stacks of ribosome-studded cisternae, while other parenchymal cells contained large clumps of lipid droplets and scanty SER tubules. A conspicuous Golgi apparatus and many dense bodies of probable lysosomal nature were also observed. These findings suggest that the adenomata are derived from the zona fasciculata and that lipid-laden and lipid-free cells are, respectively, resting and actively secreting elements.     

Acid phosphatase activity in Golgi apparatus and related structures in pars recta of rat kidney studied by thin and semi-thin section cytochemistry

Summary Using 0.5 thick (i.e., semi-thin) and conventional thin sections, observations have been made on the localization of acid phosphatase in the Golgi apparatus and related structures in the pars recta of rat kidney. In thin sections one or two Golgi cisternae located at the concave (basilar) aspect of the stack had enzymic activity. The periphery of these cisternae may be fenestrated. Coated vesicles were seen apparently free in the cytoplasm and in continuity with both reactive Golgi cisternae and smooth tubular elements. Smooth vesicular profiles with electron-lucent matrices and low enzymic activity were seen, apparently free, in the central Golgi zone. Semi-thin sections demonstrated more fully the extent of the reactive Golgi elements, their architecture and their relationships with other organelles. Within a stack the reactive Golgi cisternae were continuous with one another. A network of anastomosing tubular elements formed the periphery of the cisternae and linked some adjacent cisternae. Tubules extended considerable distances from this network in apical and lateral directions. Vesicular protuberances formed ends to the tubular extensions but free vesicles were not obvious. Apparent continuity was seen between reactive tubules and dense bodies (secondary lysosomes). Vesicular profiles with electron-lucent matrices andlow enzymic activity appeared to be continuous with the periphery of reactive Golgi cisternae and may represent the formation of primary lysosomes. This study demonstrates that semi-thin sections could be used to great advantage in the study of organelle interactions in both normal and pathological states.     

Gap junctions in the heart of the adult Protopterus aethiopicus

In thin sections and in freeze-fracture replicas small and sparse gap junctions appear to be developed on the longitudinal plasma membrane of Protopterus cardiac cells near a macula or fascia adhaerens. By thin-section electron microscopy, they had septalaminar profiles with a length between 0.042 and 0.260 micron. In freeze-fracture images they appear on the P-fracture face as maculate particle aggregations with complementary pits on the E-fracture face. Particles with a central intercellular channel could be observed. The average center-to-center distance between neighbouring particles or pits is 10.05 +/- 1.87 nm (N = 2429). The diameter of the junctional maculae in replicas lies between 0.037 and 0.229 nm. The particle packing density increases in larger maculate aggregations, while particle-free areas emerge which could be related to the degradation or reformation of gap junctions Atypical configurations of gap junctions observed in the myocardium of lower vertebrates are rarely encountered in this primitive vertebrate.     

Observations on peroxisomes in interrenal (Adrenocortical) cells of Trituras cristatus and Salamandra salamandra (Urodele Amphibians)

Intracellular organelles (average diameter: 0.3 mu) similar to peroxisomes were observed in the interrenal cells of Triturus cristatus and Salamandra salamandra. Their peroxisomal nature was demonstrated by incubating tissue sections in alkaline 3,3-diaminobenzidine. Oxidation of diaminobenzidine, a characteristic feature of peroxisomes, was observed. These organelles are devoid of a central nucleoid and are surrounded to various degrees by profiles of the smooth endoplasmic reticulum. The matrix of these organelles is sometimes in direct communication with the inside of the smooth tubules. These relationships suggest a possible origin of the peroxisomes from the smooth endoplasmic reticulum.     

Ultrastructure, histological distribution, and freeze-fracture immunocytochemistry of gap junctions in rat brain and spinal cord

The historical development of concepts of gap junctions as sites for electrical, ionic, and metabolic coupling is reviewed, from the initial discovery of gap junctions linking heart cells, to the current concepts that gap junctions represent 'electrotonic synapses' between neurons. The ultrastructure and immunocytochemistry of gap junctions in heart, brain, and spinal cord of adult rats is examined using conventional thin sections, negative staining, grid-mapped freeze-fracture replicas, and immunogold-labeled freeze-fracture replicas. We review evidence for neuronal gap junctions at 'mixed' (combined electrical and chemical) synapses throughout adult rat spinal cord. We also show immunogold labeling of connexin43 in astrocyte and ependymocyte gap junctions and of connexin32 in oligodendrocyte gap junctions. Ultrastructural and freeze-fracture immunocytochemical methods have provided for definitive determination of the number, size, histological distribution, and connexin composition of gap junctions between neurons in all regions of the central nervous systems of vertebrate species.     

Freeze-fracture of the normal and pathological megakaryocyte lineage in chronic megakaryocytic-granulocytic myelosis

Freeze-fracture and thin sections were performed on human bone marrow of chronic megakaryocytic-granulocytic myelosis (CMGM) to study the three-dimensional fine structure and maturation of normal and atypical megakaryocytes and thrombocytes. In the many normally maturing megakaryocytes the development of the demarcation membrane system (DMS) was best investigated by comparison of thin sections with freeze-fracture replicas. The DMS shows no connections with the Golgi apparatus or rough-surfaced endoplasmic reticulum, but originates from tubular infoldings of the plasma membrane. These infoldings are always in continuity with the extracellular space and form an intracellular membranous pool by branching and coalescing of flattened tubules from which finally the perforated cisternae of the DMS arise. Freeze-fracture of the normal thrombocytes confirms earlier findings. The abnormal giant platelets seen in CMGM display extensive areas of smooth membranes of a spongy structure consisting of dense tubules surrounded by the labyrinth of the surface-connected system. Their physiological significance in these atypical platelets remains unsolved.     

Definitive Evidence for the existence of tight junctions in invertebrates

Extensive and unequivocal tight junctions are here reported between the lateral borders of the cellular layer that circumscribes the arachnid (spider) central nervous system. This account details the features of these structures, which form a beltlike reticulum that is more complex than the simple linear tight junctions hitherto found in invertebrate tissues and which bear many of the characteristics of vertebrate zonulae occludentes. We also provide evidence that these junctions form the basis of a permeability barrier to exogenous compounds. In thin sections, the tight junctions are identifiable as punctate points of membrane apposition; they are seen to exclude the stain and appear as election- lucent moniliform strands along the lines of membrane fusion in en face views of uranyl-calcium-treated tissues. In freeze-fracture replicas, the regions of close membrane apposition exhibit P-face (PF) ridges and complementary E-face (EF) furrows that are coincident across face transitions, although slightly offset with respect to one another. The free inward diffusion of both ionic and colloidal lanthanum is inhibited by these punctate tight junctions so that they appear to form the basis of a circumferential blood-brain barrier. These results support the contention that tight junctions exist in the tissues of the invertebrata in spite of earlier suggestions that (a) they are unique to vertebrates and (b) septate junctions are the equivalent invertebrate occluding structure. The component tight junctional 8- to 10-nm-particulate PF ridges are intimately intercalated with, but clearly distinct from, inverted gap junctions possessing the 13-nm EF particles typical of arthropods. Hence, no confusion can occur as to which particles belong to each of the two junctional types, as commonly happens with vertebrate tissues, especially in the analysis of developing junctions. Indeed, their coexistance in this way supports the idea, over which there has been some controversy, that the intramembrane particles making up these two junctional types must be quite distinct entities rather than products of a common precursor.     

Sex differences in adrenocortical structure and function

Summary The cytological aspects of sexual dimorphism in the rat adrenal cortex and its relationship to the gonads have been investigated. The adrenal glands of mature female rats were heavier than those of males, and morphometry showed that this was almost exclusively due to conspicuous differences in the volume of cells of the zona fasciculata (ZF) and zona reticularis (ZR). Stereology demonstrated that the volume of the mitochondrial and lipid droplet compartments, as well as the surface area per cell of mitochondrial cristae and smooth endoplasmic reticulum tubules, were markedly higher in the ZF and ZR cells of female animals. Orchiectomy increased and ovariectomy decreased the adrenal weight, by eliciting hypertrophy and atrophy, respectively, of ZF and ZR cells; these effects of gonadectomy were reversed by the appropriate gonadal hormone replacement. It is suggested that the sexual dimorphism of the rat adrenal cortex may depend upon the inhibitory action of testosterone and the stimulatory effect of estradiol on the hypothalamo-hypophyseal-adrenal axis.     

Connections between mitochondria and endoplasmic reticulum in rat liver and onion stem

Summary Smooth-surfaced elements of endoplasmic reticulum contact and are attached to the outer membranes of mitochondria in rat liver and onion stem. Some connections appear as short, 150–300 Å diameter tubules that bridge the space between the conjoining elements. In liver, the smooth-surfaced endoplasmic reticulum cisternae connected to the outer mitochondrial membrane are shown to be continuous with rough-surfaced endoplasmic reticulum. Here, the smooth-surfaced endoplasmic reticulum is identified in negatively stained preparations of isolated cell fractions and in thin sections of tissues by the presence of lipoprotein particles characteristic of this cell component. In onion, the identification of endoplasmic reticulum is based on continuity with rough-surfaced endoplasmic reticulum.     

THE INTER-SERTOLI CELL TIGHT JUNCTIONS IN GERM CELL-FREE SEMINIFEROUS TUBULES FROM PRENATALLY IRRADIATED RATS: A FREEZE-FRACTURE STUDY

The tight junctions between Sertoli cells were examined by freeze-fracture in 3-month-old prenatally irradiated rats, whose seminiferous tubules are devoid of germ cells. The replicas from irradiated tubules show elaborate interdigitations of the lateral membranes of Sertoli cells and very extensive tight junctions. These junctions are characterized by a great number of continuous parallel or complex interweaving strands of intramembranous particles, preferentially associated with E fracture faces. The presence of highly cross-linked tight junctional strands is compatible with an epithelium deprived of germ cells, with a reduced need for flexibility. Anomalous ectoplasmic specializations, consisting of groups of cisternae arranged perpendicularly to the lateral surface, are found in the irradiated tubules. These structures may be involved in a storage mechanism of redundant lateral membrane resulting from the elimination of germ cells. Typical gap junctions, intercalated between the tight junctional strands, are larger and more frequently found in treated animals than in controls. These findings indicate that a very tight permeability barrier seems to be established in the irradiated testis even in the absence of germ cells. Thus, the formation and maintenance of Sertoli tight junctions do not appear to be directly dependent on the presence of germ cells. Nevertheless, the alterations detected in the tight junction architecture and in the ectoplasmic specializations indicate that maturing germ cells probably contribute to the functional organization of the blood—testis barrier in the normal testis.     

Stereological analysis of the guinea pig adrenal: effects of dexamethasone and ACTH treatment with emphasis on the inner cortex

This paper describes the morphological responses of adult male guinea pig adrenals to dexamethasone (DEX) and adrenocorticotrophic hormone (ACTH), in both qualitative and quantitative terms. Most organelles and inclusions are affected, but their responses often vary in the different cell types examined: zona fasciculata externa and interna, and zona reticularis. Following DEX the volume of lipid droplets increases in cells of zona fasciculata externa but decreases in zona reticularis; smooth endoplasmic reticulum decreases in fasciculata externa but increases in reticularis. Following ACTH, exactly the opposite occurs. This strongly suggests differing functions for these subcellular entities in each cell type, particularly for the smooth reticulum, as well as for the cells themselves. The volume of the Golgi complex markedly decreases following DEX in all cells but increases only in zona fasciculata interna and zona reticularis following ACTH. These deeper cortical cells are known to secrete at least one sulfated steroid, dehydroepiandrosterone sulfate, and these changes in the Golgi complex strengthen the suggestion that the Golgi plays a role in sulfation of steroids. Mitochondrial volume and number decrease in all cells following DEX, supporting their role in steroidogenesis. Further decreases in their volume, accompanied by increases in their number following ACTH, may be related to a proliferation of mitochondria in response to ACTH. Changes in peroxisome volume and number, following DEX and ACTH, suggest a possible role for these organelles in steroid cell metabolism. Lysosomes decrease in volume in all cells following ACTH. This does not support the recently suggested concept that they play a role in steroid secretion.     

Ultrastructural analysis of the granulosa — Luteal cell transition in the ovary of the dog

The transition from ovarian granulosa to lutein cell during the estrus cycle of 60 pregnant and non-pregnant beagle bitches was analyzed by light and electron microscopy (both 100 and 1000 KV). Early proestrus was characterized by a gradual rise in serum estrogen levels, hyperplasia of the granulosa cells, the accumulation of follicular fluid, and the development of tortuous intercellular channels. During the second half of proestrus, serum estrogen levels continued to rise, but growth, division, and differentiation of the granulosa cells was minimal. Estrus was marked by the first acceptance of the male and a well-defined LH peak In the subsequent 24 hour period, the granulosa-lutein cells hypertrophy rapidly and develop a large Golgi apparatus, small profiles of granular endoplasmic reticulum, numerous microfilaments, and large gap junctions between the cells. Mitochondria also proliferate, enlarge, and elongate, but retain lamelliform cristae. Luteinization of the cells and progesterone secretion begin just after ovulation which in turn occurs about 24 hours after the LH peak. On the third and fourth day of estrus, numerous small vesicles of agranular endoplasmic reticulum fill the extoplasm and the mitochondria swell up and round off. The vesicles rapidly fuse into whorled and flattened cisternae or anastomosing tubules of agranular endoplasmic reticulum, while the mitochondria develop tubulovesicular cristae. These structures gradually become organized with respect to the basal lamina. The Golgi apparatus is centered over the pole of the nucleus that faces the pericapillary space. Stacked and whorled cisternae of agranular ER develop in the lateral margins and avascular end of the cell while mitochondria and tubular elements of agranular ER predominate in the central medial and most basal portions of the cytoplasm. Microfilaments are ubiquitous and appear to be instrumental in this orientation process. The cell surface develops three distinct regional specializations that coincide with the underlying cellular compartments: interconnecting pleomorphic folds fill the pericapillary space; long tenous microvilli project from the lateral cell surface and form tortuous intercellular channels and canaliculi; and large gap junctions form along the margins of the cell furthest removed from the basal lamina. By the sixth day of estrus, the granulosa-luteal cell transition is nearly complete and serum progesterone levels are on the rise.     

Intercellular junctions of antennal gland epithelial cells in the crayfish,Orconectes virilis

Summary Labyrinth and nephridial canal cells of the crayfish (Orconectes virilis) antennal gland possess two types of intercellular junctions revealed by freeze-fracture studies. Apical margins of the cells are connected by long septate junctions. In replicas, these junctions consist of many parallel rows of 80–140 Å intramembrane particles situated on the PF membrane face (EF and PF fracture faces of Branton et al., 1975). Rows of pits are found on the EF fracture face and are deemed complementary to the rows of particles. Moreover, lateral margins of basal regions of the epithelial cells are attached by many intercellular junctions. These contacts are characterized in thin plastic sections by a narrow dense cytoplasmic plaque located subjacent to the plasma membrane at sites of adjoined cells, and 5 to 12 fine strands of dense material that extend across the intercellular gap between adjoined cells. In freeze-fracture replicas, EF intramembrane faces basal to the region of the plasma membrane containing septate junctions exhibit numerous discoid clusters of particles. The particle aggregates, assumed to represent freeze-cleave images of adhering junctions, range from 900 to 3,700 Å in diameter, with individual particles about 185 Å in diameter. These junctions appear to connect epithelial cell processes formed by basal infoldings of the plasma-lemma, and occur between adjacent cells as well as adjacent processes of a single cell. The discrete aggregates of particles resemble replicated desmosomes (Shienvold and Kelly, 1974) and hemi-desmosomes (Shivers, 1976); therefore, they probably do not constitute a basis for electrical coupling between antennal gland epithelial cells.Supported by the National Research Council of Canada