Seasonal changes in the endocrine cells of the gastric epithelium of Rana temporaria

By means of the light and electron microscopy methods histotopography, quantity and ultramicroscopic organization of endocrine cells (EC) have been studied in gastric epithelium of the frog (Rana temporaria) during various periods of its life: hibernation, awakening, active life. At all the periods of life 8 types of EC are revealed. Depending on the season, certain changes both in their amount and ultrastructural organization are observed. Decreasing amount of EC is noted during hibernation and their increase in spring during awakening and in summer. Quantity of enterochromaffin cells is not subjected to seasonal fluctuations. This is explained by the fact that in hibernation these cells are also at their active functional state, since they produce serotonin necessary for maintaining deep hypothermia, specific for hibernating animals.     

CYTOCHEMICAL DIFFERENCES IN KIDNEYS FROM WINTER-HIBERNATING AND AROUSED BATS (MYOTIS LUCIFUGUS), WITH PARTICULAR REFERENCE TO THE GOLGI ZONE

Kidneys from winter bats (Myotis lucifugus) were removed and fixed in cold formalin-calcium while the animals were in the following states: (a) natural hibernation; (b) arousal from hibernation for 24 hours; (c) laboratory maintained hibernation; and (d) no hibernation since the previous winter. With fixed frozen sections, the lead salt method of Wachstein and Meisel with adenosine triphosphate as substrate (pH 7.2) showed enzymic activity localized in large vacuoles and smaller vesicles or droplets in the Golgi region of distal and proximal tubular epithelial cells of kidneys from hibernating bats. No ATPase activity was detected in the basal lamellae of tubular epithelium from hibernating bats. ATPase activity in the Golgi region was not seen in cells from kidney tubules of bats aroused from hibernation 24 hours previously or of animals that had not hibernated, whereas activity for ATPase was present in the basal infoldings of tubular epithelium from these animals. Inosine di- and triphosphatase and calcium activated ATPase activities were also detected in the Golgi region of hibernating bats but were not present in the basal infoldings of tubular epithelium from active animals. There was little or no activity toward the mono- and diphosphates of adenine, thiamine pyrophosphate, and the di- or triphosphates of guanidine, cytidine, or deoxyadenosine. The loss of enzymic activity from the Golgi region of the tubular epithelium from hibernating bats and its increase in the region of the basal infoldings of tubular epithelium in aroused bats suggests that the Golgi region plays a role in the synthesis of enzymic protein usually identified with the external cell membrane.     

Ultrastructural changes in Paneth cells during hibernation in the ground squirrel Spermophilm lateralis

Summary The ultrastructure of Paneth cells from jejuno-ileal segments of the small intestine of the ground squirrel, S. lateralis, was examined under normal euthermic conditions and during the profoundly depressed metabolic conditions of natural hibernation. Paneth cells obtained from hibernating animals gave evidence of markedly reduced activity when compared to Paneth cells from euthermic animals. In hibernating animals, the nuclei were smaller, with less prominent nucleoli and with an increased proportion of heterochromatin. In hibernating animals, the rough endoplasmic reticulum was fragmentary and poorly organized, in contrast to the typical arrangement of concentric lamellae seen in euthermic animals. Although the total number of ribosomes was decreased in hibernating animals, there were proportionally more free ribosomes than in euthermic animals. Paneth cells from hibernating animals also contained a greater number of apical secretory granules which were smaller and more variable in electron density than granules from control animals. These ultrastructural features indicate that during hibernation the Paneth cell is relatively quiescent.Supported by U.S.P.H.S. grants RR 05411 and RR 05583 from the N.I.H.     

The plasma membrane transformation facilitates pregnancy in both reptiles and mammals

Mechanisms of placentation are very diverse in mammals and range from types in which the uterine epithelium is breached by the implanting blastocyst to those where the epithelium remains intact. Despite these differences in mechanisms, the initial response of the plasma membrane of uterine epithelial cells is remarkably similar across mammalian species which has led to the term 'plasma membrane transformation' to encapsulate the concept of a common beginning to implantation. Membrane phenomena similar to those of mammals have now been observed in some viviparous lizards at the ultrastructural level during early pregnancy, and we propose extending the concept of 'plasma membrane transformation' to lizards with live birth.     

Cytodifferentiation of the epithelium of the gastric mucosa glands of rats in ontogenesis

Structure of epithelium in the mucous membrane glands of the rat stomach has been studied electron microscopically during embryonal and postnatal periods of development. Time of initial appearance of various types of cells in early stages of the gastric epithelium histogenesis has been verified. Asynchronity of their appearance is demonstrated. In epithelium of the gastric glands in 20-day-old rat embryos poorly differentiated exocrine and also endocrine D- and G-cells are revealed. On the 1st day EC-, ECl-, and on the 3d day--X-endocrinocytes appear. Neighbouring exocrinocytes connect with each other by means of both nonspecific and specific intercellular contacts. Exocrine and endocrine cells connect by means of desmosomes. For the animals of each age group certain range of ultrastructural organization in variously differentiated glandulocytes (they are morphological basis of the reactive changes) is specific.     

Protein synthesis in cardiac cells and the ultrastructural dynamics of cardiomyocytes of hibernating animals during the hibernation cycle

A significant increase in protein synthesis correlating with ultrastructural dynamics of cardiomyocyte organelle convertions has been demonstrated in cardiomyocytes of ground squirrel during arousal from hibernation. In hibernating ground squirrels, the ultrastructure of protein-synthesizing organelles and of the cardiomyocyte nucleus points out to the readiness of cells to active synthesis of proteins. In the perinuclear area of cardiomyocytes abundant ribosomes, elements of endoplasmic reticulum and Golgi complex, mitochondria and high-energy substrates--glycogen and lipid inclusions--are seen. The cardiomyocyte nuclei are large, with highly convoluted borders and abundant pores, their nucleolar structure is granular, the chromatin is mainly diffuse. The potency of cardiomyocyte protein-synthesizing system of hibernating ground squirrels is realized every time at periodical arousals during hibernation. The role of cyclic changes of protein synthesis rate in adaptation of cells of hibernating mammals to functioning at various temperatures is discussed.     

Morphological and functional recovery of the canine gallbladder mucosa following two hours’ ischemia

Temporary interruption of the circulation to the canine gallbladder causes hemorrhagic necrosis of the mucosa and an abolition of active transport of glycine in vitro. The first signs of epithelial recovery following two hours' ischemia are seen in animals examined 7 days after the injury and are characterized by the appearance of a layer of cuboidal epithelial cells in which some mitotic figures are observed, accompanied by a decrease in the amount of necrosis and the presence of granulation tissue. At the same time, there is some slight resumption in glycine uptake. Two weeks after injury, the new epithelium covers a larger surface of the gallbladder wall but recovery is still incomplete. Four weeks after operation, structural and functional recovery has occurred in almost all the animals studied: the epithelium is columnar and does not differ greatly from that seen in the normal gallbladder. Mucosal folds are present although their morphological appearance is slightly altered in comparison with those of control specimens. The gallbladder wall is thickened by fibrosis. Measurements of active glycine transport in vitro confirm that the new epithelium functions normally.     

The interactions between spermatozoa and uterine epithelium in the hibernating bat,pipistrellus kuhli natt

In hibernating females of Pipistrellus kuhli, as well as in other bats previously described, the spermatozoa inseminated in autumn are stored in the uterus, both free and attached by their heads to the uterine epithelium, throughout winter. With the electron microscope the apical region of the attached sperm heads appears to be contained in plicae of the endometrial cells. Furthermore, a great number of microvilli protruding from the uterine epithelium participate in the interaction with spermatozoa. In the region of contact between the two cell types, their plasma membranes run parallel with a constant 12-nm gap. The sensitivity of these interactions to EDTA and hyaluronidase suggests an involvement of Ca⁺⁺ and proteoglycans in these bindings, while their insensitivity to trypsin tends to exclude a role of proteins. In addition, evidence of exocytosis of dense granules just at the bottom of the plicae containing the spermatozoon suggests a transfer of material from the uterine epithelium to the intercellular space, thus supporting a physiological role of these interactions.     

Light and electron microscope observations on the gastric mucosa of the frog (Rana esculenta)

Summary The structure of the frog gastric and esophageal mucosa was studied in the course of a complete hibernation period and compared with that in summer frogs (see preceding article).It appeared that especially chief cells and parietal cells are liable to cytoplasmic remodelling. Thus, in chief cells the rough endoplasmic reticulum (RER) undergoes disorganization, the number of free ribosomes increases and the Golgi system becomes transformed into a compact vesicular structure. The number of pepsinogen granules in chief cells of late winter frogs is only 20% of that in frogs studied at the onset of hibernation. The loss of pepsinogen granules is at least partly due to autophagy. In addition, lysosomes are involved in focal degradation of the cytoplasm, which may ultimately result in complete degeneration of some chief cells. The presence of zymogen granules containing fibrocyte-like cells in the tunica propria proved heterophagocytosis by these cells.In parietal cells, the area occupied by smooth endoplasmic reticulum becomes reduced. The basal cytoplasm of both chief cells and parietal cells contains numerous lipid droplets, which, in contrast to those in summer frogs, are continuous with RER cisternae. The juxtaposition of lipid droplets and mitochondria seen in summer frogs is eventually lost in hibernating animals.Apart from the appearance of supra-nuclear lipid droplets, the mucous cells of the surface epithelium show no striking alterations. However, in the glandular pits both surface mucous cells and mucous neck cells contain less mucous granules than in summer frogs.The results are discussed in connection with parallel biochemical work and available literature, and in the light of our previous studies on the exocrine pancreas in hibernating frogs.     

Study of the testis in the active and hibernating hedgehog

The structure and ultrastructure of the hedgehog's testis has been studied during active and hibernating states. The maximum activity of the hedgehog's testis manifest itself in spring and summer. In autumn they enter in a seasonal resting, in which, the interstitial cells atrophy and it can observe primary spermatocytes resting next to spermatogonia and supporting cells in the seminiferous tubules. These have a minimal diameter and contain only primary spermatocytes in hibernating period also the seminiferous epithelium that compound the walls of the tubules are about 72 microns thick in the active period and about 47 microns in hibernation. The Sertoli cells shows a small diameter in hibernation than in the active season. The nucleus has one or two nucleoli with distinguish themselves from those observed in the active season because of their closer union with the inner nuclear envelope. In autumn (resting period) the interstitial tissue regress and the Leydig cells are aggregated in blocks with little cytoplasm, minimum size nuclei and 1 or 2 nucleolus closely to the inner nuclear membrane. Like then in the ovary, the hedgehog's testis follows a seasonal cycle determined by the temperature and environmental changes and it have a power of physiological adaptation inherent of a typical hibernating animal.     

Spin label evidence for the role of lysoglycerophosphatides in cellular membranes of hibernating mammals.

The phospholipid composition of ground squirrel heart muscle changes during hibernation: more lysoglycerophosphatides are found in the hibernating state than in the active state. Phase transitions inferred from spin label motion occur in the usual manner typical of mammalian mitochondria for the mitochondria and mitochondrial lipids from active squirrels. However, a conspicuous absence of a spin label-detectable phase transition is observed in equivalent preparations from hibernating animals. The addition of lysolecithin to preparations from active squirrels removes the break and induces a straight line in the Arrhenius plot. The lack of a spin label-detectable phase transition in hibernating animals, therefore, is attributed to an increased content of lysoglycerophosphatides present in the phospholipids during hibernation.     

The ultrastructural and dynamic characteristics of erythrocyte membranes. The effect of the physiological status and temperature

Erythrocyte membranes of ground squirrels taken from hibernating, awakening and active animals with the body temperatures of 4, 30 and 36 degrees C, respectively, have been shown by freeze-fracture technique to differ in morphology of the PF surface, density and distribution patterns of intramembrane particles. The ultrastructural changes in membrane caused by temperature were confirmed by the data on alterations of dynamics parameters of the lipid bilayer and spectral characteristics of membrane proteins.     

Scanning electron microscopic study of hedgehog uteri

Ultrastructural studies of hedgehog uteri (Erinaceus europaeus L.) have been made using animals in anestrus, in estrus and in estrus after sojourn of a week with a male. In estrus and anestrus the uterine epithelium is homogeneous, regularly interrupted by orifices of glands. It is composed of microvillous cells only. Microvilli decrease in number and length in anestrus. A new type of cell, a ciliated cell, appears after copulation. Probable correlation of ultrastructural aspects of endometrium with hormonal situation is discussed.     

Seasonal changes of parafollicular and follicular cells of the dormouse thyroid (Myoxus glis): an ultrastructural and immunocytochemical study

The follicular epithelium of dormouse thyroid consists of two distinct cellular types, follicular and parafollicular cells. Parafollicular cells can be easily identified by their high cytoplasmic dye-affinity for phloxine, round to ovoid shape, basal arrangement and lack of contact with follicular colloid. The wide cytoplasmic matrix is clear and contains many secretory granules of variable electron density whose contents histochemically appears to be proteic with a lean glucidic component. Furthermore immunocytochemical reactions with antibodies against calcitonin and somatostatin showed that both hormones are co-stored in the secretory granules of all parafollicular cells. Both follicular and parafollicular cells show seasonal morphological variations in their secretory activity. Follicular cell activity is high in summer, reaches a peak in late fall or prehibernation and progressively slows down throughout hibernation. Parafollicular cells exhibit a fair synthetic activity in summer, in fall, and in the animals captured during winter hibernating sleep and killed after 12 days stay in laboratory. In winter sleep, granules with interrupted membrane and cottony contents are prevalent and the ultrastructural aspects suggest an intense discharge of secretion. The results are compared with those from other hibernating mammalians and discussed in the light of blood calcium values and seasonal balances of other metabolisms.     

Seasonal ultrastructural changes of the hypophyseal pars tuberalis in the hedgehog (Erinaceus europaeus L.)

The comparison of the hypophyseal pars tuberalis in active and in hibernating hedgehogs reveals different cytological characteristics in the specific secretory cells. In active animals, these cells show oval nuclei and the cytoplasm contains numerous secretory granules near or attached to the cell membrane, suggesting exocytotic release. In hibernating animals, the specific secretory cells are characterized by irregularly shaped and often invaginated nuclei. Clusters of secretory granules lying within the cytoplasm are often joined by lysosomes. These areas are observed to be encircled by cisterns of the endoplasmic reticulum. Degenerative processes in the structures sequestered in this way are interpreted as signs of crinophagy which has no equivalent in the cell types of the pars distalis.     

Seasonal dynamics of the gastric APUD cell count in a hibernator

Light microscopy was applied to study variation in the composition and number of apudocytes in the gastric epithelium of red-cheeked sousliks over time. During hibernation, the content of serotonin-producing EC cells was elevated. This is in agreement with serotonin involvement in the maintenance of the hibernation state. The number of non-EC cells taking part in digestion, which does not occur during hibernation, was lowered. Spring awakening was accompanied with an increase in the number of non-EC cells and the lack of significant changes in cells. In souslics, which were active in winter, the content of non-cells was high, since the animals continued feeding on, and the content of EC-cells was elevated similarly to that in hibernating animals, which could be explained by genetic dependence. The number of apudocytes in the gastric mucosa was discovered to correlate in the main with the features of their function at different seasons.     

The intensity of oxidative phosphorylation and the function of the adenylate system in the liver mitochondria of active and hibernating susliks Citellus undulatus

The state of adenylate system and intensity of oxidative phosphorylation in liver mitochondria of active and hibernating ground squirrels were studied depending on the concentration of extramitochondrial Ca2+ ([Ca2+]ex). It was shown that at [Ca2+]ex.10(-7) M, the content of ATP as well as ATP/ADP ratio are slightly lower in the mitochondria of hibernating ground squirrels than in the mitochondria of active animals. The other parameters of the adenylate system under the same conditions differ insignificantly. [Ca2+]ex increase to 10(-6) M has little effect on the parameters of the adenylate system of active animals. On the contrary, the mitochondria of hibernating ground squirrels are strongly affected: the level of ATP is 1.5-fold and the ratio of ATP/ADP is almost 2-fold decreased. At both [Ca2+]ex the intensity of oxidative phosphorylation is essentially higher in the mitochondria of active ground squirrels. With increasing [Ca2+]ex the rate of ATP synthesis decreases, and in the mitochondria of hibernating animals the decrease is more pronounced than in the mitochondria of active animals. Thus, oxidative phosphorylation and adenylate system of mitochondria from hibernating ground squirrels are more sensitive to [Ca2+]ex increase than those of the mitochondria of active animals.     

Chromatin organization in frog Purkinje neurons during the annual cycle: cytochemical and ultrastructural studies

Feulgen-DNA microdensitometric and propidium Iodide-DNA (PI-DNA) microfluorometric evaluations were made in Purkinje cells of cerebella from hibernating frogs and from active frogs. Higher Feulgen-DNA/distribution area ratios and lower PI-DNA values were found in the hibernating frog (both at high and low fluorochrome concentrations). These data indicate a higher degree of chromatin compactness. During the activity period, the Feulgen-DNA/distribution area ratios are lower and the Feulgen-DNA content is higher; the corresponding higher PI-DNA values indicate a lower degree of chromatin condensation. Histochemical and ultrastructural data show clearly different distribution patterns of chromatin and ribosomes in the two periods. The overall results underscore lower activity of neurons and a greater homogeneity within the Purkinje cell population during hibernation.     

The inhibition of succinate, beta-oxybutyrate and glutamate transport in the liver mitochondria of hibernating susliks

Studies have been made on the permeability of the inner membrane of the liver mitochondria from hibernating and active ground squirrels for succinate, glutamate, hydroxybutyrate and inorganic phosphate. The permeability was calculated from the rate of mitochondrial swelling in 100 mM ammonium salts of the substrates and phosphate. It was shown that the rate of mitochondrial swelling in hibernating animals is 2--3 times lower than in active ones, being essentially identical in a solution of ammonium phosphate. It was concluded that the permeability of the inner mitochondrial membrane for the substrates decreases in hibernating animals, remaining unaffected for phosphate. Calcium-induced activation of membrane phospholipase A2 facilitates the transport of oxidative substrates into the mitochondria of hibernating ground squirrels, significant increase in the mitochondrial respiration being simultaneously observed. The data obtained suggest that inhibition of transport of oxidative substrates is one of the main factors which account for a low respiration rate in the mitochondria of hibernating animals.     

SOME ULTRASTRUCTURAL EFFECTS OF INSULIN, HYDROCORTISONE, AND PROLACTIN ON MAMMARY GLAND EXPLANTS

The effects of insulin, hydrocortisone, and prolactin on the morphology of explants from midpregnant mouse mammary glands were studied. Insulin promotes the formation of daughter cells within the alveolar epithelium which are ultrastructurally indistinguishable from the parent cells. The addition of hydrocortisone to the medium containing insulin brings the daughter cells to a new, intermediate level of ultrastructural development by effecting an extensive increase of the rough endoplasmic reticulum (RER) throughout the cytoplasm and an increase in the lateral paranuclear Golgi apparatus. When prolactin is added to the insulin-hydrocortisone medium, the daughter cells complete their ultrastructural differentiation. There is a translocation of the RER, Golgi apparatus, and nucleus and the appearance of secretory protein granules within the cytoplasm. There is excellent correlation between the ultrastructural appearance of the alveoli and their capacity to synthesize casein.