Ultrastructure of rat pinealocytes in vitro: Influence of gonadotropic hormones and LH-RH

Summary The influence of gonadotropic hormones on the ultrastructure of rat pinealocytes in short-term organ culture was studied. Human chorionic gonadotropin (HCG), as well as pregnant mare serum gonadotropin (PMSG), caused a marked activation of pinealocytes. An hypothesis is discussed implying the presence of a feedback mechanism between the pineal organ and the hypothalamo-hypophysial system.     

Effects of blinding on the ultrastructure of mouse pinealocytes with particular emphasis on the dense-cored vesicles

Summary The mammalian pineal is thought to produce an antigonadotropic principle under conditions of reduced photoperiod, constant darkness or blinding by optic enucleation. A number of previous studies on mammalian pineals have suggested that the dense-cored vesicles present in pinealocytes may represent morphological evidence of secretory activity.In the present study the ultrastructure of pinealocytes was studied in adult Charles River CD-1 mice blinded by optical enucleation. By one month following optic enucleation the mean number of dense-cored vesicles in the cytoplasm of pinealocytes adjacent to pericapillary spaces had significantly decreased by 55% when compared with intact controls, and remained at this low level at two months and six months. A relative increase in the proportion of large agranular vesicles and an increased number of large, irregular vacuoles was observed also in the pinealocytic polar processes of blinded mice. When compared to control mice the pinealocytic Golgi regions appeared to be hypertrophied in blinded mice. The apparent stimulation of pinealocytic organelles coupled with the observed decrease in dense-cored vesicles suggest an increased synthesis and release of secretory product.Supported in part by NIH Grant No. HD 08759     

Ultrastructure of pinealocytes of the cotton rat,Sigmodon hispidus

Summary Fine structural features of pinealocytes of cotton rats (Sigmodon hispidus) were examined. Golgi complexes, mitochondria, endoplasmic reticulum and polysomes are usual organelles seen in the perikaryonal cytoplasm of pinealocytes. Many non-granulated vesicles (40 to 80 nm in diameter) and a few granulated vesicles (about 100 nm in diameter) are associated with the Golgi cisternae. Occasionally, the cisternae contain granular materials. The perikaryonal cytoplasm of pinealocytes is characterized by the presence of inclusion bodies. These bodies are usually round in shape, not bounded by a limiting membrane and composed of fine granular or filamentous materials of high electron-opacity, which are similar in appearance to the substance seen in the nucleolonema. Pinealocyte processes, filled with abundant non-granulated vesicles and some granulated vesicles, are mainly found within the parenchyma and occasionally in perivascular spaces.Supported in part by NSF grant no. PCM 77-05734 and NIH grant no. HD-10202 (Morphology Core)     

Morphologic development of neonatal rat pinealocytes in monolayer culture

Summary The morphological development of pinealocytes maintained in monolayer culture, without the neural and humoral effects present in the developing rat has been studied and compared with the development that occurs in vivo. Pinealocytes in 5 day cultures contained organelles that were similar to those present in the pineals of intact 5 day old rats. However, light and dark cells were not noted in culture, and the cultured cells did not have the dense granules noted in vivo. As pinealocytes developed in culture, cytoplasmic processes increased in length and number. By 21 days of culture age, synaptic ribbons were found to have decreased in number, the difference between light cell and dark cell cytoplasm had become more prominent, and dense-cored vesicles had become more numerous, just as in the developing gland in vivo. These results suggest that the complex neural and humoral factors impinging upon the developing neonatal pineal in the intact animal may not be necessary for some aspects of its ultrastructural differentiation.     

Subcellular localization of gonadotropic hormones in pituitary cells of the castrated pig with the use of pre-and post-embedding immunocytochemical methods

Summary Pre- and post-embedding immunocytochemical methods based on the use of specific antibodies against -subunits of porcine LH and FSH were applied to determine the changes occurring in the anterior pituitary of the pig after gonadectomy. The results showed that (1) the total number of immunoreactive gonadotropes increased from 21–25% in control animals to 24–37% in castrated animals; (2) all gonadotropes contained both LH and FSH; (3) several types of immunoreactive LH/FSH cells were revealed; and (4) the two immunocytochemical methods used with dispersed cells localized the hormones in the same subcellular sites. However, the staining intensity in the different locations varied depending on the method applied. With the post-embedding method, a dense reaction product was found in the secretory granules but the cisternae of RER and the Golgi saccules were always slightly reactive. After the pre-embedding method, the staining intensity in the RER-cisternae and in the Golgi saccules was greatly increased. Thus, the two methodological approaches used in this study have permitted to visualize immunocytochemically the gonadotropic hormones not only at the sites of their storage but also along the intracellular pathway of the secretory material, i.e., at the site of its synthesis and during its passage via the Golgi zone.     

The influence of some sympatholytic,parasympatholytic and serotonin-synthesis-inhibiting agents on the ultrastructure of the rabbit pineal organ

The influence of certain drugs on the ultrastructure of rabbit pinealocytes was studied. The results obtained after administration of p-chlorophenylalanine and p-chloroamphetamine support the hypothesis proposed earlier that the smooth endoplasmic reticulum in the light pinealocytes is involved in indoleamine synthesis. The administration of either one of the sympatholytic agents, 6-hydroxydopamine or alpha-methyl-p-tyrosine, induced typical fine structural changes corresponding to those observed after surgical sympathectomy.     

The brain-pituitary-gonadal axis in the rainbow trout,Salmo gairdneri: Gonadal hormones and the maturation of gonadotropic cells

Summary Intact and castrated juvenile male rainbow trout (Salmo gairdneri) were treated with testosterone and gonadotropic hormone (GTH) to determine the maturational effects of these hormones on the GTH-cells. Electron-microscopic studies of the GTH-cells revealed that GTH and testosterone in intact animals, and testosterone in castrated fish, caused GTH-cell maturation: These cells now displayed the same appearance as GTH-cells in adult trout, including the presence of globules, a well-developed Golgi apparatus and rough endoplasmic reticulum, all of which were absent in GTH-cells of control animals. Animals with stimulated GTH-cells also had an increased GTH content of the pituitary; release of GTH could not be demonstrated. Animals treated with GTH exhibited an accelerated development of the testes, resulting in complete gametogenesis and elevated plasma testosterone levels. These results indicate that exogenous steroids as well as endogenous gonadal steroids can stimulate the full development of GTH-cells and accelerate GTH synthesis. The significance of this stimulating effect of the gonadal hormones with respect to the development of the brain-pituitary-gonadal axis and the onset of puberty is discussed.The results were presented as a poster at the 11th conference of the European Society of Comparative Endocrinology, Jerusalem, August 10–14, 1981     

Ultrastructural changes of pituitary gonadotropic cells in estrogen-treated pregnant rats

Summary An ultrastructural study of gonadotropic pituitary cells was performed in estrogen-treated pregnant rats. Estradiol-treatment on Day 10 of pregnancy led to signs of ovulation or luteinization on Day 12 in 50% of the animals.Degranulation was observed in the FSH and LH cells of estrogen-responsive rats, whereas in the unresponsive group, the same cells were intensely granulated. The FSH cells of the control group showed signs of degranulation which could be correlated with follicular development. LH cells were sometimes degranulated.The role played by FSH and LH cells in the triggering of ovulation and luteinization by estrogen in the pregnant rat is discussed in the light of the ultrastructural observations.This work was financed by the DGRST, contract n^o 74.7.0030The authors wish to acknowledge the technical assistance of Mr. R. Dujol and Mr. F. Wolff     

The ultrastructure of pinealocytes in the pig

Summary Pinealocytes of female pigs were studied electron-microscopically and compared with those of other mammals. A prominent Golgi apparatus forming dense-cored vesicles was widely dispersed in the cytoplasm of the cell body. A very characteristic feature of the pig pinealocytes was the presence of membrane-bounded bodies showing wide variations in internal structure. Possible roles of the dense-cored vesicles and membrane-bounded bodies in secretory processes of pinealocytes are discussed.     

Effects of ovarian hormones on cell membranes in the rat uterus

Freeze-fracture techniques have been used to study tight junctions on the lateral plasma membrane of cells of the luminal epithelium of the rat uterus under various hormonal regimes. Tight junctions from ovariectomized control rats extended some 0.5 μm down the lateral membrane and the junctional strands often formed a network of closely packed, circular compartments. Following treatment of rats with estrogen for 3 days the tight junctional regional still extended 0.5 μm down the lateral membrane, but the strands ran more parallel to the apical surface. They did not enclose circular compartments. After treatment with progesterone, either alone or with estrogen in such a way as to condition the ovariectomized uterus for implantation, a third pattern of junctional organization emerged. In these animals the junctional region extended 1.1 μm down the lateral membrane and the strands frequently crosslinked, enclosing compartments of varying and irregular size and shape. Our observations suggest that ovarian hormones could regulate the contents of the uterine lumen by altering the structure extent of the tight junctions which connect the epithelial cells enclosing the lumen.     

Effects of ovarian hormones on cell membranes in the rat uterus

Histochemical techniques, including radioisotope histochemistry, have been used to investigate the nature of the surface carbohydrates at the apex of cells of the luminal epithelium of the rat uterus under various hormonal conditions. Binding of ruthenium red was quantitatively similar in ovariectomized rats without further treatment and in those given three daily injections of progesterone. Ruthenium red binding was significantly lower after 3 days treatment with estradiol, and also after 3 days treatment with progesterone with an additional dose of estradiol on day 3, a regime known to produce an epithelium receptive to the implanting blastocyst. Binding of concanavalin A (con A), whether studied by electron microscope histochemistry after incubation of tissue with con A-horseradish peroxidase, or by light microscope autoradiography after incubation with³H-con A, was not statistically different in any of the four groups of rats. The results with ruthenium red show a reduction in net negative charge of the carbohydrates on the apical cell membrane in conditions permitting implantation: this change is not due to variations in the amounts of the neutral carbohydrates, mannose and glucose, as demonstrated by con A.     

Ultrastructure of the pars distalis of the lizard Anolis carolinensis with special reference to the identification of the gonadotropic cell

Summary Five categories of granulated cells were distinguished by their ultrastructural features, and quantitative analyses were made of the pars distalis cells in normal and castrated lizards. The gonadotropin-producing cell was identified on the basis of its uniform distribution in the gland as well as from cytological changes resulting from castration. The secretory granules of the gonadotropic cell vary in size (100–500 m) and density, and lipid bodies are commonly present. Following castration, the endoplasmic reticulum proliferates, forming many small, rough-surfaced, dilated cisternae which do not coalesce greatly as in other vertebrate species. Degranulation is accompanied by hypertrophy and hyperplasia of the mitochondria and by the appearance in the cytoplasm of conspicuous clusters of microfilaments. The designated gonadotropic cell was the only class of secretory cell showing consistent changes following three weeks of castration.In addition to the uniformly distributed gonadotrope cell, two secretory cells occur mainly in the rostral half of the gland, and two in the caudal half. Tentative identification of the cell types is discussed in the light of available information on the localization of the hormones in the pars distalis of this species.Grateful recognition is given to the Electron Microscope Laboratory of the University of California, Berkeley, for use of their facilities, and to Emily Reid for her assistance with the illustrations.Member of Consejo Nacional de Investigaciones Cientificas y Técnicas.     

Ultrastructural observations on synaptic ribbons in the pineal organ of the goldfish

Summary Synaptic ribbons in the pineal organ of the goldfish were examined electron microscopically with particular attention to their topography. These structures were formed of parallel membranes, which were poorly preserved with OsO₄ fixation and could be extracted from thin sections with pronase indicating their proteinaceous nature. Synaptic ribbons were closely apposed to the plasma membrane bordering dendrites of ganglion cells, but were also related to processes of both photoreceptor and supportive cells. Their close proximity to invaginations of the plasma membrane and portions of the endoplasmic reticulum suggest that they are involved in the turnover of cytoplasmic membranes. Tubular and spherical organelles of unknown function are also described.     

The influence of noradrenaline on the process of protein/peptide secretion in the mammalian pineal organ

Summary From studies conducted with the pineal organ of the mouse, it was ascertained that for the in vitro investigation of secretory processes (synthesis and release) of proteic/peptidic compound(s), a culture time of 5 to 14 days is optimal. A 5-day organ culture was therefore chosen to study the effects of noradrenaline on these secretory processes.Addition of noradrenaline to the culture medium provokes, in pineal explants of the normal mouse and the eyeless mouse, an inhibition of the secretory process, characterized by the formation of granular vesicles. In the hamster and rat, however, opposite results were obtained. Moreover, it appears that noradrenaline, at least in the rat, may also be involved in the regulation of the ependymal-like secretory process.The present results indicate clearly that noradrenaline (thus, the sympathetic innervation) is implicated in the regulation of the production of proteic/peptidic hormonal agents, but that the effect of this neurotransmitter is species-specific. This could explain the numerous contradictory results reported in the literature.IBRO/UNESCO fellow     

The ultrastructure of the accessory sex organs of the male rat

Summary A systematic, comparative study of the accessory sex organs of the adult male rat was carried out after intra-aortic perfusion of the pelvic organs with glutaraldehyde. It has been revealed that although the epithelial cells of the different lobes of the prostate have many features in common, it is also apparent that the cell type of the various lobes have specific ultrastructural characteristics of its own, which morphologically distinguish it from the cell type of the other lobes. I.e.: the different lobes may be identified by their specific ultrastructural feature. It is also striking that the lobes, two-by-two, have so many morphological features in common that they may be divided in 3 subgroups. Based on the appearance of amount and localisation of the different organelles, the cells of the lateral lobe and the seminal vesicle are so alike that they morphologically may be classified as one group. Similarly, the coagulating gland and the dorsal lobe form another group, while the ventral lobe as a single form a third group. The few biochemical data from the different lobes which are accessible, seem suggestive to support this subgrouping.Since the various prostate lobes and the seminal vesicles have their homologies in man, further investigation both morphologically and biochemically should be concentrated upon the different groups instead of the single lobe.The study, which describes the different lobes and cell types in detail also show structures which have not been demonstrated within the prostatic epithelium before.     

Electron microscopy of the rabbit pineal organ in vitro

The results presented in this study show that the rabbit pineal organ cultured in vitro retained its in vivo fine structure for at least eight days. However, the Golgi complex in the light pinealocytes stopped forming dense core vesicles while vesicle-crowned ribbons increased in number. After addition of norepinephrine to the culture medium, the Golgi complex once more began the production of dense core vesicles. Terminals of light pinealocytic processes then often contained Golgi dense core vesicles in close contact with the cell membrane, suggesting the release of the vesicular content into the intercellular and perivascular spaces. A close topographical relationship between Golgi dense core vesicles and vesicle-crowned ribbons was observed.     

Melatonin effect on the ultrastructure of Ehrlich ascites tumor cells,lifetime and histopathology in Swiss mice

Aims

One of the models used for studying cancer is the Ehrlich ascites tumor (EAT) due to its ability to grow in liquid suspension, allowing a standard number of cells to be inoculated, growth quantification and regression of tumor mass. Among the oncostatic substances, melatonin has shown effectiveness in limiting the tumor cell proliferation. However, studies have shown contradictory effects of melatonin on the EAT. This study has investigated the melatonin effect on tumor growth, time and survival percentage, ultrastructure and metastasis of EAT cells in mice submitted or not to pinealectomy.

Main methods

Animals were inoculated with 5 × 10⁶ cells/mL and treated or not with exogenous melatonin with doses of at 150 and 300 μg/30 g animal weight for 12 days. Melatonin significantly reduced the abdominal circumference, volume of ascites liquid and EAT-cell viability, raising rates of time and mice survival percentage.

Key findings

Ultrastructurally, the melatonin treatment revealed changes in the shape of cells, the cell surface showed numerous projections, some bifurcated, cytoplasmic vacuolation, mitochondrial degeneration and nuclear fragmentation, peculiar characteristics of apoptosis. Histopathology revealed no metastasis in the liver, small intestine and large intestine in any of the animals in the experimental groups; however this process was evident in the lungs and kidneys, being inhibited by melatonin administration.

Significance

Thus, we can conclude that doses of 150 and 300 μg/30 g of melatonin for 12 consecutive days have a very effective oncostatic and cytotoxic activity on EAT cells in mice.     

Time-related changes in size of nuclei of pinealocytes in rats

Summary In a study of 96 adult male Sprague-Dawley rats, da-and time-related changes of the mean nuclear volume of pinealocytes were determined with the aim of testing the reproducibility of the karyometric findings described by Quay and Renzoni (1966). It is shown (i) that statistically significant differences exist between the central and peripheral mean volumes of pinealocyte nuclei/group of animals and time point (p<0.001), (ii) that the day and time related differences are statistically different in both regions (p<0.001), (iii) that in the center and periphery of the pineal body different diurnal patterns exist, and (iv) that the diurnal patterns are not parallel in the two regions.The question discussed is (i) whether or not probable superimposed infradian rhythms may account for the different diurnal patterns, and (ii) whether these patterns in the two regions indicate functional differences between the cortex and the medulla of the rat pineal body.Supported by the Deutsche Forschungsgemeinschaft (Grant Vo 135/4) within the Schwerpunkt-programm NeuroendokrinologieThis paper is an abridged version of a thesis submitted for obtaining the degree of Dr. med., Fachbereich Medizin, University of Mainz