Cellular changes in rat parathyroids provoked by progesterone and testosterone

Summary Male rats kept on a standard diet were treated either with progesterone or testosterone by a single intramuscular injection of preparations which are slowly absorbed and metabolized. The rats were anaesthetized 24 h after application of the hormones, perfused with glutaraldehyde, and the parathyroid glands prepared for electron microscopy. Morphometric analysis revealed that both progesterone and testosterone provoked (1) an increment in nuclear and cell volume and a concomitant increment in cell surface area, and (2) an increment in surface area of rough endoplasmic reticulum by 42% and 49%, and of the Golgi complex by 85% and 63%, respectively. Previously, we had found that oestradiol treatment led to a similar response in parathyroid cells. The conclusion is thus drawn that male and female sex hormones induce membrane synthesis resulting in an enhanced capacity for parathyroid hormone secretion since RER and Golgi complex are concerned with this secretion. It is considered probable that sex hormones have the ability fundamentally to modulate secretory activity in parathyroid cells.     

Unimpaired membrane dynamics in parathyroid cells in insulin deficient rats

The responsiveness of parathyroid cells in insulin deficient and short-term diabetic rats was investigated by morphometric analysis. Insulin deficiency was produced by intravenous injection of D-mannoheptulose and short-term (7 days) diabetes mellitus by intraperitoneal application of streptozotocin. Parathyroid glands were stimulated for parathyroid hormone secretion by decreasing the serum calcium concentration through intravenous infusion of EGTA. Parathyroid cells of controls, insulin deficient, and short-term diabetic rats responded to reduced serum calcium by a 45% increase of the cell surface area. This increase is assumed to be the result of the membrane-bound transport of parathyroid hormone from the Golgi complex and secretory granules to the plasma membrane and subsequent exocytic release of parathyroid hormone induced by the low serum calcium concentration. Therefore, the unimpaired increase in the cell surface area of parathyroid cells in insulin deficient and short-term diabetic rats indicates that insulin does not modulate the release of parathyroid hormone. It is also considered likely that synthesis of parathyroid hormone is not suppressed in short-term diabetes but that fat metabolism is disturbed leading to accumulation of lipid vacuoles.     

Quantitative assessment of cellular changes provoked by microwave enhanced fixation of parathyroids

Rat parathyroids fixed by microwave enhancement, i.e. microwave irradiation in the presence of glutaraldehyde for 8 s and postfixation with OsO4 after a delay of 5 min, were compared with parathyroids fixed by perfusion with glutaraldehyde followed by immersion in glutaraldehyde and finally in OsO4. Morphometric analysis revealed that microwave enhanced fixation led to a larger mean cell volume, to larger cell surface area, and to larger surface area in membranes of RER and secretory granules. Though it is not known by which method parathyroid cells are conserved closer to the living state it is obvious that microwave enhanced fixation retains more membranes but provokes centrifugal dislocation of membranes mimicking exocytosis.     

Quantitative aspects of membrane shifts in rat parathyroid cells initiated by decrease in serum calcium

The secretory activity of parathyroid glands in rats was stimulated by decreasing the serum Ca++ concentration through constant intravenous infusion of EGTA. The morphometric analysis of the nuclear and cytoplasmic volume and of the surface area of the rough endoplasmic reticulum, Golgi complex, secretory granules and plasma membrane revealed a membrane shift from secretory granules and Golgi complex to the plasma membrane within 1 hr of calcium depression. Subsequently, between 1 and 3 hr of calcium depression, the membrane shift was from the plasma membrane to the Golgi complex. It is considered likely that these membrane shifts are related to a rise in release of parathyroid hormone by exocytosis and a subsequent increase in retrieval of plasma membrane by endocytosis—probably through the compartment of coated pits and coated and uncoated vesicles.     

Quantitative assessment of cellular changes provoked by microwave enhanced fixation of parathyroids

Summary Rat parathyroids fixed by microwave enhancement, i.e. microwave irradiation in the presence of glutaraldehyde for 8 s and postfixation with OsO₄ after a delay of 5 min, were compared with parathyroids fixed by perfusion with glutaraldehyde followed by immersion in glutaraldehyde and finally in OsO₄. Morphometric analysis revealed that microwave enhanced fixation led to larger mean cell volume, to larger cell surface area, and to larger suface area in membranes of RER and secretory granules. Though it is not known by which method parathyroid cells are conserved closer to the living state it is obvious that microwave enhanced fixation retains more membranes but provokes centrifugal dislocation of membranes mimiking exocytosis.     

Onset of compensatory hypertrophy of interstitial tissue and Leydig cells in rats hemicastrated around the time of puberty

When rats were unilaterally castrated at 20, 30, and 40 days of age, only those rats hemicastrated at 40 days showed compensatory hypertrophy of the interstitial tissue and Leydig cells when killed 30 days after hemicastration. At the time of death, volume densities of interstitial tissue, Leydig cells, and vascular components were greater in 70-day-old hemicastrated rats than in intact rats of the same age. The total number of Leydig cells per testis in hemicastrated and intact rats was always the same at any age. Estimated Leydig cell volume in 70-day-old rats was twice that in intact rats. By contrast, the testes of 50- and 60-day-old rats at the time of death displayed essentially the same morphological features, regardless of whether animals were hemicastrated. The concentration of plasma testosterone was higher in 50-day-old controls than in hemicastrated rats. Seventy-day-old hemicastrated rats showed higher levels of plasma testosterone than controls. The level of plasma dihydrotestosterone in 60- and 70-day-old hemicastrated rats exceeded that in the controls. A significant increase in follicle-stimulating hormone was noted in 50- and 70-day-old hemicastrated rats compared to normal rats, while levels of luteinizing hormone were basically the same. The increase in Leydig cell volume, interstitial tissue volume, vascular component volume, and plasma testosterone level caused by hemicastration at 40 days of age differed from that at 20 and 30 days of age.     

Quantitative analysis of Sertoli cells in neonatally oestrogen-treated rats

On Day 1 of age rats were treated with 500 micrograms oestradiol benzoate. Oestrogen-treated rats had increased numbers of Sertoli cells per reference area or volume, whereas the total number of cells per testis was unchanged. The mean nuclear size was significantly smaller in oestrogen-treated rats than in control rats, at 22 and 45 days of age. The volume density of the heterochromatin clumps decreased from 22 to 45 days of age in control rats (68% fall), the decrease being slower in oestrogenized animals (30% fall) during the same period. The differences were significant at 45 days of age only. The relative volume occupied by the nuclear membrane infoldings was significantly less in oestrogenized rats than in control ones at the two ages considered. Nucleolar development was delayed in oestrogen-treated rats, which had lower numbers of nuclear sections showing nucleoli, as well as a decrease in the nucleolar diameter. We suggest that these Sertoli cell alterations are due to the altered gonadotrophin and testosterone concentrations induced by the steroid treatment rather than to a direct effect of oestrogen.     

Ultrastructural morphometry of parathyroid cells in rats of different ages

Summary Parathyroid glands of newborn to 1-year-old male Sprague-Dawley rats were fixed by perfusion or by immersion, and prepared for electron microscopy. Parathyroid glands fixed by immersion exhibited parenchymal cells with variable ultrastructure, indicating that these cells were in different stages of the proposed secretory cycle in parathyroid cells. In contrast, parathyroid cells of glands fixed by perfusion were uniform in ultrastructure, suggesting that all cells were in the same stage of secretory activity. Parathyroid glands of 3-, 4-, 6-, 8- and 10-week-old rats also were fixed by perfusion and analysed by electron-microscopic morphometry. These data demonstrated an increase in cell volume and in surface area of the membrane compartments concerned with parathyroid hormone secretion: these changes were not related to variations in serum calcium concentration. Both the qualitative observations and the quantitative data do not favour the idea of a secretory cycle in rat parathyroid cells.     

Morphometry and elemental analysis of rat exocrine pancreas following administration of trypsin inhibitor

The morphological responses of the exocrine pancreas of the adult male rat to soybean trypsin inhibitor (STI) were studied by ultrastructural morphometry and electron probe X-ray microanalysis. STI administered orally in drinking water for 14 days resulted in a 72% increase in the wet weight of the pancreas. This enlargement was due, largely, to an increase in acinar cell mass. Volume increases in the acinar cell mass and extra-acinar cell compartment were 72 and 30%, respectively. The estimated total number of acinar cells in the mean exocrine pancreas was 500 million in the control and 630 million in the experimental group, representing an increase of 27%. Acinar cell volume was 1,790 microns 3 for the control and 2,457 microns 3 for the STI group. The pronounced morphometric changes of the organelles in the STI group were: the mean nucleolar volume increased by 56%; the volume of zymogen granular mass per cell increased by 93%; the volume of the Golgi complex and the condensing vacuoles per cell increased by 52 and 100%, respectively, whereas the membrane area of the Golgi complex and the condensing vacuoles increased by 98 and 47%, respectively. Spectral analysis of seven elements (Na, Mg, P, S, Cl, K and Ca) showed significant changes for nuclei, zymogen granules and mitochondria following STI: nuclei showed Na, P, K increased; zymogen granules showed Na, P, S, K increased, Cl decreased; mitochondrial particles showed Mg, P, Cl, Ca increased, and the mitochondrial matrix showed S decreased. The persistent uptake of STI probably resulted in a continual release of a trophic hormone acting on pancreatic tissue components, consequently causing hyperplasia and hypertrophy of the exocrine pancreas to accommodate a heightened demand for synthesis of exportable proteins.     

COMPARISON OF TETRAPLOID AND SINGLE GENE-INDUCED GIGAS VARIANTS IN CHICKPEA (CICER ARIETINUM). II. MORPHOLOGICAL AND ANATOMICAL LEAF CHARACTERS

Two genomic variants of a chickpea (Cicer arietinum L.) parental line have been developed which exhibit gigas characters. The two genotypes were the result of a single-gene mutation (gigas) and induced tetraploidy of a single parental line. The two genotypes plus parental strain were investigated to determine the similarity-of-effect of polyploidy and this single-gene mutation on leaf anatomy and morphology. Leaves consisted of two rows of alternatively arranged leaflets. Both the tetraploid and parental lines had the same mean number of leaflets per leaf while the gigas plants had fewer, but mean total leaf surface area was greater in the gigas plants. Quantitative comparison of mesophyll and vascular tissue and air space volume density (V_v) showed that leaves of the tetraploid plants had the greatest mesophyll cell density (V_vm) and least air space density. Mesophyll cell density was equal in the parental and single-gene mutant while parental leaves had the greatest vascular tissue density. The greater mesophyll cell density values of the polyploid were due to larger mean mesophyll cell size, not to greater cell numbers per unit area. Leaf models based on tissue density and leaflet size showed tetraploid plants had the greatest productivity potential per unit of leaflet surface area. However, if models were based on a whole leaf, gigas plants had the greatest productivity potential since they had larger total leaf area. The effectiveness of using structural models to predict physiological potential in plant tissues will be tested in future studies.     

Effect of water stress on cotton leaves : I. An electron microscopic stereological study of the palisade cells

Palisade cells from fully expanded leaves from irrigated and nonirrigated, field grown cotton (Gossypium hirsutum L. cv. Paymaster 266) were subjected to a microscopic examination to evaluate the effect of water stress on subcellular structures. The water potential difference between the two treatments was 13 bars at the time of sampling. The dimensions of the palisade cells and their density per unit leaf area were determined by light microscopy. Palisade cells from stressed plants had the same diameter, but were taller than their counterparts in irrigated plants. The density of the palisade cells was the same in both treatments as was the fractional volume of the intercellular space. It was concluded that the reduced leaf area observed in the stressed plants resulted primarily from a mitotic sensitivity to water stress. Further, expansion of palisade cells was not inhibited by the stress imposed in this study.

Morphometric analysis of electron micrographs was used to evaluate the subcellular structure of palisade cells from nonstressed and stressed plants. The fractional volumes of cell walls, total cytoplasm, chloroplasts, starch granules, intrachloroplast bodies, mitochondria, peroxisomes, and central vacuoles were determined. The surface densities of grana and stroma lamellae, outer chloroplast membranes, mitochondrial cristae, endoplasmic reticulum and Golgi cisternae were also measured. The number of chloroplasts, mitochondria, and peroxisomes were determined. These data were expressed as actual volumes, areas, and numbers per palisade cell for each treatment. Palisade cells from stressed plants had thinner cell walls, larger central vacuoles and approximately the same amount of cytoplasm compared to cells from nonstressed plants. Within the cytoplasm, stressed plants had more but smaller chloroplasts with increased grana and stroma lamellae surfaces, larger mithchondria with reduced cristae surfaces, smaller peroxisomes and reduced membrane surfaces of endoplasmic reticulum and Golgi cisternae.

     

Comparison of components of the testis interstitium with testosterone secretion in hamster, rat, and guinea pig testes perfused in vitro

Components of the testis and cytoplasmic organelles in Leydig cells were quantified with morphometric techniques in hamster, rat, and guinea pig. Testosterone secretory capacity per gram of testis and per Leydig cell in response to luteinizing hormone (LH) (100 ng/ml) stimulation was determined in these three species from testes perfused in vitro. Numerous correlations were measured among structures, and between structures and testosterone secretion, to provide structural evidence of intratesticular control of Leydig cell function. Testosterone secretion per gm testis and per Leydig cell was significantly different in the three species: highest in the guinea pig, intermediate in the rat, and lowest in the hamster. The volume of seminiferous tubules per gm testis was negatively correlated, and the volumes of interstitium, Leydig cells, and lymphatic space per gm testis were positively correlated with testosterone secretion. No correlations were observed between volumes of blood vessels, elongated spindleshaped cells, or macrophages per gm testes and testosterone secretion. The average volume of a Leydig cell and the volume and surface area of smooth endoplasmic reticulum (SER) and peroxisomes per Leydig cell were positively correlated, and the volume of lysosomes and surface area of inner mitochondrial membrane per Leydig cell were negatively correlated with testosterone secretion. No correlations were observed between volume and surface area of rough endoplasmic reticulum (RER), Golgi apparatus, and lipid, and volume of ribosomes, cytoplasmic matrix, and the nucleus with testosterone secretion per Leydig cell. These results suggest that Leydig cell size is more important than number of Leydig cells in explaining the difference in testosterone-secreting capacity among the three species, and that this increase in average volume of a Leydig cell is associated specifically with increased volume and surface area of SER and peroxisomes. An important unresolved question is what is the role of peroxisomes in Leydig cell steroidogenesis.     

Effect of insulin on lipolysis in adipose tissue of rats of different ages

Several authors have not been able to find any antilipolytic effect of insulin in adipose tissue "in vitro". We investigated the possible role of cell size and/or age of donors on this phenomenon. The lipolytic rates (glycerol release per cell) were lower in the small cells of the 4-6 weeks old rats than in the larger cells of the 25-30 weeks old animals; however, the difference disappeared when the data were expressed per unit of cell surface area. Insulin (0.5-50 ng/ml) failed to inhibit both maximally and submaximally noradrenaline stimulated lipolysis in the adipocytes of the young rats, but its antilipolytic action was fully restored by using glucose-free medium. Therefore, at our experimental conditions, a glucose dependent factor, possibly involving the preferential hydrolysis of newly synthetized triglycerides, seems to blunt or to mask the insulin induced inhibition of glycerol release. Relatively higher rates of glucose metabolism and a lower lipolysis in small fat cells might explain the difference in the action of insulin on glycerol release in the adipose tissue of young rats as compared to the older ones.     

A quantitative histological study of Golgi II neurons and pale cells in different cerebellar regions of the adult and ageing mouse brain

Two types of medium to large sized neurons are present in the granular layer of the mouse cerebellum. One type has a large nucleus with a prominent nucleolus and a moderate amount of cytoplasm containing Nissl substance. This type corresponds to the classical Golgi II neuron. The second type has a much smaller nucleus (mean diameter 8.4 microns) with a darkly staining nuclear envelope which is almost invariably deeply indented by cytoplasmic intrusions. The nucleolus is smaller and less conspicuous than in Golgi II neurons. These neurons are identical to the pale cells described by Altman and Bayer (1977). The numbers of both types of neuron were estimated in the spinocerebellum, lobus simplex and nodulus in mice aged 6, 15, 22, 25, 28 and 31 months. There was no significant variation in the number of either Golgi II neurons or pale cells with age in any part of the cerebellum. The number of Golgi II neurons per mm3 was similar in all parts of the cerebellum (mean 3560 mm3). This was identical to the mean number of pale cells per mm3 in the spinocerebellum and pontocerebellum but in the nodulus pale cells were much more numerous (mean 41,170 per mm3). It is postulated that pale cells are small Golgi II neurons.     

Lipoprotn lipase content and triglyceride fatty acid uptake in adipose tissue of rats of differing body weights

Increasing body weight appears to alter lipid metabolism in adipose tissue. We have measured the content of lipoprotein lipase and the uptake of chylomicron triglyceride fatty acids in epididymal fat pads of rats of different weights. In order that the results might be expressed in terms of cell numbers, the relationship between the weights of fat pads and the numbers and volumes of fat cells isolated from them was determined. Highly significant correlations were found between fat pad weight and both the number and the volume of the individual adipocytes. In rats weighing from 140 to 350 g, the increase in the size of fat pads was attributable almost equally to increases in cell size and in cell number. Lipoprotein lipase activity was measured in acetone powders of whole fat pads and of isolated fat cell preparations. With both, lipoprotein lipase activity per cell diminished significantly as the weight of fat tissue increased, i.e., larger fat cells contained less enzyme per cell than smaller cells. The uptake of triglyceride fatty acid radioactivity was measured after incubation of fat pads with radiolabeled rat lymph chylomicrons in flasks containing either buffer alone or with added glucose or glucose plus insulin. The addition of glucose and insulin led to a mean increase of 70% in the uptake of radioactivity, but larger adipocytes were stimulated less than smaller cells. This resulted in a significant negative correlation between the weights of fat pads and the uptake of radioactivity. Enlargement of fat cells also led to a diminution in their capacity to esterify fatty acids.     

Morphometric studies on lipoprotein particles in developing rat liver and their corticosteroid-induced changes during the late gestational period

Summary Changes in lipoprotein particles in hepatocytes of the fetal rat liver have been studied morphometrically from days 15–21 of gestation. On all these days, distinct lipoprotein particles are found within the cisternae of the RER, Golgi complexes and Golgi-derived secretory vesicles. Their mean diameter is 30–31 nm. The number of Golgi complexes per hepatocyte, the lipoprotein particle number per Golgi complex and the volume density of the latter remain unchanged within the developmental period examined. The volume density of lipid droplets, however, shows a significant decrease during this time.Following corticosteroid treatment, the mean diameter of lipoprotein particles, the number of lipoprotein particles per Golgi complex, the volume density of the Golgi complex, and that of the lipid droplets increase significantly within the examined period, whereas the number of Golgi complexes per hepatocyte is reduced. These data support the view that triglyceride production in the fetal liver is directly or indirectly stimulated by corticosteroids administered to the pregnant rat, thus giving rise to larger amounts of hepatic lipoproteins and lipids.Abbreviations LP lipoprotein - VLDL very low density lipoproteins (d< 1.006 g/ml) - RER rough endoplasmic reticulum - ER endoplasmic reticulum     

Ultrastructural analysis of a local regulation of Leydig cells in the adult monkey (Macaca fascicularis) and rat

Peritubular Leydig cells located in interstitial areas surrounded by tubules at nearly the same stage of spermatogenesis were analysed. Low-power electron micrographs were used for measurement of cell profile area and higher magnification views provided volume density of SER, Golgi stacks, mitochondria, and lipids. In the adult monkey, no cyclic changes were found in Leydig cells in their size or in the volume density of their organelles. In the adult rat (63 days of age), a comparison limited to stage VII-VIII and stage XI-XII peritubular Leydig cells demonstrated a significantly higher SER content (P less than 0.01) in the former, but no other differences. The study of subadult rats (45 days of age) showed that the full development of spermatogenesis was required to detect significant changes in Leydig cell SER content. The present results provide morphological evidence for an intratesticular control of the Leydig cells of the rat but not for those of the monkey.     

Effect of cell size on epinephrine- and ACTH-induced fatty acid release from isolated fat cells

Free fatty acid release from fat cells, obtained from epididymal adipose tissue of rats of different sizes, was found to be dependent on the cell surface area, regardless of the age of the animals. The same result was found with cells of different sizes from the same animal. These results, when related to in vivo conditions, would indicate that for the same adipose tissue mass, activity would decrease with increasing cell size. On the other hand, the total activity of a given tissue would increase by increasing the size of its cells.     

Quantitative morphological assessment of parathyroid activity in response to variations in calcium concentration in vitro

Isolated parathyroid glands from gerbils incubated at low (0.5 mM), normal (1.25 mM) or high (3 mM) calcium concentration for 1 or 6 h were studied with regard to the following stereological parameters: the volume densities of the nucleus, mitochondria, endoplasmic reticulum, Golgi zone, lipid bodies, lysosomes, secretory granules and storage granules, and the surface densities of the cell membrane and endoplasmic reticulum. In glands incubated for 1 h no significant differences were seen between the experimental groups in any of the parameters investigated, however, the volume density of the Golgi zone and the surface density of the cell membrane were significantly increased in glands incubated for 6 h at low calcium concentration, compared with those incubated at high calcium concentration. The enlargement of the Golgi zone in stimulated glands was also evident when the two glands from the same animal incubated at different calcium concentrations were compared. The data suggest that the early response of parathyroid glands to altered concentration of extracellular calcium includes functional activities associated with the Golgi zone.     

Somatostatin 14 affects the pituitary-ovarian axis in infant rats

The effects of multiple somatostatin (SRIH-14) treatment on the pituitary-ovarian axis were examined in infant rats. Female Wistar rats received subcutaneously two daily 20 μg/100g b.w. doses for five consecutive days (from 11 to 15 days of age). Changes in cell volume, volume density and number per unit area (mm2) of follicle-stimulating (FSH), luteinizing (LH) and somatotropic (GH) immunolabeled cells were evaluated by stereology and morphometry. Serum FSH and LH concentrations were determined by RIA. Ovaries were analyzed by simple point counting of follicles. SRIH-14 treatment significantly reduced FSH and LH cell volume, while their volume density and number per unit area were unaltered. Serum concentrations of FSH and LH were significantly reduced. Volume and volume density of GH cells was significantly decreased after SRIH-14 treatment, while their number per unit area was unaltered. In the ovary, SRIH-14 induced a significant increase in the percentage of primordial follicles followed by a significant decrease in percentage of primary follicles. The number of healthy and atretic preantral follicles was unchanged. It can be concluded that SRIH-14 treatment during the infantile period markedly inhibits pituitary FSH, LH and GH cells. In the ovary, SRIH-14 acts by inhibiting initial folliculogenesis without affecting atretic processes.