Scanning Microscopy

Volume 7 Number 3 Article 11

9-24-1993

X-Ray Microanalysis of Chief Cells in Rat

Romuald Wroblewski Karolinska Institute

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Recommended Citation Wroblewski, Romuald (1993) "X-Ray Microanalysis of Chief Cells in Rat Parathyroid Gland," Scanning Microscopy: Vol. 7 : No. 3 , Article 11. Available at: https://digitalcommons.usu.edu/microscopy/vol7/iss3/11

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X-RAY MICROANALYSIS OF CHIEF CELLS IN RAT PARATHYROID GLAND

Romuald Wroblewski

Department of Pathology, Karolinska Institute / Hospital, S-17176 Stockholm, Sweden Telephone number: 46-8- 729 35 97 / FAX 46-8-34 58 20

(Received for publication July 20, 1993, and in revised form September 24, 1993)

Abstract Introduction

X-ray microanalysis was used to study the The parathyroid gland was the last major organ elemental composition of the rat parathyroid gland. in the human body to be discovered. Since its Analyses were performed in morphologically defined discovery by Sandstrom (1880) the parathyroid tissue cells. Use of semi-thin cryosections allowed us to has been extensively studied because of its central selectively analyze cell nucleus, cytoplasm and role in calcium regulation. In man, three types of occasionally dense granules in the cytoplasm. cells can be distinguished in the parathyroid gland - The concentration of elements (mmol/kg dry chief cells, oxyphilic cells and fat cells which appear weight) in chief cells of parathyroid gland were as first around the puberty (Munger and Roth 1963, follows: Na 83, Mg 48, P 853, S 224, Cl 164, K 551 Grimelius et al. 1981). The chief cells synthesize and and Ca 10. Significantly higher phosphorus and release (PTH), which potassium concentrations were found in the dense maintains calcium homeostasis in the body through granules as compared to cytoplasm of the parathyroid its effects on target tissues, bone and kidney. In chief cells. Choosing rat parathyroid gland gave the contrary to most other secretory cells, in parathyroid possibility to exclusively analyze chief cells. Data cells decreasing calcium concentration stimulates obtained from such a homogenous cell population secretion of PTH while increasing concentrations of will be of interest when studying human parathyroid extracellular calcium inhibit PTH secretion. The in which chief cells are mixed together with other cellular mechanism responsible for PTH secretion types of cells. has not been fully investigated but it is established that besides the changes in extracellular calcium also other cations regulate PTH release (Muff et al. 1988, Nygren et al. 1988, Brown et al. 1992, Pocotte et al. 1992). The oxyphilic cells (Castleman and Mallory 1935) are densely packed with mitochondria and produce small quantities of PTH, however, their precise role is still unknown. They appear in human parathyroid gland a few years before puberty. It has been suggested that oxyphilic cells arise from chief cells. In the rat, only chief cells are present (Rosof 1934, Engfeld t 1950). Rosof (1934) and Lever (1958) described dark and light cells within the chief cell population and suggested that differences represent different phases of secretory activity. Boquist and Lundgren (1975) found differences in morphology Key Words: Parathyroid gland, microanalysis, ionic and intracellular distribution of calcium in composition, chief cells, sodium, potassium, calcium, parathyroid cells. Whereas active chief cells of magnesium, chlorine, phosphorus, sulphur. normal parathyroid glands showed only sparse calcium-containing precipitations, this was more pronounced in stimulated chief cells of hyperplastic parathyroid gland (Boquist and Lundgren 1975). Morphological differences found within chief cells have been explained as a fixation artifact or as

875 Romuald Wr6blewski being due to differences in activity of the cells (Lever sections were stained with hematoxylin-eosin or 1958, Svensson et al. 1989). To my knowledge no periodic-acid Schiff's reagent (PAS) for light morphological studies have been performed on thin microscopical examination. cryosectioned parathyroid tissue which would reveal The cryosections used for X-ray microanalysis the closest to in vivo existing morphology. were examined in a JEOL 1200CX TEMSCAN electron X-ray microanalysis of the homogenous cell microscope equipped with a scanning attachment and population in rat parathyroid makes it possible to analyzed with a TRACOR 5500 energy dispersive X­ establish the typical elemental composition of the ray spectrometer system. chief cells. This may give information making it The specimens were examined in the scanning easier to analyze a more complex normal or transmission mode at accelerating voltage of 100 or abnormal human parathyroid gland. In humans, 120 kV. The total counting time (life-time) was 50 both in normal and in pathological conditions, in seconds. Quantitative analysis was carried out by addition to chief-, oxyphilic- and fat cells, several cell using gelatin standards containing known types which have characteristics of both chief and concentrations of mineral salts (Wroblewski et al. oxyphilic cells are present (Grimelius et al. 1981). The 1983). basic knowledge of chief cells elemental composition For statistical analysis of the data, Student's t­ is therefore of special value as it is known that in test was used. parathyroid cells extracellular ionic changes affect intracellular ionic composition and hormone release Results and Discussion (Muff et al. 1988, Pocotte et al. 1992). The aim of specimen preparation for all Scanning-transmission images of 2-4 µm thick methods of elemental analysis should be to retain the cryosections (Fig. la-b) displayed several sheets of in vivo elemental composition and distribution. This chief cells of the parathyroid gland. Loose connective can be best achieved by the use of cryo preparation tissue and capillaries containing erythrocytes were methods such as used in the present study. The use of present between the sheets of chief cells. A delicate serially cut cryosections has allowed us to correlate connective tissue capsule separated the parathyroid the elemental contents of single cells with enzyme from the adjacent tissue. Morphological and substrate histochemistry of the same cell structures observed by electron microscopy could be (Wroblewski et al. 1978, 1983, 1984). This type of correlated with the simultaneously projected light analysis is a useful pathophysiological tool, also microscopical image (Fig. 2a-b). Parathyroid cells because various pathological conditions affect exhibited minor differences in the density of the different cell types within the tissue differently cytoplasm. In most chief cells the large nucleus (Edstrom et al. 1979). X-ray microanalytical data occupied most of the cell, leaving a relatively small obtained from single cells are not influenced by the rim of cytoplasm. The nucleus often appeared more elemental content of the extracellular space, the size electron dense than the cytoplasm. As our specimen and content of which often are affected in tissues preparation technique did not include chemical undergoing pathological changes. fixation or impregnation with heavy metals, the The aim of the present study was to analyze by observed differences in the electron density were X-ray microanalysis the parathyroid chief cells of rats, directly related to the density of the organelles and to provide basic data on their elemental content in cytoplasm in vivo. Dense granules were identified in normal conditions. Such data are of great importance some cells. The size and density of these granules in work with pathological and experimental tissues varied only slightly and were more likely to depend and with the cells in culture. It is possible that the on freeze-drying procedure rather than on the elemental characteristics of chief cells will help us to original structure and metabolic state of the cell. It define the elemental content of the oxyphilic cells in was difficult to elucidate if the differences in the human parathyroid. cytoplasm density could be related to described morphological differences among chief cells (Rosof Material and Methods 1934, Lever 1958, Svensson et al. 1989) or were a result of our preparation technique, where small differences Thyroid glands with adjacent parathyroid in the section thickness in combination with the glands were quickly dissected out from five Sprague­ small size of the cells (overlying structures) might Dawley (200-250 g) adult rats of both sexes. The result in the density differences. samples were plunged into liquid Freon 22, cooled by X-ray microanalysis (XRMA) of cryosections liquid nitrogen. Samples were mounted such that including parathyroid and thyroid gland could be parathyroid and thyroid glands were cut performed separately on nuclei, cytoplasm and simultaneously. Sections (2-4µm thick) were cut on a occasionally also on dense granules (Fig. 3 a-e). The cryostat at -30°C. The sections were picked up on analytical results showed no systematic differences specially designed carbon specimen holders for X-ray between the chief cells with electron dense or microanalysis (Wroblewski et al. 1978, 1983). Adjacent electron lucid appearance within the same sheet. A

876 X-ray Microanalysis of Chief Cells in Rat Parathyroid Gland

( ~ V'.-..-~ I • ~ ..... ,

~ ~~,\ \ . ; ~ • If,

? ~ t . )f ~*-i. .. ·,.,. , • .• , ,

Fig. 2 a-b Light microscope micrographs of 4 µm cryosection of rat parathyroid and thyroid gland stained with hematoxylin-eosin. Parathyroid (PT) and thyroid (T) glands are clearly seen. In thyroid single layer of cuboidal follicular cells and a follicular lumen filled with colloid fluid (cf) are easily recognized. A connective tissue capsule separates the parathyroid gland from the thyroid gland (asterisk). staining.

typical X-ray spectrum from parathyroid chief cell is shown in Fig. 3a. It was characteristic for chief cells that peak heights for phosphorus and potassium and sulphur and chlorine respectively were of about the same size. The small dense granules displayed a certain variability in their elemental composition. Since the sections were rather thick (2-4 µm), the Fig. 1 a-b: Scanning-transmission electron analytical data on the dense granules contained micrographs of 4 µm thick cryosections of a rat contributions from the surrounding cytoplasm parathyroid gland. (a) Parathyroid chief cells are making analysis of chief cells dense granules less densely packed into groups. An extensive capillary reliable than analysis of dense granules from the network (arrows) and loose connective tissue thyroid follicular cells. It was evident that chief cell (asterisk) are present in the extracellular spaces granules had higher levels of all elements (besides separating the cords of chief cells. (b) Cell nuclei of sulphur), than the surrounding cytoplasm. In chief cells (N) occupy most of the cell volume. A comparison with the dense granules of follicular cells connective tissue capsule separates the parathyroid chief cells granules had a higher chlorine gland from the thyroid gland (asterisk). concentration and no iodine (Fig. 3 b-c).

877 Romuald Wr6blewsk.i

Analysis performed on the connective tissue between the chief cell sheets showed in addition to a. p K sodium, chlorine, phosphorus, and sulphur, a prominent potassium signal (Fig. 3d). The high phosphorus and potassium values in the connective tissues are most likely due to the inclusion of fibroblasts in the analyzed volume. Fig. 3e shows the elemental content of the follicular lumen (analyzed within the same section as in Fig. 2 a-b) with typical colloid elemental composition with high sulphur, potassium and 911017 .ol P,Ntl\<,toid eel I ' :>$ S.l2'J p iodine and low phosphorus content. b. Fig. 4 shows the elemental composition of K chief cells of parathyroid gland and cytoplasm of follicular cells of the thyroid. The fact that the content of some small dense granules was included in the measurements of the cytoplasm, should be taken into account. The high phosphorus level in the chief cells cytoplasm (853 mmol/kgdw) was probably partly due to the signals from the nuclei and to the high membrane density in the cytoplasm (Golgi apparatus and rough endoplasmic reticulum) of the chief cells. Higher phosphorus and potassium levels in chief l4S, 512 S.l@ 911017 63 PTHcell dg cells are also certainly due to the high phosphorus and potassium levels found in dense granules (Fig. C. 4). The differences in sulphur content between chief K cells and follicular cells depended on the presence of iodinated and non-iodinated thyroglobulin in the follicular cells (Wroblewski et al. 1991). A comparison of the chief cells of the parathyroid gland with the follicular cells of the thyroid gland showed that chief cells had significantly higher magnesium content. Magnesium has been shown to have effect on PTH secretion (Fiore et al. 1990). It was suggested that l4S, 512 S.Ial 0f 911017 ol T1'

Fig. 3 a-e: Characteristic X-ray spectra from different structures of from the same section including both parathyroid gland and thyroid gland. a: cytoplasm of chief cell, b: dense granule of chief cell, c: dense granule of follicular cell, d: connective tissue between 911017 ol l"Jl'Oid colloid cords of chief cells e: colloid of thyroid gland.

878 X-ray Microanalysis of Chief Cells in Rat Parathyroid Gland changes in the extracellular calcium concentrations dense granules. In the parathyroid cells, however, it (Nemeth et al. 1986, Pocotte et al. 1992) will not only was difficult to obtain more detailed information on regulate PTH secretion but also regulate PTH the elemental content of dense granules as the size of synthesis. Nemeth et al. (1986) found that changes in the parathyroid cells and their cytoplasm were relati­ the cytoplasmic calcium concentration are due to the vely small. Higher resolution of analysis (thinner sec­ release of calcium from the intracellular stores. It is tions) and a better morphological preservation are re­ unknown if calcium sensors on the chief cell plasma quired to obtain more accurate data on single granule membrane are coupled to calcium channels or basis than the method can provide in our study pumps. Therefore one can only speculate that where aggregates of granules were analyzed. This changes in the extracellular calcium may result also could be obtained, for example, by use of thinner sec­ in changes in other ions which might affect the tions from freeze-dried and low temperature embed­ secretion and synthesis of PTH. Jia et al. (1989) ded in Lowicryl material (Wroblewski 1989). described calcium activated potassium channels in Cytochemical and immunocytochemical methods the parathyroid cells. Such channels when activated may also be applied to material processed by low could cause ionic changes that might regulate the temperature embedding and may enable a further process of exocytosis. Changes in the extracellular characterization of the dense granules in the chief calcium were shown to affect numerous secondary cells of the parathyroid gland. messengers such as cAMP, inositol phosphates and The small size of the parathyroid glands was protein kinase C (Pocotte et al. 1992). one of the reasons why this organ was investigated Analysis of individual chief cells in semithin very late. For the same reason in rat, it is difficult to cryosections has allowed us to obtain results which dissect it prior to cryofixation out of the thyroid lobe. were unaffected by the content of the extracellular The location of the parathyroid gland is indicated by a space which varies markedly especially during patho­ slight difference in colour on the thyroid lobe surface. logical conditions (Wroblewski et al. 1978). The com­ Dissection could affect the morphology and induce position of the chief cells in the parathyroid gland elemental changes in the tissue. Therefore, and of the thyroid follicular cells could be analyzed cryofixation of entire rat thyroid lobe must be within the same cryosection. It was easy to analyze considered as optimal fixation method for both the thyroid follicular cells and the content of small elemental composition and morphology. Adjacent to the parathyroid, thyroid and muscle tissue might give additional information as well as serve as internal standards in normal and experimental conditions. Choosing rat parathyroid gland gave the mmol/kg dw possibility to measure characteristic elemental D Chief cells cytoplasm concentrations of the chief cells in normal 1()00 D Chief cells dense granule ■ Follicular cells cytoplasm conditions. These data will be of interest when studying the elemental content of chief cells under 800 . . different experimental conditions (such as hypo- and hypercalcemia) to find out if PTH secretion rate is 600 coupled to changes in levels of sodium, magnesium, chlorine and potassium. Changes in concentrations •• 400 of these elements may, in addition to the already described intracellular calcium changes be a factor that regulates PTH secretion and synthesis. The data will be also of interest in studies of normal and pathological human parathyroid in which chief cells are mixed together with other types of the cells. Na Mg p Cl K Ca

Fig. 4: The elemental composition of cytoplasm and Acknowledgments dense granule of parathyroid chief cells and follicular cells cytoplasm of thyroid gland. Absolute Supported by grants from the Swedish Medical concentrations (mmol/ kgd w) of sodium (Na), Research Council, the King Gustaf V Jubilee Fund magnesium (Mg), phosphorus (P), sulphur (S), and the Karolinska Institute Fund. The author is chlorine (Cl), potassium (K), calcium (Ca) are given. especially grateful to Mrs. M. Engstrom and I. May for Thin bars represent the standard error of mean of 50 their assistance. I would also like to thank prof. B.A. measurements. • denotes a statistically significant Afzelius, University of Stockholm, Department of difference (p <0.05) between cytoplasm and dense Ultrastructure Research, for letting me use analytical granules of parathyroid chief cells. equipment at his department.

879 Romuald Wr6blewski

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Editor's Note: All of the reviewer's concerns were appropriately addressed by text changes, hence there is no Discussion with Reviewers.

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