Endocrine Research
ISSN: 0743-5800 (Print) 1532-4206 (Online) Journal homepage: http://www.tandfonline.com/loi/ierc20
Alterations of Thyroid Parafollicular Cell Organization in Aged Rats Neonatally Treated with Estradiol Milka Sekulic & Mirjana Lovren To cite this article: Milka Sekulic & Mirjana Lovren (1992) Alterations of Thyroid Parafollicular Cell Organization in Aged Rats Neonatally Treated with Estradiol, Endocrine Research, 18:4, 291-306, DOI: 10.1080/07435809209111038 To link to this article: http://dx.doi.org/10.1080/07435809209111038
Published online: 12 May 2012.
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Date: 10 April 2016, At: 23:41
ENDOCRINE RESEARCH, 18(4), 291-306 (1992)
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ALTERATIONS OF THYROID PARAFOLLICULAR CELL ORGANIZATION IN AGED RATS NEONATALLY TREATED WITH ESTRADIOL Milka Sekuli6 and Mirjana Lovren Institute for Biological Research, Division of Cytology and Embryology, 1 1 0 6 0 Belgrade, 2 9 . Novembra 1 4 2 , Yugoslavia ABSTRACT Cellular and subcellular organization of parafollicular cells of aged male Wistar rats ( 1 8 0 - and 365-day-old), neonatally (3rd day) treated with a single dose ( 1 mg) of oestradiol-dipropionate (OeDP) , were studied. Based on argyrophyl properties of parafollicular cells in control and treated group, two morphometrically distinct cell types were observed (under and over 5 0 pm’ of cell area), reflecting distinct functional status of the same cell type. The first signs of hyperplastic changes in parafollicular cells were observed in 180-day-old r a t s and they were more severe in 365-day-old animals. Morphometric averages for the area of parafollicular cells and nuclei, as well as for the cell number in estradiol-treated animals were higher than in the controls, ‘but these differences were statistically insignificant. However, subcellular organization of parafollicular cells showed that besides the cells with clearly visible characteristics such as control hyperplastic cells, there were parafollicular cells with progressively more abnormalities expressed as myelin-like figures, cytoplasmic cribiriform structures and dilated endoplasmic reticulum (ER), demonstrating long-lasting effects of neonatally applied estradiol.
29 1 Copyright @ 1992 by Marcel Dekker, Inc,
292
SEKULIC AND LOVREN
INTRODUCTION
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The relationship between gonadal steroids and thyroid function was previously documented ( 1 , 2 ) , but the connection of sex steroids and parafollicular cell function is still unclear and poorly understood. Based on morphometric and ultrastructural analyses of parafollicular cells it was shown that estradiol applied during neonatal period affects these cells up to the adulthood, expressed as degranulation and hyperplasia ( 3 ) . The presence of sex hormone receptors in normal and pathological human thyroid tissue has been demonstrated ( 4 - 6 ) , although these receptors have not yet been found in parafollicular cells (7). In contrast , Greenberg et al. ( 8 ) reported that both estrogen and progesterone can stimulate i n v i t r o calcitonin secretion by a direct effect on thyroid C-cells of the rats. Several strains of rats ranging from 1 to 3 years of age exhibit progressively more severe C cell proliferative abnormalities including diffuse and nodular C cell hyperplasia and medullary thyroid carcinoma ( 9 - 1 2 ) . The incidence of C cell neoplasia in aged Long-Evans rats was significantly reduced in animals administered radiolabelled iodine ( 1 2 ) . After treatment of rats with antithyroid drugs the number of parafollicular cells was increased threefold and after a period of latency, the hyperplastic C cells developed parafollicular adenomas ( 1 3 ) . Kakudo et al. ( 1 4 ) claimed that the C cell population usually psoliferates in the thyroid of aged rats. Ljunberg and Nilsson (15) demonstrated hyperplastic and neoplastic changes both in ultimobranchial remnants and parafollicular cell system in bulls.
THYROID PARAFOLLICULAR CELLS
293
The aim of the present investigations was to find out whether neonatal estradiol treatment would express long-term effects on cellular and subcellular organization of thyroid parafollicular cells in the rat.
Downloaded by [RMIT University Library] at 23:41 10 April 2016
MATERIALS AND METHODS Male Wistar rats 180- and 365-day-old were used. On the 3rd day of life animals were 5.2. administered 1 mg OeDP dissolved in sterile olive oil. Controls were given vehicle alone. The animals were kept under standard laboratory conditions and each group consisted of 7 individuals. The thyroid glands were dissected out w i t h the trachea from the neck under ether anesthesia, fixed in Bouin’s fluid and embedded in paraffin. The paraffin blocks were serially cut into 5 pm-thick sections, stained with hematoxylin and eosin f o r routine examination and with double silver impregnation method to demonstrate argyrophyl cells ( 1 6 ) . For electron microscopic examinations pieces of the central part of thyroid lobes were immersed immediately and kept overnight in 4 % glutaraldehyde in Milloning buffer (pH 7.3), postfixed for 1 h in 1 % O s 0 4 in the same buffer, dehydrated in a graded ethanol series and embedded in Araldite. Ultrathin sections were counterstained with uranyl acetate and lead citrate and examined in a Siemens 1 0 1 EM. The sections prepared for light microscopy of each specimen were selected for morphometric evaluation. All silver positive cells with visible nuclei were measured at final magnification of picture 2099X. The areas of parafollicular cells and nuclei were outlined with
294
SEKULIt AND LOVREN
cursor on the digitizer tablet and measured by digital analyser (MOP-3, Carl Zeiss). Simultaneously, the number of parafollicular cells was determined. Thirty to 100 parafollicular cells with clearly outlined borders from each animal were used for analyses. The cells differed in size and the following morphometric parameters were determined: a. cell and nuclear maximum area in the cells over 50 pm’; b. cell and nuclear minimum c. average area of both area in the cells under 50 pm’; number of parafollicular cells and their nuclei and parafollicular cells per section. Average values and S.E.M. for parafollicular cell area and their number, as well as for nuclear area were calculated for each animal. Statistical analysis was performed using Student’s 1 test. Differences were considered significant when p < 0.05.
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a
c.
RESULTS Parafollicular Cells of Control Animals Two morphometrically distinct cell types of parafollicular cells: of small cell area (under 50 pm’) and large cell area (over 50 pm’) in 180- and 365-day-old control rats were recorded. The first signs of hyperplastic features in parafollicular cells were observed as pale small collections of eosinophilic cells between the follicular epithelium and follicular basement membrane in 180-day-old control animals. These light cell clusters were clearly visible in thyroid sections of 365-day-old rats. Staining with double silver impregnation method (17) for determination of argyrophil cells revealed parafollicular cells widely distributed into thyroid lobes with the
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THYROID PARAFOLLICULAR CELLS
295
FIGURE 1
Transversal section of thyroid gland from control 365-day-old rats. Note the clusters of smaller (sc) and larger (lc) parafollicular cells in the central part of the gland. (Pascual double silver nitrate impregnation method; X400).
highest concentration in the central part of each lobe (Fig. 1 ) . Morphometric averages for parafollicular cell number, their and nuclear area are listed in Table I. Electron microscopic examinations of the thyroid parafollicular cells in these two age groups revealed the cells with intensely dense specific granules or light cells with pale vesicles and osmiophobic secretory granules of low density (Fig. 2). In hyperplastic parafollicular cells especially in 365-day-old rats, well developed Golgi regions, numerous cytoplasmic po-
S E K U L I ~AND LOVREN
296
TABLE I. Average values of morphometric parameters of the rat thyroid parafollicular cells. Control (180 d)
Experimental group OeDP-treated Control (365 d) (180 d)
OeDP-treated (365 d) 37,0021 .OO
fin.
33.6921.48
35.2021 -52
35-94:
hhx.
67.7024.60
71.1824.30
65.862 2.29
66.0021.75
Min.
12.4920.33
13.0920.39
13.94:
0.41
14.20z0.36
b@x.
15.2021 .OO
16.3020.77
16.092 0.50
16.5920.35
0.91
1.
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~
. i
L.
~~
3.
50.6923.04
53.1922.91
50.902 1.60
51 .SO21 -37
4.
13.8420.66
14.69T0.58
15.002 0.45
15.39z0.35
5.
14.3323.37
19.25?3.93
22.33:
27.7925.68
1 -Cell area (pm');
5.77
2 - Nuclear area (pm');
3
- Ave-
rage cell area (pm'); 4 - Average nuclear area (pm'); 5 - Average number of parafollicular cells per section. Values +- S.E.M. obtained by measurement of 30-100 parafollicular cells per each animal are given. The groups consisted of seven animals. The differences were not statistically significant (p > 0.05).
lyribosomes and ribosomes connected to parallel row of ER were seen. Parafollicular Cells of Rats Neonatally Treated with OeDP Similar to the controls, in thyroid preparations of animals neonatally treated with OeDP small and l a r -
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THYROID PAMFOLLICULAR CELLS
297
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290
S E K U L I ~AND LOVREN
ge parafollicular cells were observed. Based on light microscopic analyses and argyrophilic features of parafollicular cells, no visible differences were seen as compared to control rats. Clear clusters of parafollicular cells with light cytoplasm were noticed. Morphometric averages for both estradiol-treated age groups ( 1 8 0 - and 365-day-old) are given in Table I. As seen, the values for the areas of both the cells and the nuclei were higher than in the controls, but these differences were statistically insignificant. The mean values for total area of parafollicular cells and nuclei were also higher in OeDP-treated groups in comparison with the controls, but again the difference was statistically insignificant ( p > 0.05). The average number of parafollicular cells exceeded that of the controls by 3 4 % and 2 4 % for 1 8 0 - and 365-day-old rats, respectively. However, despite this general similarity between normal and hyperplastic parafollicular cells, several differences could also be noted at the ultrastructural level. Byperplastic parafollicular cells with light, large, oval nuclei and electron-transparent cytoplasm w i t h scattered organelles and parafollicular cells with dark nuclei of irregular shape and dark cytoplasm with long cytoplasmic projections were observed (Fig. 3 ) . Also, myelin-like figures, cribiriform structures and distorted mitochondria in the cytoplasm were seen occasionally (Fig. 4a). Rarely, desmosomes between hyperplastic parafollicular cells and well developed Golgi region were observed (Fig. 4b). ER was frequently dilated. The basal lamina around parafollicular and follicular cells was often thickened. Follicular cells overlying the hyperplastic parafollicular cells were filled
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THYROID PARAFOLLICULAR CELLS
299
FIGURE 3
Parafollicular cells in close contact with follicular cells (FC) in thyroid gland of 365-day-old rat neonatally treated with estradiol. Note parafollicular cells with electron-transparent cytoplasm with scattered organelles (PC,) and parafollicular cell with dark cytoplasm, irregular shape and long cytoplasmic projections (PCz) (X15750).
with dilated ER and sometimes contained cytoplasmic crystalloid forms (Fig. 4 c ) . DISCUSSION
Two morphometrically, but not structurally
dis-
tinct cell types of parafollicular cells in the thyroid gland sections of aged rats were observed (cell area under and over 50 pm’). However, these differences in
SEKULIC AND LOVREN
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300
F I G U R E 4a
Cribiriform structures (arrows), multilamellar body (Mb), distorted mitochondria (M) and pale specific granules (sg) in cytoplasm of parafollicular cells in one-year-old rat neonatally treated with OeDP (X16380).
cell size did not reflect the existence of two distinct cell types, but rather distinct functional stages of the same cell type. During the present work, we recorded an age-related increase in the number of parafollicular cells which was higher for 55% in 3 6 5 - than in 180-day-old animals. These results could be connected to the data of several authors who reported spontaneous parafollicular cell hyperplasia in aged rats (9,lO). DeLellis et al. ( 1 1 ) described these changes of parafollicular cells in 9-12-month-old Long-Evans rats as mild diffuse C-cell hyperplasia accompanied by eleva-
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THYROID PARAFOLLICULAR CELLS
301
FIGURE 4b
Clearly visible desmosomes (De) between adjacent hyperplastic parafollicular cells, well developed Golgi region ( G o ) with newly-formed specific granules (dg) (X45000).
ted calcitonin level. It was shown also that focal or diffuse hyperplasia often precedes the development of C-cell neoplasms ( 1 7 ) . Our results demonstrate that the first signs of hyperplastic changes in parafollicular cells occur in 6-month-old Wistar rats and the appearance of hyperplastic parafollicular cells probably depends on the species of animal and on i t s genetic determinants. The mean value of parafollicular cells area in 180-day-old control rats was greater by 71% and i n 365-day-old animals by 69% as compared to 80-day-old rats
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FIGURE 4c Crystalloid forms (arrow) in follicular cell (FC) which are in close contact with adjacent parafollicular cell (PC) in the same animal as in Fig. 4a (X30400).
(3). Similar to that, the mean values for nuclear area exceeded those in 80-day-old group for 52% and 6 4 % in 1 8 0 - and 365-day-old rats, respectively ( 3 ) . It was previously shown that a large dose of female gonadal steroids administered during the critical phase of neonatal period of the rat development ( the 3rd day of life) led to a constant change in the specific brain neurons and interneuronal connections, i.5. such a treatment permanently affected neuroendocrine system ( 1 8 1 , including thyroid gland ( 1 - 3 , 2 0 ) . Taking into consideration normal appearance of hyperplastic cells in 1 8 0 - and 365-day-old animals, it
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THYROID PARAFOLLICULAR CELLS
was of interest to learn whether neonatal treatment with OeDP expresses any effect on parafollicular cells in these age groups of rats. The results of morphometric analyses of parafollicular cells in aged rats neonatally treated with OeDP showed at ultrastructural level that besides the cells with clearly visible characteristics of control cells, there were the cells with progressively more severe abnormalities such as myelin-like figures, cribiriform structures, cytoplasmic distorted mitochondria and dilated ER, thus indicating a mild diffuse and nodular C-cell hyperplasia as suggested by DeLellis et al. (11). Estrogen receptors were found in neoplastic thyroid lesions and their content was significantly higher than in normal human thyroid tissue ( 4 , 6 ) . Imai et al. ( 1 9 ) located endogenous estradiol in human thyroid cancer cells and the presence of estrogen receptors in this tissue suggested the possibility for the direct action of these hormones on the thyroid. However, neither of these authors discussed which thyroid tissue cell type was sex steroid responsive. This prompted us to examine possible long-lasting effects of neonatally administered OeDP at cellular and subcellular level of thyroid parafollicular cells in rats. As reported earlier, neonatally applied OeDP expressed long-term effects on rat thyroid follicular and mast cells ( 1 , 2 , 2 0 ) , what corroborates the reports on the presence of specific steroid receptors in thyroid tissue ( 4 - 6 ) . Our recent data ( 3 ) on morphological changes in adult rat parafollicular cells upon neonatal application of O e D P , together with the results of other authors demonstrating the presence of specific sex steroid receptors in neoplastic thyroid tissue ( 4 - 6 , 1 9 , 2 0 )
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suggested that estradiol could express certain long-term effects on parafollicular cells, as well. In the present paper we showed that in spite of morphometric similarity to control parafollicular cells, these cells of the aged rats neonatally treated with OeDP expressed the signs of degenerative changes which were more advanced t h a n age-related changes of the controls. At the moment we are not able to explain the exact mechanism(s) by which estrogens act on parafollicular cells during so prolonged periods upon their application and further examinations along these lines are in progress. ACKNOWLEDGEMENTS
We are very thankful to Dr Jelena Joksimovic of the Institute for Biological Research, Belgrade, for her continuous interest in this work and help during the preparation of the manuscript. This work was supported by Research Science Funds of Serbia, Grant No. 8 000-11. REFERENCES 1. Sekulij M, Panti6 V, Djurdjevij Dj, Mili&evi& Z . 1 9 8 0 Long lasting effect of estradiol on t h e struc-
ture of the thyroid and the level of thyroid hormones in the circulation. Vet Glasnik 34:443-449. 2. Sekuli5 M. 1 9 8 6 The thyroid glands of rats and pigs neonatally treated with gonadal steroids. Acta Vet 36~235-251. 3 . Sekulij M, Lovren M. 1 9 9 1 Changes in parafollicular
cells of rats neonatally treated with estradiol. J. Endocrinol Invest 14:577-582. 4.
Marugo M, Torre G, Bernasconi D, Fazzuoli L, Berta S , Giordano G. 1 9 8 9 Thyroid and steroid receptors J Endocrinol Invest 12:565-570.
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5. Marugo M, Torre G, Bernasconi D, Fazzuoli L, Cassulo S , Giordano G. 1 9 9 1 Androgen receptors in normal and pathological thyroids. J Endocrinol Invest 14~31-35. 6.
Miki H, Oshimo K, Inoue H, Morimoto T, Monden Y. 1 9 9 0 Sex hormone receptors in human thyroid tissues. Cancer 6 6 : 1 759-1 7 6 2
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I
7.
Weaker FJ, Herbert DC, Sheridan PJ. 1 9 8 6 Do C cells of the thyroid gland of the baboon contain estrogen receptors. Acta Anat 125:213-216.
8.
Greenberg C, Kukreja SC, Rowser EN, Hargis G K , Henderson WJ, Williams GA. 1 9 8 6 Effects of estradiol and progesterone on calcitonin secretion. Endocrinology 1 1 8 2 2 5 9 4 - 2 5 9 8 .
9. Boorman GA, van Noord MJ, Hollander CF. 1 9 7 2 Naturally occuring medullary thyroid carcinoma in the rat. Arch Path 94:35-41. 10. Boorman GA, Hollander CF. 1 9 7 6 Medullary carcinoma of the thyroid in the rat. Am J Path 83:237-240.
11. DeLellis R A , Nunnemacher G, Bitman WR, Gage1 RF, Tashjian AH, Blount M , Wolfe HJ. 1 9 7 9 C-cell hyperplasia and medullary thyroid carcinoma in the rat. Lab Invest 40: 1 40-1 54. 1 2 . Ott RA, Hofman C, Oslapas R, Nayyar R, Paloyan E. 1 9 8 7 Radioiodine sensitivity of parafollicular C cells in aged Long-Evans rats. Surgery 102:10431048.
13. Stoll R, Faucounau N, Maraud R. 1 9 7 8 Development of follicular and parafollicular adenomas in the thyroid of rats treated with thiamazole. Annales Endocrinol 39:179-189. 14.
Kakudo K, Itoh J, Takekoshi S, Watanabe K. 1 9 8 9 E f fects of synthetic salmon calcitonin on C cells of the thyroid. Acta Pathol Jpn 39:545-550.
15. Ljungberg 0, Nilsson PO. 1 9 8 5 Hyperplastic and neoplastic changes in ultimobranchial remnants and in parafollicular (C) cells in bulls: A histologic and immunohistochemical study. Vet Pathol 22:95-103.
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16. Pascual JSF. 1976 A new method for easy demonstration of argyrophyl cells. Stain Techno1 51 :231-235. 17. Capen CC, Martin SL. 1989 The effects of xenobiotics on the structure and function of thyroid follicular and C-cells. Tox Pathol 17:266-293.
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18. Panti/ V. 1981 Sensitivity of pituitary gonadotropic cell and gonads to hormones. In: Reproductive processes and contraception. Plenum Publishing Corporation pp.47-89. 19. Imai Y, Yamakawa M, Matsuda M, Kasajima T. 1989 Endogenous sex hormone and estrogen binding activity in thyroid cancer. Histol Histopathol 4:39-45. 20. Sekulij M. 1988 Ultrastructure of thyroid follicular and mast cells of rats neonatally treated with oestradiol. Iugoslav Physiol Pharmacol Acta 24: 25-32.
ISSN: 0743-5800 (Print) 1532-4206 (Online) Journal homepage: http://www.tandfonline.com/loi/ierc20
Alterations of Thyroid Parafollicular Cell Organization in Aged Rats Neonatally Treated with Estradiol Milka Sekulic & Mirjana Lovren To cite this article: Milka Sekulic & Mirjana Lovren (1992) Alterations of Thyroid Parafollicular Cell Organization in Aged Rats Neonatally Treated with Estradiol, Endocrine Research, 18:4, 291-306, DOI: 10.1080/07435809209111038 To link to this article: http://dx.doi.org/10.1080/07435809209111038
Published online: 12 May 2012.
Submit your article to this journal
Article views: 1
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ierc20 Download by: [RMIT University Library]
Date: 10 April 2016, At: 23:41
ENDOCRINE RESEARCH, 18(4), 291-306 (1992)
Downloaded by [RMIT University Library] at 23:41 10 April 2016
ALTERATIONS OF THYROID PARAFOLLICULAR CELL ORGANIZATION IN AGED RATS NEONATALLY TREATED WITH ESTRADIOL Milka Sekuli6 and Mirjana Lovren Institute for Biological Research, Division of Cytology and Embryology, 1 1 0 6 0 Belgrade, 2 9 . Novembra 1 4 2 , Yugoslavia ABSTRACT Cellular and subcellular organization of parafollicular cells of aged male Wistar rats ( 1 8 0 - and 365-day-old), neonatally (3rd day) treated with a single dose ( 1 mg) of oestradiol-dipropionate (OeDP) , were studied. Based on argyrophyl properties of parafollicular cells in control and treated group, two morphometrically distinct cell types were observed (under and over 5 0 pm’ of cell area), reflecting distinct functional status of the same cell type. The first signs of hyperplastic changes in parafollicular cells were observed in 180-day-old r a t s and they were more severe in 365-day-old animals. Morphometric averages for the area of parafollicular cells and nuclei, as well as for the cell number in estradiol-treated animals were higher than in the controls, ‘but these differences were statistically insignificant. However, subcellular organization of parafollicular cells showed that besides the cells with clearly visible characteristics such as control hyperplastic cells, there were parafollicular cells with progressively more abnormalities expressed as myelin-like figures, cytoplasmic cribiriform structures and dilated endoplasmic reticulum (ER), demonstrating long-lasting effects of neonatally applied estradiol.
29 1 Copyright @ 1992 by Marcel Dekker, Inc,
292
SEKULIC AND LOVREN
INTRODUCTION
Downloaded by [RMIT University Library] at 23:41 10 April 2016
The relationship between gonadal steroids and thyroid function was previously documented ( 1 , 2 ) , but the connection of sex steroids and parafollicular cell function is still unclear and poorly understood. Based on morphometric and ultrastructural analyses of parafollicular cells it was shown that estradiol applied during neonatal period affects these cells up to the adulthood, expressed as degranulation and hyperplasia ( 3 ) . The presence of sex hormone receptors in normal and pathological human thyroid tissue has been demonstrated ( 4 - 6 ) , although these receptors have not yet been found in parafollicular cells (7). In contrast , Greenberg et al. ( 8 ) reported that both estrogen and progesterone can stimulate i n v i t r o calcitonin secretion by a direct effect on thyroid C-cells of the rats. Several strains of rats ranging from 1 to 3 years of age exhibit progressively more severe C cell proliferative abnormalities including diffuse and nodular C cell hyperplasia and medullary thyroid carcinoma ( 9 - 1 2 ) . The incidence of C cell neoplasia in aged Long-Evans rats was significantly reduced in animals administered radiolabelled iodine ( 1 2 ) . After treatment of rats with antithyroid drugs the number of parafollicular cells was increased threefold and after a period of latency, the hyperplastic C cells developed parafollicular adenomas ( 1 3 ) . Kakudo et al. ( 1 4 ) claimed that the C cell population usually psoliferates in the thyroid of aged rats. Ljunberg and Nilsson (15) demonstrated hyperplastic and neoplastic changes both in ultimobranchial remnants and parafollicular cell system in bulls.
THYROID PARAFOLLICULAR CELLS
293
The aim of the present investigations was to find out whether neonatal estradiol treatment would express long-term effects on cellular and subcellular organization of thyroid parafollicular cells in the rat.
Downloaded by [RMIT University Library] at 23:41 10 April 2016
MATERIALS AND METHODS Male Wistar rats 180- and 365-day-old were used. On the 3rd day of life animals were 5.2. administered 1 mg OeDP dissolved in sterile olive oil. Controls were given vehicle alone. The animals were kept under standard laboratory conditions and each group consisted of 7 individuals. The thyroid glands were dissected out w i t h the trachea from the neck under ether anesthesia, fixed in Bouin’s fluid and embedded in paraffin. The paraffin blocks were serially cut into 5 pm-thick sections, stained with hematoxylin and eosin f o r routine examination and with double silver impregnation method to demonstrate argyrophyl cells ( 1 6 ) . For electron microscopic examinations pieces of the central part of thyroid lobes were immersed immediately and kept overnight in 4 % glutaraldehyde in Milloning buffer (pH 7.3), postfixed for 1 h in 1 % O s 0 4 in the same buffer, dehydrated in a graded ethanol series and embedded in Araldite. Ultrathin sections were counterstained with uranyl acetate and lead citrate and examined in a Siemens 1 0 1 EM. The sections prepared for light microscopy of each specimen were selected for morphometric evaluation. All silver positive cells with visible nuclei were measured at final magnification of picture 2099X. The areas of parafollicular cells and nuclei were outlined with
294
SEKULIt AND LOVREN
cursor on the digitizer tablet and measured by digital analyser (MOP-3, Carl Zeiss). Simultaneously, the number of parafollicular cells was determined. Thirty to 100 parafollicular cells with clearly outlined borders from each animal were used for analyses. The cells differed in size and the following morphometric parameters were determined: a. cell and nuclear maximum area in the cells over 50 pm’; b. cell and nuclear minimum c. average area of both area in the cells under 50 pm’; number of parafollicular cells and their nuclei and parafollicular cells per section. Average values and S.E.M. for parafollicular cell area and their number, as well as for nuclear area were calculated for each animal. Statistical analysis was performed using Student’s 1 test. Differences were considered significant when p < 0.05.
Downloaded by [RMIT University Library] at 23:41 10 April 2016
a
c.
RESULTS Parafollicular Cells of Control Animals Two morphometrically distinct cell types of parafollicular cells: of small cell area (under 50 pm’) and large cell area (over 50 pm’) in 180- and 365-day-old control rats were recorded. The first signs of hyperplastic features in parafollicular cells were observed as pale small collections of eosinophilic cells between the follicular epithelium and follicular basement membrane in 180-day-old control animals. These light cell clusters were clearly visible in thyroid sections of 365-day-old rats. Staining with double silver impregnation method (17) for determination of argyrophil cells revealed parafollicular cells widely distributed into thyroid lobes with the
Downloaded by [RMIT University Library] at 23:41 10 April 2016
THYROID PARAFOLLICULAR CELLS
295
FIGURE 1
Transversal section of thyroid gland from control 365-day-old rats. Note the clusters of smaller (sc) and larger (lc) parafollicular cells in the central part of the gland. (Pascual double silver nitrate impregnation method; X400).
highest concentration in the central part of each lobe (Fig. 1 ) . Morphometric averages for parafollicular cell number, their and nuclear area are listed in Table I. Electron microscopic examinations of the thyroid parafollicular cells in these two age groups revealed the cells with intensely dense specific granules or light cells with pale vesicles and osmiophobic secretory granules of low density (Fig. 2). In hyperplastic parafollicular cells especially in 365-day-old rats, well developed Golgi regions, numerous cytoplasmic po-
S E K U L I ~AND LOVREN
296
TABLE I. Average values of morphometric parameters of the rat thyroid parafollicular cells. Control (180 d)
Experimental group OeDP-treated Control (365 d) (180 d)
OeDP-treated (365 d) 37,0021 .OO
fin.
33.6921.48
35.2021 -52
35-94:
hhx.
67.7024.60
71.1824.30
65.862 2.29
66.0021.75
Min.
12.4920.33
13.0920.39
13.94:
0.41
14.20z0.36
b@x.
15.2021 .OO
16.3020.77
16.092 0.50
16.5920.35
0.91
1.
Downloaded by [RMIT University Library] at 23:41 10 April 2016
~
. i
L.
~~
3.
50.6923.04
53.1922.91
50.902 1.60
51 .SO21 -37
4.
13.8420.66
14.69T0.58
15.002 0.45
15.39z0.35
5.
14.3323.37
19.25?3.93
22.33:
27.7925.68
1 -Cell area (pm');
5.77
2 - Nuclear area (pm');
3
- Ave-
rage cell area (pm'); 4 - Average nuclear area (pm'); 5 - Average number of parafollicular cells per section. Values +- S.E.M. obtained by measurement of 30-100 parafollicular cells per each animal are given. The groups consisted of seven animals. The differences were not statistically significant (p > 0.05).
lyribosomes and ribosomes connected to parallel row of ER were seen. Parafollicular Cells of Rats Neonatally Treated with OeDP Similar to the controls, in thyroid preparations of animals neonatally treated with OeDP small and l a r -
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ge parafollicular cells were observed. Based on light microscopic analyses and argyrophilic features of parafollicular cells, no visible differences were seen as compared to control rats. Clear clusters of parafollicular cells with light cytoplasm were noticed. Morphometric averages for both estradiol-treated age groups ( 1 8 0 - and 365-day-old) are given in Table I. As seen, the values for the areas of both the cells and the nuclei were higher than in the controls, but these differences were statistically insignificant. The mean values for total area of parafollicular cells and nuclei were also higher in OeDP-treated groups in comparison with the controls, but again the difference was statistically insignificant ( p > 0.05). The average number of parafollicular cells exceeded that of the controls by 3 4 % and 2 4 % for 1 8 0 - and 365-day-old rats, respectively. However, despite this general similarity between normal and hyperplastic parafollicular cells, several differences could also be noted at the ultrastructural level. Byperplastic parafollicular cells with light, large, oval nuclei and electron-transparent cytoplasm w i t h scattered organelles and parafollicular cells with dark nuclei of irregular shape and dark cytoplasm with long cytoplasmic projections were observed (Fig. 3 ) . Also, myelin-like figures, cribiriform structures and distorted mitochondria in the cytoplasm were seen occasionally (Fig. 4a). Rarely, desmosomes between hyperplastic parafollicular cells and well developed Golgi region were observed (Fig. 4b). ER was frequently dilated. The basal lamina around parafollicular and follicular cells was often thickened. Follicular cells overlying the hyperplastic parafollicular cells were filled
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FIGURE 3
Parafollicular cells in close contact with follicular cells (FC) in thyroid gland of 365-day-old rat neonatally treated with estradiol. Note parafollicular cells with electron-transparent cytoplasm with scattered organelles (PC,) and parafollicular cell with dark cytoplasm, irregular shape and long cytoplasmic projections (PCz) (X15750).
with dilated ER and sometimes contained cytoplasmic crystalloid forms (Fig. 4 c ) . DISCUSSION
Two morphometrically, but not structurally
dis-
tinct cell types of parafollicular cells in the thyroid gland sections of aged rats were observed (cell area under and over 50 pm’). However, these differences in
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F I G U R E 4a
Cribiriform structures (arrows), multilamellar body (Mb), distorted mitochondria (M) and pale specific granules (sg) in cytoplasm of parafollicular cells in one-year-old rat neonatally treated with OeDP (X16380).
cell size did not reflect the existence of two distinct cell types, but rather distinct functional stages of the same cell type. During the present work, we recorded an age-related increase in the number of parafollicular cells which was higher for 55% in 3 6 5 - than in 180-day-old animals. These results could be connected to the data of several authors who reported spontaneous parafollicular cell hyperplasia in aged rats (9,lO). DeLellis et al. ( 1 1 ) described these changes of parafollicular cells in 9-12-month-old Long-Evans rats as mild diffuse C-cell hyperplasia accompanied by eleva-
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FIGURE 4b
Clearly visible desmosomes (De) between adjacent hyperplastic parafollicular cells, well developed Golgi region ( G o ) with newly-formed specific granules (dg) (X45000).
ted calcitonin level. It was shown also that focal or diffuse hyperplasia often precedes the development of C-cell neoplasms ( 1 7 ) . Our results demonstrate that the first signs of hyperplastic changes in parafollicular cells occur in 6-month-old Wistar rats and the appearance of hyperplastic parafollicular cells probably depends on the species of animal and on i t s genetic determinants. The mean value of parafollicular cells area in 180-day-old control rats was greater by 71% and i n 365-day-old animals by 69% as compared to 80-day-old rats
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FIGURE 4c Crystalloid forms (arrow) in follicular cell (FC) which are in close contact with adjacent parafollicular cell (PC) in the same animal as in Fig. 4a (X30400).
(3). Similar to that, the mean values for nuclear area exceeded those in 80-day-old group for 52% and 6 4 % in 1 8 0 - and 365-day-old rats, respectively ( 3 ) . It was previously shown that a large dose of female gonadal steroids administered during the critical phase of neonatal period of the rat development ( the 3rd day of life) led to a constant change in the specific brain neurons and interneuronal connections, i.5. such a treatment permanently affected neuroendocrine system ( 1 8 1 , including thyroid gland ( 1 - 3 , 2 0 ) . Taking into consideration normal appearance of hyperplastic cells in 1 8 0 - and 365-day-old animals, it
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was of interest to learn whether neonatal treatment with OeDP expresses any effect on parafollicular cells in these age groups of rats. The results of morphometric analyses of parafollicular cells in aged rats neonatally treated with OeDP showed at ultrastructural level that besides the cells with clearly visible characteristics of control cells, there were the cells with progressively more severe abnormalities such as myelin-like figures, cribiriform structures, cytoplasmic distorted mitochondria and dilated ER, thus indicating a mild diffuse and nodular C-cell hyperplasia as suggested by DeLellis et al. (11). Estrogen receptors were found in neoplastic thyroid lesions and their content was significantly higher than in normal human thyroid tissue ( 4 , 6 ) . Imai et al. ( 1 9 ) located endogenous estradiol in human thyroid cancer cells and the presence of estrogen receptors in this tissue suggested the possibility for the direct action of these hormones on the thyroid. However, neither of these authors discussed which thyroid tissue cell type was sex steroid responsive. This prompted us to examine possible long-lasting effects of neonatally administered OeDP at cellular and subcellular level of thyroid parafollicular cells in rats. As reported earlier, neonatally applied OeDP expressed long-term effects on rat thyroid follicular and mast cells ( 1 , 2 , 2 0 ) , what corroborates the reports on the presence of specific steroid receptors in thyroid tissue ( 4 - 6 ) . Our recent data ( 3 ) on morphological changes in adult rat parafollicular cells upon neonatal application of O e D P , together with the results of other authors demonstrating the presence of specific sex steroid receptors in neoplastic thyroid tissue ( 4 - 6 , 1 9 , 2 0 )
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suggested that estradiol could express certain long-term effects on parafollicular cells, as well. In the present paper we showed that in spite of morphometric similarity to control parafollicular cells, these cells of the aged rats neonatally treated with OeDP expressed the signs of degenerative changes which were more advanced t h a n age-related changes of the controls. At the moment we are not able to explain the exact mechanism(s) by which estrogens act on parafollicular cells during so prolonged periods upon their application and further examinations along these lines are in progress. ACKNOWLEDGEMENTS
We are very thankful to Dr Jelena Joksimovic of the Institute for Biological Research, Belgrade, for her continuous interest in this work and help during the preparation of the manuscript. This work was supported by Research Science Funds of Serbia, Grant No. 8 000-11. REFERENCES 1. Sekulij M, Panti6 V, Djurdjevij Dj, Mili&evi& Z . 1 9 8 0 Long lasting effect of estradiol on t h e struc-
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