0013.7227/92/1311-0436$03.00/0 Endocrinology Copyright (c‘ 1992 by The Endocrine
Mitotic Glands
Vol. 131, No. 1 Printed IR U.S.A.
Society
Activity of the Endocrine during Postnatal Life
ESPERANZA CONDE, INES MARTIN-LACAVE, RICARDO GONZALEZ-CAMPORA, AND HUGO
Cells in Rat Thyroid
JOSE C. UTRILLA, GALERA-DAVIDSON
Departments of Cellular Biology (E.C.) and Normal and Pathological J.C.U., A.M., R.G.-C., H.G.-D.), University of Seville, Seville, Spain
ANA MORENO,
Cytology and Histology
(I.M.-L.,
ABSTRACT This paper presents the results of investigations into the mitotic rates of thyroid endocrine cells in normal postnatal rats, aged l-120 days. Our study revealed considerable age-dependent shifts in the mean mitotic activity of follicular cells and C-cells. The maximum indices of endocrine cell renewal were reached during the first 10 days of life, decreasing gradually and significantly until 25 days. At 1 month, there
was a significant recuperation of the division rate for both cell types, which declined in the adult rat. These results mean that the proliferation of C-cells and follicular cells is inversely proportional to age. The preferential zone of localization of mitoses of both cell types is the central region of the thyroid lobe. The present paper provides new evidence for the postnatal origin of C-cells and follicular cells from the preexisting endocrine cells. (Endocrinology 131: 436-440, 1992)
T
are derived from follicular cells (10, 25, 26). Kameda et al. (16) considered in adult dogs that undifferentiated cells were the common precursor cells for both C-cells and follicular cells. Recently, an intermediate type of human differentiated thyroidal carcinoma with features of both medullary and follicular carcinoma has been described (27). Harach (28) suggested that some C-cells might originate from the endodermally derived ultimobranchial cells. The aims of the present report were 1) to verify the origin of C-cells and follicular cells in the postnatal rat thyroid gland from preexisting endocrine cells, 2) to investigate the possible localization of preferential zones of cell renewal in the thyroid lobe, and 3) to determinate quantitatively the mitotic activity of both endocrine cell populations from birth to 4 months of age.
HE THYROID gland has a double endocrine component: 1) the follicular cells, representing the major proportion and forming the characteristic thyroid follicles that store thyroglobulin; and 2) the C-cells, which produce calcitonin and constitute the minor proportion (1). These last are arranged in a para- or interfollicular pattern and show a very heterogeneousdistribution in the thyroid lobe (1). Another structure occasionally found in the thyroid gland is the ultimobranchial follicle (UBF), considered to be an embryonal remnant of the ultimobranchial body (UBB) (210). The origin of the C-cells from the UBB during intrauterine life has been described in the rat (3, 4, lo), but there has been no clear evidence of a postnatal relationship between C-cells and the UBF (6, 7, 10, 11). This relationship has been described only in an unusual migration of UBF in a 5-dayold rat (12). There have been studies on the variation in the percentage of C-cells (13), but not on their postnatal origin in normal thyroids. Only Wechbanjong et al. (14) reported the presenceof a few mitosesof C-cells in very young mice. The follicular cells originate in the embryo mainly from the median thyroid primordium, and probably some also arise from the UBB (10, 15-21). This relationship is maintained in postnatal life, when mixed follicles and typical thyroid microfollicles from the UBF can be observed (10, 22). Follicular cell mitoses have also been described, but the mitotic rate of these cells has not been studied during postnatal evolution. Redmond and Tuffery (23, 24) studied the mitotic rate of follicular cells in untreated and goitrogentreated rats in adult life, but did not specify the agesof the animals. Due to the scarcity of data on renewal of C-cells during the postnatal period, it has even been suggested that they
Materials
and Methods
Forty-five albino Wistar rats were divided into groups of five animals each; a group was killed 0, 5, 10, 15, 20, 25, 30, 60, and 120 days after birth. Animals of both sexes were taken at random from different litters, weaned at the age of 40 days, and then fed identical diets. The thyroid glands were removed under ether anesthesia together with a portion of the adjoining trachea and esophagus. The tissue samples were fixed in Bouin’s solution at room temperature for 3 h, embedded routinely in paraffin wax, and serially sectioned at 5 pm. Thyroid glands were oriented perpendicular to the cut surface, sectioned from base to apex, and stained with hematoxylin-eosin for estimation of the length of each thyroid lobe. Five equidistant levels were then taken along the longitudinal axis of each lobe. Each cross-section of the gland was divided into four areas in accordance with an anteroposterior order: ventral or anterior (A), midventral (B), middorsal (C), and posterior or dorsal (D). Due to the heterogenous distribution of the C-cells, sections were analyzed from the three most central levels of the lobe, where the C-cell population is greatest (1). The slides were stained for calcitonin using the peroxidaseantiperoxidase method and then with the periodic acid-Schiff technique and counterstained with Harris’s hematoxylin. Given the different proportions of endocrine cells in rat thyroid, the C-cell mitotic index was estimated from analysis of a minimum of 10 consecutive transverse sections of each of the longitudinal levels chosen
Received November 11, 1991. Address all correspondence and requests for reprints to: Dr. Ines Martin-Lacave, Department of Normal and Pathological Cytology and Histology, Faculty of Medicine, University of Seville, Avda Dr. Fedriani S/N, 41009 Seville, Spain.
436
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MITOTIC TABLE
1. Variation
in the mitotic
C-cell
ACTIVITY
population
with
OF THE
THYROID
age No. of mitotic
No. of thyroid lobes
Age (days)
5 10 15 20 25 30 60 120
TABLE
different different
2. Variation
(P < 0.05) from preceding (P < 0.005) from preceding
in the mitotic
Maximum
198 198 264 165 165 216 216 180 180
5
’ Significantly b Significantly
No./transverse
No. of sections examined
0
follicular
with
33 50 46 19 6.2 4.4 8.2 3.5 2.3
No./transverse Maximum
90 90
(P < 0.005) from preceding (P < 0.05) from preceding
follicular
f c t + t + + + +
6 14” 10 3.9* 2.3 1.6 1.2” 0.9* 0.65”
cells
section
Mean f o/10” cells
Minimum
2.1 31.4 27.1 20.2 16.7 6.8 14.5 3.2 2.3
13 25 20 11 10 8 14 I 2
72 15 70 90 90 70 75
5 different different
Mean + o/10’ cells
Minimum
age
No. of sections examined
0
a Significantly b Significantly
section
2 3 5 3 3 3 3 3 2
No. of mitotic
5 10 15 20 25 30 60 120
C-cells
value in the same column. value in the same column.
cell population
No. of thyroid lobes
Age (days)
437
GLAND
+ f + + +k -+ f t
1.5 3.8” 4.1 3.5* 2.9 1.6” 1.3” 0.35” 0.92’
value in the same column. value in the same column. low incidence of C-cells in the normal thyroid of rats (l), the figures obtained were abundant enough to allow calculation of the mitotic rate for the different animals in the same age group. The total number of resting C-cells and follicular cells was estimated from the knowledge of their volumetric fraction (1) and their number per unit surface area. The mitotic index (mean + U) for both C- and follicular cells was calculated in relation to 10,000 resting cells. Correction for variations in mitotic activity due to circadian rhythm was unnecessary because all thyroid glands were taken at the same time of day. The sections were examined with a magnification of x400. The SEM was used throughout, and Student’s t test was used to test for statistical significance between mean values. P < 0.05 and P < 0.005 were considered significant.
Results 10
ok., 0
10
.,.,., 20
30
40
.,.,.,.,.,.,.,.,., 50
60
Age
70 (days)
FIG. 1. Diagram showing the evolution and follicular cells in the thyroid gland of mitotic figures in transverse sections kind of endocrine cell.
SO
SO
100
110
120
130
of the mitotic index of C-cells at different ages. The number is related to lo4 cells of each
in each thyroid lobe. However, for the follicular cells, from a minimum of 4 transverse sections from the same For assessing the mitotic activity, all mitotic figures of follicular cells in each transverse section were counted.
estimation was levels. the C-cells and In spite of the
The endocrine cells of the rat thyroid show great variation in the mitotic index throughout their postnatal development (Tables 1 and 2 and Fig. 1). The values for both cell types fitted a potential function of the type y = uxhthroughout the various age ranges. A fit by squared minima gives the expressions: y = 194.95(“-a92)for follicular cells and y = 285.91’“-‘~0R’ for C-cells, with correlation coefficients of r = 0.94 and r = 0.92, respectively. Theseresults show that the mitotic indexes of both cell types are approximately inversely proportional to age (the exponents differ from unity by ~10%). The quantitative values coincide with histological observations of the sections examined, in which the follicular cell density of the thyroid lobe decreases gradually, with a progressive
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438
MITOTIC
ACTIVITY
OF THE
THYROID
GLAND
Endo * 1992 Voll31. No 1
FIG. 2. These sections show C-cells in different phases of the mitotic cycle (arrows) surrounded by numerous resting follicular cells. a, Prophase of a C-cell in a thyroid gland of a lo-day-old rat. b, Metaphase of a C-cell in a lo-day-old rat. c, Thyroid section from a 30-day-old rat showing a C-cell in anaphase. d, Telophase of C-cell in a &day-old rat. Immunoperoxidase staining for calcitonin, periodic acid-Schiff, Harris hematoxylin counterstaining. Magnification, X1250.
FIG. 3. Sections of rat thyroid gland showing follicular cells in different phases of the mitotic cycle (arrows) surrounded by numerous follicular cells. a, Prophase of a 30-day-old rat. b, Metaphase of a lo-day-old rat. c, Anaphase of a 25-day-old rat. d, Telophase of a 25 day-old rat. Immunoperoxidase staining for calcitonin, periodic acid-Schiff method, Harris hematoxylin counterstaining. Magnification, X1250.
in the quantity of stored colloid, while the size of the follicular cells and C-cells increases. Cells have been found at all stages of the mitotic cycle (Figs. 2 and 3), although in both cell types the highest numbers were in prophase and metaphase. The approximate ratio of different mitotic phasesat all agesstudied was: seven prophase to five metaphase to one anaphase to three telophase for both cell types. The renewal of the follicular cells, with respect to the transverse axis of the thyroid lobe, is somewhat greater in the middle zones (B-C) of the section. At the same time, increase
there is a preferential localization of mitoses of the follicular cells in the central region of the different zones considered in each section, compared with the periphery, in a ratio of approximately 6:3. The preferential zones of localization of C-cells mitosesin each section are the middorsal (C), followed by the midventral (B), in an overall ratio of approximately 4C:3B:2D:lA. At the sametime, the C-cells undergoing division were localized preferentially in the central region of each section, compared with the periphery, in a ratio of approximately 7:3. There was not statistical significance when the values obtained in
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MITOTIC one thyroid lobe.
lobe were compared
to those found
ACTIVITY
OF THE
in the other
Discussion Our study has revealed considerable age-dependent shifts in the mean mitotic activity of the follicular cells and C-cells in the rat thyroid gland. The maximum mitotic index for endocrine cells was reached during the first 10 days of life; it decreased gradually and significantly until 25 days. At 1 month, there is a significant recuperation of the division rate for both cell types, which declines subsequently in the adult rat. Puberty occurs during this period (50-60 days), but in Wistar rats vaginal opening occurs at 35.6 days, and the testes descend during the period between 15-51 days of age (29). All of these changes are related to TSH levels, which increase at about the time of puberty and then decline and remain unchanged between 6 and 12 months of age (30). The mean volume of the thyroid gland increases during postnatal development, becoming stable in young adults (14, 31, 32). However, the volume fractions of the various histological components of the thyroid exhibit different patterns in the course of postnatal development. The follicular cell fraction shows a steady and considerable decrease with age (1, 13, 33), while the C-cell fraction increases slightly with age (1). These data coincide with the evolution of the mitotic index of both cell types. According to our results, the proliferation of C-cells and follicular cells is inversely proportional to age. Hence, as the division rate is lower with time, although new divisions are produced in absolute terms, these are insufficient to maintain the initial population density. For the follicular cells, this decrease is very obvious, as they are the major histological component of the thyroid gland, while for the C-cells, which comprise a very low proportion, the overall variations are quantitatively not very significant. The mitotic index reported in the present study is below 0.8% for both endocrine cell types, which was the value reported by Leblond (34) for the thyroid gland in rats weighing 200-250 g. This mitotic rate is just sufficient to bring about an increase in cell number proportional to the increase in body weight and, therefore, contributing only to growth. This is the primary definition of these cells as an expanding cell population. Redmond and Tuffery (23) recorded a mean mitotic rate of 7.6 + 0.83 metaphases/10-4. h for male Wistar rats of 220-271 g final body weight. Both research groups used a metaphase arrest agent (colchicine and vincristine sulfate, respectively), which was injected several hours before the animals were killed. During intrauterine life, the origin of C-cells has been described in the UBB (16, 21, 35). In the postnatal period, the UBF is believed to be the vestige of the UBB (2-7, 10) and is found in most thyroid glands if the search is meticulous (1, 36). The UBF is located in the site of maximum concentration of C-cells in the thyroid lobe. However, not all UBF are associated with C-cells, nor is a greater concentration of C-cells observed around them than around the typical thyroid follicles. Thus, it has not been possible to show a clear postnatal link between C-cells and UBF in normal rats. We
THYROID
439
GLAND
have reported recently the presence not only of resting Ccells, but also mitotic figures of C-cells, in the wall of an abnormally migrated UBF in a 5-day-old rat (12,37). It seems that during fetal development, the UBB is the origin of Ccells, but from birth, the remnant of the UBB, the UBF, has exhausted the capacity for differentiation into C-cells; from this time on, the C-cells are derived exclusively from the division of preexisting C-cells. Due to the scarcity, until now, of observations of C-cells in mitosis, some researchers were of the opinion that C-cells were derived from follicular cells (25, 26). According to our results, it seems clear that the C-cell population renewal is independent from the follicular cells in the rat thyroid gland after birth. The present paper provides new evidence for the postnatal origin of C-cells and follicular cells in the preexisting endocrine cells and expands our knowledge of the natural history of these endocrine cells. Acknowledgments The authors wish to express appreciation to Dr. R. 0. Weller critically reviewing the manuscript, and to Miss Ma Dolores Jimenez the excellent technical assistance.
for for
References I, Gonzrilez-Cimpora R, Galera-David1. Conde E, Martin-Lacave son H 1991 Histometry of normal thyroid glands in neonatal and adults rats, Am J Anat 191:384-390 MC 1937 Complex IV in the dog with special emphasis on 2. Godwin the relation of the ultimobranchial body to interfollicular cells in the postnatal thyroid gland. Am J Anat 60:299-340 K 1965 Electron microscopical studies of the ultimobran3. Ishikawa chial body of the rat in embryonic life, with special emphasis on its fate: the relation to the thyroid tissue and parafollicular cells. Okajima Folia Anat Jap 41:313-335 4. Stoeckel ME, Porte A 1970 Origine embryonnaire et differentiation secretoire des cellules a calcitonine (cellules C) dans la thyroide foetal du rat. Etude au microscope electronique. Z Zellforsch 106:251-268 5. Chan AS, Conen PE 1971 Ultrastructural observations on cytodifferentiation of parafollicular cells in the human fetal thyroid. Lab Invest 25:249-259 6. Wollman SH, Neve P 1971 Postnatal development and properties of ultimobranchial follicles in the rat thyroid. Anat Ret 171:247258 7. Wollman SH, Neve P 1971 Ultimobranchial follicles in the thyroid gland of rat and mice. Recent Prog Horm Res 27:213-234 8. Wollman SH, Hilfer SR 1978 Embryonic origin of various epithelial cell types in the thyroid gland of the rat. Anat Ret 189:467-478 9. Wollman SH, Hilfer SR 1978 Embryonic origin of various epithelial cell types in the second kind of thyroid follicle in the C,H mouse. Anat Ret 191:111-122 10. Calvert R 1972 Transitional cells in the postnatal thyroid gland of the rat. Anat Ret 174:341-360 11. Swarup K, Das VK 1978 Calcitonin cells and unusual follicle in four month old wistar rats. Z Mikrosk Anat Forsch 2:212-221 12. Martin-Lacave I, Conde E, Moreno AM, Utrilla JC, Galera-Davidson H, Evidence of the occurrence of calcitonin cells in the ultimobranchial follicle of the rat postnatal thyroid. Acta Anat, in press 13. Petko M, Rigo GY, Varga ZS 1976 Quantitative changes of the Ccell population in the rat thyroid during postnatal ontogenesis. Cell Tissue Res 166:541-552 14. Wechbanjong N, McMillan PJ, Dalgleish AE 1979 Influence of development on the number of calcitonin-containing cells in the
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MITOTIC
440 mouse 15.
16.
17.
18.
19.
20. 21.
22. 23.
24.
25.
thyroid.
Am J Anat
ACTIVITY
OF THE THYROID
154:477-484 mitotic activity of the parafollicular cells of the dog thyroid in experimentally induced hypercalcemia. Arch . _ Histol Jpn32:.179-192 _ Kameda Y. Shieemoto H. Ikeda A 1980 Develoument and cvtodifferentation of C”cel1 complexes in dog fetal thyroid. An immunohistochemical study using anti-calcitonin, anti-C thyroglobulin and anti-19s thyroglobulin antisera. Cell Tissue Res 206:403-415 Kameda Y, Ikeda K, Ikeda A 1981 Uptake of radioiodine in follicles of dog C-cell complexes studied by autoradiograph and immunoperoxidase stainning. Anat Ret 200:461-470 Kameda Y 1984 Immunohistochemical studies on differentiation of the thyroid gland in rabbit fetuses and chick embryos. Histochemistry 80:23-29 Kameda Y 1987 Immunohistochemical demonstration of keratins in the cysts of thyroid gland, parathyroid glands, and C-cell complexes of the dog. Am J Anat 180:87-99 Harach HR 1987 Mixed follicles of the human thyroid gland. Acta Anat 129:27-30 Williams ED, Toyn CE, Harach HR 1989 The ultimobranchial gland and congenital thyroid abnormalities in man. J Path01 159:135-141 Calvert R 1975 Structure of rat ultimobranchial bodies after birth. Anat Ret 181:561-580 Redmond 0, Tuffery AR 1979 Mitotic rate of thyroid follicular cells in untreated and goitrogen-treated rats: variation with time of day. J Anat 129:731-741 Redmond 0, Tuffery AR 1981 Thyroid proliferation, body weight, thyrotropin and thyroid hormones in chronic antithyroid (carbimazole) treatment in rats. J Anat 133:37-47 Stux M, Thompson B, Isler H, Leblond CP 1961 The “light cells” of the thyroid gland in the rat. Endocrinology 68:292-308
Kameda Y 1970 Increased
GLAND
Endo. 1992 Vol131 -No 1
26. Sarkar SK, Isler H 1963 Origin gland.
Endocrinology
of the “light
cells”
of the thyroid
73:199-204
27. Ljungberg
28. 29.
30.
31.
32. 33.
34. 35.
36. 37.
0, Bondeson L, Bondeson AG 1984 Differentiated thyroid carcinoma, intermediate type. A new tumor entity with features of follicular and parafollicular cell carcinoma. Hum Path01 15:218-228 Harach HR 1985 Thyroid follicles with acid mucins in man: a second kind of follicles?. Cell Tissue Res 242:211-215 Baker DEJ 1979 Reproduction and breeding. In: Baker HJ, Lindsey JR, Weisbroth SH (eds) The Laboratory Rat. Biology and Diseases. Academic Press, New York, vol 1:154-168 Klug TL, Adelman RC 1979 Altered hypothalamic pituitary regulation of thyrotropin in male rats during aging. Endocrinology 104:1136-1142 Wolfe HJ, DeLellis RA, Voelkel EF 1975 Distribution of calcitonincontaining cells in the normal neonatal human thyroid gland: a correlation of morphology with peptide content. J Clin Endocrinol Metab 41:1076-1081 Brown RA, Al-Mousa M, Beck JS 1986 Histometry of the normal thyroid in man. J Clin Path01 39:475-482 Malendowicz LK, Majchrzak M 1981 Sex dimorphism in the thyroid gland. III. Morphometric studies on the rat thyroid gland in the course of postnatal ontogenesis. Endokrinologie 77:297-302 Leblond CP 1964 Classification of cell populations on the basis of their proliferative activity. Nat1 Cancer Insi Monogr 14:119-149 Pearse AGE, Carvalheria AF 1967 Cvtochemical evidence for an ultimobranchial origin of rodent thyroid C cells, Nature (Lond) 214:929-930 Juhl M 1981 Morphologv of the second kind of follicle in the guinea pig thyroid gland: Acta-Anat 110:318-326 Conde E, Moreno AM, Martin-Lacave I, Fernindez A, GaleraDavidson H, Immunocytochemical studies of the ultimobranchial tubule
in Wistar
rats, Anat
Histol
Embryol,
in press
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Mitotic Glands
Vol. 131, No. 1 Printed IR U.S.A.
Society
Activity of the Endocrine during Postnatal Life
ESPERANZA CONDE, INES MARTIN-LACAVE, RICARDO GONZALEZ-CAMPORA, AND HUGO
Cells in Rat Thyroid
JOSE C. UTRILLA, GALERA-DAVIDSON
Departments of Cellular Biology (E.C.) and Normal and Pathological J.C.U., A.M., R.G.-C., H.G.-D.), University of Seville, Seville, Spain
ANA MORENO,
Cytology and Histology
(I.M.-L.,
ABSTRACT This paper presents the results of investigations into the mitotic rates of thyroid endocrine cells in normal postnatal rats, aged l-120 days. Our study revealed considerable age-dependent shifts in the mean mitotic activity of follicular cells and C-cells. The maximum indices of endocrine cell renewal were reached during the first 10 days of life, decreasing gradually and significantly until 25 days. At 1 month, there
was a significant recuperation of the division rate for both cell types, which declined in the adult rat. These results mean that the proliferation of C-cells and follicular cells is inversely proportional to age. The preferential zone of localization of mitoses of both cell types is the central region of the thyroid lobe. The present paper provides new evidence for the postnatal origin of C-cells and follicular cells from the preexisting endocrine cells. (Endocrinology 131: 436-440, 1992)
T
are derived from follicular cells (10, 25, 26). Kameda et al. (16) considered in adult dogs that undifferentiated cells were the common precursor cells for both C-cells and follicular cells. Recently, an intermediate type of human differentiated thyroidal carcinoma with features of both medullary and follicular carcinoma has been described (27). Harach (28) suggested that some C-cells might originate from the endodermally derived ultimobranchial cells. The aims of the present report were 1) to verify the origin of C-cells and follicular cells in the postnatal rat thyroid gland from preexisting endocrine cells, 2) to investigate the possible localization of preferential zones of cell renewal in the thyroid lobe, and 3) to determinate quantitatively the mitotic activity of both endocrine cell populations from birth to 4 months of age.
HE THYROID gland has a double endocrine component: 1) the follicular cells, representing the major proportion and forming the characteristic thyroid follicles that store thyroglobulin; and 2) the C-cells, which produce calcitonin and constitute the minor proportion (1). These last are arranged in a para- or interfollicular pattern and show a very heterogeneousdistribution in the thyroid lobe (1). Another structure occasionally found in the thyroid gland is the ultimobranchial follicle (UBF), considered to be an embryonal remnant of the ultimobranchial body (UBB) (210). The origin of the C-cells from the UBB during intrauterine life has been described in the rat (3, 4, lo), but there has been no clear evidence of a postnatal relationship between C-cells and the UBF (6, 7, 10, 11). This relationship has been described only in an unusual migration of UBF in a 5-dayold rat (12). There have been studies on the variation in the percentage of C-cells (13), but not on their postnatal origin in normal thyroids. Only Wechbanjong et al. (14) reported the presenceof a few mitosesof C-cells in very young mice. The follicular cells originate in the embryo mainly from the median thyroid primordium, and probably some also arise from the UBB (10, 15-21). This relationship is maintained in postnatal life, when mixed follicles and typical thyroid microfollicles from the UBF can be observed (10, 22). Follicular cell mitoses have also been described, but the mitotic rate of these cells has not been studied during postnatal evolution. Redmond and Tuffery (23, 24) studied the mitotic rate of follicular cells in untreated and goitrogentreated rats in adult life, but did not specify the agesof the animals. Due to the scarcity of data on renewal of C-cells during the postnatal period, it has even been suggested that they
Materials
and Methods
Forty-five albino Wistar rats were divided into groups of five animals each; a group was killed 0, 5, 10, 15, 20, 25, 30, 60, and 120 days after birth. Animals of both sexes were taken at random from different litters, weaned at the age of 40 days, and then fed identical diets. The thyroid glands were removed under ether anesthesia together with a portion of the adjoining trachea and esophagus. The tissue samples were fixed in Bouin’s solution at room temperature for 3 h, embedded routinely in paraffin wax, and serially sectioned at 5 pm. Thyroid glands were oriented perpendicular to the cut surface, sectioned from base to apex, and stained with hematoxylin-eosin for estimation of the length of each thyroid lobe. Five equidistant levels were then taken along the longitudinal axis of each lobe. Each cross-section of the gland was divided into four areas in accordance with an anteroposterior order: ventral or anterior (A), midventral (B), middorsal (C), and posterior or dorsal (D). Due to the heterogenous distribution of the C-cells, sections were analyzed from the three most central levels of the lobe, where the C-cell population is greatest (1). The slides were stained for calcitonin using the peroxidaseantiperoxidase method and then with the periodic acid-Schiff technique and counterstained with Harris’s hematoxylin. Given the different proportions of endocrine cells in rat thyroid, the C-cell mitotic index was estimated from analysis of a minimum of 10 consecutive transverse sections of each of the longitudinal levels chosen
Received November 11, 1991. Address all correspondence and requests for reprints to: Dr. Ines Martin-Lacave, Department of Normal and Pathological Cytology and Histology, Faculty of Medicine, University of Seville, Avda Dr. Fedriani S/N, 41009 Seville, Spain.
436
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 28 April 2016. at 13:59 For personal use only. No other uses without permission. . All rights reserved.
MITOTIC TABLE
1. Variation
in the mitotic
C-cell
ACTIVITY
population
with
OF THE
THYROID
age No. of mitotic
No. of thyroid lobes
Age (days)
5 10 15 20 25 30 60 120
TABLE
different different
2. Variation
(P < 0.05) from preceding (P < 0.005) from preceding
in the mitotic
Maximum
198 198 264 165 165 216 216 180 180
5
’ Significantly b Significantly
No./transverse
No. of sections examined
0
follicular
with
33 50 46 19 6.2 4.4 8.2 3.5 2.3
No./transverse Maximum
90 90
(P < 0.005) from preceding (P < 0.05) from preceding
follicular
f c t + t + + + +
6 14” 10 3.9* 2.3 1.6 1.2” 0.9* 0.65”
cells
section
Mean f o/10” cells
Minimum
2.1 31.4 27.1 20.2 16.7 6.8 14.5 3.2 2.3
13 25 20 11 10 8 14 I 2
72 15 70 90 90 70 75
5 different different
Mean + o/10’ cells
Minimum
age
No. of sections examined
0
a Significantly b Significantly
section
2 3 5 3 3 3 3 3 2
No. of mitotic
5 10 15 20 25 30 60 120
C-cells
value in the same column. value in the same column.
cell population
No. of thyroid lobes
Age (days)
437
GLAND
+ f + + +k -+ f t
1.5 3.8” 4.1 3.5* 2.9 1.6” 1.3” 0.35” 0.92’
value in the same column. value in the same column. low incidence of C-cells in the normal thyroid of rats (l), the figures obtained were abundant enough to allow calculation of the mitotic rate for the different animals in the same age group. The total number of resting C-cells and follicular cells was estimated from the knowledge of their volumetric fraction (1) and their number per unit surface area. The mitotic index (mean + U) for both C- and follicular cells was calculated in relation to 10,000 resting cells. Correction for variations in mitotic activity due to circadian rhythm was unnecessary because all thyroid glands were taken at the same time of day. The sections were examined with a magnification of x400. The SEM was used throughout, and Student’s t test was used to test for statistical significance between mean values. P < 0.05 and P < 0.005 were considered significant.
Results 10
ok., 0
10
.,.,., 20
30
40
.,.,.,.,.,.,.,.,., 50
60
Age
70 (days)
FIG. 1. Diagram showing the evolution and follicular cells in the thyroid gland of mitotic figures in transverse sections kind of endocrine cell.
SO
SO
100
110
120
130
of the mitotic index of C-cells at different ages. The number is related to lo4 cells of each
in each thyroid lobe. However, for the follicular cells, from a minimum of 4 transverse sections from the same For assessing the mitotic activity, all mitotic figures of follicular cells in each transverse section were counted.
estimation was levels. the C-cells and In spite of the
The endocrine cells of the rat thyroid show great variation in the mitotic index throughout their postnatal development (Tables 1 and 2 and Fig. 1). The values for both cell types fitted a potential function of the type y = uxhthroughout the various age ranges. A fit by squared minima gives the expressions: y = 194.95(“-a92)for follicular cells and y = 285.91’“-‘~0R’ for C-cells, with correlation coefficients of r = 0.94 and r = 0.92, respectively. Theseresults show that the mitotic indexes of both cell types are approximately inversely proportional to age (the exponents differ from unity by ~10%). The quantitative values coincide with histological observations of the sections examined, in which the follicular cell density of the thyroid lobe decreases gradually, with a progressive
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FIG. 2. These sections show C-cells in different phases of the mitotic cycle (arrows) surrounded by numerous resting follicular cells. a, Prophase of a C-cell in a thyroid gland of a lo-day-old rat. b, Metaphase of a C-cell in a lo-day-old rat. c, Thyroid section from a 30-day-old rat showing a C-cell in anaphase. d, Telophase of C-cell in a &day-old rat. Immunoperoxidase staining for calcitonin, periodic acid-Schiff, Harris hematoxylin counterstaining. Magnification, X1250.
FIG. 3. Sections of rat thyroid gland showing follicular cells in different phases of the mitotic cycle (arrows) surrounded by numerous follicular cells. a, Prophase of a 30-day-old rat. b, Metaphase of a lo-day-old rat. c, Anaphase of a 25-day-old rat. d, Telophase of a 25 day-old rat. Immunoperoxidase staining for calcitonin, periodic acid-Schiff method, Harris hematoxylin counterstaining. Magnification, X1250.
in the quantity of stored colloid, while the size of the follicular cells and C-cells increases. Cells have been found at all stages of the mitotic cycle (Figs. 2 and 3), although in both cell types the highest numbers were in prophase and metaphase. The approximate ratio of different mitotic phasesat all agesstudied was: seven prophase to five metaphase to one anaphase to three telophase for both cell types. The renewal of the follicular cells, with respect to the transverse axis of the thyroid lobe, is somewhat greater in the middle zones (B-C) of the section. At the same time, increase
there is a preferential localization of mitoses of the follicular cells in the central region of the different zones considered in each section, compared with the periphery, in a ratio of approximately 6:3. The preferential zones of localization of C-cells mitosesin each section are the middorsal (C), followed by the midventral (B), in an overall ratio of approximately 4C:3B:2D:lA. At the sametime, the C-cells undergoing division were localized preferentially in the central region of each section, compared with the periphery, in a ratio of approximately 7:3. There was not statistical significance when the values obtained in
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MITOTIC one thyroid lobe.
lobe were compared
to those found
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in the other
Discussion Our study has revealed considerable age-dependent shifts in the mean mitotic activity of the follicular cells and C-cells in the rat thyroid gland. The maximum mitotic index for endocrine cells was reached during the first 10 days of life; it decreased gradually and significantly until 25 days. At 1 month, there is a significant recuperation of the division rate for both cell types, which declines subsequently in the adult rat. Puberty occurs during this period (50-60 days), but in Wistar rats vaginal opening occurs at 35.6 days, and the testes descend during the period between 15-51 days of age (29). All of these changes are related to TSH levels, which increase at about the time of puberty and then decline and remain unchanged between 6 and 12 months of age (30). The mean volume of the thyroid gland increases during postnatal development, becoming stable in young adults (14, 31, 32). However, the volume fractions of the various histological components of the thyroid exhibit different patterns in the course of postnatal development. The follicular cell fraction shows a steady and considerable decrease with age (1, 13, 33), while the C-cell fraction increases slightly with age (1). These data coincide with the evolution of the mitotic index of both cell types. According to our results, the proliferation of C-cells and follicular cells is inversely proportional to age. Hence, as the division rate is lower with time, although new divisions are produced in absolute terms, these are insufficient to maintain the initial population density. For the follicular cells, this decrease is very obvious, as they are the major histological component of the thyroid gland, while for the C-cells, which comprise a very low proportion, the overall variations are quantitatively not very significant. The mitotic index reported in the present study is below 0.8% for both endocrine cell types, which was the value reported by Leblond (34) for the thyroid gland in rats weighing 200-250 g. This mitotic rate is just sufficient to bring about an increase in cell number proportional to the increase in body weight and, therefore, contributing only to growth. This is the primary definition of these cells as an expanding cell population. Redmond and Tuffery (23) recorded a mean mitotic rate of 7.6 + 0.83 metaphases/10-4. h for male Wistar rats of 220-271 g final body weight. Both research groups used a metaphase arrest agent (colchicine and vincristine sulfate, respectively), which was injected several hours before the animals were killed. During intrauterine life, the origin of C-cells has been described in the UBB (16, 21, 35). In the postnatal period, the UBF is believed to be the vestige of the UBB (2-7, 10) and is found in most thyroid glands if the search is meticulous (1, 36). The UBF is located in the site of maximum concentration of C-cells in the thyroid lobe. However, not all UBF are associated with C-cells, nor is a greater concentration of C-cells observed around them than around the typical thyroid follicles. Thus, it has not been possible to show a clear postnatal link between C-cells and UBF in normal rats. We
THYROID
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have reported recently the presence not only of resting Ccells, but also mitotic figures of C-cells, in the wall of an abnormally migrated UBF in a 5-day-old rat (12,37). It seems that during fetal development, the UBB is the origin of Ccells, but from birth, the remnant of the UBB, the UBF, has exhausted the capacity for differentiation into C-cells; from this time on, the C-cells are derived exclusively from the division of preexisting C-cells. Due to the scarcity, until now, of observations of C-cells in mitosis, some researchers were of the opinion that C-cells were derived from follicular cells (25, 26). According to our results, it seems clear that the C-cell population renewal is independent from the follicular cells in the rat thyroid gland after birth. The present paper provides new evidence for the postnatal origin of C-cells and follicular cells in the preexisting endocrine cells and expands our knowledge of the natural history of these endocrine cells. Acknowledgments The authors wish to express appreciation to Dr. R. 0. Weller critically reviewing the manuscript, and to Miss Ma Dolores Jimenez the excellent technical assistance.
for for
References I, Gonzrilez-Cimpora R, Galera-David1. Conde E, Martin-Lacave son H 1991 Histometry of normal thyroid glands in neonatal and adults rats, Am J Anat 191:384-390 MC 1937 Complex IV in the dog with special emphasis on 2. Godwin the relation of the ultimobranchial body to interfollicular cells in the postnatal thyroid gland. Am J Anat 60:299-340 K 1965 Electron microscopical studies of the ultimobran3. Ishikawa chial body of the rat in embryonic life, with special emphasis on its fate: the relation to the thyroid tissue and parafollicular cells. Okajima Folia Anat Jap 41:313-335 4. Stoeckel ME, Porte A 1970 Origine embryonnaire et differentiation secretoire des cellules a calcitonine (cellules C) dans la thyroide foetal du rat. Etude au microscope electronique. Z Zellforsch 106:251-268 5. Chan AS, Conen PE 1971 Ultrastructural observations on cytodifferentiation of parafollicular cells in the human fetal thyroid. Lab Invest 25:249-259 6. Wollman SH, Neve P 1971 Postnatal development and properties of ultimobranchial follicles in the rat thyroid. Anat Ret 171:247258 7. Wollman SH, Neve P 1971 Ultimobranchial follicles in the thyroid gland of rat and mice. Recent Prog Horm Res 27:213-234 8. Wollman SH, Hilfer SR 1978 Embryonic origin of various epithelial cell types in the thyroid gland of the rat. Anat Ret 189:467-478 9. Wollman SH, Hilfer SR 1978 Embryonic origin of various epithelial cell types in the second kind of thyroid follicle in the C,H mouse. Anat Ret 191:111-122 10. Calvert R 1972 Transitional cells in the postnatal thyroid gland of the rat. Anat Ret 174:341-360 11. Swarup K, Das VK 1978 Calcitonin cells and unusual follicle in four month old wistar rats. Z Mikrosk Anat Forsch 2:212-221 12. Martin-Lacave I, Conde E, Moreno AM, Utrilla JC, Galera-Davidson H, Evidence of the occurrence of calcitonin cells in the ultimobranchial follicle of the rat postnatal thyroid. Acta Anat, in press 13. Petko M, Rigo GY, Varga ZS 1976 Quantitative changes of the Ccell population in the rat thyroid during postnatal ontogenesis. Cell Tissue Res 166:541-552 14. Wechbanjong N, McMillan PJ, Dalgleish AE 1979 Influence of development on the number of calcitonin-containing cells in the
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MITOTIC
440 mouse 15.
16.
17.
18.
19.
20. 21.
22. 23.
24.
25.
thyroid.
Am J Anat
ACTIVITY
OF THE THYROID
154:477-484 mitotic activity of the parafollicular cells of the dog thyroid in experimentally induced hypercalcemia. Arch . _ Histol Jpn32:.179-192 _ Kameda Y. Shieemoto H. Ikeda A 1980 Develoument and cvtodifferentation of C”cel1 complexes in dog fetal thyroid. An immunohistochemical study using anti-calcitonin, anti-C thyroglobulin and anti-19s thyroglobulin antisera. Cell Tissue Res 206:403-415 Kameda Y, Ikeda K, Ikeda A 1981 Uptake of radioiodine in follicles of dog C-cell complexes studied by autoradiograph and immunoperoxidase stainning. Anat Ret 200:461-470 Kameda Y 1984 Immunohistochemical studies on differentiation of the thyroid gland in rabbit fetuses and chick embryos. Histochemistry 80:23-29 Kameda Y 1987 Immunohistochemical demonstration of keratins in the cysts of thyroid gland, parathyroid glands, and C-cell complexes of the dog. Am J Anat 180:87-99 Harach HR 1987 Mixed follicles of the human thyroid gland. Acta Anat 129:27-30 Williams ED, Toyn CE, Harach HR 1989 The ultimobranchial gland and congenital thyroid abnormalities in man. J Path01 159:135-141 Calvert R 1975 Structure of rat ultimobranchial bodies after birth. Anat Ret 181:561-580 Redmond 0, Tuffery AR 1979 Mitotic rate of thyroid follicular cells in untreated and goitrogen-treated rats: variation with time of day. J Anat 129:731-741 Redmond 0, Tuffery AR 1981 Thyroid proliferation, body weight, thyrotropin and thyroid hormones in chronic antithyroid (carbimazole) treatment in rats. J Anat 133:37-47 Stux M, Thompson B, Isler H, Leblond CP 1961 The “light cells” of the thyroid gland in the rat. Endocrinology 68:292-308
Kameda Y 1970 Increased
GLAND
Endo. 1992 Vol131 -No 1
26. Sarkar SK, Isler H 1963 Origin gland.
Endocrinology
of the “light
cells”
of the thyroid
73:199-204
27. Ljungberg
28. 29.
30.
31.
32. 33.
34. 35.
36. 37.
0, Bondeson L, Bondeson AG 1984 Differentiated thyroid carcinoma, intermediate type. A new tumor entity with features of follicular and parafollicular cell carcinoma. Hum Path01 15:218-228 Harach HR 1985 Thyroid follicles with acid mucins in man: a second kind of follicles?. Cell Tissue Res 242:211-215 Baker DEJ 1979 Reproduction and breeding. In: Baker HJ, Lindsey JR, Weisbroth SH (eds) The Laboratory Rat. Biology and Diseases. Academic Press, New York, vol 1:154-168 Klug TL, Adelman RC 1979 Altered hypothalamic pituitary regulation of thyrotropin in male rats during aging. Endocrinology 104:1136-1142 Wolfe HJ, DeLellis RA, Voelkel EF 1975 Distribution of calcitonincontaining cells in the normal neonatal human thyroid gland: a correlation of morphology with peptide content. J Clin Endocrinol Metab 41:1076-1081 Brown RA, Al-Mousa M, Beck JS 1986 Histometry of the normal thyroid in man. J Clin Path01 39:475-482 Malendowicz LK, Majchrzak M 1981 Sex dimorphism in the thyroid gland. III. Morphometric studies on the rat thyroid gland in the course of postnatal ontogenesis. Endokrinologie 77:297-302 Leblond CP 1964 Classification of cell populations on the basis of their proliferative activity. Nat1 Cancer Insi Monogr 14:119-149 Pearse AGE, Carvalheria AF 1967 Cvtochemical evidence for an ultimobranchial origin of rodent thyroid C cells, Nature (Lond) 214:929-930 Juhl M 1981 Morphologv of the second kind of follicle in the guinea pig thyroid gland: Acta-Anat 110:318-326 Conde E, Moreno AM, Martin-Lacave I, Fernindez A, GaleraDavidson H, Immunocytochemical studies of the ultimobranchial tubule
in Wistar
rats, Anat
Histol
Embryol,
in press
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