Number and Size of Rat Thyroid C Cells: No Effect of Pinealectomy P.J. McMILLAN, U. HEIDBUCHEL, AND L. VOLLRATH Department of Anatomy, Johannes Gutenberg-University, Mainz, Federal Republic of Germany (U.H., L. V) and Department ofdnatomy, Loma Linda University, Loma Linda, CA 92350 (PJ.M.) ABSTRACT A method for the estimation of the size and total number of calcitonin-containing cells (C cells) in the rat thyroid gland has been devised. The total area, the number of C cells per unit area, and the areal fraction of C cells were determined for the C cell region using ste serial sections. From these data it was estimated that from 0.3 x lo6 to 1.0 x 102C cells were evenly divided between the two thyroid lobes. Approximately 150 pm3 of cytoplasm were associated with each of these cells. In comparison with sham-operated rats, pinealectomy had little effect on the number of C cells. In a n experiment terminated in the summer, there was a statistically insignificant decrease 6 weeks postsurgery; no effect was seen at 12 weeks. On the other hand, a slight increase in the number of C cells was seen in January, 12 weeks postsurgery. The volume of cytoplasm per cell was not altered by pinealectomy. As C cells are restricted to what can be called the C cell region of the thyroid gland (McMillan et al., 1974; Wolfe et al., 1974), assessment of total C cell number and average C cell volume requires a technique which takes into consideration both the extent of the C cell region and the density of the C cells within this region. This paper describes a technique that permits estimation of quantitative data hitherto not available, i.e., C cell number and size in the different thyroid lobes of the rat. This method was used to test the influence of pinealectomy on C cells. The need for this study derives from the fact that apart from the pineal’s role in the reproductive system of photoperiodic mammals (Reiter, 1982; Hoffmann et al., 1981) its precise influence on other endocrine systems remains to be established (cf. Vollrath, 1981). One of the systems reported to be influenced by the pineal gland is the parafollicular or C cells of the thyroid, which by way of their calcitonin secretion play a n important role in calcium homeostasis. Previous studies dealing with the effects of the pineal gland on the C cells have yielded somewhat contradictory results (see Discussion). Moreover, one aspect in particular has not been adequately studied, i.e., how C cell number is affected by pinealectomy. This kind of information, however, is essential for correctly interpreting both qualitative and quantitative findings resulting from the experimental manipulation of the pineal. In a n electron microscopic study, KrstiC (1969) observed more C cells two months after pinealectomy, but the number of C cells had not actually been counted. Two other papers claimed that pinealectomy increases C cell numbers (Miline et al., 1968; Csaba and Barath, 1974), but nonspecific staining reactions were used and objective quantification was lacking. The methods described in this study overcome these shortcomings in two ways: 1) highly sensitive irnmunohistological staining reaction is used to demonstrate C cells, and 2) C cell number and size are quantitated 0 1985 ALAN R. LISS, INC. morphometrically. Pinealectomy was found to have no effect on C cell size and a n inconsistent effect on C cell number. MATERIALS AND METHODS Male rats of the Wistar (Han) strain (200-225 g) were used in this study. They were maintained, four to a cage, at a temperature of 22 & 2°C under LD 12:12 (lights on 7 A.M. to 7 P.M.), with free access to water and food (Altromin rat pellets). Two experiments were performed. In experiment 1 (May-July, 1980)40 rats were randomly assigned to four groups. One group of 10 animals was pinealectomized and another was sham-operated (Nembutal anesthesia) 6 and 12 weeks prior to sacrifice. In the sham-operated animals, the dura mater anterior and to the right of the confluence of sinuses was incised, but the pineal gland was left undisturbed. At the time of sacrifice the presence or absence of the pineal gland was established by inspection. In experiment I1 (November-February, 1981) 12 rats were pinealectomized and 12 were sham-operated as above, 12 weeks prior to sacrifice. At the end of the experiments the rats were anesthetized and the trachea with attached thyroid and parathyroid were fixed in Bouin’s fixative, in the first experiment by immersion and in the second experiment by perfusion. The tissue was embedded in butylmethacrylate-paraffin(Engen and Wheeler, 1978), serially sectioned at 5 pm and every 20th section immunohistologically stained for calcitonin and counterstained with hematoxylin. The immunohistological procedure followed that of Sternberger (19741, using 10%normal goat serum to block nonspecific stain- Received October 12, 1984; accepted December 13,1984. Address reprint requests to Dr. Paul J. McMillan. 168 P.J. McMILLAN, U. HEIDBUCHEL, AND L. VOLLRATH ing, anticalcitonin sera (which were gifts from L.J. Deftos and B.A. Roos), a peroxidase-antiperoxidase complex obtained from Litton Bionetics Inc., and diaminobenzidine as the chromogenic agent. The calcitonin antiserum was used a t dilutions of 1:400 and 1:3200 containing 1% normal goat serum. Controls consisted of normal rabbit serum and anticalcitonin serum adsorbed with synthetic human calcitonin (300 pg/ml) (which was a gift from Ciba to Dr. F. Fischer). In experiment 11, the pituitary gland, adrenal glands and testes were also taken and weighed. Quantitation In addition to C cell number and volume (see below), data were obtained for the volumes of the thyroid, the portion of the thyroid that contains C cells and the parathyroids by circumscribing the respective organ and region profiles on every sample section by means of a Kontron MOP digitizer. Volumes were calculated by multiplying the total area traced on all sections by the interval between sections (0.1 mm). To minimize the time required to obtain a n estimate of C cell number, a sampling method was devised. It was assumed that a reasonable but maximal (not corrected for section thickness) estimate could be obtained if all of the C cell nuclei in all of the sample sections (25-35) were counted and the result multiplied by 20 (the number of sections between sample sections). An estimate of the number of nuclei in the sample sections is given by the C cell density (number of C cells per unit area) times the total sample area. This times 20 would give the desired count. It was found that a n adequate estimate of the C cell density could be obtained from three sections equally spaced through the C cell region. The adequacy of this sample was tested by determining the cell density for every sample section for each of three randomly selected rats. From the 20-30 density estimates for each thyroid lobe, the mean density and standard deviation (SD) were calculated. Then density estimates were obtained, based on three randomly selected sections. Altogether 90 sets of three (15 sets per lobe) were evaluated. Of these estimates, 96%were less than one SD away from the mean and 63% less than one half the SD away from the mean. Therefore, three sections evenly spaced through the C cell region were accepted as a n adequate sample. In the second experiment, estimation of the volume occupied by calcitonin-containing cytoplasm per cell involved a two-step process. The areal fraction of stained cytoplasm in the C cell region was first determined. Then that estimate of the C cell volume fraction was multiplied by the volume of the C cell region as determined previously and divided by the total number of C cells to give the volume per cell. An image-array processor (Gould-DeAnza 5000) was used to estimate the areal fraction (number of pixels in the C cell intensity “window”/total number of pixels). It was measured by systematically scanning the entire C cell area of three representative sections. A correction for dirt and nonspecific staining was obtained by measuring the areal fraction in the intensity window of thyroid regions that did not contain C cells. This correction averaged 5% of the corresponding C cell fraction. Statistical Analysis Significance of group differences was determined using single classification analysis of variance after logarithmic transformation of the data. For correlation studies, the nonparametric Spearman’s coefficient of rank correlation was used. RESULTS With the immunological method used, C cells appear as dark, polymorphic structures with dark blue nuclei typically lying between follicular cells and the basement membrane (Fig. la). The specificity of the reaction is demonstrated by the fact that cells do not stain when either normal rabbit serum or anticalcitonin serum adsorbed with calcitonin is used in place of the specific antiserum (Fig. lb). The serum has been previously checked for cross-reactivities and specificity (Deftos, 1971; Roos and Deftos, 1976). C cells occur singly or adjacent to other C cells. Clusters consisting of more than two to four C cells are rare. C cell profiles range from being polygonal to cells with two small cylindrical processes. The length of the processes is about twice the width of the C cell body. The volume of calcitonin-stained cytoplasm per C cell in the 12-week winter experiment was the same for both control (146_+38pm3) and experimental (148532 pm3) rats. C cells were found to be concentrated in the middle and caudal portions of the thyroid lobes. They were generally absent from the periphery of each lobe (Fig. 2) and were never found in the rostal pole or isthmus. No statistically significant differences exist between the number of C cells in the right and left thyroid lobes. The total number of C cells per thyroid averaged 0.6 x lo6 (range, 0.3-1.5 x lo6) or 63,000 per mm3 of thyroid tissue. By comparing the volume of the thyroid in Figure 3 with the volume of the C cell region in Figure 4, it can be seen that on the average only 20-25 percent of the thyroid volume contains C cells. In agreement with a previous study (McMillan et al., 19741, no immunoreactive cells were found in the parathyroid glands. Figures 3-5 depict thyroid volume, volume of C cell region, and C cells. per gland in sham-operated and pinealectomized rats. Due to some incomplete series of sections, the number of animals given in each group differs from that in Materials and Methods. One animal in the sham series had some C cells anomalously located in the thymus (McMillan et al., 1982). The total volume of the C cell region and the number of C cells in the thyroid gland of this animal were at the mean of those of the other rats in this group in spite of the fact that these were greatly reduced on the affected side. Data for this animal have been included in the data for volume of the C cell region but excluded from the final data on C cell numbers and volume. Since the measured parameters for this anomalous rat were at the mean of the other rats, inclusion or exclusion only alters the N used for statistical analysis. To be conservative it was excluded from the final data. Variable results were obtained in comparing the number of C cells in control and experimental animals (Fig. 5). Six weeks postsurgery in the summer experiment, the number of C cells was insignificantly reduced (F-ratio = 4.0, P = 0.06) and at 12 weeks no difference was seen (F-ratio = 1.4, P = .26). 169 THYROID C CELLS AND PINEALECTOMY Fig. 1. Thyroid. Immunoperoxidase stain for calcitonin (a); adjacent section reacted with primary antiserum which was absorbed with calcitonin 6). Both are counterstained with hematoxylin. Magnification ~ 3 0 0 . *I E €12 Ijl( 'I1 s S Pr Px 6wks l2wks l2wks Fig. 3. Thyroid volume in summer experiment (6 and 12 weeks, left) and winter experiment (12 weeks, right). Parathyroid volume, pituitary weight, adrenal weight and testes weight were not significantly (P > 0.05) affected by pinealectomy. DISCUSSION The data obtained in this study on rat C cells will be of value in future experimental and comparative studies. Separate assessment of C cell numbers in the right and left thyroid lobes revealed that there are no statistically significant differences. Hence calcitonin meaFig. 2. Cross-section of rat thyroid at the level of the parathyroid surements carried out in one thyroid lobe can be immunohistcchemically stained for calcitonin and counterstained correlated with C cell number in the other, and hemithylightly with hematoxylin. The C cell region is outlined. Magnification roidectomy will remove roughly half of the C cell popux32. lation, the total population being on the order of 700,000 cells per gland. One exception (due to anomalous develIn the winter experiment, however, a marginally signif- opment) to the even distribution of C cells has been icant increase (F-ratio = 4.7, P = 0.04) was observed 12 observed (McMillan et al., 1982). The number of C cells weeks postsurgery. per mm3 of thyroid tissue is 63,000 in the rat but 30,600 No correlation was found to exist between any of the in 8-week-old mice (Wechbanjong et al., 1979). For huC cell parameters and thyroid and parathyroid volumes. mans no such data are available, but judging from vis- 170 P.J. McMILLAN, U.HEIDBUCHEL, AND L. VOLLRATH ual inspection (McMillan et al., 1974) C cells are much scarcer than in rats and mice. The estimation of the volume of C cell cytoplasm (150 pm3) provides for the first time a baseline for comparison in studies of the control of calcitonin storage. This estimate must be considered as a n upper limit, because of the thickness of the sections used. The estimation of C cell volume was only performed on perfusion-fixed tissue. This avoids the probability that the superficial and deep cells would shrink differentially in immersion fixation. The pinealectomy experiments described in this study were carried out since, according to the literature, the pineal gland is capable of influencing two important components of the calcium-regulating system of the body, the parathyroid glands and the C cells. With respect to the parathyroid glands, it had been found that pinealectomy is followed by hypertrophy and hyperplasia of parathyroid cells, whereas administration of pineal substances leads to a n atrophy (KrstiC, 1966, 1967, 1968; Miline and KrstiC, 1966), pointing to a n inhibiting effect of the pineal gland on the parathyroids. By contrast, Kiss et al. (1969)observed a n atrophy of the parathyroid and a decrease of 3H-methionine incorporation which suggests that the pineal may stimulate the parathyroid gland. Feedback of parathormone on the pineal gland is suggested by the observation that in guinea pigs the majority of pinealocytes can be influenced by microiontophoretically applied parathormone (Semm et al., 1981). Dramatic effects of the pineal gland can be demonstrated in parathyroidectomized rats. While parathyroidectomy alone does not lead to severe functional changes, animals that are additionally pinealectomized undergo convulsive attacks and most of them die within 14 hours (Reiter and Morgan, 1972; Reiter et al., 1972, 1973a and b, 1975; Pomerantz and Reiter 1973). As the effect of pinealectomy o n parathyroid volume had not been investigated in the previous studies, this aspect could be conveniently examined in the present study. According to our results, there is no statistically significant difference in parathyroid volume either 6 or 12 weeks after pinealectomy. This suggests that if hypertrophy and hyperplasia of the intrinsic cells do occur after pinealectomy (see above) they must be very slight. Studies dealing with the interactions of the pineal gland and the calcitonin-producing cells of the thyroid point to mutual inhibition. Pinealectomy has been re- ported to increase 1)C cell numbers (Miline et al., 1968; KrstiC, 1968; Csaba and Barath, 19741, 2) the nucleocytoplasmic ratio in favour of the cytoplasm, and 3) the number of electron-dense secretory granules (Csaba and Barath 19751, although it should be noted that Soriano (1973) found a decreased secretory activity of the C cells after pinealectomy. Pineal inhibition by calcitonin reduced the activity of a number of histochemically demonstrable enzymes in the pineal gland (KrstiC and Tarsoly, 1972). Moreover, in the guinea pig, the majority of pinealocytes responsive to microiontophoretically applied calcitonin were inhibited as assessed electrophysiologically (Semm et al., 1981). In view of these findings, it was somewhat surprising to see that none of the C cell parameters investigated in the present study showed consistent differences after pinealectomy when compared to sham-operated controls. Despite these relatively negative data, it should be noted that in the two sets of experiments opposing tendencies were present: a decrease in C cell numbers in the summer and a n increase in the winter. In addition, it should be taken into account that as C cell numbers are highly variable between animals, a very large number of experimental animals would be required to demonstrate positively a n effect of pinealectomy, especially if seasonal differences exist. Nevertheless, under the conditions of these experiments, we believe that the pineal exerts little influence on the number or size of C cells. We are all the more confident with this conclusion, as the data were obtained by applying a combination of a highly specific immunohistological technique and a morphometric method specifically designed for coping with the uneven distribution of C cells within the thyroid. That the C cell number can be expected to change at all has been repeatedly demonstrated. C cells show a n increase during development (Wechbanjong et al., 1979; Alumets et al., 1980), with age (Peng et al., 19761, and when chronically stimulated with vitamin D and calcium (Young and Capan 1969). Whether C cells can be experimentally decreased in number remains to be seen. The problem of seasonal differences in C cells in nonhibernating species has received little attention in contrast to hibernators (cf. Nunez et al., 1967; Pearse and Welsch, 1968; Nunez and Gershon, 1972). According to Petko (1978), the area occupied by C cells in the rat "E E 6wks l2wks l2wks Fig. 4. Volume of C cell region in summer experiment (6 and 12 weeks, left) and winter experiment (12 weeks, right). 6hs l2wk1 12wkr Fig. 5. Number of C cells in summer experiment (6 and 12 weeks, left) and winter experiment (12 weeks, right). THYROID C CELLS AND PINEALECTOMY thyroid is large in autumn and small in spring; unfortunately, no experiments were carried out in the summer and winter. In the present study, statistically significant differences that could be related to season were not found for any of the parameters investigated. It should be noted, however, that in our study, which was not aimed at seasonal differences, the experiments were carried out in seasons different from those of Petko’s study. ACKNOWLEDGMENTS The financial support of the Deutsche Forschungsgemeinschaft is gratefully acknowledged. Fr. Sabina Soda’s expert technical assistance is very appreciated, a s is the secretarial help of Ms. Cherry Wendtland and Ms. Terry McMillan. LITERATURE CITED Alumets, J., R. Hakanson, G. Lundqvist, F. Sundler, and J. Thorell (1980) Ontogeny and ultrastructure of somatostatin and calcitonin cells in the thyroid gland of the rat. Cell Tissue Res., 206~193-201. Csaba, G., and P. Barath (1974) The effect of pinealectomy on the parafollicular cells of the rat thyroid. Acta Anat., 88t137-146. Csaba, G., and P. Barath (1975) Morphological changes of the thymus and thyroid gland after postnatal extirpation of pineal body. Endocrinol. Exp., 9t59-67. Deftos, L.J. (1971) Immunoassay for human calcitonin. I. Method. Metabolism, 20~1122-1128. Engen, P.C., and R. Wheeler (1978)N-butyl methacrylate and paraffin as an embedding medium for light microscopy. Stain Technol., 53: 17-22. Hoffmann, K., H Illnerova, and J. Vanecek (1981) Effect of photoperiod and of one minute light at night-time on the pineal rhythm on Nacetyltransferase activity in the Djungarian hamster, Phodopus sungorus. Biol. Reprod., 24t551-556. Kiss, J., D. Banhegyi and G. Csaba (1969) Endocrine regulation of blood calcium 11. Relationship between the pineal body and the parathyroid glands. Acta Med. Hung., 26:363-370. Krstic, R. (1966) Die Treffermethode in der Messung der Parathyreozytenaktivitat nach der Epiphysektomie. Experientia, 22:336-337. KrstiC, R. (1967) Uber Veranderungen der Epithelkorperchen nach Epiphysektomie. Z. Zellforsch. Mikrosk. Anat., 77%-24. KrstiC, R. (1968) The influence of epiphyseal extract on the parathyroid glands of the rat. Z. Zellforsch. Mikrosk. Anat., 89t73-79. Krstic, R. (1969) Quantitative investigations of the dark and light parafollicular cells in the rat. Z. Anat. Entwickl. -Gesch., 129t353358. Krstic, R., and E. Tarsoly. (1972) Histochemical enzyme studies of the effect of calcitonin on the rat pineal body. Experientia, 28:12341235. McMillan, P.J., W.M. Hooker, L.J. Deftos (1974) Distribution of calcitonincontaining cells in the human thyroid. Am. J. Anat., 140t7379. McMillan, P.J., U. Heidbiichel and L. Vollrath (1982) Anomalous occurrence of immunoreactive calcitonin cells in the thymus of the rat. Cell Tiss. Res., 222:629-634. Miline, R., and R. KrstiC (1966) Sur l’histophysiologie correlative de la glande pineale et des glandes parathyroides. Z. Zellforsch. Mikrosk. Anat., 69:428-437. Miline, R., M. SCepoviC and R. KrstiC (1968) Influence de I’epiphysectomie sur les cellules parafolliculaires de la glande thyroid. CR. 171 Assoc. Anat., 139t893-898. Nunez, E.A. and M.D. Gershon (1972) Synthesis and storage of serotonin by parafollicular (C) cells of the thyroid eland of active. prehibernating and hibernating bats. EndochnoloG, 90:1008-1024; Nunez, E.A., R.P. Gould, D.W. Hamilton, J.S. Hayward and S.J. Holt (1967) Seasonal changes in the fine structure of the basal granular cells of the bat thyroid. J. Cell. Sci. 2t401-415. Pearse, A.G.E. and U. Welsch (1968) Ultrastructural characteristics of the thyroid C cells in the summer, autumn and winter states of hedgehog (Erinaceus europaeus I.), with some reference t o other mammalian species. Z. Zellforsch. Mikrosk. Anat., 92t596-609. Peng, T.C., Cooper and S.C. Garner (1976) Thyroid and blood thyrocalcitonin concentrations and C cell abundance in strains of rats at different ages. Proc. Soc. Exp. Biol. Med., 253268-272. Petko, M. (1978) Seasonal variations of rat thyroid C cell population. Acta Biol. Acad. Sci. Hung., 29:367-374. Pomerantz, G., and R.J. Reiter (1973) Age dependent changes in the susceptibility of parathyroidectomized, pinealectomized rats to seizures. Exp. Neurol., 4Ot254-257. Reiter, R.J. (1982) Neuroendocrine effects of the pineal gland and of melatonin. Frontiers in Neuroendocrinolom, 7:287-316. Reiter, R.J., and W.W. Morgan (1972) Attempt to characterize the convulsive response of parathyroidectomized rats to pineal gland removal. Physiol. Behav., 9:203-208. Reiter, R.J., D.E. Blask, J.A. Talbot, and M.P. Barnett (1973a) Nature and the time course of seizures associated with the surgical removal of the pineal gland from parathyroidectomized rats. Exp. Neurol., 38:386-397. Reiter, R.J., W.W. Morgan, and J.A. Talbot (197313) Seizures in rats induced by pinealectomy: influence of diazepam, chlordiazepoxide, diphenylhydantoin and pineal substances. Arch. Intern. Pharm. Therap., 202~219-230. Reiter, R.J., L.Y. Johnson, and L. Bigelow (1975)Effects of drugs which inhibit tryptophan hydroxylose or dopamine-0-hydroxylase on pinealectomy-induced seizures in parathyroidectomized rats. Gen. Pharm., (253-57. Reiter, R.J., S. Sorentino, Jr., and R.A. Hoffman (1972)Muscular spasms and death in thyroparathyroidectomized rats subjected to pinealectomy. Life Sci. I, 11t123-133. Roos, B.A. and L.J. Deftos (1976) Radioimmunoassay of calcitonin in plasma, normal thyroid and medullary thyroid carcinoma of the rat. J. Lab. Clin. Med., 88t173-182. Semm, P., C. Demaine, and L. Vollrath (1981) Electrical responses of pineal cells to thyroid hormones and parathormone: a microelectrophoretic study. Neuroendocrinology, 33:212-217. Soriano, F.M. (1973) Ultrastructura de las celulas parafoliculares del tiroides de la rata blanca despues de la pinealectomia. Ann. Anat., 22:311-320. Sternberger, L.A. (1974) Immunocytochemistry. Prentice-Hall, Inc., Englewood Cliffs, NJ, pp. 104-169. Vollrath, L (1981) The pineal organ. In: Hdb. Mikr. Anat. Mensch VU 7. A. Oksche and L. Vollrath, eds. Springer Verlag, Berlin-Heidelberg-New York, pp. 1-478. Wechbanjong, N., P.J. McMillan, and A.E. Dalgleish. (1979) Influence of development on the number of calcitonin-containing cells in the mouse thyroid. Am. J. Anat., 154t477-488. Wolfe, H.J., E.F. Voelkel, and A.H. Tashjian, Jr. (1974) Distribution of calcitonin-containing cells in the normal adult human thyroid gland A correlation of morphology with peptide content. J. Clin. Endocrinol. Metab., 38r688-694. Young, D.M. and C.C. Capen. (1969) Thyrocalcitonin: Response to experimental hypercalcemia induced by vitamin D in cows. In: Calcitonin 1969. Proceedings of the Second International Symposium. S. Taylor and G . Foster, eds. Heinemann Medical Books Ltd., London, pp. 141-153.