Steroids 84 (2014) 22–29

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Morpho-functional characteristics of rat fetal thyroid gland are affected by prenatal dexamethasone exposure Milica N. Manojlovic´-Stojanoski ⇑,1, Branko R. Filipovic´ 1, Nataša M. Nestorovic´, Branka T. Šošic´-Jurjevic´, Nataša M. Ristic´, Svetlana L. Trifunovic´, Verica Lj. Miloševic´ Institute for Biological Research ‘‘Siniša Stankovic´’’, University of Belgrade, 142 despota Stefana Blvd., 11060 Belgrade, Serbia

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Article history: Received 9 September 2013 Received in revised form 24 February 2014 Accepted 4 March 2014 Available online 20 March 2014 Keywords: Thyroid Fetal development C cells Follicular cells Dexamethasone Stereology

a b s t r a c t Thyroid hormones (TH) and glucocorticoids strongly contribute to the maturation of fetal tissues in the preparation for extrauterine life. Influence of maternal dexamethasone (Dx) administration on thyroid glands morpho-functional characteristics of near term rat fetuses was investigated applying unbiased stereology. On the 16th day of pregnancy dams received 1.0 mg/Dx/kg/b.w., followed by 0.5 mg/Dx/kg/ b.w. on the 17th and 18th days of gestation. The control females received the same volume of saline. The volume of fetal thyroid was estimated using Cavalieri’s principle; the physical/fractionator design was applied for the determination of absolute number of follicular cells in mitosis and immunohistochemically labeled C cells; C cell volume was measured using the planar rotator. The functional activity of thyroid tissue was provided from thyroglobulin (Tg) and thyroperoxidase (TPO) immunohistochemical staining. Applying these design-based modern stereological methods it was shown that Dx treatment of gravid females led to a significant decrease of fetal thyroid gland volume in 19- and 21-day-old fetuses, due to decreased proliferation of follicular cells. The Tg and TPO immunohistochemistry demonstrated that intensive TH production starts and continues during the examined period in control and Dx-exposed fetuses. Under the influence of Dx the absolute number of C cells was lower in both groups of near term fetuses, although unchanged relation between the two populations of endocrine cells, follicular and C cells suggesting that structural relationships within the gland are preserved. In conclusion maternal glucocorticoid administration at the thyroid gland level exerts growth-inhibitory and maturational promoting effects in near term rat fetuses. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The thyroid gland contains two major types of endocrine cells, follicular cells, which form thyroid follicles for storing thyroglobulin, and a minor population of C cells, which primarily produce calcitonin (CT). In contrast to follicular cells, C cells are heterogeneously distributed in the thyroid lobes [1]. The thyroid gland originates partly from the pharyngeal endoderm which differentiates into follicular (thyroid hormone (TH)-producing) cells. The second type of progenitors derive from the most caudal pharyngeal pouches and are subsequently incorporated into the thyroid bud where they differentiate into C cells [2]. In fetal rats, significant growth and rapid structural and functional development of the thyroid gland happen during the last third of gestation. The first appearance of follicles, iodine organifi⇑ Corresponding author. Tel.: +381 11 2078 323; fax: +381 1 2761 433. 1

E-mail address: [email protected] (M.N. Manojlovic´-Stojanoski). M. Manojlovic´-Stojanoski and B. Filipovic´ contributed equally to this work.

http://dx.doi.org/10.1016/j.steroids.2014.03.006 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

cation and thyroid hormonogenesis occur in parallel with a marked increase of thyroid-stimulating hormone (TSH) in the fetal circulation and expression of TSH receptors (TSHR) in thyroid tissue [3]. Since immunohistochemical appearance of TSH cells in the rat pituitary is recorded on day 17 of fetal development, and their number continues to grow until the second week of life [4], maturational changes in the thyroid gland lead to a significant increase in the blood concentration of TH during the perinatal period. During this transient window in time numerous tissues are sensitive to TH action which induces indispensable, permanent changes in their structure and functions. Iodothyronine deiodinase (D1, D2, D3) enzymes provide biologically active triiodothyronine (T3) to developing tissues by activating and/or deactivating systemic serum TH. Three types of deiodinases differ in tissue distribution, substrate specificity and sensitivity to inhibiting compounds [5]. Action of D2 and D3 preserves the safe level of T3 in the developing brain and the pituitary, while the activity of D3 in the utero-placental unit protects fetal tissues against high maternal thyroxine (T4) concentrations [5].

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Little is known about the activity of C cells in fetal rat thyroids. An earlier study detected CT in the thyroid gland of 17.5-day-old rat fetuses [6] and the rise in plasma CT levels between 19.5 and 20.5 days of gestation corresponded to increased C cell activity [7]. As glucocorticoids have a potent influence on maturation of fetal lung and other tissues they have been used for more than 40 years in human pregnancies at risk of preterm delivery. Antenatal glucocorticoids mimic the actions of the endogenous rise in plasma glucocorticoids, reducing complications, such as neonatal respiratory distress syndrome, and most importantly, neonatal mortality [8]. Synthetic glucocorticoids, such as dexamethasone (Dx), are poorly metabolized by placental 11b-hydroxysteroid dehydrogenase 2 (11b-HSD2), an enzyme which largely prevents maternal glucocorticoids from reaching the fetus, pass into the fetal circulation and shape fetal development [9]. Although elevated glucocorticoid levels during pregnancy are associated with in utero growth retardation, metabolic, cardiovascular and immune adaptations under the influence of glucocorticoids are fundamental to successful reaction to birth-related stress and the environmental challenges of the neonate [10,11]. Nevertheless, as an established programming concept, all these adaptations predispose organisms to diseases in adulthood [11]. Prenatal alterations in the maternal glucocorticoid environment reflect fetal, neonatal and adult hypothalamic–pituitary–thyroid (HPT) axis activity and function [12,13]. Maternal Dx administration influences TH level and peripheral deiodination of TH in ovine fetuses [12]. In addition, alterations in the maternal glucocorticoid milieu during pregnancy have been found to affect functioning of the HPT axis in both female and male adult rat offspring at all levels (hypothalamic TRH expression, pituitary TSH level, and thyroid hormonogenesis) [13,14]. Additionally, thyrocytes express glucocorticoid receptor (GR), and this signalling system contributes to differentiation of thyroid cells [15]. Thus, it was hypothesized that maternal glucocorticoid administration during pegnancy affects fetal thyroid gland. To address this hypothesis, our study aim was to determing structural changes of thyroid gland, as well as immunohistochemical properties of follicular and C cells in 19and 21-day-old fetuses after maternal exposure to Dx.

2. Material and methods 2.1. Animals Adult female Wistar rats, weighing 260 ± 10 g, bred in the laboratory of the Institute for Biological Research, Belgrade, were used. The animals were kept in a light (lights on 06.00–20.00 h) and temperature (22 ± 2 °C) controlled room. Rat chow and tap water were available ad libitum. Female rats were examined daily and only those showing regular 4-day cycles were included. The presence of spermatozoa in vaginal smears the morning after caging with a fertile male in the night of proestrus was indicative of pregnancy and this day was considered as day 1 of gestation. Dams were randomized into a control and an experimental group, each consisting of six animals. On day 16 of pregnancy experimental dams received 1.0 mg Dx (Dexamethasone phosphate – Krka, Novo Mesto, dissolved in 0.9% saline)/kg b.w. subcutaneously, followed by 0.5 mg Dx/kg b.w./day on days 17 and 18 of gestation. The control gravid females received the same volume of saline vehicle. Exposure to tapering regime of Dx i.e., reduction of Dx doses has been used in order to minimize adverse effects of glucocorticoid administration. The applied doses of Dx are equivalent to doses used as anti-inflammatory therapy in clinical practice [16–18]. The same effects of subcutaneus and intramuscular application of Dx are established, so the consequences of these routes of Dx administration are comparable [18,19]. Fetuses from control and experimen-

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tal females were sacrificed under ether narcosis on days 19 and 21 of gestation and they are referred to as 19-day-old and 21-day-old fetuses. Each group (n = 6) was formed from randomly selected male fetuses (based on ano-genital distance), taking into account that fetuses were taken from different mothers. All animal procedures complied with the EEC Directive (86/609/ EEC) on the protection of animals used for experimental and other scientific purposes, and were approved by the Ethical Committee for the Use of Laboratory Animals of the Institute for Biological Research ‘‘Siniša Stankovic´’’, University of Belgrade. 2.2. Tissue preparation and immunohistochemistry The laryngeal area, including the thyroid gland, was immediately excised, fixed in Bouin’s solution for 48 h and dehydrated in increasing concentrations of ethanol and xylene. After embedding in Histowax (Histolab Product AB, Göteborg, Sweden), each tissue block was serially sectioned at 3 lm thickness on a rotary microtome (RM 2125RT Leica, Wetzlar, Germany). Sakura Tissue-Tek Accu-Edge Low-Profile microtome blades for extremely thin sectioning were used. The slices were placed on silica coated glass slides (SuperFrost Plus, Prohosp, Denmark). CT-immunopositive thyroid C-cells and thyroglobulin (Tg)immunopositive cells were localized using the peroxidase–antiperoxidase (PAP) method. Endogenous peroxidase activity was blocked by incubation in 9 mM hydrogen peroxide solution in methanol for 15 min at ambient temperature. Nonspecific background staining was prevented by incubation of the sections with nonimmune, normal porcine serum diluted in phosphate-buffered saline (PBS; pH 7.4) for 60 min. For detection of CT in the C cells anti-human CT antiserum (Dakopatts, Copenhagen, Denmark) diluted 1:500 were incubated for 60 min and served as the primary antibody; for Tg detection rabbit-anti-human antibody diluted 1:500 was employed for 120 min (Dakopatts, Copenhagen, Denmark). After washing in PBS, sections were incubated for another 60 min with the secondary antibody, polyclonal swine-anti-rabbit IgG/HRP (Dako, Glostrup, Denmark), and rinsed again with PBS for 10 min. Antibody localization was visualized using 0.05% 3,3-diaminobenzidine tetrachloride (DAB) liquid substrate chromogen system (Dako). Sections were thoroughly washed under running tap water, counterstained with hematoxylin and mounted in DPX. Thyroperoxidase (TPO) localization was also determined. After dewaxing and hydration the sections were exposed to microwaves (800 W) in 0.05 M citrate buffered saline (pH 6.0) for 10 min for antigen retrieval. The subsequent immunohistochemical procedure was described before. Mouse monoclonal antbody directed against human TPO (Santa Cruz Biotechnology INC, CA, USA; diluted 1:25) was applied as the primary antibody, and sections were incubated overnight, while polyclonal rabbit anti-mouse IgG/HRP (Dako, Glostrup, Denmark) was used as the secondary antibody. 2.3. Stereological measurements All stereological analyses were carried out using a workstation comprising a microscope (Olympus, BX-51) equipped with a microcator (Heidenhain MT1201) to control movement in the z-direction (0.2 lm accuracy), a motorized stage (Prior) for stepwise displacement in the x–y directions (1 lm accuracy), and a CCD video camera (PixeLink) connected to a 19’’ PC monitor (Dell). The whole system was controlled by a newCAST stereological software package (VIS – Visiopharm Integrator System, version 2.12.1.0; Visiopharm; Denmark). The main objectives were planachromatic 10 dry lenses and a 100 oil lens. Control of the stage movements and the interactive test grids (uniformly spaced points test grids

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and rectangular unbiased disector frame) were provided by newCAST software running on a Dell computer. 2.4. Estimation of thyroid volume Thyroid volumes were determined using Cavalieri’s principle [20]. Thus, a number between 1 and 20 was obtained from a random number table for sampling the first section, in order to ensure a random position for the start. Thereafter every 20th section from each tissue block was analyzed to enable systematic uniform random sampling. The same sections were used in the subsequent estimation of C-cell number and number of dividing cells by the physical dissector method. At the monitor, a final magnification of 300 allowed easy and accurate recognition of tissue boundaries. The number of points falling within the boundaries of the tissue were counted and summed for the whole thyroid gland. Mean section thickness was estimated using the block advance (BA) method [21] and we found that true section thickness was 2.9 lm. Thyroid volume (Vpt) was then estimated as: _

V pt ¼ aðpÞ  BA 

n X Pi

40  40 lm (1600 lm2) was used in order to ensure 150–200 cells per animal. After defining tissue boundaries, meander sampling was set to analyze 50% of the tissue. The X–Y step of meander sampling movements was 56.57 lm (stepx,y = 56.57 lm). Therefore, sampling fraction 2 for estimation of absolute cell number was f2 = 0.5. The fields of vision were randomly selected and the percentage of tissue analyzed was controlled by the software. C-cells were counted if their nuclei appeared within the unbiased counting frame applied to the reference section, they were not intersected by exclusion boundaries and did not appear in the ‘‘look up’’ section [22,23]. The same rules for estimation of follicular cell number in mitosis were applied (Fig. 1). The average counts were 209 and 165 C-cells and 219 and 159 proliferating follicle cells (mitotic cells) per thyroid of control and experimental 19-dayold fetuses respectively. For thyroids of 21-day-old control and Dx fetuses, 292 and 217 C-cells and 176 and 136 cells in mitosis, were counted respectively. Raw counts (Q) of C-cells, as well as dividing cells were multiplied by the reciprocals of the sampling fractions to estimate the total number per thyroid gland. The number of C-cells and mitotic cells = Q(C cell; dividing cell)(1/f1)  (1/f2) = Q 1/0.1  1/0.5.

i¼1

where a(p) is the area associated with each sampling point (21474.8 lm2); BA is the block advance i.e. the mean distance between two consecutively studied sections (58 lm); n is the number of sections studied for each thyroid; and RPi is the sum of points hitting a given target. 2.5. Quantification of C-cells and cells in mitosis (dividing cells) A fractionator/physical dissector design with two levels of sampling was used to estimate the number of C-cells and follicular cells in mitosis in 19- and 21-day-old fetuses from control and Dx treated mothers. Using criteria given by Van Diest et al. [20] dividing cells were identified as cells with absent nuclear membrane, and clear, hairy appearance of condensed chromatin that is either clotted, or in a plane forming the mitotic figures (Fig. 1.) Sampling was systematically uniform from the random start [21,22]. Sections designated as section pairs were first captured into a super image. A 100 oil-immersion objective was used for counting the cells. One section in the pair became the reference section and the other the ‘look up’ section. Subsequently, the analysis was performed in both directions with the reference section also becoming the ‘‘look up’’ section. This doubles the first sampling fraction from 1/20th to 1/10th (sampling fraction 1 (f1) = 1/10). The average numbers of section pairs analyzed per group of thyroids were 14 ± 1 for 19-day old control and 12 ± 1 for Dx exposed fetuses, 16.5 ± 1 for 21-day-old control and 14 ± 1 for Dx exposed fetuses, respectively. For estimation of C-cells as well as dividing follicular cell number an unbiased counting frame measuring

2.6. Volume of C cells For evaluation of individual cell size the planar rotator was used as the unbiased local estimator at an objective magnification of 100. The C cell volume was measured on cells sampled and counted by the physical dissector, applying unique counting rules. 2.7. Statistical analysis Stereological data obtained from each rat were averaged per experimental group and the mean and standard deviations were calculated for six animals per group. Data were evaluated for normality of distribution by the Kolmogorov–Smirnov test. Two-way analysis of variance (ANOVA) with treatment (control and Dx treatment), and fetal age (19- and 21-day-old fetuses) as factors was performed. To determine the significance of differences between the groups post hoc the Bonferroni test was used. The significance of differences between control and treated 21-dayold fetuses for volume occupied by follicular cells, C cells and colloid, was estimated by Student’s t-test. A probability value of 5% or less was considered as statistically significant. 3. Results 3.1. Histological analysis The thyroid gland of 19-day-old fetuses is not fully differentiated i.e. follicular cells were arranged in follicle structures but colloid

Fig. 1. Identification and counting of dividing cells (arrows) by physical dissector. Marked cells were counted as dividing cells as they appeared within the unbiased counting frame of the reference section and did not appear in the ‘‘look up’’ section, and not intersected by exclusion boundaries (red line). Scale bar = 20 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Representative micrographs of fetal thyroid glands of 19- and 21-day-old fetuses from control (a and c) and Dx treated (b and d) dams. Numerous follicular cells in mitosis (arrowhead) and immunostained C cells (arrow) were present in thyroid gland of 19- and 21-day-old control fetuses (a and c); in Dx exposed fetuses number of proliferating follicular cells and C cells was decreased (b and d). In 21-day-old fetuses from control and Dx treated mothers follicles with centrally positioned colloid (⁄) were clearly visible (c and d). Scale bar = 13 lm.

accumulation in the central lumen was minor. Numerous mitotic figures were present in the follicular cells. Thyroid C cells were isolated in an intrafollicular position in the center of the thyroid gland. In 21-day-old fetuses follicles were prominent and follicular cells formed a continuous layer around the central mass of colloid; follicular cells in mitosis were still abundant. Numerous C cells of different shapes were present, some with cell processes extending towards the interfollicular capillaries. The C cells were located in intrafollicular and interfollicular positions (Fig. 2A and C). The thyroids of near term fetuses from Dx-treated gravid females appeared smaller than those of the corresponding control group (in area and volume). There were fewer dividing cells, as well as less abundant C cells after Dx treatment of dams in both examined periods in comparison to the corresponding controls but, as in the controls, clearly visible differentiated follicles and C cells were present in 21-day-old foetuses (Fig. 2B and D).

Immunohistochemical staining for Tg in thyroid glands from 19-day-old fetuses revealed that it was mainly present within the follicular cells, but accumulation in the lumen had already started. Two days later significant structural maturation of thyroid gland had occurred. Much Tg was translocated from the cells into the lumen. Immunohistochemical positivity for Tg was strongest on the apical side of follicular cells. Resorptive vacuoles were detected in colloid (Fig. 3A). Maternal Dx application did not influence Tg immunolabelling. As in controls, Tg staining was observed in the cytoplasm of follicular cells, while accumulation in the outer space was minor in 19-day-old fetuses. In 21-dayold fetuses from Dx treated mothers differentiated follicles were strongly labelled for Tg especially at the apical pole of follicular cells. Resorptive vacuoles were present in colloid (Fig. 3B). TPO staining was detected in the follicular cell cytoplasm of 19-day-old fetuses with increased expression at 21 days, The

Fig. 3. Representative micrographs of Tg immunostained follicular cells of 19- (a) and 21-day-old-fetuses (b) from control dams. Resorptive vacuoles (arrow) presence was observed in accumulated follicular colloid. Strong Tg immunohistochemistry was detected mainly in the cytoplasm of follicular cells while accumulation in outer space was minor in 19-day-old control fetuses (a). In thyroids of 21-day-old fetuses follicles were differentiated with increased amount of accumulated colloid (b). Scale bar = 8 lm.

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Fig. 4. Representative micrographs of TPO immunostained follicular cells of 21-day-old-fetuses from control (a) and Dx treated dams (b). TPO immunohistochemcal staining was detected in the follicular cell cytoplasm. Especially intensive TPO immunostaining was observed in border area between the apical membrane of follicular cells and the colloid in 21-day-old fetuses from control dams (arrowhead) (a). The same pattern of TPO immunostaining was determined in the 21-day-old fetal follicles after maternal Dx treatment (b). Scale bar = 20 lm.

Fig. 5. Volume of thyroid gland (mm3) of 19- and 21-day-old fetuses and volume of thyroid gland tissue components (follicular cells, colloid and C cells) of 21-day-old fetuses from control (C) and Dx treated (Dx) dams. Results are given as means ± SD (n = 6); ⁄p < 0.05, Dx vs. C; ap < 0.05, between controls. Data were assessed by two-way ANOVA followed by Bonferroni test for thyroid gland volume estimation; Student’s t-test was applied for estimation differences between thyroid gland tissue components in 21-dayold fetuses. ⁄p < 0.05, Dx vs. C.

localization was also cytoplasmic, with occasional intense immunopositivity on the apical surface of follicular cells. The same pattern of TPO immunostaining was observed in the fetal follicles after maternal Dx treatment. Especially strong TPO immunopositivity was detected in the border area between the apical membrane of follicular cells and colloid in 21-day-old fetuses of Dx treated dams (Fig. 4A and B). 3.2. Morphometric parameters of thyroid gland volume, number of proliferating cells, C cell number and volume and numerical density of C cells When thyroid gland volumes of the fetal groups were compared, significant main effects of the treatment (F(1,20) = 45,46, p < 0.001) and fetal age (F(1,20) = 35,85, p < 0.001) were revealed by two-way ANOVA, but no significant interaction between them.

Subsequent post hoc analysis showed that fetal thyroid gland volume increased significantly with advancing gestational age. After maternal Dx application thyroid gland volumes of both 19- and 21-day-old fetuses were significantly smaller than the respective values for control fetuses (Fig. 5). The structural differentiation of the thyroid gland allows measurement of the volume of the main tissue fractions only in 21day-old fetuses. Maternal Dx treatment led to a significant decrease (p < 0.05) in the volume occupied by follicular cells, C cells and colloid (Fig. 5). Main effects of fetal exposure to Dx (F(1,20) = 50,08, p < 0.001) and fetal age (F(1,20) = 21,81, p < 0.001) on the number of follicular cells in mitosis were observed, but with no significant interaction between them. Post hoc comparison revealed significantly fewer follicular cells in mitosis in 21-day-old control fetuses compared with 19-day-old control fetuses. Marked reduction of proliferating

Fig. 6. Absolute number of follicular cells in mitosis and absolute number of C cells in thyroid gland of near term fetuses from control (C) and Dx treated (Dx) dams. Results are given as means ± SD (n = 6); ⁄p < 0.05, Dx vs. C; ap < 0.05, between controls. Data were assessed by two-way ANOVA followed by Bonferroni test.

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Fig. 7. Numerical density of C cells (1/mm3) and volume of C cells (lm3) of near term fetuses from mothers treated with saline (C) or Dx (Dx). Results are given as means ± SD (n = 6); Data were assessed by two-way ANOVA followed by Bonferroni test.

cells was also noted at both fetal ages when the groups from Dx treated mothers were compared with the appropriate control group (Fig. 6). Both main effects of fetal Dx exposure (F(1,20) = 91,73, p < 0.001) and fetal age (F(1,20) = 117,44, p < 0.001), as well as treatment x fetal age interaction (F(1,20) = 6,54, p < 0.018) were statistically significant concerning the absolute number of C-cells. C cell number increased with advancing fetal age, but there were fewer after Dx exposure in both 19- and 21-day-old fetuses when compared to the corresponding controls (Fig. 6). No treatment and fetal age effects were observed on numerical density of C cells (absolute number of cells expressed per volume unit) or on C cell volume, nor were there significant interactions between the treatment and fetal age (Fig. 7).

4. Discussion The results obtained by applying an unbiased stereological approach revealed that Dx treatment of gravid females led to a significant decrease of fetal thyroid gland volume, due to decreased proliferation of follicular cells. Under the influence of Dx the absolute number of C cells was lower in both groups of near term fetuses, although C cell number expressed per unit thyroid gland volume was unchanged, suggesting that structural relationships within the gland are preserved. The functional characterisation of thyroid tissue achieved using immunohistochemical Tg and TPO staining, demonstrated that intensive TH production starts and continues during the examined period in both control and Dx exposed fetuses. Dx treatment of gravid females was applied from gestational days 16 to 18, a period of intensive structural and functional changes of the fetal HPT axis [3,4]. In rats, as short gestation species (term = 22 days), near term period is developmentally equivalent to the 2nd trimester compared to human fetuses, time period when active thyroid secretion starts [24]. Synthetic glucocorticoids, such as Dx, readily cross the enzymatic placental barrier and strongly influence developmental processes in fetuses [11]. We showed previously that the number of pituitary TSH cells was significantly decreased in 21-day-old fetuses after maternal Dx application, but circulating TSH concentration reached the control value, demonstrating a maturational promoting effect of Dx at the pituitary level [25]. Thereby, since the peripheral TSH level was unchanged, it could be excluded as the primary regulator of changes in the thyroid gland. Therefore, it may be assumed that direct action of Dx could be responsible for the alterations found in this study. Expression of GR mRNA in the thyroid gland [15], allows direct influence of Dx. Establishment of the definitive structure and function of fetal thyroid glands occurs between days 19 and 21 of gestation, when follicles are formed and synthesis of TH takes place. Known antiproliferative effects of glucocorticoids become apparent from day

16 of gestation, as shown by the marked decrease of follicular cell proliferation and consequently reduction of thyroid gland volume recorded in 19- and 21-day-old fetuses. Additionally, in 21-dayold fetuses under the influence of Dx significant decrease of all tissue fractions i.e. follicular cells, colloid and C cells were observed. Thyroid gland volume discriminated groups of control fetuses from those exposed to Dx, so representing the principal parameter that reflects changes in all the other analyzed parameters. Numerous studies have shown that prenatal treatment with Dx leads to fetal growth retardation; pituitary development is affected and the volume of the gland and number of hormone-producing cells (ACTH, TSH, gonadotropes) are reduced [26,27]. In addition, administration of Dx to pregnant rats induces a decrease of adrenal gland weight, volume of adrenal cortex and medulla, as well as lower proliferative activity of cortical and chromaffin cells during the fetal and neonatal period [27]. For functional characterisation of thyroid tissue Tg and TPO were labelled. These major thyroid-specific proteins stimulate an array of cellular events, including iodide production and organification, as well as thyroid hormonogenesis [3]. The characterization of the functional status of thyroid follicles through the immunohistochemical detection of the proteins involved in iodine transport and organification has been issue of several papers, and the link is established. Namely, immunohistochemical expression of TPO and other functional proteins and follicle activity are highly correlated [28–30]. Histological analysis of 19-day-old fetuses showed a large amount of Tg localized predominantly in follicular cells. After 48 h colloid filled the follicle interior, with strong Tg staining that was also present in the apical surface of follicular cells, from where Tg is released. Therefore, from day 21 of gestation the fetal thyroid gland is mature and capable of intensive TH synthesis in order to control the energy balance efficiently. Our histological analysis revealed that maternal Dx application did not influence the intensity, position or amount of Tg immunopositivity in near term fetuses. Moreover, the presence of resorptive vacuoles in the colloid, in both control and experimental fetuses, indicated intense resorption of colloid, confirming an ongoing process of hormonogenesis. The cytoplasmic TPO immunohistochemical staining of follicular cells, and marked TPO immunopositivity that was occasionally displayed in the border area of the apical membrane of follicular cells and colloid, indicated that TH synthesis intensified with advancing gestation. Under the influence of Dx even stronger TPO immunohistochemical positivity was located at the apical pole of follicular cells, i.e. more TPO, and consequently greater TH production. Published data on mammals, birds and reptiles show that glucocorticoids act as stimulators of T3 plasma level during the fetal period affecting tissue deiodination in a tissue-specific and age-dependent manner [31–33]. In ovine fetuses maternal Dx administration induced a rise in circulating T3 concentration by forcing hepatic T3 production, due to increased hepatic

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deiodination of T4 to T3, and by decreasing T3 degradation in the kidney and placenta, while T4 level was unaffected [12]. At the same time fetal glucocorticoid exposure strongly affects fetal thyroid gland structure, as presented. The immunohistohemical appearance of functional markers of TH synthesis, such as Tg and TPO, could indicate that the fetal thyroid is able sustain T4 production at the control level despite the significantly smaller volume of the TH producing tissue fraction in the gland. As in adults, the position of C cells during fetal development is predominantly in the central region of the thyroid lobe, the socalled C cell region [34]. Therefore, counting C cells by a physical fractionator approach, on systematically uniformly selected samples provides an accurate evaluation of the absolute number of C cells, taking into account their uneven distribution in the thyroid gland. Significantly fewer C cells were recorded in 19- and 21day-old Dx exposed fetuses, most probably due to a decreased proliferation rate, starting from day 16 of pregnancy. Although the absolute C cell number was lower, the numerical density of C cells was not altered, because application of Dx to gravid females led to a proportional decrease in both the number of C cells and thyroid gland volume. Moreover, C cell volume was not significantly changed. Considering that the interstitium i.e. connective tissue and blood vessels, in near term rat fetuses occupies a negligible part of the thyroid gland volume, the numerical density of C cells indicated a direct relationship between follicular and C cells during development. Consequently, it could be concluded that structural relations in the gland remained unchanged under the influence of Dx. The intrathyroidal relation between follicular and C cells and their mutual paracrine influence are of essential significance for development and functioning of the gland [35]. In adults it has been demonstrated that these two endocrine cell types maintain the same numerical relation in the thyroid gland, independently of its functional state (hyperstimulated versus hypothyroid) [36]. Fetal C cell volume increases slightly with advancing gestation in order to achieve complete maturation, probably representing enhancement of storage capacity in the cells, but maternal treatment with Dx did not influence C cell size in either examined period. In parallel with growth retardation, antenatal glucocorticoid administration influences the developing HPT axis at different levels. In the pituitaries of Dx exposed fetuses, a significantly smaller number of TSH cells achieved TSH production that was at the control level [25]. The results obtained here demonstrate that prenatal Dx treatment promoted maturational processes in the thyroid gland of 19- and 21-day-old fetuses. While thyroid gland size was markedly reduced, the immunohistochemical Tg and TPO staining pointed to intensive thyroid hormonogenesis. In turn, during critical time periods of development TH influence the accretion, differentiation and metabolism of many tissues and cell types, promoting maturational processes in near term fetuses. More precisely, glucocorticoids induce TH activation in the rat brain, enabling crucial structural and functional maturation during the perinatal period [37]. Enhancement of gluconeogenic enzyme activities in liver and kidney may also be achieved under the influence of prenatal TH, which is controlled by glucocorticoids, natural or synthetic [38]. Additionally, fetal thyroid mediated effects of glucocorticoids accelerated some aspects of fetal lung maturation [39]. Thereby short term benefits are achieved, maximizing the chance of survival of newborns with low birth weight. However, growth inhibition and the mentioned structural changes of the HPT axis under the influence of glucocorticoids determine the future capacity of HPT axis action [13,14]. It should be noted that even moderate transient changes in thyroid function during development may have lasting consequences on TH dependent tissues. The maternal glucocorticoid environment induces

permanent alterations in the physiology of the HPT axis in adult offspring of both sexes and, as a consequence, metabolic disorders may appear representing a long term risk [11,40]. In conclusion, the significantly reduced thyroid gland volume and decreased absolute number of C cells in near term rat fetuses, established after maternal Dx administration, might be the basis of the programming phenomenon, and could lead to decreased ability of an organism to adapt and react to different internal and external challenges during the life cycle. Acknowledgements This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant number 173009. We would like to thank Company Krka FARMA d.o.o. Belgrade, Serbia for kind donation of Dexamethasone phosphate. The authors wish to express their gratitude to Mrs. Ann Judith Nikolic´, Ph.D. for language correction on manuscripts. References [1] Conde E, Martin-Lacave I, Gonzalez-Campora R, Galera-Davidson H. Histometry of normal thyroid glands in neonatal and adult rats. Am J Anat 1991;191:384–90. [2] Fagman H, Nilsson M. Morphogenesis of the thyroid gland. Mol Cell Endocrinol 2010;323:35–54. [3] Brown RS, Shalhoub V, Coulter S, Alex S, Joris I, De Vito W, Lian J, Stein GS. Developmental regulation of thyrotropin receptor gene expression in the fetal and neonatal rat thyroid: relation to thyroid morphology and to thyroidspecific gene expression. Endocrinology 2000;141:340–5. [4] Taniguchi Y, Yasutaka S, Kominami R, Shinohara H. Proliferation and differentiation of thyrotrophs in the pars distalis of the rat pituitary gland during the fetal and postnatal period. Anat Embryol 2001;203:249–53. [5] Gereben B, Zavacki AM, Ribich S, Kim BW, Huang SA, Simonides WS, Zeold A, Bianco AC. Cellular and molecular basis of deiodinase-regulated thyroid hormone signaling. Endocr Rev 2008;29:898–938. [6] Garel JM, Besnard P, Rebut-Bonneton C. C cell activity during the prenatal and postnatal periods in the rat. Endocrinology 1981;109:1573–7. [7] Stoeckel ME, Porte A. Embryonic origin and secretory differentiation of calcitonin cells (C cells) in the fetal rat thyroid. Electron microscopic study. Z Zellforsch Mikrosk Anat 1970;106:251–68. [8] Crowley PA. Antenatal corticosteroid therapy: a meta-analysis of the randomized trials, 1972 to 1994. Am J Obstet Gynecol 1995;173:322–35. [9] Nathanielsz PW. Animal models that elucidate basic principles of the developmental origins of adult diseases. ILAR J 2006;47:73–82. [10] Fowden AL, Li J, Forhead AJ. Glucocorticoids and the preparation for life after birth: are there long-term consequences of the life insurance? Proc Nutr Soc 1998;57:113–22. [11] Harris A, Seckl J. Glucocorticoids, prenatal stress and the programming of disease. Horm Behav 2011;59:279–89. [12] Forhead AJ, Jellyman JK, Gardner DS, Giussani DA, Kaptein E, Visser TJ, Fowden AL. Differential effects of maternal dexamethasone treatment on circulating thyroid hormone concentrations and tissue deiodinase activity in the pregnant ewe and fetus. Endocrinology 2007;148:800–5. [13] Slone-Wilcoxon J, Redei EE. Maternal–fetal glucocorticoid milieu programs hypothalamic–pituitary–thyroid function of adult offspring. Endocrinology 2004;145:4068–72. [14] Carbone DL, Zuloaga DG, Lacagnina AF, Handa RJ. Prepro-thyrotropin releasing hormone expressing neurons in the juxtaparaventricular region of the lateral hypothalamus are activated by leptin and altered by prenatal glucocorticoid exposure. Brain Res 2012;1477:19–26. [15] Zhang XW, Li Y, Wang ZL, Li P. Glucocorticoid receptor subunit gene expression in thyroid gland and adenomas. Acta Oncol 2006;45:1073–8. [16] Flagel SB, Vázquez DM, Watson Jr SJ, Neal Jr CR. Effects of tapering neonatal dexamethasone on rat growth, neurodevelopment, and stress response. Am J Physiol Regul Integr Comp Physiol 2002;282:R55–63. [17] Nahaczewski AE, Fowler SB, Hariharan S. Dexamethasone therapy in patients with brain tumors – a focus on tapering. J Neurosci Nurs 2004;36:340–3. [18] Kilic I, Dagdeviren E, Kaya E. Effects of neonatal dexamethasone or methylprednisolone on rat growth and neurodevelopment. Pediatr Int 2008;50:489–94. [19] Neal Jr CR, Weidemann G, Kabbaj M, Vázquez DM. Effect of neonatal dexamethasone exposure on growth and neurological development in the adult rat. Am J Physiol Regul Integr Comp Physiol 2004;287:375–85. [20] Van Diest PJ, Baak JPA, Matze-Cok P, Wisse-Brekelmans ECM, van Galen CM, Kurver PH, Bellot SM, Fijnheer J, van Gorp LH, Kwee WS, et al. Reproducibility of mitosis counting in 2469 breast cancer specimens: results from multicenter morphometric mammary carcinoma project. Hum Pathol 1992;23:603–7. [21] Gundersen HJ, Jensen EB. The efficiency of systematic sampling in stereology and its prediction. J Microsc 1987;147:229–63.

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