THE ANATOMICAL RECORD 252:600–607 (1998) Immunohistochemical Study of Fibronectin and Thyroglobulin in the Thyroid Gland of Female Rats After Exposure to Radioactive Iodine V.S. USENKO,1,2* E.A. LEPEKHIN,2 I.N. KORNILOVSKA,1 V.V. LYZOGUBOV,1 E.O. APOSTOLOV,1 I.S. RALETS,1 AND M. WITT3 1Morphological Laboratory BIONTEC, 320000 Dnepropetrovsk, Ukraine 2Center for Molecular Physiology, National Academy of Science, 320625, Dnepropetrovsk, Ukraine 3Department of Anatomy, Technical University, 01307, Dresden, Germany ABSTRACT The aim of this work was to study the effect of a dose of 150 µCi 131I on the barrier properties of the thyroid epithelium in pregnant female rats. Thirty-five female Wistar rats were divided into a control and four experimental groups (each distinguished by the time of 131I injection: group I—no less then 12 days before mating; groups II, III, and IV—on 5th, 10th, and 16th days of gestation, respectively). The thyroid glands were fixed in Bouin’s fluid, embedded in paraffin, and stained immunohistochemically for thyroglobulin and fibronectin. In group IV the appearance of follicles with fibronectin-positive colloid demonstrates the penetration of blood plasma into the follicular lumen. There are more fibronectin positive follicles in group III. Regardless of the nature of the follicles’ contents, numerous thyrocytes with an intensive fibronectin positive reaction begin to appear in the follicles. In group II the number of fibronectin positive follicles and thyrocytes is clearly reduced, and in group I only a few remain. In group IV there is a noticeable reduction in the quantity of colloid inside the follicles and often an absence of any thyroglobulin positive reaction. There are thyrocytes in which thyroglobulin positive granules localized in the basal zone. There is thyroglobulin positive staining in the stroma and blood vessels. In group II thyroglobulin is no longer found in the stroma. Small doses of 131I provoke a serious breakdown in the thyroid epithelium’s barrier properties, although these changes are of a transient nature. The central zone of the thyroid gland reacts more actively and dynamically to exposure to radioactive iodine than the peripheral zone. Anat. Rec. 252:600–607, 1998. r 1998 Wiley-Liss, Inc. Key words: thyroid; 131I; hypothyroidism; fibronectin; thyroglobulin; epithelium; barrier; rat The study of maternal hypothyroidism induced by small doses of 131I is a question of particular topical interest in endocrinology (Cox et al., 1990; De Ona et al., 1991; Radiobiology, 1997). Radioactive iodine is widely used in the therapy and diagnostics of thyroid gland pathology. Furthermore, in view of the threat of various types of nuclear accidents, it is possible that large areas of land will be polluted by radioactive iodine isotopes as happened after the Chernobyl disaster. There are very few strictly morphological studies on the influence of radioiodine on the undamaged thyroid gland r 1998 WILEY-LISS, INC. (Nunez et al., 1966; Greig et al., 1970; Thurston et al., 1982; Liu et al., 1986; Artemova et al., 1976; O’Niell et al., 1968; Trolldenier et al., 1967a,b). A considerable propor- Grant sponsor: Government of the Free State of Saxonia; Grant number: AZ 1–6220.3–02/41. *Correspondence to: Dr. Vasilij S. Usenko, BIONTEC, Komsomolskaja Str. 52A/88, 320000 Dnepropetrovsk, Ukraine. E-mail: [email protected] Received 2 March 1998; Accepted 22 June 1998 THYROID GLAND AFTER RADIOACTIVE IODINE EXPOSURE tion of the available data on the effect of 131I on the body are concerned with the purely physiological changes observed in patients or in experimental animals. There is no information on the effect of 131I on the morphology of the thyroid gland of pregnant female rats and their offspring. There is no doubt that the study of the state of the mother and fetus during a pregnancy, which develops in conditions of radioactive iodine pollution, is of particular topical importance. Of the thyroid gland’s numerous morphological parameters, it is the condition of the follicular epithelium and, in particular, its barrier function, which is of most importance not only for diagnostic, but also for prognostic practice. A considerable increase in thyroglobulin penetration from the follicle cavity into the stroma can have fatal consequences because thyroglobulin is one of the main autoantigens produced during the progression of autoimmune disease of the thyroid gland (Berezin et al., 1993; Scherbaum, 1987). Thus, a change in the localization of thyroglobulin could be evidence of a change in the properties of the follicular epithelium, although this problem has not yet been researched. On the other hand, one of the most prevalent extracellular matrix proteins is fibronectin. Fibronectin is involved in the organization of the extracellular matrix via its interactions with the collagens and the proteoglycans and binds to basal membrane proteins such as the laminins. The insoluble fibronectin complex of the extracellular matrix originates either from the exudation of soluble fibronectin from plasma or from the local synthesis and secretion of a cellular form (Samuel et al., 1994). Thyrocytes have the capacity to synthesize the cellular form of fibronectin, whilst at the same time the properties of the actual cells themselves are determined by the interaction of thyrocytes with the components of the basal membrane, fibronectin included (Demeure et al., 1994; Ruoslahti, 1984; Samuel et al., 1994). Soluble fibronectin is known, together with other plasma proteins, to percolate through the damaged epithelium in case of a breakdown in the latter’s barrier properties (Casscells et al., 1990; Jin et al., 1991). However, there are no available data on the study of fibronectin in the thyroid gland after exposure to radioactive iodine. This work is a fragment of an extensive research program devoted to studying different aspects of the effect of small doses of radioiodine on pregnant female rats and their offspring. The aim of the present investigation was to study the effect of a dose of 150 µCi 131I (5.5 MBq) on the localization of thyroglobulin and fibronectin, and on the barrier properties of the thyroid epithelium in the thyroid gland of female rats who had given birth after being exposed to radioactive iodine either before pregnancy or at different stages of pregnancy. MATERIALS AND METHODS Thirty-five female Wistar rats (170 to 220 g body weight) were used. The animals were kept at 26°C and 60% air humidity in a controlled dark/light cycle (10 hr dark/14 hr light) with food and water available ad libitum. Females and males were mated from 7 a.m. to 9 a.m. Day 0 of gestation was calculated from the moment a large amount of sperm was found in the vaginal smears. We employed a rat hypothyroidism model using the minimum effective doses of 131I described by Reilly et al. 601 (1986). One group of animals (seven females) was used as a control and was not treated at all. The experimental rats were given intramuscular injections of 0.05 ml of physiological saline containing 150 µCi 131I (5.5 MBq), which corresponds to the absorbed dose of 0.5 Gy (Greig et al., 1970). Unlike the Reilly model, the rats were not given intraperitoneal injections, but rather intramuscular injections with the aim of lessening the direct radioactive activity on the embryos and ovaries, and excluding the possibility of mechanical damage. All the treated females were divided into four groups in accordance with the time of injection of 131I (group I—no less then 12 days before mating; groups II, III, and IV—on 5th, 10th, and 16th days of gestation, respectively). It is worth noting that the exposure to radioactive iodine 131I during pregnancy was effected 1 day before the onset of certain critical stages in the rat embryonic development. Group II. The 5th day of embryonic development is the eve of the implantation of the embryo and when the embryoblast splits into two layers: the epiblast and the hypoblast (Edwards, 1968). Group III. The 10th day of embryonic development is on the eve of thyroid primordia by means of evagination from foregut. It is also the stage of neurulation (Edwards, 1968; Phillips et al., 1959). Group IV. The 16th day of embryonic development is the eve of the beginning of follicle formation, of iodine concentration as well as thyroxin production (Phillips et al., 1959; Luciano et al., 1979; Remy et al., 1980). Decapitation of all animals was performed under hexenal anesthesia (30 mg per kg, ip) from 3 to 20 hr after giving birth. Then, rats were bled and dissected. The right lobes of the rats’ thyroid glands were fixed in Bouin’s fluid, routinely dehydrated, and embedded in paraffin. Paraffin sections (5 µM) were cut and mounted on gelatin-chromealum-coated slides, two or four sections per slide. The concentrations of plasma T4 and TSH were measured using the EIA test (Medix Biochemica, Finland). The staining techniques used were Mallory-Slinchenko’s method (Slinchenko, 1964), immunohistochemical staining with the use of antibodies to fibronectin (FN) and thyroglobulin (TG). Rabbit polyclonal antibody versus bovine thyroglobulin (Serva, Heidelberg, Germany) was obtained in our laboratory. Western immunoblotting of the SDS-fraction from rat thyroid gland with TG-antiserum detects one main band of 300 kD polypeptide and one minor band of about 35 kD polypeptide. TG rabbit polyclonal antiserum was used at a dilution of 1/8,000, FN polyclonal antibody (Sigma, St. Louis, MO)— dilution 1/200 and monoclonal antiserum vs. cells’ fibronectin (cFN; Sigma)—dilution 1/200. An indirect avidin-biotinperoxidase method was used for immunochemical staining (Lacey, 1989). Peroxidase was visualized by incubating the sections in 0.05% of 3.3-diaminobenzidine tetrahydrochloride solution containing 0.01% hydrogen peroxide. The specificity of staining was controlled by substituting the primary antibodies with a buffer as a negative control and by using skin samples as a positive control. After being rinsed in water, the sections were lightly counterstained with hematoxylin. Statistical analysis included descriptive statistics for each group. The significance of the difference between the mean values of control and treated 602 USENKO ET AL. Fig. 1. Control rat’s thyroid gland. An intensive FN-positive reaction in the blood vessels (long arrows) and the basal membranes thyroid epithelium (short arrows). Connective tissue cell with FN-positive cytoplasm and a sharply positive staining of the cell contour (arrowhead). Immunohistochemical FN staining with hematoxylin counter staining. ⫻50. groups was calculated by both the Student’s t-test and the Mann-Whitney U-test. RESULTS A measurement of the hormone level in the female rats’ blood in the first postnatal days showed a significant reduction in the concentration of thyroxin in all experimental groups (mean 43%) as compared to the control group (U-test and t-test P ⬍ 0.05). Parallel to this, the level of TSH in the females of all experimental groups showed a dramatic increase—about 792% (U-test and t-test P ⬍ 0.05). The Control Group Fibronectin. It is essential to note that the polyclonal antiserum vs. human FN used in this study should, according to the data sheet, connect with both the plasma and cellular forms of human FN. However, a parallel staining of sections of the control rats’ thyroid glands using both polyclonal antiserum vs. human FN and monoclonal antiserum vs. cellular FN showed that the polyclonal antiserum did not stain those structures which were stained by the monoclonal antiserum. This proves that polyclonal antiserum in the rat thyroid gland connects only with plasma FN. The use of polyclonal antiserum vs. FN in the sections of the thyroid gland of the control animals reveals that the most intensive presence of FN is found in the blood vessels. With regard to other structures FN can be found predominantly in the basal membranes of the thyroid epithelium. There is no expression of FN in the epithelium itself or in the interfollicular contacts. Furthermore, FN positive staining reveals separate, relatively thin fascicles of fibres in the organ’s stroma. In some sections one occasionally finds connective tissue cells with FN-positive cytoplasm and a sharply positive staining of the cell contour (Fig. 1). The most intensive FN reaction is in the central zone of the thyroid gland lobe and as a rule this reaction decreases Fig. 2. Control rat’s thyroid gland. An intensive TG-positive reaction in the colloid inside the follicles (arrows) of the thyroid gland, and with the apices of the thyrocytes (arrowhead). Immunohistochemical TG staining with hematoxylin counter staining. ⫻50. sharply as one moves closer to the capsule. The capsule of the thyroid gland itself may have a highly diverse FN reaction across its different sections and is stained extremely unevenly. The visualization of cellular FN using monoclonal antiserum showed that it is found in thyrocytes in the form of intensively stained structures which are spindle-shaped, reticular, or granular in form. These structures are located in the apical part of the perinuclear zone. Examination of tangential sections which pass through the apical parts of thyrocytes sometimes reveals a positive staining of cell contours. A slightly positive FN staining gives a heterochromatin result in the nuclei of some thyrocytes leaving karyolemma and nucleoli unstained. The blood vessel content was the only substance in the sections of the rat thyroid gland which connected with both polyclonal and monoclonal antiserum. However, the cellular FN reaction was extremely weak. From the above one can conclude that the polyclonal antiserum we used exposes only plasma FN in the rat thyroid gland; a finding of particular importance for successfully realizing the goal of our research. Thyroglobulin. In the thyroid gland of the control female rats which had given birth, the antiserum vs. TG connected with the colloid inside the follicles of the thyroid gland, and with the apices of the thyrocytes. At the same time, the most intensive staining was observed in the zone where the epithelium and colloid contacted (Fig. 2). The cytoplasm of the apical zone of thyrocytes also often showed a positive TG result. Individual thyrocytes with intensively diffusely stained cytoplasm are very rarely found. These cells have pyknotic nuclei and the structure of their cytoplasm is seriously damaged. It is obvious that this staining is caused by TG imbibition of the matrix of the dead cell. Groups of follicles with a sharply increased level of resorption in the colloid are sometimes found where the colloid has a frothy appearance and a sharply positive TG reaction, whilst in the apical parts of thyrocytes large TG-positive drops of colloid are visible. THYROID GLAND AFTER RADIOACTIVE IODINE EXPOSURE 603 Fig. 3. Treated rat’s thyroid gland on the 6th day after exposure 131I (group IV). Connective tissue swelling. An intensive FN-positive reaction in the stroma. Immunohistochemical FN staining. ⫻50. Treated Animals: Group IV Fibronectin. On the 5th/6th day after exposure to radioactive iodine the connective tissue of the thyroid gland is swollen and has a sharply FN-positive reaction (Fig. 3) which in its intensity is comparable with the FN reaction in the blood vessels. At the same time, in the connective tissue one finds bubble-like areas of varying sizes in which there is no FN. In the swollen layers of connective tissue there are inflamed fascicles of fibres with an intensive dark-brown staining, this being evidence of a high concentration of FN in them. No FN positive cells were found in the stroma. The clear demarcation of zones in the lobe of the thyroid gland is preserved. The largest swelling and an FN positive staining is found in the central zone of the lobe. The organ’s capsule is substantially swollen and loose in appearance. At the same time it has a relatively uniform FN positive reaction. With regard to thyroid gland parenchyma, it is predominantly in the lobe’s central zone that one finds follicles, the total contents of which display a sharply positive diffuse FN reaction. Thyroglobulin. In the thyroid gland of the female rats exposed to radioactive iodine on the 16th day of pregnancy, i.e., 5–6 days before birth, one found considerable polymorphism in relation to the morphology of TG in the follicles. The follicles were reduced in size. The contents of a large number of follicles, predominantly small, do not reveal a clear TG reaction. Many follicles have colloid which is reticular or granular in structure. A close analysis of the sections of follicles which where stained using antibodies vs. TG and plasma FN showed that the colloid in these follicles contains both FN and TG (Fig. 4A,B). A high level of variety was observed in the presence and nature of TG localization in the thyrocyte cytoplasm. A substantial number of thyrocytes which show morphological signs of degeneration have cytoplasm with an intensive, uniform TG-positive staining. The nuclei of these thyrocytes are quite often displaced into the cell’s apical zone, deformed, or swollen in appearance whilst the cytoplasm is compressed. Those thyrocytes which show no sign Fig. 4. Treated rat’s thyroid gland on the 6th day after exposure 131I (group IV). Close, successive sections of the thyroid lobe: A: Immunohistochemical FN staining. FN-positive colloid with reticular structure (arrowheads). Staining of the connective tissue fibre (short arrows) and vessel contents (long arrows). ⫻100. B: Immunohistochemical TG staining with hematoxylin counter staining. The colloid is the same as in A, follicles retain TG-positive staining, have a reticular structure (arrowheads). ⫻100. of crude degenerative change mostly have TG-positive granules in the apical zone or are filled with stained granules. However, there are also thyrocytes in which clearly distinguishable concentrations of TG positive granules are only evident in the basal zone (Fig. 5). As well as the changes in the localization of TG in the parenchyma of the thyroid gland one also finds this protein in the stroma. In the stromal layers there is localized TG saturation of the connective tissue in close proximity to the thyrocytes with an intensive, diffuse TG positive staining. Furthermore, there are individual intensive TG-positive granules. The contents of the blood vessels exhibit a particularly weak uniform TG positive reaction. Group III Fibronectin. At day 11–12 after exposure to 131I there is a weaker indication of swelling in the connective tissue of the thyroid gland. However, the connective tissue layers remain loose with thick, clearly visible, fibres and a diffuse 604 USENKO ET AL. Fig. 5. Treated rat’s thyroid gland on the 6th day after exposure 131I (group IV). Immunohistochemical TG staining with hematoxylin counter staining. Distinct TG-positive colloid with signs of resorption (C). Presence of intensive TG positive reaction in the basal zone of numerous thyrocytes (arrowheads) and uniform diffuse reaction spread over whole cell (arrow). ⫻250. Fig. 6. Treated rat’s thyroid gland on the 12th day after exposure 131I (group III). Loosening of the organ’s connective tissue. FN positive thyrocytes with damaged structure (long arrows). FN positive vacuoles of varying size in colloid (short arrows). Immunohistochemical FN staining with hematoxylin counter staining. ⫻250. FN positive reaction. The capsule is dense with a uniformly weak positive staining. There are follicles in all zones of the section the contents of which show a strongly positive FN reaction. In the cavity of such follicles FN is either diffusely distributed or represented in the form of granules or reticular structures. The follicles with FN positive contents often have thyrocytes with FN saturated cytoplasm. A significant number of follicles whose contents do not have a total FN positive reaction also contain thyrocytes with an intensive FN positive cytoplasm reaction. In the colloid, the apices of such cells often contain differently sized vacuoles which show a FN positive reaction or a local diffuse positive staining of the colloid (Fig. 6). Evidence of zonality in this group can be seen by the fact that the various forms of damage do not effect those follicles which are located directly under the capsule. At the same time, in the center of the lobe the structures of the epitheliomers and interfollicular contacts are quite seriously damaged and difficult to identify. Thyroglobulin. On days 11–12 after exposure, the follicles in the thyroid gland of the females who received radioactive iodine have a predominantly granular colloid, which is stained with varying intensity. The localization of TG-positive follicles in the section of the thyroid gland’s lobe matches the localization of FN-positive follicles. A large number of TG positive granules are found in the cytoplasm of thyrocytes. Small drops may be uniformly distributed in the cytoplasm, whilst large ones are mostly found in the apical zones. Degenerating thyrocytes with an intensive TG-positive staining are sometimes found. The presence of TG in the stroma structures is no longer observed. pronounced and have a coarse fibrous structure. The connective tissue on the whole shows a more uniform but less intensive positive FN reaction than for the animals of the previous group. The organ’s capsule is FN positive, fibrous, thickened, and solid. Isolated follicles with FNpositive colloid of reticular structure were preserved mainly in the lobe’s peripheral zone. Individual FN-positive thyrocytes are still found in the epithelium of follicles. Thyroglobulin. On days 16–17 after exposure to radioactive iodine certain differences in the localization of TG are even more noticeable. In most follicles the colloid is subject to almost total resorption and the remains of TG positive material is only preserved in the center of the cavity. The cube-shaped thyrocytes of these follicles develop a relatively smoothly contoured luminar surface. Follicles filled with TG-positive colloid can be found only on the organ’s periphery (Fig. 7). However, dying thyrocytes saturated in TG are still present. Group II Fibronectin. On days 16–17 after exposure to radioactive iodine the stroma’s connective tissue looks more organized. The connective tissue layers and septa are well Group I Fibronectin. Within 30 to 40 days after the female rats were exposed to radioactive iodine the stroma of the thyroid gland is in many ways similar in structure to that of the control animals. The thickness of the stromal layers is close to normal and their positioning clearly shows the usual zonal division. However, the connective tissue is distinguished from its normal state by the high density of fibres with a FN positive reaction. The organ’s capsule is FN positive and fibrous. (Fig. 8). Isolated follicles with an FN-positive colloid are still found. There is an insignificant number of FN-positive thyrocytes. The morphology and nature of staining on the organ’s periphery is close to that of the control animals. Thyroglobulin. The most significant differences in comparison with the control are observed in this group. There is virtually no TG-positive colloid, except for a few individual follicles. At the same time, there is a large number of colloidal drops with intensive TG staining in the THYROID GLAND AFTER RADIOACTIVE IODINE EXPOSURE Fig. 7. Treated rat’s thyroid gland on the 16th day after exposure 131I (group II). Very rarely follicles with intensive TG positive colloid can be found in the lobe’s peripheral zone. Immunohistochemical TG staining with hematoxylin counter staining. ⫻100. Fig. 8. Treated rat’s thyroid gland on 30th day after exposure 131I (group I). Intensive FN positive reaction in the stroma of central zone of lobe. Follicles rarely contain homogeneous FN positive colloid (arrowhead). Thyroid capsule is loose. Nature of staining of some areas in periphery of thyroid lobe is similar to that of control. Immunohistochemical FN staining with hematoxylin counter staining. ⫻100. apical zones of the thyrocytes. The same picture, irrespective of zone, is repeated all over the parenchyma. DISCUSSION The application of a dose of 150 µCi 131I (5.5 MBq) to pregnant female rats at different stages of pregnancy led to the onset of a hyperthyroid state which was defined on the first day after birth. The level of T4 in these animals is reduced by almost half whilst the content of TSH in the antiserum of the blood increases approximately 10-fold. The dose used in the experiment is equal to 0.5 Gy of the absorbed dose (Greig et al., 1970), which is five times less than the minimum dose needed to cause the development of clinical symptoms of radiation damage in rats (Bajenov 605 et al., 1990). This dose is close to that received by those Soviet citizens living on the territories polluted by 131I as a result of the Chernobyl nuclear accident (ÑEC Report EUR 15248, 1993). It is obvious that the localization of plasma FN in the thyroid gland of the control animals is exclusively restricted to stromal structures such as blood vessels, basal membranes, fibres of the connective tissue, and its individual cells. The ground substance of the connective tissue, contains the normal minimum level of plasma FN. TG is found in the follicle lumen and in the cytoplasm of thyrocytes. Thus, the immunohistochemically exposed localization of plasma FN and TG in the control animals fully corresponds to earlier published data (Birembaut et al., 1980; Rosai et al., 1992). On the 5 to 6 days after exposure to radioactive iodine the nature of the distribution of plasma FN changes sharply. The connective tissue is swollen, enlarged, and all its components are saturated with plasma FN. It is obvious that such a change is the result of radiation damage to the organ’s exchange vessels, a sharp increase in the permeability of their endothelium, and the filtration of a significant amount of blood plasma. At the same time, the appearance of FN positive follicles demonstrates the penetration of blood plasma into the follicular lumen, and consequently of a breakdown in the barrier function of the thyroid epithelium on the 5th to 6th day after exposure to a small dose of radioactive iodine. There are even more FN positive follicles by day 11–12. At the same time, regardless of the nature of the follicle contents, numerous thyrocytes with an intensive FN positive reaction begin to appear in the follicles. As a rule, they all show signs of degeneration. A similar picture is observed in heart myocytes subjected to experimental ischemia. (Casscells et al., 1990; Froen et al., 1995). The colloid can remain intact above the apices of these thyrocytes, but more often the colloid is saturated in FN, or resorbent vacuoles with fibronectin positive contours are situated here. However, by day 16–17 the number of FN positive follicles and thyrocytes is sharply reduced, and by day 30–40 only a few remain, thus giving clear evidence of the restoration of the thyroid epithelium’s barrier functions. By day 5–6 after exposure there is already a noticeable reduction in the quantity of colloid inside the follicles and often an absence of any TG-positive reaction. On the one hand, its production could be suppressed by the influence of radioactive iodine. On the other hand, a substantial number of thyrocytes in the apical and other parts of the cytoplasm contain numerous TG-positive granules, thus providing evidence of active colloid resorption by these cells. However, TG resorption in thyrocytes is a normal phenomenon. Far more important is the fact that very shortly after exposure to radioactive iodine, numerous thyrocytes appear with signs of imminent death and a sharply positive homogenous TG reaction. The discovery of diffuse TGpositive staining of the connective tissue close to the basal surface of these thyrocytes provides evidence of the possibility of TG filtration into the stroma through the matrix of dead cells or of its direct penetration through the damaged contacts between thyrocytes. Furthermore, in the thyroid stroma TG is often present in the form of individual drops. The discovery of thyrocytes in which TG-positive granules are localized in the basal zone strongly suggests that a possibility that those thyrocytes with damaged polariza- 606 USENKO ET AL. tion secrete TG not into the follicle lumen, but rather through the basal membrane into the connective tissue; although according to available data, a breakdown in the polarity of thyrocytes in vivo has not been described whilst a change in the polarity of follicular cells has been found to be possible under the influence of various factors in vitro (Nilsson et al., 1988). In any case, the discovery of a weak TG positive reaction in blood vessels on the 5th to 6th day after exposure indicates that the above mentioned forms of damage combine to ensure the entry of a significant amount of TG into the blood. These data give morphological confirmation of the assumption that the increased production of autoantibodies after 131I induced thyroid gland damage is the result of a massive input of iodized thyroglobulin into the blood (Nevstrueva et al., 1972). With regard to the distribution of TG in the thyroid gland in the later stages, one should note that after 16 to 17 days TG is no longer found in the stroma or in the vessel contents. There is a sharp decrease in the number of cells which have TG saturated cytoplasm. This shows the noticeable restoration of the thyroid epithelium’s barrier functions. Thus, even small doses of 131I provoke a serious breakdown in the thyroid epithelium’s barrier properties, although these changes are of a transient nature. The study of the change in the localization of TG and FN as a result of exposure to radioactive iodine or other damaging factors can serve as a method for evaluating the influence of these factors on the barrier properties of the thyroid epithelium. However, in this period the quantity of colloid continues to decrease and in the majority of follicles colloid remains are only preserved in the center of the cavity. After 30 to 40 days, remains of colloid are found only in isolated follicles, this being evidence of the significant predominance of colloid resorption over colloid production. 131I is cleared with a half-life of 3.38 ⫾ 0.01 days (Reilly et al., 1986). Taking into account the fact that female rats of this experimental group were exposed to the radioactive iodine no less than twelve days before mating (more than the 10-fold time of effective half-life of the 131I up to beginning of embryo’s thyroid iodine intake), the development of fetuses in this group took place in a state of 131I-induced maternal hypothyroidism without the influence of direct effects of radioactive iodine. This group is the second control group for the evaluation of direct effects 131I on embryogenesis. It is important to note that colloid resorption and the appearance and disappearance of TG-positive and FNpositive thyrocytes and follicles occurs considerably earlier in the central zone of the lobe of the thyroid gland than it does in the peripheral zone. Furthermore, the changes in the peripheral zone are of a far less pronounced nature. 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