Radioactive Iodine-induced hypothyroidism interferes with the maturation of reproductive organs during puberty in immature female rats

Abstract

Animal and human studies suggest that thyroid hormone may have critical roles in the development of the ovary. For example, thyroid deficiency disrupts the ovarian microarchitecture and menstrual cycle in neonate and adult women, respectively. Therefore, it is conceivable that thyroid deficiency might disrupt sexual maturation during the peri-pubertal period. To investigate the impact of radioactive iodine-induced thyroid deficiency on reproductive organs throughout puberty, immature female rats were given water containing radioactive iodine (0.37 MBq/g body weight) twice, on postnatal days 22 and 29. Radioactive iodine-induced hypothyroidism was revealed by low free thyroxin levels. Thyroid deficiency delayed the onset of vaginal opening, reduced ovarian weight and the number of medium-sized follicles and led to elongated uteri. However, there was no effect on the estrous cycle or absolute uterus weight. We conclude that radioactive iodine-induced thyroid deficiency delays sexual maturation and alters normal ovarian growth in peri-pubertal rats.

Introduction

Juvenile hypothyroidism is one of most common thyroid disorder among children [1]. Hypothyroidism in children and adolescents is commonly caused by an autoimmune disease such as Hashimoto’s thyroiditis [2], as well as by radioactive iodine therapy for Graves’ disease [3], which appears to be increasing in the pediatric population. However, many cases involve idiopathic subclinical hypothyroidism associated with a variety of different clinical problems [4]. Juvenile hypothyroidism is thus not easy to recognize early because of its diverse clinical presentations, including normal to subnormal skeletal growth [5, 6], which may depend on the extent and duration of hormone deficiency. Given the fact that thyroid hormones are essential regulators of the growth, metabolism and function of various organs [7], thyroid hormone deficiency is especially important throughout puberty.

Adolescence is a critical period for the growth and maturation of the reproductive organs; it is characterized by extensive morphological and functional changes [8], suggesting that thyroid deficiency could have an even more pronounced impact on the reproductive system at that time than it does in adults. Human studies have generally been of small size and have been flawed by unreliable assessments of the extent and duration of thyroid deficiency. Furthermore, most studies of thyroid deficiency have been carried out in adults [animal or human] and cannot be generalized to children and adolescents. In addition, clinical experiments have revealed gender differences in pubertal responses to thyroid deficiency [9]. Impairment of the endocrine processes that regulate menarche (pubertal onset) could have lasting detrimental effects on reproductive function. Hence, understanding the impact of thyroid hormone deficiency on the reproductive organs is of vital importance to adult well-being.

Previous studies suggest that hypothyroidism causes abnormal pubertal development in both males and females [10, 11]; however, studies have not shown that it can directly affect ovarian growth during puberty. Therefore, the aim of this study was to investigate whether thyroid hormone deficiency directly affects ovarian growth and sexual maturation in immature female rats, and to observe the gross and microscopic features of any observed effects.

Materials and methods

Animals and experimental design

Two-week-old female Sprague–Dawley rats (n = 12) were obtained from Samtako Biokorea (Kyunggi, Korea) and allowed to acclimate. At weaning (21 days of age), the animals were assigned to groups according to body weight in order to eliminate variation in mean body weight between groups and individual animals were housed in separate plastic cages under controlled conditions (22–24 °C, humidity 40–50%, 12 h light–dark cycle), with free access to food and water. Animal care was consistent with institutional guidelines, and the Hanyang University ACUC committee approved all procedures involving animals (HY-IACUC-2013-0110A).

The rats weighed approximately 42.5 ± 2.2 g on the first day of the experiment, and were randomly assigned to 2 groups (n = 6/group), a control (CT) and a radioactive iodine (RAI)-treated group, based on their body weights to eliminate any difference in mean body mass between the groups. RAI (KAERI, Daejeon, Korea) was dissolved in water and fed via gavage (0.37 MBq/g body weight) to ensure its complete consumption; a control group received the same volume of distilled water on PD (postnatal day) 22 and 29.

Animals were examined for any clinical signs or toxicity. Body weights were recorded to the nearest 0.1 g at one week-intervals using an electronic scale (Dretec, Seoul, Korea). The treatment duration covered the juvenile (PND21-35) and peripubertal (PND35-55) [12] periods of female rats in order to ensure an accurate assessment of effects on the growth and maturation of the reproductive system. The day before animals were killed, body fat and lean body mass were measured by dual-energy X ray absorptiometry (DXA) (Discovery W QDR series, Hologic Inc., Bedford, MA, USA), using a dedicated small animal software package. All the animals were killed with isoflurane (Forane solution; Choongwae Pharma, Seoul, Korea) following ethical protocols and procedures 3 weeks after the last RAI treatment. Terminal blood samples were collected by heart puncture and sera were stored at − 70 °C.

Pubertal study

As an index of sexual maturation, animals were examined daily for vaginal opening (VO) from PD 25. Age was recorded on the day VO was first detected. As soon as complete VO had been confirmed, vaginal smears were taken daily (between 9:00 and 10:00 A.M.). Vaginal lavage was collected by repeated pipetting of 0.9% saline into the vagina. The lavage fluid was applied to a clean glass slide and the smear was viewed immediately under low magnification (× 100). Cytology was evaluated and the stage of the estrous cycle was determined by the method of Everett [13]. The interval between VO and the first estrus smear was recorded for all the rats, because the first estrus indicates the time of first ovulation. Cycle regularity was evaluated for an approximately 10-days period according to validated published criteria [14]. Briefly, females that displayed the characteristic estrous stages during each cycle with the classic cycle duration of 4–5 days were classified as having regular cycles. Females with aberrant periods between each estrous stage or with abnormal frequencies of a given stage were classified as having irregular cycles.

Measurement of organ weights

To investigate the effect of RAI on the pubertal growth of reproductive organs in vivo, the uteri and ovaries were dissected, and cleaned of fat and connective tissue. Ovaries (paired) and uteri were then weighed to the nearest 0.001 g with an electronic scale (Adventure™ electronic balances, AR1530, OHAUS Corp., USA) and their morphology was grossly evaluated. Then, the ovaries were fixed in 10% buffered formalin (pH 7). In addition, uterine length was measured from both horns.

Ovarian histology and follicle numbers

After approximately 24 h of fixation, ovaries were processed for paraffin embedding and sectioning. Serial sections of 5 µm thickness were made from the mid portion of the ovary and stained with hematoxylin and eosin. Numbers of healthy follicles that had formed at different stages were counted as previously described [15]. Secondary follicles were defined as containing larger oocytes and more than one layer of granulosa cells (GCs), along with theca cells. Tertiary follicles included antral follicles. Atretic follicles characterized by degenerating oocytes, disorganized GC layers, and folded zona pellucida were excluded. Follicles in which the nuclei of oocytes were visible were counted on the largest cross section of each ovary by the same trained and blinded examiner using a light microscope (DM4000B, Leica, Heidelberg, Germany). Six serial sections were traced for each ovary and combined to obtain a value per animal. Average numbers of each follicle population are presented as mean ± standard deviation (SD).

Assay of hormones

To confirm that RAI treatment caused hypothyroidism, serum levels of free thyroxine (FT₄) were measured using a commercially available radioimmunoassay kit (OCFD03-FT4) (Cisbio Bioassays, Codolet, France). Intra- and inter-assay coefficients of variance for FT₄ were ≤ 15%. The limit of detection of FT₄ under the test conditions was 0.5 pg/ml. All samples were run in duplicate.

Statistical analysis

Data are expressed as means ± standard deviations (SD). Data were analyzed with IBM SPSS Statistics 21 for Windows (IBM Corp., Armonk, NY, USA). Statistical significance for two-group comparisons was determined with the Mann–Whitney U-test, and significance was accepted at p < 0.05.

Results

Total body mass and amount of body fat

Hypothyroidism in the RAI group was confirmed by a significantly decreased level of free T₄ (CT, 1.42 ± 0.06 vs. RAI, 0.11 ± 0.01 ng/dL) (p < 0.05). To determine whether RAI-induced hypothyroidism was associated with changes in body composition, total body mass (TBM), body fat and lean body mass (LBM) were analyzed using DXA (Table 1). Throughout the experimental period, no treatment-related clinical signs were observed. TBM as an indicator of general growth was significantly reduced in the treated group (p < 0.05), as were total body fat and LBM (p < 0.05). However, the proportions of body fat and LBM did not differ between the control and RAI-fed groups, indicating that there was a parallel reduction in body fat and LBM in the RAI-fed rats.

Table 1.

Effect of RAI-induced hypothyroidism on body weight gain and body fat determined by DXA

Groups	CT	RAI
TBM (g)	192.4 ± 3.9	141.9 ± 9.7*
Fat
Weight (g)	25.1 ± 2.5	20.9 ± 3.3*
% of body mass	14.3 ± 4.3	14.4 ± 1.3
LBM
Weight (g)	164.6 ± 11.4	118.4 ± 7.3*
% of body mass	82.5 ± 4.3	82.3 ± 1.3

Values are expressed as mean ± SD of 6 animals per group. Body weight gain (g) = terminal body weight – Values are means ± SD of six rats per group. TBM, total body mass; fat %, total body fat divided by TBM; LBM, lean body mass; LBM %, lean body mass divided by TBM. CT, control; RAI, radioactive iodine-treated group. ^*p < 0.05 vs. CT. Statistical significance for two-group comparisons was determined with the Mann–Whitney U-test, and significance was accepted at p < 0.05

Onset of puberty

To investigate whether RAI-induced hypothyroidism affected the development of puberty, indexes of female puberty, such as VO and first estrus, were compared [8, 14]. Mean age at complete VO was PD31.7 ± 2.1 (range 30–34) for the control and PD35.7 ± 2.1 (range 34–38) for the RAI-fed group (p < 0.05) (Fig. 1a). The day of first estrus was somewhat delayed in the RAI-fed group (PD46.6 ± 4.2) compared to the controls (PD44.0 ± 2.0) (Fig. 1b), although the difference was not statistically significant. On the other hand, only one animal in each group had at least one estrous cycle, while all the others displayed irregular cycles (data not shown).

Fig. 1.

Effects of radioactive iodine-induced hypothyroidism on the onset of female puberty. Onset of Puberty was determined by a mean age at vaginal opening and b mean age at first estrus. Values are expressed as mean ± SD of six rats per group. The cell types present in vaginal swabs were used to identify stages of the estrus cycle from vaginal opening to the end of the experiment. V.O vaginal opening, PD postnatal day, CT control, RAI radioactive iodine-treated group. ^*p < 0.05 vs. CT

Growth of reproductive organs

The results of the effects of RAI-induced hypothyroidism on reproductive organ weights are summarized in Figs. 2 and 3. Ovarian weights were about 30% lower in the RAI-treated rats than the controls (Fig. 2a; CT, 39.3 ± 4.9; RAI, 28.0 ± 8.7 mg; p < 0.05). Ovarian weights relative to body weights were comparable in the two groups (CT, 19.8 ± 2.4; RAI, 19.23 ± 4.5 mg/100 g body weight) (Fig. 2b). Uterine weights varied greatly between individuals, but absolute uterine weights were comparable in the groups (Fig. 3a). Relative uterine weights appeared to be higher in RAI group (CT, 180.8 ± 26.5; RAI, 254.3 ± 64.9 mg/100 g body weight) (p < 0.05) (Fig. 3b). In addition, we measured and summed the lengths of both uterine horns, and found that uterine length was significantly greater in the RAI group (CT, 71.6 ± 14.5 vs. RAI, 91.7 ± 8.4 mm) (p < 0.05) (Fig. 3c).

Fig. 2.

Effect of radioactive iodine-induced hypothyroidism on ovarian maturation. a Absolute ovarian weights correspond to the means of the weights of left and right ovaries. b Relative weights are ovarian weights (mg) per 100 g of terminal body weight. Values are means ± SD of six rats per group. c Follicle counts in the ovaries of control and RAI-treated rats. Data are presented as mean ± SD of follicle numbers. Six serial sections were traced for each ovary and the results were combined to obtain the value for each animal. Prm primordial follicles, PF primary follicles, SF secondary follicles, TF tertiary follicles, POF preovulatory follicles, CT control, RAI radioactive iodine-treated group. ^*p < 0.05 vs. CT

Fig. 3.

Effect of radioactive iodine-induced hypothyroidism on uterine growth. a Absolute uterine weights. b Uterine weights (mg) per 100 g terminal body weight. Values are means ± SD of six rats per group. c Uterine length given by the sum of the lengths of the two uterine horns. CT control, RAI radioactive iodine-treated group. ^*p < 0.05 vs. CT

Ovarian follicle development

Ovaries were analyzed to assess the characteristics and numbers of follicles at the different stages of development (Fig. 2c). No significant differences were observed in numbers of primordial and primary follicles between the groups. However, the number of follicles at more than the secondary stage was lower in the RAI group; in particular, a significant difference in the number of secondary follicles was noted (p < 0.05).

Discussion

In the present study we demonstrate that RAI-induced hypothyroidism in immature female rats delayed sexual maturation along with reduced ovarian size and aberrant follicular growth. To the best of our knowledge, there have been no previous reports of the influence of RAI-induced hypothyroidism on ovarian and uterine growth during puberty.

Adolescence is a critical period for growth and maturation of the reproductive organs, and is characterized by extensive morphological and functional changes [8], suggesting that thyroid hormone deficiency might have an even more pronounced impact on the reproductive system at that time than it does in adults. The adolescence period in rats lasts from 21 to 45 days of age [12]. Therefore, the 28-day period (PND 22–49) of the female pubertal assay spans the time during which the ovary develops and the rat brain begins to respond to the positive feedback of estrogen, resulting in the LH surge and the occurrence of the first estrous cycle and ovulation [8]. In this study, we applied RAI to PD22 and PD29, and observed them for three weeks during which covered the pubertal age for SD rats. This is the critical period for various neuroendocrine developments, when the hypothalamus-pituitary–gonadal axis is still immature and therefore the levels of sex hormone in the body are relatively low, as well as a critical window for increased susceptibility to insults. The RAI dosage chosen was based on dosages that had been shown to destroy all thyroid tissue without compromising the general health of rats [16].

Increased body size including body fat is one of the major physical changes that characterize normal pubertal development. In this study we analyzed body mass and percentage of body fat using DXA. RAI-treated animals developed significantly reduced body weight and fat mass compared to the controls (Table 1). Several animal studies have reported reduced body weight in hypothyroid rats [17, 18]. On the other hand, juvenile hypothyroidism usually results in increased weight gain [5, 10, 19], although decreased appetite can be a common symptom in children [6]. These species differences (human vs. rat) in the effects of thyroid deficiency may be attributed to the presence of functionally active brown fat in the rat, which responds to alternative thermogenesis in hypothyroid conditions [19]. In addition, RAI may cause damage to the salivary glands [20], thereby decreasing food intake, although food intake was not considered in this study.

A number of animal and human studies have pointed to some effect of body fat on the initiation of puberty at least in girls [21], although effects of weight and adiposity are not sufficient for puberty to occur. In fact, juvenile hypothyroidism mostly delays pubertal onset and progression [11]. Similarly, a number of animal studies have shown delayed vaginal opening by about 3–5 days in hypothyroid rats [22, 23]. Likewise, we found 4 days’ delay in RAI-treated animals (Fig. 1a). Therefore, our results also suggest that reduced body weight and fat deposition in RAI-induced hypothyroid rats may contribute to the delay of puberty. Age at first estrus is considered an end point for assessing completion of the pubertal process in the rat [14]. We observed first estrus at PD 44 in the controls, whereas RAI-treated animals delayed first estrus to PD 46.6, although this difference was not statistically significant, due perhaps to the small number of animals (Fig. 1b). The first estrus follows the first preovulatory surge of gonadotropins [8, 24] and disruption of the normal estrous cycle is an indicator of alteration in the function of the hypothalamo-pituitary-gonadotropin axis. Hypothyroid rats frequently have irregular estrus cycles regardless of when hypothyroid is induced [25, 26]. We also evaluated the estrus cycle, but no difference was found between groups (data not shown). It is known that normal estrous cycles are usually appeared after 54 days of age in female SD rats [27]. We evaluated animals by 51 days of age and they seemed still too immature to define any differences in estrous cycle between groups.

Reproductive organ weights can serve as crucial benchmarks for assessing pubertal progression. In SD rats, mean ovary weight increases rapidly to PD 42, after which it only increases slowly [8], although there are fluctuations depending on the estrous cycle. Here we measured organ weights at PD 51, when ovarian weights had stabilized, and demonstrated that RAI treated animals had a reduced absolute ovarian weight (Fig. 2a). A previous study reported a striking reduction in ovary weight in hypothyroid rats [23]. Several clinical cases have reported large ovarian cysts in juvenile hypothyroid patients [28, 29], but this may have been the effect of ovulatory dysfunction rather than increased ovarian weight. In fact, ovulatory dysfunction is common in hypothyroid patients [30].

The increase in ovary weight during normal puberty is mainly correlated with expansion in both the size and number of large follicles [31]. The ovarian follicle is the basic structural and functional unit of the ovary and histological examination of the ovary (including of the number of growing follicles) may be a good index of ovarian toxicity. Because the ovary contains more tertiary and Graafian follicles as it matures [31], we counted the number of growing follicles and observed a profound decrease in number of secondary follicles in the RAI-treated animals (Fig. 2c). In addition, numbers of tertiary (CT 12.3 ± 8.5; RAI 7.0 ± 6.9) and preovulatory follicles (CT 2.0 ± 3.5; RAI 0.0 ± 0.0) also fell in the RAI-treated animals, although statistical significance was not attained due to the high individual variation (Fig. 2c). Similarly, it has been reported that thyroid deficiency reduced numbers of healthy developing follicles at all stages in rats [23, 32]. Thus, thyroid deficiency may play a role in disrupting ovarian and follicular growth, thereby contributing to ovulatory dysfunction.

In normal pubertal girls, uterine size and endometrial thickness increase while the shape of the uterus changes from tubular to pear-shaped [33]. Similar changes were also reported during puberty in immature SD rats, along with increased estrogen [34]. Since thyroid hormone receptors are present in the uterus [22, 35], it is conceivable that thyroid deficiency leads to aberrant uterine growth. Considering uterine shape changes from tubular to pear-shape as rats mature [34], significant increases in uterine length in RAI treated animals (CT 180.78 ± 26.54, RAI 254.33 ± 64.88) (p < 0.05) (Fig. 3c) indicate delayed uterine maturation. On the other hand, uterine weight relative to body weight also increased (CT 71.57 ± 14.5, RAI 91.66 ± 8.4 mg/100 g body weight) (p < 0.05) (Fig. 3b). This contrasts with the reduced uterine weight observed in adult animals after exposure to propylthiouracil [36]. Given that adolescence is characterized by extensive morphological and functional changes in reproductive organs [8], its vulnerability to thyroid deficiency seems to differ from that of adults. There are no data on the effects of thyroid deficiency on the uterus during puberty, and studies in adults usually focus on the endometrium. So far as we know, this study is the first to define the effects of thyroid deficiency on uterine growth in peripubertal female rats.

Our results clearly demonstrate that thyroid deficiency delays sexual maturation and alters normal ovarian growth during puberty. However, RAI exposure can also affect the growth of reproductive organs. Further study is needed to decide whether RAI exposure itself or thyroid deficiency affects ovarian and uterine growth.

Funding

This work was supported by the research fund of Hanyang University (HY-2017).

Declarations

Conflict of interest

All authors state that they have no conflicts of interest with any financial organization regarding the material discussed in the manuscript.