Thyroid hormones act at all the levels of regulation within the reproductive system . Granulosa cells and ovarian stromal cells express thyroid hormone receptors. Thus, thyroid hormones play an essential role in ovarian physiology . Moreover, other investigators  detected that the ovarian surface epithelium is a physiologically important target for thyroid hormone.
Some researchers  stated that thyroid hormones might directly influence ovarian function through steroid hormone receptors, and that ovulation failure was linked with severe hypothyroidism. Association of ovarian cancer with hyperthyroidism was reported by the researchers . Other reports  added that hypothyroidism and hyperthyroidism are more common in women than in men. Some investigators  clearly detected estrogen receptor (ER) and androgen receptor (AR) in granulosa and theca cells of the ovary.
This study was planned according to the facts that the mechanisms by which thyroid hormones impact ovarian function remain poorly understood , together with the belief of the researchers  that the effect of ovarian steroid hormones are mediated through interaction with specific receptors.
Our study aimed to simulate the clinical conditions of hypothyroidism and hyperthyroidism in adult female albino rats, and to detect the possible changes in ovarian estrogen and androgen hormone receptors associated with such clinical conditions.
Materials and methods
This study used 36 adult female albino rats. Their ages ranged between 3 and 4 months (mean: 3.5±0.2 months) and their mean weight ranged between 200 and 250 g (mean: 221±12g). Animals were confirmed to have regular estrous cycles.
The experiment was carried out in the animal house of Kasr El-Aini Hospital. Rats were divided into three groups. Each group was housed in a separate cage and received tap water and was fed ad libitum.
This group included 12 rats that received 0.5 ml saline solution once daily. Six rats received the saline orally, and the other half received it by intraperitoneal injection for 4 weeks.
This group consisted of 12 rats that received daily orally (using a tube) propylthiouracil (Thyrocil, Amoun pharmaceutical Co., Cairo, A.R.E.) dissolved in saline solution for 4 weeks. The dose was 50 mg/kg in rabbits , this was adjusted according to animal species using Paget equation to be 18.75 mg/kg in rats . Propylthiouracil is one of the drugs that prevent iodotyrosine formation .
This group consisted of 12 rats that received a daily intraperitoneal dose of 40 μg/kg L-thyroxine (Eltroxin, GlaxoSmithKline, Cairo, A.R.E.) dissolved in saline solution for 4 weeks .
Vaginal smears were performed at the end of the fourth week from the start of the experiment to all rats included in the study to select those showing predominating leukocytes over cornified and epithelial cells that coincides with the diestrus phase of the estrus cycle. An inverted phase contrast microscope (Olympus, Tokyo, Japan) was used . Rats not showing the diestrus phase were excluded from the study.
Before sacrifying the selected rats, retro orbital blood samples were collected by capillary tubes. Serum thyroxine level was obtained by radioimmunoassay in the Chemical Pathology Department of Kasr El-Aini Hospital, Cairo, Egypt.
The selected rats were killed by decapitation; right-sided ovaries were obtained and transversely bisected to examine the maximum area, then fixed in 10% formol saline, embedded in paraffin, and cut at 7 μm thickness. The sections were subjected to:
- (1) Hematoxylin and eosin stain .
- (2) Immunohistochemical stains.
Ovarian sections were incubated with the primary mouse monoclonal anti-ERα antibody (Thermo Scientific, UK) . Moreover, these sections were subjected to staining with rabbit polyclonal anti-AR antibody (Thermo Scientific, UK) . Tissue sections were counterstained with Mayer's hematoxylin. Negative controls were obtained by skipping the step of applying the primary antibody.
Using Leica Qwin 500 LTD (Cambridge, UK) image analysis, the number of corpora lutea in hematoxylin and eosin stained sections was counted under the microscope in 10 low-power fields. The optical density of ER-immunoreactive and AR-immunoreactive surface germinal epithelial cells and theca and granulosa lutein cells was measured using mean gray in 10 low-power fields.
Statistical analysis of the measured data was carried out using analysis of variance test followed by Bonferroni pair-wise comparison . P values of less than 0.05 were considered to be statistically significant.
Vaginal smears performed at the end of the experiment indicated that among the six control rats receiving oral saline, three were in the diestrus phase of the estrus cycle, and that among the other six receiving intraperitoneal saline, four showed the diestrus phase. With regard to the hypothyroid group, eight rats only were in the diestrus phase, whereas among the hyperthyroid group, seven were in the diestrus phase.
Hormonal assay results (Table 1)
The recorded means of thyroxine values were 2.9, 1.3, and 8.5 ng/dl in the control, hypothyroid, and hyperthyroid groups, respectively.
Hematoxylin and eosin results
Examination of some ovarian sections from the control group revealed the presence of primordial follicles, atretic follicles, and corpora lutea. The ovarian medulla and blood vessels were also noted.
Sections revealed few corpora lutea, atretic follicles, and dilated congested blood vessels. Vacuolated cells of interstitial tissue were detected.
The ovarian sections examined showed numerous corpora lutea, atretic follicles, and markedly dilated congested blood vessels.
Estrogen receptor (Fig. 7): Strong ER immunoreactive nuclei of the surface germinal epithelium were recorded. The theca lutein cells exhibited strong nuclear and weak cytoplasmic immunoreactions. Weak cytoplasmic reaction was detected in the granulosa lutein cells and negative immunoreaction was detected in stromal cells.
Androgen receptor (Fig. 8): The surface germinal epithelium showed negative AR immunoreaction. The theca lutein cells showed strong nuclear and cytoplasmic immunoreactions. Moderate cytoplasmic reaction was observed in the granulosa lutein cells, with negative immunoreaction in stromal cells.
Estrogen receptor (Fig. 9): Moderate ER immunoreactive nuclei of the surface germinal epithelium were recorded. The theca lutein cells revealed strong nuclear and weak cytoplasmic immunoreactions. Very weak cytoplasmic reaction was noted in the granulosa lutein cells, whereas stromal cells revealed negative immunoreaction.
Androgen receptor (Fig. 10): The surface germinal epithelium showed negative AR immunoreaction. Strong nuclear and cytoplasmic immunoreactions were observed in the theca lutein cells. The granulosa lutein cells exhibited strong cytoplasmic reaction. Negative immunoreaction was detected in stromal cells.
Estrogen receptor (Fig. 11): Strong ER immunoreactive nuclei of the surface germinal epithelium were observed. The theca lutein cells revealed strong nuclear and weak cytoplasmic immunoreactions. Moderate nuclear and cytoplasmic reactions were noted in the granulosa lutein cells, with negative immunoreaction in stromal cells.
Androgen receptor (Fig. 12): Nuclei of the surface germinal epithelium showed strong AR immunoreaction. Strong nuclear and cytoplasmic immunoreactions were observed in the theca lutein cells. Moderate cytoplasmic reaction was noted in the granulosa lutein cells, whereas stromal cells revealed negative immunoreaction.
The mean number of corpora lutea revealed a high significant increase (P<0.0005) in the hyperthyroid group versus the control and the hypothyroid groups (Table 2 and Histogram 1). With regard to ER immunoreactivity in the surface germinal epithelial cells, a high significant decrease (P<0.007) in the mean optical density of ER immunoreactive nuclei was recorded in the hypothyroid group versus the control group with a nonsignificant difference between the hyperthyroid and control groups. A high significant increase (P<0.008) and a high significant decrease (P<0.012) in the mean optical density of ER immunoreactivity in the theca lutein cells were recorded in hyperthyroid and hypothyroid groups, respectively versus the control group. A high significant decrease (P<0.0005) in the mean optical density of ER immunoreactivity in the granulosa lutein cells was estimated in both the hypothyroid and hyperthyroid groups versus the control group with a nonsignificant difference between both experimental groups (Table 3 and Histogram 2). In contrast, AR immunoreactive nuclei of the surface germinal epithelial cells were observed only in the hyperthyroid group. The mean optical density of AR immunoreactivity of the theca lutein cells showed a high significant increase (P<0.004) and a high significant decrease (P<0.004) in the hypothyroid and hyperthyroid groups, respectively versus the control group. The mean optical density of AR immunoreactivity of the granulosa lutein cells revealed a high significant increase (P<0.0005 and 0.015) in both the hypothyroid and hyperthyroid groups, respectively versus the control group (Table 4and Histogram 3). Therefore, a negative correlation was documented between ER and AR immunoreactivity in the hypothyroid and hyperthyroid groups (Histogram 4).
This study simulates the clinical conditions of hypothyroidism and hyperthyroidism, and records the changes occurring in the ovary of female adult albino rats in the diestrus phase, this phase was chosen because some studies  mentioned that granulosa cells at early stages of follicular development do not actively participate in thyroid hormone binding. The histological and immunohistochemical findings of this study will be discussed.
Dilated congested blood vessels were observed in both hypothyroid and hyperthyroid rats; this finding could be attributed to the increased vascular supply needed for the growing corpora lutea and the increase in their functional activity .
Numerous corpora lutea were detected in the hyperthyroid group that was significantly increased versus control and hypothyroid groups. Similar to the previous finding, investigators  detected inhibition of folliculogenesis and ovulation with corpora lutea that are smaller in number and diameter in hypothyroidism. Other researchers  added that in thyroidectomy, there was decreased responsiveness of ovarian granulosa cells to follicle stimulating hormone that increased serum prolactin level consequently inhibiting normal luteinizing hormone secretion. However, recent investigators  recorded that hypothyroidism did not influence the number of corpora lutea. Therefore, it could be suggested that the reverse might occur in hyperthyroid conditions stimulating ovulation, and hence corpora lutea formation.
In both hypothyroid and hyperthyroid groups, many atretic follicles were seen. This could be explained by the researchers  who reported that ovarian follicles were not well developed in hypothyroid animals, suggesting that thyroid hormones could have a direct effect on growth of ovarian follicles. In hyperthyroidism, the ovaries contained follicles of abnormal size, form, and number .
In the hypothyroid group, vacuolated stromal cells were detected most probably representing those of the interstitial gland. This finding was explained by the researchers  as stromal hyperthecosis; microscopically, it was noted as hypercellular stroma with luteinization of stromal cells that appear with a vacuolated cytoplasm. This is a disorder of ovarian stroma and was recorded in women with polycystic ovarian disease (PCOD).
With regard to the immunohistochemical results, the control group demonstrated strong ER immunoreactivity in nuclei of the surface germinal epithelium and theca lutein cells; weak cytoplasmic immunoreaction was recorded in both theca and granulosa lutein cells. This was in part supported by the researchers  who reported that ERα was detected in the germinal epithelium. Other investigators  observed no immunostaining in rat ovary with anti-ERα antibody.
In the hypothyroid group, moderate ER immunoreaction was recorded in nuclei of the surface epithelium. There was a significant decrease in ER immunoreactivity in the hypothyroid group versus control in all the studied compartments namely surface germinal epithelium, theca, and granulosa lutein cells. Studies [20,26] recorded a decrease in serum levels of estrogen in thyroidectomy and hypothyroidism. On the contrary, other researchers [21,27] observed increased serum concentration of estradiol in hypothyroidism with increasing expression of ER.
In the hyperthyroid group, strong ER immunoreactivity was observed in nuclei of the surface germinal epithelium. A significant increase in ER immunoreactivity of the theca lutein cells and decrease in the granulosa lutein cells versus control group were confirmed. These findings were in agreement with some investigators  who detected that excess thyroid hormone increased expression of ERα in the ovarian surface epithelium that was strongly associated with ovarian cancer. Moreover, other researchers  reported upregulation of ERα by thyroid hormone in rat pituitary cell line. A recent review  added that serum estrogen and ovarian conversions of androgen-to-estrogen were increased in thyrotoxicosis. On the contrary, other investigators  reported that thyroid hormone inhibited estradiol production in all culture conditions. The recorded changes in hyperthyroidism might clinically give rise to oligomenorrhea, which may progress to amenorrhea as reported by some researchers .
With regard to AR immunoreactivity of the control group, negative AR immunoreaction was reported in the surface germinal epithelium, whereas strong nuclear and cytoplasmic immunoreactions were detected in theca lutein cells and only moderate cytoplasmic immunoreaction of granulosa lutein cells was observed. In agreement with the previously mentioned results, some investigators [7,24] recorded AR immunoreactivity in the nuclei of ovarian cells, principally the granulosa and theca cells, and suggested that thecal androgen could be a paracrine modulator of granulosa cell function.
AR immunoreactivity of the hypothyroid group revealed negative immunoreaction in the surface germinal epithelium. Morphometrically, AR immunoreactivity was proved to be significantly increased versus the control group. This finding was consistent with the researchers  who suggested that AR upregulation could be mediated by the accumulation of endogenous testosterone. This accumulation might be attributed to inhibition of the aromatase enzyme and blockade of androgen conversion to estrogen. Such explanation was supported by the investigators  who reported that thecal cells synthesize androstenedione that is converted to estradiol by the cytochrome P450 aromatase enzyme of the granulosa cells. In contrast, some investigators  reported low testosterone levels in hypothyroid rats that could be associated with decreased AR on the basis of the suggestions of the other researchers .
The suggestion of testosterone accumulation in the hypothyroid group might give rise clinically to manifestations of PCOD reported by the researchers  including hirsutism, acne, and male pattern alopecia. Microscopically, follicular atresia, follicular cysts, and anovulation with stromal hyperthecosis are changes of PCOD that were recorded in the hypothyroid group of this study.
With regard to AR immunoreactivity of hyperthyroid rats, strong immunoreactions were recorded in both surface germinal epithelia. AR immunoreactions were significantly decreased in the theca cells and significantly increased in the granulosa lutein cells versus the controls. The decrease in AR immunoreactivity could be explained by increased conversion of androgen-to-estrogen by the aromatase enzyme, which could also explain the increased AR immunoexpression in granulosa lutein cells, as it is going to be converted to estrogen by aromatase. On the contrary, other investigators  demonstrated that thyroid hormone increased basal androgen secretion, with consequent increase in AR based on the results of some researchers .
With regard to stromal cells, they were negative for both ER and AR immunoreactivity in all the studied groups including the control group. This was consistent with the researchers  who detected ER and AR immunoreactivity only in granulosa and theca cells.
In conclusion, our data clearly demonstrated a negative correlation between ER and AR immunoreactivity in the experimental groups. In other words, a decrease in ER and an increase in AR immunoreactivities were detected in hypothyroid rats, which was vice versa in hyperthyroid rats. Therefore, it could be suggested that thyroid hormones are important to maintain normal estrous cycle and hence in the regulation of ovarian hormones. The mechanism by which thyroid hormones influence ovarian function remain to be elucidated. However, it might be through their effect on aromatase enzyme, which is the key regulator in ovarian hormone production reflected on the level of steroid hormone receptor expression.
1. Wakim AN, Polizotto SL, Buffo MJ, Marrero MA, Burholt DR. Thyroid hormones in human follicular fluid and thyroid hormone
receptors in human granulosa cells. Fertil Steril. 1993;59:1187–1190
2. Wakim AN, Paljug WR, Jasnosz KM, Alhakim N, Brown AB, Burholt DR. Thyroid hormone
receptor messenger ribonucleic acid in human granulosa and ovarian stromal cells. Fertil Steril. 1994;62:531–534
3. Rae MT, Niven D, Ross A, Forster T, Lathe R, Critchley HOD, et al. Steroid signalling in human ovarian surface epithelial cells: the response to interleukin-1α determined by microarray analysis. J Endocrinol. 2004;183:19–28
4. Krassas GE. Thyroid disease and female reproduction. Fertil Steril. 2000;74:1063–1070
5. Ness RB, Grisso JA, Cottreau C, Klapper J, Vergona R, Wheeler JE, et al. Factors related to inflammation of the ovarian epithelium and risk of ovarian cancer. Epidemiology. 2000;11:111–117
6. Doufas AG, Mastorakos G. The hypothalamic-pituitary-thyroid axis and the female reproductive system. Ann N Y Acad Sci. 2000;900:65–76
7. Zurvarra FM, Salvetti NR, Mason JI, Velazquez MML, Alfaro NS, Ortega HH. Disruption in the expression and immunolocalisation of steroid receptors and steroidogenic enzymes in letrozole-induced polycystic ovaries in rat. Reprod Fertil Develop. 2009;21:827–839
8. Boelaert K, Franklyn JA. Thyroid hormone
in health and disease. J Endocrinol. 2005;187:1–15
9. Drummond AE, Britt KL, Dyson M, Jones ME, Kerr JB, O'Donnell L, et al. Ovarian steroid receptors and their role in ovarian function. Mol Cell Endocrinol. 2002;191:27–33
10. Türkoglu V, Yegin E. Effects of propylthiouracil-induced hypothyroidism on plasma lipid table in rabbits. Turk J Vet Anim Sci. 2000;24:149–152
11. Paget GE, Barnes JMLaurence DR, Bacharach AL. Toxicity tests. Evaluation of drug activities: Pharmacometrics. 1964 . London Academic Press:135
12. Dadan J, Zbucki RRL, Sawicki B, Winnicka MM, Puchalski Z. Activity of the thyroid parafollicular (C) cells in rats with hyperthyroidism immunohistochemical investigations. Annals Academiae Medicae Bialostocensis. 2004;49:135–137
13. Marcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol. 2002;62:609–614
14. Kiernan JA Histological and histochemical methods: theory and practice. 2001:3rd ed London, New York & New Delhi A Hodder Arnold Publication 111–162.
15. Salvetti NR, Acosta JC, Gimeno EJ, Muller LA, Mazzini RA, Taboada AF, Ortega HH. Estrogen receptors alpha and beta and progesterone receptors in normal bovine ovarian follicles and cystic ovarian disease. Vet Pathol. 2007;44:373–378
16. Armitage P, Berry GD Statistical methods in medical research. 19943rd ed London Wiley-Blackwell
17. Aghajanova L, Lindeberg M, Carlsson IB, Stavreus Evers A, Zhang P, Scott JE, et al. Receptors for thyroid-stimulating hormone and thyroid hormones in human ovarian tissue. Reprod Biomed Online. 2009;18:337–347
18. Gaytan F, Morales C, Bellido C, Aguilar E, Sanchez Criado JE. Proliferative activity in the different ovarian compartments in cycling rats estimated by the 5-bromodeoxyuridine technique. Biol Reprod. 1996;54:1356–1365
19. Armada Dias L, Carvalho JJ, Breitenbach MM, Franci CR, Moura EG. Is the infertility in hypothyroidism mainly due to ovarian or pituitary functional changes? Braz J Med Biol Res. 2001;34:1209–1215
20. Hatsuta M, Abe K, Tamura K, Ryuno T, Watanabe G, Taya K, Kogo H. Effects of hypothyroidism on the estrous cycle and reproductive hormones in mature female rat. Eur J Pharmacol. 2004;486:343–348
21. Hapon MB, Gamarra Luques C, Jahn GA. Short term hypothyroidism affects ovarian function in the cycling rat. Reprod Biol Endocrinol. 2010;8:14–24 Art. No. 14.
22. Skjoldebrand Sparre L, Kollind M, Carlstrom K. Ovarian ultrasound and ovarian and adrenal hormones before and after treatment for hyperthyroidism. Gynecol Obstet Invest. 2002;54:50–55
23. Robbins SL, Kumar VKumar V, Abbas AK, Fausto N, Aster J. Female genital tract. Robbins & Cotran pathologic basis of disease. 2010 Philadelphia Saunders:728–729
24. Pelletier G, Labrie C, Labrie F. Localization of oestrogen receptor alpha, oestrogen receptor beta and androgen receptors in the rat reproductive organs. J Endocrinol. 2000;165:359–370
25. Hiroi H, Inoue S, Watanabe T, Goto W, Orimo A, Momoeda M, et al. Differential immunolocalization of estrogen receptor
alpha and beta in rat ovary
and uterus. J Mol Endocrinol. 1999;22:37–44
26. Krassas GE, Poppe K, Glinoer D. Thyroid function and human reproductive health. Endocr Rev. 2010;31:702–755
27. Hapon MB, Simoncini M, Via G, Jahn GA. Effect of hypothyroidism on hormone profiles in virgin, pregnant and lactating rats and on lactation. Reproduction. 2003;126:371–382
28. Rae MT, Gubbay O, Kostogiannou A, Price D, Critchley HO, Hillier SG. Thyroid hormone
signaling in human ovarian surface epithelial cells. J Clin Endocrinol Metab. 2007;92:322–327
29. Fujimoto N, Jinno N, Kitamura S. Activation of estrogen response element dependent transcription by thyroid hormone
with increase in estrogen receptor
levels in a rat pituitary cell line, GH3. J Endocrinol. 2004;181:77–83
30. Gregoraszczuk EL, Slomczynska M, Wilk R. Thyroid hormone
inhibits aromatase activity in porcine thecal cells cultured alone and in coculture with granulosa cells. Thyroid. 1998;8:1157–1163
31. Rubin R, Strayer DSRubin R, Strayer DS. The endocrine system. Rubin's pathology: clinicopathologic foundations of medicine. 20075th ed Philadelphia Lippincott Williams & Wilkins:941–954
32. Manneras L, Cajander S, Holmang A, Seleskovic Z, Lystig T, Lonn M, Stener Victorin E. A new rat model exhibiting both ovarian and metabolic characteristics of polycystic ovary
syndrome. Endocrinology. 2007;148:3781–3791
33. Bao B, Kumar N, Karp RM, Garverick HA, Sundaram K. Estrogen receptor
-beta expression in relation to the expression of luteinizing hormone receptor and cytochrome P450 enzymes in rat ovarian follicles. Biol Reprod. 2000;63:1747–1755
34. Gregoraszczuk EL, Skalka M. Thyroid hormone
as a regulator of basal and human chorionic gonadotrophin-stimulated steroidogenesis by cultured porcine theca and granulosa cells isolated at different stages of the follicular phase. Reprod Fertil Dev. 1996;8:961–967