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Receptor Cross Talk and Interplay between Melatonin and Ovarian Thyroid Axis in a Letrozole Induced Polycystic (Pco) Rat

The objective of present study was to establish the interrelationship between thyroid and melatonin during anovulatory/letrozole induced polycystic ovarian condition on female Wister rats. Rats were procured and after acclimatization 20 rats were divided in 4 groups with 5 rats in each. They were divided as Control, Letrozole induced PCO rat (1 mg/kg BW/d), and melatonin alone (200 μg/100 g BW/d). The experiment was conducted for the duration of 28 d. Assessment of gravimetric, hormonal profile and thyroid histology and relative expression of MT1, MT2, and ERα, (thyroid, ovary) Dio2, TRα (Ovary) done followed by standard protocol. Histological observation showed shrinkages in thyroid follicles in PCO rats however exogenous melatonin maintained the cellular architecture and normal thyroid weight. PCO rats showed significantly high circulating testosterone but significant decreased in estrogen and progesterone level. Circulatory gonadotropins (LH, FSH) were noted significantly high in PCO rats. Melatonin injection to the PCO rats however reversed to the control level and restored. Circulatory TSH level in PCO rats were noted suppressed where as T3 and T4 were non-significantly increased suggesting a reciprocal relation between melatonin and thyroxine. Thyroid tissue of PCO rats expressed MT1 and MT2 in way alternate and opposite way being MT1 as up regulated whereas down regulation of MT2. Ovarian tissue of PCO rats showed reverse receptor expression to that of thyroid tissue being MT1 down regulated and MT2 was noted unregulated. Parallel relation was noted between ERα and TRα receptor expression in thyroid and ovarian tissue respectively. PCO rats resulted in up regulation of Dio2, receptor expression in a non-significant manner. Therefore, present finding suggests a fine interplay and cross talk via melatonin its two receptor MT1 , MT2 with ERα, TRα, and Dio2 thyroid and ovarian tissue as the case between ovarian thyroid axis hence maintaining a physiological trade-offs between theses gland with a tonic regulation to maintain melatonin and thyroid homeostasis during polycystic pathogenicity.


Introduction
Melatonin (N-acetyl-5methoxytryptamine) is an indoleamine and principle secreting hormone of pineal gland. This neurohormone is recognized to regulate the seasonal reproduction, the phasing of mammalian circadian rhythm [1,2]. Beside playing the circadian oscillator it has an important role as antioxidant [3,4] being a strong free radical scavenger [5], antiaging [6] and potential anti carcinogenic activities [7], including suppressive effect on secretion and growth processes of the thyroid gland [8,9].
Melatonin regulates its physiological role via its membrane bound receptor MT1 and MT2 [10]. Both the receptors MT1 and MT2 are 7 transmembrane Gi/o protein coupled receptor which works via cAMP, Protein Kinase C (PKC) and Diacyleglicerol Mediated Pathway (DAG) [9,11]. Human MT1 and MT2 receptor showed approximately 60% overall and 75% homology of amino acid with each other within the transmembrane domain.
The thyroid gland is one of the most important glands regulating iodine metabolism. It metabolizes iodine for the synthesis and secretion of its T3 and T4 hormone. The thyroidal anomalies have shown effects on the ovarian physiology. Ovarian atrophy was reported in induced hypothyroid rats [12,13]. The reports of clinical evidences suggest that women suffering from thyroidal disorders are most frequently associated with disturbances in reproductive, ovarian cycles and impaired fertility. The animal studies in monkey uterus have shown long term steroids influence to the thyroid hormones, Thyroid Stimulating Hormone (TSH) as well as Thyrotropin Releasing Hormone (TRH) receptors [13]. It is also reported that formation of ovarian follicular cysts are also may be caused by the influence of hypothyroidism as it increases ovarian sensitivity to gonadotropin action thus may be followed by consequences like irregular menses, breakthrough bleeding, low endometrial thickness and sometimes non proliferative endometrium [13]. Further ovarian atrophy has been reported following thyroid suppressive drug [14]. Report suggests decreased number of primordial antral and Graafian follicles in hypothyroid rats without any significant changes in reproductive behavior [15]. The induced neonatal hypothyroidism showed disturbed folliculogenesis and absence of corpora lutea [16,17]. The thyroid hormone was reported to be synergized with FSH to exert direct stimulatory effects on grannulosa cell function, LH/CG (Chorionic gonadotropin) receptor formation and activation of steroidogenic enzymes [14]. Ovary is known as the prime female reproductive and endocrine organ because of the presence of reproductive steroid (estrogen and progesterone) secreting cells. This syndrome (PCO) may broadly be categorized or classified as clinical, endocrinological, and metabolic. The clinical category evidences irregular reproductive cycle, acne, alopecia, hirsutism, frequent abortions, anovulation which finally leads to infertility. The endocrinological category shows elevated

Journal of Clinical & Cellular Immunol
gonadal steroids, LH [4] and prolactin levels. The metabolic category features lipid abnormality, obesity, insulin resistance and type 2 diabetes [18]. Diseases like hypothyroidism, glucose intolerance, diabetes and cardiovascular may have links with PCOS [19]. Previous studies on women with PCOS showed thyroid volume is associated with age, anthropometry, smoking, iodine status and hyperinsulinemia [20].
Increasing problem of PCOD among human population in the present era becoming alarming day by day and may be one of the causes of infertility. Previous studies proved that hypothyroidism is a very common metabolic and hormonal disorder associated with reproductive abnormalities like menstrual disorders, amenorrhea, frequent abortions and infertility. The autoimmune thyroiditis is a most prevalent cause for hypothyroidism and found in females with PCOS condition [21]. PCOS was previously hypothesized to be responsible for the irregularity of gonadal steroid thus causing autoimmune thyroiditis [21].
The exogenous melatonin proved to be potential to protect the ovarian condition against PCOS [4]. A trade-off relation between the thyroxine and melatonin in Indian palm squirrel has already being proposed by research group of Rai in 2005 [22]. Reproductive abnormalities during PCOS have received much attention but till date least attention has been given to understand the metabolic irregularities and dysfunction during PCOS condition. Therefore, the study was designed to elucidate the physiological cross talk between ovarian and thyroid tissue during pathogenesis of letrozole induced polycystic ovarian (PCO) rat model. The study is designed with an objective to explore the functional correlation between these twoglands following exogenous melatonin treatment by assessing cellular, hormonal and receptor (MT1, MT2, ERα, TRα, Dio2) expression PCO rat.

Animal model
The animal experiment and all procedures were carried out in accordance with guidelines for care and use of laboratory animals of institutional animal ethical committee (IAEC), Guru Ghasidas Vishwavidyalaya, Bilaspur (C.G) India (Registration number: 994/GO/ERe/S/06/CPCSEA). Female adult albino rats (Wister strain) weighing 150 10 g of approximately same age were procured from Defence Research Development Establishment (DRDE) Gwalior. They were housed in polypropylene cages with proper bedding, feeding and water ad libitum. After an adaptation period of two weeks rats were randomly divided into following experimental groups (Table 1).

Induction of polycystic ovary by oral dose of letrozole
Letrozole concentration 1 mg/kg/bodyweight/d was noted appropriate and sufficient to induce polycystic condition in female rats as observed with the histological preparation and therefore the same was selected for further experiment. Vaginal smear of PCO induced female rats showed presence of cornified cells denoting failure of at least two consecutive estrus cycles and leading to persistent (PE) estrus cycles [4].

Treatment of exogenous melatonin on letrozole induced PCO rats
Melatonin solution was made by dissolving it in few drops (approx. 100 µl) of ethanol and then diluted with normal saline (0.9% NaCl) up to the desired concentration. Group I comprising control rats were given orally 1% CMC/d with the help of oral gauge. Group II and III female rats were orally supplemented with Letrozole (1 mg/kg body weight/d) to induce PCO condition. Group III females, with PCOS condition, received melatonin (200 µg/100 g body weight/d). Group IV rats were given melatonin alone treatment (200 µg/100 g body weight/d). The duration of the experiment was for 28 d (Table I). At the end of the experiment animals of each group were sacrificed following complete anaesthesia. Thyroid glands of all experimental groups were dissected and cleaned subjected to thyroid weight analysis. One pair of thyroid gland of each group was fixed in Bouins solution for histological block preparation and sectioning. Blood serum was separated by centrifugation ant 3000 rpm for 15 min and was stored in deep freeze (-20ºC) for ELISA of melatonin, thyroxine (T4), triiodothyronin (T3), Thyroid Stimulating Hormone (TSH), Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH). Further, thyroid and ovary tissues were treated to assay receptor expression of MT1, MT2, TRα, ERα and Dio2.

Gravimetric analysis of thyroid tissue
Thyroid gland was dissected out in normal saline and adherent fat was removed. The weight of organ was noted and expressed in mg.

Histological Preparation of thyroid tissue
Thyroids of all four groups were previously fixed in Bouin's fixative for 24 h. Then the tissues were washed in running tap water followed by the process of dehydration by alcohol grades. The dehydrated tissues were cleared with the clearing agent xylene followed by the

qRT-PCR
qRT-PCR was done for the expression assay of MT1, MT2, ERα, Dio2, TRα by first extracting the total mRNA, followed by cDNA synthesis. The primers were purchased from Imperial life sciences (P) limited ( Table 2).

Gene Produ ct Forward
Reverse Table 2. Gene products along with their primers

Statistical analysis
Statistical analysis was performed using two tailed student t-test and one way analysis of variance. Calculations were performed using commercial software SPSS.16 (SPSS, Chicago, IL, USA). At a probability value P<0.05, P<0.01 and p<0.001 were considered to be statistically significant.

Gravimetric analysis
Effect of exogenous melatonin on weight of thyroid gland: The Letrozole induced PCO rats showed significant (P<0.001, 0.01) decrease in the weight of thyroid. Exogenous melatonin to PCO rats showed recovery in the tissue weight significantly (P<0.001, 0.01) comparable to that of control group. However, melatonin alone maintains the healthy weight of thyroid compared to the control ( Figure 1).

Histological Observation
Effect of exogenous melatonin on cellular architecture of thyroid follicle: PCOS condition resulted in significant decrease in the number of thyroid follicle. PCOS rat receiving melatonin injection showed significant recovery in cellular architecture and the number of thyroid follicle comparable to the normal condition. (Figure 2) melatonin alone resulted in healthy thyroidal architecture.  Thyroid hormones (TSH, T3 T4): PCO condition resulted in suppression of circulatory TSH level. Melatonin treatment to PCO rats showed further suppression of serum circulatory TSH levels ( Figure 4). Non-significant elevation in serum circulatory level of the T3 and T4 levels was noted in letrozole induced PCO rats. Melatonin treatment to PCO could not restore T3 and T4 circulatory levels to of the normal rat ( Figures 5 and 6).     Gonadal Hormones (Testosterone, estrogen, Progesterone): PCO rats resulted in significantly high serum circulatory testosterone but estrogen and progesterone levels were noted significantly decreased. Melatonin supplementation to the PCO rats were resulted in normal circulatory level of gonadal steroid (Figures 9-11).   Effect of exogenous melatonin on MT1, MT2, ERα receptor expression on thyroid: Letrozole induced PCO condition showed a significant (P<0.01) up regulation of MT1 but down regulation of MT2 receptor expression on thyroid tissue, Injection of melatonin however, down regulated the relative expression comparable to the normal level (Figures 12 and 13).     Receptor expression for Dio2 on ovarian tissue of PCO rats were found down regulated. PCO rats treated with exogenous melatonin however resulted in non-significant restoration ( Figure 17). Ovarian tissue of PCO rats showed upregulation in TRα receptor expression when compared to control. However supplementation of melatonin to PCO could not restored significantly the TRα receptor expression (Figure 18).

Discussion
The Polycystic Ovarian Syndrome (PCOS) has many hypothesis and speculations about its pathophysiology which may further lead to metabolic diseases such thyroid dysfunctions. In present study letrozole induced PCOS female rats resulted a significant decrease in thyroid gland weight. Further, histological photomicrograph of thyroid gland evidenced in decrease in the cellular density and scanty of thyroid follicles in letrozole induced PCOS female rats. However the treatment of exogenous melatonin showed a normalization of the condition by increasing the density and quantity of the thyroid follicle comparable to the normal condition. The exogenous melatonin treatment to the normal rats reduced the number of thyroid follicles in the thyroid gland. Decrease in thyroid gland weight can be correlated with the decrement of lesser cellular density of thyroid follicle in PCO rats as compared to the control group. PCO condition might have resulted stress induced by hyperandrogenism during PCO condition and hence led to thyroid follicular damage. Melatonin supplementation to PCO rats resulted in restoration of thyroid gland weight as well as the thyroid cellularity. The melatonin being virtue of its strong antioxidant and free radical scavenger might have rescued and protected the thyroid gland during PCO pathogenicity. [9]. The thyroid gland weight might be decreased due the oxidative stress produced by the PCOS condition [4]. Melatonin which is a potent antioxidant is synthesized and received by the thyroid gland that may regulate the thyrocyte function and thyroid volume [9]. Our finding is in agreement of the previous reports suggesting thyroid volume correlation and variation of circulatory gonadotropin levels during in the PCOS endocrinopathy [20]. Our finding also presents the disturbance in the pulsatility and ration of gonadotropins (LH and FSH). The changes in the gonadotropin level might be expected to impact negatively to the thyroid follicles. Results suggests the sensitivity of the thyroid gland for exogenous melatonin which in turn be acting on the hypophyseal-pituitary ovarian axis and hence regularizing the serum circulatory gonadotropin level in PCO rats receiving melatonin injections and therefore thyroid follicle density restored.
comparable to the normal level. The decreased T4 secretion might be resulted by the PCOS condition as it was previously reported that hypo and hyperthyroidism is related with the reproductive abnormality [23,13]. Earlier finding suggest impairment of ovarian function in hypothyroid condition which is reversible by the treatment of exogenous thyroxin injections supporting the present ovarian-thyroid correlation confirming that PCO condition may cause thyroid dysfunction specifically hypothyroidism. The exogenous melatonin reducing the T4 level in PCOS condition might have effect on recovering the reproductive capability of female rats. The tradeoff relation between melatonin and thyroxine might be the key mediator providing a tonic regulation to maintain thyroid hormone homeostasis during PCOS condition following melatonin injection [22]. Nonetheless, it has also been reported that melatonin administration influences the reproductive physiology by controlling iodothyroninedeiodenase followed by circulating thyroxine levels [9].
The mammalian reproductive function depends upon the Hypothalamus-Pituitary-Gonadal Axis (HPG) which regulates the gonadotropins (LH and FSH) from anterior pituitary via gonadotrophs. Considering the neuroendocrine and reproductive connection of PCOS, in the present experiment we measured the gonadotropin and melatonin levels in the circulation in all experimental groups. The asymmetrical ratio of gonadotropins was noted in the PCOS condition. The LH ratio was noted higher in the PCOS condition whereas the FSH was noted to be down regulated significantly. The melatonin supplementation in the PCOS rats regained the normal ratio of the gonadotropin circulation. The regulation of gonadotropins in the PCOS condition by melatonin can be hypothesized that melatonin inhibits the secretion of the GnRH from the hypothalamus [24]. The letrozole is an aromatase inhibitor that prevents the conversion from testosterone to estradiol in the ovary, this might intern affect the gonadotropin secretion thus the variation of the circulatory level [4]. As a result we get a significantly high elevation on the testosterone level. In case of hypothyroidism it is reported that the gonadotropins, gonadal steroids and Sex Hormone Binding Globulin (SHBG) gets a higher surge. As other reports also says that the hyperthyroid may also cause gonadotropin dysfunction that may explain the reason behind the gonadotropin dysfunction and a surge in thyroxine level in case of letrozole induced PCOS condition [13]. However the exact mechanism of the regulation by melatonin is not known. The melatonin level was showed a down regulation in the circulation in the PCOS condition. The decrease might be hypothesized and linked with the increased thyroxine level at the same condition. The thyroxine secretion and melatonin shows a trade-off relation [22] that might be responsible for the down regulation of the melatonin level in circulation. The up regulation of melatonin and the down regulation of thyroxine were noted in the treatment of exogenous melatonin in PCOS condition confirming trade-off relation between the two hormones.
In the present work opposite and crisscross expression of MT1 and MT2 was noted on thyroid follicle during PCO pathogenicity. The MT1 expression on thyroid tissue during PCO condition was upregulated whereas MT2 showed a down regulation but not significant. Interestingly ovarian tissue also showed similar trade for MT1 and MT2 receptor expression respectively. The MT2 (Figure 16) receptor expression reciprocates with the expression pattern of MT1 receptor expression in ovary. The affinity binding might have result the reciprocal relationship between MT1 and MT2 receptor gene expression in case of PCOS condition.
A parallel relation was noted towards the expression of ERα on thyroid tissue and TRα on ovarian tissue. Presence of Dio2 receptor was noted on the ovarian tissue which was upregulated during PCO condition. The comparative receptor expression of thyroid ERα and ovarian Dio2 and TRα has a parallel relationship in spite of these functional association are with thyroid and ovary respectively. Presence of MT1 and MT2 receptor and variation in there expression in PCO rat indicates there involvement in regulation of ovarian function via melatonin. However, MT1 and MT2 expression are regulating thyroid function in trade off manner similar to circulating melatonin and thyroxine. Previous studies revealed presence of MT1 receptors along with AANAT and HIOMT activity on thyroid follicular cells which further supports our present results [25]. Further, literatures provide evidences that melatonin mediates it impact either via MT1/MT2 receptor of thyroid cells or directly influencing thyroid physiology through TTF1 and PAX 8 pathway [9] but these pathway don not involve MT1/MT2 proteins. Possibly TSH, T4 and PAX8 are in an agreement to control the thyroidal function as the increased T4 indicates the role of TSH. The endogenous melatonin either pineal or non-pineal origin along with other physiological agents might be involved in regulation of the thyroid function [26]. The main hormone responsible for PCOS condition is estrogen focusing this fact we observed relative expression of ERα receptor on thyroid tissue. The receptor expression for ERα showed up regulated during PCO pathogenesis. Present result can be correlated with variation in circulatory thyroxine level being decreased in letrozole induced PCO rats. Upregulation of ERα during PCO pathogenicity indicates influence toward HPT axis regarding further synthesis of thyroxine therefore is the signal for ERα mediated cell proliferation as a consequence cascade to elevated expression of the same.
The decrease in the circulatory levels of T3, T4 and TSH insisted to check the relative expressions of TRα and Dio2 enzyme in the ovary as these two polypeptides are the key regulatory molecule to regulate the functions of thyroid hormones in the target tissue. Dio2 shows a greater relative expression in case of letrozole induced PCOS condition. Ovarian tissue needs more triiodothyronine in case of PCOS condition as a result of the decreased secretion of T3. Dio2 coverts more T4 to T3 to regulate the homeostasis of the HPG and HPT axis cross talk. The expression of TRα also shows significant increase in the relative expression in case of PCOS condition comparable to the normal rat. The increased expression of thyroid receptor also indicates the correlation between these two tissues. This result initiates a logical evidence and explanation about the crosstalk between thyroid and ovarian axis. In nut shell, as depicted in the results the present study shows a close relation between thyroid and ovarian machinery. In addition we found a relation between gonadotropins and thyroid hormone interplay with underlying regulation by melatonin in thyroid physiology and a hint of redox control mechanism. However, further studies may show the perfect machinery for the melatonin receptor pathway in the thyroid gland in induced PCOS condition. Nevertheless, further investigations are required to note the underlying mechanism of melatonin and PCOS in thyroidal physiology and pathologic conditions.