Receptor Mediated Effect of Melatonin in Pituitary Regulated Thyroid Secretions of Hyperthyroid Mice

Animal Procurement and Maintenance

Five mice (n = 5) were colonized at each polycarbonate cage (43 cm x 27 cm x 14 cm) to avoid the crowding effect. The experimental animals were settled at animal house with suitable laboratory temperature of 25+2°C and maintained 12 hrs light and 12 hrs dark cycles. Mice feed and sufficient water was provided to the mice for their proper health growth.

Experimental Design

For analyzing the receptor mediated actions of melatonin in pituitary regulated thyroid hormones secretions in hyperthyroidism, experimental animals were categorized into four groups, five mice (n = 5) were settled for each experimental group.

Control (Con) group: Saline treatment (0.01%), 0.1 ml/day was given to this group of mice.

Melatonin (Mel) group: melatonin was provided subcutaneously to this group of mice at the concentration of 25 µg/100 g body weight per day for consecutive 30 days at evening time, 16:30 hrs-17:00hrs.

Hyperthyroid (T₄) group: L-thyroxine was provided subcutaneously to this group of mice at the concentration of 0.6 µg/g body weight per day for consecutive 30 days.

(T₄ + Mel) group: Both L-thyroxine and melatonin were given to this group of mice consecutively for 30 days.

Sample Collection and Processing

Experimental animals were anaesthetized and after sacrificing them blood serum was collected from blood samples of each group. Collected blood serum were kept at -20^oC until analyzed. The part of pituitary and thyroid glands were dissected out from each groups of mice and stored at freeze (-20^oC) for performing western blotting for melatonin receptors of pituitary and TSH receptors of thyroid gland. The part of each tissue from each experimental group was fixed in Bouin’s fixative for performing immunohistochemistry.

Hormonal Analysis

Commercial ELISA kits (Diagnostic AutomatationInc, CA, USA) were used for analyzing the hormones (T₃, T₄ and TSH) concentrations.

Immunohistochemical Staining

Immunohistochemical staining was done to observing the localization of melatonin receptors¹¹ in pituitary gland. 5mm thin paraffin sections were kept on slides coated by 3% gelatin. Alcohol grades were used over them for deparaffinising and rehydrating. Then tissue sections were taken in PBS and then kept in methanol (with 0.3% H₂O₂) for blocking the activity of peroxide. Sections washed with PBS (0.1M Na₂HPO₄, NaH₂PO₄, 0.9% NaCl, pH=7.4) and then for 2 hrs kept in blocking solution. The sections were incubated with primary antibodies of melatonin receptors at 4^oC overnight. Next day sections were washed by PBS for 3 times and incubated with secondary antibody. After this incubation the sections were again washed for 3 times by PBS and then incubated to preformed AvidinBiotin reagent for 30 minutes. The immune interactions were detected by using DAB in in 0.01 M Tris-Cl (pH = 7.6) and 0.1% H₂O₂ and counterstained with Ehrlich’s haematoxylin. The samples were dehydrated, mounted by DPX and observed followed by taking microphotographs under 40 X of Olympus microscopes BX 41.

Western Blot analysis

Experimental tissue samples were homogenized lysed and then quantified the proteins in the studied samples by following Lowry method. SDS-PAGE was used for resolving aliquots containing 100 mg proteins followed by electro transfer to nitrocellulose membrane (Santa Cruz Biotech, USA). Primary antibodies of MT1, MT2 and TSH-R were taken for immune detection. Antibody of β-actin was taken and dissolved in PBS containing 5% skimmed milk and 0.01% Tween-20. That was followed by incubation with secondary antibodies conjugated with horse-radish peroxidase. Super Signal West Pico Chemiluminescent Substrate was used for detecting the immune responsiveness. The protein bands were quantified by measuring the optical density by Scion Image Analysis Software. Expression of value (% control value) was measured by taking the ratios of the specific signal with the signal of β-actin¹⁶.

Statistical Analysis

Data was analyzed by ANOVA and experimental results were taken significant by Tukey’s HSD multiple range test. For calculating the data and for preparing the graph Microsoft Excel program and SPSS were obtained.

Serum T3 level

Significant (p<0.01) decrease of T₃ hormone level was observed in melatonin injected mice group, whereas, it was found significantly (p<0.01) increased in L-thyroxine induced hyperthyroid group of mice as compare to control group of mice. Melatonin administration in hyperthyroid condition in mice significantly (p<0.01) decreased the T₃ hormone concentration as compare to hyperthyroid group of mice (Figure 1).

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Figure 1.

T₃ hormone level of blood serum of different experimental groups of mice. Mean ± SEM was represented by Histogram. The experimental changes were significantly considered when p< 0.05. **p<0.01: Control group vs. Melatonin group, Control group vs. T₄-treated (hyperthyroid) group; ^##p<0.01: T_4-treated (hyperthyroid) group vs. T₄-treated (hyperthyroid) +Melatonin treated group.

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Serum T4 level

In melatonin treated mice, T₄ hormone level was significantly (p<0.01) decreased and in L-thyroxine induced hyperthyroid group of mice, significant (p<0.01) increase of T₄ level was noted in comparison to control mice group. But in hyperthyroid along with melatonin treatment group, significant (p<0.01) decrease of T₄ concentration was observed as compare to hyperthyroid mice group (Figure 2).

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Figure 2.

T₄ hormone level of blood serum of different experimental groups of mice. Mean ± SEM was represented by Histogram. The experimental changes were significantly considered when p<0.05. **p<0.01: Control group vs. Melatonin group, Control group vs. T₄-treated (hyperthyroid) group; ^##p<0.01: T_4-treated (hyperthyroid) group vs. T₄-treated (hyperthyroid) +Melatonin treated group.

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Serum TSH level

The level of TSH was remain unchanged in melatonin treated mice whereas it was found significantly (p<0.01) increased in hyperthyroid group of mice as comparison with control mice. But in hyperthyroid along with melatonin treatment group, TSH level was found to be decreased significantly (p<0.01) as compare to hyperthyroid mice group (Figure 3).

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Figure 3

TSH hormone level of blood serum of different experimental groups of mice. Mean ± SEM was represented by Histogram. The experimental changes were significantly considered when p<0.05. **p<0.01: Control group vs. Melatonin group, Control group vs. T₄-treated (hyperthyroid) group; ^##p<0.01: T₄-treated (hyperthyroid) group vs. T₄-treated (hyperthyroid) + Melatonin treated group.

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TSH Receptor Protein Expression in Thyroid Gland

Significantly (p<0.01) increased expression of TSH receptor was observed in melatonin treated mice group and also in hyperthyroid mice group as compare to control mice group. But in hyperthyroid along with melatonin injected group of mice, TSH receptor expression was observed significantly (p<0.01) decreased in comparison to hyperthyroid mice group (Figure 4).

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Figure 4.

TSH receptor protein expression of pituitary gland. As loading control β-actin was used. Percent expressions of proteins were shown at lower panel done by Scion Image analysis software. Mean ± SEM was represented by Histogram. The experimental changes were significantly considered when p<0.05. **p<0.01: Control group vs. Melatonin group, Control group vs. T₄-treated (hyperthyroid) group; ##p<0.01: T₄-treated (hyperthyroid) group vs. T₄-treated (hyperthyroid) +Melatonin treated group.

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Immunohistochemical Observation of MT1 and MT2 Receptors in Pituitary Gland

In pituitary gland, MT1 and MT2 receptors immunoreactivity were observed in pars distalis region as shown (Figure 5).

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Figure 5.

Immunohistochemistry of MT1 receptor (a) and MT2 receptor (b) of pituitary gland, observed under microscope in 40X magnification. In control (c) section, DAB was not detected. Microphotographs were taken by Olympus Microscope.

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MT1 Receptor Protein Expression in Pituitary Gland

In both melatonin treatment group and hyperthyroid group of mice, MT1 receptor protein expression of pituitary was noted significantly (p<0.01) increased as compare to control group of mice. But in hyperthyroid along with melatonin administered group of mice, MT1 expression of pituitary was found to be decreased significantly (p<0.01) in comparison to L-thyroxine treated hyperthyroid mice group (Figure 6).

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Figure 6.

MT1 receptor protein expression of pituitary gland. As loading control β-actin was used. Percent expressions of proteins were shown at lower panel done by Scion Image analysis software. Mean ± SEM was represented by Histogram. The experimental changes were significantly considered when p<0.05. **p<0.01: Control group vs. Melatonin group, Control group vs. T₄-treated (hyperthyroid) group; ##p<0.01: T₄-treated (hyperthyroid) group vs. T₄-treated (hyperthyroid) +Melatonin treated group.

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MT2 Receptor Protein Expression in Pituitary Gland

MT2 receptor protein expression of pituitary was significantly (p<0.01) increased in melatonin treated mice and also in L-thyroxine administered hyperthyroid mice group as compare to control. Whereas, MT2 expression of pituitary was noted significantly (p<0.01) reduced in hyperthyroid along with melatonin treated mice group in comparison to hyperthyroid mice group (Figure 7).

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Figure 7.

MT2 receptor protein expression of pituitary gland. As loading control β-actin was used. Percent expressions of proteins were shown at lower panel done by Scion Image analysis software. Mean ± SEM was represented by Histogram. The experimental changes were significantly considered when p<0.05. **p<0.01: Control group vs. Melatonin group, Control group vs. T₄-treated (hyperthyroid) group; ##p<0.01: T₄-treated (hyperthyroid) group vs. T₄-treated (hyperthyroid) +Melatonin treated group.

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