In this study, white New Zealand rabbits and Balb/C mice aged 8-12 weeks were used. All procedures were conducted according to the guidelines of the animal care and ethics committee of Avicenna Research Institute.

Wet vaginal smear was obtained by intra vaginal injection of Phosphate Buffered Saline (PBS). Stages of estrous cycle were determined by cellular morphology and ratio of epithelial cells to leukocytes in each slide. Five mice were studied in each four stages of estrous cycle: proestrous, estrous, metestrous and diestrous.

Following determination of estrous cycle, mice were sacrificed and their uterine horns were excised. Left uterine horn was washed completely with PBS and cut open from anti-mesometrial axis. Exposed endometrial tissue was then scraped with scalpel. Endometrial tissue was rapidly homogenized using pellet pestle (Sigma, USA) and dissolved in RNA-Bee (Biosite, Sweden).

As our positive control for thyroglobulin expression, thyroid gland of one mouse was excised and homogenized similarly in RNA-Bee solution. Total RNA from tissue homogenates was then extracted according to standard protocols. Briefly, after chloroform was added to and mixed with RNA-Bee solution, total RNA of tissue was collected from liquid supernatant and precipitated by 2 steps alcohol precipitation. Extracted RNA was dissolved in 20–50 µl of sterile water and stored in −20°C for further analysis.

Ten micro liters of total RNA from each tissue were heated to 65°C and immediately cooled on ice. cDNA Mix including 5x buffer, 2 mM dNTP mix (Roche, Germany), 2 mM Random hexamer (Cybergene, Sweden) and 20 u/µl RT M-MuLV in a final volume of 10 µl distilled water was added to the RNA. The mixture was then incubated in 42°C for 60 minutes. The cDNA was then kept at −20°C.

For each specimen, 3 PCR reactions were set up using 2 sets of primers for TG and 1 set for housekeeping gene GAPDH (Glycer al-dehyde 3-phosphate dehydrogenase). Each PCR reaction contained 10x PCR buffer (Roche, Germany), 2.5 mM MgCl2, 0.4 mM dNTP mix and 0.04 u/µl Taq DNA polymerase (all from Roche, Germany) and 0.4 µM of each primer set. One microliter of cDNA mixture was added to each reaction and incubated in thermocycler with the following thermal profile: 94°C for 2 minutes for initial denaturation, 30 cycles of 94°C, 60°C and 72°C each for 30 seconds and final extension in 72°C for 6 minutes.

Sequence of the primers used is demonstrated in Table 1.

First and second sets of primers were designated to amplify 207 bp (TG1) and 510 bp (TG2) fragments of TG mRNA, respectively. The third set of primers amplified a 309 bp segment of GAPDH gene as internal control. PCR products were electrophoresed on 1% agarose gel and the amplified bands were visualized and documented by UV transilluminator (UVP, USA).

White New Zealand rabbits were injected IM with bovine TG (Sigma, Sweden) plus freund adjuvant every 2 weeks. Before and after each injection, blood samples were collected and titers of anti TG antibody were assessed by indirect ELISA. Briefly, TG was coated on wells and after blocking, rabbit serum was added to them in serial dilutions. Following incubation and washing, HRP conjugated sheep antirabbit antibody (Avicenna Research Institute, Iran) was added to the wells.

Finally, optical density at 450 nm was measured after addition of horseradish peroxidase (HRP) substrate, TMB (3, 3′, 5, 5′-Tetra methylbenzidine).

Following the 5^th injection, massive bleeding was performed and the antiTG antibody was purified over TG affinity chromatography column. Purity of the resultant antibody was assessed by electrophoresis on 10% polyacrylamide gel and its reactivity was confirmed by immunohistochemistry of mouse thyroid tissue.

Samples from uterine, ovary and fallopian tubes of 20 mice (5 mice in each phase of estrous cycle) and the thyroid from one mouse were snap frozen in OCT (Jung, Denmark). Frozen section of tissues, 5 µm in thickness, were cut and transformed on glass slides. After initial steps of fixation and blocking of non-specific binding sites by 10% normal sheep serum, immunostaining was carried out by indirect method.

Briefly, slides were incubated with anti-TG antibody (5 µg/ml) for 90 minutes, after then endogenous peroxidase was quenched by 0.3% H2O2. 1:250 dilution of sheep anti-rabbit antibody (Avicenna Research Institute) was then added for 45 minutes followed by Diaminobenzidine (DAB) (Roche, Germany). Afterwards, slides were counterstained with hematoxylin, dehydrated in increasing grades of ethanol and mounted in enthelan (Merck, Germany). In negative control slides, primary antibody was substituted by the same concentration of non-immune rabbit immunoglobulin.

As a housekeeping gene, GAPDH was used as an internal control for mRNA extraction and cDNA synthesis. As expected, 309 bp band of amplified GAPDH mRNA was found in all endometrial and thyroid samples. On the contrary, neither 207 bp nor 510 bp bands corresponding to the amplified segments of TG mRNA with TG1 & TG2 primer sets, respectively, were detected in any endometrial samples but they were both strongly positive in thyroid tissue. Therefore, we concluded that TG gene is not expressed in endometrial tissue of Balb/C mice at any stage of estrous cycle (Figure 1).

Despite the fact that we used bovine TG for immunization, the resulting polyclonal antibody had excellent reactivity with mouse TG because of high molecular homology between species. Based on ELISA assay, high titers of antiTG antibody were reached after the third round of injection.

Polyacrylamide gel electrophoresis of the purified antibody showed a dominant band of 150 kD molecular weight which corresponded to IgG isotype. In order to confirm the reactivity of the purified antibody, we used IHC staining on both frozen and paraffin embedded sections of thyroid which showed specific binding in both methods (Figure 2).

In order to study the expression of thyroglobulin at the protein level in female reproductive organs, we performed IHC staining of endometrium, ovary and fallopian tubes at different stages of estrous cycle using our homemade antiTG antibody.

As shown in Figure 3, none of the tissues of reproductive organs had detectable levels of TG, while sections of thyroid were all strongly positive for expression of TG. IHC staining confirmed our RT-PCR results and proved that TG is expressed at neither RNA nor protein level in any reproductive organs of cycling Balb/C mice.