Testosterone Reduces Tight Junction Complexity and Down-regulates Expression of Claudin-4 and Occludin in the Endometrium in Ovariectomized, Sex-steroid Replacement Rats

Materials and Methods

Animals and hormone treatment. Three-month-old adult female Sprague-Dawley (SD) rats (n=6), weighing 225±25 g, were caged under standard conditions (lights on 06:00 to 18:00 h; room temperature 25±2°C; 5-6 animals per cage). Animals were fed with rat chow (Harlan, Germany) and tap water ad libitum. All experimental procedures were approved by the University of Malaya Institutional Ethics Committee (2013-07-15/FIS/R/NS). Estrogen, testosterone, flutamide, finasteride and peanut oil were obtained from Sigma–Aldrich (Saint Louis, MO, USA). Rats were divided into five groups with six rats in each group. Bilateral ovariectomy was performed 21 days prior to steroid treatment to eliminate the effect of endogenous sex steroids hormones (18). Drugs were dissolved in peanut oil and injected subcutaneously behind the neck scruff in a volume of 0.1 ml.

In this study, ovariectomized rats were treated with a sex-steroid regime to mimic the hormonal changes in early pregnancy. The regime included the injection of 1.0 μg/kg/day estrogen on days 1 and 2, 1.0 μg/kg/day estrogen and 4 mg/kg/day progesterone on day 3, no treatment on days 4 and 5, and 16 mg/kg/day progesterone and 0.5 μg/kg/day estrogen between days 6-8 according to the established protocol by Kennedy et al. (19). Vehicle-treated animals received daily injections for 8 days of 0.1 ml peanut oil. Testosterone at 1 mg/kg/day, the dose regarded as supra-physiological in females (20),was given for 3 days (days 6-8) that was considered the period of uterine receptivity. Testosterone was given with flutamide (5 mg/kg/day) or finasteride (1 mg/kg/day). Both inhibitors were administered 30 min prior to testosterone injection. The rats from all groups were sacrificed and uterine horns were harvested 1 day after the last day of treatment.

Transmission electron microscopy (TEM). Uteri were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. 2 mm-cross sections of each uterine horn were cut and incubated overnight at 4°C in 2.5% glutaraldehyde buffer. Following removal of the buffer, the samples were rinsed three times, 15 min each, in 0.1 M phosphate buffer. Samples were then incubated in 1% osmium in 0.1 M phosphate buffer, rinsed and dehydrated in series of ethanol (70-100%). Samples were incubated twice for 5 min in propylene oxide and then transferred to a rotor for 1 h at room temperature in 1:1 mixture of propylene oxide and epon (47% Embed 812, 31% dodenyl succinic anhydride, 19% nadic methyl anhydride, 3% benzyldimethylamine; Electron Microscopy Sciences, Hatfield, PA, USA). This was followed by overnight incubation in 1:2 propylene oxide-epon, and finally 100% epon for 2-3 h. Individual uterine samples were embedded in 100% epon in silicon flat embedding molds, and capsules were polymerized at 60°C for 48 h. Ultrathin transverse sections (70 nm) were prepared by using a diamond knife (Diatome, Hatfield, PA, USA) on a MT 6000-XL ultramicrotome, captured on 300-mesh copper grids, and stained with 2% uranyl acetate. The ultrathin sections were observed under TEM (Libra 120; Zeiss, Oberkochen, Germany) to assess the changes in tight junction morphology. The junctional regions of two randomly chosen villi were examined in each specimen.

Immunofluorescence detection of protein distribution. Uterine tissues were cut into 5-mm sections, deparaffinized in xylene, and rehydrated in decreasing concentrations of ethanol. Trisodium citrate (pH 6.0) was used for antigen retrieval, while 10% H₂O₂ in phosphate buffered saline (PBS) was used to neutralize endogenous peroxidase. Uterine sections were blocked in 10% normal rabbit serum (sc-2338; Santa Cruz Biotechnology, Santa Cruz, CA, USA) prior to incubation with goat IgG polyclonal primary antibody to claudin-4 (sc-17664) and goat IgG polyclonal primary antibody to occludin (sc-8145; Santa Cruz Biotechnology). The antibodies were diluted at 1:100 in PBS with 1.5% normal blocking serum at room temperature for 1 h. After rinsing three times with PBS, sections were incubated with rabbit anti-goat IgG–fluorochrome-conjugated secondary antibody (sc-2777; Santa Cruz Biotechnology) at a dilution of 1:250 in PBS with 1.5% normal blocking serum at room temperature for 45 min. The slides were rinsed three times with PBS and were mounted with Ultracruz mounting medium (Santa Cruz Biotechnology). Counterstaining with 4,6-diamidino-2-phenylindole was used to visualize the nuclei. All images were viewed under Nikon Eclipse 80i (Nikon, Tokyo, Japan) camera that was attached to a fluorescent microscope. Negative controls were performed by omitting the primary antibodies specific to claudin-4 and occludin or by using non-immune IgG; in these sections, no staining was observed.

Real-time PCR (qPCR) quantification of messenger RNA (mRNA). Whole uterine tissues were kept in RNALater solution (Ambion, Carlsbad, CA, USA) prior to RNA extraction. RNA was extracted by using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) and its concentration was assessed by 260/280 UV absorption ratios (GeneQuant 1300; Biochrom, Cambridge, UK). Gene expression of claudin-4 (Cldn4) and occludin (Ocln) was evaluated using two-step real-time PCR. In this study, TaqMan1 RNA-to-CT 1-Step Kit (Ambion) was used. Firstly, cDNA was reversely transcribed to RNA by using high capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA). Amplifications on samples with no reverse transcriptase acted as control. The amplified region of cDNA was probed with TaqMan fluorescence-labeled probe. The TaqMan probe had a sensitivity of 100% and specificity of 96.67% (21) and was capable of detecting as few as 50 copies of RNA/ml (22) and as few as 5-10 molecules (23). The specificity of primer and probe ensured that expression of target DNA was specifically evaluated. Validation was performed in silico by using whole rat genome and in-vitro by using whole rat cDNA (Applied Biosystem) to ensure that specific sequences were detected. Thus, additional sequencing was not required.

The assay used TaqMan® Rn01196224_s1 for Cldn4 and Rn00580064_m1 for Ocln (Applied Biosystems) which amplified 86 bp from the whole mRNA length of 1,824bp of Cldn4 and 4,148 for Ocln. In this study, glyceraldehyde 3-phosphate dehydrogenase (Gapdh) (Rn99999916_s1) and hypoxanthine phosphoribosyltransferase-1 (Hprt1) (Rn01527840-m1) (Applied Biosystems) were used as reference or house-keeping genes as their expression in uterine tissue was found to be most stable throughout the estrus cycle (24).

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Figure 1.

Ultramorphological appearance of uterine tight junctions in ovariectomized rats. Transmission electron microscopy images showing tight junction (TJ) morphology in the endometrium of different experimental groups. C: Vehicle control; N: normal sex-steroid replacement regime; T: testosterone; FLU: flutamide; FIN: finasteride. NP: normal early pregnant rat; M: microvilli; V: vacuole. Scale bar: 40 μm; n=6 per treatment group.

PCR amplification program included 2 min at 50°C with uracil N-glycosylase, 20 s of 95°C activation with ampliTaq gold DNA polymerase and 1 min denaturation at 95°C for 20 s and annealing/extension at 60°C for 1 min. Denaturing and annealing was performed for 40 cycles. Negative controls were performed which include omission of reverse transcriptase or omission of cDNA. All measurements were normalized by using GenEx software (MultiD, Odingatan, Sweden) followed by Data Assist v3 (Applied Biosystems) that was used to calculate the fold changes in RNA. Data were analyzed according to the comparative cycle threshold (CT) (2^^ΔΔCT) method. The relative quantity of target in each sample was determined by comparing the normalized target quantity in each sample to the average normalized target quantity of references.

Statistical analysis. Statistical differences were evaluated by Student's t-test and analysis of variance (ANOVA). A probability level of less than 0.05 (p<0.05) was considered as significant. Post-hoc statistical power analysis was performed for all experiments and all values obtained were >0.8, which were considered adequate.