Modulation of small leucine-rich proteoglycans (SLRPs) expression in the mouse uterus by estradiol and progesterone

Swiss female mice, aged 3-5 months, were used in this study. Animals were housed in a 12-h light: 12-h dark, temperature-controlled (22°C) environment, with free access to food and water. The stages of the estrous cycle were determined by vaginal smears. Animals in estrus and diestrus (physiological control of E2 and P4 levels, respectively), and ovariectomized animals submitted or not to hormone treatment were anesthetized with an intraperitoneal injection of tribromoethanol (Avertin^®) (Aldrich Chemical Company, Inc., Milwaukee, Wi, USA; 0,025 mL/g body weight). The uteri were subsequently removed, cut with razor blades and immediately immersed in a fixative solution or in RNAlater solution (Sigma-Aldrich, St. Louis, MO, USA). National guidelines for laboratory animal care were followed, and all experiments were approved by the Institute of Biomedical Sciences Animal Ethics Committee (authorization number, 144/2002).

The general protocol adopted by us [23] was adapted from Domino and Hurd [24]. During standardization of this experimental protocol, the established doses were 10 μg of 17β-estradiol (Sigma-Aldrich) and 0.5 mg of medroxyprogesterone acetate (MPA) (Pharmacia & Upjohn), a progesterone analog. The choice was based on vaginal smear features and uterine morphology, which were similar to what was observed in the cycling mice [21]. Mice were ovariectomized and divided into five experimental groups, as follows:

1. Tissue collection twenty days after ovariectomy without hormone treatment.

2. Pre-treatment with daily priming doses of E2, diluted in mineral oil (Schering-Plough), for three days, followed by a two-day rest and daily injections of E2 during four consecutive days.

3. Pre-treatment with daily priming doses of E2, followed by a resting period and daily injections of MPA, diluted in distilled water, during four consecutive days.

4. Same pre-treatment and resting period as the previous groups, followed by daily injections of both E2 and MPA during four consecutive days.

5. Control group received injections of vehicle alone (mineral oil) for three days, followed by a two-day rest and daily oil injections during four consecutive days.

All injections were sub-cutaneous in a 100 μl volume. Twenty four hours after the last injection, the mice were anesthetized and the uterine samples were collected as described above.

The samples were fixed at 4°C for 3 h in Methacarn (absolute methanol, chloroform and glacial acetic acid; 6:3:1), rinsed with absolute ethanol, and embedded in Paraplast (Oxford, St. Louis, MO, USA) at 60°C. 5 μm sections were adhered onto glass slides pre-coated with 0.1% poly-L-lysine (Sigma, St. Louis, MO, USA) and then dried at 37°C.

The immunoperoxidase staining was performed as previously described [21]. Sections were treated with 3% (v/v) H₂O₂ in PBS (30 min) to block endogenous peroxidase activity. Each of the succeeding steps was followed by a thorough rinse in PBS. All steps were performed in a humidified chamber. Samples were pretreated with Chondroitinase ABC from Proteus vulgaris (Seikagaku, Tokyo, Japan), diluted in 20 mM pH 6,0 Tris-HCl buffer (1 h at 37°C). Nonspecific staining was blocked by incubating the sections for 1 h with normal rabbit serum (for lumican) or goat serum (for the other molecules), diluted 1:1 (v/v) in PBS - 10% BSA (w/v) (room temperature). Sections were then incubated with primary antibodies (Table 1) diluted in PBS containing 0.3% (v/v) Tween 20, overnight (4°C). After extensive rinsing in PBS all sections were incubated for 1 h at room temperature with the specific biotin-conjugated secondary antibody (Table 1) diluted in PBS, for 1 h at room temperature. After rinsing in PBS, sections were incubated with Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The peroxidase reaction was visualized using 0.03% (w/v) 3,3'-diaminobenzidine in PBS with 0.03% (v/v) H₂O₂. In order to achieve standardization of the immunoreactions, for each antibody, the slides were simultaneously incubated with DAB, and reaction was immediately interrupted with PBS after a specific period of time (1-5 minutes, depending on the antibody). Afterwards, sections were lightly counterstained with Mayer's haematoxilin (Merck, Darmstadt, Germany). For each immunohistochemical reaction, control reactions were performed by omitting the primary antibody step from the protocol. In addition, paraffin sections of mouse embryos were used as positive and negative control.

The immunostained sections were examined in a Nikon Eclipse E600 microscope and the images were captured using a digital camera (Cool SNAP-Procf color; Roper Scientific, Trenton, NJ, USA) and Image Pro Plus software (Media Cybernetics, Silver Spring, MD, USA).

Table 1 lists the antibodies used in the present study. Decorin, biglycan and fibromodulin antibodies (LF-113, LF-159 and LF-150, respectively) were raised in rabbit and recognize the core protein of each macromolecule. These antibodies were previously tested by Western blot by the producers. Detail of the procedures made by Larry Fisher (National Institute of Dental and Craniofacial Research, NIH, Bethesda, USA) may be found in Fisher et al [23].

The anti-mouse lumican antibody (R&D Systems, #AF2745) is an IgG produced in goats, immunized with purified, NS0-derived, recombinant mouse Lumican (rmLumican). The mouse lumican specific IgG was purified by affinity chromatography. The specificity was tested by direct ELISA and Western blot.

For the molecular biology experiments, the phases of estrus (highest estrogen levels) and diestrus (highest progesterone levels) were chosen to represent the estrous cycle.

Upon use, uterine samples (n = 6 per group), excised with sterilized razor blades and stored in RNAlater solution at -20°C, were transferred to sterile microfuge tubes containing ceramic beads and 500 μl of Trizol reagent (Invitrogen, Calbard, CA, USA). Samples were homogenized and total RNA extracted using the Precellys 24 homogenizer (Bertin Technologies, Saint-Quentin-en-Yvelines, France), following the manufacturer's instructions. RNA quantity and quality were assessed with a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., USA). Reverse transcription (RT-PCR) was performed with AffinityScript QPCR cDNA Synthesis kit (Stratagene, Cedar Creek, TX, USA) and 1 μg of total RNA. Different reference genes were tested and Ywhaz (Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) was chosen as internal control for showing the most uniform expression across groups in the PCR amplification experiments. The relative expression of the SLRPs mRNA was determined as described previously [25]. The relative levels of mRNA of the tested genes were estimated in duplicate samples by fluorescence quantified with the ABI Prism 7500 sequence detector (Applied Biosystems, Ontario, Canada). Reactions were performed in a total volume of 25 μl containing 20 ng of cDNA and 450 nM primers in a reaction buffer containing SYBR Green PCR master mix (Stratagene). All C_q (quantification cycle, according to the MIQE guidelines [26]) values were normalized to the expression of the reference gene and the results were expressed as fold-induction relative to the expression of the calibrator sample (estrus), arbitrarily set to 1. Primers were designed with NCBI's primer designing tool to span an exon-exon junction, in order to limit amplification only to mRNA. Primer sequences are given in Table 2.

Multiple comparisons were performed by one-way analysis of variance (ANOVA) followed by the Student Newman Keuls post-test to determine significant differences among all groups, using Prism 4.0 (GraphPad Software, San Diego, CA, USA). All results are expressed as the mean ± standard error of the mean (SEM). Values of p less than or equal to 0.05 were considered statistically significant.

Ovariectomy did not suppress the deposition of decorin in the uterine tissues. In the non-treated groups (ovx and oil), the immunoreaction was present in the deep stroma and only traces were observed in the superficial stroma. In the myometrium, the immunoreaction was present in the external muscle layer (EML) and connective tissue between layers, but was not observed in the ECM of the internal muscle layer (IML) (Figure 1A).

In the E2-treated group, strong immunoreaction was present in the whole endometrial stroma, observed as a network of thin brownish filaments. The immunoreaction was more intense around the luminal and glandular epithelia, as well as around blood vessels. The staining was also strong in the myometrium, especially around bundles of smooth muscle cells in the EML (Figure 1B).

In the MPA-treated group, the immunoreaction was strong in the deep stroma, however only traces were observed in the subepithelial stroma, as previously observed in diestrus [20]. In the myometrium, the reaction was present in the ECM and inside muscle cells of both layers, and in the connective tissue between layers (Figure 1C).

In the E2+MPA-treated group, the immunoreaction was present in the whole stroma, as a network of thin filaments, except in the subepithelial stroma at the cleft regions of the luminal epithelium branches. In the myometrium, the immunostaining was moderate in the ECM of the IML and strong in the EML and connective tissue between layers (Figure 1D).

During the estrous cycle, there were no significant differences between groups. Ovariectomy significantly increased decorin mRNA expression (≈4 fold) (p < 0.001) when compared to estrus. Hormone replacement promoted a significant reduction in decorin expression in the E2-treated group (≈4 fold) (p < 0.001) and MPA and E2+MPA-treated groups (≈2 fold) (p < 0.01), when compared to the non-treated ovariectomized group. Moreover, decorin expression in the MPA and E2+MPA-treated groups was significantly higher than in diestrus and estrus, respectively (≈2 fold) (p < 0.01) (Figure 2).

In the ovx and oil groups, the deposition of biglycan was not observed in the endometrium and myometrium. However, the immunoreaction was detected in the cytoplasm of mononucleated leukocytes (Figure 3A).

In the E2-treated group, the immunoreaction was present in the endometrial stroma mainly around the luminal and glandular epithelia. In the myometrium, the staining was observed in the ECM of both smooth muscle layers, especially the IML, and in the connective tissue between them, particularly around blood vessels (Figure 3B).

In the MPA-treated group, a diffuse brownish staining was observed in the endometrial stroma. In the myometrium, the immunoreaction was moderate in both layers. It was also present in the connective tissue between layers, especially around blood vessels (Figure 3C).

In the E2+MPA-treated group, biglycan was distributed as a network of thin brownish filaments, especially in the subepithelial stroma, where leukocytic infiltration was observed. In the myometrium, the immunoreaction was more conspicuous in the IML and connective tissue between layers, whereas a weak staining was observed in the EML (Figure 3D).

As observed for decorin, there was no significant difference in biglycan mRNA levels between estrus and diestrus. However, after ovariectomy there was a drastic reduction in biglycan expression (≈3 fold) (p < 0.001), when compared to estrus. A significant increase was observed after hormone treatment with MPA (≈3 fold) (p < 0.05) and E2+MPA (≈5 fold) (p < 0.001), when compared to the non-treated ovariectomized group. Treatment with E2 alone did not promote significant differences in biglycan mRNA levels in relation to the ovariectomized group. However, it was significantly lower than in estrus (≈2 fold) (p < 0.05) (Figure 4).

In the ovx and oil groups, a faint immunoreaction was observed in the scarce extracellular spaces of the endometrial stroma, especially in the region of basement membrane of the luminal epithelium, as well as in the deep stroma. In the myometrium, the deposition of lumican was weak in the connective tissue between layers, in the EML and in the outer lining of the uterus, the mesothelium (Figure 5A).

In the E2-treated group, lumican was widely distributed in the whole endometrial stroma, in both layers of the myometrium, around bundles of smooth muscle cells, and in the connective tissue between layers (Figure 5B).

In the MPA-treated group, the immunoreaction was present in the whole endometrial stroma as a diffuse brownish staining, particularly in the deep stroma. Under the luminal epithelium, only traces of immunoreaction were observed. In the myometrium, the immunostaining was present only in the EML and connective tissue between layers (Figure 5C).

In the E2+MPA-treated group, strong immunoreaction was present in the whole endometrial stroma. In the myometrium, the immunodeposition of lumican was observed in the connective tissue between layers and a weak staining was present in both myometrial layers, especially in the EML (Figure 5D).

There were no significant differences in lumican expression between estrus and diestrus. After ovariectomy there was a ≈2 fold increase in lumican mRNA levels when compared to estrus. Hormone treatments did not significantly alter lumican expression in the uterus, when compared to the non-treated ovariectomized group, maintaining a ≈2 fold increase in relation to estrus (p < 0.05) (Figure 6).

In the ovx and oil groups, fibromodulin immunostaining was not observed in the ECM of the endometrium and myometrium. However, some mononucleated leukocytes were immunoreactive in the endometrial stroma, as previously observed during the estrous cycle (Figure 7A).

In the E2-treated group, the immunoreaction was present in the apical region of the luminal epithelium and in the cytoplasm of mononucleated leukocytes observed in the endometrial stroma. Strong immunostaining was present in both myometrial layers, and around blood vessels of the connective tissue between layers (Figure 7B).

In the MPA-treated group, the immunoreaction was present in the luminal epithelium. The glandular secretion was also immunoreactive to fibromodulin. In the myometrium, the immunoreaction was strong in both muscle layers, particularly the EML (Figure 7C).

In the E2+MPA-treated group, the staining pattern was similar to that observed in the MPA group (Figure 7D).

Fibromodulin mRNA expression was significantly higher in diestrus than in estrus (≈2 fold) (p < 0.05). Ovariectomy promoted a conspicuous increase in fibromodulin mRNA levels, when compared to estrus (≈7 fold) (p < 0.001). In addition, fibromodulin expression was significantly reduced after E2 treatment (≈7.5 fold) (p < 0.001), when compared to the non-treated ovariectomized group. Moreover, MPA and E2+MPA groups showed significantly augmented expression when compared to diestrus (≈2 fold) (p < 0.01) and estrus (≈2.5 fold) (p < 0.05), respectively (Figure 8).