E2 (17β-estradiol), P4 (progesterone), and mifepristone (RU486) were obtained from Sigma-Aldrich (USA). ICI 182780 was purchased from Tocris (UK).

Mature (14 weeks old) and immature (14 days old) female Sprague-Dawley rats were obtained from Koatech (Korea). All animals were housed in polycarbonate cages and acclimatized to an environmentally controlled room (temperature: 23 ± 2℃, relative humidity: 50 ± 10%, frequent ventilation, and a 12-h light-dark cycle) before use. The mature rats were monitored for 14 days and daily vaginal smears were obtained and examined using a low-power light microscope. After this period of observation, the rats (n = 12) were separated according to estrous cycle stage: proestrus, estrus, and diestrus.

For hormone and antagonist treatments, immature female rats (n = 30) with matched body weight were divided into six groups (five rats each a group). The steroid hormones E2 (1.2 µg) or/and P4 (120 µg) were dissolved in 50 µL of 10% ethanol (Sigma, USA) for an immature rat. And two steroid solutions were administered to the rats by subcutaneous injections. The animals were treated with E2 (40 µg/kg BW), P4 (4 mg/kg BW), or E2 and P4 for 3 days, and were then euthanized 12 h after the final injection.

To determine the effects of E2, 10 immature rats were divided into two groups that were treated daily for 3 days with subcutaneous injections of the estrogen receptor antagonist ICI 182780 (10 mg/kg BW) 30 min prior to injection with either E2 (positive control, n = 5, 40 µg/kg BW) or ethanol (negative control, n = 5). To measure the effects of P4, 10 immature rats were divided into two groups that were treated daily for 3 days with subcutaneous injections of the progesterone antagonist RU486 (10 mg/kg BW) 30 min prior to injection with either P4 (positive control, n = 5, 4 mg/kg BW) or ethanol (negative control, n = 5). The Ethics Committee of Chungbuk National University (Korea) approved all experimental procedures involving animals.

The rats were euthanized under deep ether anesthesia, and uteri were rapidly excised and washed in cold, sterile 0.9% NaCl. Total RNA extraction from uteri with TRIzol reagent (Invitrogen, USA) is a common method of total RNA extraction according to the criteria of [10], and the concentration of RNA was set by pure, concentrated of RNA to an optical density of 1.0 at 260 nm using a Perkin Elmer (MBA2000; USA) Spectrophotometer. The concentration of stock solutions were then calculated and used for all subsequent dilutions. Total RNA (1 µg) was then reverse transcribed to first-strand complementary DNA (cDNA) using moloney murine leukemia virus reverse-transcriptase (iNtRON Bio, Korea) and random primers (9-mers; TaKaRa Bio, Japan). Enpp2 cDNA was amplified in a 20 µg PCR reaction containing 0.5 U Taq polymerase (iNtRON Bio, Korea), 1.5 mM MgCl₂, 2 mM dNTP, and 100 pmol of the specific primers. The oligonucleotide primers used for producing Enpp2 cDNA were 5'-AGC TGC CTG GAC TTC ACT CA-3' (sense) and 5'-GCA GGT ATG TCT TGA GGG TCA-3' (antisense). PCR reactions were carried out with denaturation at 95℃ for 30 sec, annealing at 58℃ for 30 sec, and extension at 72℃ for 30 sec. The 1A gene was used as an internal control to normalize total cDNA; this is the cytochrome c oxidase subunit 1 which is a key enzyme for aerobic metabolism. The primers for 1A were 5'-CCA GGG TTT GGA ATT ATT TC-3' (sense) and 5'-GAA GAT AAA CCC TAA GGC TC-3' (antisense). Enpp2 and 1A genes were quantified by performing 30 and 25 cycles, respectively. PCR products (8 µL) were separated on a 2.3% agarose gel and photographed using a Gel Doc EQ (Bio-Rad, USA).

Uteri were rapidly excised and washed in cold, sterile 0.9% NaCl. Soluble proteins were extracted using Proprep (iNtRON, Korea) according to the manufacturer's instructions. Sixty µg of total protein per lane was separated by SDS-PAGE on a 7.5% gel and then transferred to polyvinylidene fluoride transfer membranes (Perkin Elmer, USA) with a TransBlot Cell (TE-22; Hoefer, USA) according to the manufacturer's protocol. The blots were blocked in Tris-buffered saline with Tween 20 (TBS-T) containing 5% skim milk for 2 h at room temperature and then incubated with primary antibodies against Enpp2 (rabbit polyclonal, 1 : 500; Cayman Chemicals, USA) or beta-actin (mouse monoclonal, 1 : 4,000; Santa Cruz Biotech, USA). After washing in TBS-T (Tris-buffered saline with containing 5% Tween20) buffer, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (anti-rabbit, 1 : 2,500, Santa Cruz Biotech, USA; or anti-mouse, 1 : 5,000, Santa Cruz Biotech, USA) for 2 h at room temperature. After washing using the 10 mL of TBS-T, antibody binding was visualized by incubating the blots in enhanced chemiluminescence chemiluminescence reagent (Amersham Biosciences, UK) and subsequent exposure to Biomax Light film (Kodak, USA) for periods ranging from 1 to 5 min. Signal specificity was confirmed by incubating the blots without either primary antibody. All bands were normalized to the β-actin immunoreactive bands on the same membrane after stripping (stripping solution is composed with SDS (10 g) and Tris (3.785 g). The density of each band was measured with ImageJ software (NIH, USA). Density measurements for each band were performed with NIH ImageJ software. Background samples from an area near each lane were subtracted from each band to obtain mean band density. Data were analyzed for significant differences by one-way ANOVA (Prism 4 Graph Pad; Graph Pad Software, USA).

The localization of Enpp2 protein was determined using immunohistochemistry. Uteri were embedded in paraffin and then sectioned. Sections (5-µm thick) were then deparaffinized in xylene and hydrated in decreasing concentrations of ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in TBS-T for 30 min. Nonspecific reactions were blocked by incubating the sections in 10% normal goat serum (Vector, USA) for 2 h at room temperature. The sections were subsequently incubated overnight at 4℃ with a polyclonal rabbit antibody (1 : 500, Cayman Chemicals, USA) against Enpp2 dissolved in 10% normal goat serum. After washing with TBS-T, the sections were incubated with biotinylated secondary antibody (anti-rabbit IgG, 1 : 1,000; Vector Laboratories, USA) for 1 h at 37℃ and further incubated with ABC-Elite (Vector, USA) for 30 min at 37℃. Diaminobenzidine (Sigma, USA) was used as a chromogen, and the sections were counterstained with hematoxylin before being mounted in mounting solution (Cytoseal-60; Richard Allen Scientific Kalamazoo, USA).

Data were analyzed using a nonparametric one-way ANOVA using Tukey's test followed by Dunnett's test for multiple comparisons to the negative controls. Data were ranked according to these tests. All statistical analyses were performed using SPSS for Windows (SPSS, USA). p-values < 0.05 were considered statistically significant.

To investigate expression of Enpp2 in rat uterus during the estrous cycle, rat uteri from each stage of the estrous cycle were classified according to vaginal cell morphology. Total RNA was extracted and RT-PCR was carried out using specific primers. The level of Enpp2 mRNA was significantly increased during diestrus compared to other stages of the cycle and was decreased during estrus (Fig. 1). Western blot analysis was performed to measure Enpp2 protein expression in rat endometrium throughout the estrous cycle. Enpp2 protein was highly expressed during diestrus, but the expression was reduced during proestrus and estrus (Fig. 2). These results indicate that the expression of uterine Enpp2 mRNA and protein are tightly regulated throughout the estrous cycle and may be influenced by steroid hormone fluctuations during this cycle.

To investigate the effects of E2 and P4 on Enpp2 expression, immature rats were injected daily with E2 and/or P4 for 3 days. Uterine expression of Enpp2 mRNA and protein was significantly decreased by E2 compared to the vehicle (Figs. 3 and 4). To investigate the ER-mediated effects of E2 on uterine Enpp2 transcription, ICI 182780, an ER antagonist, was injected into immature rats 30 min prior to E2 treatment. Pretreatment with the ER antagonist completely reversed the E2-induced reduction of Enpp2 mRNA and protein expression (Figs. 3 and 4). This finding suggests that ERs are involved in E2-mediated regulation of Enpp2 expression in the uterus.

To examine the localized expression of Enpp2 protein in the uterus, we performed immunohistochemistry using an anti-Enpp2 antibody on tissue sections obtained during different stages of the estrous cycle (proestrus, estrus, and diestrus). Enpp2 protein was found in stromal cells during the proestrus and estrous stages. Enpp2 protein expression was also observed in endometrial luminal epithelial cells and the endometrial glands during diestrus (Fig. 5).