All experimental procedures involving animals were conducted in accordance with the Guide for Care and Use of Research Animals in Teaching and Research and approved by the Institutional Animal Care and Use Committee of Yonsei University and the National Institute of Animal Science. Sexually mature crossbred female gilts were assigned randomly to either cyclic or pregnant status. The reproductive tracts of the gilts were obtained immediately after slaughter on day 0 (the onset of estrous), 3, 6, 9, 12, 15, or 18 of the estrous cycle and day 12, 15, 30, 60, 90, or 114 of pregnancy (n = 3 – 6 gilts/d/status). Pregnancy was confirmed by the presence of apparently normal spherical to filamentous conceptuses in uterine flushings on days 12 and 15 and the presence of embryos and placenta on the later days of pregnancy. Chorioallantoic tissues were obtained from days 30, 60, 90, and 114 of pregnancy (n = 3 – 4 gilts/d). Endometrium, dissected free of myometrium, was collected from the middle portion of each uterine horn, snap-frozen in liquid nitrogen, and stored at −80°C prior to RNA extraction. For in situ hybridization, cross-sections of endometrium were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) for 24 h and then embedded in paraffin as previously described [13].

Total RNA was extracted from endometrial and conceptus tissues using TRIzol reagent (Invitrogen Life Technology, Carlsbad, CA, USA), according to the manufacturer’s recommendations. The quantity of RNA was assessed spectrophotometrically, and the RNA integrity was validated following electrophoresis in 1% agarose gel.

Four micrograms of total RNA were treated with DNase I (Promega, Madison, WI, USA) and reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen, USA) to obtain cDNAs. The cDNA templates were then diluted at a 1:4 ratio with sterile water and amplified by polymerase chain reaction (PCR) using Taq polymerase (Takara Bio, Shiga, Japan). The PCR conditions and sequences of primer pairs are listed in Table 1. The PCR products were separated on 2% agarose gel and visualized by ethidium bromide staining. The identity of each amplified PCR product was verified by sequence analysis after cloning into the pCRII vector (Invitrogen, USA).

The level of S100A7A expression in endometrial and chorioallantoic tissues was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) using the Applied Biosystems StepOnePlus System (Applied Biosystems, Foster City, CA, USA) with the SYBR Green method. The Power SYBR Green PCR Master Mix (Applied Biosystems, USA) was used for PCR reactions. The final reaction volume of 20 μL included 2 μL of cDNA, 10 μL of 2X Master mix, 2 μL of each primer, and 4 μL of ddH₂O. The PCR conditions and sequences of primer pairs are listed in Table 1. The results are reported as expression relative to that detected on day 12 of the estrous cycle or that detected in control explant tissues after normalization of the transcript amount to the endogenous porcine ribosomal protein L7, ubiquitin B, and TATA binding protein controls by the 2^−ΔΔCT method, as previously described [14].

Nonradioactive in situ hybridization was performed to determine the localization of S100A7A expression in the uterine endometrium, as previously described, with some modifications [6,15]. Sections (5 μm thick) were rehydrated through successive baths of xylene, 100% ethanol, 95% ethanol, diethylpyrocarbonate (DEPC)-treated water, and DEPC-treated PBS. Tissue sections were boiled in citrate buffer (pH 6.0) for 10 min. After washing in DEPC-treated PBS, they were digested using 5 μg/mL proteinase K (Sigma, St. Louis, MO USA) in TE buffer (100 mM Tris-HCl, 50 mM ethylenediaminetetraacetic acid, pH 7.5) at 37°C. After postfixation in 4% paraformaldehyde, tissue sections were incubated twice for 15 min each in PBS containing 0.1% active DEPC and were equilibrated for 15 min in 5× saline sodium citrate (SSC). The sections were prehybridized for 2 h at 68°C in a hybridization mix (50% formamide, 5× SSC, 500 μg/mL herring sperm DNA, 250 μg/mL yeast tRNA). Sense and antisense riboprobes for each gene were generated using partial cDNAs cloned into pCRII vectors by linearizing with appropriate restriction enzymes and labeling with digoxigenin (DIG)-UTP using a DIG RNA Labeling kit (Roche, Indianapolis, IN, USA). The probes were denatured for 5 min at 80°C and added to the hybridization mix. The hybridization reaction was carried out overnight at 68°C. Prehybridization and hybridization reactions were performed in a box saturated with a 5× SSC 50% formamide solution to avoid evaporation, and no coverslips were used. After hybridization, sections were washed for 30 min in 2× SSC at room temperature, 1 h in 2× SSC at 65°C, and 1 h in 0.1× SSC at 65°C. Probes bound to the section were detected immunologically using sheep anti-DIG Fab fragments covalently coupled to alkaline phosphatase and nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (toluidine salt) as a chromogenic substrate, according to the manufacturer’s protocol (Roche, USA).

To determine the effects of steroid hormones, E₂ and progesterone (P₄), and IL1B on S100A7A expression in the endometrium, endometrial explant tissues obtained from gilts on Day 12 of the estrous cycle were cultured as previously described [13,16]. The endometrium was dissected from the myometrium and placed into warm phenol red-free Dulbecco’s modified Eagle’s medium/F-12 culture medium (DMEM/F-12; Sigma, USA) containing penicillin G (100 IU/mL) and streptomycin (0.1 mg/mL). The endometrium was minced into small pieces (2 to 3 mm³) with scalpel blades, and aliquots of 500 mg were placed into T25 flasks with serum-free modified DMEM/F-12 containing 10 μg/mL insulin (Sigma, USA), 10 ng/mL transferrin (Sigma, USA), and 10 ng/mL hydrocortisone (Sigma, USA). Endometrial explants were cultured immediately after mincing in the presence of ethanol (control), E₂ (10 ng/mL; Sigma, USA), P₄ (30 ng/mL; Sigma, USA), P₄+E₂, P₄+E₂+ICI182,780 (ICI; an estrogen receptor antagonist; 200 ng/mL; Tocris Bioscience, Ellisville, MO, USA), or P₄+E₂+RU486 (RU; a progesterone receptor [PGR] antagonist; 30 ng/mL; Sigma, USA) for 24 h with rocking in an atmosphere of 5% CO₂ in air at 37°C. To determine the effect of IL1B on endometrial S100A7A expression, explant tissues were treated with 0, 1, 10, or 100 ng/mL IL1B (Sigma, USA) in the presence of both E₂ (10 ng/mL) and P₄ (30 ng/mL) at 37°C for 24 h. Explant tissues were then harvested, and total RNA was extracted for real-time RT-PCR to determine S100A7A mRNA level. These experiments were conducted using endometria from three gilts on day 12 of the estrous cycle, and treatments were performed in triplicate using tissues obtained from each of the three gilts.

Data from real-time RT-PCR for S100A7A expression were subjected to analysis of variance using the General Linear Models procedures of SAS (Cary, NC, USA). As sources of variation, the model included day, pregnancy status (cyclic or pregnant, days 12 and 15 post-estrus), and their interactions to evaluate steady-state levels of S100A7A mRNA. Data from real-time RT-PCR performed to assess the effects of day of the estrous cycle (day 0, 3, 6, 9, 12, 15, and 18) and pregnancy (day 12, 15, 30, 60, 90, and 114) in the endometrium, the effects of day of pregnancy in chorioallantoic tissue (day 30, 60, 90, and 114), and the effect of IL1B dose in explant culture were analyzed by least squares regression analysis. Data from real-time RT-PCR to assess the effects of steroid hormone in explant culture were analyzed by preplanned orthogonal contrasts (control vs E₂; control vs P₄; P₄ vs P₄+E₂; P₄+E₂ vs P₄+E₂+ICI; and P₄+E₂ vs P₄+E₂+RU). Data are presented as mean with standard error of the mean. A p-value <0.05 was considered significant.

We examined the relative abundance of S100A7A mRNA in the endometrium using real-time RT-PCR to determine whether the expression of S100A7A changed in the endometrium during the estrous cycle and pregnancy in pigs. As shown in Figure 1, the expression of S100A7A in the endometrium did not change during the estrous cycle, while it changed during pregnancy, with the greatest abundance on day 12 of pregnancy (linear effect of day, p<0.05). On days 12 and 15 post-estrus, S100A7A expression was affected by day (p<0.01), pregnancy status (p<0.01), and day×status (p< 0.01), and the expression of S100A7A was greater on day 12 of pregnancy than day 12 of the estrous cycle (p<0.05).

Next, we performed in situ hybridization analysis to determine which cell type(s) express S100A7A mRNA in the endometrium. As shown in Figure 2, the expression of S100A7A mRNA was primarily localized to luminal epithelial (LE) cells and superficial glandular epithelial (GE) cells in the endometrium during the estrous cycle and pregnancy with strong signal intensity in LE cells on day 12 of pregnancy. In skin tissue used as a positive control, the expression of S100A7A mRNA was localized to stromal and hair follicle cells.

Having determined that S100A7A mRNA was detected in the endometrium in a stage-specific and cell type-specific manner, we then assessed whether conceptus tissues express S100A7A during early pregnancy. We performed RT-PCR using cDNAs from conceptuses from days 12 and 15 and detected S100A7A in conceptus tissues on both days of pregnancy (Figure 3A). Real-time RT-PCR analysis was performed to determine whether the expression of S100A7A changes in chorioallantoic tissues during pregnancy. The expression of S100A7A in chorioallantoic tissues during mid- to term pregnancy increased toward term pregnancy (quadratic effect of day, p<0.01) (Figure 3B).

Next, we determined the factor(s) regulating S100A7A expression in the endometrium based on the results indicating that the abundance of S100A7A mRNA in the endometrium was greatest on day 12 of pregnancy. Because estrogen and IL1B of conceptus origin are secreted into the uterine lumen with the greatest abundance on day 12 of pregnancy and the expression of many endometrial genes is regulated estrogen and IL1B of conceptus origin and/or P₄ from the CL in pigs [4], we hypothesized that E₂, P₄, and/or IL1B may affect the expression of S100A7A in the endometrium. As shown in Figure 4A, S100A7A mRNA level was increased by E₂ (Control vs E₂, p<0.001; P₄ vs P₄+E₂, p<0.01) but not by P₄. The E₂-induced increase in S100A7A mRNA was inhibited by ICI, an estrogen receptor antagonist (P₄+E₂ vs P₄+E₂+ICI, p<0.05). IL1B also increased the expression of S100A7A in a dose-dependent manner in endometrial tissues (linear effect of dose, p<0.01), as shown in Figure 4B.