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 (No. YWC-P120) and the National Institute of Animal Science (No. 2015-137). Sexually mature crossbred female gilts of similar age (6 to 8 months) and weight (100 to 120 kg) were assigned randomly to either cyclic or pregnant status. Gilts assigned to the pregnant uterus status group were artificially inseminated with fresh boar semen at the onset of estrus (day 0) and 12 h later. The reproductive tracts of gilts were obtained immediately after slaughter on either days 0 (onset of estrous behavior), 3, 6, 9, 12, 15, or 18 of the estrous cycle (21-day cycle: days 0 to 3, estrus; days 3 to 6, metestrus; days 6 to 15, diestrus; days 15 to 0, proestrus) and either days 10, 12, 15, 30, 60, 90, or 114 of pregnancy (n = 3 – 6/d/status). Pregnancy was confirmed by the presence of apparently normal spherical to filamentous conceptuses in uterine flushings on days 10, 12, and 15 and the presence of embryos and placenta on the later days of pregnancy. Uterine flushings were obtained by introducing and recovering 25 mL phosphate-buffered saline (PBS; pH 7.4) into each uterine horn. Chorioallantoic tissues were obtained from days 30, 60, 90, and 114 of pregnancy (n = 3 – 4/d). Endometrial tissues from prepubertal gilts (n = 8; approximately 6 months of age) that had not undergone the estrous cycle with no corpus luteum formed were obtained from a local slaughterhouse. Endometrial tissue, dissected free of the 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 PBS (pH 7.4) for 24 h and then embedded in paraffin as described previously [21].

Total RNA was extracted from endometrial, conceptus, and chorioallantoic tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. The quantity of RNA was assessed spectrophotometrically, and the integrity of RNA was validated by electrophoresis in 1% agarose gels. Four micrograms of total RNA from endometrial, conceptus, and chorioallantoic tissues were treated with DNase I (Promega, Madison, WI, USA) and reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen, USA) to obtain complementary DNAs (cDNAs). cDNA templates were then diluted 1:4 with nuclease-free water and amplified by polymerase chain reaction (PCR) using Taq polymerase (Takara Bio, Shiga, Japan) and specific primers based on porcine MMP2, MMP8, MMP9, MMP12, and MMP13 mRNA sequences. PCR conditions, sequences of primer pairs for MMP2, MMP8, MMP9, MMP12, and MMP13, and expected product sizes are listed in Table 1. PCR products were separated on 2% agarose gels 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).

To analyze expression of MMP2, MMP8, MMP9, MMP12, and MMP13 mRNAs in the endometrial and chorioallantoic tissues, real-time reverse transcription polymerase chain reaction (RT-PCR) was performed using the Applied Biosystems StepOnePlus System (Applied Biosystems, Foster City, CA, USA) and SYBR Green. Complementary DNAs were synthesized from 4 μg total RNA isolated from different uterine endometrial tissues, and newly synthesized cDNAs (total volume of 21 μL) were diluted 1:4 with nuclease-free water and then used for PCR. 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 (100 nM), and 4 μL of dH₂O. PCR conditions and sequences of primer pairs are listed in Table 1. Results are reported as expression relative to the level detected on day 12 of the estrous cycle for endometrial tissues, day 30 of pregnancy for chorioallantoic tissues, or the control group in endometrial explant tissues after normalization of the transcript amount to the endogenous porcine ribosomal protein L7 (RPL7), ubiquitin B (UBB), and TATA-binding protein (TBP) controls by the 2^−ΔΔCT method [22].

A nonradioactive in situ hybridization procedure was performed as described previously (Braissant and Wahli, 1998) with some modifications. Sections (5 μm thick) were rehydrated through successive baths in xylene, 100% ethanol, 95% ethanol, and diethylpyrocarbonate (DEPC)-treated water. 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 100 mM Tris-HCl and 50 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5, at 37°C. After postfixation in 4% paraformaldehyde, tissue sections were incubated twice for 15 min in PBS containing 0.1% active DEPC and equilibrated for 15 min in 5× saline sodium citrate (SSC). Sections were prehybridized for 2 h at 68°C in a hybridization mix (50% formamide, 5× SSC, 500 μg/mL herring sperm DNA, and 250 μg/mL yeast tRNA). Sense and antisense MMP2, MMP8, and MMP13 riboprobes labeled with digoxigenin (DIG)-UTP were denatured for 5 min at 80°C and added to the hybridization mix. Hybridization reactions were carried out overnight at 68°C. Prehybridization and hybridization reactions were performed in a box saturated with a 5× SSC and 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 chromogenic substrate, according to the manufacturer’s protocol (Roche, Basel, Switzerland). Images were captured using an Eclipse TE2000-U microscope (Nikon, Seoul, Korea) and processed with Adobe Photoshop CS6 software (Adobe Systems, Seattle, WA, USA).

Gelatinase activity in endometrial tissue homogenates was determined by gelatin zymography. Endometrial tissues were homogenized in lysis buffer (1% [v/v] Triton X-100, 0.5% [v/v] Nonidet P-40, 50 mM NaCl, 10 mM Tris, 1 mM EDTA, 0.2 mM Na₃VO₃, 0.2 M phenylmethylsulphonyl fluoride, 0.5 μg/mL NaF, and a proteinase inhibitor cocktail [Roche, Switzerland]) at a ratio of 100 mg tissue per 1 mL buffer, and cellular debris was removed by centrifugation. Proteins (50 μg) were loaded in each lane and electrophoresed on 8% SDS-PAGE gels containing 0.3% gelatin under non-reducing conditions, followed by incubation overnight at 37°C in 50 mM Tris buffer (pH 7.5) with 1% (v/v) Triton X 100, 5 mM CaCl₂, 1 μM ZnCl₂, and 1 μM 4-aminophenylmercuric acetate. Gels were stained with Coomassie blue for 1 h and destained with tap water until bands could be clearly visualized. Gel images were captured using the Gel Doc EZ Gel Documentation System (Bio-Rad, Hercules, CA, USA) and processed with Adobe Photoshop CS6 software (Adobe Systems, USA).

Endometrial explant tissues were obtained from prepubertal gilts to determine the effects of ovarian steroid hormones or from gilts on day 12 of the estrous cycle to determine the effect of conceptus-derived cytokines on the expression of MMP2, MMP8, MMP9, MMP12, and MMP13 mRNAs in the endometrium as described previously [23,24]. 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). Endometrial tissue was minced into small pieces using scalpel blades (2 to 3 mm³), 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 0, 5, 50, 500 pg/mL estradiol-17β (E2; Sigma, USA) or 0, 0.3, 3, 30 ng/mL progesterone (P4; Sigma, USA) or 0, 1, 10, or 100 ng/mL of IL-1β (IL1B) (Sigma, USA) or IFN-γ (IFNG) (R&D Systems, Minneapolis, MN, USA) in the presence of 30 ng/mL P4, 10 ng/mL E2, and 10 ng/mL IL1B in an atmosphere of 5% CO₂ in air at 37°C. Doses were chosen to encompass the full range of physiological levels of E2, P4, IL1B, and IFNG [25]. Explant tissues were then harvested, and total RNA was extracted for real-time RT-PCR to determine the expression of MMP2, MMP8, MMP9, MMP12, and MMP13 mRNAs.

Data from real-time RT-PCR to assess MMP2, MMP8, MMP9, MMP12, and MMP13 expression during the estrous cycle and pregnancy were analyzed by ANOVA using the general linear models procedures of SAS (Cary, NC, USA). Data from real-time RT-PCR to assess the effects of day of the estrous cycle (days 0, 3, 6, 9, 12, 15, and 18) and pregnancy (days 10, 12, 15, 30, 60, 90, and 114) in the endometrium and chorioallantoic tissue (days 30, 60, 90, and 114) and the effect of E2, P4, IL1B, and IFNG on MMP2, MMP8, MMP9, MMP12, and MMP13 expression were analyzed by least-squares regression analysis. Data are presented as means with standard error of the mean. Differences were considered significant if p<0.05.

To determine whether MMP2, MMP8, MMP9, MMP12, and MMP13 are expressed in the endometrium during the estrous cycle and pregnancy in pigs, we measured their relative abundance in the endometrium during the estrous cycle and pregnancy using real-time RT-PCR analysis (Figure 1). During the estrous cycle, the abundance of MMP12 and MMP13 mRNAs, but not MMP2, MMP8, and MMP9 mRNAs, changed in the endometrium with the highest levels observed on day 18 (cubic effect of the day; p<0.01 for MMP12, p<0.05 for MMP13) (Figure 1A). During pregnancy, the abundance of MMP2, MMP9, MMP12, and MMP13 mRNAs, but not MMP8 mRNA, changed in the endometrium (linear effect of the day; p<0.01) (Figure 1B).

Next, we determined whether conceptuses during the peri-implantation period of pregnancy express MMP2, MMP8, MMP9, MMP12, and MMP13 by RT-PCR using cDNAs from conceptuses from days 12 and 15 of pregnancy. MMP2 and MMP13 mRNA expression was detected in conceptus tissues on both days of pregnancy, while MMP9 mRNA was detected only on day 12 and MMP8 and MMP12 mRNAs were not detected (Figure 2A). We also performed real-time RT-PCR analysis to determine if the expression of MMP2, MMP8, MMP9, MMP12, and MMP13 changed in chorioallantoic tissues from day 30 to term pregnancy. The expression of MMP2, MMP8, and MMP12 mRNAs, but not MMP9 and MMP13 mRNAs, changed during pregnancy (linear effect of the day for MMP2 and MMP12, cubic effect of the day for MMP8; p<0.05) (Figure 2B).

To determine which cell type(s) express MMP2, MMP8, and MMP13 mRNAs in the endometrium, we performed in situ hybridization analysis (Figure 3). MMP2 mRNA was localized to stromal cells in the endometrium during the estrous cycle and pregnancy, and interestingly, luminal epithelial (LE) cell-specific MMP2 expression was also detected on day 15 of pregnancy. In addition, MMP2 expression was primarily localized to stromal cells in chorioallantoic tissue during pregnancy (Figure 3A). MMP8 and MMP13 expression was localized to epithelial and stromal cells in the endometrium during the estrous cycle and pregnancy, and also to epithelial and stromal cells in chorioallantoic tissue during pregnancy (Figures 3B and 3C).

Having determined that MMPs were expressed in the endometrium during the estrous cycle and pregnancy, we further determined if MMP2 protein with enzymatic activity was present in the endometrium on days 12 and 15 of the estrous cycle and pregnancy using gelatin zymography (Figure 4). Clear single band of MMP with gelatinase activity and a molecular weight of approximately 75 kDa, which corresponds to pro-MMP2 [26], was detected in each lane.

Because the endometrium is a major target tissue of the steroid hormones E2 and P4 [25] and the expression of some MMPs varied during the estrous cycle and pregnancy, we investigated whether E2 and P4 affected the expression of MMP2, MMP8, MMP9, MMP12, and MMP13 in endometrial tissues. To assess the individual effects of E2 or P4 and to exclude the possibility that these hormones influenced endometrial tissues during the estrous cycle, we utilized endometrial tissues from prepubertal gilts that had not yet undergone an estrous cycle. When we cultured endometrial explant tissues with increasing doses of E2 or P4, we found that E2 increased the expression of MMP8 and MMP12 (linear effect of dose; p<0.01) (Figure 5A), and P4 decreased the expression of MMP12 (linear effect of dose; p<0.01) (Figure 5B). The expression of MMP2, MMP9, and MMP13 in endometrial tissues was not affected by E2 or P4 treatment.

Because the expression of MMP2 in the endometrium were greatest at the implantation period, which corresponds to the time when the implanting conceptus secretes a significant amount of IL1B and IFNs [3,25], we postulated that IL1B and/or IFNG may affect the expression of MMPs in the endometrium during early pregnancy. We treated endometrial explant tissues from day 12 of the estrous cycle with increasing doses of IL1B or IFNG. IL1B increased the expression of MMP2, MMP8, MMP9, and MMP13, but not MMP12, in a dose-dependent manner (linear effect of dose; p<0.05) (Figure 6A), while IFNG increased the expression of MMP2, but not others, in endometrial explant tissues (linear effect of dose; p<0.01) (Figure 6B).