Among those, we selected, for the microarray and RNA-seq analyses, four PG transporters, ABCC1 (also known as MRP1), ABCC9 (also known as sulfonylurea receptor 2 [SUR2]), SLCO4C1 (also known as OATP4C1), and SLCO5A1 (also known as OATP5A1), because these have been shown to be differentially expressed in the uterine endometrium during early pregnancy [10,11].
SLCO4C1 and SLCO5A1 are also expressed in many tissues including kidney, liver, and brain [15,16].
Although it has been shown that expression of ABCC1 gene is induced by estrogen and progesterone in human trophoblast cells , regulatory mechanisms of the expression of PG transporter genes, ABCC1, ABCC9, SLCO4C1, and SLCO5A1 are not well understood.
Therefore, to understand the expression and regulation of PG transporters in porcine uterine endometrium during the estrous cycle and pregnancy, we evaluated i) expression of ABCC1, ABCC9, SLCO4C1, and SLCO5A1 mRNAs in the uterine endometrium during the estrous cycle and pregnancy, in the conceptus of early pregnancy, and in chorioallantoic tissues during pregnancy; ii) localization of ABCC1 and ABCC9 mRNAs in the uterine endometrium; and iii) effects of steroid hormones, IL1B, and interferon gamma (IFNG) on ABCC1, ABCC9, SLCO4C1, and SLCO5A1 mRNA expression in the endometrial tissues.
Total RNA extraction and cloning of porcine ABCC1, ABCC9, SLCO4C1, and SLCO5A1 cDNAs
The 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 ABCC1, ABCC9, SLCO4C1, and SLCO5A1 mRNA sequences.
The levels of expression of ABCC1, ABCC9, SLCO4C1, and SLCO5A1 genes in endometrial and chorioallantoic tissues were analyzed by real-time RT-PCR using the Applied Biosystems StepOnePlus System (Applied Biosystems, Foster City, CA, USA) and SYBR Green method.
To determine the effect of steroid hormones, estradiol-17[beta] ([E.sub.2]) and [P.sub.4], and IL1B on the expression of AB CC1, AB CC9, SLCO4C1, and SLCO5A1 mRNAs in the uterine endometrium, endometrial explant tissues obtained from gilts on D12 of the estrous cycle were cultured as previously described .
Data from real-time RT-PCR for ABCC1, ABCC9, SLCO4C1, and SLCO5A1 expression during the estrous cycle and pregnancy were subjected to least squares analysis of variance using the general linear models procedures of SAS (Cary, NC, USA).
To determine the expression of mRNAs for PG transporters ABCC1, ABCC9, SLCO4C1, and SLCO5A1 in the porcine uterine endometrium during the estrous cycle and pregnancy, we performed real-time RT-PCR analysis (Figure 1).