Identification of appropriate reference genes for qPCR analyses of placental expression of SLC7A3 and induction of SLC5A1 in porcine endometrium

Abstract

IntroductionEndometria and placentae undergo developmental changes that affect the stability of genes used as references for normalization of qPCR data. We identified genes that are stable within the porcine endometrium and placenta throughout pregnancy, and elucidated the temporal/spatial mRNA localization of the glucose and arginine transporters, solute carrier family (SLC) 5A1 and SLC7A3, respectively. Materials and MethodsqPCR was performed for 10 genes within porcine endometria from Days 5, 11, and 15 of the estrous cycle and 11, 15, 25, 40, 60, and 85 of pregnancy; and chorioallantois from Days 30, 35, 45, 50, 60, and 85. Gene stability was analyzed using GeNorm and NormFinder algorithms. qPCR and in situ hybridization determined temporal/spatial localization of SLC5A1 and SLC7A3 at the uterine-placental interface. ResultsThe geometric mean of TATA-binding protein (TBP), hypoxanthine phosphoribosyl transferase 1 (HPRT1), and tubulin alpha 1B (TUBA1B) provides acceptable reference values for porcine placenta. The geometric mean of TBP, beta actin (ACTB), and succinate dehydrogenase complex subunit A flavoprotein (SDHA) is acceptable for endometria. SLC5A1 is induced by estrogen in endometrial luminal epithelium (LE) on Days 12 and 13 of pregnancy. SLC7A3 is expressed in the chorion. Discussion and ConclusionUsing appropriate reference genes resulted in complementary results between qPCR and in situ hybridization techniques for SLC5A1 and SLC7A3 mRNAs. SLC5A1 is induced in uterine LE by estrogen of trophectoderm origin when the blastocyst is free-floating and dependent on glucose from the endometrium, and SLC7A3 is expressed by the established placenta to support fetal growth.