S100As are calcium-binding proteins with two EF-hand calcium-binding motifs. In several studies, S100A proteins are described to play important roles in pro-inflammatory responses including damage-associated molecular pattern (DAMP) signaling and in the establishment of pregnancy. However, the role of S100As have not been determined in the uterine endometrium during the estrous cycle in pigs. Thus, this study was performed to investigate expression and regulation of S100A8, S100A9, and S100A12 in the uterine endometrial tissues during the estrous cycle in pigs. Real-time RT-PCR analysis showed that S100A8, S100A9, and S100A12 mRNAs were expressed in the uterine endometrium during the estrous cycle with higher levels on days 15 and 18 of the estrous cycle than other days of cycle. To investigate the effects of steroid hormones, estradiol (E2) and progesterone (P4), on expression of S100A8, S100A9, and S100A12 mRNAs, endometrial tissue explants from immature pigs were treated with steroid hormones. Levels of S100A8, S100A9, and S100A12 were increased by the treatment of P4, and the increased levels of S100A8, S100A9, and S100A12 by P4 were not inhibited by the treatment of progesterone receptor antagonist, RU486. However, levels of S100A8, S100A9, and S100A12 were decreased by treatment of MEK inhibitor, U0126. These results exhibited that S100As were expressed in the uterine endometrium during the estrous cycle in a cyclic stage-specific manner, and their expression was affected by P4. These suggest that S100As may play an important role in endometrial function during the proestrous period of the estrous cycle in pigs. [Supported by the Next Generation Biogreen 21 program (#PJ01119103), Rural Development Administration, and by Korea Research Foundation (#2015R1D1A1A01058356)]

For the establishment and maintenance of successful pregnancy the maternal immune system must tolerate semi-allogenic fetus during pregnancy. Several mechanisms explaining immune tolerance have been proposed. Among those, it has been suggested that the CD40/CD40L system is involved in immune tolerance in several tissues. However, expression and function of CD40/CD40L in the maternal-fetal interface during pregnancy have not been studied in pigs. Thus, this study determined expression and localization of CD40 and CD40L in the uterine endometrium during pregnancy in pigs. We obtained uterine endometrial tissue samples from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy. Quantitative real-time PCR analysis showed that levels of CD40L mRNA expression during pregnancy increased on D15 of pregnancy and decreased thereafter whereas levels of CD40 mRNA was highest on D30 of pregnancy. Localization of CD40 and CD40L proteins by immunohistochemistry showed that CD40 was localized to vascular endothelial cells with strongest signal intensity on D15 of pregnancy, and CD40L was localized to luminal epithelial cells on D15 of pregnancy and amniotic membrane during mid- to late pregnancy. To determine the effect of IFNG on CD40 and CD40L expression, we took advantage of endometrial explant culture using tissues from D12 of the estrous cycle, and found that CD40 was up-regulated by IFNG in a dose-dependent manner. These results showed that CD40 and CD40L were expressed in the uterine endometrium in a cell-type and stage-specific fashion during pregnancy, and IFNG induced CD40, indicating that the CD40/CD40L system may be important for establishment and maintenance of pregnancy in pigs. [Supported by the Next Generation BioGreen21 Program (#PJ01110301), Rural Development Administration]

Interleukin-12 (IL12) and IL23 are members of the IL12 family and secreted from antigen presenting cells (APCs) such as dendritic cells and macrophages. IL12 and IL23 are composed of two subunits of a sharing subunit, IL12B, and a unique subunit, IL12A for IL12 and IL23A for IL23. IL12 is involved in induction of T helper (Th) type 1 response, whereas IL23 is associated with the differentiation of naive T cells into Th17 cells. It has shown that IL12, a proinflammatory cytokine, is down-regulated during pregnancy for successful establishment and maintenance pregnancy and increases in plasma levels in women with preeclampsia. IL23 decreases IL12 expression to change the immune microenvironment at the maternal fetal interface in humans and mice. In the present study we determined the expression of IL12 and IL23 and their receptors in the endometrium and placenta during pregnancy. Real-time RT-PCR analysis showed levels of IL12A and IL12B mRNAs in the endometrium were high during early pregnancy, but maintained low during mid- to term pregnancy. During pregnancy, levels of IL12RB1 mRNA in the endometrium showed a biphasic pattern with the highest levels on Days 15 and 60 of pregnancy, while levels of IL12RB2 mRNA did not change. Levels of IL23A and IL23R mRNAs in the endometrium decreased toward term pregnancy. Immunohistochemical analysis showed that IL12A protein was localized specifically to scattered cells in endometrial stroma, but barely detected during mid- to term pregnancy. Conceptuses from early pregnancy expressed IL12, IL23, and their receptors, except IL12RB2, and corioallantoic tissues during mid- to late pregnancy expressed IL12, IL23, and their receptors, but not IL23R. These results showed that IL12 and IL23 and their receptors were expressed at the maternal-conceptus interface, suggesting that IL12 and IL23 may play a key role in regulating maternal immune environment for the establishment and maintenance of pregnancy in pigs.

S100 protein family is small calcium-binding proteins with two EF-hand motifs and comprises more than 20 proteins in human. Although S100A proteins are known to play important roles in proinflammatory responses including damage-associated molecular pattern (DAMP) signaling and in the establishment of pregnancy, the expression of S100As have not been determined in the uterine endometrium during the estrous cycle in pigs. Thus, this study was performed to investigate expression and localization of S100A8, S100A9, and S100A12 in the uterine endometrial tissues during the estrous cycle in pigs. Real-time RT-PCR analysis showed that S100A8, S100A9, and S100A12 mRNAs were expressed in the uterine endometrium during the estrous cycle with higher levels on days 15 and 18 of the estrous cycle than the other days of cycle. Immunohistochemistry analysis showed that S100A9 and S100A12 proteins were mainly localized to the immune cells in the uterine endometrium. Especially, S100A9- and S100A12-positive immune cells were detected in the uterine blood vessels on day 15 of the estrous cycle, and also localized to stroma near to luminal epithelium on days 0 and 18 of the estrous cycle. These results showed that S100As were expressed in the uterine endometrium during the estrous cycle in a cyclic stage-specific manner, and these proteins were localized to the immune cells in the endometrium. These suggest that immune cells expressing S100A proteins may be recruited into the endometrium during the estrous cycle and play an important role in regulating endometrial function in pigs.