Inkyu Yoo

Comprehensive Analysis of Prostaglandin Metabolic Enzyme Expression During Pregnancy and the Characterization of AKR1B1 as a Prostaglandin F Synthase at the Maternal-Conceptus Interface in Pigs

ABSTRACT Prostaglandins (PGs) are important lipid mediators regulating various reproductive processes in many species. In pigs, the expression pattern of PGE2 and PGF2α metabolic enzymes and the regulatory mechanism controlling PGE2 and PGF2α levels in the uterus during pregnancy are not completely understood. This study determined endometrial expression of the genes (PLA2G4A, PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, CBR1, and HPGD) involved in PGE2 and PGF2α metabolism during the estrous cycle and pregnancy and measured levels of PGE2 and PGF2α in uterine endometrial tissues and uterine flushings at the time of conceptus implantation in pigs. Except PTGES3, expression of the genes studied changed in a pregnancy-stage-specific manner, and localization of PTGES, AKR1B1, CBR1, and HPGD mRNAs were cell-type specific in the uterine endometrium. Levels of both PGE2 and PGF2α in uterine endometrial tissues and uterine lumen were higher on Day 12 of pregnancy than those of the estrous cycle and affected by different morphology of spherical and filamentous conceptuses. Furthermore, we determined that endometrial expression of AKR1B1, known to encode a PGF2α synthase in other species, was increased by estrogen and interleukin-1beta and that AKR1B1 exhibited PGF2α synthase activity in the porcine uterine endometrium. These results in pigs indicate that the PGE2 and PGF2α metabolic enzymes are expressed stage specifically in the endometrium during pregnancy and regulate the abundance of PGE2 and PGF2α in the uterus at the time of implantation and that AKR1B1 may act as a major PGF synthase in the endometrium during early pregnancy.

Prostaglandin Transporters, ABCC4 and SLCO2A1, in the Uterine Endometrium and Conceptus During Pregnancy in Pigs

ABSTRACT Prostaglandins (PGs) are involved in many reproductive activities including luteolysis, maternal recognition of pregnancy, endometrial gene expression, conceptus development, and parturition in domestic animals. However, mechanisms by which PGE2 and PGF2alpha are modulated in the uterine endometrium and expression of ABCC4 and SLCO2A1, responsible for efficient transport of PGs across the cell membrane, in the endometrium during the estrous cycle and pregnancy are not fully understood in pigs. Therefore, we determined expression of ABCC4 and SLCO2A1, genes involved in transport of PGE2 and PGF2alpha in the uterine endometrium during the estrous cycle and pregnancy in pigs. ABCC4 and SLCO2A1 mRNAs were expressed in the uterine endometrium, most abundantly on Day 12 of pregnancy and during late pregnancy. Expression of ABCC4 mRNA and protein was localized mainly to uterine luminal epithelial (LE) and glandular epithelial (GE) cells, and expression of SLCO2A1 mRNA and protein was expressed primarily in uterine LE and blood vessels. Expression of ABCC4 and SLCO2A1 mRNAs was also detected in conceptuses during early pregnancy. In addition, explant culture experiments showed that increasing doses of interleukin 1B (IL1B) with estrogen and progesterone increased levels of ABCC4 and SLCO2A1 mRNAs in the uterine endometrium. These results indicate that expression of genes responsible for transport of PGE2 and PGF2alpha are dynamically regulated in the uterine endometrium during pregnancy and that ABCC4 and SLCO2A1 play critical roles in supporting the establishment and maintenance of pregnancy by regulating PG transport at the maternal–fetal interface in pigs.

Analysis of Stage-Specific Gene Expression Profiles in the Uterine Endometrium during Pregnancy in Pigs

The uterine endometrium plays a critical role in regulating the estrous cycle and the establishment and maintenance of pregnancy in mammalian species. Many studies have investigated the expression and function of genes in the uterine endometrium, but the global expression pattern of genes and relationships among genes differentially expressed in the uterine endometrium during gestation in pigs remain unclear. Thus, this study investigated global gene expression profiles using microarray in pigs. Diverse transcriptome analyses including clustering, network, and differentially expressed gene (DEG) analyses were performed to detect endometrial gene expression changes during the different gestation stages. In total, 6,991 genes were found to be differentially expressed by comparing genes expressed on day (D) 12 of pregnancy with those on D15, D30, D60, D90 and D114 of pregnancy, and clustering analysis of detected DEGs distinguished 8 clusters. Furthermore, several pregnancy-related hub genes such as ALPPL2, RANBP17, NF1B, SPP1, and CST6 were discovered through network analysis. Finally, detected hub genes were technically validated by quantitative RT-PCR. These results suggest the complex network characteristics involved in uterine endometrial gene expression during pregnancy and indicate that diverse patterns of stage-specific gene expression and network connections may play a critical role in endometrial remodeling and in placental and fetal development to establish and maintenance of pregnancy in pigs.

Analysis of Cysteine-X-Cysteine motif chemokine ligands 9, 10, and 11 and their receptor CXCR3 and their possible role on recruitment of immune cells at the maternal-conceptus interface in pigs†

Abstract Chemokines play critical roles in the establishment and maintenance of pregnancy in animals. Cysteine-X-cysteine motif chemokine ligand 9 (CXCL9), CXCL10, and CXCL11 are involved in recruiting immune cells by binding to their shared receptor, CXC receptor 3 (CXCR3), in a variety of tissues. This study examined the expression and regulation of chemokines CXCL9, CXCL10, and CXCL11, their receptor CXCR3, and their role at the maternal-conceptus interface in pigs. The endometrium expressed CXCL9, CXCL10, CXCL11, and CXCR3 stage specifically during pregnancy, with the greatest abundance on Day 15 of pregnancy. It was noted that their expression was primarily localized to stromal cells, endothelial cells, or vascular smooth muscle cells in the endometrium. Interferon-γ increased the abundance of CXCL9, CXCL10, CXCL11 mRNAs, but not CXCR3, in endometrial explants. Furthermore, recombinant CXCL9 (rCXCL9), rCXCL10, and rCXCL11 proteins increased migration of cultured peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. Recombinant CXCL9 and rCXCL10 caused migration of CD4+, CD8+, CD4+CD8+ T cells, and natural killer (NK) cells, and rCXCL11 increased migration of CD4+ T and NK cells in PBMCs. The present study demonstrated that interferon-γ-induced CXCL9, CXCL10, and CXCL11, and their receptor CXCR3 were expressed in the uterus in stage- and cell-type specific manners and increased the migration of T and NK cells, which showed the greatest endometrial infiltration on Day 15 of pregnancy. These results suggest that CXCL9, CXCL10, and CXCL11 may play an important role in the recruitment of immune cells into the endometrium during the implantation period in pigs. Summary Sentence Chemokines CXCL9, 10, and 11 induced by interferon-gamma of conceptus origin in the endometrium are involved in the recruitment of immune cells at the maternal-conceptus interface in pigs.

Chemokine (C-C Motif) Ligand 28 and Its Receptor CCR10: Expression and Function at the Maternal-Conceptus Interface in Pigs

ABSTRACT Many chemokines are present at the maternal-fetal interface and play important roles in the establishment and maintenance of pregnancy. Our study demonstrates that a chemokine, chemokine (C-C motif) ligand 28 (CCL28), is expressed in the uterine endometrium during early pregnancy in pigs. Thus, we investigated expression of CCL28 and its receptors, chemokine (C-C motif) receptor types 3 (CCR3) and 10 (CCR10), in the uterine endometrium during the estrous cycle and pregnancy and the function of CCL28 at the maternal-fetal interface during early pregnancy. Levels of CCL28 mRNAs were highest on Day 10 of pregnancy and decreased thereafter during pregnancy, and CCL28 was localized mainly to endometrial glandular epithelial cells. The presence of the CCL28 protein in uterine flushings was confirmed on Day 12 of the estrous cycle and pregnancy. Endometrial tissues expressed CCR3 and CCR10 during pregnancy. The CCR10 protein was localized to endometrial luminal and glandular epithelial cells, chorionic epithelial cells, and the allantoic membrane during pregnancy. Conceptuses during early pregnancy expressed CCL28 and CCR10, but not CCR3, and chorioallantoic tissues expressed CCR10 at increasing levels towards term. Treatment with recombinant CCL28 increased the proliferation and migration of a porcine trophectoderm cell line. These results indicated that the CCL28 chemokine and its receptors, CCR3 and CCR10, are expressed at the maternal-conceptus interface, and CCL28 induces the proliferation and migration of trophectoderm cells through CCR10, suggesting that CCL28 may play a critical role in the establishment and maintenance of pregnancy in pigs.

Calcium extrusion regulatory molecules: differential expression during pregnancy in the porcine uterus

Calcium ions in the uterine endometrium are essential for the establishment and maintenance of pregnancy, but the cellular and molecular mechanisms of calcium ion regulation in the endometrium are not fully understood. Our previous study in pigs found that calcium regulatory molecules, transient receptor potential, vanilloid type 6 and calbindin-D9K, are expressed in the uterine endometrium during the estrous cycle and pregnancy. However, we did not determine the expression of calcium extrusion regulatory molecules, plasma membrane calcium ATPases (ATP2Bs), sodium/calcium exchangers (SLC8As), or potassium-dependent sodium/calcium exchangers (SLC24As), in the uterine endometrium and conceptuses. Thus, in this study we determine whether ATP2Bs, SCL8As, and SLC24As are expressed in the uterine endometrium during the estrous cycle and pregnancy and in conceptuses during early pregnancy. Real-time RT-PCR analysis showed that ATP2Bs, SLC8As, and SLC24As were expressed in the uterine endometrium in a pregnancy status- and stage-specific manner. Conceptuses during early pregnancy also expressed these molecules. In situ hybridization analysis showed that ATP2B1, SLC8A1, and SLC24A4 were localized mainly to luminal and glandular epithelium and stromal cells in the endometrium during pregnancy. These results indicate that calcium extrusion regulatory molecules are expressed in the uterine endometrium during the estrous cycle and pregnancy and in conceptuses during early pregnancy, indicating that calcium extrusion regulatory molecules may play important roles in the establishment and maintenance of pregnancy by regulating calcium ion concentration in the uterine endometrium in pigs.

Analysis of legumain and cystatin 6 expression at the maternal-fetal interface in pigs.

Cathepsins (CTSs), a family of lysosomal cysteine proteases, and their inhibitors, cystatins (CSTs), play a critical role in endometrial and placental tissue remodeling during the establishment and maintenance of pregnancy in many species including rodents, sheep, cow, and pigs. In this study, we determined expression of legumain (LGMN), a cathepsinmember, and its inhibitor, CST6, at the maternal–fetal interface in pigs. Expression of both LGMN and CST6 mRNAs increased during mid‐ to late pregnancy in the uterine endometrium. LGMN and CST6 mRNAs localized to luminal epithelial cells (LE) and glandular epithelial cells (GE) and to the chorionic membrane (CM), with a strong intensity in GE and the CM for LGMN and in the CM for CST6 during pregnancy. LGMN protein was detected at molecular weights (MW) of approximately 50,000 and 37,000, and the abundance of the37,000‐MW LGMN protein increased during mid‐ to latepregnancy. CST6 protein was also highly expressed in the uterine endometrium in mid‐ to latepregnancy. LGMN protein localized to LE, GE, and the CM during pregnancy. LGMN and CST6 were aberrantly expressed in the uterine endometrium from gilts with somatic cell nuclear transfer‐derived conceptuses at term compared to those of gilts carrying conceptuses derived from natural mating. These results demonstrated that LGMN and CST6 were expressed in the uterine endometrium in a cell‐type and stage‐specific manner, suggesting that the LGMN and CST6 system at the maternal–fetal interface may play an important role in the establishment and maintenance of pregnancy in pigs. Mol. Reprod. Dev. 80: 570–580, 2013.

Characterization of interferon α and β receptor IFNAR1 and IFNAR2 expression and regulation in the uterine endometrium during the estrous cycle and pregnancy in pigs

Type I interferons (IFNs) bind to the heterodimeric receptor composed of IFN-α/β receptor 1 (IFNAR1) and IFN-α and β receptor 2 (IFNAR2) to transmit signals into the cell. It is well known that IFN-δ (IFND), a type I IFN, is secreted by the conceptus during early pregnancy in pigs. However, expression and regulation of IFNAR1 and IFNAR2 in the porcine uterine endometrium are not well understood. Thus, we analyzed the expression and regulation of IFNAR1 and IFNAR2 in the uterine endometrium during the estrous cycle and pregnancy and conceptus and chorioallantoic tissues during pregnancy in pigs. The IFNAR1 and IFNAR2 mRNAs were expressed in the uterine endometrium, and their levels on Day 12 of pregnancy were higher than those on Day 12 of the estrous cycle and highest during pregnancy. The IFNAR1 and IFNAR2 mRNAs were also expressed in conceptuses during early pregnancy, in chorioallantoic tissues during mid-to-term pregnancy, and in endometrial epithelial cells and chorionic membrane during mid-to-late pregnancy. The abundance of IFNAR1 and IFNAR2 mRNAs was increased by interleukin-1β (IL1B), and the abundance of IFNAR2 was increased by estradiol in endometrial tissue explants. Thus, IFNAR1 and IFNAR2 mRNAs were expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner, and their expression was affected by estradiol and/or IL1B. These results suggest that endometrial and conceptus IFNAR1 and IFNAR2 may mediate the action of type I IFNs during the implantation period for the establishment and maintenance of pregnancy in pigs.

Expression and regulation of prostaglandin transporters, ATP-binding cassette, subfamily C, member 1 and 9, and solute carrier organic anion transporter family, member 2A1 and 5A1 in the uterine endometrium during the estrous cycle and pregnancy in pigs

Objective Prostaglandins (PGs) function in various reproductive processes, including luteolysis, maternal pregnancy recognition, conceptus development, and parturition. Our earlier study has shown that PG transporters ATP-binding cassette, subfamily C, member 4 (ABCC4) and solute carrier organic anion transporter family, member 2A1 (SLCO2A1) are expressed in the uterine endometrium in pigs. Since several other PG transporters such as ABCC1, ABCC9, SLCO4C1, and SLCO5A1 are known to be present in the uterine endometrium, this study investigated the expression of these PG transporters in the porcine uterine endometrium and placenta. Methods Uterine endometrial tissues were obtained from gilts on day (D) 12 and D15 of the estrous cycle and days 12, 15, 30, 60, 90, and 114 of pregnancy. Results ABCC1, ABCC9, SLCO4C1, and SLCO5A1 mRNAs were expressed in the uterine endometrium, and levels of expression changed during the estrous cycle and pregnancy. Expression of ABCC1 and ABCC9 mRNAs was localized mainly to luminal and glandular epithelial cells in the uterine endometrium, and chorionic epithelial cells during pregnancy. Conceptuses during early pregnancy and chorioallantoic tissues from mid to late pregnancy also expressed these PG transporters. Estradiol-17β increased the expression of ABCC1 and SLCO5A1, but not ABCC9 and SLCO4C1 mRNAs and increasing doses of interleukin-1β induced the expression of ABCC9, SLCO4C1, and SLCO5A1 mRNAs in endometrial explant tissues. Conclusion These data showed that several PG transporters such as ABCC1, ABCC9, SLCO4C1, and SLCO5A1 were expressed at the maternal-conceptus interface, suggesting that these PG transporters may play an important role in the establishment and maintenance of pregnancy by regulating PG transport in the uterine endometrium and placenta in pigs.

Maternal-Conceptus Interactions: Mediators Regulating the Implantation Process in Pigs

For successful embryo implantation, the communication of the maternal endometrium with the conceptus trophec-toderm is required essentially. In pigs, conceptuses undergo morphological change in length to enlarge the physical contact area with the maternal endometrium and secrete estrogen to induce the maternal recognition of pregnancy during the peri-implantation period. Conceptus-derived estrogen prevents luteolysis by conversion in direction of PGF2α secretion from the uterine vasculature to the uterine lumen as well as it affects on expression of the uterine endo-metrial genes. In addition to estrogen, conceptuses release various signaling molecules, including cytokines, growth factors, and proteases, and, in response to these signaling molecules, the maternal uterine endometrium also syn-thesizes many signaling molecules, including hormones, cytokines, growth factors, lipid molecules, and utilizes ions such as calcium ion by calcium regulatory molecules. These reciprocal interactions of the conceptus trophectoderm with the maternal uterine endometrium make development and successful implantation of embryos possible. Thus, signaling molecules at the maternal-conceptus interface may play an important role in the implantation process. This review summarized syntheses and functions of signaling molecules at the maternal-conceptus interface to further understand mechanisms of the embryo implantation process in pigs.