Interleukin-10 and its receptors at the maternal-conceptus interface: expression, regulation, and implication for T helper 2 cytokine predominance and maternal immune tolerance in the pig, a true epitheliochorial placentation species†.

The appropriate balance between pro-inflammatory and anti-inflammatory cytokines is important for the maternal immune tolerance during pregnancy in mammals. Among the various cytokines, interleukin (IL)-10 (IL10) plays an essential role in anti-inflammatory responses, while IL12 is involved in pro-inflammatory responses during pregnancy. However, the roles of IL10 and IL12 in the endometrium during pregnancy have not been studied in pigs. Thus, we investigated the expression of IL10, IL12 (IL12A and IL12B), and their receptors (IL10RA, IL10RB, IL12RB1, and IL12RB2) at the maternal-conceptus interface. IL10, IL12, and their receptors were expressed in the endometrium during the estrous cycle and pregnancy in a pregnancy stage-specific manner. During pregnancy, IL10 expression increased on Day 15, whereas the expression of IL12A and IL12B decreased after the implantation period. IL10 protein was localized to luminal epithelial (LE), stromal cells, and macrophages; IL10RA protein to LE, endothelial, stromal, and T cells; and IL10RB mRNA to LE cells in the endometrium. IL10 and IL10RA proteins and IL10RB mRNA were also localized to chorionic epithelial (CE) cells. In endometrial explants, the expression of IL10RA and IL10RB was induced by estradiol-17ß, IL-1ß, and/or interferon-γ. Heme oxygenase 1, an IL10-inducible factor, was expressed in the endometrium with the highest levels on Day 30 of pregnancy and was localized to LE and CE cells. These results in pigs suggest that conceptus-derived signals change the endometrial immune environment by regulating the expression of IL10 and IL10 receptors at the maternal-conceptus interface and that IL10 may provide anti-inflammatory conditions for the maternal immune tolerance.

Calcium-binding proteins S100A8, S100A9, and S100A12: expression and regulation at the maternal-conceptus interface in pigs†.

Among the many calcium-binding proteins, S100A8, S100A9, and S100A12 play important roles in inflammation, innate immunity, and antimicrobial function, but their expression, regulation, and function at the maternal-conceptus interface in pigs are not fully understood. Therefore, we determined the expression and regulation of S100A8, S100A9, S100A12, and their receptor AGER at the maternal-conceptus interface in pigs. We found that S100A8, S100A9, and S100A12 mRNAs were expressed in the endometrium during the estrous cycle and pregnancy, with the greatest levels on Day (D) 12 of pregnancy, and AGER appeared at greater levels on D15 and D30 of pregnancy than on other days. The expression of S100A8, S100A9, and S100A12 was predominantly localized to epithelial cells in the endometrium, and they were detected in early-stage conceptus and later chorioallantoic tissues during pregnancy. AGER expression was localized to endometrial epithelial and stromal cells and chorionic epithelial cells. In endometrial explant tissues, the expression of S100A8, S100A9, and S100A12 was induced by estrogen, S100A8 by interleukin-1ß, and AGER by interferon-γ. We further found that on D12 of pregnancy, the expression of S100A8, S100A9, and S100A12 decreased significantly in the endometria of gilts carrying conceptuses derived from somatic cell nuclear transfer. These results indicate that the expression of S100A8, S100A9, and S100A12 is dynamically regulated in response to conceptus-derived signals at the maternal-conceptus interface, suggesting that S100A8, S100A9, and S100A12 could play a critical role in regulating endometrial epithelial cell function and conceptus implantation to support the establishment and maintenance of pregnancy in pigs.

Uterine epithelial expression of the tumor necrosis factor superfamily: a strategy for immune privilege during pregnancy in a true epitheliochorial placentation species.

The maternal immune system tolerates semi-allogeneic placental tissues during pregnancy. Fas ligand (FASLG) and tumor necrosis factor superfamily 10 (TNFSF10) are known to be components of maternal immune tolerance in humans and mice. However, the role of FASLG and TNFSF10 in the tolerance process has not been studied in pigs, which form a true epitheliochorial type placenta. Thus, the present study examined the expression and function of FASLG and TNFSF10 and their receptors at the maternal-conceptus interface in pigs. The endometrium and conceptus tissues expressed FASLG and TNFSF10 and their receptor mRNAs during pregnancy in a stage-specific manner. During pregnancy, FASLG and TNFSF10 proteins were localized predominantly to endometrial luminal epithelial cells with strong signals on Day 30 to term and on Day 15, respectively, and receptors for TNFSF10 were localized to some stromal cells. Interferon-γ (IFNG) increased the expression of TNFSF10 and FAS in endometrial tissues. Co-culture of porcine endometrial epithelial cells over-expressing TNFSF10 with peripheral blood mononuclear cells yielded increased apoptotic cell death of lymphocytes and myeloid cells. In addition, many apoptotic T cells were found in the endometrium on Day 15 of pregnancy. The present study demonstrated that FASLG and TNFSF10 were expressed at the maternal-conceptus interface and conceptus-derived IFNG increased endometrial epithelial TNFSF10, which, in turn, induced apoptotic cell death of immune cells. These results suggest that endometrial epithelial FASLG and TNFSF10 may be critical for the formation of micro-environmental immune privilege at the maternal-conceptus interface for the establishment and maintenance of pregnancy in pigs.

Analysis of interferon-γ receptor IFNGR1 and IFNGR2 expression and regulation at the maternal-conceptus interface and the role of interferon-γ on endometrial expression of interferon signaling molecules during early pregnancy in pigs.

It has long been known that pig conceptuses produce interferon-γ (IFNG) at the time of implantation, but the role of IFNG and its mechanism of action at the maternal-conceptus interface are not fully understood. Accordingly, we analyzed the expression and regulation of IFNG receptors IFNGR1 and IFNGR2 in the endometrium during the estrous cycle and pregnancy in pigs. Levels of IFNGR1 and IFNGR2 messenger RNA (mRNA) expression changed in the endometrium, with the highest levels during mid pregnancy for IFNGR1 and on Day 12 of pregnancy for IFNGR2. The expression of IFNGR1 and IFNGR2 mRNAs was also detected in conceptuses during early pregnancy and chorioallantoic tissues during mid to late pregnancy. IFNGR1 and IFNGR2 mRNAs were localized to endometrial epithelial and stromal cells and to the chorionic membrane during pregnancy. IFNGR2 protein was also localized to endometrial epithelial and stromal cells, and increased epithelial expression of IFNGR2 mRNA and protein was detectable during early pregnancy than the estrous cycle. Explant culture studies showed that estrogen increased levels of IFNGR2, but not IFNGR1, mRNAs, while interleukin-1ß did not affect levels of IFNGR1 and IFNGR2 mRNAs. Furthermore, IFNG increased levels of IRF1, IRF2, STAT1, and STAT2 mRNAs in the endometrial explants. These results in pigs indicate that IFNGR1 and IFNGR2 are expressed in a stage of pregnancy- and cell-type specific manner in the endometrium and that sequential cooperative action of conceptus signals estrogen and IFNG may be critical for endometrial responsiveness to IFNs for the establishment of pregnancy in pigs.

Changes in calcium levels in the endometrium throughout pregnancy and the role of calcium on endometrial gene expression at the time of conceptus implantation in pigs.

Calcium plays an essential role in regulating many cellular functions, including proliferation, differentiation, and apoptosis. In spite of its importance in the establishment and maintenance of pregnancy, changes in calcium levels at the maternal-conceptus interface during pregnancy and its action on endometrial gene expression are not well understood. Thus, we examined changes in calcium levels in the endometrium during pregnancy, calcium deposition at the maternal-conceptus interface during pregnancy, and the role of calcium on the expression of endometrial genes related to conceptus implantation during early pregnancy in pigs. The amounts of endometrial calcium increased during mid- to late pregnancy, and calcium deposition was mainly localized to endometrial and chorionic epithelial cells at the maternal-conceptus interface during pregnancy and conceptus tissues during early pregnancy. The amounts of total recoverable calcium in uterine flushings were greater on Day 12 of pregnancy than Day 12 of the estrous cycle, and estrogen increased absorption of calcium ions by endometrial tissues. Increasing endometrial calcium levels by treatment with A23187, a calcium ionophore, decreased the expression of the estrogen-responsive endometrial genes AKR1B1, ESR1, FGF7, IL1RAP, LPAR3, S100G, SPP1, and STC1 and increased the expression of genes related to prostaglandin synthesis and transport, namely PTGES, PTGS2, and SLCO5A1. These data suggest that calcium ions at the maternal-conceptus interface play a critical role in the establishment and maintenance of pregnancy in pigs by regulating the expression of endometrial genes involved in conceptus implantation, as well as the attachment of endometrial epithelial and conceptus trophectoderm/chorionic epithelial cells during pregnancy.

Leukemia inhibitory factor and its receptor: expression and regulation in the porcine endometrium throughout the estrous cycle and pregnancy.

OBJECTIVE: Leukemia inhibitory factor (LIF) binds to a heterodimeric receptor composed of LIF receptor (LIFR) and glycoprotein 130 (GP130) to transmit signals into the cell. LIF plays an important role in reproduction by regulating immune response, decidualization, and implantation in several species. However, the expression of LIF and LIFR in the endometrium throughout the estrous cycle and pregnancy in pigs is not fully understood. METHODS: We analyzed the expression of LIF and LIFR in the endometrium on days 0 (estrus), 3, 6, 9, 12, 15, and 18 of the estrous cycle, and days 12, 15, 30, 60, 90, and 114 of pregnancy, in conceptuses on days 12 and 15, and in chorioallantoic tissues on days 30, 60, 90, and 114 of pregnancy in pigs. We also determined the effects of estrogen and progesterone on the expression of LIF and LIFR in endometrial tissues. RESULTS: The expression of LIF increased in the endometrium during the late diestrus phase of the estrous cycle and during mid- to late- pregnancy, while the expression of LIFR increased during early pregnancy. The expression of LIF was induced by increasing doses of estrogen, whereas the expression of LIFR was induced by increasing doses of progesterone. CONCLUSION: These results indicate that the expression of LIF and its receptor LIFR in the endometrium is regulated in a stage-specific manner during the estrous cycle and pregnancy, suggesting that LIF and its receptor signaling system may play critical roles in regulating endometrial function in pigs.

Unique epithelial expression of S100A calcium binding protein A7A in the endometrium at conceptus implantation in pigs

Objective: S100A7A, a member of the S100 protein family, is involved in various biological processes, including innate immunity, antimicrobial function, and epithelial tumorigenesis. However, the expression and function of S100A7A in the endometrium during the estrous cycle and pregnancy are not well understood in pigs. Therefore, this study determined the expression and regulation of S100A7A at the maternal-conceptus interface in pigs. Methods: We obtained endometrial tissues from pigs throughout the estrous cycle and pregnancy, conceptus tissues during early pregnancy, and chorioallantoic tissues during mid- to late pregnancy and analyzed the expression of S100A7A in these tissues. We also determined the effects of steroid hormones, estradiol-17ß (E2) and progesterone, and interleukin-1ß (IL1B) on S100A7A expression in endometrial tissues. Results: We found that S100A7A was expressed in the endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-dependent manner and was localized to endometrial LE and superficial GE cells with strong intensity in LE cells on Day 12 of pregnancy. Early stage conceptuses and chorioallantoic tissues from Day 30 to term pregnancy also expressed S100A7A. The expression of S100A7A was increased by E2 and IL1B in endometrial tissues. Conclusion: S100A7A was expressed at the maternal-conceptus interface at the initiation of implantation in response to conceptus-derived estrogen and IL1B and could be a unique endometrial epithelial marker for conceptus implantation in pigs. These findings provide an important insight into the understanding of conceptus-endometrial interactions for the successful establishment of pregnancy in pigs.

Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

Cysteine-X-cysteine (CXC) motif chemokine ligand 12 (CXCL12) and its receptor, CXC chemokine receptor type 4 (CXCR4), are involved in regulating the proliferation, migration, and survival of trophoblast cells and the maternal immune response in humans and mice. The present study examined the expression, regulation, and function of CXCL12 and CXCR4 at the maternal-conceptus interface during pregnancy in pigs. The endometrium expressed CXCL12 and CXCR4 mRNAs with the greatest CXCL12 abundance on Day 15 of pregnancy. CXCL12 protein was localized mainly in endometrial epithelial cells, while CXCR4 protein was localized in subepithelial stromal cells, vascular endothelial cells, and immune cells in blood vessels in the endometrium during the estrous cycle and pregnancy. CXCL12 protein was detected in uterine flushing on Day 15 of pregnancy. The conceptus during early pregnancy and chorioallantoic tissues during mid-to-late pregnancy expressed CXCL12 and CXCR4. Interferon-γ increased the abundance of CXCL12, but not CXCR4 mRNA in endometrial explants. Recombinant CXCL12 (rCXCL12) protein dose-dependently increased migration of cultured porcine trophectoderm cells and peripheral blood mononuclear cells (PBMCs). Furthermore, rCXCL12 caused migration of T cells, but not natural killer cells, in PBMCs. This study revealed that interferon-γ-induced CXCL12 and its receptor, CXCR4, were expressed at the maternal-conceptus interface and increased the migration of trophectoderm cells and T cells at the time of implantation in pigs. These results suggest that CXCL12 may be critical for the establishment of pregnancy by regulating trophoblast migration and T cell recruitment into the endometrium during the implantation period in pigs.

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

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.

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-17ß increased the expression of ABCC1 and SLCO5A1, but not ABCC9 and SLCO4C1 mRNAs and increasing doses of interleukin-1ß 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.

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

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 type 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.

Prostaglandin transporters ABCC4 and SLCO2A1 in the uterine endometrium and conceptus during pregnancy in pigs.

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.

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.

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.

Complement system molecules: Expression and regulation at the maternal-conceptus interface during pregnancy in pigs.

The complement system, composed of complement components and complement control proteins, plays an essential role in innate immunity. Complement system molecules are expressed at the maternal-conceptus interface, and inappropriate activation of the complement system is associated with various adverse pregnancy outcomes in humans and rodents. However, the expression, regulation, and function of the complement system at the maternal-conceptus interface in pigs have not been studied. In this study, we investigated the expression, localization, and regulation of complement system molecules at the maternal-conceptus interface in pigs. Complement components and complement control proteins were expressed in the endometrium, early-stage conceptus, and chorioallantoic tissues during pregnancy. The expression of complement components acting on the early stage of complement activation increased in the endometrium on Day 15 of pregnancy, with greater levels on that day compared with the estrous cycle. Localization of several complement components and complement control proteins was cell-type specific in the endometrium. The expression of C1QC, C2, C3, C4A, CFI, ITGB2, MASP1, and SERPING1 was increased by IFNG in endometrial explant tissues. Furthermore, cleaved C3 fragments were detected in endometrial tissues and uterine flushings on Day 15 of the estrous cycle and Day 15 of pregnancy, with greater levels on Day 15 of pregnancy. These results suggest that complement system molecules in pigs expressed at the maternal-conceptus interface play important roles in the establishment and maintenance of pregnancy by regulating innate immunity and modulating the maternal immune environment during pregnancy.

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.

Matrix metalloproteinases: expression and regulation in the endometrium during the estrous cycle and at the maternal-conceptus interface during pregnancy in pigs.

OBJECTIVE: Matrix metalloproteinases (MMPs) are a family of endoproteases produced by various tissues and cells and play important roles in angiogenesis, tissue repair, immune response, and endometrial remodeling. However, the expression and function of MMPs in the pig endometrium during the estrous cycle and pregnancy have not been fully elucidated. Thus, we determined the expression, localization, and regulation of MMP2, MMP8, MMP9, MMP12, and MMP13 in the endometrium throughout the estrous cycle and at the maternal-conceptus interface during pregnancy in pigs. METHODS: Endometrial tissues during the estrous cycle and pregnancy and conceptus and chorioallantoic tissues during pregnancy were obtained and the expression of MMPs was analyzed. The effects of steroid hormones and cytokines on the expression of MMPs were determined in endometrial explant cultures. RESULTS: Expression levels of MMP12 and MMP13 changed during the estrous cycle, while expression of MMP2, MMP9, MMP12, and MMP13 changed during pregnancy. Expression of MMP2, MMP8, and MMP13 mRNAs was cell type-specific at the maternal-conceptus interface. Gelatin zymography showed that enzymatically active MMP2 was present in endometrial tissues. In endometrial explant cultures, estradiol-17ß induced the expression of MMP8 and MMP12, progesterone decreased the expression of MMP12, interleukin-1ß increased the expression of MMP2, MMP8, MMP9, and MMP13, and interferon-γ increased the expression of MMP2. CONCLUSION: These results suggest that MMPs expressed in response to steroids and cytokines play an important role in the establishment and maintenance of pregnancy by regulating endometrial remodeling and processing bioactive molecules in pigs.

Spatiotemporal expression and regulation of peptidase inhibitor 3 and secretory leukocyte protease inhibitor at the maternal-fetal interface in pigs.

OBJECTIVE: Two serine protease inhibitors, peptidase inhibitor 3 (PI3) and secretory leukocyte protease inhibitor (SLPI), play important roles in protease inhibition and antimicrobial activity, but their expression, regulation, and function at the maternal-fetal interface in pigs are not fully understood. Therefore, we determined the expression and regulation of PI3 and SLPI in the endometrium throughout the estrous cycle and at the maternal-fetal interface in pigs. METHODS: Endometrial tissues during the estrous cycle and pregnancy, conceptus tissues during early pregnancy, and chorioallantoic tissues during mid to late pregnancy were obtained, and the expression of PI3 and SLPI was analyzed. The effects of the steroid hormones estradiol-17ß (E2) and progesterone (P4) on the expression of PI3 and SLPI were determined in endometrial explant cultures. RESULTS: PI3 and SLPI were expressed in the endometrium during the estrous cycle and pregnancy, with higher levels during mid to late pregnancy than during the estrous cycle and early pregnancy. Early-stage conceptuses and chorioallantoic tissues during mid to late pregnancy also expressed PI3 and SLPI. PI3 protein and SLPI mRNA were primarily localized to endometrial epithelia. In endometrial explant cultures, the expression of PI3 was induced by increasing doses of P4, and the expression of SLPI was induced by increasing doses of E2 and P4. CONCLUSION: These results suggest that the PI3 and SLPI expressed in the endometrium and conceptus tissues play an important role in antimicrobial activity for fetal protection against potential pathogens and in blocking protease actions to allow epitheliochorial placenta formation.

Antimicrobial peptides ß-defensin family: Expression and regulation in the endometrium during the estrous cycle and pregnancy in pigs.

Members of the ß-defensin (DEFB) family, which are antimicrobial peptides and humoral components of the innate immune system, protect the surfaces of various host tissues by killing a broad range of microorganisms and are involved in immunomodulatory actions. The expression of these DEFB members changed during the estrous cycle and pregnancy in a stage-specific manner. The expression of DEFBs was also detected in conceptus and chorioallantoic tissues during pregnancy. DEFB1 and DEFB3 proteins and DEFB2 mRNA were localized primarily to endometrial epithelial cells during early pregnancy. Increasing doses of progesterone upregulated DEFB2 and EP2C expression in endometrial explant tissues. These results showed that members of the DEFB family were expressed stage-specifically at the maternal-conceptus interface in pigs, suggesting that the DEFB family plays important roles at the maternal-conceptus interface in regulation of innate immunity by protection of the maternal endometrial and conceptus tissues from pathogens to preserve fertility in pigs.

Inhibitors of apoptosis: expression and regulation in the endometrium during the estrous cycle and at the maternal-conceptus interface during pregnancy in pigs.

OBJECTIVE: Caspase-mediated apoptosis plays a crucial role in the regulation of endometrial and placental function in females. Caspase activity is tightly controlled by members of the inhibitors of apoptosis proteins (IAPs) family. However, the expression and regulation of IAPs at the maternal-conceptus interface has not been studied in pigs. Therefore, we determined the expression of IAP family members baculovirus IAP repeat-containing 1 (BIRC1) to BIRC6 at the maternal-conceptus interface in pigs. METHODS: We obtained endometrial tissues from pigs at various stages of the estrous cycle and pregnancy, conceptus tissues during early pregnancy, and chorioallantoic tissues during mid- to late pregnancy and analyzed the expression of IAPs. Furthermore, we determined the effects of the steroid hormones estradiol-17ß (E2) and progesterone on the expression of IAPs in endometrial explant tissue cultures. RESULTS: During the estrous cycle, BIRC2 and BIRC5 expression varied cyclically, and during pregnancy, endometrial BIRC1, BIRC2, BIRC3, BIRC4, and BIRC5 expression varied in a stage-specific manner. Conceptus and chorioallantoic tissues also expressed IAPs during pregnancy. The BIRC2 and BIR3 mRNAs were localized to luminal epithelial cells, and BIRC4 proteins to glandular epithelial cells in the endometrium. Exposure of endometrial tissues to E2 increased the expression of BIRC6, while progesterone increased the expression of BIRC1, BIRC4, and BIRC6 in a dose-dependent manner. CONCLUSION: These results indicated that IAPs were expressed in the endometrium during the estrous cycle and at the maternal-conceptus interface during pregnancy in a stage-specific manner. In addition, steroid hormones were found to be responsible for the expression of some IAPs in pigs. Together, the results suggested that IAPs may play important roles in endometrial and placental functions by regulating caspase action and apoptosis at the maternal-conceptus interface.

Antimicrobial peptides cathelicidin, PMAP23, and PMAP37: Expression in the endometrium throughout the estrous cycle and at the maternal-conceptus interface during pregnancy and regulation by steroid hormones and calcitriol in pigs.

The endometrium, which regulates the reproductive cyclicity and the establishment and maintenance of pregnancy, is a type of mucosal tissue and is involved in the regulation of immunity. Antimicrobial peptides, called cathelicidins, play critical roles in innate immunity by killing various microbes and modulating host immune properties. The expression and regulation of cathelicidin in female reproductive tissues have been studied in some species, but not determined in pigs, which form a true epitheliochorial placenta. Thus, we determined the expression of Cathelicidin (the conserved common cathelin domain for all porcine cathelicidin family members), PMAP23, and PMAP37 in the endometrium throughout the estrous cycle and pregnancy. The expression of Cathelicidin, PMAP23, and PMAP37 in the endometrium changed during the estrous cycle and pregnancy in a stage-specific manner. The mRNA expression of Cathelicidin, PMAP23, and PMAP37 was also detected in conceptus and chorioallantoic tissues during pregnancy. Cathelicidin protein was mainly localized to luminal epithelial cells and some immune cells in blood vessels and to chorionic epithelial cells of chorioallantoic membranes during mid-to late pregnancy. The expression of Cathelicidin was induced by calcitriol but increasing doses of estradiol-17ß and progesterone did not affect the expression of Cathelicidin, PMAP23, or PMAP37 in endometrial tissues. These results indicate that members of the cathelicidin family were dynamically expressed at the maternal-conceptus interface in pigs, suggesting that the cathelicidin family may play important roles in the regulation of innate immunity by mediating antimicrobial activity to protect the maternal and/or conceptus tissues to conserve pregnancy capacity in pigs.