Characterization of the interleukin-1beta system during porcine trophoblastic elongation and early placental attachment

Conceptus-uterine communication is established during trophoblastic elongation when the conceptus synthesizes and releases estrogen, the maternal recognition signal in the pig. Interleukin-1beta (IL-1beta) is a differentially expressed gene during rapid trophoblastic elongation in the pig. The current investigation determined conceptus and endometrial changes in gene expression for IL-1beta, IL-1 receptor antagonist (IL-1Rant), IL-1 receptor type 1 (IL-1RT1), and IL-1 receptor accessory protein (IL-1RAP) in developing peri- and postimplantation conceptuses as well as uterine endometrium collected from cyclic and pregnant gilts. Conceptus IL-1beta gene expression was enhanced during the period of rapid trophoblastic elongation compared with earlier spherical conceptuses, followed by a dramatic decrease in elongated Day 15 conceptuses. IL-1RT1 and IL-1RAP gene expression was greater in Day 12 and 15 filamentous conceptuses compared with earlier morphologies while IL-1Rant gene expression was unchanged by conceptus development. The uterine lumenal content of IL-1beta increased during the process of trophoblastic elongation on Day 12. Uterine IL-1beta content declined on Day 15, reaching a nadir by Day 18 of pregnancy. IL-1beta gene expression in porcine conceptuses was temporally associated with an increase in endometrial IL-1RT1 and IL-1RAP gene expression in pregnant gilts. Endometrial IL-1beta and IL-1Rant gene expression were lowest during Days 10-15 of the estrous cycle and pregnancy. The temporal expression of IL-1beta during conceptus development and the initiation of conceptus-uterine communication suggests conceptus IL-1beta synthesis plays an important role in porcine conceptus elongation and the establishment of pregnancy in the pig.     

Regulation of expression and role of leukemia inhibitory factor and interleukin-6 in the uterus of early pregnant pigs

Cytokines, which are generally involved in the process of inflammation, may also play a critical role in conceptus implantation. We examined: (1) the expression profiles of leukemia inhibitory factor (LIF) and interleukin (IL)-6 mRNA and their protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus-exposed medium on LIF and IL-6 synthesis in the endometrium; (3) the profiles of IL6R and LIFR mRNA expression in pig conceptuses collected on Days 10 to 18 of pregnancy; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells. The expression of LIF mRNA in the endometrium increased between Days 10 and 12 in both cyclic and pregnant gilts, and tended to be higher in Day 12 pregnant animals compared with nonpregnant ones. The LIF protein content in the uterine lumen peaked on Day 12 of pregnancy, and was higher than on Day 12 of the estrous cycle. Endometrial IL-6 mRNA expression was upregulated on Day 12 in pregnant gilts compared with nonpregnant animals. Moreover, a higher content of IL-6 protein was observed in pregnant than in cyclic gilts. The addition of conceptus-exposed medium resulted in up-regulation of LIF and IL6 mRNA expression, and increased IL-6 content in endometrial slices. In conceptuses, increased mRNA expression was detected on Days 10 to 14 for IL6R and on Day 14 for LIFR, when compared with other days studied. LIF and IL-6 stimulated the attachment and proliferation of trophoblast cells in vitro. In summary, LIF and IL-6 are important components of embryo-uterine interactions during early pregnancy in the pig, and may contribute to successful conceptus implantation.     

Inhibition of TRAF6 ubiquitin-ligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation

TRAF6 (TNF receptor associated factor 6) plays a pivotal role in NFKB activation and macroautphagy/autophagy activation induced by TLR4 (toll like receptor 4) signaling. The objective of this study was to determine the functional role of PRDX1 (peroxiredoxin 1) in NFKB activation and autophagy activation. PRDX1 interacted with the ring finger domain of TRAF6 and inhibited its ubiquitin-ligase activity. The inhibition on TRAF6 ubiquitin-ligase activity by PRDX1 induced the suppression of ubiquitination of an evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) essential for NFKB activation and BECN1 (beclin 1) required for autophagy activation. An inhibitory effect of PRDX1 on TRAF6 was clearly evidenced in PRDX1-knockdown (PRDX1KD) THP-1, PRDX1KD MDA-MB-231, and PRDX1KD SK-HEP-1 cells. PRDX1KD THP-1 cells showed increases of NFKB activation, pro-inflammatory cytokine production, NFKB-dependent gene expression induced by TLR4 stimulation, and resistance against Salmonella typhimurium infection. Additionally, migration and invasion abilities of PRDX1KD MDA-MB-231 and PRDX1KD SK-HEP-1 cancer cells were significantly enhanced compared to those of control cancer cells. Taken together, these results suggest that PRDX1 negatively regulates TLR4 signaling for NFKB activation and autophagy functions such as bactericidal activity, cancer cell migration, and cancer cell invasion by inhibiting TRAF6 ubiquitin-ligase activity.

Abbreviations: 3-MA: 3-methyladenine; BECN1: beclin 1; CHUK/IKKA: conserved helix-loop-helix ubiquitous kinase; ECSIT: ECSIT signalling integrator; ELISA: enzyme-linked immunosorbent assay; NFKB: nuclear factor kappa-light-chain-enhancer of activated B cells; IB: immunoblotting; IKBKB/IKKB: inhibitor of nuclear factor kappa B kinase subunit beta; IL1B: interleukin 1 beta; IL6: interleukin 6; IP: immunoprecipitation; LPS: lipopolysaccharide; MAP1LC3/LC3: microtuble associated protein 1 light chain 3; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MAPK14/p38: mitogen-activated protein kinase 14; mROS: mitochondrial reactive oxygen species; PRDX1: peroxiredoxin 1; PRDX6: peroxiredoxin 6; RELA/p65: RELA proto-oncogene, NF-kB subunit; TRAF6 TNF: receptor associated factor 6.     

Porcine endometrial expression of kininogen,factor XII,and plasma kallikrein in cyclic and pregnant gilts

Establishment of pregnancy in the pig is accompanied by a localized uterine acute inflammatory response and increase in uterine blood flow. Following rapid trophoblast elongation on Day 12 of pregnancy there is an increase in tissue kallikrein activity and release of bradykinin into the uterine lumen, suggesting the kallikrein-kininogen-kinin system is active in the porcine uterus. The present study investigated endometrial expression and presence of the various factors of the kallikrein-kininogen-kinin system. Endometrial L- and H-kininogen gene expression as well as presence of kininogens in the uterine flushings was evaluated throughout the estrous cycle and early pregnancy in the pig. The possible involvement of plasma kallikrein and Factor XII, activators of the kallikrein-kininogen-kinin system, were evaluated through analysis of gene expression in endometrial and conceptus tissues. Gene expression for plasma kallikrein, Factor XII, and H-kininogen were detected in endometrium but not early conceptus tissues. Factor XII and H-kininogen gene expression were similar across the days of the estrous cycle and early pregnancy. Endometrial plasma kallikrein gene expression was low but increased on Day 15 of the estrous cycle, whereas expression was similar across the days of early pregnancy. In comparison to cyclic gilts, endometrial L-kininogen gene expression increased fourfold on Days 15 and 18 of pregnancy. Both L- and H-kininogen were detected in the uterine flushings of cyclic and pregnant gilts. Presence of L- and H-kininogen in the porcine uterus and endometrial gene expression of plasma kallikrein and Factor XII provide evidence that the kallikrein-kininogen-kinin system is biologically active during establishment of pregnancy in the pig.     

Regulation of prostacyclin synthase expression and prostacyclin content in the pig endometrium

Prostaglandins (PGs) are critical regulators of a number of reproductive processes, including embryo development and implantation. In the present study, prostacyclin (PGI₂) synthase (PGIS) mRNA and protein expression, as well as 6-keto PGF_1α (a PGI₂ metabolite) concentration, were investigated in the pig uterus. Endometrial tissue and uterine luminal flushings were obtained on Days 4 to 18 of the estrous cycle and pregnancy. Additionally, conceptuses were collected and examined for PGIS mRNA expression and 6-keto PGF_1α concentration. Regulation of PGI₂ synthesis in the porcine endometrium by steroids, conceptus products, and cytokines was studied in vitro and/or in vivo. Endometrial PGIS protein level increased on Days 12 and 16 in pregnant but not in cyclic gilts. Moreover, higher PGIS protein expression on Day 12 of pregnancy was accompanied by a greater content of 6-keto PGF_1α in the endometrium. The concentration of 6-keto PGF_1α in uterine luminal flushings increased substantially on Days 16 and 18 in pregnant gilts and was higher than in cyclic animals. Greater PGIS mRNA expression and PGI₂ metabolite concentration were detected in Day 12 and 14 conceptuses, respectively. Incubation of endometrial explants with conceptus-conditioned medium resulted in upregulation of PGIS protein expression and increased PGI₂ secretion. Moreover, PGIS mRNA and protein expression were upregulated in the endometrium collected from gravid uterine horn on Day 14 of pregnancy. In summary, PGIS is differentially expressed in the endometrium of cyclic and pregnant gilts resulting in higher PGI₂ synthesis in pregnant animals. Porcine conceptuses are important regulators of endometrial PGIS expression and PGI₂ release during the implantation period.     

Platelet-activating factor (PAF)-like activity, localization of PAF receptor (PAF-R) and PAF-acetylhydrolase (PAF-AH) activity in bovine endometrium at different stages of the estrous cycle and early pregnancy

PAF-like activity in the endometrium increased from days 2-4 to day 12 and day 20 in both cyclic and pregnant cows. There was an increase in platelet aggregation induced by PAF-like activity in the endometrium of pregnant animals on day 20 as compared to cyclic animals at the same point in time. Two major bands of PAF-R protein at 67 kDa and 97 kDa were detected by Western blot analysis. PAF-R was localized mainly in luminal and glandular epithelium of the endometrium, but the staining was markedly increased in the endometrium of pregnant cows on day 20 compared to cyclic animals on the same day. The purified PAF-AH from the endometrium is similar to in plasma. In cyclic cattle, no changes in PAF-AH activity of endometrium were observed, whereas a decrease in enzyme activity occurred in pregnant cows on day 20 as compared to cyclic animals on the same day. We suggest that the bovine endometrium produces PAF-like activity, expresses the PAF-R and possesses a PAF-AH activity which varies during pregnancy.     

Regulatory mechanism for expression of IL1B receptors in the uterine endometrium and effects of IL1B on prostaglandin synthetic enzymes during the implantation period in pigs

During the implantation period, the porcine conceptus secretes interleukin-1beta (IL1B) that may be involved in the establishment of pregnancy in pigs. However, the regulatory mechanism for IL1B receptor expression and the function of IL1B in the uterine endometrium are not well elucidated. In this study, we determined IL1B receptor expression in the uterine endometrium of pigs during pregnancy. IL1B receptor subtypes, IL1 receptor type I (IL1R1) and IL1 receptor accessory protein (IL1RAP) were expressed in the uterine endometrium with the expression being most abundant on Day 12 of pregnancy primarily in the luminal and glandular epithelial cells. Expression of IL1R1 mRNA increased in response to IL1B in a dose-dependent manner, and expression of IL1RAP mRNA increased in response to both IL1B and estradiol, indicating that expression of endometrial IL1B receptors was regulated cooperatively by IL1B and estrogen of conceptus origin. During the peri-implantation period, the porcine uterine endometrium actively synthesizes and secretes prostaglandins (PGs). IL1B increased expression of PTGS1 and PTGS2 genes that are rate-limiting for PG synthesis in the uterine endometrium. Collectively, the results indicated that IL1B regulates expression of IL1R1 and IL1RAP and stimulates expression of PTGS1 and PTGS2 that are considered to be the most rate-limiting enzymes for endometrial synthesis of PGs during the peri-implantation period of pregnancy in pigs.     

Platelet-activating factor (PAF)-like activity, localization of PAF receptor (PAF-R) and PAF-acetylhydrolase (PAF-AH) activity in bovine endometrium at different stages of the estrous cycle and early pregnancy*

PAF-like activity in the endometrium increased from days 2-4 to day 12 and day 20 in both cyclic and pregnant cows. There was an increase in platelet aggregation induced by PAF-like activity in the endometrium of pregnant animals on day 20 as compared to cyclic animals at the same point in time. Two major bands of PAF-R protein at 67 kDa and 97 kDa were detected by Western blot analysis. PAF-R was localized mainly in luminal and glandular epithelium of the endometrium, but the staining was markedly increased in the endometrium of pregnant cows on day 20 compared to cyclic animals on the same day. The purified PAF-AH from the endometrium is similar to in plasma. In cyclic cattle, no changes in PAF-AH activity of endometrium were observed, whereas a decrease in enzyme activity occurred in pregnant cows on day 20 as compared to cyclic animals on the same day. We suggest that the bovine endometrium produces PAF-like activity, expresses the PAF-R and possesses a PAF-AH activity which varies during pregnancy.     

Analysis of osteopontin at the maternal-placental interface in pigs

Noninvasive, epitheliochorial placentation in the pig follows a prolonged preimplantation period characterized by migration, spacing and elongation of conceptuses, and secretion of estrogen for maternal recognition of pregnancy. Osteopontin (OPN) is an extracellular matrix protein that binds integrins to promote cell-cell attachment and communication. OPN appears to play a key role in conceptus implantation and maintenance of pregnancy in sheep; however, a role for OPN in the porcine uterus has not been established. Therefore, this study examined OPN expression and function in the porcine uterus and conceptus (embryo/fetus and associated extraembryonic membranes). Northern and slot blot hybridization detected an increase in endometrial OPN expression between Days 25 and 30, and levels remained elevated through Day 85 of pregnancy. In situ hybridization localized OPN mRNA to discrete regions of the uterine luminal epithelium (LE) on Day 15 of pregnancy and to the entire LE thereafter. Glandular epithelial (GE) expression of OPN mRNA was first detected on Day 35 of pregnancy and increased through Day 85. Both 70- and 45-kDa forms of OPN protein were detected in cyclic and pregnant endometrium by Western blotting. OPN protein was localized to the LE and GE by immunofluorescence; however, only the 70-kDa OPN was detected in uterine flushings. OPN protein was present along the entire uterine-placental interface after Day 30 of pregnancy. In addition, OPN mRNA and protein were localized to immune-like cells within the stratum compactum of the endometrium in both Day 9 cyclic and pregnant gilts. Incubation of OPN-coated microbeads with porcine trophectoderm and uterine luminal epithelial cells induced Arg-Gly-Asp (RGD)-dependent integrin activation and transmembrane accumulation of cytoskeletal molecules at the apical cell surface as assessed by immunofluorescence detection of talin or alpha-actinin as markers for focal adhesions. These results suggest that OPN, expressed by uterine epithelium and immune cells, may interact with receptors (i.e., integrins) on conceptus and uterus to promote conceptus development and signaling between these tissues as key contributors to attachment and placentation in the pig.     

The effect of tumor necrosis factor α (TNFα), interleukin 1β (IL1β) and interleukin 6 (IL6) on endometrial PGF2α synthesis, metabolism and release in early-pregnant pigs

Cytokines produced by the porcine uterus and embryos may be involved in the regulation of endometrial prostaglandin synthesis, metabolism, and release. We studied the effect of tumor necrosis factor α (TNFα), interleukin 1β (IL1β) and interleukin 6 (IL6) on: 1) endometrial release of prostaglandin F₂α (PGF₂α), 2) expression of the terminal enzyme of PGF₂α synthesis - PGF synthase mRNA (PGFS mRNA), 3) secretion of PGF₂α metabolite - 13,14-dihydro-15-keto PGF₂α (PGFM) by the endometrium and 4) presence and activity of endometrial NAD-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The effects of cytokines were determined on days 10-11 and days 12-13, e.g., before and during maternal recognition of pregnancy, and on days 15-16, e.g., during the peri-implantation period and compared with its effect in cyclic gilts on corresponding days of the estrous cycle. TNFα did not affect endometrial release of PGF₂α in pregnant and cyclic pigs. IL1β enhanced endometrial PGF₂α release on days 12-13 and 15-16 in pregnant and cyclic pigs, respectively. IL6 increased PGF₂α release mainly on days 15-16 of pregnancy. Expression of PGFS mRNA was decreased by IL1β on days 12-13 of pregnancy (P < 0.05) and increased in response to IL1β, TNFα and IL6 on 12-13 (P < 0.05) and 15-16 (P < 0.01) of the estrous cycle. IL1β increased release of PGFM in gravid pigs on days 12-13, 15-16 and in non-gravid pigs 10-11 and 15-16 of the cycle. On days 15-16 of pregnancy TNFα and IL6 increased endometrial secretion of PGFM. We determined that in porcine endometrium NAD-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is present. In gravid pigs, the highest expression of endometrial 15-PGDH occurred during days 12-13 of pregnancy, while in non-gravid pigs during days 10-11 of the estrous cycle. These data provide new evidence that TNFα, IL1β, IL6 are involved in the regulation of endometrial synthesis, release and metabolism of PGF₂α to protect CL during early pregnancy or to facilitate its regression in cyclic females.     

Immunocytochemical localization of transforming growth factor α, epidermal growth factor and epidermal growth factor receptor in the cat endometrium and placenta

This study was undertaken to determine the immunocytochemical localization of transforming growth factor α, epidermal growth factor and epidermal growth factor receptor in the endometrium of ovariectomized cats treated with oestradiol-17β and/or progesterone and in the endometrium and placenta of pregnant cats. Specific immunostaining was observed for all three antibodies. Moderate immunostaining for transforming growth factor α was observed in the epithelium of ovariectomized and oestrogen-treated cats. Dark epithelial staining was observed throughout pregnancy. The epithelial cells in progesterone-treated and peri-implantation animals contained dense deposits of reaction product, which were not reduced in intensity when immunoabsorbed antiserum was used. For epidermal growth factor, light--moderate epithelial staining was observed in ovariectomized and steroid-treated animals, and this increased in pregnant cats. Stromal staining for both the transforming and the epidermal growth factors was limited in steroid-treated animals and increased as pregnancy continued. Dark staining for epidermal growth factor receptor was observed in the epithelium and stroma in all the animals studied. The tips of surface epithelial convolutions in the non-implantation sites were always more darkly stained than in other regions of the surface epithelium. Staining in the placental trophoblast was limited to the syncytiotrophoblast for the two growth factors and the cytotrophoblast for the receptor during most of pregnancy and was absent late in pregnancy. The placental maternal giant cells contained specific immunoreactivity for all the immunogens from the middle of pregnancy to term. This study demonstrates that the two growth factors and the epidermal growth factor receptor are present in the endometrium and placenta of cats and suggests that these growth factors may play an autocrine/paracrine role during reproduction     

Expression of VEGF-receptor system in conceptus during peri-implantation period and endometrial and luteal expression of soluble VEGFR-1 in the pig

In view of the importance of vascular events observed during gestation, it was hypothesized that the VEGF-receptor system plays a critical role during early pregnancy and maternal recognition of pregnancy in pigs. This hypothesis was tested by examining the expression of the VEGF-receptor system in the porcine conceptus. Additionally, the endometrium, corpus luteum (CL) and embryos were studied for the expression of soluble VEGF receptor 1 (sVEGFR-1), the strong endogenous antagonist of VEGF. The expression patterns show that VEGF164 mRNA levels increase gradually in line with conceptus development, whereas VEGF120 and VEGFR-2 remain unchanged during the peri-implantation period. Interestingly, elevated VEGFR-1 expression was observed in conceptuses on days 15-16 of gestation (P < 0.05). Comparison of the endometrial sVEGFR-1 mRNA expression revealed up-regulation on days 12 and 15-16 of pregnancy (P < 0.01 and P < 0.05, respectively). Furthermore, increased sVEGFR-1 levels were observed on day 12 of the estrous cycle in the CL (P < 0.05). Concluding, it seems that conceptus-derived VEGF164 plays crucial role in peri-implantation vascular events in pigs. These results support a potential role of VEGFR-1 in the proper growth and development of porcine conceptus during pregnancy. Moreover, expression patterns of sVEGFR-1 in the endometrium of pregnant pigs suggest that it may participate in vascular remodeling important for successful implantation. Finally, luteal sVEGFR-1 may be involved in the maintenance of CL function whenever pregnancy occurs in pigs.