Bovine Endometrial Research Articles

ATP Induces Interleukin-8, Intracellular Calcium Release, and ERK1/2 Phosphorylation in Bovine Endometrial Cells, Partially through P2Y Receptors

The bovine endometrium has an important defensive role in the postpartum period that acts when an inflammatory process associated with tissue damage or infection by bacteria is produced. Endometrial cells release cytokines and chemokines that recruit inflammatory cells, which release danger-associated molecular patterns (DAMPs), such as adenosine triphosphate (ATP), and initiate and regulate the inflammatory response. However, the role of ATP in bovine endometrial cells is unclear. The aim of this study was to determine the effect of ATP on interleukin-8 (IL-8) release, intracellular calcium mobilization, ERK1/2 phosphorylation, and the role of P2Y receptors, in bovine endometrial cells. Bovine endometrial (BEND) cells were incubated with ATP and the IL-8 release was determined by the ELISA assay. ATP of 50 and 100 μM significantly increased IL-8 released in BEND cells (50 μM: 23.16 ± 3.82 pg/mL, p = 0.0018; 100 μM: 30.14 ± 7.43 pg/mL, p = 0.0004). ATP (50 μM) also induced rapid intracellular calcium mobilization in Fura-2AM-loaded BEND cells, as well as ERK1/2 phosphorylation (ratio 1.1 ± 0.04, p = 0.0049). Suramin (50 μM), a pan-antagonist of P2Y receptors, partially reduced the intracellular calcium mobilization, ERK1/2 phosphorylation (ratio 0.83 ± 0.08, p = 0.045), and IL-8 release (9.67 ± 0.02 pg/mL, p = 0.014) induced by ATP. Finally, BEND cells expressed higher mRNA levels of P2Y1 and P2Y2 purinergic subtype receptors, and lower levels of P2Y11 and P2Y12 receptors, as determined by RT-qPCR. In conclusion, these results showed that ATP activates pro-inflammatory responses in BEND cells, which are partially mediated via P2Y receptors, and BEND cells express the mRNA of subtypes of P2Y receptors, which could have a key role in bovine endometrial inflammation.

Effects of non-esterified fatty acids on relative abundance of prostaglandin E2 and F2α synthesis-related mRNA transcripts and protein in endometrial cells of cattle in vitro

Cows nearing parturition have a negative energy balance (NEB), which is closely associated with lesser fertility. The NEB results in greater fat mobilisation and production of a large amount of non-esterified fatty acid (NEFA). Prostaglandins (PG), especially prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), have important functions in regulating reproductive function. There, however, is little known about how the synthesis and release of PG are affected by NEFA. In this study, there was a focus on effects of NEFA on PG secretion as well as relative abundances of mRNA transcript and protein for PG synthetases and PG receptors in bovine endometrial (BEND) cells. Proliferation rate of BEND cells decreased in a concentration-dependent manner as NEFA increased in the media. The concentrations of PGE2 and PGF2α in NEFA treatment groups also decreased, while the ratio of PGE2/PGF2α and the relative abundances of proteins and mRNA that regulate PG synthesis and PG receptor mRNA transcripts and protein were greater as the NEFA concentration increased. Collectively, when there were large NEFA concentrations in the medium, there was a lesser release of PGE2 and PGF2α, however, there was a greater ratio of PGE2/PGF2α and relative abundances of mRNA transcripts and protein for PG synthetases and PG receptors in BEND cells, which changed the internal milieu and physiological function of the uterus with possible effects on fertility after calving. These findings provide important information that will help for further investigation of associations between NEB and fertility in dairy cows during the non-lactation to lactation-transition period.

Protective effects of astaxanthin on lipopolysaccharide-induced inflammation in bovine endometrial epithelial cells†.

Astaxanthin (AST), a natural antioxidant carotenoid, has been shown to exert anti-inflammatory effects. However, to our knowledge, no study has specifically addressed the potential protective effects of AST against bovine endometritis. The purpose of this study was to examine whether treatment with AST could protect endometrial epithelial cells against lipopolysaccharide (LPS)-induced inflammatory injury. Treatment of bovine endometrial (BEND) epithelial cell line with AST reduced LPS-induced production of interleukin-6 and tumor necrosis factor-alpha, increased the cellular activity of superoxide dismutase and catalase, decreased the proportion of apoptotic cells, and promoted the production of insulin-like growth factor and epithelial growth factor. The effects of AST were mediated through the downregulation of B-cell lymphoma 2 (Bcl-2) associated X, apoptosis regulator (Bax), and cleaved caspase-3 and through the upregulation of Bcl-2. Moreover, AST significantly increased the expression of the tight junction proteins (TJP) claudin, cadherin-1, and TJP1, which play an essential role in the maintenance of host endometrial defense barrier against pathogen infection. Collectively, these results demonstrated that treatment with AST protected against oxidative stress, prevented cell apoptosis, promoted BEND cells viability, and increased the production of growth factors, in addition to activating the endometrial defense barrier. Therefore, AST is a promising therapeutic agent for the prevention and treatment of endometritis. This finding is of utmost importance in the present times when the excessive use of antibiotics has resulted in the development of antibiotic-resistant bacteria.

Functional expression of the free fatty acids receptor-1 and -4 (FFA1/GPR40 and FFA4/GPR120) in bovine endometrial cells.

Endometrial epithelial cells play a key defensive role as part of the innate immune response of cow uterus. An association between risk of acquiring infectious diseases and increased levels of free fatty acids postpartum has been suggested, and the use of omega-3 fatty acids such as docosahexaenoic acid (DHA) has been proposed as a beneficial strategy to improve immunity and fertility. The goal of our study was to demonstrate the presence of free fatty acid (FFA)-1 and 4 receptors in endometrial cells and to investigate their role on DHA interference in lipopolysaccharide (LPS)-induced inflammatory endometrial activation. We demonstrated that the bovine endometrial (BEND) cells line and bovine endometrium express both FFA1 and FFA4 receptors. FFA1 and FFA4 receptors were localized in the epithelium lining the endometrial cavity and in endometrial glands whereas in BEND cells a characteristic cell membrane localization of both receptors was observed. DHA, a FFA4 natural agonist, increased intracellular calcium mobilization in BEND cells, but the FFA1 agonists oleic and linoleic acids did not increase this response. DHA-induced intracellular calcium mobilization was inhibited by the FFA4 and FFA1 antagonists AH7614 and GW1100, respectively. DHA significantly reduced LPS-induced prostaglandin E2 (PGE2) production, but none of the antagonists reduced the effect produced by DHA. On the contrary, linoleic acid increased LPS-induced PGE2 production. In conclusion, endometrial cells express FFA4 and FFA1 receptors, and DHA induces intracellular calcium release via FFA4 and FFA1 receptors. DHA reduces PGE2, but this response was not mediated by FFA4 or FFA1 receptors.

Proanthocyanidins Protect against β-Hydroxybutyrate-Induced Oxidative Damage in Bovine Endometrial Cells.

Metabolic diseases, such as ketosis, are closely associated with decreased reproductive performance (such as delayed estrus and decreased pregnancy rate) in dairy cows. The change of β-hydroxybutyrate (BHBA) concentration in dairy cattle is an important mechanism leading to ketosis, and its blood concentration in ketotic cows is always significantly higher than in nonketotic cows. Many studies indicated that BHBA can induce oxidative damage in liver and other organs. Proanthocyanidins (PCs) have gained substantial attention in the last decade as strong antioxidative substances. This study aimed to demonstrate a protective effect of PCs against BHBA-induced oxidative stress damage in bovine endometrial (BEND) cells by activating the nuclear erythroid2-related factor2 (Nrf2) signaling pathway. Our research show that PCs could significantly increase activities of catalase (CAT) and glutathione peroxidase (GSH-PX), glutathione (GSH) content, and antioxidant capacity (T-AOC), while significantly decreasing malondialdehyde (MDA) content in BEND cells. Both mRNA and protein expression levels of Nrf2 were significantly increased in BEND cells, and glutamate–cysteine ligase catalytic subunit (GCLC), heme oxygenase 1 (HO-1), manganese superoxide dismutase (Mn-SOD), and NAD(P)H quinone dehydrogenase 1 (NQO-1) were also significantly increased. These results indicate that PCs can antagonize BHBA-induced oxidative damage by activating the Nrf2 signaling pathway to exert an antioxidant effect.

Ephrin A1 promotes proliferation of bovine endometrial cells with abundant expression of proliferating cell nuclear antigen and cyclin D1 changing the cell population at each stage of the cell cycle.

Ephrin A1 has a role in a variety of biological events, including cell proliferation, differentiation, migration, and angiogenesis. Ephrin A1 expression is abundant in trophoblasts and endometrial cells during the implantation period; however, its intracellular activities have not yet been reported in bovine endometrial (BEND) epithelial cells. The aim of this study was to identify the functional role of ephrin A1 in BEND cells, which have served as a good model system for investigating the regulation of signal transduction following treatment with interferon-τ (IFNT) in vitro. Supplementation of ephrin A1 to BEND cells increased cell proliferation and increased levels of proliferating cell nuclear antigen and cyclin D1 protein in BEND cell nuclei. To investigate intracellular mechanisms regulated by ephrin A1, we performed Western blot analysis focused on mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling, which are significantly involved in the successful maintenance of pregnancy. Ephrin A1 dose-dependently increased phosphorylation of extracellular signal-regulated kinases (ERK)1/2, c-Jun N-terminal kinases (JNK), P38, protein kinase B (AKT), P70S6K, S6, and cyclin D1, and the activated proteins were suppressed by pharmacological inhibitors including wortmannin (a PI3K inhibitor), U0126 (an ERK1/2 inhibitor), and SP600125 (a JNK inhibitor). Among ephrin A1 receptors, abundant expression of EPHA2 and EPHA4 messenger RNA was detected in BEND cells by reverse transcription polymerase chain reaction analysis. Furthermore, tunicamycin-induced endoplasmic reticulum (ER) stress was inactivated by ephrin A1 treatment of BEND cells. Our findings suggest that ephrin A1 promotes the development of BEND cells and likely enhances uterine capacity and maintenance of pregnancy by activating MAPK and PI3K signaling cascades and by restoring ER stress.

C-C motif chemokine ligand 23 abolishes ER stress- and LPS-induced reduction in proliferation of bovine endometrial epithelial cells.

To reduce embryonic losses in domestic animals for economic production of livestock meat and milk, chemokines and their receptors are required for proper implantation and placentation during early pregnancy. In this study, we investigated the effects of C-C-motif chemokine ligand 23 (CCL23) on the proliferation of bovine endometrial (BEND) epithelial cells. CCL23 treatment improved BEND cell proliferation by enhancing PCNA and cyclin D1 expression via activation of the PI3K/AKT and MAPK signaling pathways. In addition, a combination of CCL23 and tunicamycin reversed the ER stress-induced reduction in cell proliferation and the decreased expression of UPR-mediated signaling proteins, including IRE1α, PERK, and ATF6α. Moreover, it regulated the lipopolysaccharide-induced inflammation in BEND cells by inhibiting the expression of pro-inflammatory cytokines (IL-6 and IL-8), and by restoring intracellular Ca2+ levels. These findings demonstrate that CCL23 improves endometrial development and uterine receptivity required for implantation and placentation during early pregnancy.

C-C motif chemokine ligand 2 regulates lps-induced inflammation and ER stress to enhance proliferation of bovine endometrial epithelial cells.

Chemokines play an important role in regulating the complex immune system at the maternal-fetal interface during pregnancy. Among various chemokines, CC motif chemokine ligand 2 (CCL2) plays a role in the recruitment of immune regulatory cells to implantation sites within the endometrium. In cattle, CCL2 is abundantly expressed in the uterine endometrium. However, its intracellular signaling has not been identified. In this study, we examined the effects of CCL2 on bovine endometrial (BEND) cell proliferation. CCL2 stimulated BEND cell proliferation by abundant expression of PCNA, accumulation of cells in the G2/M phase, and activation of the PI3 K/AKT and MAPK signaling pathways. Moreover, CCL2 reduced endoplasmic reticulum stress and restored the inflammation-induced reduction in BEND cell proliferation by regulating the unfolded protein response genes and cytokines. Collectively, these results demonstrated that CCL2 plays a pivotal role in reproductive tissues and may support maternal-fetal interface to improve efficiency of pregnancy.

Fibroblast growth factor 2 induces proliferation and distribution of G2 /M phase of bovine endometrial cells involving activation of PI3K/AKT and MAPK cell signaling and prevention of effects of ER stress.

Fibroblast growth factor 2 (FGF2) is abundantly expressed in conceptuses and endometria during pregnancy in diverse animal models including domestic animals. However, its intracellular mechanism of action has not been reported for bovine endometrial cells. Therefore, the aim of this study was to identify functional roles of FGF2 in bovine endometrial (BEND) cell line which has served as a good model system for investigating regulation of signal transduction following treatment with interferon-tau (IFNT) in vitro. Results of present study demonstrated that administration of FGF2 to BEND cells increased their proliferation and regulated the cell cycle through DNA replication by an increase of PCNA and Cyclin D1. FGF2 also increased phosphorylation of AKT, P70S6K, S6, ERK1/2, JNK, and P38 in BEND cells in a dose-dependent manner, and expression of each of those transcription factors was inhibited by their respective pharmacological inhibitor including Wormannin, U0126, and SP600125. In addition, the increase in proliferation of BEND cells and activation of the protein kinases in response to FGF2 was suppressed by BGJ398, a FGFR inhibitor. Furthermore, proliferation of BEND cells was inhibited by tunicamycin, but treatment of BEND cells with FGF2 restored proliferation of BEND cells. Consistent with this result, the stimulated unfolded protein response (UPR) regulatory proteins induced by tunicamycin were down-regulated by FGF2. Results of this study suggest that FGF2 promotes proliferation of BEND cells and likely enhances uterine capacity and maintenance of pregnancy by activating cell signaling via the PI3K and MAPK pathways and by restoring ER stress through the FGFR.

CLA and EPA inhibit LPS-induced prostaglandin release from bovine endometrial cells through an NF-κB-dependent signaling mechanism

Although their action in antagonizing arachidonic acid metabolism is a key antiinflammatory effect of conjugated linoleic acid (CLA) and omega-3 polyunsaturated fatty acids (n-3 PUFA), these molecules have other antiinflammatory effects that might occur upstream of altered eicosanoid production. We examined the effects of two CLA isomers (c9,t11 and t10,c12 isomers), n-3 PUFA (EPA and DHA), and κNF-κB inhibitor (ammonium pyrrolidine dithiocarbamate (PDTC)) on lipopolysaccharide (LPS)-induced secretion of prostaglandins (PGE 2 and PGF 2a ) by bovine endometrial (BEND) cells. LPS increased PGE 2 and PGF 2a concentrations in BEND cell- conditioned media in a concentration- and time-dependent manner. The c9,t11 CLA isomer and EPA decreased prostaglandin response to LPS. Addition of PDTC, alone or in combination with c9,t11 CLA or EPA, inhibited LPS-induced eicosanoid release into the culture medium. Results indicate that the c9,t11 CLA isomer and EPA inhibit the endometrial prostaglandin release in cattle and that these molecules may act through an NF-κB signaling mechanism.

Phospholipase A2 regulation of bovine endometrial (BEND) cell prostaglandin production

BackgroundProstaglandins (PG), produced by the uterine endometrium, are key regulators of several reproductive events, including estrous cyclicity, implantation, pregnancy maintenance and parturition. Phospholipase A2 (PLA2) catalyzes the release of arachidonic acid from membrane phospholipids, the rate-limiting step in PG biosynthesis. The bovine endometrial (BEND) cell line has served as a model system for investigating regulation of signaling mechanisms involved in uterine PG production but information concerning the specific PLA2 enzymes involved and their role in regulation of this process is limited. The objectives of this investigation were to evaluate the expression and activities of calcium-dependent group IVA (PLA2G4A) and calcium-independent group VI (PLA2G6) enzymes in the regulation of BEND cell PG production.MethodsCells were grown to near-confluence and treated with phorbol 12, 13 dibutyrate (PDBu), interferon-tau (IFNT), the PLA2G4A inhibitor pyrrolidine-1 (PYR-1), the PLA2G6 inhibitor bromoenol lactone (BEL) and combinations of each. Concentrations of PGF2alpha and PGE2 released into the medium were determined. Western blot analysis was performed on cellular protein to determine effects of treatment on expression of PLA2G4A, PLA2G6 and PLA2G4C. PLA2 assays were performed on intact cells by measuring arachidonic acid and linoleic acid release and group-specific PLA2 activity assays were performed on cell lysates.ResultsBEND cells produced about 10-fold more PGE2 than PGF2alpha under resting conditions. Production of both PGs increased significantly in response to PDBu-stimulation. PYR-1 significantly diminished production of both PGs by resting cells and abolished the stimulatory effect of PDBu. BEL stimulated production of both PGs. IFNT reduced both PGE2 and PGF2alpha production by resting cells and diminished PDBu stimulation of PG production. Conversely, IFNT did not significantly reduce BEL stimulation of PG production. Cellular expression of PLA2G4A was enhanced by PDBu and this response was diminished by IFNT. Expression of PLA2G6 was not observed to be affected by treatments and no PLA2G4C expression was observed. Arachidonic acid release from intact cells was significantly increased by PDBu and this effect was attenuated by PYR-1 but not by BEL. Release of linoleic acid from intact cells was stimulated by PDBu and inhibited by BEL but not PYR-1. Group specific PLA2-activity assays demonstrated both PLA2G4A and PLA2G6 activity.ConclusionResults from this study demonstrate that PGE2 and PGF2-alpha production by BEND cells is mediated by the activity and expression of PLA2G4A. Interferon-tau treatment diminished expression of PLA2G4A and PG production. BEND cells were shown to express PLA2G6 but, unlike primary or early passage luminal bovine endometrial cells, stimulation of PLA2G6 activity was not associated with increased PG production.

Cytokines and growth factors inhibit tumor necrosis factor alpha–induced up-regulation of fibronectin binding on bovine endometrial cells

To design a high-throughput cell assay to identify molecules modulating adhesion induced by tumor necrosis factor alpha (TNF-alpha) of endometrial cells to mesothelium. Prospective study. Biotech company. Bovine endometrial (BEND) and human mesothelial cells. Endometrial cells were treated with TNF-alpha and different proteins. TNF-alpha increased binding of fibronectin-coated fluorescein isothiocyanate (FITC) beads. The ability of various proteins to inhibit TNF-alpha-induced fibronectin-bead binding was measured. Treatment of BEND cells with TNF-alpha increased binding of fibronectin-coated beads. Addition of TNF-alpha-binding protein abrogated the effect of TNF-alpha in a dose-dependent manner. The initial screen of 1014 proteins identified interferon-alpha2 (IFN-alpha2), inteleukin-17 (IL-17), transforming growth factor beta (TGF-beta), and platelet-derived growth factor (PDGF) as inhibiting TNF-alpha-induced bead binding. Interferon-alpha2, IL-17, and TGF-beta inhibited bead-binding with an IC50 (ng/mL, mean +/- SD) of 0.15 +/- 0.11, 0.098 +/- 0.008, and 5.91 +/- 0.72, respectively. All three isoforms of PDGF (AA, AB, and BB) were also found to inhibit TNF-alpha-induced bead-binding, with IC50s (ng/mL) of 1.8 +/- 0.45, 10.0 +/- 1.49, and 1.72 +/- 0.73, respectively. We describe a novel high-throughput cell-based assay for endometrial cell binding to fibronectin. We show that IFN-alpha, IL-17, TGF-beta, and PDGF have inhibitory actions on adhesion of endometrial cells to fibronectin.

Conjugated Linoleic Acid Reduces Phorbol Ester-Induced Prostaglandin F2α Production by Bovine Endometrial Cells

Recent interest in conjugated linoleic acid (CLA) research stems from the well-documented anticarcinogenic, antiatherogenic, antidiabetic, and antiobesity properties of CLA in animal models. The objective of this study was to examine the effects of 2 CLA isomers (cis-9,trans-11 and trans-10,cis-12) on phorbol 12,13-dibutyrate (PDBu)-induced PGF2α production in cultured bovine endometrial (BEND) cells. Confluent BEND cells were incubated in the absence (control) or presence of 100μM each of linoleic acid, cis-9,trans-11 CLA, or trans-10,cis-12 CLA for 24h. After incubation, cells were rinsed and then stimulated with PDBu (100 ng/mL) for 6h. Compared with untreated cells, PDBu stimulated PGF2α secretion (+25-fold) within 6h. The increases in PGF2α secretion were paralleled by signifi-cant induction of prostaglandin endoperoxide synthase-2 (PGHS-2) mRNA (+63-fold) and protein (+1.6-fold) expression. In spite of stimulatory effects on PGHS-2 and peroxisome proliferator-activated receptor δ (PPARδ) mRNA responses, CLA greatly decreased PGF2α production by PDBu-stimulated BEND cells. There was no evidence for PDBu or CLA modulation of PPARδ protein synthesis in cultured BEND cells. Results indicated that CLA modulation of PGF2α production by BEND cells was not mediated through PGHS-2 or PPARδ gene repression.

Differential Effects of n-3 and n-6 Fatty Acids on Prostaglandin F2α Production by Bovine Endometrial Cells

Recent studies have implicated n-3 polyunsaturated fatty acids in the reduction of eicosanoid production in the bovine uterus. The objective of this study was to determine whether the effect of eicosapentaenoic acid (EPA; C20:5, n-3) on PGF2α production by bovine endometrial (BEND) cells is influenced by the quantity of linoleic acid (C18:2, n-6) in the incubation medium. Confluent BEND cells were incubated in the absence (control) or presence of 100μMof EPA for 24h. After incubation, cells were rinsed and then stimulated with phorbol 12,13-dibutyrate (PDBu; 100 ng/mL) for 6h. Additional sets of culture dishes were treated with a combination of EPA and increasing n-6/n-3 fatty acid ratios for 24h and then challenged with PDBu for 6h. The PDBu stimulated PGF2α secretion and upregulated steady-state concentrations of prostaglandin endoperoxide synthase-2 and peroxisome proliferator-activated receptor delta mRNA within 6h. Preincubation of BEND cells with EPA for 24h decreased PGF2α response to phorbol ester, but had no detectable effects on prostaglandin endoperoxide synthase-2 or peroxisome proliferator-activated receptor delta mRNA abundance in PDBu-stimulated BEND cells. The inhibitory effect of EPA on PGF2α production was reverted in BEND cells treated with an increasing n-6-to-n-3 fatty acid ratio. Findings indicate that the net inhibition of endometrial PGF2α bioynthesis by n-3 fatty acids may vary depending on the ratio of n-6 to n-3 fatty acids in the uterus.

Isolation and sequence of an interferon-tau-inducible, pregnancy- and bovine interferon-stimulated gene product 15 (ISG15)-specific, bovine ubiquitin-activating E1-like (UBE1L) enzyme.

Bovine (bov) interferon-stimulated gene product 15 (ISG15) is produced in the endometrium in response to conceptus-secreted interferon (IFN)-tau. ISG15 conjugates to endometrial proteins through an enzymatic pathway that is similar to ubiquitinylation. Ubiquitin-activating enzyme 1-like protein (UBE1L) initiates enzymatic conjugation by forming a thioester bond with ISG15, thus preparing it for transfer to the next series of enzymes. The bovUBE1L has not been described. We hypothesized that bovUBE1L was induced by pregnancy and IFN-tau in the endometrium. A 110-kDa protein was purified from bovine endometrial (BEND) cells based on affinity with recombinant (r) glutathione S-transferase (GST)-ISG15. This protein was digested in gel with trypsin. Seven peptides were purified using HPLC, sequenced using liquid chromatography-mass spectroscopy-mass spectroscopy and found to share 43-100% identity with human UBE1L. The full-length bovUBE1L cDNA was isolated from a BEND cell cDNA library, sequenced, and found to share 83% identity with human UBE1L cDNA. Northern blot revealed two mRNAs that were detected in greater (P<0.05) concentrations in endometrium from Day 17-21 pregnant versus nonpregnant cows. Western blots using antihuman UBE1L antibody revealed a similar pattern of pregnancy-associated expression of UBE1L protein in the uterus. The bovUBE1L mRNA was localized, using in situ hybridization, primarily to glandular and luminal epithelium, with more diffuse localization to stroma of the endometrium from pregnant cows. Because bovUBE1L was purified through its interaction with rGST-ISG15 and shares significant amino acid and cDNA sequence identity with human UBE1L, it is concluded that it mediates conjugation of ISG15 to uterine proteins in response to the developing and attaching conceptus.

Inhibition of phorbol ester-induced PGF 2α secretion by IFN-τ is not through regulation of protein kinase C

Inhibitory effect of IFN-τ on phorbol ester (PdBu)-induced PGF 2α secretion was hypothesized to be manifested by the regulation of protein kinase C (PKC) in bovine endometrial (BEND) cells. Following 12 h stimulation with PdBu, cells were unresponsive to freshly added PdBu. Pretreatment of cells with a PKC inhibitor abolished PGF 2α secretion in response to PdBu. Therefore, PdBu induction of PGF 2α secretion is through activation of PKC. The α, ɛ, ι and λ isotypes of the PKC family were identified by Western blotting. Cells were then treated with medium alone (control), PdBu or PdBu + IFN-τ for 3 or 6 h. The PdBu-induced secretion of PGF 2α was suppressed by IFN-τ. At 3 and 6 h, PKCα and PKCɛ were detected both in the cytosolic and membrane fractions of unstimulated cells. There was a clear reduction of PKCα in the cytoplasm induced by PdBu and PdBu + IFN-τ at 3 and 6 h. The total abundance (cytoplasm and membrane fractions) of PKCα was lower in the PdBu + IFN-τ than PdBu alone. These temporal responses indicate a PKCα responsiveness of BEND cells to PdBu and PDBu + INF-τ with some evidence that IFN-τ causes a slight but detectable reduction in PKCα when added with PdBu. However, IFN-τ-induced decrease in the total abundance of PKCα was not enough to affect negatively the translocation of the PKCα to the membrane. Therefore, IFN-τ's ability to suppress secretion of PGF 2α is unlikely due to an interference with the PdBu-induced activity of PKC.

Differential effects of interferon-tau on the prostaglandin synthetic pathway in bovine endometrial cells treated with phorbol ester.

Rationale for these experiments was to evaluate the dose effects of bovine interferon-tau (IFN-tau) on the prostaglandin secretory pathway of immortalized bovine endometrial (BEND) cells in response to phorbol 12, 13-dibutyrate (PdBu) and to characterize similar responses in primary bovine uterine epithelial cells as a biomonitor of embryo-induced antiluteolytic effects on the endometrium. The BEND cells were treated with PdBu (0 or 100 ng/mL) and IFN-tau (0 or 50 ng/mL) for 6 h. The PdBu stimulated secretions of PGF2alpha and prostaglandin E2 (PGE2). Co-treatment of cells with IFN-tau blocked PdBu-induced secretion of both PGF2alpha and PGE2. Treatment with PdBu for 6 h induced expression of prostaglandin H synthase-2 mRNA, prostaglandin H synthase-2 protein, and prostaglandin E synthase mRNA, which were blocked with concurrent addition of IFN-tau. Doses of IFN-tau (0.05, 0.5, 1, 5, 10, and 20 microg/mL) were used with PdBu (0 and 100 ng/mL). The IFN-tau alone failed to stimulate secretion of PGF2alpha and PGE2, whereas IFN-tau doses <5 microg/mL suppressed PdBu-stimulated secretions of PGF2alpha and PGE2. Uterine epithelial cells were isolated from cows at d 17 after estrus and were cultured to confluence in serum-free medium. Cells were treated with IFN-tau (0, 50, or 500 ng/mL) and PdBu (0 or 100 ng/mL) before media were collected after 24 h for PGF2alpha and PGE2 analyses. Treatment of primary uterine epithelial cells with PdBu induced PGF2alpha secretion, and IFN-tau (50 and 500 ng/mL) caused a reduction in PGF2alpha secretion induced by PdBu. In the absence of PdBu, IFN-tau increased basal secretion of PGF2alpha. Concentrations of PGE2 increased in response to PdBu, and the 50-ng/mL dose of IFN-tau had a stimulatory effect on PGE2 concentrations compared with the 500-ng/mL dose in the absence of PdBu. Phorbol ester-induced gene transcription as related to prostaglandin synthesis is regulated by IFN-tau in vitro.

Interferon-tau induces degradation of prostaglandin H synthase-2 messenger RNA in bovine endometrial cells through a transcription-dependent mechanism.

A series of experiments were undertaken to examine the effects of interferon (IFN)-tau on regulation of prostaglandin H synthase (PGHS)-2 mRNA in bovine endometrial (BEND) cells as a means to elucidate the actions of IFN-tau to maintain pregnancy. The objective was to determine if IFN-tau mediates posttranscriptional regulation of PGHS-2 mRNA. Cells were treated with phorbol 12,13-dibutyrate (PdBu) for 3 h to induce PGHS-2 mRNA expression. Actinomycin D (0 or 1 microg/ml) or the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580 (1 microM), were added at 3 h, followed by addition of IFN-tau (0 or 50 ng/ml) at 3.5 h and extraction of RNA at 4.5 h. The concentrations of PGHS-2 mRNA were stable between 3 and 4.5 h regardless of actinomycin D. Simultaneous treatment of PdBu-treated cells with actinomycin D and SB203580 (1 microM) decreased PGHS-2 mRNA. Addition of IFN-tau (50 ng/ml) reduced PGHS-2 mRNA, which was not observed when actinomycin D was present. Concurrent treatments of cells with SB203580 and IFN-tau (5 ng/ml) decreased concentrations of PGHS-2 mRNA in an additive manner. Although IFN-tau reduced PGHS-2 mRNA concentrations, phosphorylation of p38 MAPK was induced by IFN-tau, PdBu, and PdBu combined with IFN-tau after 10 min of treatment. Both the p38 MAPK inhibitor and IFN-tau decreased prostaglandin F(2alpha) secretion, and decreases were additive when the two were given together. In summary, activation of p38 MAPK by PdBu is required for continued presence of PGHS-2 mRNA and secretion of prostaglandin F(2alpha) in BEND cells. Interferon-tau mediates a transcription-dependent mechanism, which induces degradation of PGHS-2 mRNA. However, the consequences of an IFN-tau-induced activation of p38 MAPK warrant further investigation, because inhibition of p38 MAPK caused a degradation of PGHS-2 mRNA.

Bovine Somatotropin Attenuates Phorbol Ester-Induced Prostaglandin F2α Production in Bovine Endometrial Cells

The recent observation that bovine somatotropin (bST) treatment at a timed insemination improves pregnancy rates in lactating dairy cows raises the possibility that growth hormone (GH) may modulate the endocrine and biochemical cross talk between the conceptus and maternal uterus at the time of pregnancy establishment in cattle. The objective of this study was to characterize the cellular and molecular mechanisms by which exogenous GH affects phorbol esterinduced prostaglandin F2α (PGF2α) production in cultured bovine endometrial (BEND) cells. Serum-deprived BEND cells were incubated with or without recombinant bovine GH (rbGH), insulin-like growth factor (IGF)-I, recombinant bovine interferon (rbIFN)-τ or a combination of rbGH + rbIFN-τ for 3h and then treated with phorbol 12,13-dibutyrate (PDBu) for an additional 6h. Exogenous PDBu increased PGF 2α secretion and steady-state levels of COX-2 mRNA within 3h. Priming of BEND cells with rbGH reduced PGF2α response to PDBu, whereas cotreatment with IGF-I amplified PDBu induction of PGF2α Preincubation of cell monolayers with rbIFN-τ suppressed PGF2α and COX-2 mRNA responses to PDBu. Inhibitory effects of rbGH and rbIFN-τ on PDBu-induced PGF2α production were additive. Results provide the first direct evidence that supplemental bST may interact with conceptus-secreted IFN-τ to modulate PGF2α secretion at the critical time of maternal recognition of pregnancy.

Uterine-conceptus interactions and reproductive failure in cattle

The dialogue between trophectoderm cells of the conceptus and epithelial cells of the endometrium is critical to CL maintenance and embryo survival. The signal transduction mechanisms by which bovine interferon (IFN)-τ regulates cyclooxygenase (COX)-2 expression and secretion of prostaglandin F 2α (PGF 2α) in bovine endometrial (BEND) cells is examined. Stimulation of Protein Kinase C with a phorbol ester (phorbol 12, 13 dibutyrate [PDBu]) activates COX-2 gene expression and PGF 2α secretion via the mitogen-activated protein kinase (MAPK) pathway. Interferon-τ attenuates PDBu activation of PGF 2α secretion, but this inhibitory effect appears to be independent of the MAPK pathway. Embryonic IFN-τ, acting through a Type I IFN receptor, activates the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway resulting in activation or repression of interferon-stimulated genes. Experimental evidence is provided that IFN-τ regulation of STATs regulates gene expression of COX-2 in a manner that decreases secretion of PGF 2α. Maternal regulation of the antiluteolytic pathway is discussed relative to the ability of the polyunsaturated fatty acid, eicosapentaenoic (EPA), to decrease endometrial secretion of PGF 2α and progesterone to increase both conceptus development and IFN-τ secretion.