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The fungicide boscalid induces thyroid histopathological and hormone effects in the rat, secondary to liver enzyme induction. To assess the human relevance of liver enzyme induction presumably leading to thyroid hormone disruption, a species comparative in vitro study on T4-glucuronidation was conducted. Currently no guidelines how to evaluate Phase II induction are in place. Therefore, we investigated the optimal conditions to evaluate the Phase I and II induction potential of boscalid in primary rat (PRH) and human (PHH) hepatocytes. Endpoints included mRNA gene expression and enzyme activities (CYPs and UGTs), measured after 3 (D3) and 7 (D7) days of exposure to reference compounds and to 5, 10 and 20 μM boscalid, focusing on T4-glucuronidation. Basal CYP activities and T4 glucuronidation were similar or higher on D7 than D3. The highest induction responses of CYPs were on D3, whereas UGT induction and T4-glucuronidation increases were highest on D7. Boscalid induced CYP1A, CYP2B and CYP3A mRNA and/or increased related activities in PRH and PHH. Species differences in the induction pattern of UGT genes by reference inducers (ß-naphthoflavone (BNF), 5-pregnen-3ß-ol-20-one-16a-carbonitirile (PCN), rifampicin (RIF) and phenobarbital (PB)) and boscalid were seen: UGT1A1, UGT1A3 and UGT1A9 were predominantly induced in PHH, while UGT2B1 was predominantly induced in PRH. Basal activity levels for T4-glucuronidation were very low in humans and an order of magnitude higher in rat, for this reason increases in activities were assessed as delta activity to the control. Significant increases in T4-glucuronidation occurred with boscalid in rat but not in human hepatocytes.

Neospora caninum is one of the most efficient transplacentally transmitted pathogens in cattle and is a cause of abortion in this domestic species. The invasion and proliferation of Neospora caninum in the placenta and its dissemination to the foetus are crucial events in the outcome of an infection. In the bovine placenta, the placentomes are formed by maternal caruncles, which are delimited by a maternal epithelium and foetal cotyledons, which are delimited by an epithelial layer named the trophoblast. These epithelia form a physical barrier against foetal infection. Furthermore, trophoblast cells act as an innate immune defence at the foetal-maternal interface. Neospora caninum invades and proliferates in trophoblast cells in vitro, but it is unknown whether host cell modulation events, which affect the immune response and other processes in the trophoblast, occur. In this work, we investigated the transcriptomic modulation by Neospora caninum infection in the bovine trophoblast cell line F3. In addition, two Neospora caninum isolates with marked differences in virulence, Nc-Spain1H and the Nc-Spain7, were used in this study to investigate the influence of these isolates in F3 modulation. The results showed a clear influence on extracellular matrix reorganisation, cholesterol biosynthesis and the transcription factor AP-1 network. Interestingly, although differences in the transcriptome profiles induced by each isolate were observed, specific isolate-modulated processes were not identified, suggesting very similar regulation in both isolates. Differential expression of the N. caninum genes between both isolates was also investigated. Genes involved in host cell attachment and invasion (SAG-related and microneme proteins), glideosome, rhoptries, metabolic processes, cell cycle and stress response were differentially expressed between the isolates, which could explain their variability. This study provides a global view of Neospora caninum interactions with bovine trophoblast cells and of the intra-specific differences between two Neospora caninum isolates with biological differences.

FXR agonists have demonstrated very promising clinical results in the treatment of liver disorders such as primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and nonalcoholic steatohepatitis (NASH). NASH, in particular, is one of the last uncharted white territories in the pharma landscape, and there is a huge medical need and a large potential pharmaceutical market for a NASH pharmacotherapy. Clinical efficacy superior to most other treatment options was shown by FXR agonists such as obeticholic acid (OCA) as they improved various metabolic features including liver steatosis as well as liver inflammation and fibrosis. But OCA's clinical success comes with some major liabilities such as pruritus, high-density lipoprotein cholesterol (HDL

Background Bovine neosporosis, one of the main causes of reproductive failure in cattle worldwide, poses a challenge for the immune system of pregnant cows. Changes in the Th-1/Th-2 balance in the placenta during gestation have been associated with abortion. Cotyledon and caruncle cell layers form the maternal-foetal interface in the placenta and are able to recognize and induce immune responses against Neospora caninum among other pathogens. The objective of the present work was to elucidate the immunomodulation produced by high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates of N. caninum in bovine trophoblast (F3) and caruncular cells (BCEC-1) at early and late points after infection. Variations in the mRNA expression levels of toll-like receptor-2 (TLR-2), Th1 and Th2 cytokines (IL-4, IL-10, IL-8, IL-6, IL-12p40, IL-17, IFN-γ, TGF-β1, TNF-α), and endothelial adhesion molecules (ICAM-1 and VCAM-1) were investigated by RT-qPCR, and protein variations in culture supernatants were investigated by ELISA. Results A similar pattern of modulation was found in both cell lines. The most upregulated cytokines in infected cells were pro-inflammatory TNF-α (P

To histologically determine number and size of skin cores and hair fragments resulting from intramuscular injection techniques using equine cadaver skin explants. Clipped (clip) and nonclipped (no.clip) skin specimens, of the hamstring region (n = 10 horses), were obtained from freshly euthanized horses and punctured with 18G needles (18G), 18G needles with stylet (18GM), and 22G needles (22G). Needles were flushed with 0.2 mL of sterile saline solution. The saline was collected on slides and stained (Pappenheim). Skin cores, produced with 18G, were fixed with formalin solution (4%) and embedded in paraffin. Sections were stained with Masson's trichrome. Light microscopy was used for evaluation. Each different injection technique resulted in the identification of skin cores within a subset of slides obtained from the different 10 skin samples. Displaced skin cores were identified more frequently in clip samples than no.clip samples; 18G needle punctures resulted in the highest number and largest surface area of skin cores in both no.clip and clip samples as compared to 22G and 18GM punctures ( P

The local immune system in the oviduct has a unique ability to deal with pathogens, allogeneic spermatozoa, and the semi-allogeneic embryo. To achieve this, it seems likely that the oviduct possesses an efficient and strictly controlled immune system that maintains optimal conditions for fertilization and early embryo development. The presence of a proper sperm and/or embryo-oviduct interaction begs the question of whether the local immune system in the oviduct exerts beneficial or deleterious effects on sperm and early embryo; support or attack?. A series of studies has revealed that bovine oviduct epithelial cells (BOECs) are influenced by preovulatory levels of Estradiol-17β, progesterone, and LH to maintain an immunologic homeostasis in bovine oviduct, via inhibition of proinflammatory responses that are detrimental to allogenic sperm. Under pathologic conditions, the mucosal immune system initiates the inflammatory response to the infection; the bacterial lipopolysaccharide (LPS) at low concentrations induces a proinflammatory response with increased expression of TLR-4, PTGS2, IL-1β, NFκB1, and TNFα, resulting in tissue damage. At higher concentrations, however, LPS induces a set of anti-inflammatory genes (TLR-2, IL-4, IL-10, and PTGES) that may initiate a tissue repair. This response of BOECs is accompanied by the secretion of acute phase protein, suggesting that BOECs react to LPS with a typical acute proinflammatory response. Under physiological conditions, polymorphonuclear neutrophils (PMN) are existent in the oviductal fluid during preovulatory period in the bovine. Interestingly, the bovine oviduct downregulates sperm phagocytosis by PMN via prostaglandin E2 (PGE2) action. In addition, the angiotensin-endothelin-PGE2 system controlling oviduct contraction may fine-tune the PMN phagocytic behavior to sperm in the oviduct. Importantly, a physiological range of PGE2 supplies anti-inflammatory balance in BOEC. Our recent results show that the sperm binding to BOECs further shift the local immunity toward anti-inflammatory conditions with upregulation of IL-10, TGFβ, and PGE2. In addition, this local environment leads PMN to express anti-inflammatory cytokines. In conclusion, the oviduct displays mucosal immunity that maintains an anti-inflammatory environment under physiological conditions that supports the sperm. Under pathologic condition, however, the oviduct supplies the innate immunity that may attack the sperm. Moreover, the oviduct-sperm interaction further suppresses the innate immune cells and strengthens the anti-inflammatory balance in the oviduct. Therefore, the oviduct immunity ensures sperm viability before fertilization.

The gross anatomic features (cotyledonary type) and histologic classification (synepitheliochorial) of the bovine placenta have been known for many years. Thorough ultrastructural analysis as well as a variety of descriptive studies dealing with the localization of cytoskeletal filaments, extracellular matrix, growth factor systems, steroid hormone receptors, and major histocompatibility complex have contributed further significant knowledge. However, this knowledge was not sufficient to solve clinical placenta-based problems, such as retained fetal membranes. Owing to the complexity of the fetomaternal interface in vitro, culture systems have been developed. As trophoblast giant cells (TGC) are thought to be key players in the cattle placenta, most cell culture models attempt to overcome the pitfall of losing the entire TGC population in vitro. Nevertheless, distinct cell line-based in vitro systems such as cell monolayers or 3-dimensional (co-culture) spheroids were generated for the fetal (trophoblast) and maternal (uterine epithelium) placental compartments. Monolayers have been used to study for example, growth factor or hormonal signaling and TGC formation, whereas spheroids served as models for, for example, trophoblast attachment, uterine epithelium depolarization, and also TGC formation. In the future, the use of more improved culture models might lead to better treatments of retained fetal membranes and increased prevention of embryonic loss. In addition, the in vitro models could shed more light on the mechanisms of the differentiation of uninucleate trophoblast into TGC.

Evidence is emerging that the interaction between male seminal fluid and female tissues promotes fertility, pregnancy, and health of offspring. This includes the acceleration of ovulation in a species known as a spontaneous ovulator, the domestic pig. Earlier studies revealed that seminal plasma acts by a local mechanism in the female pig. The aim of the present study was to examine local short-term and mid-term effects of seminal plasma (SP) on mRNA expression of immunoregulatory genes and transcripts associated with follicle- and oocyte maturation. In the porcine animal model, effects on mRNA expression in the female tract and preovulatory follicles were examined. SP suppressed mRNA expression of Prostaglandin-Endoperoxide Synthase 2 (PTGS2) ipsilateral to the infused uterine horn which was associated with a lower presence of immune cells in the uterine epithelium and lower PTGS2 immunoreaction. Depending on the sampling time (2 h vs. 17 h) and hormonal status, SP altered significant correlative relations of mRNA expression between PTGS2 and the transcripts Tumor Necrosis Factor Alpha, Tumor Necrosis Factor Alpha-Induced Protein 6 and Pentraxin 3 in uterus, granulosa and cumulus cells. A modulatory effect of SP on the oocyte gene network comprising eight oocyte transcripts was observed: uterine exposure to SP induced positive correlations (r >0.08, p

Epidermal growth factor (EGF) has been shown to be involved in control of the oviductal microenvironment. To elucidate the potential mechanisms responsible for the detrimental effect of heat stress and to identify the relation with the endocrine status, the effects of EGF on the level of phosphorylated mitogen-activated-protein kinase (MAPK) and proliferation of bovine oviductal epithelial cells (OECs) exposed to different cyclic ovarian steroidal environments (luteal phase (LP), follicular phase (FP) and postovulatory phase (PO)) and temperatures (mild heat stress (40 C) and severe heat stress (43 C)) were investigated. Western blot was performed to evaluate phosphorylated MAPK, while proliferation was analyzed by MTT assay. Stimulation of OECs with EGF alone or with EGF in the PO and FP environments significantly increased the amount of phosphorylated MAPK, with MAPK 44 phosphorylation being highest during exposure to PO conditions. These effects were not observed in the LP. Heat treatment completely blocked effects of EGF on phosphorylated MAPK. Additionally, severe heat stress led to a significantly lower basal level of phosphorylated MAPK. PD98059 (MAPK inhibitor) completely abolished EGF-stimulated MAPK phosphorylation and OECs proliferation. Overall the results indicate that EGF has the potential to increase the amount of phosphorylated MAPK in OECs and therefore could be involved in regulation of the bovine oviductal microenvironment. However, these regulatory mechanisms may be compromised in the presence of heat stress (high ambient temperature), leading to low fertility rates and impaired embryo survival.

Ruminants are the main source of human infections with the obligate intracellular bacterium Coxiella (C.) burnetii. Infected animals shed high numbers of C. burnetii by milk, feces, and birth products. In goats, shedding by the latter route coincides with C. burnetii replication in epithelial (trophoblast) cells of the placenta, which led us to hypothesize that epithelial cells are generally implicated in replication and shedding of C. burnetii. We therefore aimed at analyzing the interactions of C. burnetii with epithelial cells of the bovine host (1) at the entry site (lung epithelium) which govern host immune responses and (2) in epithelial cells of gut, udder and placenta decisive for the quantity of pathogen excretion. Epithelial cell lines [PS (udder), FKD-R 971 (small intestine), BCEC (maternal placenta), F3 (fetal placenta), BEL-26 (lung)] were inoculated with C. burnetii strains Nine Mile I (NMI) and NMII at different cultivation conditions. The cell lines exhibited different permissiveness for C. burnetii. While maintaining cell viability, udder cells allowed the highest replication rates with formation of large cell-filling Coxiella containing vacuoles. Intestinal cells showed an enhanced susceptibility to invasion but supported C. burnetii replication only at intermediate levels. Lung and placental cells also internalized the bacteria but in strikingly smaller numbers. In any of the epithelial cells, both Coxiella strains failed to trigger a substantial IL-1β, IL-6 and TNF-α response. Epithelial cells, with mammary epithelial cells in particular, may therefore serve as a niche for C. burnetii replication in vivo without alerting the host's immune response.

Background Neospora caninum, one of the main causes of abortion in cattle, is very effective at crossing the placental barrier and placental damage is crucial in the pathogenesis of abortion. Bovine trophoblast and caruncular cell layers are key cellular components in the maternal-foetal interface in placentomes, playing a fundamental role in placental functionality. Methods We studied tachyzoite adhesion, invasion, proliferation and egress of high- (Nc-Spain7) and low- (Nc-Spain1H) virulence N. caninum isolates in established cultures of bovine caruncular epithelial (BCEC-1) and trophoblast (F3) cells. The parasite invasion rate (pInvR) and the cell infection rate (cInfR) were determined by immunostaining plaque assay at different time points and multiplicities of infection (MOIs), respectively. In addition, tachyzoite growth kinetics were investigated using real-time PCR (qPCR) analysis and immunostaining plaque assay at different times. Results Neospora caninum invaded and proliferated in both cell lines. The pInvR was higher in F3 compared to BCEC-1 cells for the Nc-Spain7 isolate (P

SUMMARY Little is known about the local production and function of alpha 1-acid glycoprotein (AGP), an acute-phase protein, in the female reproductive tract. This study aimed to investigate the regulation and immune function of AGP in cultured bovine oviduct epithelial cells. Analysis by Western blotting and immunohistochemistry revealed that bovine oviduct tissue expresses AGP protein in epithelial cells and the smooth muscle layer. Stimulation of bovine oviduct epithelial cells in culture with either progesterone (1 ng/ml) or lipopolysaccharide (LPS, 10 ng/ml) induced both mRNA expression and secretion of AGP. Estradiol (1 ng/ml), progesterone (1 ng/ml), and luteinizing hormone (10 ng/ml), which are observed during the peri-ovulatory period in oviduct tissues (steroids) or in circulation (luteinizing hormone), suppressed LPS-induced expression and secretion of AGP, which in turn induced the expression of Toll-like receptor-4 (TLR-4) and interleukin-1β (IL-1B), but suppressed TLR-2 and tumor necrosis factor-α (TNFA) expression. AGP also inhibited LPS-induced TLR-2 and TNFA expression, but had no effect on LPS-induced TLR-4 and IL-1B expression. These findings suggest that oviductal epithelial cells can participate in antimicrobial processes through the secretion of AGP, which is partly regulated by ovarian steroids. Moreover, oviductal AGP may regulate the response of epithelial cells, thereby reducing the expression of the acute pro-inflammatory cytokine TNFA, which could contribute to the local homeostasis during the acute response to endotoxin release in the oviduct's anti-infection process. Mol. Reprod. Dev. 81: 861–870, 2014. © 2014 Wiley Periodicals, Inc.

A key metabolic alteration in tumor cells is an elevated glycolysis rate, resulting in the production of increased amounts of protons and lactate leading to lactic acidosis within the tumor microenvironment. The tumor-promoting effects of lactic acidosis are versatile, ranging from stimulation of proliferation, angiogenesis and metastasis to the reshaping of the tumor immune environment to a tumor-permissive phenotype. In tumors lactate is transported across the plasma membrane by the monocarboxylate transporters 1 and 4 (MCT 1/4) in symport with a proton. While MCT1 is expressed almost ubiquitously at low levels, MCT4 expression is regulated by hypoxia-inducible factor 1α (Hif1α), the central player in the cellular response to hypoxia. Thus, MCT4 is present in hypoxic, highly glycolytic cells. Given their central role in prevention of intracellular acidification and lactate transport, MCTs appear to be indispensable for glycolytic tumor growth. Since MCT4 has the strongest implication on the development and aggressiveness of diverse cancer types it has emerged as a new target for the treatment of solid tumors. We have identified MCT4 specific inhibitors structurally based on an internal compound library using cell-based lactate export assays. A subsequent extensive medicinal chemistry program was able to significantly improve activity and selectivity of the primary structures. Cellular target engagement was verified by cell-based assays like cellular thermal shift assay (CETSA). These efforts resulted in the identification of highly potent, orally bioavailable and selective MCT4 inhibitors (MCT4 IC50 < 20nM; MCT1 IC50 > 100µM) with good physicochemical properties and beneficial pharmacokinetics in mouse. In vivo efficacy and tumor lactate modulation were evaluated in NCI-H358 xenograft and MC38 syngeneic mouse models. Thereby we explore the potential of our MCT4 inhibitor to act as a single anti-cancer agent or to synergize with other established therapies like angiogenesis inhibitors or cancer immunotherapeutics. Citation Format: Nina Hambruch, Barbara Herkert, Christian Gege, Aurelie Mallinger, Johannes Fabian, Olaf Kinzel, Yansong Wang, Gero Fink, Michael Albers, Anette Funk, Annika Bittman-Waetzig, Tilly Fleckenstein, Floriane Braun, Claus Kremoser. Identification of a potent, orally bioavailable and selective MCT4 Inhibitor for the treatment of solid Warburg tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3588.

application/pdf, We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E2 (PGE2) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE2 at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.

Calgranulin A (S100A8) and B (S100A9) are found at high levels in inflamed tissue and have been associated with acute and chronic inflammatory disorders. Calgranulins are discussed as damage-associated molecular patterns (DAMPs). To analyze the role of calgranulins for inflammatory responses, bovine S100A8 and S100A9 were cloned, successfully expressed and FPLC-purified. Both molecules did not induce NF-κB activation in boTLR4-transfected HEK293 cells and stimulation of bovine monocytes with both proteins did not result in interleukin 1β (IL-1β) secretion or an upregulated mRNA expression of selected genes (IL1B, TNF, CXCL8, IL10, IL12). However, Interferon γ (IFN-γ) primed bovine monocytes released significantly higher amounts of IL-1β after stimulation with S100A8, S100A9, and co-stimulation with adenosine triphosphate (ATP). In IL-4/IL-13-primed monocytes, the IL-1β release was completely abrogated. The results imply that TLR4/MyD88/NF-κB-independent S100A8/A9-mediated activation of the inflammasome in cattle is favored in a Th1 environment and that S100A8 and S100A9 act as a DAMP in cattle.

application/pdf, This study aimed to investigate the role of epithelial cells in regulating innate immunity in bovine oviduct epithelial cell (BOEC) culture. We studied the effect of Escherichia coli lipopolysaccharide (LPS) and its interaction with ovarian steroids, estradiol (E2) and progesterone (P4), and luteinizing hormone (LH) at concentrations observed during the preovulatory period on immune responses in BOEC culture. Immunohistochemistry of oviduct tissue showed intensive expression of Toll-like receptor-4 (TLR-4) and TLR-2 in epithelial cells. A dose of 10 ng/ml LPS stimulated TLR-4, cyclooxygenase-2 (COX-2), nuclear factor kappa B inhibitor A (NFKBIA), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) expression, indicating an early pro-inflammatory response. A dose of 100 ng/ml LPS did not induce expression of these genes but stimulated TLR-2, IL-10,IL-4 and microsomal prostaglandin E synthase-1 (mPGES-1) expression and PGE2 secretion, indicating an anti-inflammatory response. Ovarian steroids and LH completely block LPS (10 ng/ml)-induced TLR-4, IL-1β and TNF-α expression as well as LPS (100 ng/ml)-induced TLR-2 expression. Taken together, this study suggests the existence of an early signaling system to respond to infection in the BOEC. In addition, ovarian steroids and LH may play a critical role in inducing homeostasis and in controlling hyperactive pro-inflammatory responses detrimental to epithelial cells, sperm and the embryo.

application/pdf, Sperm are allogenic to the female genital tract; however, oviducts provide optimal conditions for survival and capacitation of these non-self cells until fertilization. Recently, we showed that oviduct-conditioned media and prostaglandin E-2 (PGE(2)) suppress sperm phagocytosis by polymorphonuclear neutrophils (PMNs) under physiological conditions. We hypothesized that sperm binding to bovine oviduct epithelial cells (BOECs) could change the local innate immunity via PGE2. As the first step to obtain basic information, sub-confluent BOEC monolayers were co-cultured with swim-up sperm for 2 h. BOECs with viable bound sperm were cultured for an additional 3, 6, 12, or 24 h. Then, we confirmed the impact of the sperm-BOEC binding on both BOECs and PMN gene expression. Immunohistochemistry revealed that BOECs strongly express TGFB1 and IL10 in the oviduct. Sperm binding to BOECs in culture induced the anti-inflammatory cytokines (TGFB1 and IL10) and PGE(2) production by BOECs. Exogenous PGE(2) in vitro suppressed pro-inflammatory cytokine expression (TNF and IL1B) in BOECs. Moreover, pre-exposure of PMNs to BOEC-conditioned media suppressed the TNF expression, but the BOEC media co-cultured with sperm stimulated PMNs to express TGFB1 and IL10, with increasing PGE(2) secretion. Of note, exogenous PGE(2) led PMNs in vitro to decrease their TNF expression and increase anti-inflammatory cytokines expression. Our findings strongly suggest that BOECs provide an anti-inflammatory environment under physiological conditions and the sperm-BOEC binding further strengthens this milieu thus suppresses PMNs in the bovine oviduct. PGE(2) is likely to drive this stable anti-inflammatory environment in the oviduct.

The concept of a network within the family of adhesion/growth regulatory galectins implies distinct spatio-temporal expression profiles. To test this assumption immunohistochemically for bovine placenta, placentomal (P) and interplacentomal tissues (IP) were collected at a slaughterhouse from the three stages of pregnancy (early gestation = day 30–130; mid gestation = day 130–220; late gestation = day 220–275). The specimens were snap-frozen or fixed in Bouin’s solution, then embedded in paraffin. Gene expression for galectins-1, -3, -4 and -9 in P and IP of late gestational stages was monitored by RT-PCR. Galectin-type-specific antibodies were used for immunohistochemical localization. In IP, galectin-1 was present in stroma cells and early gestational trophoblast giant cells (TGC), whereas galectin-3 was confined to uterine epithelial cells. In contrast, both galectins were found in epithelia of P tissue. Uterine epithelial cells and blood vessel walls were positive for galectin-4, while galectin-9 was detected predominantly in uterine epithelial cells and late gestational TGC. Our study thus reveals individual profiles among the galectins tested, an indication for specific functions exerted by each protein in the bovine endometrium and placenta.

Matrix metalloproteinases (MMPs) and counteracting tissue inhibitors of metalloproteinases (TIMPs) are balancing extracellular matrix (ECM) formation and degradation. The latter is believed to be an important aspect for the detachment of fetal membranes postpartum when loosening the feto-matemal connection which is a prerequisite to avoid placental retention a common disease in cows leading to considerable economic loss. Membrane-type (MT) MMPs have been suggested as potential activators controlling ECM remodelling. In particular, MT1-MMP (MMP-14) is able to degrade ECM substrates and activate MMP-2 through binding TIMP-2 at the cell surface. Since the connection between the trophoblast and the maternal caruncular epithelium is supported by integrin receptors bound to ECM, we hypothesize that impaired modulation of the ECM by TIMPs/MMPs participates in the aetiology of bovine retained fetal membranes. To analyse this involvement, placentomes were collected from cows after term parturition and timely release of fetal membranes (n = 4) and cows with retained fetal membranes after various treatments for the induction of parturition using progesterone antagonist (aglepristone), PGF(2 alpha) analogue, glucocorticoid, and after elective caesarean sections (each group n = 3). The expression of MMP-14, MMP-2 and of TEMP-2 was examined by real-time-PCR, irnmunohistochemistry, Western blot and zymography. The relative mRNA expression levels of MMP-14 remained unchanged, while the expression levels of TIMP-2 and MMP-2 partly increased in animals with induced parturition and retention of fetal membranes compared to animals without placental retention. MMP-14 protein was expressed in cells of the uninucleated trophoblast, the fetal mesenchyme and maternal stoma. TIMP-2 was present exclusively in trophoblast giant cells, while MMP-2 could be detected in uninucleated trophoblast cells and the fetal mesenchyme. The presence of the activated enzyme was confirmed by zymography. In conclusion, MMP-14, MMP-2 and TIMP-2 are co-localized in the fetal compartment and therefore could influence the timely release of fetal membranes in cattle. (c) 2011 Elsevier Inc. All rights reserved.

Drug treatment is critical in pregnant cows due to the possibility of a maternal-to-fetal drug transfer across the placenta. Since the (syn)epitheliochorial bovine placental barrier includes an intact uterine epithelium, which in general limits drug transfer to the fetal trophoblast, the establishment of a species- and organ-specific in vitro model like the bovine caruncular epithelial cell line 1 (BCEC-1) for testing bovine placental drug transport is desirable. P-glycoprotein (P-gp or ABCB1) is an important efflux carrier that limits drug permeability across blood-tissue barriers such as the placenta and transports a wide range of structurally unrelated compounds including many drugs commonly used in veterinary medicine. The aim of the present study was to elucidate the suitability of BCEC-1 as an appropriate in vitro model for P-gp mediated drug transport in the bovine placenta. P-gp mRNA expression was detected by RT-PCR in BCEC-1 and placental tissue. Additionally, the carrier protein was localised in the apical membrane of BCEC-1 by immunofluorescence staining with the mouse monoclonal antibody C494. Drug transport in BCEC-1 was investigated by FACS analysis using the fluorescent P-gp substrate Rhodamine 123. Inhibition of Rhodamine 123 efflux by the P-gp inhibitors Verapamil and PSC833 confirmed functional expression of P-gp in BCEC-1. Furthermore, transport measurements in the transwell-system revealed a basal-to-apical net flux of the P-gp substrate digoxin at concentrations ranging from 10nM to 10 μM. This transwell digoxin flux was inhibited by Verapamil. In conclusion, P-gp is functionally expressed in BCEC-1 and mediates a basal-to-apical flux of digoxin indicating dominant apical localization of P-gp in this cell culture model. Therefore, BCEC-1 may be an appropriate in vitro model to study drug transport across the maternal epithelium as part of the epitheliochorial placental barrier of the cow.

Differentiation and restricted invasion/migration of trophoblast cells are crucial for feto-maternal communication in the synepitheliochorial placenta of cattle. EGF is expressed in the bovine placenta and likely regulates these cell properties. As cell migration and motility rely on the degradation of extracellular matrix we hypothesize that EGF is involved in the regulation of the MMP-9/TIMP-1 balance and thus could influence trophoblast migration, tissue remodeling, and the release of the fetal membranes after parturition. The aim of this in vitro study was to examine EGF-mediated effects on cell motility, proliferation, and MMP-9 and TIMP-1 expression in cultured bovine trophoblast cells. We used a trophoblast cell line (F3) derived from bovine placentomes to examine the influence of EGF on MMP-9 and TIMP-1 expression by semiquantitative RT-PCR and MMP activity by zymography. Migration assays were performed using a Boyden chamber and cell motility was measured by time-lapse analyses. To identify the involved signaling cascades, phosphorylation of mitogen-activated protein kinase (MAPK) 42/44 and Akt was detected by Western blot. EGF treatment increased both the abundance of MMP-9 and TIMP-1 mRNAs and the proteolytic activity of MMP-9. Furthermore, EGF stimulated proliferation and migration of F3 cells. Addition of specific inhibitors of MAPK (PD98059) and/or PI3K (LY294002) activation abolished or reduced EGF-induced effects in all experiments. In conclusion, EGF-mediated effects stimulate migration and proliferation of bovine trophoblast cells and may be involved in bovine placental tissue remodeling and postpartum release of fetal membranes.

Introduction Impaired placental maturation has been associated with retention of fetal membranes, which is a major reproductive disease in cattle. This maturation includes alterations in all tissue compartments of the placenta, specifically of epithelial and stroma cells and extracellular matrix. It is believed to be controlled by hormones, adhesion molecules and proteolytic enzymes. To investigate if the proteolytic enzyme heparanase and its substrates, the syndecans (SDCs) could be involved in the release of fetal membranes, their expression in bovine placentomes was analyzed. Methods Placentomes were taken from gestational day 35 until term, directly after spontaneous parturition, after preterm caesarean section, and after chemically induced parturition. Heparanase and SDCs were localized by immunohistochemistry and the respective mRNAs were quantified by qRT-PCR. Heparanase expression was additionally quantified by Western blot. Results Heparanase, SDC1 and SDC4 displayed significant changes in expression and localization depending on gestational progress and mode of parturition. All three proteins showed an expression at the end of gestation, together with an altered, predominant localization in fetal and maternal epithelia. After physiological parturition, the placentomal tissue stained weaker for all syndecans. This change in staining pattern could not be observed after induced preterm parturition. SDC2 expression did not change during the course of gestation. Discussion The changing placental expression patterns of heparanase, SDC1 and SDC4 indicate that these molecules might be involved in fetomaternal communication and placental maturation in cattle. The matrix degrading properties of heparanase could assist in a timely reduction of fetomaternal adhesion and thus promote separation of the membranes after parturition.

The neurotrophin receptor p75 interacts with the GTPase Ras. Unstimulated it inactivates Ras while ligand binding induces Ras activation. We developed an inhibitory peptide (ip75RBD) which interferes with the binding domain of Ras of the intracellular domain of p75. ip75RBD inhibits the binding of Ras to the receptor in vitro. It is membrane-permeable and inhibits ligand-induced Ras activation via p75 in vivo but does not influence Ras activation by the stimulated receptor tyrosine kinases Trk and the epidermal growth factor receptor EGFR. The activation of the neutral sphingomyelinase by stimulated p75 is slightly delayed but not inhibited by the peptide. p75-mediated neuronal death induced by NGF or aggregated beta-amyloid1–42 is reduced. We conclude that ip75RBD specifically blocks the Ras binding site of p75 and can be used to analyze p75-induced Ras signaling.

Introduction The feto–maternal interface during bovine implantation was studied in vivo and using three-dimensional bovine endometrial (BCECph) and trophoblast spheroids (CCS), each with underlying fibroblasts. Methods The expression of ezrin and cytokeratin 18 (CK18) was analyzed via immunohistochemistry (IHC), RT-PCR and western blotting in bovine endometrium (GD 18-44) with in vivo (VIVO) and in vitro-produced embryos (VITRO). BCECph were stimulated with cotyledon-conditioned media (CCM) and analyzed by TEM/SEM and IHC. CCS were stained (IHC) for TGC markers, to test if spheroidal trophoblast cells had differentiated into TGC. Results At GD 20, caruncular epithelium (CE) and uterine glands (UG) showed a loss of cytosolic ezrin and CK18 followed by a complete loss of both proteins. At GD 35 both reappeared in CE and UG. The endometrial expression pattern did not differ between VIVO and VITRO. RT-PCR and western blotting confirmed the presence of ezrin and CK18. All spheroids had an outer polarized, cytokeratin and ezrin positive epithelium (CE or trophoblast) with apical microvilli. Stimulation of BCECph with CCM induced similar changes in ezrin expression as observed in endometrial tissue. However, no ultrastructural alterations were found by transmission electron microscopy. Absence of TGC-specific glycoproteins in CCS indicated that TGC differentiation was not induced by three-dimensional culture conditions. Discussion Ezrin and CK18 are downregulated during implantation in cattle. The expression changes represent a temporal depolarization, which could be important for an establishment of bovine pregnancy. Our in vitro experiments demonstrate that the trophoblast could contribute to this change in vivo.

Within the testis, Sertoli cell (SC) junctional complexes between somatic SC create a basal and apical polarity within the seminiferous epithelium, restrict movement of molecules between cells, and separate the seminiferous epithelium into a basal and adluminal compartments. This barrier consists of membrane integrated proteins known as tight, adherens, and gap junctions, which promote cell-cell contact along the blood-testis-barrier (BTB). Nevertheless, these junctions, which form the basis of the BTB are structures whose function and dynamic regulation is still poorly understood. Thus, in this study, through the use of immunohistochemistry (IHC), semi quantitative western blot (WB) analysis, and real-time-quantitative-PCR (qRT-PCR) we focused on the expression pattern of the main testicular tight junction protein, occludin, in SC. For this, the established transgenic SC specific connexin 43 (Cx43) knockout (SCCx43KO) mouse line was used; both knockout (KO) and wildtype (WT) males of different ages from juvenile to adult were compared. The object was to elucidate a possible role of Cx43 on the expression pattern and regulation of occludin. This conditional KO mouse line lacks the gap junction gene Gja1 (coding for Cx43) only in SC and reveals impaired spermatogenesis. The qRT-PCR indicates an increase in occludin mRNA in adult KO mice. These results correspond to the occludin protein synthesis of adult mice. Additionally, during puberty, occludin localization at the BTB barrier in KO mice is delayed. Our study demonstrates spatiotemporal alterations in occludin mRNA- and protein-expression, indicating that Cx43 might act as a regulator for BTB formation (and function).

Within the testis, Sertoli cell (SC) junctional complexes between somatic SC create a basal and apical polarity within the seminiferous epithelium, restrict movement of molecules between cells, and separate the seminiferous epithelium into a basal and adluminal compartment. This barrier consists of membrane integrated proteins known as tight, adherens, and gap junctions, which promote cell-cell contact along the blood-testis-barrier (BTB). Nevertheless, these junctions, which form the basis of the BTB are structures whose function and dynamic regulation is still poorly understood. Thus, in this study, through the use of immunohistochemistry (IHC), semi quantitative western blot (WB) analysis, and real-time-quantitative-PCR (qRT-PCR) we focused on the expression pattern of the main testicular tight junction protein, occludin, in SC. For this, the established transgenic SC specific connexin 43 (Cx43) knockout (SCCx43KO) mouse line was used; both knockout (KO) and wildtype (WT) males of different ages from juvenile to adult were compared. The object was to elucidate a possible role of Cx43 on the expression pattern and regulation of occludin. This conditional KO mouse line lacks the gap junction gene Gja1 (coding for Cx43) only in SC and reveals impaired spermatogenesis. The qRT-PCR indicates an increase in occludin mRNA in adult KO mice. These results correspond to the occludin protein synthesis of adult mice. Additionally, during puberty, occludin localization at the BTB barrier in KO mice is delayed. Our study demonstrates spatiotemporal alterations in occludin mRNA- and protein-expression, indicating that Cx43 might act as a regulator for BTB formation (and function).

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in several aspects of the immune response. MIF appears to play important roles in materno-fetal immuno-tolerance during placental establishment, modulation and growth as studied in epitheliochorial porcine and hemochorial human and mouse placentae. Here we studied the bovine placenta being multiplex, villous and synepitheliochorial with a low degree of invasion, to see if MIF could be involved. Placental tissues sampled from 12 cows at 9 stages of gestation (days 18-250), and endometrial tissues from two non-pregnant animals were processed for immunohistochemistry. Bovine MIF was detected by Western blot using anti-human MIF monoclonal antibodies. An immunoreactive band of approximately 12kDa confirmed similarities between bovine and human MIFs. Compared to the non-pregnant stage with very faint staining, the caruncular epithelium during pregnancy showed stronger staining for MIF. The intercaruncular epithelium in non-pregnant endometrium showed some reaction apically with increasing intensity at uterine gland openings; in contrast, at day 18 of gestation this staining was markedly increased. During gestation both caruncular and trophoblast epithelium of the placentomes were positive with different intensity in relation to the gestational stage. In the uterine glands, some strongly stained cells were present. The mature binucleated trophoblast giant cells were negative throughout pregnancy. During reestablishment of vascularisation, the vasculature in the caruncular area showed MIF reactivity. While supporting involvement of MIF in different placental types, the spatio-temporal variation in the bovine placenta suggests a regulatory role for MIF mainly in the interhemal barrier and during vascular development.

Background The equine periodontium provides tooth support and lifelong tooth eruption on a remarkable scale. These functions require continuous tissue remodeling. It is assumed that multipotent mesenchymal stromal cells (MSC) reside in the periodontal ligament (PDL) and play a crucial role in regulating physiological periodontal tissue regeneration. The aim of this study was to isolate and characterize equine periodontal MSC. Tissue samples were obtained from four healthy horses. Primary cell populations were har-vested and cultured from the gingiva, from three horizontal levels of the PDL (apical, midtooth and subgingival) and for comparison purposes from the subcutis (masseteric region). Colony-forming cells were grown on uncoated culture dishes and typical in vitro characteristics of non-human MSC, i.e. self-renewal capacity, population doubling time, expression of stemness markers and trilineage differentiation were analyzed. Results Colony-forming cell populations from all locations showed expression of the stemness markers CD90 and CD105. In vitro self-renewal capacity was demonstrated by colony-forming unit fibroblast (CFU-F) assays. CFU-efficiency was highest in cell populations from the apical and from the mid-tooth PDL. Population doubling time was highest in subcutaneous cells. All investigated cell populations possessed trilineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages. Conclusions Due to the demonstrated in vitro characteristics cells were referred to as equine subcutaneous MSC (eSc-MSC), equine gingival MSC (eG-MSC) and equine periodontal MSC (eP-MSC). According to different PDL levels, eP-MSC were further specified as eP-MSC from the apical PDL (eP-MSCap), eP-MSC from the mid-tooth PDL (eP-MSCm) and eP-MSC from the subgingival PDL (eP-MSCsg). Considering current concepts of cell-based regenerative therapies in horses, eP-MSC might be promising candidates for future clinical applications in equine orthopedic and periodontal diseases.

Introduction: In this study, we aimed to form spheroids with the bovine placental trophoblast cell line F3. Spheroids are 3-dimensional culture models which can be used to conduct versatile in vitro and in vivo experiments. Materials and Methods: The spheroids were generated using the hanging drop technique, 25% methocel and matrigel. The F3 spheroids were characterized morphologically by light microscopy and transmission (TEM) and scanning electron microscopy (SEM) and immunohistochemistry (ezrin, vimentin, cytokeratin, placental lactogen). The fluorescent dyes calcein and ethidium homodimer were used to determine the viability of the spheroidal F3 cells by immunofluorescence microscopy. Results: The cell line F3 only formed spheroids by the hanging drop technique when matrigel was added. The trophoblast spheroids were delimited and fully covered by extracellular matrix (light microscopy/TEM/SEM). Cells contributing to spheroids could not be discriminated from each other (light microscopy). The outer spheroidal layer consisted of cells which possessed an apical pole with microvilli that were directed to the outside (light microscopy/TEM). All of the spheroidal F3 cells expressed ezrin, vimentin and cytokeratin, but not placental lactogen. The spheroid core contained degenerating cells whilst the F3 cells of the outer rim were viable (TEM/immunofluorescence microscopy). Discussion: We have established a 3-dimensional spheroid model for the bovine placental trophoblast cell line F3. The developed culture model might prove valuable for future in vitro studies on the differentiation of bovine trophoblast cells.

In the bovine placenta, multinucleate trophoblast giant cells (TGC), evolving from uninucleate trophoblast cells, are crucial for feto-maternal interaction as they show endocrine activity and the ability to migrate and fuse with caruncular epithelial cells. In contrast to caruncular epithelial cells, the isolation and culture of bovine trophoblast cells is complicated because they cease to express their specific products, like placental lactogen (PL), during prolonged culture. In the present study, we aimed to establish a bovine cotyledonary trophoblast cell line targeting our long term goal to develop an in vitro model for the bovine placenta. Therefore, the functional activity of important signalling pathways was tested. Primary trophoblast cells were isolated from a bovine cotyledon of a male fetus and successfully subcultured and cryopreserved. The obtained cell line, termed F3, showed epithelial morphology and characteristic binuclear giant cells in small numbers through all passages. The trophoblastic origin of F3 cells was verified by amplification of a Y-chromosome specific DNA-sequence and the presence of PL mRNA. Immunofluorescence demonstrated that F3 cells were continuously positive for zonula occludens-2 (ZO-2), cytokeratin and vimentin, whereas they expressed the TGC specific marker PL only in the first two passages. F3 cell growth was accelerated in medium supplied with epidermal growth factor (EGF). EGF-stimulated proliferation was mediated through activation of Ras and the phosphorylation of mitogen-activated protein kinase (MAPK) 42 and 44. In conclusion, the F3 cell line shows several in vivo characteristics of bovine cotyledonary trophoblast cells. The response to EGF stimulation indicates that EGF plays a role during bovine placentation, and illustrated that F3 cells may provide a valuable tool for further mechanistic studies elucidating the feto-maternal interplay.