Analysis of connexin 43, connexin 45 and N-cadherin in the human sertoli cell line FS1 and the human seminoma-like cell line TCam-2 in comparison with human testicular biopsies.

BACKGROUND: Germ cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression. METHODS: FS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines' representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling. RESULTS: Cx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells. CONCLUSION: The junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression.

Boscalid shows increased thyroxin-glucuronidation in rat but not in human hepatocytes in vitro.

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 Phase I and Phase II induction potential of boscalid in primary rat (PRH) and human (PHH) hepatocytes. Endpoints included mRNA gene expression and enzyme activities (cytochrome P450 isozymes [CYPs] and uridine diphosphate-glucuronosyltransferases [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-16α-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.

Establishment and functional characterization of a murine primary Sertoli cell line deficient of connexin43.

The Sertoli cell (SC) specific connexin43 (Cx43) knockout (SCCx43KO) mouse line is ideal to gain insight into the mechanistic gap junction formation in SC and the seminiferous epithelium. A method for developing primary SC cultures from these mice was established, validated and successfully characterized via polymerase chain reaction, immunohistochemistry, immunofluorescence (IF), and Western blots (WB). It was evident that both knockout (KO) and wild-type (WT) primary cell cultures were similar in morphology. These highly pure SC cultures were subjected to cell proliferation assays indicating no notable proliferation in cultures of both genotypes. Measurements of cell monolayer integrity indicated significant increases in transepithelial electrical resistance and consequently in tight junction expression of the KO cultures. Using semi-quantitative WB and IF, tight junction protein claudin-11 was analyzed. These results support a role for Cx43 in regulating blood-testis barrier (BTB) function, composition, and dynamics in vitro. Thus, the SC deficient Cx43 cell cultures may provide a valuable in vitro tool for a better understanding of the mechanistic role of Cx43 in spermatogenesis and BTB assembly.

Nonsteroidal FXR Ligands: Current Status and Clinical Applications.

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 (HDLc) lowering, low-density lipoprotein cholesterol (LDLc) increase, and a potential for drug-induced liver toxicity. Some of these effects can be attributed to on-target effects exerted by FXR, but with others it is not clear whether it is FXR- or OCA-related. Therefore a quest for novel, proprietary FXR agonists is ongoing with the aim to increase FXR potency and selectivity over other proteins and to overcome at least some of the OCA-associated clinical side effects through an improved pharmacology. In this chapter we will discuss the historical and ongoing efforts in the identification and development of nonsteroidal, which largely means non-bile acid-type, FXR agonists for clinical use.

Permissiveness of bovine epithelial cells from lung, intestine, placenta and udder for infection with Coxiella burnetii.

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-1ß, 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.

Effect of epidermal growth factor (EGF) on the phosphorylation of mitogen-activated protein kinase (MAPK) in the bovine oviduct in vitro: Alteration by heat stress.

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.

An acute-phase protein as a regulator of sperm survival in the bovine oviduct: alpha 1-acid-glycoprotein impairs neutrophil phagocytosis of sperm in vitro.

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.

Regulation of innate immune function in bovine oviduct epithelial cells in culture: the homeostatic role of epithelial cells in balancing Th1/Th2 response.

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 1ß (IL-1ß) 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-1ß 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.

Isolation and characterization of multipotent mesenchymal stromal cells from the gingiva and the periodontal ligament of the horse.

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 harvested 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.

Immune response profile of caruncular and trophoblast cell lines infected by high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates of Neospora caninum.

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-ß1, 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 < 0.05-0.0001) and IL-8 (P < 0.05-0.001) whereas regulatory IL-6 (P < 0.05-0.001) and TGF-ß1 (P < 0.05-0.001) were downregulated in both cell lines. The measurement of secreted IL-6, IL-8 and TNF-α confirmed the mRNA expression level results. Differences between isolates were found in the mRNA expression levels of TLR-2 (P < 0.05) in both cell lines and in the mRNA expression levels (P < 0.05) and protein secretion of TNF-α (P < 0.05), which were higher in the trophoblast cell line (F3) infected with the low-virulence isolate Nc-Spain1H. CONCLUSIONS: Neospora caninum infection is shown to favor a pro-inflammatory response in placental target cells in vitro. In addition, significant immunomodulation differences were observed between high- and low-virulence isolates, which would partially explain the differences in virulence.

Seminal plasma modulates the immune-cytokine network in the porcine uterine tissue and pre-ovulatory follicles.

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<0.05) of maturation promoting factors among each other and with cumulus cells on the side of the treated horn. In conclusion, SP orchestrates the gene network regulating the bidirectional communication between oocytes and surrounding somatic cells. The modulation of the immune-cytokine network of the female reproductive system could contribute to the previously reported SP-induced acceleration of ovulation in the porcine species.

Transcriptome modulation of bovine trophoblast cells in vitro by Neospora caninum.

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.

Differential susceptibility of bovine caruncular and trophoblast cell lines to infection with high and low virulence isolates of Neospora caninum.

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 < 0.05), and higher for the Nc-Spain7 than the Nc-Spain1H in F3 cells (P < 0.01). The cInfR was also higher in F3 cells than in BCEC-1 cells for both isolates (P < 0.0001), and the cInfR for the Nc-Spain7 isolate was higher than for the Nc-Spain1H isolate in both cell lines (P < 0.05). Tachyzoite growth kinetics showed tachyzoite exponential growth until egress at 58 hpi for both isolates in F3, whereas Nc-Spain1H showed a non-exponential growth pattern in BCEC-1. Asynchronous egress of both isolates was observed from 22 h post-infection onwards in BCEC-1. In addition, the tachyzoite yield (TY58h) was higher in F3 than in BCEC-1 infected by both isolates (P < 0.0001), highlighting better replication abilities of both parasites in F3. Nc-Spain7 showed shorter doubling times and higher TY58h compared to Nc-Spain1H in F3 cells; adhesion, invasion and proliferation mechanisms were very similar for both isolates in BCEC-1. CONCLUSIONS: Our results indicate a highly similar behavior of high- and low-virulence isolates in their interactions with maternal caruncular cells and suggest an important role of foetal trophoblasts in the pathogenesis of N. caninum infection.

Sternal bone marrow derived equine multipotent mesenchymal stromal cells (MSCs): investigations considering the sampling site and the use of different culture media.

Aspiration of equine sternal bone marrow is required for the cultivation of bone marrow-derived multipotent mesenchymal stromal cells (BM-MSCs) for regenerative therapies. For bone marrow aspiration as well as for MSC cultivation, there is a need to optimize techniques and protocols to enhance MSC harvest at minimized culture times. In a comparative study bone marrow aspirates from sternebra 4 and 5 were collected at two different positions within the sternebrae, either from 10 mm or from 30 mm dorsal from the ventral margin of the sternebrae. Accuracy of the puncture depth was confirmed by ultrasonography and computed tomography. Isolated MSCs were cultivated using media supplemented with three alternative sera, i.e. fetal calf serum, standardized horse serum and autologous serum. Due to morphological characteristics (spherical shape, only thin layer of hyaline cartilage at the ventral site, reliable bone marrow aspiration from only 10 mm intraosseous depth), sternebra 5 appeared most suitable for bone marrow aspiration. Cultivation and expansion of BM-MSCs was most efficient using fetal calf serum.

Sperm Binding to Oviduct Epithelial Cells Enhances TGFB1 and IL10 Expressions in Epithelial Cells as Well as Neutrophils In Vitro: Prostaglandin E2 As a Main Regulator of Anti-Inflammatory Response in the Bovine Oviduct.

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 E2 (PGE2) 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 PGE2 production by BOECs. Exogenous PGE2 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 PGE2 secretion. Of note, exogenous PGE2 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. PGE2 is likely to drive this stable anti-inflammatory environment in the oviduct.

Loss of connexin43 (Cx43) in Sertoli cells leads to spatio-temporal alterations in occludin expression.

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).

Recombinant bovine S100A8 and A9 enhance IL-1ß secretion of interferon-gamma primed monocytes.

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 1ß (IL-1ß) 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-1ß after stimulation with S100A8, S100A9, and co-stimulation with adenosine triphosphate (ATP). In IL-4/IL-13-primed monocytes, the IL-1ß 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.