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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.

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.

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.

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.

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.

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.

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.