Establishing a model of primary bovine hepatocytes with responsive growth hormone receptor expression.

The liver becomes resistant to growth hormone before parturition in dairy cows (uncoupling of the somatotropic axis). However, the mechanism of growth hormone insensitivity has not been fully described. The aim of the present study was to improve a previous model of adult bovine hepatocytes in a sandwich culture system to ensure growth hormone receptor (GHR) expression. First, we modified the protocol for hepatocyte retrieval and tested the effect of short (18 min) and long (up to 30 min) warm ischemia on hepatocyte viability. Second, we used medium additives that affect GHR expression in vivo-insulin (INS), dexamethasone (DEX), both (INS+DEX), or no hormone additives (CTRL)-to ensure the functionality of hepatocytes, as measured by lactate dehydrogenase activity and urea concentration in the medium. We also used reverse transcriptase PCR of hepatocytes to evaluate expression of albumin (ALB), hepatocyte nuclear factor 4α (HNF4A), nuclear factor-κ-B-inhibitor α (NFKBIA), cytosolic phosphoenolpyruvate carboxykinase (PCK1), and vimentin (VIM) mRNA. Moreover, we analyzed the expression of GHRtot (GHR), GHR1A, insulin-like growth factor-1 (IGF1), and IGF binding protein-2 (IGFBP2) in response to exposure to media with the different compositions. Modification of the protocol (changes in rinsing and perfusion times, buffer composition, and the volume and standardization of collagenase) led to increased cell counts and cell viability. Short warm ischemia with the modified protocol significantly increased cell count (4.7 × 107 ± 1.9 × 107 vs. 3.5 × 106 ± 1.5 × 106 vital cells/g of liver) and viability (79.1 ± 8.4 vs. 37.1 ± 8.9%). Therefore, we gathered hepatocytes from the liver after short warm ischemia with the modified protocol. For these hepatocytes, lactate dehydrogenase activity was lower in media with INS and with DEX than in media with INS+DEX or CTRL; urea concentrations were highest at d 4 for INS+DEX. As well, HNF4A and ALB were more highly expressed in hepatocytes cultured with INS and INS+DEX than in those cultured with DEX or CTRL, and the substitution of DEX suppressed VIM and NFKBIA expression but increased PCK1 expression. The expression of GHR, GHR1A, and IGF1 was suppressed by dexamethasone (DEX and INS+DEX), whereas INS distinctly increased GHR, GHR1A, and IGF1 mRNA expression. Hepatocytes in a sandwich culture showed influenceable GHR expression; this study provides a model that can be used in studies examining factors that influence the expression and signal transduction of GHR in dairy cows.

IFNτ mediates chemotaxis, motility, metabolism and CK18 downregulation in bovine trophoblast cells in vitro via STAT1 and MAPK42/44 signaling.

INTRODUCTION: IFNτ is the ruminant pregnancy recognition signal. In the study we investigated the autocrine influence of IFNτ on bovine F3 trophoblast cells. In detail chemotaxis, motility, metabolism, cell polarisation (CK18; ezrin) and the underlying classical (STAT1) and non-classical (MAPK42/44) signaling pathways were examined. METHODS: Cellular signaling was analysed by densitometric Western blot (STAT1, MAPK42/44, proteinkinase B) and RT-PCR (IFNAR1, -2). Cellular assays were carried out for chemotaxis (agarose spot assay), cell motility (live cell imaging), metabolism (MTT) and cell polarisation (CK18; ezrin). In vivo-produced conceptuses of gestational days (GD) 20-39 underwent immunohistochemistry (CK18; ezrin) to set the in vitro findings (cell polarisation) in proportion to the in vivo situation. RESULTS: IFNτ (10-1000 ng/ml) mediated dose-dependent effects. 10 ng/ml IFNτ induced chemotaxis and motility, whilst 1000 ng/ml led to reduced chemotaxis, motility and a 92-fold activation of MAPK44. Stimulation of cells with 10-1000 ng/ml IFNτ promoted metabolism (1.4-fold), increased the gene expression of IFNAR1/2 (24 h) and downregulated CK18 but not ezrin. All described in vitro effects were significant. Signaling, motility and metabolism could be blocked by specific inhibitors (PD98059, LY294002). CK18 and ezrin expression patterns in the trophoblast of in vivo conceptuses differed depending on GD. DISCUSSION: IFNτ is a major factor for bovine F3 trophoblast cells and mediates a variety of cellular actions ranging from chemotaxis to polarisation. IFNτ exerts its effects via classical (STAT1) and non-classical (MAPK42/44) signaling pathways in a dose-dependent way. We hypothesize that (dose-dependent) IFNτ regulation of the cellular effects could also be essential for bovine elongation and implantation.

Bovine placentomal heparanase and syndecan expression is related to placental maturation.

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 bovine placenta in vivo and in vitro.

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.

Changes in endometrial ezrin and cytokeratin 18 expression during bovine implantation and in caruncular endometrial spheroids in vitro.

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.

Galectin fingerprinting detects differences in expression profiles between bovine endometrium and placentomes as well as early and late gestational stages.

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.

Expression of matrix metalloproteinase (MMP)-2, MMP-14 and tissue inhibitor of matrix metalloproteinase (TIMP)-2 during bovine placentation and at term with or without placental retention.

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-maternal 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(2α) analogue, glucocorticoid, and after elective caesarean sections (each group n = 3). The expression of MMP-14, MMP-2 and of TIMP-2 was examined by real-time-PCR, immunohistochemistry, 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 stroma. 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.

Formation of bovine placental trophoblast spheroids.

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.

Epidermal growth factor (EGF) induces motility and upregulates MMP-9 and TIMP-1 in bovine trophoblast cells.

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.

EGF stimulates proliferation in the bovine placental trophoblast cell line F3 via Ras and MAPK.

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.