Tumor necrosis factor alpha system in the bovine oviduct: a possible mechanism for embryo transport

Active contractile pattern of the oviduct occurs during the periovulatory period for the movement of the gamete/embryo, which is strictly regulated by endocrine and paracrine/autocrine factors. In this review, an involvement of tumor necrosis factor alpha (TNFalpha) in the regulation of cow oviductal contraction is discussed. Oviductal epithelial cells express TNFalpha ligand and it's both receptor types; high expression during the follicular and postovulatory stages, while low expression during luteal stage and thus, TNFalpha system in the cow oviduct is most active during the periovulatory period. The immune cells present in large numbers in the oviduct during the periovulatory period of the estrus cycle, and these cells are also considered as another potential source for the TNFalpha in the oviduct. Using in vitro models, TNFalpha clearly stimulated local production and release of contraction related substances such as prostaglandins (PGs), endothelin-1 (ET-1) and angiotensin II (Ang II). Since these substances have been shown to activate directly the oviductal contraction in vitro, TNFalpha appears to stimulate the oviductal contraction during the periovulatory period and contribute to create an optimal local environment suitable for gamete/embryo transport. In addition, the ability of embryo to act as a source of TNFalpha in the oviduct cannot be excluded. To support this idea, the embryo at 2-4 cells stages indeed express TNFalpha, so that the minute quantities of TNFalpha secreted by the embryo may further acts locally to enhance the production of PG, ET-1 and Ang II in the oviduct, which may result in an active oviductal contraction in the microenvironment around the embryo. This may ensure the embryo to migrate into the uterus at the optimal time.     

Isolation and monolayer culture of bovine oviduct epithelial cells

Oviduct epithelia obtained from 32 cows were cultured. The oviducts were classified into follicular and luteal phases and divided into ampulla and isthmus regions. The epithelial cells were dissociated by enzyme digestion and cultured in plastic dishes with Dulbecco's modified Eagle's medium/Ham's F12 (1:1) containing 10% calf serum. After enzyme treatment, the epithelial suspension showed free ciliated and non-ciliated cells, and cell mass. The non-ciliated cells contained secretory granules in the cytoplasm. The cell mass was composed of ciliated and secretory cells. The cell mass adhered to the dish within 12-24 hr, while the free ciliated cells attached on Day 2 of the culture. The cells grew into confluent monolayers on Day 4. The cell monolayers contained ciliated and non-ciliated cells. The monolayered non-ciliated cells showed a few secretory granules. When the cells were further cultured without subculturing, ciliary activity diminished on Day 5 and was rarely detected on Day 9. When the cells were subcultured on Day 3, ciliary movement was detected on the monolayers for only 2 days. Cell mass that did not adhere to the dish and remained floating in the medium formed ball-like structures on Day 2. Active ciliary beating was observed on the cells that were cultured in the medium supplemented with 10(-5) and 10(-9) M estradiol-17 beta, however, the ciliary activity diminished on Day 5. No difference in the cell growth was observed between the follicular and luteal phases or between the ampulla and isthmus regions.     

Microvascular distribution and vascular endothelial growth factor expression in bovine cystic follicles

The aim of this study was to examine the distribution of microvessels in the theca and the expression of vascular endothelial growth factor (VEGF) in the theca and granulosa of cystic follicles. Paraffin sections of cystic follicles were stained with Bandeiraea simplicifolia-I (BS-I) to visualize the endothelial cells of microvessels. The other sections were immunostained with anti-VEGF antibody. The mRNA expression of VEGF in the theca interna of cystic and healthy follicle was determined by RT-PCR. In the theca interna, cystic follicles with granulosa cells had significantly greater microvessel number density (the number of microvessels per given field) and area (area occupied by microvessels per given area) than healthy follicles in various sizes (<3, 4–8, >9 mm). Loss of granulosa cells from cystic follicles resulted in a similar number density, but significantly smaller area of microvessels in the theca interna. There was no significant difference in the microvessel number density and area of the theca externa between the types of follicle. VEGF protein was expressed in the granulosa and theca interna of healthy and cystic follicles. These results demonstrate that cystic follicles have a highly developed vasculature network in the theca interna, especially in cystic follicles containing granulosa cells. It is also suggested that VEGF is highly expressed in the cystic follicle as well as healthy follicle, which may be associated with advanced vasculature and the accumulation of follicular fluid in cystic follicles.     

Effects of leptin on the release of luteinizing hormone, growth hormone and prolactin from cultured bovine anterior pituitary cells

The effects of leptin on the release of luteinizing hormone (LH), growth hormone (GH) and prolactin (PRL) were studied in cultured bovine anterior pituitary (AP) cells in vitro. The AP cells were obtained from fully‐fed Japanese Black steers and were incubated for 3 h with 10^?13 to 10^?7 mol/L of leptin after incubating in Dulbecco's modified Eagle's Medium for 3 days. Leptin significantly increased the concentration of LH in the culture medium by 45 and 44% at doses of 10^?8 and 10^?7 mol/L, respectively, compared with the controls (P < 0.05). Leptin significantly increased the concentration of GH in the culture medium by 14 and 12% at doses of 10^?8 and 10^?7 mol/L, respectively (P < 0.05). Leptin also significantly increased the concentration of PRL in the culture medium by 26% compared with the controls at a dose of 10^?7 mol/L (P < 0.05). These results show that leptin stimulates the release of LH, GH and PRL by acting directly on bovine AP cells from fully‐fed steers.     

The influence of ketone bodies and glucose on interferon, tumor necrosis factor production and NO release in bovine aorta endothelial cells

Bovine aorta endothelial cells (BAECs) were used to determine the effect of ketone bodies and glucose on in vitro interferon (IFN), tumor necrosis factor (TNF) and nitric oxide (NO) production. BAECs were incubated for 4 and 24h with the ketone bodies: 3.8mmol/l beta-hydroxybutyrate (BHB), 1mmol/l acetoacetate (AcAc) and 5. 2mmol/l acetone (Ac), used separately or in a mixture together with cytokine inducers: Newcastle disease virus (NDV) and lipopolysaccharide (LPS). BHB alone (but not AcAc or Ac) and a mixture of ketone bodies caused a significant decrease in IFN titers induced by NDV and LPS and in TNF titers induced by LPS. Glucose used at concentrations of 5.55, 3.33 and 1.66mmol/l did not influence cytokine production.NO measured by the nitrite content in culture medium was released spontaneously from BAECs. A slight enhancement of NO release was observed after infection of BAECs with NDV; however, treatment with LPS caused inhibition of the release. The mixture of ketone bodies used with NDV or LPS enhanced NO release. However, when cells were incubated in the medium with 1. 66mmol/l glucose (mimicking low plasma glucose level in ketotic cows) a significant decrease in NO release was observed. This enhancing effect of ketone bodies and inhibition by low glucose in the final effect balanced each other, and the amounts of NO released in the medium with 1.66mmol/l glucose and with the mixture of ketone bodies resembled those produced at 3.33mmol/l glucose without ketone bodies. The significance of these effects of ketone bodies and glucose concentrations on cytokine and NO production in the immunity of ketotic cows has been discussed.     

Effect of vascular endothelial growth factor on maturation,fertilization and developmental competence of bovine oocytes

To examine the effect of Vascular Endothelial Growth Factor (VEGF) on the maturation of bovine oocytes, human recombinant VEGF(165) was used in 3 experiments. In Exp. 1, bovine cumulus oocyte complexes (COCs) were matured for 22 hr in modified Synthetic Oviduct Fluid (m-SOF) supplemented with 0 (control) or 5 ng/ml of VEGF. Maturation rate increased (P<0.05) from 78.2% in the control to 90.5% in the VEGF treated group. In Exp. 2, bovine COCs were matured in m-SOF and co-incubated with sperm in modified BO medium, each supplemented with or without 5 ng/ml VEGF. Normal fertilization rate was improved (P<0.05) from 63.0% (control) to 79.8% or 82.3% with VEGF during maturation or both maturation and fertilization. In Exp. 3, bovine COCs were matured the same way as in Exp. 1, then co-incubated with sperm for 6 hr and cultured for 162 hr in m-SOF without VEGF. Cleavage rate and development rate to the 4- to 8-cell stage were examined at 42 hr post-co-incubation and development rate to blastocyst was examined at 162 hr post-co-incubation. Cleavage, the development to the 4- to 8-cell stage and blastocyst rates (82.0%, 70.3% and 45.1%, respectively) were significantly higher (P<0.05) in the VEGF group than those in the control (67.3%, 52.5% and 33.3%, respectively). These results indicate that VEGF has a beneficial effect on the maturation of bovine oocytes.     

Expression of epidermal growth factor receptor and vascular endothelial growth factor in malignant canine epithelial nasal tumours*

Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) signalling pathways play a role in carcinogenesis. Inhibition of EGF receptor (EGFR) and of VEGF is effective in increasing the radiation responsiveness of neoplastic cells both in vitro and in human trials. In this study, immunohistochemical evaluation was employed to determine and characterize the potential protein expression levels and patterns of EGFR and VEGF in a variety of canine malignant epithelial nasal tumours. Of 24 malignant canine nasal tumours, 13 (54.2%) were positive for EGFR staining and 22 (91.7%) were positive for VEGF staining. The intensity and percentage of immunohistochemically positive neoplastic cells for EGFR varied. These findings indicate that EGFR and VEGF proteins were present in some malignant epithelial nasal tumours in the dogs, and therefore, it may be beneficial to treat canine patients with tumours that overexpress EGFR and VEGF with specific inhibitors in conjunction with radiation.     

Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor associated with tumor cell proliferation in canine cutaneous squamous cell carcinomas and trichoepitheliomas

The expression of 5 markers associated with angiogenesis was studied in canine squamous cell carcinomas (SCCs) (n = 19) and canine trichoepitheliomas (TCPs) (n = 24). SCCs were assigned histologic grades, and tissue sections from both tumor types were immunohistochemially stained for the expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR-2), as well as intratumoral microvessel density (iMVD), tumor proliferation index (PI), and tumor apoptotic index (AI), using antibodies against VEGF, VEGFR-2, von Willebrand's factor, Ki-67 antigen, and the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate end-labeling method (TUNEL), respectively. VEGF and VEGFR-2 were detected in 17/19 (89.4%) and 19/19 (100%) SCCs and in 17/24 (70.8%) and 20/24 (83.3%) TCPs, respectively. In SCCs, there was substantial correlation between histologic grade and PI (r = 0.51); and moderate correlation between VEGF and histologic grade (r = 0.43), VEGFR-2 and histologic grade (r = 0.47), VEGF and PI (r = 0.47), and VEGFR-2 and PI (r = 0.47) (Spearman rank correlation coefficient). In TCPs, there was substantial correlation between VEGF and PI (r = 0.51) and a moderate correlation between VEGFR-2 and iMVD (r = 0.36). The median iMVD of SCCs (15.5) was significantly higher than the median iMVD of TCPs (9.05) (P value < .05). It was concluded that VEGF and VEGFR-2 may promote tumor cell proliferation in TCPs and SCCs. An autocrine pathway for VEGF probably operates in canine SCCs and TCPs, as VEGF and VEGFR-2 expression was found in most tumors and was associated with evidence for tumor cell proliferation.     

Adherence of Haemophilus somnus to tumor necrosis factor-alpha-stimulated bovine endothelial cells in culture.

Vascular thrombosis and tissue infarction is a principal lesion in Haemophilus somnus septicemia known also as thrombotic meningoencephalitis. This study was undertaken to examine whether tumor necrosis factor-alpha (TNF-alpha) can influence the adherence of H. somnus to cultured bovine aortic endothelial cells (BAEC). Confluent BAEC were exposed to 0-100 nM of human recombinant TNF-alpha for 12-48 h. Suspensions of different strains of H. somnus (approximately 1.5-3 x 10(8) labelled with [methyl-3H]-thymidine, were added to BAEC and incubated for 1.5 h. Initial studies with one pathogenic (P) strain and one non-pathogenic (NP) strain revealed that both strains adhered to normal endothelial cells but minimally to subendothelial matrix remaining after removal of BAEC. Adherence to BAEC was reduced by an excess of unlabelled H. somnus of the same strain. Adherence was enhanced for both strains by exposure of BAEC to TNF-alpha in a manner that increased with TNF-alpha concentration and with duration of exposure to TNF-alpha prior to addition of bacteria. A survey of adherence of six live P strains and six NP strains demonstrated considerable variation but no difference in adherence between P and NP strains to normal or to TNF-alpha-stimulated BAEC. However, TNF-alpha consistently increased adhesion of each strain to BAEC. Both P and NP strains caused more severe cytotoxic changes in TNF-alpha-treated BAEC. Tumor necrosis factor-alpha also increased adhesion of formalin-killed bacteria of P and NP strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active immunization of heifers against luteinizing hormone-releasing hormone, human chorionic gonadotropin and bovine luteinizing hormone

Seventy crossbred heifers were allotted randomly to 10 treatment groups. Treatments consisted of active immunization against ovalbumin (OV) conjugates of luteinizing hormone-releasing hormone (LHRH), human chorionic gonadotropin (hCG) and bovine luteinizing hormone (bLH) with each of three adjuvants. The adjuvants were complete Freund's adjuvant (CFA), M103(6) and 6VR6. Control animals were immunized against OV alone using CFA. Bulls were placed with the heifers following immunization to allow comparison of pregnancy rates between groups. Blood samples were collected weekly for 14 wk to determine antibody concentrations. Significant levels of circulating LH or LHRH antibodies were detected in heifers immunized with each of the hormone conjugates. Complete Freund's adjuvant was the most effective for stimulating antibody response to these antigens; however, M103 was equally effective when used with bLH or hCG conjugates. None of the heifers in the bLH-OV-CFA, bLH-OV-M103 or LHRH-OV-CFA immunization groups was pregnant at slaughter, whereas 71% of the OV-CFA control heifers were pregnant. Fertility suppression may be achieved in the bovine by active immunization against any of these three hormone conjugates. However, the duration of this study (8 wk after immunization) does not allow evaluation of the duration of effectiveness of each of the treatments.     

The inhibitory effect of interferon gamma and tumor necrosis factor alpha on intracellular multiplication of Neospora caninum in primary bovine brain cells

Primary culture of bovine brain cells was examined for its susceptibility to Neospora caninum infections, and this model was used to investigate the effects of bovine interferon gamma (IFN-gamma) and tumor necrosis factors alpha (TNF-alpha) on tachyzoite growth. Tachyzoites of N. caninum grew well in this culture, and tachyzoite growth in astroglia and microglia were confirmed by immunocytochemical staining. IFN-gamma inhibited the tachyzoite growth, and this inhibition was not reversed by the addition of nitric oxide antagonist. TNF-alpha, to a lesser extent, also inhibited the tachyzoite growth. Th-1 type cytokines may play an important role in host defense mechanisms in N. caninum infection.     

Expression and characterization of a recombinant soluble form of bovine tumor necrosis factor receptor type I

A recombinant soluble bovine tumor necrosis factor receptor type I (sboTNF-RI) was expressed in the methylotrophic yeast Pichia pastoris and evaluated for its ability to inhibit bovine tumor necrosis factor alpha (TNF-alpha) cytotoxicity. A cDNA encoding the extracellular domain of bovine TNF-RI was placed under the control of the powerful and tightly regulated alcohol oxidase1 (AOX1) gene promoter of the pPICZa A vector and the resulting construct integrated into the 5' region of the alcohol oxidase genes of GS115 and KM71 strains of Pichia. Soluble bovine TNF-RI was secreted into the medium following induction of the AOX1 gene promoter with methanol, and purified to greater than 95% purity by ion-exchange chromatography. In in vitro assays, the purified recombinant sboTNF-RI will block the cytolytic activity of bovine TNF-alpha on WEHI 164 cells clone 13 by 50% when used at a concentration of 170 microg/ml, and by nearly 90% when used at a concentration of 310 microg/ml. Results of this study suggest that recombinant sboTNF-RI may have therapeutic value as a TNF inhibitor in cattle with coliform mastitis.     

The luteotrophic function of galectin-1 by binding to the glycans on vascular endothelial growth factor receptor-2 in bovine luteal cells

The corpus luteum (CL) is a temporary endocrine gland producing a large amount of progesterone, which is essential for the establishment and maintenance of pregnancy. Galectin-1 is a β-galactose-binding protein that can modify functions of membrane glycoproteins and is expressed in the CL of mice and women. However, the physiological role of galectin-1 in the CL is unclear. In the present study, we investigated the expression and localization of galectin-1 in the bovine CL and the effect of galectin-1 on cultured luteal steroidogenic cells (LSCs) with special reference to its binding to the glycans on vascular endothelial growth factor receptor-2 (VEGFR-2). Galectin-1 protein was highly expressed at the mid and late luteal stages in the membrane fraction of bovine CL tissue and was localized to the surface of LSCs in a carbohydrate-dependent manner. Galectin-1 increased the viability in cultured LSCs. However, the viability of LSCs was decreased by addition of β-lactose, a competitive carbohydrate inhibitor of galectin-1 binding activity. VEGFR-2 protein, like galectin-1, is also highly expressed in the mid CL, and it was modified by multi-antennary glycans, which can be recognized by galectin-1. An overlay assay using biotinylated galectin-1 revealed that galectin-1 directly binds to asparagine-linked glycans (N-glycans) on VEGFR-2. Enhancement of LSC viability by galectin-1 was suppressed by a selective inhibitor of VEGFR-2. The overall findings suggest that galectin-1 plays a role as a survival factor in the bovine CL, possibly by binding to N-glycans on VEGFR-2.     

Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 in canine simple mammary gland adenocarcinomas

The expression of 5 markers associated with angiogenesis, proliferation, and apoptosis was studied in 26 canine simple mammary gland adenocarcinomas (SMGAs). The adenocarcinomas were graded histologically, and tissue sections were immunohistochemically stained for the expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), intra-tumor microvessel density, and tumor proliferation (PI) using antibodies against VEGF, VEGFR-2, von Willebrand factor, and Ki-67 antigen, respectively. Apoptotic indices (AI) were determined by an apoptosis assay. Markers VEGF and VEGFR-2 were detected in 96% and 100% of SMGAs, respectively. A high correlation between histologic grade and PI (r = 0.73), a moderate correlation between VEGF and histologic grade (r = 0.33), and between VEGF and PI (r = 0.42) were found. There was a significant difference in median PI among the 3 histologic grade groups (r < 0.05). Vascular endothelial growth factor may stimulate tumor cell proliferation through an autocrine loop, since VEGF and VEGFR-2 were expressed in most tumors.     

Angiogenic activity of swine granulosa cells: effects of hypoxia and vascular endothelial growth factor Trap R1R2, a VEGF blocker

The possible role played by hypoxia and vascular endothelial growth factor (VEGF) in the regulation of follicular angiogenesis was studied in a three-dimensional fibrin gel model. Granulosa cells from follicles >5mm were subjected to normoxia (19% O2), partial (5% O2) or total (1% O2) hypoxia and their culture media were collected and used to stimulate porcine Aortic Endothelial Cells (AOC) included in the fibrin matrix. A suspension of AOC on microcarrier beads was pipetted in a fibrinogen solution (1 mg/ml PBS) before the addition of 1250 IU thrombine (250 microl) to catalize the gel formation. Granulosa cell conditioned media were tested in the presence or absence of VEGF Trap R1R2 (150 ng/ml), a potent VEGF inhibitor, that had its efficacy tested by adding VEGF (100 ng/ml) to AOC culture. Endothelial cell proliferation was measured at 48, 96, 144, 192 h by means of Scion Image Beta. A significant (p < 0.01) increase of AOC proliferation at each time of measurement was induced by culture media from granulosa cells subjected to partial (except at the end of the first 48 h) and total hypoxia compared to control and normoxia conditions, and by VEGF. VEGF Trap significantly (p < 0.01) inhibited the stimulatory effect of media conditioned by granulosa cells cultured in hypoxic conditions. These data suggest that hypoxia stimulates angiogenic activity of granulosa cells possibly by means of VEGF which could represent the main effector in promoting endothelial cell proliferation.     

The effects of luteinizing hormone as a suppression factor for apoptosis in bovine luteal cells in vitro

The fate of the corpus luteum, a transient endocrine gland formed and degraded during an oestrous cycle, is decided by various physiological factors, such as luteinizing hormone (LH). As a stimulator of progesterone, LH is known to maintain corpus luteum functional and structural integrity by inhibiting apoptosis, a programmed cell death. Therefore, we aim to investigate its action during the mid-luteal phase hypothesized that LH suppresses the death mechanism of bovine luteal steroidogenic cells (LSC) by analysing the expression of proteins involved. Cultured bovine LSC obtained from corpus luteum were treated for 24 hr with recombinant TNF and IFNG in the presence or absence of LH. The result showed that LH proved to have a protective effect by increased cell viability (p < .05) and prevented DNA fragmentation (p < .05), as demonstrated by the WST-1 colorimetric assay and TUNEL assay. Expression analysis of mRNA and protein levels showed that LH altered the expression of BCL2 (p < .05), CASP3 (p < .05), FAS (p < .05), and BAX (p < .05) to support cell survival. In conclusion, our study suggests that LH prolongs the corpus luteum life span through the anti-apoptotic mechanism by increasing cell viability and suppressing apoptosis-related genes and protein expression.