Expressions of VEGF and its receptors in rat corpus luteum during interferon alpha administration in early and pseudopregnancy

It is accepted that angiogenesis plays an important role in the development of the corpus luteum (CL) and is probably necessary for normal lutein cell function. A number of drugs currently being tested in clinical trials as possible angiogenesis inhibitors were not originally developed with the intention of suppressing tumor angiogenesis. Interferon alpha (IFN-alpha) is one of the notable examples of such 'accidental angiogenesis inhibitors' and daily administration of IFN-alpha is known to suppress tumor growth, tumor vascularization, and down-regulation of various growth factors. We investigated the effects of IFN-alpha treatment on the expression of vascular endothelial growth factor (VEGF), and its receptors KDR and Flt-1, and CD34 in CL during the first week of pseudopregnancy and pregnancy in hormonally induced rat ovaries by immunohistochemistry and Western blot techniques. Basal body temperatures of the drug-treated rats, as an indicator of treatment effect, were determined daily and were increased significantly when compared to controls (38.03 +/- 0.18 vs. 36.6 +/- 0.1 degrees C), respectively. The effect of IFN-alpha treatment was minimal when the entire week was evaluated, however, the expression of VEGF decreased at 3rd, 5th, and 7th days of both pregnancy and pseudopregnancy, when compared to the 1st day, whereas there was not a such alteration in the untreated rats regarding these days. The daily subcutaneous administrations of 672.500 U IFN-alpha2b had minimal effects on the expressions of VEGF, and its two receptors KDR and Flt-1 in either pregnant or pseudopregnant corpora lutea utilizing HSCORE.     

Location of cell cycle regulators cyclin B1, cyclin A, PCNA, Ki67 and cell cycle inhibitors p21, p27 and p57 in human first trimester placenta and deciduas

Although placental development and implantation depend on the coordination of trophoblast proliferation, differentiation and invasion, little is known about the cell cycle regulators that govern the control of these events. The hypothesis that the coordinated expression of cell cycle progression and inhibition factors will determine whether cytotrophoblasts proliferate or undergo cell cycle arrest or cell cycle exit allowing subsequent differentiation was tested. The cell cycle promotors cyclin A, cyclin B1, PCNA, Ki67 and the cell cycle inhibitors p21, p27 and p57 were immunolocalized in tissue sections of first trimester pregnancies (weeks 6 and 9–12). Double staining with cytokeratin 7 allowed unambiguous identification of extravillous cytotrophoblast (EVT) in the decidua. Villous cytotrophoblasts were immunolabelled for Ki67 and cyclin A but only few were stained with anti-cyclin B1. The syncytiotrophoblast was devoid of immunoreactivity for any of the cell cycle progression factors. It expressed especially p21, whereas p27 and p57 were predominantly found in villous cytotrophoblasts. PCNA, Ki67, cyclin A and cyclin B1 were immunolocalized in proximal and distal EVTs of anchoring villi and in EVT which had invaded the upper decidual segments. All EVTs strongly expressed p27 and p57, but not p21. These data clearly suggest different functions for p21, p27 and p57 in placental development with distinct roles for p21 and p57 in syncytiotrophoblast and EVT differentiation, respectively. p27 appears to be involved in both the processes. The results may also challenge the concept of differential mitotic activity in the proximal and distal parts of the first trimester cytotrophoblast cell column, but more functional studies are clearly needed. The presence of p27 and p57 in EVT cells, which invade the deciduas deeply, may account for the loss of mitogenic potential of these cells.     

Zoledronic acid is synergic with vinblastine to induce apoptosis in a multidrug resistance protein-1 dependent way: an in vitro study

We have explored the action of zoledronic acid, which has an apoptotic effect and is used as an agent for treating skeletal metastases and osteoporosis, in the presence of vinblastine, and whether this effect is associated with MRP-1 (multidrug resistance protein-1) expression. HEK (human embryonic kidney) 293 cells were transfected to form the multidrug resistant cell line designated 293MRP (MRP-1 expressing HEK293 cells). Both lines were treated with varying concentrations of vinblastine and zoledronic acid. Apoptosis was determined by the TUNEL (deoxyuridine triphosphate nick end-labeling) method. The type of treatment, MRP-1 expression status, and the type of treatment with respect to MRP-1 expression status significantly affected (P < 0.001) the degree of apoptosis. The largest increase in cytotoxicity was noted in HEK293 cells, when 100 micromol zoledronic acid was added to 4 microg/ml vinblastine (an increment of 80.3%, P < 0.001). This preliminary work shows that zoledronic acid acts synergistically with vinblastine to induce apoptosis in an MRP-1 dependent way.     

Human sperm maintain their shape following decondensation and denaturation for fluorescent in situ hybridization: shape analysis and objective morphometry

The relationship between abnormal sperm morphology and chromosomal aberrations has been of interest. Thus far, however, studies have focused on frequencies of sperm with either abnormal morphology or aneuploidies in semen samples, not on detection of individual spermatozoa exhibiting both abnormal morphology and aneuploidy. To assess the feasibility of simultaneous evaluation of both attributes in an individual sperm cell, we investigated whether sperm shape is preserved after decondensation and denaturation, procedures that are required for fluorescent in situ hybridization (FISH). On 21 slides, 395 sperm were fixed, photographed, and then digitized by the computer-assisted Metamorph morphometry program for individual evaluation before decondensation. To establish whether sperm of various shapes would behave in similar manners, the cells were also classified, according to their head shapes, into symmetrical (n = 115), asymmetrical (n = 115), irregular (n = 115), and amorphous (n = 50) categories. Following decondensation and subsequent denaturation, sperm that had been photographed initially were relocalized and digitized for morphometry. Head area, perimeter, long axis, short axis, shape factor, and tail length were evaluated in each of the 395 sperm in both the native and decondensed states. After the decondensation and denaturation protocol of the FISH procedure, the sperm exhibited a proportional increase in dimensions as compared to their original sizes. Their initial shapes were preserved with high fidelity whether the sperm were in the symmetrical, asymmetrical, irregular, or amorphous categories. Hybridization with the chromosome probes had no further effect on sperm shape or size. We provide images to demonstrate how these findings facilitate studies about the relationship between sperm shape and chromosomal content or aberrations in individual spermatozoa.     

Localization of vascular endothelial growth factor in the zona pellucida of developing ovarian follicles in the rat: a possible role in destiny of follicles

There is increasing evidence that in many species angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), may have important roles in folliculogenesis. The aim of this study is to determine the localization of VEGF and its receptors, Flt-1 and KDR, and bFGF expression in the rat ovary and to evaluate their distributions throughout the different follicular stages. Out of 20 virginal female rats, 10 were studied during the natural ovarian cycle without any ovulation induction. The other 10 were superovulated and their ovaries were studied by western analysis and immunohistochemistry. Granulosa cells (GC) and oocytes of primordial follicles were negative for VEGF. In early primary follicles, VEGF was present in the oocyte but its immunoreactivity was weak, while newly developing zona pellucida (ZP) of primary follicles was negative for VEGF. Subsequently, with the commencement of antral spaces between GC of the secondary follicle, ZP of some secondary follicles became strongly positive for VEGF, forming a continuous ring around the oocyte. In preovulatory mature follicles granulosa and theca interna (TI) cells showed a weak immunoreactivity for VEGF. Western blot analyses have also demonstrated that VEGF, a 26-kDa protein, was present in follicles. Moreover, in ovulated cumulus–oocyte complex we observed a halo-like immunoreactivity of VEGF around the fully mature oocyte. The immunoreactivity for Flt-1 and KDR receptors in growing follicles was mostly limited to GC and TI cells. Anti-bFGF did not exhibit any immunoreactivity in ZP of follicles at any stage. Its expression was weak in GC of the follicles at different stages, whereas, it could be localized to some extent in the blood capillaries of TI of antral follicles and in blood vessels localized in the stroma. Interestingly, VEGF immunoreactivity in the ZP of some secondary follicles is very striking. Accordingly, the possibility that VEGF may be an important regulatory molecule for the dominant follicle selection or atresia should be considered.     

Meconium enhances platelet-activating factor and tumor necrosis factor production by rat alveolar macrophages

Meconium aspiration syndrome (MAS) frequently results in inactivation of surfactant, persistent pulmonary hypertension (PPHN) and respiratory failure among newborn infants. Inflammation and inflammatory mediators play an important role in MAS. Since alveolar macrophages are thought to be very important cells in the pathogenesis of various inflammatory diseases, we evaluated whether meconium could stimulate rat alveolar macrophages to generate platelet-activating factor (PAF) and tumor necrosis factor (TNF)-alpha in vitro. We also examined the response to A23187 (calcium ionophore), 1-0-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (synthetic PAF) and dexamethasone on meconium-induced release of PAF and TNF-alpha. PAF and TNF-alpha concentrations from supernatant fluid were measured after high-performance liquid chromatography purification by specific radioimmunoassay, and TNF-alpha concentrations were determined by using an enzyme-linked immunosorbent assay. Our results showed that alveolar macrophages exposed to meconium could enhance PAF and TNF-alpha production in a dose (0.1, 1, 5 and 10%, P<0.01)-dependent way. In the presence of A23187, the capability of meconium to stimulate PAF production was further enhanced in the supernatant fluids. Furthermore, treatment with synthetic PAF significantly increased the generation of TNF-alpha in response to meconium. On the other hand, dexamethasone effectively inhibited both PAF and TNF-alpha production stimulated by 5% meconium (P<0.01, P<0.01; respectively). We suggest that alveolar macrophages and PAF, TNF-alpha play an important role in the pathogenesis of lung injury and severe complications in MAS. Furthermore, the protective effect of glucocorticoids in MAS could be due, at least in part, to a suppression of PAF and TNF-alpha generation.     

Do glucose transporters have other roles in addition to placental glucose transport during early pregnancy?

Human placenta regulates the transport of maternal molecules to the fetus. It is known that glucose transport occurs via glucose transporters (GLUTs) in the feto–placental unit. Data on the expression of GLUTs during implantation are very scarce. Moreover, the question of how the decidual leukocytes obtain the energy for their activation during implantation mechanism is still under investigation. We studied the distributions of GLUT1, GLUT3, and GLUT4 in tissue sections of first trimester pregnancies the human maternal–fetal interface. GLUT1 was present in apical microvilli of the syncytiotrophoblast, in cytotrophoblast, and in vascular patterns of the villous core, whereas GLUT3 was localized in cytotrophoblasts of placental villi and in some fetal endothelial cells. Moreover, the proliferating cells of the proximal cell columns were also immunopositive for GLUT1 and GLUT3. We did not observe any positive immunoreactivity for GLUT4 in placental and decidual tissues. Essentially, GLUT3 and also to some extent GLUT1 was present in maternal leukocytes and platelets. In conclusion, our results suggest that the glucose taken up via GLUT1 and GLUT3 from the maternal circulation might not only be needed for placental functions but also for successful implantation by trophoblast invasion, proliferation and also by having a role to support energy for maternal leukocytes.     

In vitro pollen germination in Hypericum perforatum L. and Hypericum rumeliacum Boiss

In vitro pollen germination and pollen tube growth investigations are valuable tools used in identification of the effects of environmental factors and genotypic differences on pollen viability, pollen germination and tube elongation. In this study pollen viability, in vitro pollen germination capacity, abnormality ratios and tube length in germinated pollens of Hypericum perforatum L. and H. rumeliacum Boiss. were investigated. Both of these species has spheroid-shaped and tricolporate pollen grains. The diameters of Hypericum perforatum and H. rumeliacum pollens were found as 24 +/- 3 microm and 19 +/- 2 microm, respectively. Pollen viability of H. perforatum and H. rumeliacum was found as 83% and 72%, respectively. The germination percentages were found as 12.85% for H. perforatum and 64.42% for H. rumeliacurm. Tube lengths in germinated pollens of both taxa were measured approximately as 95.25 +/- 38 microm in H. perforatum and 165.92 +/- 53 microm in H. rumeliacium 4 h after inoculation. In germinated pollen grains of H. perlbratum and H. rumeliacumn abnormality percentages were determined as 13.23% and 43.97%, respectively. In germinated pollens of these two species, highly significant (P < 0.00001) differences in in vitro germination percents and abnormality percents were observed. Abnormalities such as swollen tube tip, branched tube, spiralled tube and excessive tube formation were observed in pollen tubes. The results of this study showed that there were obvious differences in pollen germinability between these two species growing under the same environmental conditions.     

Evaluation of Two ELISA and Two Indirect Hemagglutination Tests for Serodiagnosis of Pulmonary Hydatid Disease

To establish a definite diagnosis for pulmonary hydatid disease, combination of radiology and serology is useful. In this study, 19 preoperative sera from patients with surgically confirmed pulmonary hydatidosis, 40 sera from patients with other parasitosis and pulmonary diseases, and 20 sera from healthy donors were evaluated using 4 different serological tests, i.e., the commercial ELISA (ELISA-kit) test, the ELISA (ELISA-lab) test prepared in our laboratory, the commercial indirect hemagglutination assay kit (IHA-kit) test, and the IHA test using sensitized sheep red blood cells with tannic acid (IHA-TA). The ELISA-kit was the most sensitive (84.2%) and the most specific test (100.0%). The ELISA-kit also demonstrated the highest positive (100.0%) and negative (95.2%) predictive values. The sensitivity of the ELISA-lab test, that we prepared, was found to be 73.6%, whereas the IHA-kit test and the IHA-TA test were found to be 73.6% and 68.4%, respectively. The specificity of these tests was 96.6%, 98.3%, and 83.3%, respectively. When all 4 tests were assessed together, it was found that the sensitivity had risen to 94.7%. When the ELISA-kit was assessed with the IHA-kit and IHA-TA together, it was found that the sensitivity was 89.5% and 84.2%, respectively. Likewise, the combination of the ELISA-lab and IHA-kit or IHA-TA allowed us to achieve a sensitivity of 84.2% in cases of pulmonary echinococcosis. In conclusion, the diagnosis would be imminent if least 2 tests were applied together.