The purpose of the present study was to investigate the stability of vascular endothelial growth factor (VEGF) in plasma samples and the influence of ovarian hyperstimulation on systemic levels of VEGF. Stability assays for VEGF in plasma samples revealed significant increases following even short incubations of samples at room temperature (< or = 2 h, p < 0.001). To investigate a possible impact of controlled ovarian hyperstimulation (COH) on peripheral VEGF levels, serial blood collection over one menstrual cycle was performed in unstimulated as well as in gonadotropin-stimulated cycles for in vitro fertilisation/embryo transfer (IVF/ET) (10 women each). Peripheral levels for VEGF were significantly higher in gonadotropin stimulated cycles as compared to non-stimulated cycles (p < 0.001). There was no significant difference between follicular phase and luteal phase levels in either group. VEGF levels tended to correlate with the number of follicles detected by vaginal sonography prior to oocyte aspiration (p = 0.051). In conclusion, VEGF levels are elevated in gonadotropin-stimulated IVF/ET cycles as compared to natural cycles.

Vascular endothelial growth factor (VEGF) is a potent stimulator of vascular proliferation and permeability. Ovarian granulosa cells have been identified as a major source of the cytokine and r-hCG was able to stimulate VEGF mRNA expression in vitro. In this study we have investigated the immediate effect of ovulation induction with hCG on peripheral VEGF levels in 6 women with primary infertility enrolled in the IVF/ET program. The patients underwent a 24-hour continuous blood withdrawal with sampling intervals of 15 minutes starting from 5 hours before ovulation induction with 10.000 IU hCG. Ovulation induction with hCG had no significant immediate effect on mean peripheral VEGF levels. However, VEGF plasma levels did exhibit significant episodic fluctuations with rapid increases every 90-120 minutes without any relation to circulating hCG levels. Taken together, the results of this study suggest that VEGF is released episodically and that systemic VEGF levels are not acutely altered by ovulation induction with hCG.

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Four myeloma cell lines and four hybridomas were examined for the presence of mycoplasmas by the fluorescence method using bisbenzimide 33258 (Hoechst). The cell suspensions under examination were cultivated for 3 days together with Vero cells as indicators and then examined by fluorescence microscopy. Mycoplasma contamination was detected in 2 myeloma lines. The results were confirmed by culture methods and by scanning electron microscopy. Mixed cultures, bisbenzimide, scanning electron microscopyFluorescence (Chen, 1977) is often used for the detection of mycoplasmas and other contaminant microorganisms in cell cultures. The method is based on chemical bound of the fluorochrome bisbenzimide 33258 (Hoechst) to DNA. The fluorescence microscopy visualizes not only nuclear' DNA of the examined cells, but also DNA present in the contaminant microorganisms.In our laboratory, routine examinations of monolayer cell cultures using fluorescence method' by Chen (1977), modified by Machatkova et al. (1986) are performed. In correctly stained preparations, only the nuclei of examined cells and organellae of contaminant microorganisms. containig DNA show bright, greenish-yellow fluorescence. Weak fluorescence, sometimes observed in the cytoplasm of examined cells is considered to be an artifact. Mycoplasmas and bacteria are the' most frequent contaminant microorganisms, but we have demonstrated that mould and yeast: contaminations are also detectable by the fluorescence method (Fischer et al. 1989).While the fluorescence method is reliable and, above all, much faster than culture methods for the examination of monolayer cell cultures, it cannot be used for direct examinations of semi-sus-. pension cultures of myeloma cells and hybridomas. Staining with bisbenzimide results in a very' intensive fluorescence of myeloma and hybridoma cells and fluorescence of the much smallercontaminant microorganissms can be overlooked easily (Smirnova and Fridlyanskaya 1985) .. Regarding the threat of mycoplasma contamination of myeloma cell lines and hybridomas maintained in our laboratory (Granatova et al. 1988), we tried to detect mycoplasms in these semi-sus--pension cultures by the fluorescence method using Vero cells as indicators. Materials and MethodsMyeloma cells and hybridomas (Table 1) were cultured in an open system in H~aeus thermo-· stat at 37°C in an atmosphere containing 5 % COg. The medium RPM! 1640 (USOL, Prague)· supplemented with nonessential amino acids (USOL, Prague) was used. The content of an ampule was added to 1 OOOml medium containing· 2 mM/ml L-glutamine, 2 mM/ml· sodium pyruvate, 40 I'g/ml gentamycine and 10 % bovine fetal serum (Cooperative farm, Sokolnice). Before reseeding~.the cells were released from the walls of Mueller flasks by agitating. The reseeding ration was 1 :3., Vero indicator cells (ATCC.CCL 81), isolated from kidney of Cercopithecus aethiops, were cul-

Fischer 0., M. Machatkova, L. Pecuchova, D. Zendulkova and Z. H 0 fin 0 va: Establishment and Characteristics ofa Cell linefrom FetalBovine Thyroid (FBIT). Acta vet. Bmo, 63, 1994: 81-87.Characteristics of a cell line from fetal bovine thyroid were tested in the 150th passage. The line was adapted to growth in the minimal essential medium supplemented with 10 % bovine fetal semm and it had an epithelioid morphology. After seeding of lxlO" cellslml, the culture density was higher than lxl()f> cellslrnl on day 6. The FBTY line was heteroploid. The average number of chromosomes was 48.8 ± 10.0 and only 1 % of mitoses had a diploid number of chromosomes (2n = 60). Species specificity (Bos taurus) was confirmed by indirect immunofluorescence. Besides pronounced fluorescence with rabbit antisera against bovine proteins, weak reactions with antisera against ovine, caprine and swine proteins were observed. The FBTY line was susceptible to two strains of parainfluenza 3 virus, six strains of infectious bovine rhinotracheitis virus and bovine adenoviruses of the serovars I, 2, 3, and 8. • line ECTC derived from fetal bovine thyroid gland which was, in passage 150, susceptible to all known serovars of bovine adenoviruses, including members of the subgroup II which are difficult to replicate. Mitosis, indirect immunofluorescence, bovine adenovirusIn 1981, Marie Machatkova in the Veterinary Research Institute, Bmo, established a cell line from fetal bovine thyroid gland and the results are described in this paper.The characteristics of this cell line (FBTY) were tested in passage 150.Replications of bovine adenoviruses in FBTY and some other bovine cell cultures were compared by P e c uchova (1993). Materials and Methods Establishment and cultureThyroid gland of a nine-month-old male bovine fetus was collected under sterile conditions immediately after exsanguination (Machatkova and PospBil 1975) and transported to the laboratory in minimal essential medium (MEM SEVAC, Institute of Sera and Vaccines, Praha) supplemented with 200 I. U. of penicillin and 200 \lg of streptomycin per m!. Tissue fragments were washed in the medium and cultured in 60 mm plastic Petri dishes in 5 ml of the EPL SEVAC medium (Institute of Sera and Vaccines, Praha) containing 10 % bovine fetal serum, 100 I. U. of penicillin and 100 \lg of streptomycin per m!. The cells were cultured at 37°C in an atmosphere containing 5 % CO 2 , The EPL was replaced after 72h by a 1: 1 mixture of EPL+MEM subsequently changed every other day.As soon as the attached fragments were surrounded by growing cells, the cells were released by 0.02 % versene with 0.25 % trypsin Difco (1:250), reseeded into Mueller's flasks and cultured further in MEM with 10 % bovine fetal serum, 100 I. U. of penicillin and 100 \lg of streptomycin per ml in 300 ml and 500 rnl Roux flasks, Legroux flasks and Mueller's flasks.In passage 85, the cells were frozen and kept for 8 years (1981)(1982)(1983)(1984)(1985)(1986)(1987)(1988)(1989) in liquid nitrogen. When reseeded after passage 85,...