A new method to estimate 3D cell parameters from 2D microscopy images.

Optical microscopy has been a basic and standard technique in cell biology research for decades. Microscopy techniques function well for thin, optically transparent cultures and allow for the imaging of thicker biological specimens. There is no better method of in vitro cell observation and analysis, hence microscopic techniques are extensively used and constitute an optimal tool for cell culture studies. This paper proposes an original methodology of optical microscopy data processing based on the phase contrast technique during cell culture monitoring. By exploiting images recorded during cell proliferation, a surface reconstruction was performed based on assumption, it can be considered that the local brightness of the image depends on the cells' thickness and thus the obtained results can be interpreted in the form of a surface that represents a three-dimensional structure, which allowed for a quantitative description of the cell evolution. The 3D data obtained enabled the investigation of parameters describing the morphology of the cells and the topology of their proliferation. These parameters included cell sizes in plane but also in the direction perpendicular to it, cell volume changes, their spatial distribution, as well as anisotropy and directivity. The method presented provides data carrying information similar to that obtained using a holographic microscope, e.g. A HoloMonitor (Phase Holographic Imaging PHI Inc.), or from confocal scanning microscopy with the "z-stack" mode. The techniques of bright field or phase contrast cell observation are, however, much cheaper, and widely available when compared to holographic microscopy, for instance. Besides, these also enable monitoring of cell activity over time, i.e. the study and quantitative description of dynamic changes in the cells. The proposed approach uses generally available free tools such as ImageJ software with BoneJ and Particle Analyzer plugins. The methodology is suitable for even a basic microscope, it can be easily implemented as a script, and thus data processing can be significantly shortened, the methodology can be automated, and also applied for data processing in real time.

Effect of sperm subpopulation's kinetics on human fertilization in vitro.

The present study was carried out to investigate the relationship between sperm subpopulation kinetics on in vitro fertilization rate. The ability of human sperm to achieve fertilization oocytes was investigated in relation to particular motility parameters obtained on a computer aided sperm analysis system base. Analysis covers velocity straight linear (VSL), cross beat frequency (CBF), lateral head displacement (LHD) and homogeneity of progressive motility velocity (HPMV) of fresh semen and semen after density gradient selection. Investigation was based on sperm samples from 82 infertile couples undergoing IVF. Two subpopulations were extracted from each sample using the clustering method with respect to VSL parameter: a slow and rapid one. Comparison of obtained results before and after selection shows no significant change of subpopulations percentage. However, this method of selection strongly influences motility parameters of both subpopulations. There was found a positive correlation for VSL, LHD and HPMV and a negative correlation for CBF parameters found in slow fraction of fresh semen and percentage of fertilized oocytes. On the other hand, rapid subpopulation parameters for fresh semen and parameters found for both subpopulations in semen after selection did not correlate with one. This means that information of slow sperm subpopulation kinetics carries important prognostic value of IVF success. Since the current prognosis factors ignore motility parameters of slow sperms, our results show the importance of such an analysis.

Predictive value of selected sperm parameters for classical in vitro fertilization procedure of oocyte fertilization.

A proportion of fertilized oocytes during classical in vitro fertilization (IVF) procedure was analysed depending on the following factors: number of mature oocytes, seminological criteria such as sperm morphology in raw semen and after its selection in a density gradient (six structural defects of a male gamete were taken into consideration), sperm concentration, motility parameters according to World Health Organization criteria and the functional tests: hypo-osmotic swelling assay and acrosomal reaction induced by calcium ionophore. Evaluation of DNA content in sperm by image cytometry and determination of malonyldialdehydes in seminal plasma were also performed. Seventy-nine semen samples from patients undergoing IVF were assessed. Apart from significant correlations obtained for selected semen parameters and proportion of fertilized eggs, logistic regression analysis showed that the best predictive factors for oocyte fertilization were normal morphology of sperm before and after gradient selection, grade B and C of sperm movement in raw semen, and DNA content after density gradient centrifugation, which all accounted for 76.7% of fertilization predictive value.

Effect of bromocriptine on cell apoptosis and proliferation in GH3 cell culture.

OBJECTIVES: In our study, with the use of GH3 cells line we decided to examine 1) what is the relation between the dose of bromocriptine and the development of apoptosis in GH3 cells 2) whether the induction of apoptosis is accompanied by alterations in bcl-2 and p53 content and 3) whether dibutyryl-cAMP or phorbol esters affect the initiation of apoptosis in GH3 cells. RESULTS: The current study demonstrated the absence of alterations in GH3 cells incubated for 24 h with bromocriptine at the concentrations of up to 15 micromol/l. Apoptotic and necrotic changes were observed after 48 h incubation with bromocriptine at the concentrations of 25 micromol/l. The ratio of necrotic to apoptotic cells increased at 40 micromol/l of bromocriptine concentration. An inhibitory effect of bromocriptine on cell proliferation was also observed. Phorbol-12-myristate-13-acetate (PMA), at concentrations ranging between 25 ng to 200 ng/ml, reduced the amount of apoptotic cells. CONCLUSIONS: Application of dibutyryl-cAMP at the concentration of 1 to 8 mmol/l resulted in an inhibition of apoptosis, followed by an increase in the number of cultured cells. Ultrastructural studies showed evident apoptotic lesions in the cells.

Plasma membrane translocation of phosphatidylserine in human spermatozoa.

The purpose of the study was to examine the phosphatidylserine translocation in human spermatozoa membrane during capacitation. Material consisted of human semen from normozoospermic men. Spermatozoa were stained with fluorescein-labelled annexin V. The presence and distribution of annexin V binding sites were analysed using the fluorescence microscope. Within first 60 min afterejaculation, 5-39% viable annexin V-positive spermatozoa were detected. The annexin V binding sites were found mainly in the midpiece. After 4 to 8 h of incubation of spermatozoa in capacitation medium (BMI), the number of cells positively stained with annexin V increased. After capacitation, the localisations of phosphatidylserine was changed and the annexin V binding sites were found also in the acrosomal region but never in the equatorial area. The process of the phosphatidylserine translocation observed during our experiments may reflect changes of the plasma membrane occurring during capacitation or, less likely, apoptosis of spermatozoa.

Temperature dependent hysteresis-like changes of motility parameters in human spermatozoa.

We studied the impact of temperature changes on selected parameters of normozoospermic spermatozoa motility. The examinations were carried out in the temperature range of 11 degrees C to 21 degrees C both during cooling and heating. We found that a phenomenon of hysteresis, i.e. alternate means of changes in velocity straight linear and lateral head displacement was obtained both at cooling and at heating. This phenomenon was not found for other parameters examined.

Expression of progesterone membrane receptor in spermatozoa from normozoospermic and oligozoospermic men.

The aim of the study was to assess the expression of progesterone membrane receptors in human spermatozoa before and after capacitation. The sperm of 16 men with normozoospermia and 48 men with oligozoospermia was examined. Progesterone-BSA complex labelled with fluorescein isothiocyanate (P-BSA-FITC) was applied to visualise the progesterone receptors. The spermatozoa were capacitated with BM1 medium. In freshly ejaculated sperm from normozoospermic men, P-BSA-FITC staining (bright fluorescence of acrosome region) was observed in 50.1+/-5.1% of spermatozoa. Following capacitation in BM1 medium, the percentage of P-BSA-FITC stained spermatozoa increased to 69.9+/-3.3%. In sperm from slight oligozoospermia the percentage of P-BSA-FITC stained cells before and after capacitation was 48.2+/-8.5% and 67.9+/-4.2%, respectively. In men with severe oligozoospermia the percentage of P-BSA-FITC stained cells was 11.9+/-1.7% and 11.9+/-1.9%, respectively. It is supposed that progesterone membrane receptors in human spermatozoa are gradually exposed during capacitation. Disturbances in the expression of progesterone membrane receptors might be involved in male infertility.

Short-term pre-surgical treatment with somatostatin analogues, octreotide and lanreotide, in acromegaly.

Eighteen patients with symptoms of active acromegaly were treated with somatostatin analogues for 4 weeks before surgery. Both before and after the treatment, levels of growth hormone (GH), prolactin (PRL), insulin growth factor -I (IGF-I), luteotropin (LH), folliculostimulin (FSH) and subunit alpha of glycoprotein hormones were estimated. Glucose tolerance test, magnetic resonance imaging (MRI) examination, sight acuity and field of vision tests were also performed. The same tests were performed on ten control patients with clinically and biochemically active acromegaly, subjected to surgery but not treated with somatostatin analogues. In six patients treated with somatostatin analogues GH levels decreased significantly to less than 5 ng/ml and in two patients remained elevated while in 10 patients GH level decreased and ranged from 6.1 to 42.9 ng/ml. In 13 patients we observed a decrease in IGF-I to normal levels (<400 ng/dl) and in 3 patients we noted a decrease to levels slightly higher than normal. There was also a slight decrease in alpha subunit concentration. In the glucose inhibition test 4 patients demonstrated normalized GH levels. In patients with elevated PRL and TSH levels, treatment with somatostatin analogues induced their decrease. No changes were observed in levels of LH and FSH. After therapy MRI examination disclosed a decrease in tumor volume in two patients (by 20 and 25%, respectively) and no changes in tumor size in 16 patients. The two patients with a decreased tumor volume also showed normalized glucose tolerance tests. All patients manifested an improved clinical condition. Neurosurgeons disclosed a decreased tumor consistency which greatly facilitated surgical procedure. Our studies documented favourable effects of somatostatin analogues on the assayed hormone levels, and on the general condition of the patients as well as on the course of the surgical procedure itself.

[Investigation of occurrence of deletion of the DAZ gene among men with azoospermia and severe oligozoospermia]. / Badania nad wystepowaniem delecji w genie DAZ u mezczyzn z azoospermia i cieza oligozooospermia.

A genetic etiology has been proposed for severe forms of idiopathic male infertility and involvement of variety of genes is critical for the regulation of spermatogenesis. The role of DAZ gene is supported by its exclusive expression in the testis and deletions of DNA sequence within this gene are associated with azoospermia or severe oligozoospermia. The objective of our study was to validate the PCR strategy, using primers based on cDNA sequence, for the detection of genomic aberration among males with azoospermia or severe oligozoospermia who carry mutations in the DAZ gene. Using the PCR technology, deletion of the DAZ gene in one infertile male with azoospermia was detected. No deletion was detected in any of the remaining 15 patients.

[Kinetics of the changes in free calcium ions concentration in human spermatozoa under the effect of progesterone]. / Kinetyka zmian stezenia wolnych jonów wapniowych w plemnikach ludzkich pod wplywem progesteronu.

In the studies, changes in concentration of free calcium ions were analysed in human spermatozoa, as affected by steroid hormones. The experiments were performed on human sperm preparations originating from healthy donors which fulfilled criteria of WHO (1992). The cells separated by the swim-up technique were loaded with the Fura-2AM (4 mM). The changes in concentration of Ca2+ were monitored employing the ratio-imaging technique (MagiCal, Applied Imaging, England). We examined the influence of progesterone, hydrocortisone, RU 486, testosterone, 17 b-estradiol, pro-BSA on Ca2+ concentration. The performed experiments demonstrated that already a few seconds (5-150s) exposure to progesterone and prog-BSA induced a dose dependent increase in Ca2+ concentration in normal spermatozoa. The cell characteristics (no transcription), reaction time, dose dependent and the reaction to pro-BSA, show that progesterone acts through the membrane receptors.

[Studies on DNA content in sperm of men with oligozoospermia]. / Badania nad zawartoscia DNA w plemnikach mezczyzn z oligozoospermia.

The aim of our study was to analysis the ploidy of spermatozoa from oligozoospermic individuals. The study was performed in 48 patients with oligozoospermia. Spermatozoal DNA was analysed using MagiCal imaging cytometry. The percentage of aneuploid cells was determined using CYT-2 program. The results indicated aneuploidy in 73% of the examined patients. The amount of cells with abnormal DNA content ranged from 30 to 100%. Monitoring of the DNA in human spermatozoa may have practical applications in establishing the reasons for infertility as well as for screening of the semen for assisted reproduction techniques.

[Investigations on the pathogenesis of primary varicose veins]. / Badania nad patogeneza pierwotnych zylaków konczyn dolnych.

The data from the clinical course and epidemiology of primary varicose veins of lower limb suggest that sex hormones can directly influence the development of the disease through their intracellular receptor localised in cells of venous wall. The purpose of this study was to determine the stereometric differences in the structure of healthy and varicose veins of lower limb and to determine the presence and localisation of oestrogen and progesterone receptors in the cells of vein. The segments of greater saphenous vein obtained from the 8 women operated for varicose vein were used for the study. The segments of the greater saphenous vein obtained from 8 women that underwent femoro-popliteal venous bypass procedure were used as control group. The vein samples for stereometric analysis were preserved in Buin's solution, embedded in paraffin and then evaluated with automatic analyser MagiCal. To determine the presence of oestrogen and progesterone receptors the immunohistochemic analysis LAB with monoclonal antibodies produced by DAKO was used. The decreased smooth muscle fraction in venous wall, thickening of adventitia, the change of the smooth muscle cells to stroma cells ratio in the muscular layer of venous wall and change of muscular layer to adventitia ratio were observed in varicose veins in comparison with control group. The oestrogen receptors were found in the nuclei of the smooth muscle cells and endothelium. The progesterone receptors were localised in nuclei of smooth muscle cells and cells of subendothelial layer. It seems that quantitative analysis of sex hormones receptor in the venous wall could be useful in the determination of patients with increased risk of the development of primary varicose veins.