The presence of biochemical signs of apoptosis in ejaculated spermatozoa suggests that apoptosis may be one of the pathways for sperm death. The relationship between the phosphatidylserine (PS) externalization and the presence of the active form of caspase-3 (CP-3) was studied in human spermatozoa after exposure to hydrogen peroxide (H(2)O(2)). Semen from 27 normozoospermic men was examined, as the neat semen, after swim-up isolation and after H(2)O(2) incubation, for the translocation of PS, activation of caspase-3 and mitochondrial membrane potential. The percentage of vital spermatozoa expressing PS translocation was lower than the percentage of vital spermatozoa with the active form of the caspase-3, either in neat (4.9 +/- 2.3% versus 19.7 +/- 6.2%, P < 0.001) or in swim-up semen (2.2 +/- 2.3% versus 4.8 +/- 2.9%, P < 0.01). After swim-up isolation, the percentage of vital spermatozoa with active caspase-3 decreased (P < 0.01). After H(2)O(2) stimulation of the swim-up semen fraction, a decrease in the mitochondrial membrane potential was observed (P < 0.001). Only the midpiece revealed PS translocation after H(2)O(2) stimulation, and it was also the only part to reveal the presence of the active form of caspase-3. All spermatozoa expressing the PS translocation revealed the presence of the active form of caspase-3.

Abstract. Degeneration of the intervertebral disc (IVD) is the main cause of age-related damage of spinal tissues. Using multipotent mesenchymal stromal cells (MSCs) regenerative medicine intends to restore the IVD components of annulus fibrosus (AF) and nucleus pulposus (NP). In the present study NP cells (NPCs) and MSCs obtained from adolescent patients suffering from scoliosis were used. IVDs and vertebrae were obtained during surgery and subsequently processed in order to establish cultures of NPCs and MSCs. The two cell types were co-cultured in 1-µm pore size insert system (indirect co-culture) or on one surface (direct co-culture). Prior to co-culture in these systems one of the cell types was stained by lipophilic fluorescent dye DiD (red). The results demonstrated that regardless of the cell type, the flow of DiD from stained to non-stained cells was more efficient in the direct co-culture in comparison with the insert system. Moreover, in the direct system the DiD flow was more efficient from MSCs towards NPCs compared with that in the opposite direction. These data indicated that the membrane interchange between the two cell types was asymmetric. To discriminate the subpopulation of cells that underwent membrane interchange, cells were double stained with DiD and DiO (green). In the first part of the experiment NPCs were stained by DiO and MSCs by DiD. In the second, NPCs were stained by DiD and MSCs by DiO. The cells were co-cultured in the direct system for 8 days and subsequently analyzed by flow cytometry and confocal microscopy.This analysis revealed that >50% of cells were stained by the DiO and DiD dyes. NPCs and MSCs formed structures similar to tunnelling nanotubes (TnT). In conclusion, the formation of TnT-like structures is able to promote, phenotypic changes during the direct co-culture of NPCs with MSCs.

DiO and DiD are lipophilic cell labelling dyes used in the staining of cells in vivo and in vitro. The aim of the present study was to quantify the asymmetrical distribution of dyes in co-cultured cells and to measure the intercellular transfer of DiO and DiD. DiO and DiD were applied separately to stain two identical populations of SW-1353 human chondrosarcoma cells that were subsequently co-cultured (homotypic co-culture). The intercellular migration of dyes in the co-cultured cells was measured by flow cytometry and recorded under a fluorescent microscope. DiD and DiO caused no effect on the proliferation of cells, the degradation rate of the two dyes was comparable and crossover effects between dyes were negligible. The results of the present study suggested that asymmetrical intercellular migration of DiD and DiO was responsible for the asymmetrical distribution of these dyes in co-cultured cells. To take advantage of the lipophilic dyes migration in the double-stained co-cultured cells we suggest to apply mixed-dyes controls prior to the flow cytometric analysis. These controls are performed by staining cells with a 1:1 mix of the two dyes and would enable the estimation of the intensity of intercellular contact in co-culture systems. A 1:1 premix of DiO and DiD was applied to estimate cellular effect on intercellular exchange of lipid dyes in co-cultures incubated with cycloheximide and cytochalasin B. The cellular effect contributed 6–7% of intercellular migration of the lipophilic dyes, DiO and DiD. The majority of the observed intercellular transfer of these dyes was due to non-cellular, passive transfer.

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The aim of this study was to prospectively investigate the spermatozoa ultrastructure in relation to the results of in vitro fertilization -embryo transer (IVF-ET).Forty-nine consecutive couples admitted for IVF-ET were prospectively evaluated for electron microscopic spermatozoa morphology and the outcome of IVF-ET. Thirty-four couples revealed successful fertilization, defined as presence of two pronuclei 14-16 hours after spermatozoa administration, while the remaining 15 formed the failure group. Spermatozoa fixed with 2.5% glutaraldehyd and embedded in Spurr's resin were analyzed with JAM 100 S transmission electron microscope (TEM) for the following ultrastructure abnormalities: head deformity, cytoplasmic residues, chromatin condensation failures, acrosomal alterations, neck defects, midpiece defects, principal piece and end-piece defects and immature forms.Successful IVF-ET couples revealed a significantly higher percentage of normal spermatozoa utrastructure (32.0 AE 13.1% versus 17.1 AE 13.4%, p < 0.001). Failed IVF-ET couples represented a significantly higher percentage of chromatin condensation failures (9.8 AE 5.1% versus 5.7 AE 5.3%, p < 0.05) and tail defects (16.7 AE 11.5% versus 7.2 AE 7.2%, p < 0.001). A positive correlation between normal ultrastructure spermatozoa percentage and fertilized oocytes percentage was found (r ¼ 0.35, p < 0.05).Our data suggest that spermatozoa TEM findings correlate with IVF-ET results. Ultrastructural estimation of spermatozoa can improve the diagnosis of male fertility and may explain some reasons of failure in assisted reproduction methods. We consider systematic TEM spermatozoa examination in cases with failed IVF-ET prior to intracytoplasmic sperm injection (ICSI). KEYWORDS in vitro fertilization, spermatozoa, ultrastructureAbbreviations: IVF-ET: in vitro fertilization -embryo transfer; TEM: transmission electron microscope; ICSI intracytoplasmic sperm injection.

The aim of this paper is to present methods for a fully computerised analysis of spermatocyte movement. The technique were designed for processing pure images of spermatozoa captured from a light microscope. The techniques described allowed for the reduction of background light inhomogeneity and for the correct detection of moving cells and involved densitometric equalization of background inhomogeneity and implementation of the dynamic threshold, adopted for the recognition of objects. The method for relating cells (found in each frame processed) to movement trajectories used heuristic rules, describing the behaviour of moving cells. This permitted samples containing high numbers of spermatocytes within the observed area to be processed with maximum accuracy.

Oxytocin enhanced basal corticosterone secretion by dispersed rat adrenal zonae fasciculata and reticularis cells, the maximum effect being observed at a concentration l0_9-10_8 M. Video-imaging analysis revealed the existence of a small population of isolated cells (about 5%), responding to the addition of oxytocin (10_8 M) by a marked rise in the cytosolic free Ca" concentration. An interesting phenomenon was also noted, suggesting the propagation of the oxytocin-evoked signal from activated to non-activated cells.The role of oxytocin in the regulation of adrenocortical function is far from being fully understood (see 4, for review). The presence of oxytocin has been demonstrated in the adrenal glands of many mammalian species, including the rat (1): immunoreactive oxytocin is distributed throughout the entire cortex with the most intense staining in zona glomerulosa, while in the medulla only norepinephrine cells are immunopositive. Such a localization of oxytocin suggests the involvement of this peptide in the regulation of adrenal cortex function. Accordingly, Hinson er a/. (2) demonstrated a direct stimulatory effect of oxytocin on aldosterone secretion. Here we report findings showing that oxytocin exerts a direct glucocorticoid secretagogue effect on dispersed rat adrenal zonae fasciculata and reticularis cells (inner adrenocortical cells), by activating intracytoplasmatic Ca2+ redistribution in a small population of responsive cells. MATERIALS AND METHODSThe adrenal glands removed from adult female Wistar rats were decapsulated, and isolated inner adrenocortical cells were obtained by sequential collagenase digestion and mechanical dispersion (8). The viability of isolated cells was checked by the trypan-blue exclusion test and found to be higher than 92%.Dispersed cells obtained from 6 rats were pooled to obtain a single cell suspension, and 6 cell preparations were employed. Aliquots of each cell suspension (105 cells/ml) were incubated with oxytocin (Gedeon-Richter, Budapest, Hungary). The incubation was carried out for 60 min in a shaking bath at 37°C in an atmosphere of 95% O2 and 5% CO2. Corticosterone was measured in the medium by a specific radioimmunoassay (5). Intraand inter-assay variations were 6% and 8%, respectively.

Summary The dynamic development of technical sciences and informatics makes now possible acquisition of microscopic images of histological sections, not only using digital cameras, but also through specialized devices called scanners. The digitalized images stored in a computer storage device are called virtual slides and, together with special software, are known as virtual microscopy. The virtual slides can be analyzed on a computer screen by panoramic viewing or using a detailed image examination at higher magnification. In many research and education institutions in both the U.S. and Europe, the virtual microscopy is used for teaching and training purposes. In the academic year of 2009/10, Department of Histology and Embryology, University of Medical Sciences in Poznan, as one of the first in Poland, has created a virtual database for educational purposes. This database created by archiving the traditional images of histological slides in the form of digital images. So far, more than 130 virtual slides have been acquired and catalogued in 24 thematic folders, available for medical students participating in histology, embryology and cell biology courses. Telepathology is the second branch which uses virtual microscopy. Virtual microscope allows to discuss and resolve medical/diagnostic problems with the use of telecommunication systems and information technology. The existing internet platforms offer access to virtual microscopes and virtual slides. In June, 2011 the Center of Morphologic Images Archivization and Digital Database of Microscopic Pictures in the Department of Histology and Embryology, Poznan University of Medical Sciences has launched an online platform (www.caom.pl), aimed to provide the central database of scanned histological sections of physiological tissues, and pathological, rare and sporadic lesions, including tumor