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.