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BACKGROUND: Apoptosis plays an important role in regulating spermatogenesis. However, the biological significance of apoptosis in ejaculated sperm is not yet clear. This study set out to investigate how apoptosis correlates with semen quality and the presence of seminal leukocytes. METHODS: Fifty-seven semen samples from the male partners of infertile couples were classified as normal or abnormal according to World Health Organization guidelines. Flow cytometry was used to evaluate sperm populations and seminal leukocytes. Preliminary flow cytometry analysis using 6-carboxyfluoresceindiacetate (6-CFDA), which identifies live cells, and propidium iodide (PI), which stains only dead cells, was performed in order to pinpoint the sperm region accurately. Having thus gated the sperm population, bivariate Annexin V/PI analysis was then carried out in order to measure the percentage of apoptotic and necrotic sperm and the apoptotic index (the ratio between apoptotic:live sperm). Leukocytes were counted by the standard peroxidase test and by flow cytometry using monoclonal antibody (mAb) anti-CD45 or anti-CD53. RESULTS: No significant differences in the apoptotic index and the percentage of live and apoptotic sperm were detected between the subjects with normal and abnormal semen. A significant inverse correlation between sperm concentration and the apoptotic index was observed only in the normal sperm group. There was no correlation between the concentration of leukocytes, detected either by peroxidase or by mAb anti-CD45 or anti-CD53, either with the percentage of apoptotic sperm or with the apoptotic index. In contrast, the ratio between CD45 positive leukocytes and sperm showed a significant correlation with the apoptotic index. A weaker correlation was found when leukocytes were counted by peroxidase, while no correlation was observed using mAb anti-CD53. CONCLUSIONS: Sperm apoptosis did not seem to be correlated with semen quality. In the absence of genito-urinary infection, one of the main functions of seminal leukocytes is probably to provide for the removal of apoptotic sperm.

The feasibility of allogenic implants of chondrocytes in alginate gels was tested for the reconstruction in vivo of artificially full-thickness-damaged articular rabbit cartilage. The suspensions of chondrocytes in alginate were gelled by the addition of calcium chloride solution directly into the defects giving in situ a construct perfectly inserted and adherent to the subchondral bone and to the walls of intact cartilage. The tissue repair was controlled at 1, 2, 4 and 6 months after the implant by NMR microscopy, synchrotron radiation induced X-ray emission to map the sulfur of glycosaminoglycans and by histochemistry. Practically a complete repair of the defect was observed 4-6 months from the implant of the chondrocytes with the recovery of a normal tissue structure. Controls in which Ca-alginate alone was implanted developed only a fibrous cartilage.

Vascular invasion of calcified cartilage, during endochondral ossification, is initiated and sustained by invasive cells (endothelial cells and macrophages) which degrade the tissue by releasing lytic enzymes. Concurrently, reactive oxygen species (ROS) are also released by these cells and we hypothesize that ROS also contribute to the degradation of the tissue. As a preliminary approach to this problem, the antioxidant activities and the effect of ROS on hypertrophic cartilage and chondrocytes (HCs) were investigated. Compared to resting or articular chondrocytes, HCs exhibited higher catalase but lower SOD specific activities and lower PHGPx concentration, thus revealing a defence activity specific against H2O2. Moreover, dose-dependent depletion of ATP occurred after few minutes of exposure to ROS, and a long-term treatment (16 h incubation with ROS) promoted the release of LDH activity and a significant variation of the poly- to mono-unsaturated fatty acid ratio. Finally, the incubation of HCs with low ROS doses induced the release of sedimentable alkaline phosphatase activity (matrix vesicles). How the obtained results fit the in vivo occurring events is discussed.

Objective: The objective of this study was to find a correlation between magnetic resonance (MR) appearance and biochemical composition of the normal articular cartilage by comparing the laminar aspects with the distribution of the two principal matrix components: proteoglycans and collagen. Design: T 2 -weighted MR microimages of porcine cartilage-bone plugs, excised from both the habitually loaded and habitually unloaded regions of the proximal end of the humerus, were obtained using a spin-echo sequence. Proteoglycans (PGs) were monitored by histology and by measuring the uronate and the sulfur content of the tissue; a histological method and the chemical determination of hydroxyproline were used for the evaluation of the collagen content. Results: The ‘loaded' cartilage exhibited the expected MR laminar appearance whereas the ‘unloaded' tissue appeared to be more homogeneous. The PG content in the ‘loaded' cartilage, was found to be 2.4 times higher than in the habitually unloaded tissue, exhibiting an increasing trend from the articular surface to the bone. In the ‘unloaded' cartilage the uronate distribution was more uniform with a higher concentration in the intermediate zone. The mean collagen content of both cartilage regions was found to be about 39% of the tissue dry weight. Histology and hydroxyproline distribution pattern showed that collagen was particularly concentrated at the surface and in a central zone of the ‘loaded' cartilage whereas in the ‘unloaded' tissue collagen was evident only at the surface. In accordance with the collagen distribution, transverse relaxation (T 2 ) times in ‘loaded' cartilage showed a minimum value at the articular surface and another minimum in a central region. On the contrary, the average T 2 value of the ‘unloaded' tissue was high at the surface and decreased rapidly in the deeper zones. Conclusion: These results demonstrate that the MR appearance of articular cartilage correlates with the collagen content, but not with that of PGs, of the different zones. Other matrix components might, however, influence the MR appearance by contributing to the macromolecular organization of the tissue.

Some aspects of the physiology of chondrocytes from horse articular cartilage were studied, since this animal model can be helpful in understanding arthritic processes. The replicative ability of articular chondrocytes, measured by the incorporation of [ 3 H]thymidine, and their capacity of proteoglycan production, evaluated from the incorporation of [ 35 S]sulfate, are very low. In addition, these cells do not differentiate in vitro as shown by the constant specific activity of alkaline phosphatase measured at different times in culture. Two types of potassium channels were identified by patch clamp experiments in the cell-attached configuration, one characterized by a conductance of 40 pS and the other of 100 pS. No active K + channels were found at V pip = 0. It was shown by Furs-2 experiments that the low replicative ability is paralleled by a modest variation of the intracellular calcium concentration after a mitogenic stimulus. 31 P NMR experiments, both on slices of whole articular cartilage and on isolated cells, demonstrate that chondrocytes derive their energy mainly from the glycolytic pathway.