The mechanism of copper effect on the spermatozoa in vitro
Zuzana Kňažická 1
Norbert Lukáč 1
1 Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic
|Section:||Cellular metabolism, physiology, molecular biology and genetics|
Copper as an important trace and essential element for the organism has various effects on the male reproductive system, which can be strongly reflected in the process of spermatogenesis. The aim of the present study was to evaluate the dose- and time-dependent effects of copper on the motility, viability, structural and functional characteristics of spermatozoa. Furthermore, also expand the knowledge of its impact on the fertilization potential of the spermatozoa.
Bovine semen samples were obtained from adult breeding bulls (SBS, Nitra – Luzianky). Spermatozoa were cultivated with various concentrations (1000; 500; 250; 125; 62.50; 31.20; 15.60; 7.80 or 3.90 µmol.dm-3) of copper (CuSO4.5H2O) in the laboratory at room temperature (22-25 ˚C). The motility analysis was carried out using a CASA system - SpermVisionTM program and Annexin-V-FLOUS was used for detection of the membrane integrity of spermatozoa. The morphological analysis we assessed the abnormal spermatozoa forms (Giemsa staining). The viability of the cells we assessed by the MTT (metabolic activity) assay.
The initial spermatozoa motility in the presence of copper in physiological saline solution showed significantly (P<0.001) decreased values at concentrations ≥ 250 µmol.dm-3 and concurrently they have a cytotoxic effect on the mitochondrial complex of spermatozoa. The low concentrations (≤ 7.80 µmol.dm-3 of CuSO4.5H2O) stimulated the mitochondrial activity of cells and maintained of spermatozoa motility (Time 2 h). The long-term cultivation (Time 24 h) significantly (P<0.001) reduced the average motility values in all experimental groups compared to control group. Annexin-V fluorescence reaction was detected at the highest concentrations (500; 1000 µmol.dm-3 CuSO4.5H2O) at the beginning of incubation. The group with the highest concentration (1000 µmol.dm-3 CuSO4.5H2O) and the longest time of exposure (6 h) caused significant distabilization of lipid membrane systems not only in the mitochondrial segment, but also on the head of spermatozoa (acrosomal and postacrosomal part), what subsequently led to the destruction of membranes in these parts of the spermatozoa. The total percentage of pathological abnormal spermatozoa was significantly higher in the group with the highest concentration of copper (9.63 %) in comparison to control group (4.79 %) after 24 h. Predominant morphological abnormalities were separated flagellum 2.96 % (P<0.05), flagellum torso 2.90 % (P<0.001), knob twisted flagellum 1.10 % (P<0.001) and broken flagellum 1.06 % (P<0.001) in connection with segment (mid-piece) of spermatozoa.
Based on our results, we can concluded, that toxic and cytotoxic effect of copper appeared on the individual cell structure of spermatozoa depends on time. In functional aspects, all of these subtile changes could disrupt mechanism of spermatozoa motility.