Biochemical and physiological changes in bovine sperm during extended incubation under aerobic and anaerobic conditions
There are two main systems for storing diluted bovine semen for artificial insemination. It is either cryo-preserved in liquid nitrogen or held in liquid form at ambient temperature. During storage at ambient temperature, reactive oxygen species generated in the presence of oxygen are a major cause of cellular damage eventuating in the loss of fertility of sperm cells. This has been known for many years and current production systems for storage of semen in the liquid state attempt to minimise oxidative stress. This is achieved by reducing oxygen tension in the diluent through nitrogen gassing and by removal of hydrogen peroxide by catalase. However, the complete elimination of oxidative damage is not achieved because of diffusion of oxygen back into the diluent during re-dilution and packaging of individual insemination doses. The present study was conducted to broaden the understanding of the biochemical mechanisms contributing to the drop in fertility during in vitro storage of sperm at ambient temperature, in relation to the oxidative state of the storage medium. To this aim, the survey was undertaken to determine the biochemical and physiological changes occurring in sperm during extended incubations (storage) under conditions of either complete exclusion of oxygen from the diluent, reduced oxygen tension, normal atmospheric conditions or elevated exposure to reactive oxygen species. Velocities of bovine sperm in a medium containing glucose were found to be similar under fully anaerobic and aerobic conditions. Sperm were not able to sustain motility under anaerobic conditions when glycolysis was inhibited, but regained motility when re-aerated. This demonstrated that immobilisation when glycolysis was inhibited was due to lack of oxygen and that conditions under which motility was analysed were truly anaerobic. Sperm motility parameters were not significantly different in the presence and absence of 4 μmol/1 antimycin A and 4 μmol/1 rotenone when glucose was present in the medium showing that glycolysis alone could fully sustain motility. After each incubation, functionality of sperm mitochondria was assayed by washing sperm into a medium that supported respiration but not glycolysis and motility was visually assessed. All sperm samples were highly motile in this medium indicating that their mitochondria were functional. When glycolysis was inhibited, antimycin and rotenone abolished motility immediately after addition. Thus it was concluded that bovine sperm can maintain similar levels of motility aerobically and anaerobically if a glycolysable substrate is available. Changes in protein tyrosine phosphorylation, viability and motility were studied as a function of extended storage of bovine sperm in vitro at ambient temperature (18-20°C). Over a period of 8 days of storage at ambient temperature there was a time-dependent decline in sperm motility. This decline, to some extent, was reversed by incubation of stored sperm with theophylline, indicating that it was due to a decline in activation rather than the deterioration of sperm. Two soluble proteins (Mᵣ=67 000 and Mᵣ=36 000) displayed tyrosine phosphorylation the sum of which declined with the time of storage. Tyrosine phosphorylation of a number of proteins from whole cell extracts increased in a time-dependent manner during in vitro storage. The influence of the oxygenation state of the storage medium had profound effects on tyrosine phosphorylation of proteins from whole cell extracts. Tyrosine phosphorylation of proteins from whole cell extracts increased considerably during anaerobic storage while there was no significant change during aerobic storage. This increase in phosphorylation under anaerobic conditions was reversed when sperm were transferred from an anaerobic to an aerobic environment, indicating that the oxygenation state of the medium regulates both protein tyrosine kinases and phosphatases. In addition, sperm stored under aerobic conditions for five days retained the ability to phosphorylate proteins when transferred to an anaerobic environment. Sperm motility and viability declined more rapidly under aerobic compared with anaerobic conditions. Results from the flow cytometry experiments showed that the integrity of the sperm membrane was maintained better under anaerobic than under aerobic storage conditions. It was demonstrated that exogenous hydrogen peroxide either directly added to the diluent or generated through the enzymatic oxidation of phenylalanine was detrimental to sperm motility and the integrity of the plasma membrane. The assessment of sperm DNA susceptibility to in situ acid denaturation by the Sperm Chromatin Structure Assay did not detect any difference between sperm stored under aerobic and anaerobic conditions in a standard diluent. Exposure to exogenous hydrogen peroxide was detrimental to chromatin stability, increasing the DNA susceptibility to in situ acid denaturation. Inclusion of a general deoxyribonuclease inhibitor aurintricarboxylic acid in the diluent dramatically decreased sperm chromatin stability under both aerobic and anaerobic conditions, suggesting that this inhibitor had a damaging effect on sperm DNA. The effects of storage time and the oxygenation state of the storage medium on motility, viability and in vitro fertility of stored sperm were investigated. In vitro fertility of sperm declined with the time of storage and there was no significant effect of the oxygenation state of the medium on in vitro fertility of stored sperm. The results of in vitro fertility trials suggests some form of sperm competition or oocyte selection of sperm based on its the ability to induce normal development. In conclusion, the oxygenation state of the diluent in which sperm are stored in vitro has a significant effect on sperm metabolism, tyrosine phosphorylation signalling pathways and maintenance of viability. The impact of oxygenation state of the diluent on the in vivo fertility of stored sperm requires further investigation.
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