Physiology of peroxidation precesses in mammalian sperm

Reactive oxygen species are produced during normal mitochondrial respiration processes. Intracellular homeostasis is among other factors dependent on the balance between ROS production and antioxidant systems. When this balance is threatened or ceases to exist pathologies can arise (cancer, degenerative diseases, and reproductive disorders). In this thesis we focus on how molecular peroxidation affects the fertility characteristics of bovine cryopreserved sperm. Firstly it is described how new methodologies can be used to detect sperm plasma membrane susceptibility to peroxidative damage before and after cryopreservation processes. These techniques are useful for selection of sires to be used in insemination programmes. Furthermore it is shown that cholesterol oxidation products (COPs) are already present in ejaculated sperm, whilst for other lipid molecules such as phospholipid- phosphatidylcholine, peroxidation is observed after cryopreservation. One of the most toxic COPs- triol was almost absent on sperm lipid extracts. However, exposure to pro-oxidants dramatically increased the total COPs content, but surprisingly did not elevate the concentration of triol. In addition the oxidative challenge increased levels of triol on sperm membrane reconstituted lipid vesicles suggesting that sperm cells may metabolize this toxic COP. Furthermore, COPs may play signaling roles on pathways that drive sperm to capacitation and acrosome reaction, contributing for acquisition of fertilizing ability. Several methodologies that are currently available not only on lipid peroxidation detection but also for protein and DNA oxidation are described in detail in this thesis. It is also shown how determined oxidation conditions influence frozen-thawed fertilizing ability and subsequent embryo development in vitro. Frozen-thawed sperm was exposed prior to IVF during three hours to three different oxidative treatments, (a) 100 µM H2O2; (b) 500 µM H2O2 and (c) 500 µM H2O2 and 20/100 µM Fe2+/Ascorbate. These pro-oxidants are known for their peroxidative properties namely in lipids and DNA. Two control groups were considered, (d) where sperm was equally incubated for 3 h without oxidants and (e) where IVF procedure occurred according to protocol without any previous incubation. The results showed that fertilization took place in all conditions, although incubations (c) showed the lowest cleavage rate compared to all the other groups followed by incubation (b) that had significantly lower cleavage rates than incubations (d) and (e). Embryo development was impaired for condition (c) where a 2-cell block was observed, and condition (b) had lower percentage of Day 9 blastocysts than both control incubations [(d) and (e)]. Flow cytometry analysis showed that DNA oxidation was observed for incubations with highest concentration of oxidants (b) and (c), whereas plasma membrane oxidation was highest from incubation (c). On the other hand percentages of viable and acrosome intact sperm cells were lower only between sperm samples that were incubated for 3 hours and group (e), despite the presence or absence of oxidants. Equally mitochondrial and intracellular oxidations were highest in incubation (c). These results suggest that frozen-thawed sperm can withstand a certain degree of oxidative damage without impairing its acquisition of fertilizing ability (capacitation, acrosome reaction) or impair fertilization itself. And the oocyte plays a very important role in repairing damage that is brought by the sperm upon fertilization and allowing further embryo development.