*2.2. Capacitation and Sperm-Egg Interaction*

Another redox-regulated aspect of sperm function that has received little attention is sperm-zona interaction. When human spermatozoa are incubated with ferrous ion promoters to enhance lipid peroxidation, there is, at levels of peroxidation that are still compatible with full viability and motility, a dramatic increase in the ability of these cells to bind to the zona pellucida [8]. This phenomenon has been used to enhance levels of fertilization in a mouse in vitro fertilization system in which the induction of sublethal levels of lipid peroxidation was shown to significantly increase the number of spermatozoa binding to the zona pellucida [49]. How the induction of peroxidative damage to the sperm plasma membrane enhances sperm-zona binding is unknown. It does not appear to involve a generalized increase in sperm adhesiveness because no amount of lipid peroxidation will enhance zona binding if the latter has been precoated with an anti-zona antibody (unpublished observations). During fertilization, the oocyte is also thought to generate ROS and may play an active role in the process of zona hardening, a proposed component of the block-to-polyspermy. Zona hardening is thought to be induced by peroxidases released during the cortical granule reaction enhancing the creation of molecular cross-links within zona pellucida with the aid of a hydrogen peroxide burst associated with fertilization [50]. This mechanism has been elegantly demonstrated in the case of the sea urchin fertilization envelope where the dual oxidase, Udx1, has been shown to generate the hydrogen peroxide associated with the hardening of this membrane [51]. The existence of an analogous process in mammalian oocytes seems likely; however, while these cells are known to contain a variety of oxidases capable of generating ROS [52], their role during fertilization remains largely unexplored. Whatever mechanisms are involved, it is clear that many aspects of sperm biology including tyrosine phosphorylation, cholesterol exclusion, hyperactivation and sperm-egg interaction are redox regulated.

As a result, capacitating spermatozoa in either the female reproductive tract or in vitro can be thought of as under physiological oxidative stress. In order to protect the spermatozoa during this critical time in their life history, sophisticated antioxidant defense mechanisms have developed, involving such key players as glutathione-*S*-transferase omega 2 and peroxiredoxin 6 [53,54]. As a consequence of such strategies, spermatozoa can engage in a redox-regulated capacitation cascade without fear of succumbing to the oxidative stresses involved. However, if a spermatozoon should fail to find an egg and the capacitation period is prolonged, even these defensive mechanisms are ultimately overwhelmed and the cell, now in a state of "over-capacitation", enters a senescence pathway culminating in the enhanced release of ROS from the mitochondria and cell death [24]. Thus, capacitation and senescence can be regarded as components of redox-regulated continuum [55].

### *2.3. Inadequate Antioxidant Protection from Seminal Plasma*

As a biological response to oxidative stress, seminal plasma has evolved one of the most powerful antioxidant fluids known to man, replete with a range of antioxidant enzymes and small molecular mass free radical scavengers that, combined, generate a level of total antioxidant power that is estimated to be 10× higher than blood [56]. This antioxidant cocktail includes catalase, superoxide dismutase (SOD), glutathione peroxidase, glutathione-S-transferase and peroxiredoxins as well as water-soluble (uric acid, hypotaurine, tyrosine, polyphenols, vitamin C, ergothionine and glutathione) and fat-soluble (all-*trans*-retinoic acid, all-*trans*-retinol, α-tocopherol, carotenoids and coenzyme Q10) scavengers [56–58]. There are now many studies indicating that there is a consistent negative relationship between the levels of antioxidant protection provided by seminal plasma and the appearance of male infertility as well as the incidence of miscarriage [33,59]. A recent development in this field has been the suggestion that the measurement of oxidation-reduction potential (ORP) in human semen samples is predictive of oxidative stress [60]. It will be of interest to determine whether such ORP measurements are providing diagnostic information over and above the measurement of total antioxidant potential. It will also be

fascinating to determine whether the decreased seminal antioxidant protection observed in cases of male infertility is a result of local [61] or systemic [62] pro-oxidant factors.
